WO2020002694A1 - Microrna targeting agent for treatment of heart disease - Google Patents

Microrna targeting agent for treatment of heart disease Download PDF

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Publication number
WO2020002694A1
WO2020002694A1 PCT/EP2019/067497 EP2019067497W WO2020002694A1 WO 2020002694 A1 WO2020002694 A1 WO 2020002694A1 EP 2019067497 W EP2019067497 W EP 2019067497W WO 2020002694 A1 WO2020002694 A1 WO 2020002694A1
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mir27b
mice
cre
acid agent
mlc2v
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French (fr)
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Jaya Krishnan
Corinne BISCHOF
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United Kingdom Research and Innovation
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United Kingdom Research and Innovation
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Priority to CN201980055950.5A priority Critical patent/CN112638475B/en
Priority to JP2020573348A priority patent/JP2021529202A/en
Priority to US17/256,206 priority patent/US20210155930A1/en
Priority to EP19737488.7A priority patent/EP3813941A1/en
Publication of WO2020002694A1 publication Critical patent/WO2020002694A1/en
Anticipated expiration legal-status Critical
Priority to JP2024085628A priority patent/JP2024112962A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA

Definitions

  • the present invention relates to the treatment and prevention of heart disease by administering oligonucleic acid agents that modulate the activity or expression of microRNAs. More precisely, the invention provides methods for treating or preventing heart disease by inhibiting the expression and/or activity of the microRNA miR27b-5p.
  • HCM hypertrophic cardiomyopathy
  • AS aortic stenosis
  • HCM and AS epitomize the cardiac overgrowth phenotype and are characterized by endoreplication, resulting in polyploidy and multinucleation (cells with two or more nuclei), and pathologic cardiomyocyte growth in an environment of depressed mitochondrial ATP synthesis.
  • energetically deficient cardiomyocytes exhibiting a reduced capacity to generate energy and cofactors though mitochondrial oxidation, are able to drive anabolic processes to support cardiomyocyte hypertrophy.
  • mitochondrial myopathies are predominantly characterized by cardiac overgrowth.
  • ADP and inorganic phosphate as downregulated in HCM and AS biopsies.
  • HIF hypoxia-inducible factor
  • MIR27B the stress dependent-regulated hypoxia-inducible factor
  • Inhibition of ATP5A1 suppresses mitochondrial ATP synthase activity, leading to accumulation of intra-mitochondrial ADP that is channeled and serves as a cofactor for MTHFD1 L, a mitochondria localized rate-limiting enzyme of the 1 -carbon pathway regulating formate and purine biosynthesis.
  • MTHFD1 L Activation of MTHFD1 L by ADP in cardiomyocytes accelerates de novo nucleotide synthesis and drives cardiomyocyte endoreplication and cell hypertrophy through parallel activation of AMPK-driven E2F. Consequently, MIR27B inactivation in vitro and in mice attenuates pre-existing heart failure in response to surgery-mediated aortic stenosis (transaortic constriction (TAC)), while cardiac-specific MIR27B expression results in spontaneous cardiac overgrowth. Analyses of the downstream components, ATP5A1 and MTHFD1 L in mice, further support a central role for the HIF1 oMIR27B-ATP5A1 -MTHFD1 L axis in cardiac polyploidization and cell size control.
  • the objective of the present invention is to provide means and methods for treatment of heart disease, particularly cardiomyopathy. This objective is attained by the subject matter of the claims of the present specification.
  • the present invention is based upon the discovery that the inhibition of miRNA miR27b-5p through administration of an oligonucleic acid agent (SEQ ID NO 001 ) enables the modification of pathological growth of the myocardium via regulators such as ATP synthase, which drives mitochondrial ADP build-up and its redirection to methylenetetrahydrofolate dehydrogenase 1 L (MTHFD1 L) and de novo nucleotide biosynthesis.
  • SEQ ID NO 001 oligonucleic acid agent
  • miRNA in the context of the present invention relates to pri-, pre- and mature miRNA.
  • miRNA27b-3p There are two mature subspecies of the stem-loop sequence of miRNA27b, namely miRNA27b-3p and miRNA27b-5p.
  • “Capable of forming a hybrid” in the context of the present invention relates to sequences that under the conditions existing within the cytosol of a mammalian cell, are able to bind selectively to their target sequence.
  • Such hybridizing sequences may be contiguously reverse- complimentary to the target sequence, or may comprise gaps, mismatches or additional non- matching nucleotides.
  • the minimal length for a sequence to be capable of forming a hybrid depends on its composition, with C or G nucleotides contributing more to the energy of binding than A or T/U nucleotides, and the backbone chemistry.
  • oligonucleic acid agent in the context of the present specification refers to an oligonucleotide capable of specifically binding to and leading to a significant reduction of the physiological role of miR27b-5p.
  • oligonucleic acid agents of the present invention are antisense oligomers made of DNA, DNA having phosphorothioate modified linkages in their backbone, ribonucleotide oligomers, RNA comprising bridged or locked nucleotides, particularly wherein the ribose ring is connected by a methylene bridge between the 2’-0 and 4’-C atoms, RNA having phosphorothioate modified linkages in their backbone or any mixture of deoxyribonucleotide and ribonucleotide bases as an oligomer.
  • antisense oligonucleotide or oligonucleotide agent in the context of the present specification refers to any oligonucleotide capable of specifically binding to and leading to a significant reduction of the physiological role of miR27b-5p.
  • antisense oligonucleotides of the present invention are antisense oligomers made of DNA, DNA having phosphorothioate modified linkages in their backbone, ribonucleotide oligomers, RNA comprising bridged or locked nucleotides, particularly wherein the ribose ring is connected by a methylene bridge between the 2’-0 and 4’-C atoms, RNA having phosphorothioate modified linkages in their backbone or any mixture of deoxyribonucleotide and ribonucleotide bases as an oligomer.
  • oligonucleic acid agent and antisense oligonucleotide or oligonucleotide agent are used interchangeably in the present specification.
  • the antisense oligonucleotide of the invention comprises analogues of nucleic acids such as phosphotioates, 2’O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl) -glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2 ⁇ , 4’C methylene bridged RNA building blocks).
  • nucleic acids such as phosphotioates, 2’O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl) -glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2 ⁇ , 4’C methylene bridged RNA building blocks).
  • the antisense sequence may be composed partially of any of the above analogues of nucleic acids, with the rest of the nucleotides being“native” ribonucleotides occurring in nature, or may be mixtures of different analogues, or may be entirely composed of one kind of analogue.
  • gapmer is used in its meaning known in the field of molecular biology and refers to an antisense oligonucleotide complementary to its target sequence, that comprises a central block of a deoxyribonucleotide oligomer flanked by short ribonucleotide oligomers.
  • the flanking ribonucleotide oligomers consist of nuclease and protease resistant ribonucleotides.
  • the nuclease and protease resistant ribonucleotides comprise 2’-0 modified ribonucleotides, in particular bridged nucleic acids with a bridge between the 2’-0 and 4’-C of the ribose moiety.
  • nucleotides in the context of the present invention are nucleic acid or nucleic acid analogue building blocks, oligomers of which are capable of forming selective hybrids with miRNA oligomers on the basis of base pairing.
  • the term nucleotides in this context includes the classic ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymin), cytidine, the classic deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine.
  • nucleic acids such as phosphotioates, 2 ⁇ - methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2 ⁇ , 4’C methylene bridged RNA building blocks).
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • the hybridizing sequence may be composed of any of the above nucleotides, or mixtures thereof.
  • sequence identity and percentage of sequence identity refer to the values determined by comparing two aligned sequences.
  • Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981 ), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).
  • sequence identity values refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.
  • a first aspect of the invention provides an oligonucleic acid agent directed against the microRNA miR27b-5p for use in a method of treatment or prevention of heart disease.
  • the oligonucleic acid agent of the invention is directed against, or capable of forming a hybrid with, the miRNA miR27b-5p (SEQ ID NO 002).
  • the oligonucleic acid agent is directed against miR27b-5p miRNA in its mature form.
  • the oligonucleic acid agent is capable of reducing miR27b-5p miRNA levels in a cell by an amount (expressed in percentage) of at least 10-20%, at least 30-40%, at least 50-60%, at least 70-80%, at least 90-98%, or at least 99% when the oligonucleic acid agent is introduced into a mammalian cell.
  • the oligonucleic acid agent for use in a method of treatment or prevention of heart disease comprises, or essentially consists of, the sequence ACC AAT CAG CTA AGC T (SEQ ID NO 001 ).
  • oligo nucleic acid agents of the invention are defined by their sequence; however, the skilled person understands that by exchanging one or two positions, particularly while increasing binding to the mRNA through introduction of nucleotide analogues, sufficient specificity of binding may be attained to achieve the inventive effect.
  • the oligonucleic acid agent comprises a sequence hybridizing to miR27b-5p.
  • the agent sequence is at least 95% identical, particularly 96%, 97%, 98%, 99% or 100% identical to SEQ ID 001.
  • the hybridizing sequence comprises deoxynucleotides, phosphothioate deoxynucleotides, LNA and/or PNA nucleotides or mixtures thereof.
  • the oligonucleic acid agent is an antisense oligonucleotide.
  • the oligonucleic acid agent comprises or is essentially composed of LNA moieties and comprises about 20 or fewer nucleotides.
  • the oligonucleic acid agent is essentially composed of LNA moieties and is described by the sequence ACC AAT CAG CTA AGC T (SEQ ID NO 008).
  • the nucleoside analogues of SEQ ID NO 008 are linked by phosphate esters.
  • the nucleoside analogues of SEQ ID NO 008 are linked by phosphothioate esters.
  • the oligonucleic acid agent for use in a method of treatment or prevention of heart disease comprises, or essentially consists of one or several peptide nucleic acid (PNA) moieties.
  • PNA peptide nucleic acid
  • the present invention includes a method of treating or preventing heart disease in a subject in need thereof comprising administering to the subject an oligonucleic acid agent directed against the miRNA miR27b-5p.
  • Inhibition of the miRNA miR27b-5p can be effected by sequence specific chemically modified oligonucleotides.
  • the oligonucleic acid agent of the invention can comprise locked nucleic acid (LNA), in which the nucleic acid's ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon, which locks the ribose in the 3'-endo conformation.
  • the oligonucleic acid agent of the invention may comprise PNA moieties. LNA and PNA have a higher binding energy to base-matched DNA or RNA, resulting in tighter binding. Such binding may add in antisense-based inhibition of the complementary miR27b-5p miRNA target.
  • the oligonucleic acid agent of the invention may include a phosphonate, a phosphorothioate or a phosphate ester phosphate backbone modification.
  • the oligonucleic acid agent of the invention may include ribonucleotides.
  • the oligonucleic acid agent of the invention may include deoxy ribonucleotides.
  • the hybridizing sequence of the oligonucleic acid agent comprises ribonucleotides, deoxynucleotides, phosphothioate deoxynucleotides, phosphothioate ribonucleotides and/or 2’-0-methyl-modified phosphothioate ribonucleotides.
  • the oligonucleic acid agent comprises ribonucleotides and deoxyribonucleotides, in particular modified ribonucleotides and modified deoxyribonucleotides.
  • a non-limiting example of a modification of deoxyribonucleotides and ribonucleotides are phosphorothioate modified linkages in the oligonucleotide backbone.
  • a non-limiting example of a modification of ribonucleotides is a 2’-0 to 4’-C bridge.
  • the 2’-0/4’-C bridge is a five-membered, six-membered or seven membered bridged structure.
  • the oligonucleic acid agent is a gapmer characterized by a central DNA block, the sequence of which is complementary to the miR27b-5p miRNA, and which is flanked on either side (5’ and 3’) by nuclease-resistant LNA sequences which are also complementary to the miR27b-5p miRNA.
  • the central DNA block contains the RNase H activating domain, in other words is the part that lead the target DNA to be hydrolyzed.
  • the flanking LNA is fully phosphorothioated.
  • the oligonucleic acid agent comprises 12 - 20 nucleotides. In certain particular embodiments, the oligonucleic acid agent comprises 14-16 nucleotides. In certain embodiments, the hybridizing sequence of the oligonucleic acid agent according to the invention comprises 14,15 or 16 nucleotides.
  • the central deoxyribonucleotide oligomer block of the gapmer comprises at least 5 deoxyribonucleotides. In certain embodiments, the central deoxyribonucleotide oligomer block of the gapmer comprises 5 to 10 deoxyribonucleosides linked by phosphate ester bonds or thiophosphate ester bonds.
  • the central deoxyribonucleotide oligomer block of the gapmer comprises a phosphate backbone between the deoxyribonucleosides.
  • the oligonucleic acid agent comprises, or essentially consists of, a central block of 5 to 10 deoxyribonucleotides linked by phosphate ester bonds flanked on either side by 2’-0 modified ribonucleotides or PNA oligomers.
  • the oligonucleic acid agent comprises, or essentially consists of, a central block of 5 to 10 deoxyribonucleosides flanked by LNA nucleoside analogues.
  • said LNA nucleoside analogues are linked by phosphothioate moieties.
  • the oligonucleic acid agent of the invention comprises or essentially consists of the sequence ACCA-atcagcta-AGCT (SEQ ID NO 005), wherein the capital letters signify nucleoside analogues, particularly LNA, more particularly LNA linked by phosphothioate esters, and the lower case letters signify DNA nucleosides linked by phosphate esters, and the link between a nucleoside analogue and a DNA nucleoside is selected from phosphate ester and thiophosphate.
  • the oligonucleic acid agent is a ribonucleic agent, particularly a siRNA or shRNA.
  • RNA interference agent in the context of the present specification refers to a ribonucleotide oligomer that causes the degradation of its enhancer RNA (eRNA) target sequence.
  • RNAi agents of the invention comprise, or consist of,
  • RNA oligomer or precursor thereof comprising a sequence tract complementary to the targeted enhancer RNA molecule
  • sequence tract complementary to the targeted enhancer RNA molecule is a contiguous sequence tract 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 nucleotides in length.
  • the RNAi agents of the invention include, but are not limited to, small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs and non-coding RNAs or the like, Morpholinos (phosphodiamidate morpholino oligomers) and Dicer substrate siRNAs (DsiRNAs, DsiRNAs are cleaved by the RNAse III class endoribonuclease Dicer into 21 - 23 base duplexes having 2-base 3’-overhangs), UsiRNAs (UsiRNAs are duplex siRNAs that are modified with non-nucleotide acyclic monomers, termed unlocked nucleobase analogues (UNA), where the bond between two adjacent carbon atoms of ribose is removed), self- delivering RNAs (sdRNAs) including rxRNATM (RXi Pharmaceuticals, Westborough, MA, USA).
  • siRNAs small interfering RNAs
  • the RNAi agents of the invention comprise analogues of nucleic acids such as phosphotioates, 2’O-methylphosphothioates, peptide nucleic acids (PNA; N-(2- aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha- carbon of the glycine) or locked nucleic acids (LNA; 2 ⁇ , 4’C methylene bridged RNA building blocks).
  • nucleic acids such as phosphotioates, 2’O-methylphosphothioates, peptide nucleic acids (PNA; N-(2- aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha- carbon of the glycine) or locked nucleic acids (LNA; 2 ⁇ , 4’C methylene bridged RNA building blocks).
  • the hybridizing sequence may be composed partially of any of the above nucleotides, with the rest of the nucleotides being“native” ribonucleotides occurring in nature, or may be mixtures of different analogues, or may be entirely composed of one kind of analogue.
  • the oligonucleic agent is conjugated to, or encapsulated by, a nanoparticle, a virus and a lipid complex.
  • the oligonucleic acid agent is a gapmer comprising a central deoxyribonucleotide oligomer block flanked by nuclease resistant ribonucleotide analogues on either (5’ and 3’) side.
  • a method for treating or preventing heart disease in a patient in need thereof comprising administering to the patient an oligonucleic acid agent according to the invention.
  • a dosage form for the prevention or treatment of heart disease comprising the oligonucleic acid agent according to the invention.
  • Dosage forms may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository.
  • parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms.
  • a pharmaceutically acceptable carrier and/or excipient may be present.
  • the amount of the oligonucleic acid agent sufficient to reduce expression of miR27b-5p miRNA is of 1 nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less, 5 picomolar or less, 2, picomolar or less and 1 picomolar or less in the environment of the cell.
  • SEQ ID NO 001 (miR27b-5p targeting sequence ; 5’-3’, and nucleotide chemistry):
  • SEQ ID NO 005 ACCA-atcagcta-AGCT (upper case: nucleoside analogue, particularly LNA linked by thiophosphate; lower case: DNA; boundary: phosphate or thiophosphate)
  • SEQ ID NO 006 (Primer sequence forward; mir27b): GCATGCTGATTTGTGACTTGAG-3’
  • SEQ ID NO 007 (Primer sequence reverse; mir27b): 5 -CCTCTGTTCTCCAAACTGCAG-3'.
  • SEQ ID NO 008 (miR27b-5p targeting sequence ; 5’-3’, LNA):
  • FIG 1A -B show the control of FiFo ATP synthase on endoreplication and multinucleation in pathologic growth.
  • HCM hypertrophic cardiomyopathy
  • AS aortic stenosis
  • FIG 1C-D shows biopsies of left ventricles from patients with HCM and aortic stenosis and healthy controls (c) or sham- and TAC-operated mice (d) assessed for denoted protein expression by immunoblotting.
  • FIG 1 E-F show the ADP/ATP ratio in ventricular biopsies from HCM and aortic stenosis patients versus healthy controls (e), and in ventricular biopsies from mice subjected to TAC versus sham-operated animals (f).
  • AS aortic stenosis
  • HCM hypertrophic cardiomyopathy
  • FIG 1 H shows heart sections stained as in FIG. 1 G and assessed for the percentage of mono- , bi-, or multinucleated cardiomyocytes.
  • FIG 1J shows heart sections stained as in (i) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes. (2 sections/heart were analyzed with 2 fields/section used for quantification. In total, 3 hearts were analyzed per group).
  • FIG 1 K shows schematic representation of the AAV9-fl/fl-shAtp5a1 virus before and after Cre- mediated recombination (left panel) and of the experimental timeline (right panel).
  • FIG 1 L shows an immunoblot of ventricular lysates from Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 using antibodies against denoted proteins.
  • FIG 1 M shows an ADP/ATP ratio measured in left ventricular samples of Mlc2v-cre + and Mlc2v- cre mice transduced with AAV9-fl/fl-shAtp5a1 1 1 weeks after AAV9 injection.
  • FIG 10 shows heart sections stained as in FIG1 N assessed for the ratio of mono-, bi-, or multinucleated cardiomyocytes.
  • FIG 1P shows representative images of left ventricles of Mlc2v-cre + and M!c2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 11 weeks after AAV9 injection.
  • FIG 1Q shows representative images of H&E-stained histological sections of Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 1 1 weeks after AAV9 injection.
  • FIG 1 R-S show left ventricular weight/body weight (LVW/BW) (R) and ejection fraction (S) of Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 11 weeks after AAV9 injection.
  • LVW/BW left ventricular weight/body weight
  • S ejection fraction
  • FIG 2A shows the expression of MIR27B in patients with hypertrophic cardiomyopathy (HCM) or aortic stenosis (AS)
  • FIG 2B shows the schematic representation of the experimental timeline of Mlc2v-cre mice transduced with AAV9-fl/fl-mir27b.
  • FIG 2C shows representative images of left ventricles of Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl-mir27b viruses.
  • FIG 2D shows representative images of H&E-stained histological sections of Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-mir27b viruses.
  • FIG 2E-F show LVW/BW (e) and ejection fraction (f) of Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl-mir27b viruses.
  • FIG 2G show the relative expression of mature miR27b-5p and miR27b-3p in Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl-mir27b viruses.
  • FIG 2H shows an immunoblot of ventricular lysates from Mlc2v-cre + and Mlc2v-cre mice transduced as in FIG 2C-G using antibodies against denoted proteins.
  • FIG 2J shows heart sections stained as in FIG 2I and assessed for the ratio of mononucleated to multinucleated cardiomyocytes.
  • FIG 2K shows a schematic representation of the antagomir study in sham- and TAC-operated mice.
  • FIG 2 L-M show representative images of left ventricles (FIG 2L) and H&E-stained (FIG 2M) histological sections.
  • FIG 2N-P show longitudinal monitoring of LVW/BW (FIG 2N), left ventricular internal diameter at end systole (LVID;s) (FIG 20) and ejection fraction (FIG 2P) of C57BL/6J mice subjected to sham or TAC surgery and treated with either scrambled (scrLNA) or miR27b-5p LNAs.
  • FIG 2Q-T show the relative expression of mature miR27b-5p (FIG 2Q), miR27b-3p (FIG 2R), Nppa and Nppb (FIG 2S) and Atp5a1 mRNA (FIG 2T) in C57BL/6J mice treated with scrLNA or miR27b-5p LNAs and subjected to either sham or TAC surgery by qPCR.
  • FIG 2U shows an immunoblot of ventricular lysates from sham- and TAC operated C57BL/6J mice treated with either scrLNA or miR27b-5p LNAs using antibodies against denoted proteins.
  • FIG 2 V shows left ventricular longitudinal sections from sham- or TAC operated mice treated with either scrLNA or miR27b-5p LNAs stained for DAPI (blue), laminin (green) and oactinin (red) and imaged by confocal microscopy.
  • FIG 2W shows heart sections stained as in (v) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes.
  • FIG 2X shows Kaplan-Meier survival curves comparing mortality between mice subjected to sham or TAC surgery and treated with scrLNA or miR27b-5p LNAs. No mortality was observed in sham-operated animals.
  • FIG 3A shows a schematic representation of de novo purine biosynthesis pathway showing the contribution of glycolysis and 1 -carbon metabolism.
  • FIG 3B shows the relative amount of formate in NRCs transduced and treated as denoted.
  • FIG 3C shows relative mitochondrial ADP/ATP ratio in NRCs transduced and treated as denoted.
  • FIG 3E shows the relative amount of [ 14 C]carbon derived from [ 14 C]glucose, [ 14 C]serine and [ 14 C]glycine incorporated into nucleic acids in NRCs transduced and treated as denoted.
  • FIG 4A-F show NRCs transduced and treated as indicated were stained with propidium iodide (PI) and assessed for polyploidy by flow cytometry coupled to imaging (a,c,e) and multinucleation quantified from images (b,d,f).
  • PI propidium iodide
  • FIG 4G-I show an evaluation of [ 3 H]leucine incorporation in NRCs transduced and treated as indicated. Data is represented as incorporated radioactivity relative to control NRCs (set as 1 .0).
  • FIG 5A shows a schematic representation of the experimental timeline of Mlc2v-cre mice transduced with AAV9-fl/fl-shMthfd1 l and subjected to sham or TAC surgery.
  • FIG 5B shows left ventricular longitudinal sections from sham- or TAC-operated Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl-shMthfd1 l viruses were stained for DAPI (blue), laminin (green) and a-actinin (red), and imaged by confocal microscopy. (3 sections/heart were analyzed with 2-4 fields/section imaged with representative z-stack fields shown. In total, 3 hearts were analyzed per group). Arrows indicate nucleation of cardiomyocytes.
  • FIG 5C shows heart sections stained as in (b) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes.
  • FIG 5D shows representative images of left ventricles of sham or TAC-operated Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl-sh Mthfd 11 viruses.
  • FIG 5E shows representative images of H&E-stained histological sections of sham or TAC- operated Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl-sh Mthfd 11 viruses.
  • FIG 5F-G shows LVW/BW (f) and ejection fraction (g) of sham or TAC-operated Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl-sh Mthfd 11 viruses.
  • FIG 5H shows an immunoblot of ventricular lysates from sham or TAC-operated Mlc2v-cre + and Mlc2v-cre mice transduced as in (b-g) using antibodies against denoted proteins.
  • FIG 5I shows a schematic representation of the experimental timeline of Mlc2v-cre mice co- transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11.
  • FIG 5K shows heart sections stained as in (j) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes.
  • FIG 5I shows representative images of left ventricles of Mlc2v-cre + and Mlc2v-cre mice co- transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11.
  • FIG 5M shows representative images of H&E-stained histological sections of Mlc2v-cre + and Mlc2v-cre mice co-transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11.
  • FIG 5N-0 shows LVW/BW (n) and ejection fraction (o) of Mlc2v-cre + and Mlc2v-cre mice co- transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11.
  • FIG 5P shows an immunoblot of ventricular lysates from Mlc2v-cre + and M!c2v-cre mice transduced as in j-p using antibodies against denoted proteins.
  • FIG. 6A shows an immunoblot of NRCs ectopically expressing a constitutively active form of AMPKal using antibodies against denoted proteins.
  • FIG. 6B shows an immunoprecipitation (IP) with either control IgG or anti-AMRKa antibody from NRCs ectopically expressing a constitutively active form of AMPKal .
  • IPs were blotted using p-AMPKa and p-Rb antibodies.
  • FIG. 6C shows an immunoblot of NRCs ectopically expressing HIFI aAODD and treated with either DMSO or the AMPK inhibitor compound C (CC) using antibodies against denoted proteins.
  • FIG. 6D shows an immunoprecipitation with either control IgG or anti-AMRKa antibody from NRCs ectopically expressing HIFI aAODD and treated with either DMSO or the AMPK inhibitor CC.
  • the IPs were blotted using p-AMRKa and p-Rb antibodies.
  • FIG. 6E shows an immunoblot of ventricular lysates from sham- or TAC operated mice treated with either scrLNA or miR27b-5p LNAs using antibodies against p-Rb and total Rb.
  • FIG. 6F shows an immunoblot of ventricular lysates from Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl-mir27b viruses using antibodies against p-Rb and total Rb.
  • FIG. 6G shows an immunoblot of ventricular lysates from Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl-shAtp5a1 viruses using antibodies against p-Rb and total Rb.
  • FIG. 6H shows an immunoblot of ventricular lysates from sham- or TAC-operated Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl-shMthfd1 l viruses using antibodies against p-Rb and total Rb.
  • FIG. 6I shows an immunoblot of ventricular lysates from Mlc2v-cre + and Mlc2v-cre mice co- transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11 using antibodies against p-Rb and total Rb.
  • FIG. 6J shows an immunoblot of ventricular lysates from HCM and aortic stenosis patients and healthy controls using antibodies against p-Rb and total Rb.
  • FIG. 6K shows a heat map of relative expression of denoted genes in ventricular lysates from sham- or TAC operated mice treated with either scrLNA or miR27b-5p LNAs.
  • FIG. 6L shows a heat map of relative expression of denoted genes in ventricular lysates from Mlc2v-cre + and Mlc2v-cre mice injected with either AAV9-fl/fl-mir27b
  • FIG. 6M shows a heat map of relative expression of denoted genes in ventricular lysates from Mlc2v-cre + and Mlc2v-cre mice subjected to sham or TAC surgery and injected with AAV9- fl/fl-sh Mthfd 11 viruses.
  • FIG. 6N shows a heat map of relative expression of denoted genes in ventricular lysates from HCM and aortic stenosis patients and healthy controls.
  • FIG 60 shows the transfection of E2F luciferase reporter in NRCs infected and treated as indicated. Data is normalized to control infected NRCs (set as 1.0).
  • FIG. 6P shows the transfection of E2F luciferase reporter in NRCs infected as indicated. Data is normalized to control infected NRCs (set as 1 .0).
  • FIG 6Q shows the proposed mechanism of FiFo ATP synthase function in cardiac ploidy and growth control.
  • Pathologic stress leads to the stabilization and accumulation of HIF1 a and HIF1 a -dependent induction of glycolytic genes and the microRNA MIR27B.
  • MIR27B binds to the 3’UTR of the alpha subunit of FiFo ATP synthase (ATP5A1 ) thereby targeting it for degradation.
  • ATP5A1 the alpha subunit of FiFo ATP synthase
  • the ADP/ATP ratio is elevated inside the mitochondrial matrix which, on one hand, activates the energy sensor AMPK.
  • pAMPK in turn hyperphosphorylates Rb resulting in the release of the transcription factor E2F and induction of cyclins and CDKs expression.
  • the accumulating mitochondrial ADP is rechanneled towards MTHFD1 L, an enzyme involved in the mitochondrial one-carbon metabolism that uses ADP as a cofactor to catalyze the conversion from CHO-THF to format.
  • Formate that is particularly derived from serine serves as a 1 -carbon donor for the de novo synthesis of purines.
  • the increased de novo formation of purines as substrates for nucleic acid synthesis and the induction of cyclins and CDKs promote DNA replication, multinucleation and cardiac growth.
  • CHO-THF denotes formyl-tetrahydrofolate
  • CH2-THF denotes methylenetetrahydrofolate
  • 3-PG denotes 3-phosphoglycerate.
  • FIG 7 e Cardiomyocytes were isolated from sham- and TAC-operated mice and stained for DAPI and oactinin. Cells were imaged by confocal microscopy and a representative z-stack image from 3 mice per group is shown. Scale bar is 20 pm.
  • FIG 7 h,i Representative images of H&E (h) and picrosirius red (i) stained histological sections of sham- and TAC-operated mice. Scale bar is 500 pm.
  • FIG 8d Cardiomyocytes were isolated from Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl-shAtp5a 1 viruses and stained for DAPI and a-actinin. Cells were imaged by confocal microscopy and a representative z-stack image from 3 mice per group is shown. Scale bar is 20 pm.
  • FIG 8 h Representative images of picrosirius red-stained histological sections of Mlc2v-cre + and Mlc2v-cre ⁇ mice transduced with AAV9-fl/fl-shAtp5a1 viruses. Scale bar is 500 pm.
  • FIG 8i Relative expression of Col1a1, Col3a1 and TGFbl mRNA in left ventricular lysates of Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 viruses.
  • Data is normalized to Mlc2v-cre mice (set as 1 .0).
  • FIG 8k ADP/ATP ratio in iPSC-hCM transduced with lentivirus expressing nsRNA or shATP5A1 .
  • n 3 biological replicates per group; shown is mean ⁇ SD; ** p ⁇ 0.01 ; two-tailed unpaired t-test).
  • FIG 8l,m Quantification of ADP (I) and ATP (m) levels from iPSC-hCM transduced with lentivirus expressing nsRNA or shATP5A1 .
  • n 3 for both groups; shown is mean ⁇ SD; ** p ⁇ 0.01 ; two-tailed unpaired t-test).
  • FIG 8n iPSC-hCM transduced with lentivirus expressing nsRNA or shATP5A1 were stained for DAPI and phalloidin, and imaged by confocal microscopy. Representative fields from 3 biological replicates are shown. Scale bar is 20 pm.
  • FIG 8r NRCs transduced with nsRNA or shAtp5a1 were assessed for Atp5a1 protein levels by immunoblotting. Loading is normalized to cardiac actin.
  • Ventricular lysates of sham- or TAC-operated control Hifla fl/fl
  • Hifla cKO mice left panel
  • control Vhl fl/fl
  • Vhl cKO mice right panel
  • Loading is normalized to cardiac actin.
  • FIG 9 d,e Quantification of ATP amount (d) and ejection fraction (e) in left ventricular biopsies from control ( Hifla fl/fl) and ventricle-specific Hifla conditional knockout ( Hifla cKO) mice subjected to sham or TAC surgery (d).
  • n 5 mice per group for (d) and (e); shown is mean ⁇ SEM; * , % p ⁇ 0.05; one-way ANOVA and Bonferroni correction).
  • FIG 9 f,g Quantification of ATP amount in left ventricular biopsies (f) and ejection fraction (g) of ventricle-specific V hi conditional knockout (V hi cKO) and respective control (V hi fl/fl) mice (g).
  • V hi cKO ventricle-specific V hi conditional knockout
  • V hi fl/fl mice respective mice per group for (f) and (g); shown is mean ⁇ SEM; * p ⁇ 0.05; ** p ⁇ 0.01 ; two- tailed unpaired t-test).
  • FIG 9 h Expression of miRNAs containing putative HRE(s) in their promoter (p ⁇ 0.0001 between technical quadruplicates, and p ⁇ 0.05 of the biological triplicates with average signal intensity of 1 -fold over background) from ventricular lysates of Hifla cKO mice subjected to TAC surgery versus TAC-operated control animals, or Vhl cKO mice versus control animals.
  • FIG 9 k-m Relative expression levels of AmpO (k), mature miR27b-3p, miR23b-3p and miR24- 3p (I), and miR27b-5p, miR23b-5p and miR24-5p (m) from ventricular lysates of wildtype C57BL/6J mice subjected to sham or TAC surgery.
  • Data is normalized to sham-operated control mice (set as 1 .0).
  • FIG 9 v Sequence of the human, monkey, possum, rat and mouse mir27b promoter harboring a conserved HRE located 162 bp upstream of the precursor mir27b transcription start site. HRE is shown in red, with the core HRE motif capitalized.
  • FIG 9 z NRCs transduced with nsRNA or shVhl were assessed for Hifl a and Vhl protein levels by immunoblotting. Loading is normalized to cardiac actin.
  • FIG 10 a Target gene recognition motif in the 3’UTR of human and mouse Atp5aJ
  • the miR27b-5p seed sequence and corresponding target region on Atp5a1 are indicated by alignment.
  • FIG 10 c NRCs transduced with empty control vector or ectopic mir27b were assessed for
  • FIG 10 i Immunoblot for Hifl a and Atp5a1 expression in NRCs transduced with lentivirus expressing empty control vector or HIFI aAODD and treated with either scrambled control (scrLNA) or miR27b-5p LNAs. Loading is normalized to cardiac actin.
  • FIG 10 j,k Relative expression of mature miR27b-5p (j) and miR27b-3p (k) in NRCs treated with PBS (mock) or T3 in the presence of a scrLNA or miR27b-5p LNA.
  • FIG 10 m NRCs treated as in (I) were assessed for Atp5a1 protein levels by immunoblotting. Loading is normalized to cardiac actin.
  • FIG 10 p NRCs transduced with empty control vector or ectopic ATP5A1 were assessed for Atp5a1 protein levels by immunoblotting. Loading is normalized to cardiac actin.
  • FIG 11 a Relative expression of Nppa and Nppb mRNA in Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-mir27b 1 1 weeks after AAV9 injection.
  • Data is normalized to Mlc2v- ere mice transduced with AAV9-fl/fl-mir27b (set as 1 .0).
  • FIG 11 b LVID;s in Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-mir27b 1 1 weeks after AAV9 injection.
  • FIG 11 c Relative expression of Atp5a1 mRNA in Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-mir27b 1 1 weeks after AAV9 injection by qPCR.
  • Data is normalized to Mlc2v- cre mice transduced with AAV9-fl/fl-mir27b (set as 1 .0).
  • FIG 11 d Left ventricular sections from Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9- fl/fl-mir27b were stained with H&E and imaged by light microscopy 1 1 weeks after AAV9 injection. (3 sections/heart with 3-5 fields/section were surveyed with representative fields shown. In total, 3 hearts were surveyed per group). Scale bar is 200 pm.
  • FIG 11 e Cardiomyocytes were isolated from Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl-mir27b viruses and stained for DAP I and a-actinin. Cells were imaged by confocal microscopy and a representative z-stack image from 3 mice per group is shown. Scale bar is 20 pm.
  • FIG 11 g Representative images of picrosirius red-stained histological sections of Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-mir27b viruses. Scale bar is 500 pm.
  • FIG 11 j Left ventricular sections from C57BL/6J mice subjected to sham or TAC surgery and treated with either scrLNA or miR27b-5p LNAs were stained with H&E and imaged by light microscopy. (3 sections/heart with 2-4 fields/section were surveyed with representative fields shown. In total, 3 hearts were surveyed per group). Scale bar is 200 pm.
  • FIG 11 k Cardiomyocytes were isolated from C57BL/6J mice subjected to sham or TAC surgery and treated with either scrLNA or miR27b-5p LNAs and stained for DAPI and oactinin. Cells were imaged by confocal microscopy and a representative z-stack image from 3 mice per group is shown. Scale bar is 20 pm.
  • FIG 11 m Representative images of picrosirius red-stained histological sections of C57BL/6J mice subjected to sham or TAC surgery and treated with either scrLNA or miR27b-5p LNAs. Scale bar is 500 pm.
  • FIG 11 n Relative expression of Col1a1, Col3a1 and TGFbl mRNA in left ventricular lysates of C57BL/6J mice treated with scrLNA or miR27b-5p LNAs and subjected to either sham or TAC surgery by qPCR.
  • Data is normalized to sham-operated mice treated with scrLNA (set as 1 .0).
  • FIG. 12 b Immunoblot of NRCs transduced with lentivirus expressing empty control vector or MTHFD1 L using antibodies against Mthfd 11. Loading is normalized to cardiac actin.
  • FIG 15 a Principal Component Analysis (PCA) of denoted cohorts.
  • FIG 15 d Barplot of the saturation profiles in the denoted four cohorts (mean +/- SD). Lipids were grouped according to the number of double bonds (db), and all species exceeding 6 db were gathered in a single group (6+).
  • Analyzed lipids include the following 19 lipid classes cholesteryl esters (CE), ceramides (Cer), cholesterols (Choi), cardiolipins (CL), diacylglycerols (DAG), lyso-phosphatides (LPA), lyso-phosphatidylcholines (LPC), Lyso- phosphatidylehanolamines (LPE), ether linked LPE (LPE-O ), lyso-phosphatidylinositols (LPI), phosphatidylcholines (PC), ether linked PC (PC-O ), phosphatidylethanolamines (PE), ether linked PE (PE-O ), phosphatidylglycerols (PG), phosphatidylinositols (PI), phosphatidylserines (PS), sphingomyelins (SM) and triacylglycerols. (adj. p-value ⁇ 0.001
  • FIG 15 f Mean mol% abundance of lipid species in left ventricular biopsies of scrLNA treated TAC-operated mice minus mean mol% abundance of lipid species in sham-controls with.
  • FIG 15 g Mean mol% abundance of lipid species in TAC-operated mice injected with miR27b- 5p LNA minus mean mol% abundance of lipid species in sham-controls with repressed (LNA) miR27b-5p.
  • FIG 15 h Relative expression of indicated mRNAs in C57BL/6J mice treated with scrLNA or miR27b-5p LNAs and subjected to either sham or TAC surgery by qPCR.
  • Data is normalized to sham-operated mice treated with scrLNA (set as 1 .0).
  • FIG 16 a,b Quantification of ADP (a) and ATP (b) levels in NRCs treated with PBS or 10 mM ADP for 3 days.
  • n 4 biological replicates per group; results shown are the mean ⁇ SD; * , % p ⁇ 0.05; one-way ANOVA followed by Bonferroni correction).
  • FIG 16 c,d Quantification of ADP (c) and ATP (d) levels in NRCs treated with PBS or 20 nM oligomycin.
  • n 3 biological replicates per group; results shown are the mean ⁇ SD; * , % p ⁇ 0.05; one-way ANOVA followed by Bonferroni correction).
  • FIG 16 e NRCs treated with PBS or 10 mM ADP for 3 days were stained for DAPI and oactinin and imaged by confocal microscopy. Representative fields of three independent experiments are shown. Scale bar is 5 pm.
  • FIG 16 f NRCs treated with PBS or oligomycin were stained for DAPI and oactinin and imaged by confocal microscopy. An average of 4 fields per condition and experiment were imaged and representative fields of three independent experiments are shown. Scale bar is 50 pm.
  • FIG 17 a-d NRCs transduced with the indicated lentiviruses were stained for the cardiac- specific marker a-actinin, DAPI and phospho-Histone H3 (p-Histone H3) for assessment of cell mitosis and imaged by confocal microscopy.
  • p-Histone H3 positive cardiomyocytes were quantified. 3 biological replicates with 3-4 fields/replicate were analyzed per condition with representative fields shown. Scale bar is 100 pm.
  • FIG 17 f Immunoblot of NRCs transduced with lentivirus expressing nsRNA or shMthfdl l using an antibody against Mthfdl l. Loading is normalized to cardiac actin.
  • FIG 17 g,h, NRCs transduced with the indicated lentiviruses were stained for the cardiac- specific marker a-actinin, DAPI and phospho-Histone H3 (p-Histone H3) for assessment of cell mitosis and imaged by confocal microscopy.
  • p-Histone H3 positive cardiomyocytes were quantified. 3 biological replicates with 3-4 fields/replicate were analyzed per condition with representative fields shown. Scale bar is 100 pm.
  • FIG 18 a Left ventricular sections from sham- or TAC-operated Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-sh Mthfd 11 were stained with H&E and imaged by light microscopy 1 1 weeks post AAV 9 injection. (3 sections/heart with 2-4 fields/section were surveyed with representative fields shown. In total, 3 hearts were surveyed per group). Scale bar is 200 pm.
  • FIG 18 b Left ventricular internal dimension at systole (LVID;s) in sham- or TAC-operated M!c2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-sh Mthfd 11 1 1 weeks post AAV9 injection.
  • LVID left ventricular internal dimension at systole
  • FIG 18 c,d Relative expression of Nppa and Nppb (c) and Mthfdll mRNA (d) from ventricular lysates obtained from sham- or TAC-operated Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-sh Mthfd 11 1 1 weeks post AAV9 injection. Data is normalized to sham-operated Mlc2v-cre mice (set as 1 .0).
  • FIG 18 e Cardiomyocytes were isolated from sham- or TAC-operated Mlc2v-cre + and Mlc2v- cre mice transduced with AAV9-fl/fl-shMthfd1 l and stained for DAPI and oactinin. Cells were imaged by confocal microscopy and a representative z-stack image from 3 mice per group is shown. Scale bar is 20 pm.
  • FIG 18 g Representative images of picrosirius red-stained histological sections of sham- or TAC-operated Mlc2v-cre + and Mlc2v-cre mice transduced with AAV9-fl/fl-shMthfd1 l. Scale bar is 500 pm.
  • FIG 18 h Relative expression of Col1a1, Col3a1 and TGFbl mRNA in left ventricular lysates of C57BL/6J in sham or TAC-operated Mlc2v-cre + and Mlc2v-cre mice injected with AAV9-fl/fl- shMthfdl l viruses. Data is normalized to sham-operated Mlc2v-cre mice (set as 1 .0).
  • FIG 18 i Left ventricular sections from Mlc2v-cre + and Mlc2v-cre mice co-transduced with AAV9-fl/fl-shAtp5a 1 and AAV9-fl/fl-shMthfd1 l were stained with H&E and imaged by light microscopy 1 1 weeks post AAV 9 injection. (3 sections/heart with 3-5 fields/section were surveyed with representative fields shown. In total, 3 hearts were surveyed per group). Scale bar is 200 pm.
  • FIG 18 j Cardiomyocytes were isolated from Mlc2v-cre + and Mlc2v-cre mice co-transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11 viruses and stained for DAPI and oactinin. Cells were imaged by confocal microscopy and a representative z-stack image from 3 mice per group is shown. Scale bar is 20 pm.
  • FIG 18 p Representative images of picrosirius red-stained histological sections of Mlc2v-cre + and Mlc2v-cre mice co-transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-shMthfd1 l viruses. Scale bar is 500 pm.
  • FIG 19 c NRCs transduced with lentivirus expressing empty vector control or HIFI aAODD and transfected as indicated were stained for DAPI, Ki67 and oactinin, and imaged by confocal microscopy. An average of 4 fields per condition and experiment were imaged and representative fields of three independent experiments are shown. Scale bar is 50 pm.
  • FIG 19 h NRCs transfected with non-silencing or Cdknl b siRNAs were stained for DAPI, Ki67 and oactinin, and imaged by confocal microscopy. An average of 4 fields per condition and experiment were imaged and representative fields of three independent experiments are shown. Scale bar is 50 pm.
  • FIG 20 b Relative expression of ALDH1L1 mRNA in left ventricular biopsies from hypertrophic cardiomyopathy (HCM) and aortic stenosis (AS) patients versus healthy controls .
  • Data is shown as 2 A -dCt relative to HPRT1.
  • FIG 20 d Immunoblot of left ventricular lysates from HCM and aortic stenosis patients and healthy controls using a methylated lysine antibody. Loading is normalized to cardiac actin.
  • FIG 20 e-j Immunoblot of left ventricular lysates from mice treated as indicated using a methylated lysine antibody. Loading is normalized to cardiac actin.
  • IVS intraventricular septum thickness at diastole (d) and systole (s); LVID, left ventricular internal diameter at diastole (d) and systole (s); LVPW, left ventricular posterior wall thickness at diastole (d) and systole (s); FS, fractional shortening; EF, ejection fraction; LVW/BW, left ventricular weight/body weight; HW/BW, heart weight/body weight. Values shown are mean ⁇ s.e.m.; * P ⁇ 0.05; ** P ⁇ 0.01 ; two-tailed unpaired t-test. Table 2. Echocardiographic analysis of Mlc2v-cre-/-cre+ mice injected with AAV9-fl/fl-mir27b viruses
  • IVS intraventricular septum thickness at diastole (d) and systole (s); LVID, left ventricular internal diameter at diastole (d) and systole (s); LVPW, left ventricular posterior wall thickness at diastole (d) and systole (s); FS, fractional shortening; EF, ejection fraction; LVW/BW, left ventricular weight/body weight; HW/BW, heart weight/body weight. Values shown are mean ⁇ s.e.m.; * P ⁇ 0.05; ** P ⁇ 0.01 ; two-tailed unpaired t-test.
  • HW/BW post sacrifice
  • LVID left ventricular internal diameter at diastole (d) and systole (s)
  • LVPW left ventricular posterior wall thickness at diastole (d) and systole (s)
  • FS fractional shortening
  • EF ejection fraction
  • LVW/BW left ventricular weight/body weight
  • HW/BW heart weight/body weight.
  • Values shown are mean ⁇ s.e.m.; * P ⁇ 0.05; ** P ⁇ 0.01 ; TAC scrLNAvs. sham scrLNA; miR27b- 5p LNA sham vs. miR27b-5p LNA TAC; two-tailed unpaired t-test.
  • IVS intraventricular septum thickness at diastole (d) and systole (s); LVID, left ventricular internal diameter at diastole (d) and systole (s); LVPW, left ventricular posterior wall thickness at diastole (d) and systole (s); EF, ejection fraction; LVW/BW, left ventricular weight/body weight; HW/BW, heart weight/body weight. Values shown are mean ⁇ s.e.m.; * P ⁇ 0.05; ** P ⁇ 0.01 TAC c2v-cre-vs. TAC Mlc2v-cre+; two-tailed unpaired t-test. Table 5. Echocardiographic analysis of Mlc2v-cre-/-cre+ mice co-injected with AAV9- I- shAtp5a1 and AAV9-fl/fl-shMthfd1l viruses
  • IVS intraventricular septum thickness at diastole (d) and systole (s); LVID, left ventricular internal diameter at diastole (d) and systole (s); LVPW, left ventricular posterior wall thickness at diastole (d) and systole (s); FS, fractional shortening; EF, ejection fraction; LVW/BW, left ventricular weight/body weight; HW/BW, heart weight/body weight. Values shown are mean ⁇ s.e.m.; * P ⁇ 0.05; ** P ⁇ 0.01; two-tailed unpaired t-test. Examples
  • FiFn ATP synthase repression drives cardiometabolic endoreolication and pathologic growth
  • TAC Fig. 1 a,b and Fig. 7a
  • ATP5A1 mRNA and protein was consistently reduced in both human and mouse cardiac hypertrophy and correlated significantly with upregulation of the hypertrophic markers natriuretic peptide A ( NPPA ), natriuretic peptide B ( NPPB ), and echocardiographically measured disease indicators of cardiac morphology and function (Fig. 1 a-d and Fig. 7a).
  • AMP-activated protein kinase AMP-activated protein kinase
  • ACC Acetyl-CoA- Carboxylase
  • the TAC protocol mimics human aortic stenosis through surgical constriction of the mouse aorta, resulting in increased blood velocity into the ventricle to impose a pressure overload stress leading to pathologic cardiac growth and fibrosis indicated by increased picrosirius red staining and expression of the fibrotic marker genes collagen type I alpha 1 (Col1a1), collagen type I alpha 3 (Col3a1) and transforming growth factor beta 1 (TGF1b) as encountered in human aortic stenosis (Barrick, C.J., et al., 2007, Am J Physiol Heart Circ Physiol, 292, 21 19-2130). (Fig. 7 g-j).
  • AAV9 harboring shRNAs targeting Atp5a1 was delivered to Mlc2v-Cre transgenic mice (Chen, J., et al., 1998, Development, 125, 1943-1949). that express Cre recombinase specifically in the ventricular myocardium (Fig. 1 k).
  • AAV9-fl/fl-shAtp5a1 delivery led to repressed Atp5a1 mRNA and protein expression (Fig. 11 and Fig. 8a), with concomitant activation of Ampk and augmented phosphorylation of its downstream target Acc (Fig. 11), increased ADP:ATP levels (Fig. 1 m and Fig.
  • Fig. 8b cardiomyocyte multinucleation and overgrowth combined with significantly reduced cardiac contractility and fibrosis (Fig. 1 n-s, Fig. 8c-i and Table 1 ).
  • iPSC induced pluripotent stem cell
  • NRC primary neonatal rat cardiomyocytes
  • Human and rat cardiomyocytes were stained with DAPI to visualize nuclei, and phalloidin to label filamentous actin and outline the cell surface.
  • Fig. 8j-y knockdown of ATP5A1 with shRNAs in human and rat heart cells resulted in increased ADP:ATP ratio (Fig.
  • HIF1a regulates FiFn ATP synthase activity via mir27b-5o
  • HRE hypoxia response element
  • mir27b is contained within the mir23b-27b-24 cluster in intron 15 of the aminopeptidase O ⁇ AmpO) gene in humans and mice (Zhou, Q., et at. , 201 1 , Proc Natl Acad Sci U S A ,108, 8287-8292).
  • TAC TAC in mice resulted in a slight induction of AmpO, largely unchanged expression of mature miR23b-3p/5p and miR24-3p/5p, and a -2.5-fold induction of miR27b- 3p/5p (Fig. 9k-m).
  • Atp5a1 harbors a miR27b-binding site in the 3’UTR, conserved between human and mice (Fig. 10a), which upon disruption by site-directed mutagenesis, results in de-repression of mir27b- mediated inhibition (Fig. 10b).
  • Fig. 10a ectopic mir27b expression in NRC resulted in pronounced repression of Atp5a1 mRNA and protein (Fig. 10c-e).
  • miR27b-5p was unique by its capacity to inhibit wildtype Atp5a1 UTR-reporter expression (Fig. 10b).
  • Atp5a1 inactivation also mimicked the effects of ectopic mir27b expression, while concomitant ATP5A1 overexpression with a flag-tagged construct (Fig. 10p) rescued the diminished ATP synthase activity upon ectopic HIFI aAODD expression (Fig. 10o).
  • ATP synthase activity is highly dependent on Atp5a1 expression levels.
  • MIR27B expression correlates with human cardiac pathology
  • MIR27B expression was elevated in left ventricular biopsies of HCM and AS patients compared to healthy subjects, thus inversely correlating with ATP5A1 expression (Fig. 1 a).
  • AAV9 carrying pre-mir27b AAV9-fl/fl-mir27b
  • Mlc2v-Cre transgenic mice to achieve ectopic mir27b expression specifically in the ventricular myocardium.
  • mice 10b,f-p led us to interrogate miR27b-5p function in mice exhibiting severe heart failure - a disease state commonly associated with ATP synthase repression and ATP depletion.
  • C57BL/6J mice were randomly assigned to two groups, with the groups subjected to either sham or TAC surgery and further subdivided into treatment groups with scrambled LNA (scrLNA) or miR27b-5p LNA (Fig. 2k).
  • scrLNA scrambled LNA
  • Fig. 2k miR27b-5p LNA
  • TAC operated mice treated with scrLNA displayed a further decline in cardiac function and heart failure as evidenced by pronounced hypertrophy development, ventricular dilatation and reduced cardiac ejection fraction (Fig. 2l-p and Table 3).
  • TAC mice treated with miR27b-5p LNA exhibited improved cardiac function and partial reversion of hypertrophic growth (Fig. 2l-p and Table 3).
  • Analysis of miR27b-3p and miR27b-5p expression confirmed specific inactivation of the miR27b-5p species (Fig. 2q,r), correlating with reduced hypertrophic marker gene expression (Fig. 2s) and increased Atp5a1 mRNA and protein expression compared to scrambled LNA treated TAC operated mice, mimicking expression levels in sham-treated mice (Fig.
  • Mitochondrial ADP drives purine biosynthesis
  • MTHFD1 L utilizes ADP as a rate-limiting cofactor for the hydrolysis of 10-formyltetrahydrofolate (CHO-THF) to formate (Fig. 3a).
  • CHO-THF 10-formyltetrahydrofolate
  • Strikingly ectopic Hifl a, miR27b or shAtp5a1 expression resulted in increased levels of glucose and glycolytic intermediates (Dihydroxyacetonephosphate, 3-Phosphoglycerate) pointing to increased glucose uptake and glycolysis.
  • elevated levels of serine and glycine, intermediates of the purine biosynthesis pathway (Ribose-5-phosphate, FGAR) as well as purine and pyrimidine derivatives (Inosine, Xanthine, dUTP, GTP, Uridine) were observed.
  • concomitant miR27b-5p inactivation, depletion of Mthfd 11 or ectopic ATP5A1 expression partially reversed these effects (Fig. 3d).
  • ectopic expression of MTHFD1 L alone was not sufficient to induce the de novo purine biosynthesis pathway, supporting the view that mitochondrial ADP serves preferably as a substrate for ATP Synthase and that the induction of mitochondrial formate biosynthesis is highly dependent on repression of ATP5A1 (Fig. 3d). Similar results were observed with T3, resulting in highly miR27b- dependent growth (Fig. 13a), formate production (Fig. 13b) and ADP/ATP increase (Fig. 13c). LC-MS based analysis of the metabolome revealed increased production of purine and pyrimidine derivatives as a function of miR27b expression (Fig. 13d).
  • ADP rechanneling connects energetic compromise to stress-induced biosynthesis pathways.
  • Purines are essential for nucleic acid synthesis, endoreplication and cell growth. Its de novo synthesis can be traced to the glucose that enters the cell and undergoes glycolysis to form 3- phosphoglycerate (3-PG), and further metabolized to generate serine and (indirectly) glycine via the serine biosynthesis pathway, which then serve as key 1 -carbon donors through incorporation of its carbon into the purine ring (Fig. 3a). As the carbon atom incorporated into the purine ring essentially traces back to glucose carbons, we followed the flux of carbon into the purine ring of nucleic acids through labeling of glucose, serine and glycine, respectively (Fig. 3e).
  • the metabolite changes observed in cardiac left ventricles closely paralleled changes observed in vitro above, including the elevation of purine precursors such as N-Formylglycinamide ribonucleotide (FGAR) and 5- Formamidoimidazole-4-carboxamide ribotide (FAICAR) in scrLNA treated TAC mice compared to TAC-operated animals treated with miR27b-5p LNA or scrLNA treated mice subjected to sham surgery.
  • FGAR N-Formylglycinamide ribonucleotide
  • FICAR 5- Formamidoimidazole-4-carboxamide ribotide
  • the purines xanthine and inosine were significantly lower abundant in miR27b-5p treated TAC mice compared to TAC-operated mice treated with scrLNA (Fig. 14a).
  • Hierarchical clustering revealed that the metabolic signature of left ventricular biopsies from TAC-treated mice injected with miR27b-5p LNAs was altered compared to scrLNA injected TAC-mice, but comparable to that of of sham-operated scrLNA and miR27b-5p LNA treated mice (Fig. 14b).
  • TAC-operated left ventricular biopsies of miR27b-5p LNA injected mice revealed lower levels of free fatty acids and other lipid species compared to TAC scrLNA controls (Fig. 14a).
  • Fig. 15a-g we have in addition investigated the lipidome in the above-named in vivo samples.
  • PCA Principal component analyses
  • lipid catabolism was markedly elevated in miR27b-5p LNA-treated mice, as evidenced by enrichment of long chain fatty acids in TAC-operated hearts and treated with scrLNA compared to miR27b-5p LNA-treated TAC hearts (Fig. 15a-g). Furthermore, an increased expression of lipid catabolism mediators was observed in miR27b-5p LNA-treated TAC hearts (compared to mice subjected to TAC and treated with scrLNA), corresponding to an increased palmitate oxidation capacity in miR27b-5p LNA-treated TAC hearts (Fig. 15h,i).
  • ADP levels were indeed elevated in cells treated with oligomycin or ADP.
  • the inventors assessed the sufficiency for oligomycin or ADP to induce endoreplication and multinucleation, as visualized by immunofluorescent staining with DAPI and skeletal a-Actinin to quantify the fraction of multinucleated cells (Fig. 16e-h), and cardiomyocyte hypertrophy by cell size quantification (Fig. 16i,j).
  • the inventors analyzed cardiomyocyte ploidy and cell size in Hifl a, miR27b, Atp5a1 and Mthfdl l gain- and loss of function settings (Fig. 4a-i and Fig. 17a-h). As shown by imaging coupled flow cytometry and flow cytometry of propidium iodide (PI) stained DNA, ectopic HIF1 a, mir27b or shAtp5a1 expression was sufficient to increase the population of multinucleated polyploid cells (Fig. 4a-f), an effect reverted upon simultaneous miR27b-5p LNA-mediated inactivation (Fig. 4a, b), ectopic ATP5A1 expression (Fig.
  • PI propidium iodide
  • cardiomyocytes were stained for phosphorylated histone 3 at serine 10 (p-Histone H3) and imaged by confocal microscopy (Fig. 17a-h).
  • Phosphorylated histone H3 marks condensed chromosomes that are characteristic of karyokinetic cells.
  • ectopic HIFI aAODD expression led to increased phosphorylated histone H3 stained nuclei which was rescued upon parallel miR27b-5p inhibition with LNAs (Fig. 17a,b). Furthermore, mir27b overexpression or Atp5a1 inhibition similarly led to increased numbers of karyokinetic cells compared to corresponding controls (Fig. 17c,d).
  • shRNA-mediated Mthfdl l inhibition led to efficient depletion of its mRNA and protein (Fig. 17e,f) and resulted in comparable phospho- Histone H3 staining as control cells (Fig. 17g,h).
  • MTHFD1L is a key modulator of pathologic cardiac growth
  • Mlc2v-cre ⁇ and cre + mice subjected to sham or TAC surgeries with AAV9 harboring short- hairpin RNAs targeting Mthfdl l (AAV9-fl/fl-shMthfd1 l) (Fig. 5a).
  • AAV9-fl/fl-shMthfd1 l AAV9-fl/fl-shMthfd1 l
  • Mlc2v-cre ⁇ mice developed pathologic cardiac hypertrophy and systolic dysfunction after TAC surgery, evident by increased left ventricular weighbbody weight ratio, increased left ventricular internal diameter in diastole (LVId) and eleveated hypertrophic marker genes and reduced ejection fraction compared to sham-operated Mlc2v-cre ⁇ mice. (Fig. 5d-g, Fig. 18b-d and Table 4). Strikingly, the percentage of multinucleated cells was significantly lower in TAC operated Mlc2v-cre + mice compared to similarly treated Mlc2v-cre ⁇ littermates (Fig. 5b, c and Fig. 18a,e,f).
  • mice co-injected with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-shMthfd11 showed no increase in multinucleated cells (Fig. 5j,k and Fig. 18i-k), normal ventricular size (Fig. 5l,m and Fig. 181), left ventricular weight:body weight ratio (Fig. 5n), hypertrophic marker gene expression (Fig. 18m) and systolic left ventricular function (Fig. 5o and Table 5), despite efficient inhibition of Atp5a1 (Fig. 5p and Fig. 18n) when Mthfd 11 was inhibited simultaneously (Fig.
  • ATP5A1 integrates metabolic and growth signaling to drive pathologic growth
  • AMPK activation has been shown to drive inhibition of the tumor suppressor Retinoblastoma protein (Rb) via phosphorylation at Ser804.
  • Rb restricts DNA replication and cell cycle progression from G1 to S through its sequestration and inhibition of E2F transcription factors, which are composed of dimers E2F protein and a dimerization partner (DP) protein (E2F-DP).
  • E2F-DP dimerization partner protein
  • Rb phosphorylation results in the release and de-repression of E2F transcription factors from the E2F-DP complex, culminating in E2F target gene induction and cell cycle re-entry.
  • HIF1 omiR27b-ATP5A1 -MTHFD1 L axis creates a metabolic context permissive for cell cycle re-entry and endomitosis in post-mitotic adult cardiomyocytes
  • the inventors interrogated possible cooperation of metabolic and growth factor signaling in cardiac pathologic hypertrophy.
  • the inventors confirmed direct interaction of AMPK with Rb in our setting through co- immunoprecipitation of AMPK and Rb.
  • the AMPKa subunit was ectopically expressed in cardiomyocytes and subsequently immunoprecipitated.
  • Pull-down products revealed co-precipitation of AMPKa with phosphorylated-Rb (Fig, 6a, b).
  • the inventors confirmed this interaction through pull-down of endogenous AMPK in cells expressing ectopic HIFI aAODD.
  • AMPK immunoprecipitated with phosphorylated Rb in cardiomyocytes ectopically expressing HIFI aAODD but not upon simultaneous inhibition of AMPK with compound C (CC), a potent AMPK inhibitor (Fig, 6c, d).
  • E2F1 -target genes Cyclin A1 , A2, D1 , E1 ( Ccnal , Ccna2, Ccndl, Ccnel ) and Cyclin-dependent kinase ⁇ (Cdk1)
  • Fig. 6k-n Cyclin-dependent kinase ⁇
  • E2F luciferase reporter assays performed in NRC further confirmed E2F transcriptional activity under ectopic expression of HIFI aAODD, mir27b or Atp5a1 depletion (Fig. 6o,p). Finally, to functionally assess the impact of cell cycle regulators on cardiomyocyte endomitosis and multinucleation the inventors inactivated Ccndl (cyclin D1 ) and Ccnel (cyclin E1 ), two key downstream components of Rb activation in cardiomyocytes expressing ectopic HIFI aAODD.
  • Hifla fl/fl mice were obtained from Randall S. Johnson (University of California, San Diego, USA) and V hi fl/fl mice were kindly provided by Rudolf Jaenisch (Massachusetts Institute of Technology, USA).
  • mice The respective ventricular-specific mouse lines described in this manuscript were generated by crossing loxP-flanked Hifla (Hifla fl/fl) (Ryan, H.E., ef a/., 2000, Cancer Res, 60, 4010-4015) or Vhl (Vhl fl/fl) (Haase, V.H., ef a/., 2001 , Proc Natl Acad Sci U S A, 98, 1583-1588) mice to myosin light-chain Mlc2v-Cre transgenic mice.
  • the data presented represent studies with male mice aged 3-20 weeks of the C57BL/6J background. By 3-20 weeks the inventors were referring to the whole duration of the animal experiments.
  • the AAV9 viruses were administered to 3-week old mice. As theAAV9 vectors have a lag phase of approximately 6-10 weeks until it reaches maximal expression, surgeries were performed at 9-1 1 weeks and hearts harvested at least 1 1 weeks after AAV9 administration as shown in the experimental outlines in Fig. 1 k, Fig. 2b, k and Fig. 5a, i. In TAC experiments, mice were 10-12 weeks old at the beginning of the experiment. Only mice of a similar age (+/- 1 week) were used in the corresponding experiments. In experiments with the Mlc2v-cre mice, littermates and mice of a similar age (+/- 1week) were used. After baseline echocardiography mice were randomly assigned to groups, AAV injections, LNA delivery and echocardiography was performed blinded.
  • mice were maintained at the MRC Clinical Sciences Centre (Imperial College London), Institute of Molecular Health Sciences (ETH Zurich) and/or the Cardiovascular Assessment Facility (CAF), Department of Medicine, Department of Medicine, University of Lausanne in a specific pathogen-free facility. Maintenance and animal experimentation were in accordance with the Swiss Federal Veterinary Office (BVET) guidelines.
  • BVET Swiss Federal Veterinary Office
  • Human left ventricular biopsies of HCM patients were obtained from left ventricular papillary muscle of explanted hearts.
  • the myocardial samples were acquired directly in the operating room during the surgery and immediately washed in precooled cardioplegic solution (1 10 mM NaCI, 16mM KCI, 16mM MgCh, 16mM NaHCOs, 1 .2 mM CaCh, 1 1 mM glucose) followed by rapid snap-freezing in liquid nitrogen. Healthy heart samples were obtained from left ventricles of donor hearts. Sample weight was approximately 20-150 mg. Clinical data pertaining to these subjects are shown in a previous publication 16 .
  • mice 9-14 week old mice were subjected to transaortic banding (TAC) through constriction of the descending aorta as described (Kassiri, Z., et al., 2005, Circ Res, 97, 380-390). The mice were monitored up to 9 weeks after surgery and their heart dimensions and functions were determined by echocardiography.
  • TAC transaortic banding
  • In vivo miRCURY LNA Power Inhibitors were injected intraperitoneally (i.p.) into C57BL/6J mice at a dose of 10 mg/kg for 4 consecutive days at 49 days post surgery as described in Fig. 2k.
  • the following In vivo miRCURY LNA Power Inhibitors were purchased from Exiqon: i-mmu-miR-27b-5p (199900). i-Cel-controIJnh (199900) was used as scrambled control LNA.
  • Transthoracic echocardiography was performed using the MS400 (18-38MHz) probe from Vevo 2100 color doppler ultrasound machine (VisualSonics). Mice were lightly anesthetized with 1-1.5% isoflurane, maintaining heart rate at 400-550 beats per minute. The mice were placed in decubitus dorsal on a heated 37°C platform to maintain body temperature. A topical depilatory agent is used to remove the hair and ultrasound gel is used as a coupling medium between the transducer and the skin. Hearts were imaged in the 2D mode in the parasternal long-axis view.
  • an M-mode cursor was positioned perpendicular to the inter- ventricular septum and the posterior wall of the left ventricle at the level of the papillary muscles.
  • left ventricular internal end-diastolic and end-systolic chamber LVID;d and LVID;s
  • %EF Fractional shortening was assessed from M-mode based on the percentage changes of left ventricular end-diastolic and end-systolic diameters. %EF is derived from the formula of (LV vol;d - LV vol;s)/ LV vol;dx100. At the end of the duration of the experiment, the animals were sacrificed and the heart weight-to-body weight ratio was measured.
  • NRC primary neonatal rat cardiomyocytes
  • the plating medium was changed to maintenance medium (88% DMEM, 9% M199, 1% HS, 2% glutamine and 1% P/S) 24 h after isolation of NRCs.
  • Cardiomyocytes were treated with 3,3',5-Triiodo-L-thyronine (T3, T5516, Sigma Aldrich) for 6 days at a concentration of 15 nM.
  • NRCs were stimulated with exogenous ADP (A2754, Sigma Aldrich) at a concentration of 10 mM for 3 days.
  • Oligomycin A (75351 , Sigma Aldrich) and the AMPK inhibitor Compound C were applied at concentrations of 20 nM for 2 days and 5 mM for 24 h, respectively.
  • NRC were randomly chosen for treatment the day after isolation.
  • the left ventricle of adult mouse hearts was cut into pieces of 1 mm 3 and fixed for 2 h with 4% paraformaldehyde (PFA)/PBS.
  • the biopsies were digested with 1000 U collagenase type II (17101 -015, Gibco) in HBSS for approximately 42 h at 37°C while rotating. Appropriate concentrations of isolated adult cardiomyocytes were centrifuged at 600 rpm for 1 min onto gelatin-coated microscope slides using a cytospin (SCA-0030, Shandon Southern).
  • iPSC-CM Human induced pluripotent stem cell-derived human cardiomyocytes
  • Mitochondria were isolated using the Mitochondria Isolation Kit for Cultured Cells (ab1 10171 , Abeam). Briefly, 1 .2x10 6 NRCs were seeded per 6 cm dish and cells collected by scraping. After a freeze-thaw step to weaken the cell membrane, the cells were resuspended to a protein concentration of 5 mg/ml_ in Reagent A and incubated for 10 min on ice. The cells were homogenized with 30 strokes in a Dounce Homogenizer and centrifuged at 1000 g for 10 min at 4°C.
  • the pellet was resuspended in the same volume of Reagent B than was used for Reagent A and rupturing and centrifugation was repeated. The supernatants after the two centrifugation steps were combined and centrifuged at 12000 g for 15 min at 4°C. Pellet was resuspended in 80 pl_ Reagent C supplemented with protease inhibitors and stored at -80°C. Mitochondrial ADP:ATP quantification was performed using the EnzyLight ADP/ATP Ratio Assay Kit (ELDT-100, Bioassay Systems) as recommended by the manufacturer.
  • HEK-293T cells were transfected at 80-90% confluence with polyethylenimine (PEI) transfection reagent.
  • PEI polyethylenimine
  • 10 pg transgene, 7.5 pg pMD2.G and 6.5 pg psPAX2 were mixed with 2 ml serum-free DMEM and 45 pg PEI per 10 cm dish.
  • RT room temperature
  • the DNA/PEI-complexes were added slowly to the cells cultured in DMEM containing 0.5% FCS and L-glutamine.
  • Medium was changed to NRC maintenance medium 4 h after transfection.
  • Lentiviruses were harvested 48 h after transfection and stored at -80°C. NRC were infected 20 h after isolation and incubated at 37°C/5% CO2 overnight.
  • an shRNA targeting Mthfdl l was cloned into a pSico vector where the U6 promoter-driven shRNA expression is controlled in a Cre dependent manner.
  • the shRNAs targeting Mthfdl l were designed using the online software pSico Oligomaker (MIT, version 1 .5). The following shRNA sequence was used: AAV9-fl/fl-sh Mthfdl l,
  • LNA locked nucleic acids
  • miRNA mimics miRNA mimics
  • siRNAs siRNAs
  • miRCURY LNA Power Inhibitors were added directly to the cell culture medium at a final concentration of 50 nM and fresh LNAs added every second day.
  • the following miRCURY LNA Power Inhibitors were purchased from Exiqon: i-mmu-miR27b-5p (4101712-101 ). Negative Control A (199006-101 ) was used as non-targeting LNA.
  • miRIDIAN microRNA Mimics were transfected into NRCs using Lipofectamine 2000 (Invitrogen). Per well in a 96-well plate, 0.4 pL Lipofectamine 2000 was mixed with 25 pL OptiMEM (Invitrogen). After incubation of 5 min at RT, the mixture was added to an Eppendorf tube containing 6.25 nM or 12.5 nM miRNA mimics in 50 pL OptiMEM and incubated for 20 min at RT to form the complexes. 50 pL of the complexes was added to the cells cultured in 100 pL medium without antibiotics. The medium was replaced 4 h after transfection and cells were harvested 48 h after transfection.
  • miRIDIAN microRNA Mimics were purchased from Dharmacon: mmu-miR27b-3p (C-310380-05-0005), mmu-miR27b-5p (C- 310810-01 -0005). miRIDIAN microRNA Mimic Negative Control #1 (CN-001000-01 ) was used as negative control.
  • siRNAs were transfected at a final concentration of 50 nM into NRCs using the DharmaFECT 1 transfection reagent (Dharmacon) as recommended by the manufacturer. Gene expression was analysed 72 h after transfection. The following ON-TARGETplus SMARTpool siRNAs from Dharmacon were used: Ccndl (L-089285-02-0005), Ccnel (L-101575-02-0005) and Cdknl b (L-090938-02-0005). Non-targeting siRNA pool (D-001810-10-05) was used as control.
  • pCMV6 MTHFD1 L (RC223034, Origene) or pEBG-AMPKi -3i 2 (#27632, addgene) were transiently transfected into primary neonatal rat cardiomyocytes 2 days after isolation using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were harvested and analyzed 48 h after transfection.
  • the pLenti-HIFI aAODD puro lentiviral expression vector was generated by subcloning the HIFI aAODD fragment from a pcDNA3-HA-HIF1 aAODD (401-603) plasmid (Huang, L.E., et al., Proc Natl Acad Sci U S A, 95, 7987-7992). into pLenti-pgk puro vector as described previously (Troilo, A., et al. ,2014, EMBO Rep, 15, 77-85). As a corresponding control the pLenti-pgk puro empty vector was used.
  • the miR27b overexpression construct was generated by amplification of the precursor mir27b sequence and the flanking sequence of 158 bp from either end of the mir27b precursor transcript from mouse genomic DNA.
  • the sequence was cloned into the pLKO.1 CMV puro construct provided by A. Ittner (ETH Zurich), by using EcoRI and Sail restriction sites.
  • As a control vector the empty pLKO.1 CMV puro construct was used.
  • a 1 .0 kilobyte (kb) fragment of the mir23b, mir24-1 and mir27b promoter was amplified from mouse genomic DNA and cloned into the pGL3 luciferase reporter vector (Stratagene) between the Xhol and Hindlll restriction sites. Mutation of the HRE in the mir27b promoter was generated by recombinant PCR (Elion, E.A., et al., 2007, CurrProtoc Mol Biol, Chapter 3, Unit 3 17). Sense and Antisense primers were designed bearing HRE mutations, which were used to amplify the mutant 5’ and 3’ regions of the mir27b promoter, respectively.
  • the 5’ and 3’ products generated from the respective PCR reactions were mixed at a 1 :1 ratio and the entire fragment was amplified using primers targeting the 5’ and 3’ ends of the promoters.
  • the wildtype constructs and the mutation of the HRE in the mir27b promoter were confirmed by DNA sequencing (Microsynth).
  • UTR Luciferase Construct 3’ UTR of Atp5a1 was amplified from mouse cDNA and cloned into the pmirGLO Dual- Luciferase miRNA Target Expression Vector (Promega). Mutation of mir27b binding site on the 3’ UTR of Atp5a1 was generated by recombinant PCR. Sense and antisense primers were generated bearing mir27b binding site mutations, which were used to amplify the mutant 5’ and 3’ regions of the 3’UTR, respectively.
  • the 5’ and 3’ products generated from the respective PCR reactions were mixed at a 1 :1 ratio and the entire sequence was amplified using primers targeting the 5’ and 3’ ends of the Atp5a1 3’UTR.
  • the mutation of the mir27b binding site in the 3’ UTR of Atp5a1 was confirmed by sequencing (Microsynth).
  • shRNAs targeting Mthfdl l were designed using the online software BLOCK-iT RNAi Designer (Life Technologies) and compared against the rat genome using BLAST.
  • the shRNA was cloned into the pLKO.1 vector.
  • Sense and antisense oligomers were resuspended to a concentration of 20 mM in Annealing Buffer (100 mM NaCI, 50 mM HEPES, pH 7.4).
  • 5 pL of sense and antisense oligomers were mixed and annealed by incubating them in a beaker containing boiling water and letting it cool to room temperature.
  • the annealed oligomers were ligated into the pLKO.1 vector between the Agel and EcoRI restriction sites.
  • TRC pLKO.1 shRNA vectors were used: Atp5a1 (shAtp5a1 , TRCN0000076239), Hifl a (shHifl a, TRCN0000232220) and Vhl (shVhl, TRCN0000436052).
  • pLKO.1 vector containing non-silencing shRNA nsRNA; SHC002, Sigma
  • pLenti ATP5A1 (RC214840L1 )
  • pCMV6 MTHFD1 L (RC223034) plasmids were from Origene.
  • pEBG-AMPKcd (1 -312) was a gift from Reuben Shaw (Addgene plasmid # 27632).
  • RNA samples were harvested in Trizol (Invitrogen) and total RNA isolated as recommended by the manufacturer. 750 ng RNA were reverse transcribed into cDNA using RNA to cDNA EcoDry Premix (random hexamers) kit (Clontech, Cat No 639545) following the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) reactions were set up using iTaq Universal SYBR Green Supermix (Biorad, Cat No 1725121 ) according to manufacturer’s recommendations and run on a PikoReal Real-Time PCR machine (Thermo Scientific). Ct values were normalized against the housekeeping gene HprU. The qRT-PCR primer sequences are shown in Table 6.
  • miR23b-3p (ID000400), miR23b-5p (ID243680_mat) miR24-3p (ID000402), miR24-5p (ID 000488), miR27b-3p (ID000409), miR27b-5p (ID002174), snoRNA (ID001718) and snoRNA202 (ID001232).
  • ChIP assays were performed with material from NRCs and the assays carried out using the ChIP-IT Kit (Active Motif) according to the manufacturer’s instructions and analyzed by qRT- PCR. ChIP was performed with a ChIP-grade antibody against Hifl a (mouse, ab1 , Abeam). In silico promoter analyses and alignments were performed using Matlnspector and DiAlignTF (Genomatix). Primer sequences used for mir27b in the ChIP were 5'- G CAT G CT GATTT GT G ACTT GAG-3’ (SEQ ID NO 006) and 5'-
  • the microRNA microarrays were performed on 3 biological replicates of V hi cKO mice and three control mice ( 7?/ fl/fl), and on 3 biological replicates of Hifla cKO mice subjected to TAC and three controls subjected to TAC surgery (TAC Hif1a M ⁇ ), respectively. Cardiac dimensions and function were confirmed in all mice by echocardiography.
  • Total RNA was isolated from left ventricle and miRNAs were labelled using the miRCURY LNA microRNA Power Labelling Kit (Exiqon) and hybridized on miRNA arrays (miRXplore) that carry 1 194 DNA oligonucleotides with the reverse-complementary sequence of the mature miRNAs.
  • arrays cover 728 human, 584 mouse, 426 rat and 122 viral miRNAs, each spotted on the arrays in quadruplicate.
  • the Cy5-labelled miRNAs were normalized to a reference pool of miRNAs that were simultaneously labeled with Cy3. All the data are represented as ratios of logarithmic values between the diseased and control animals and deposited under GSE62418.
  • NRCs 4x10 5 NRCs were seeded per well in a 3cm dish and the assay was performed using the NADP/NADPH Quantitation Kit (MAK038, Sigma) as recommended by the manufacturer. Data was normalized to protein amount in the cell lysate by the Bradford assay.
  • ATP was separated and quantified on an anion exchange column (Nucleosil 4000-7 PEI, 50/4 from Macherey-Nagel) with a linear gradient (0-1 .5 M NaCI in 10 mM Tris-HCI, pH 8.0) using an HPLC system equipped with two independent UV-visible spectrometers (Shimadzu,). Elution of samples was monitored at 259 and 220 nm. The 220 nm wavelength was used to detect possible traces of contaminants.
  • the assay was performed using the ADP/ATP Ratio Assay Kit (ab65313, Abeam) as recommended by the manufacturer with small modifications. Briefly, the heart tissue was frozen and stored in liquid nitrogen immediately after the harvest. The tissue was powdered with a mortar and suspended in lysis buffer (10 mI/mg of tissue powder) for 5 min at room temperature. After the centrifugation at 10000 g for 1 min, the supernatant was used for the assay. Data was normalized to protein amount in the supernatant by the Bradford assay.
  • 4x10 4 NRCs were plated in a white 96-well plate and cultured for 3 days.
  • 40 ng of wildtype or mutant pmirGLO Atp5a1 3’UTR construct was co-transfected with 6.25, 12.5 or 25 nM control or miR27b mimics using Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer.
  • Luciferase activity was measured 24 h after transfection using the Dual Luciferase Reporter Assay System (Promega) as recommended by the manufacturer on a FLUOstar Omega Microplate Reader (BMG Labtech).
  • Luciferase activity was measured 36 h after transfection using the Dual Luciferase Reporter Assay System (Promega) as recommended by the manufacturer. E2F transactivation activities were measured using the Cignal Reporter Assay Kit (CCS-003L, Qiagen) according to the manufacturer’s instructions using the Dual Luciferase Reporter Assay System (Promega).
  • Heart tissue was solubilized in blue wonder sample buffer (3.7 M urea, 134.6 mM Tris pH 6.8, 5.4% (v/v) sodium dodecyl sulphate (SDS), 2.3% (v/v) NP-40, 4.45% (v/v) b-mercapto-ethanol, 4% (v/v) glycerol, 60 mg/L bromphenol blue) and proteins denatured for 5 min at 95°C after homogenization using an Ultra-Turrax T10 tissue homogeniser (IKA). NRCs were washed twice with ice-cold PBS and harvested in blue wonder sample buffer.
  • blue wonder sample buffer 3. M urea, 134.6 mM Tris pH 6.8, 5.4% (v/v) sodium dodecyl sulphate (SDS), 2.3% (v/v) NP-40, 4.45% (v/v) b-mercapto-ethanol, 4% (v/v) glycerol, 60 mg/L bromphenol blue
  • 0.5x10 7 transfected (pEBG-AMPK-1-312) or transduced (HIFI aAODD-lentiviruses) NRC per condition were lysed in TNN cell lysis buffer (50 mM Tris-HCI pH 7.5, 250 mM NaCI, 5 mM EdTA, 0.5% NP-40, 50 mM NaF, 0.5 mM EGTA, 1 mM PMSF, 1 mMDTT, and protease inhibitor cocktail) for 30 min.
  • Magnetic dynabeads (Invitrogen) were incubated with anti- AMPKa antibody or IgG control antibody (ab171870) for 10 min at room temperature.
  • AMPK was then immunoprecipitated from cell lysate by incubation for 3 hours at 4°C, IgG control antibody (ab171870). Finally, reaction was terminated by boiling beads in 6x Laemmli buffer for 5 min. Samples were resolved in 10% SDS-PAGE, and Western blot analysis was performed with phospho-RbSer800/804 (Cell Signaling Technology) and phospho-AMPKa Thr172 (Cell Signaling).
  • Immunofluorescent stainings were performed as described previously (Krishnan, J., et al., 2009, Cell Metab, 9, 512-524). After fixation of NRCs with 4% PFA/PBS, the cells were permeabilized and incubated with primary antibodies diluted in 2% (v/v) HS for 1 h at RT.
  • DAPI 4',6-diamidino-2-phenylindole
  • Phalloidin 555 A34055, Molecular Probes
  • AlexaFluor 647 anti-mouse A-1 1001 , Thermo Fisher Scientific
  • AlexaFluor 488 anti- mouse A-21235, Thermo Fisher Scientific
  • Hearts were embedded in optimal cutting temperature (OCT) compound and sectioned at 10 pm. Sections were fixed for 10 min with 4% PFA/PBS and after 2 washes with PBS for 2 min blocked for 1 h with 2% HS/PBS for 1 h at room temperature. After permeabilisation for 10 min with 0.2% Triton X-100/PBS, the sections were washed 3 times with PBS for 5 min and incubated with primary antibodies against sarcomeric oactinin (A781 1 , Sigma Aldrich, 1 :800) and Laminin (ab1 1575, Abeam, 1 :300) diluted in 2% (v/v) HS overnight in a humidified chamber at 4°C.
  • OCT optimal cutting temperature
  • Fluorescent images were acquired with the SP8 confocal microscopy (Leica) using a 20x magnification. The entire z-axis was imaged and z-stack image generated from approximately 0.2 pm steps. To determine the number of nuclei per myocyte the inventors included only myocytes that had been cut along their longitudinal axis. Nuclei that were surrounded by the extracellular matrix stain laminin were excluded from the analysis. Quantification of nuclei was performed blinded.
  • [ 3 H]leucine incorporation assay was used to measure de novo protein synthesis as an indirect readout for cell growth(Fukuzawa, J., et at. , 2000, Hypertension, 35, 1 191 -1 196).
  • 4x10 5 cells were seeded per 3 cm dish. After 3 days, cells were serum-starved overnight. Next day, cells were cultured in leucine-free medium for 4h, followed by culturing in maintenance medium containing labeled L-[4,5- 3 H(N)]isoleucine (specific activity 30-60 Ci/mmol, ART0233, American Radiolabelled Chemicals) at a concentration of 0.5 pCi/mL for 20 h.
  • cardiomyocytes were incubated overnight in MEM (Invitrogen) supplemented with MEM Vitamin Solution (Invitrogen) and containing 0.6 pCi/mL radiolabelled serine at carbon 3 ([3- 14 C]serine, specific activity 50-62 mCi/mmol, NEC827050UC, Perkin Elmer).
  • MEM Invitrogen
  • MEM Vitamin Solution Invitrogen
  • glycine incorporation cells were incubated in BME (Invitrogen) containing 1 pCi/mL uniformly radiolabelled glycine ([ 14 C(U)]glycine, specific activity >100mCi/mmol, NEC276E250UC, Perkin Elmer) for 4 h.
  • Fatty acid oxidation rate in animal tissues using [1 - 14 C]-palmitic acid was determined as described previously (Huynh, F.K., et al., 2014, Methods Enzymol, 542, 391 -405). with the exception that 0.7% BSA/500 mM palmitate/2 pCi 14 C-palmitate was used per reaction. Radioactivity was measured for 5 min in the Liquid Scintillation Analyzer Tri-Carb 2800TR (Perkin Elmer). Scintillation counts were normalized to protein amount.
  • NRCs 1 x10 6 NRCs were plated on a 6 cm dish (Nunc) and after 4-6 days cells were harvested by trypsinization. Cells were washed 1 x with PBS and centrifuged at 80 g for 5 min. Cells were resuspended in 500 pL PBS and 5 mL cold 70% (v/v) ethanol (kept at -20°C) was added immediately. The fixed cells were kept at 4°C for up to 1 week. Prior to flow cytometry analysis, cells were centrifuged and washed with PBS.
  • the cells were resuspended in 500 pL propidium iodide solution (69 pM propidium iodide in 38 mM sodium citrate, pH 7.4) containing 40 pg/mL RNase and incubated for 1 h at 37°C. Samples were run on the ImageStream (Amnis), a flow cytometer coupled to fluorescence image acquisition to obtain representative images of the flow cytometry data. The multinucleation was quantified from the ImageStream images.
  • NRCs were cultured per 3 cm dish.
  • the whole cell culture plates were snap frozen in liquid nitrogen after the cells were washed with 75 mM Ammonium carbonate (Sigma), adjusted to pH 7.4 with acetic acid.
  • the metabolites were extracted with cold extraction buffer (-20°C) containing acetonitrile:methanol:water in a 40:40:20 ratio.
  • Untargeted analysis of metabolites by flow injection-time-of-flight mass spectrometry as previously described (Fuhrer, T., et ai, 201 1 , Anal Chem, 83, 7074-7080).
  • Data was processed and analyzed with Matlab.
  • Metabolomics analysis performed by Metabolon (Fig. 12d) was performed as previously described (Cimen, I., et al., 2016,. Sci Trans! Med, 8, 358ra126).
  • Lipids were extracted using a two-step chloroform/methanol procedure(Ejsing, C.S., et ai, 2009, Proc Natl Acad Sci U S A, 106, 2136-2141 ).
  • Samples were spiked with internal lipid standard mixture containing: cardiolipin 16:1/15:0/15:0/15:0 (CL), ceramide 18:1 ;2/17:0 (Cer),, hexosylceramide 18:1 ;2/12:0 (HexCer), lyso-phosphatidate 17:0 (LPA), lyso-phosphatidylcholine 12:0 (LPC), lyso-phosphatidylethanolamine 17:1 (LPE), lyso-phosphatidylglycerol 17:1 (LPG), lyso-phosphatidylinositol 17:1 (LPI), lyso- phosphatidylserine 17:1 (LPS), phosphati
  • MS and MSMS data were combined to monitor CE, DAG and TAG ions as ammonium adducts; PC, PC 0-, as acetate adducts; and CL, PA, PE, PE 0-, PG, PI and PS as deprotonated anions.
  • MS only was used to monitor LPA, LPE, LPE 0-, LPI and LPS as deprotonated anions; Cer, HexCer, SM, LPC and LPC O- as acetate adduct and cholesterol as ammonium adduct of an acetylated derivative.

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Abstract

The present invention relates to an oligonucleic acid agent directed against the microRNA miR27b-5p for use in a method of treatment or prevention of heart disease.

Description

MicroRNA Targeting Agent for Treatment of Heart Disease
The present invention relates to the treatment and prevention of heart disease by administering oligonucleic acid agents that modulate the activity or expression of microRNAs. More precisely, the invention provides methods for treating or preventing heart disease by inhibiting the expression and/or activity of the microRNA miR27b-5p.
Background of the invention
The mechanism through which stress signaling promotes endoreplication, and thus the replication of DNA during S phase of the cell cycle without subsequent completion of mitosis and/or cytokinesis, to raise the cell size threshold is unknown. While the analysis of cell cycle regulation, growth signaling pathways, and transcriptional and translational networks has provided insight into upstream control networks, the more fundamental question of the underlying energetics in which the aforementioned regulators can function remains largely unaddressed. The energetic state of a cell not only determines ploidy, which is by definition the number of sets of chromosomes in a cell, but also cell size and function to establish an environment permissive for specific outputs that are co-opted by signaling cascades and transcriptional regulators to implement specific growth or functional endpoints. Emerging evidence links deregulated ADP:ATP homeostasis to the development of human hypertrophic cardiomyopathy (HCM) and aortic stenosis (AS). HCM and AS epitomize the cardiac overgrowth phenotype and are characterized by endoreplication, resulting in polyploidy and multinucleation (cells with two or more nuclei), and pathologic cardiomyocyte growth in an environment of depressed mitochondrial ATP synthesis. It is paradoxical that energetically deficient cardiomyocytes, exhibiting a reduced capacity to generate energy and cofactors though mitochondrial oxidation, are able to drive anabolic processes to support cardiomyocyte hypertrophy. In accord, mitochondrial myopathies are predominantly characterized by cardiac overgrowth.
Given that cardiac hypertrophy is metabolically characterized by depressed mitochondrial ATP synthesis we commenced on systematically profiling the expression of all subunits of the ATP synthase complex in patient biopsies of human HCM and AS. We identified ATP5A1 (encoding for the a-subunit of the ATP synthase catalytic Fi complex which forms together with the b- subunit, the catalytic domain through which the central stalk rotates to regenerate ATP from
ADP and inorganic phosphate) as downregulated in HCM and AS biopsies. Mechanistically, we demonstrate that ATP5A1 repression is mediated by the stress dependent-regulated hypoxia-inducible factor (HIF)1 a driven induction of MIR27B. Inhibition of ATP5A1 suppresses mitochondrial ATP synthase activity, leading to accumulation of intra-mitochondrial ADP that is channeled and serves as a cofactor for MTHFD1 L, a mitochondria localized rate-limiting enzyme of the 1 -carbon pathway regulating formate and purine biosynthesis. Activation of MTHFD1 L by ADP in cardiomyocytes accelerates de novo nucleotide synthesis and drives cardiomyocyte endoreplication and cell hypertrophy through parallel activation of AMPK-driven E2F. Consequently, MIR27B inactivation in vitro and in mice attenuates pre-existing heart failure in response to surgery-mediated aortic stenosis (transaortic constriction (TAC)), while cardiac-specific MIR27B expression results in spontaneous cardiac overgrowth. Analyses of the downstream components, ATP5A1 and MTHFD1 L in mice, further support a central role for the HIF1 oMIR27B-ATP5A1 -MTHFD1 L axis in cardiac polyploidization and cell size control. In accord, activation of this axis correlates with human pathologic cardiac hypertrophy. These findings reveal a stress-dependent pathway connecting deregulated mitochondrial ATP homeostasis with pathophysiologic endoreplication in cardiomyocytes, resulting in multinucleation and morphologic growth.
As the inventors could identify cardiometabolic endoreplication as a hitherto unknown mechanism dictating pathologic growth progression in the energetically deficient myocardium through miR27b-5p overexpression, there is a need in the art for treatment methods that can inhibit the expression of miR27b-5p for prevention of pathological growth through endoreplication and de novo nucleotide biosynthesis.
Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods for treatment of heart disease, particularly cardiomyopathy. This objective is attained by the subject matter of the claims of the present specification.
Description
The present invention is based upon the discovery that the inhibition of miRNA miR27b-5p through administration of an oligonucleic acid agent (SEQ ID NO 001 ) enables the modification of pathological growth of the myocardium via regulators such as ATP synthase, which drives mitochondrial ADP build-up and its redirection to methylenetetrahydrofolate dehydrogenase 1 L (MTHFD1 L) and de novo nucleotide biosynthesis.
The term miRNA in the context of the present invention relates to pri-, pre- and mature miRNA.
There are two mature subspecies of the stem-loop sequence of miRNA27b, namely miRNA27b-3p and miRNA27b-5p.
“Capable of forming a hybrid” in the context of the present invention relates to sequences that under the conditions existing within the cytosol of a mammalian cell, are able to bind selectively to their target sequence. Such hybridizing sequences may be contiguously reverse- complimentary to the target sequence, or may comprise gaps, mismatches or additional non- matching nucleotides. The minimal length for a sequence to be capable of forming a hybrid depends on its composition, with C or G nucleotides contributing more to the energy of binding than A or T/U nucleotides, and the backbone chemistry.
The term oligonucleic acid agent in the context of the present specification refers to an oligonucleotide capable of specifically binding to and leading to a significant reduction of the physiological role of miR27b-5p. Examples of oligonucleic acid agents of the present invention are antisense oligomers made of DNA, DNA having phosphorothioate modified linkages in their backbone, ribonucleotide oligomers, RNA comprising bridged or locked nucleotides, particularly wherein the ribose ring is connected by a methylene bridge between the 2’-0 and 4’-C atoms, RNA having phosphorothioate modified linkages in their backbone or any mixture of deoxyribonucleotide and ribonucleotide bases as an oligomer.
The term antisense oligonucleotide or oligonucleotide agent in the context of the present specification refers to any oligonucleotide capable of specifically binding to and leading to a significant reduction of the physiological role of miR27b-5p. Examples of antisense oligonucleotides of the present invention are antisense oligomers made of DNA, DNA having phosphorothioate modified linkages in their backbone, ribonucleotide oligomers, RNA comprising bridged or locked nucleotides, particularly wherein the ribose ring is connected by a methylene bridge between the 2’-0 and 4’-C atoms, RNA having phosphorothioate modified linkages in their backbone or any mixture of deoxyribonucleotide and ribonucleotide bases as an oligomer.
The terms oligonucleic acid agent and antisense oligonucleotide or oligonucleotide agent are used interchangeably in the present specification.
In certain embodiments, the antisense oligonucleotide of the invention comprises analogues of nucleic acids such as phosphotioates, 2’O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl) -glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2Ό, 4’C methylene bridged RNA building blocks). The antisense sequence may be composed partially of any of the above analogues of nucleic acids, with the rest of the nucleotides being“native” ribonucleotides occurring in nature, or may be mixtures of different analogues, or may be entirely composed of one kind of analogue.
The term gapmer is used in its meaning known in the field of molecular biology and refers to an antisense oligonucleotide complementary to its target sequence, that comprises a central block of a deoxyribonucleotide oligomer flanked by short ribonucleotide oligomers. The flanking ribonucleotide oligomers consist of nuclease and protease resistant ribonucleotides. In certain embodiments, the nuclease and protease resistant ribonucleotides comprise 2’-0 modified ribonucleotides, in particular bridged nucleic acids with a bridge between the 2’-0 and 4’-C of the ribose moiety. “Nucleotides” in the context of the present invention are nucleic acid or nucleic acid analogue building blocks, oligomers of which are capable of forming selective hybrids with miRNA oligomers on the basis of base pairing. The term nucleotides in this context includes the classic ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymin), cytidine, the classic deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. It further includes analogues of nucleic acids such as phosphotioates, 2Ό- methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2Ό, 4’C methylene bridged RNA building blocks). The hybridizing sequence may be composed of any of the above nucleotides, or mixtures thereof.
In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to the values determined by comparing two aligned sequences. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981 ), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).
One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 1 1 , Extension 1 ; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1 .-2; Gap costs: Linear. Unless otherwise stated, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.
Detailed description of the invention
A first aspect of the invention provides an oligonucleic acid agent directed against the microRNA miR27b-5p for use in a method of treatment or prevention of heart disease.
In other words, the oligonucleic acid agent of the invention is directed against, or capable of forming a hybrid with, the miRNA miR27b-5p (SEQ ID NO 002). In certain embodiments, the oligonucleic acid agent is directed against miR27b-5p miRNA in its mature form.
In certain embodiments, the oligonucleic acid agent is capable of reducing miR27b-5p miRNA levels in a cell by an amount (expressed in percentage) of at least 10-20%, at least 30-40%, at least 50-60%, at least 70-80%, at least 90-98%, or at least 99% when the oligonucleic acid agent is introduced into a mammalian cell.
In certain embodiments, the oligonucleic acid agent for use in a method of treatment or prevention of heart disease comprises, or essentially consists of, the sequence ACC AAT CAG CTA AGC T (SEQ ID NO 001 ).
The oligo nucleic acid agents of the invention are defined by their sequence; however, the skilled person understands that by exchanging one or two positions, particularly while increasing binding to the mRNA through introduction of nucleotide analogues, sufficient specificity of binding may be attained to achieve the inventive effect.
In certain embodiments, the oligonucleic acid agent comprises a sequence hybridizing to miR27b-5p. The agent sequence is at least 95% identical, particularly 96%, 97%, 98%, 99% or 100% identical to SEQ ID 001. In certain embodiments, the hybridizing sequence comprises deoxynucleotides, phosphothioate deoxynucleotides, LNA and/or PNA nucleotides or mixtures thereof.
Antisense composed partially or entirely of nucleoside analogues
In certain embodiments, the oligonucleic acid agent is an antisense oligonucleotide.
In certain embodiments, the oligonucleic acid agent comprises or is essentially composed of LNA moieties and comprises about 20 or fewer nucleotides.
In certain embodiments, the oligonucleic acid agent is essentially composed of LNA moieties and is described by the sequence ACC AAT CAG CTA AGC T (SEQ ID NO 008). In a particular embodiment, the nucleoside analogues of SEQ ID NO 008 are linked by phosphate esters. In a particular embodiment, the nucleoside analogues of SEQ ID NO 008 are linked by phosphothioate esters.
In certain embodiments, the oligonucleic acid agent for use in a method of treatment or prevention of heart disease comprises, or essentially consists of one or several peptide nucleic acid (PNA) moieties.
In certain embodiments the present invention includes a method of treating or preventing heart disease in a subject in need thereof comprising administering to the subject an oligonucleic acid agent directed against the miRNA miR27b-5p. Inhibition of the miRNA miR27b-5p can be effected by sequence specific chemically modified oligonucleotides. In certain embodiments, the oligonucleic acid agent of the invention can comprise locked nucleic acid (LNA), in which the nucleic acid's ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon, which locks the ribose in the 3'-endo conformation. Similarly, the oligonucleic acid agent of the invention may comprise PNA moieties. LNA and PNA have a higher binding energy to base-matched DNA or RNA, resulting in tighter binding. Such binding may add in antisense-based inhibition of the complementary miR27b-5p miRNA target.
In certain embodiments, the oligonucleic acid agent of the invention may include a phosphonate, a phosphorothioate or a phosphate ester phosphate backbone modification.
In certain embodiments, the oligonucleic acid agent of the invention may include ribonucleotides. Optionally, the oligonucleic acid agent of the invention may include deoxy ribonucleotides.
In certain embodiments, the hybridizing sequence of the oligonucleic acid agent comprises ribonucleotides, deoxynucleotides, phosphothioate deoxynucleotides, phosphothioate ribonucleotides and/or 2’-0-methyl-modified phosphothioate ribonucleotides.
In certain embodiments, the oligonucleic acid agent comprises ribonucleotides and deoxyribonucleotides, in particular modified ribonucleotides and modified deoxyribonucleotides. A non-limiting example of a modification of deoxyribonucleotides and ribonucleotides are phosphorothioate modified linkages in the oligonucleotide backbone. A non-limiting example of a modification of ribonucleotides is a 2’-0 to 4’-C bridge.
In certain embodiment the 2’-0/4’-C bridge is a five-membered, six-membered or seven membered bridged structure.
Antisense aaomers
In certain embodiments, the oligonucleic acid agent is a gapmer characterized by a central DNA block, the sequence of which is complementary to the miR27b-5p miRNA, and which is flanked on either side (5’ and 3’) by nuclease-resistant LNA sequences which are also complementary to the miR27b-5p miRNA. The central DNA block contains the RNase H activating domain, in other words is the part that lead the target DNA to be hydrolyzed. In certain embodiments, the flanking LNA is fully phosphorothioated.
In certain embodiments, the oligonucleic acid agent comprises 12 - 20 nucleotides. In certain particular embodiments, the oligonucleic acid agent comprises 14-16 nucleotides. In certain embodiments, the hybridizing sequence of the oligonucleic acid agent according to the invention comprises 14,15 or 16 nucleotides.
In certain embodiments, the central deoxyribonucleotide oligomer block of the gapmer comprises at least 5 deoxyribonucleotides. In certain embodiments, the central deoxyribonucleotide oligomer block of the gapmer comprises 5 to 10 deoxyribonucleosides linked by phosphate ester bonds or thiophosphate ester bonds.
In certain embodiments, the central deoxyribonucleotide oligomer block of the gapmer comprises a phosphate backbone between the deoxyribonucleosides.
In certain embodiments, the oligonucleic acid agent comprises, or essentially consists of, a central block of 5 to 10 deoxyribonucleotides linked by phosphate ester bonds flanked on either side by 2’-0 modified ribonucleotides or PNA oligomers. In certain embodiments, the oligonucleic acid agent comprises, or essentially consists of, a central block of 5 to 10 deoxyribonucleosides flanked by LNA nucleoside analogues. In certain particular embodiments, said LNA nucleoside analogues are linked by phosphothioate moieties.
In certain embodiments, the oligonucleic acid agent of the invention comprises or essentially consists of the sequence ACCA-atcagcta-AGCT (SEQ ID NO 005), wherein the capital letters signify nucleoside analogues, particularly LNA, more particularly LNA linked by phosphothioate esters, and the lower case letters signify DNA nucleosides linked by phosphate esters, and the link between a nucleoside analogue and a DNA nucleoside is selected from phosphate ester and thiophosphate.
Figure imgf000008_0001
In certain embodiments, the oligonucleic acid agent is a ribonucleic agent, particularly a siRNA or shRNA.
An RNA interference (RNAi) agent in the context of the present specification refers to a ribonucleotide oligomer that causes the degradation of its enhancer RNA (eRNA) target sequence.
In certain embodiments, the RNAi agents of the invention comprise, or consist of,
- a single-stranded or double-stranded interfering ribonucleic acid oligomer or precursor thereof, comprising a sequence tract complementary to the targeted enhancer RNA molecule; or
- a single-stranded or double-stranded antisense ribonucleic or deoxyribonucleic acid, comprising a sequence tract complementary to the targeted enhancer RNA molecule. In certain embodiments, the sequence tract complementary to the targeted enhancer RNA molecule is a contiguous sequence tract 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 nucleotides in length.
In certain embodiments, the RNAi agents of the invention include, but are not limited to, small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), microRNAs and non-coding RNAs or the like, Morpholinos (phosphodiamidate morpholino oligomers) and Dicer substrate siRNAs (DsiRNAs, DsiRNAs are cleaved by the RNAse III class endoribonuclease Dicer into 21 - 23 base duplexes having 2-base 3’-overhangs), UsiRNAs (UsiRNAs are duplex siRNAs that are modified with non-nucleotide acyclic monomers, termed unlocked nucleobase analogues (UNA), where the bond between two adjacent carbon atoms of ribose is removed), self- delivering RNAs (sdRNAs) including rxRNA™ (RXi Pharmaceuticals, Westborough, MA, USA).
In some embodiments, the RNAi agents of the invention comprise analogues of nucleic acids such as phosphotioates, 2’O-methylphosphothioates, peptide nucleic acids (PNA; N-(2- aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha- carbon of the glycine) or locked nucleic acids (LNA; 2Ό, 4’C methylene bridged RNA building blocks). The hybridizing sequence may be composed partially of any of the above nucleotides, with the rest of the nucleotides being“native” ribonucleotides occurring in nature, or may be mixtures of different analogues, or may be entirely composed of one kind of analogue.
In certain embodiments, the oligonucleic agent is conjugated to, or encapsulated by, a nanoparticle, a virus and a lipid complex.
In certain embodiments, the oligonucleic acid agent is a gapmer comprising a central deoxyribonucleotide oligomer block flanked by nuclease resistant ribonucleotide analogues on either (5’ and 3’) side.
Within the scope of the present invention is a method for treating or preventing heart disease in a patient in need thereof, comprising administering to the patient an oligonucleic acid agent according to the invention.
Similarly, a dosage form for the prevention or treatment of heart disease is provided, comprising the oligonucleic acid agent according to the invention.
Dosage forms may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. Alternatively, parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and/or excipient may be present. In certain embodiments, the amount of the oligonucleic acid agent sufficient to reduce expression of miR27b-5p miRNA is of 1 nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less, 10 picomolar or less, 5 picomolar or less, 2, picomolar or less and 1 picomolar or less in the environment of the cell.
Sequences
SEQ ID NO 001 (miR27b-5p targeting sequence ; 5’-3’, and nucleotide chemistry):
ACC AAT CAG CTA AGC T SEQ ID NO 002 (miR27b-5p)
G C AG AAC UUAGCCACUGU G AA
SEQ ID NO 003 (AAV9-fl/fl-sh Mthfd 11 ; sense):
T G AAT G GTGT CAG AG AATTTTT CAAG AG AAAATT CT CT G ACACCATT CTTTTTT C SEQ ID NO 004 (AAV9- I- shMthfdl l; antisense):
T CGAGAAAAAAGAAT GGT GT CAGAGAATTTT CT CTT GAAAAATT CT CT GACACCATT CA
SEQ ID NO 005 ACCA-atcagcta-AGCT (upper case: nucleoside analogue, particularly LNA linked by thiophosphate; lower case: DNA; boundary: phosphate or thiophosphate)
SEQ ID NO 006 (Primer sequence forward; mir27b): GCATGCTGATTTGTGACTTGAG-3’
SEQ ID NO 007 (Primer sequence reverse; mir27b): 5 -CCTCTGTTCTCCAAACTGCAG-3'.
SEQ ID NO 008: (miR27b-5p targeting sequence ; 5’-3’, LNA):
ACC AAT CAG CTA AGC T
Wherever alternatives for single separable features are laid out herein as“embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.
The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.
Brief description of the figures
FIG 1A -B show the control of FiFo ATP synthase on endoreplication and multinucleation in pathologic growth. Expression of genes coding for ATP synthase subunits and markers of cardiac hypertrophy and dysfunction ( NPPA and NPPB) in left ventricular biopsies from hypertrophic cardiomyopathy (HCM) and aortic stenosis (AS) patients versus healthy controls and in ventricular biopsies from mice subjected to transaortic constriction (TAC) versus sham- operated animals. The echocardiographically obtained measurements of the inter-ventricular septum diameter (IVSD;d) and left-ventricular posterior wall diameter in diastole (LVPWD;d), as well as the fractional shortening (%FS) and ejection fraction (%EF) of TAC or sham operated animals is shown.
FIG 1C-D shows biopsies of left ventricles from patients with HCM and aortic stenosis and healthy controls (c) or sham- and TAC-operated mice (d) assessed for denoted protein expression by immunoblotting.
FIG 1 E-F show the ADP/ATP ratio in ventricular biopsies from HCM and aortic stenosis patients versus healthy controls (e), and in ventricular biopsies from mice subjected to TAC versus sham-operated animals (f).
FIG 1 G shows left ventricular longitudinal sections from patients with aortic stenosis (AS) (n=3) and hypertrophic cardiomyopathy (HCM) (n=3) and healthy controls (n=3) stained for DAPI (blue), laminin (green) and oactinin (red), and imaged by confocal microscopy.
FIG 1 H shows heart sections stained as in FIG. 1 G and assessed for the percentage of mono- , bi-, or multinucleated cardiomyocytes.
FIG 11 shows left ventricular longitudinal sections from sham- (n=3) and TAC (n=3) -operated mice stained for DAPI (blue), laminin (green) and oactinin (red), and imaged by confocal microscopy.
FIG 1J shows heart sections stained as in (i) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes. (2 sections/heart were analyzed with 2 fields/section used for quantification. In total, 3 hearts were analyzed per group).
FIG 1 K shows schematic representation of the AAV9-fl/fl-shAtp5a1 virus before and after Cre- mediated recombination (left panel) and of the experimental timeline (right panel).
FIG 1 L shows an immunoblot of ventricular lysates from Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 using antibodies against denoted proteins.
FIG 1 M shows an ADP/ATP ratio measured in left ventricular samples of Mlc2v-cre+ and Mlc2v- cre mice transduced with AAV9-fl/fl-shAtp5a1 1 1 weeks after AAV9 injection.
FIG 1 N shows left ventricular longitudinal sections from Mlc2v-cre+ (n=3) and Mlc2v-cre (n=3) mice transduced with AAV9-fl/fl-shAtp5a1 were stained for DAPI (blue), laminin (green) and o actinin (red), and imaged by confocal microscopy.
FIG 10 shows heart sections stained as in FIG1 N assessed for the ratio of mono-, bi-, or multinucleated cardiomyocytes. FIG 1P shows representative images of left ventricles of Mlc2v-cre+ and M!c2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 11 weeks after AAV9 injection.
FIG 1Q shows representative images of H&E-stained histological sections of Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 1 1 weeks after AAV9 injection.
FIG 1 R-S show left ventricular weight/body weight (LVW/BW) (R) and ejection fraction (S) of Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 11 weeks after AAV9 injection.
FIG 2A shows the expression of MIR27B in patients with hypertrophic cardiomyopathy (HCM) or aortic stenosis (AS)
FIG 2B shows the schematic representation of the experimental timeline of Mlc2v-cre mice transduced with AAV9-fl/fl-mir27b.
FIG 2C shows representative images of left ventricles of Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-mir27b viruses.
FIG 2D shows representative images of H&E-stained histological sections of Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-mir27b viruses.
FIG 2E-F show LVW/BW (e) and ejection fraction (f) of Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-mir27b viruses.
FIG 2G show the relative expression of mature miR27b-5p and miR27b-3p in Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-mir27b viruses.
FIG 2H shows an immunoblot of ventricular lysates from Mlc2v-cre+ and Mlc2v-cre mice transduced as in FIG 2C-G using antibodies against denoted proteins.
FIG 2I shows left ventricular longitudinal sections from Mlc2v-cre+ (n=3) and Mlc2v-cre (n=3) mice transduced with AAV9-fl/fl-mir27b, stained for DAPI (blue), laminin (green) and oactinin (red) and imaged by confocal microscopy.
FIG 2J shows heart sections stained as in FIG 2I and assessed for the ratio of mononucleated to multinucleated cardiomyocytes.
FIG 2K shows a schematic representation of the antagomir study in sham- and TAC-operated mice. Baseline echocardiography measurement was performed at day = -5, sham or TAC surgery at day = 0, scrLNA and miR27b-5p LNAs delivered intraperitoneal (i.p) at day = 49-52 post surgery and monitored by echocardiography at the indicated time points.
FIG 2 L-M show representative images of left ventricles (FIG 2L) and H&E-stained (FIG 2M) histological sections. FIG 2N-P show longitudinal monitoring of LVW/BW (FIG 2N), left ventricular internal diameter at end systole (LVID;s) (FIG 20) and ejection fraction (FIG 2P) of C57BL/6J mice subjected to sham or TAC surgery and treated with either scrambled (scrLNA) or miR27b-5p LNAs.
FIG 2Q-T show the relative expression of mature miR27b-5p (FIG 2Q), miR27b-3p (FIG 2R), Nppa and Nppb (FIG 2S) and Atp5a1 mRNA (FIG 2T) in C57BL/6J mice treated with scrLNA or miR27b-5p LNAs and subjected to either sham or TAC surgery by qPCR.
FIG 2U shows an immunoblot of ventricular lysates from sham- and TAC operated C57BL/6J mice treated with either scrLNA or miR27b-5p LNAs using antibodies against denoted proteins.
FIG 2 V shows left ventricular longitudinal sections from sham- or TAC operated mice treated with either scrLNA or miR27b-5p LNAs stained for DAPI (blue), laminin (green) and oactinin (red) and imaged by confocal microscopy.
FIG 2W shows heart sections stained as in (v) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes.
FIG 2X shows Kaplan-Meier survival curves comparing mortality between mice subjected to sham or TAC surgery and treated with scrLNA or miR27b-5p LNAs. No mortality was observed in sham-operated animals.
FIG 3A shows a schematic representation of de novo purine biosynthesis pathway showing the contribution of glycolysis and 1 -carbon metabolism.
FIG 3B shows the relative amount of formate in NRCs transduced and treated as denoted.
FIG 3C shows relative mitochondrial ADP/ATP ratio in NRCs transduced and treated as denoted.
FIG 3D shows a heat map of relative metabolite abundance in NRCs transduced and treated as indicated. Depicted are metabolites with log2(fold change)>0.5 compared to control treatment and adjusted p value<0.01 in at least one treatment group compared to corresponding control (n=4 biological replicates per group).
FIG 3E shows the relative amount of [14C]carbon derived from [14C]glucose, [14C]serine and [14C]glycine incorporated into nucleic acids in NRCs transduced and treated as denoted.
FIG 4A-F show NRCs transduced and treated as indicated were stained with propidium iodide (PI) and assessed for polyploidy by flow cytometry coupled to imaging (a,c,e) and multinucleation quantified from images (b,d,f).
FIG 4G-I show an evaluation of [3H]leucine incorporation in NRCs transduced and treated as indicated. Data is represented as incorporated radioactivity relative to control NRCs (set as 1 .0). FIG 5A shows a schematic representation of the experimental timeline of Mlc2v-cre mice transduced with AAV9-fl/fl-shMthfd1 l and subjected to sham or TAC surgery.
FIG 5B shows left ventricular longitudinal sections from sham- or TAC-operated Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-shMthfd1 l viruses were stained for DAPI (blue), laminin (green) and a-actinin (red), and imaged by confocal microscopy. (3 sections/heart were analyzed with 2-4 fields/section imaged with representative z-stack fields shown. In total, 3 hearts were analyzed per group). Arrows indicate nucleation of cardiomyocytes.
FIG 5C shows heart sections stained as in (b) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes.
FIG 5D shows representative images of left ventricles of sham or TAC-operated Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-sh Mthfd 11 viruses.
FIG 5E shows representative images of H&E-stained histological sections of sham or TAC- operated Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-sh Mthfd 11 viruses.
FIG 5F-G shows LVW/BW (f) and ejection fraction (g) of sham or TAC-operated Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-sh Mthfd 11 viruses.
FIG 5H shows an immunoblot of ventricular lysates from sham or TAC-operated Mlc2v-cre+ and Mlc2v-cre mice transduced as in (b-g) using antibodies against denoted proteins.
FIG 5I shows a schematic representation of the experimental timeline of Mlc2v-cre mice co- transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11.
FIG 5J shows left ventricular longitudinal sections from Mlc2v-cre+ (n=3) and Mlc2v-cre (n=3) mice co-transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11 were stained for DAPI (blue), laminin (green) and oactinin (red), and imaged by confocal microscopy.
FIG 5K shows heart sections stained as in (j) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes.
FIG 5I shows representative images of left ventricles of Mlc2v-cre+ and Mlc2v-cre mice co- transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11.
FIG 5M shows representative images of H&E-stained histological sections of Mlc2v-cre+ and Mlc2v-cre mice co-transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11.
FIG 5N-0 shows LVW/BW (n) and ejection fraction (o) of Mlc2v-cre+ and Mlc2v-cre mice co- transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11.
FIG 5P shows an immunoblot of ventricular lysates from Mlc2v-cre+ and M!c2v-cre mice transduced as in j-p using antibodies against denoted proteins.
FIG. 6A shows an immunoblot of NRCs ectopically expressing a constitutively active form of AMPKal using antibodies against denoted proteins.
FIG. 6B shows an immunoprecipitation (IP) with either control IgG or anti-AMRKa antibody from NRCs ectopically expressing a constitutively active form of AMPKal . The IPs were blotted using p-AMPKa and p-Rb antibodies.
FIG. 6C shows an immunoblot of NRCs ectopically expressing HIFI aAODD and treated with either DMSO or the AMPK inhibitor compound C (CC) using antibodies against denoted proteins.
FIG. 6D shows an immunoprecipitation with either control IgG or anti-AMRKa antibody from NRCs ectopically expressing HIFI aAODD and treated with either DMSO or the AMPK inhibitor CC. The IPs were blotted using p-AMRKa and p-Rb antibodies.
FIG. 6E shows an immunoblot of ventricular lysates from sham- or TAC operated mice treated with either scrLNA or miR27b-5p LNAs using antibodies against p-Rb and total Rb.
FIG. 6F shows an immunoblot of ventricular lysates from Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-mir27b viruses using antibodies against p-Rb and total Rb.
FIG. 6G shows an immunoblot of ventricular lysates from Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-shAtp5a1 viruses using antibodies against p-Rb and total Rb.
FIG. 6H shows an immunoblot of ventricular lysates from sham- or TAC-operated Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-shMthfd1 l viruses using antibodies against p-Rb and total Rb.
FIG. 6I shows an immunoblot of ventricular lysates from Mlc2v-cre+ and Mlc2v-cre mice co- transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11 using antibodies against p-Rb and total Rb.
FIG. 6J shows an immunoblot of ventricular lysates from HCM and aortic stenosis patients and healthy controls using antibodies against p-Rb and total Rb.
FIG. 6K shows a heat map of relative expression of denoted genes in ventricular lysates from sham- or TAC operated mice treated with either scrLNA or miR27b-5p LNAs.
FIG. 6L shows a heat map of relative expression of denoted genes in ventricular lysates from Mlc2v-cre+ and Mlc2v-cre mice injected with either AAV9-fl/fl-mir27b
FIG. 6M shows a heat map of relative expression of denoted genes in ventricular lysates from Mlc2v-cre+ and Mlc2v-cre mice subjected to sham or TAC surgery and injected with AAV9- fl/fl-sh Mthfd 11 viruses. FIG. 6N shows a heat map of relative expression of denoted genes in ventricular lysates from HCM and aortic stenosis patients and healthy controls.
FIG 60 shows the transfection of E2F luciferase reporter in NRCs infected and treated as indicated. Data is normalized to control infected NRCs (set as 1.0).
FIG. 6P shows the transfection of E2F luciferase reporter in NRCs infected as indicated. Data is normalized to control infected NRCs (set as 1 .0).
FIG 6Q shows the proposed mechanism of FiFo ATP synthase function in cardiac ploidy and growth control. Pathologic stress leads to the stabilization and accumulation of HIF1 a and HIF1 a -dependent induction of glycolytic genes and the microRNA MIR27B. MIR27B, in turn, binds to the 3’UTR of the alpha subunit of FiFo ATP synthase (ATP5A1 ) thereby targeting it for degradation. As a consequence of F1F0 ATP synthase inhibition, the ADP/ATP ratio is elevated inside the mitochondrial matrix which, on one hand, activates the energy sensor AMPK. pAMPK in turn hyperphosphorylates Rb resulting in the release of the transcription factor E2F and induction of cyclins and CDKs expression. On the other hand, the accumulating mitochondrial ADP is rechanneled towards MTHFD1 L, an enzyme involved in the mitochondrial one-carbon metabolism that uses ADP as a cofactor to catalyze the conversion from CHO-THF to format. Formate, that is particularly derived from serine serves as a 1 -carbon donor for the de novo synthesis of purines. The increased de novo formation of purines as substrates for nucleic acid synthesis and the induction of cyclins and CDKs promote DNA replication, multinucleation and cardiac growth. CHO-THF denotes formyl-tetrahydrofolate, CH2-THF denotes methylenetetrahydrofolate, 3-PG denotes 3-phosphoglycerate.
FIG 7 a shows a Pearson’s correlation coefficient of ATP synthase subunit genes presented in Fig. 1 a (left panel) against expression of NPPB. (n=4 for healthy controls; n=10 for AS and n=1 1 for HCM).
FIG 7 b shows quantification of ATP levels from ventricular lysates of sham- and TAC-operated animals. (n=5 for sham and n=6 for TAC; shown is mean ± SEM; *** p < 0.001 ; two-tailed unpaired t-test).
FIG 7 c, Left ventricular sections from HCM (n=3) and aortic stenosis (n=3) patients and healthy controls (n=3) were stained with haematoxylin and eosin (H&E) and imaged by light microscopy. (2 sections/heart with 2 fields/section were surveyed with representative fields shown). Scale bar is 200 pm.
FIG 7 d, Left ventricular sections from sham- (n=3) and TAC (n=3)-operated mice were stained with H&E and imaged by light microscopy. (3 sections/heart with 3-5 fields/section were surveyed with representative fields shown). Scale bar is 200 pm. FIG 7 e, Cardiomyocytes were isolated from sham- and TAC-operated mice and stained for DAPI and oactinin. Cells were imaged by confocal microscopy and a representative z-stack image from 3 mice per group is shown. Scale bar is 20 pm.
FIG 7 f, Adult cardiomyocytes stained as in (e) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes. (At least 130 cells were quantified from n=3 mice per group).
FIG 7 g, HW/BW in sham- and TAC-operated mice. (n=5 for sham and n=6 for TAC; * p < 0.05; two-tailed unpaired t-test).
FIG 7 h,i, Representative images of H&E (h) and picrosirius red (i) stained histological sections of sham- and TAC-operated mice. Scale bar is 500 pm.
FIG 7 j, Relative expression of Col1a1, Col3a1 and TGFbl mRNA in left ventricular lysates of sham and TAC-operated mice. Data is normalized to sham-operated animals (set as 1 .0). (n=5 for sham and n=6 for TAC; results shown are the mean ± SEM; * p < 0.05; two-tailed unpaired t-test)
FIG 8 a, Relative expression of Atp5a1 mRNA in Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 1 1 weeks after AAV9 injection. Data is normalized to Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 (set as 1 .0). (n=8 for Mlc2v-cre and n=9 for Mlc2v-cre+, shown is mean ± SEM; *** p < 0.001 ; two-tailed unpaired t-test).
FIG 8b, Quantification of ATP levels from ventricular lysates of Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 (n=8 for Mlc2v-cre and n=9 for Mlc2v-cre+, shown is mean ± SEM; * p < 0.05; two-tailed unpaired t-test).
FIG 8c, Left ventricular sections from Mlc2v-cre+ (n=3) and Mlc2v-cre (n=3) mice injected with AAV9-fl/fl-shAtp5a 1 viruses were stained with H&E and imaged by light microscopy. (3 sections/heart with 3-5 fields/section were surveyed with representative fields shown). Scale bar is 200 pm.
FIG 8d, Cardiomyocytes were isolated from Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-shAtp5a 1 viruses and stained for DAPI and a-actinin. Cells were imaged by confocal microscopy and a representative z-stack image from 3 mice per group is shown. Scale bar is 20 pm.
FIG 8e, Adult cardiomyocytes stained as in (d) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes. (At least 150 cells were quantified from n=3 mice per group).
FIG 8f, Relative expression of Nppa and Nppb mRNA in Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 1 1 weeks after AAV9 injection. Data is normalized to Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 (set as 1 .0). (n=8 for Mlc2v-cre and n=9 for Mlc2v-cre+ ; shown is mean ± SEM; * p < 0.05; two-tailed unpaired t-test).
FIG 8g, LVID;s in Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 1 1 weeks after AAV9 injection. (n=8 for Mlc2v-cre and n=9 for Mlc2v-cre+, shown is mean ± SEM; ** p < 0.01 ; two-tailed unpaired t-test).
FIG 8 h, Representative images of picrosirius red-stained histological sections of Mlc2v-cre+ and Mlc2v-cre~ mice transduced with AAV9-fl/fl-shAtp5a1 viruses. Scale bar is 500 pm.
FIG 8i, Relative expression of Col1a1, Col3a1 and TGFbl mRNA in left ventricular lysates of Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-shAtp5a1 viruses. Data is normalized to Mlc2v-cre mice (set as 1 .0). (n=8 for Mlc2v-cre and n=9 for Mlc2v-cre+ mice; results shown are the mean ± SEM; * p < 0.05; two-tailed unpaired t-test).
FIG 8j, Relative expression of ATP5A1 mRNA in iPSC-derived human cardiomyocytes (iPSC- hCM) transduced with lentivirus expressing non-silencing shRNA (nsRNA) or shRNA against ATP5A1 (shATP5A1 ). Data is normalized to control NRCs expressing nsRNA (set as 1.0). (n=3 biological replicates per group; results shown are the mean ± SD; *** p < 0.001 ; two-tailed unpaired t-test).
FIG 8k, ADP/ATP ratio in iPSC-hCM transduced with lentivirus expressing nsRNA or shATP5A1 . (n=3 biological replicates per group; shown is mean ± SD; ** p < 0.01 ; two-tailed unpaired t-test).
FIG 8l,m, Quantification of ADP (I) and ATP (m) levels from iPSC-hCM transduced with lentivirus expressing nsRNA or shATP5A1 . (n=3 for both groups; shown is mean ± SD; ** p < 0.01 ; two-tailed unpaired t-test).
FIG 8n, iPSC-hCM transduced with lentivirus expressing nsRNA or shATP5A1 were stained for DAPI and phalloidin, and imaged by confocal microscopy. Representative fields from 3 biological replicates are shown. Scale bar is 20 pm.
FIG 8o, Quantification of percentage of multinucleated cells in iPSC-hCM treated as in (n) (n=3 biological replicates with approximately 100 cells analyzed per experiment and condition; shown is mean ± SD; * p < 0.05; two-tailed unpaired t-test).
FIG 8p, iPSC-hCM treated as in (n) were assessed for cell size using ImageJ. (n=3 independent experiments with approximately 100 cells analyzed per experiment and condition; shown is mean ± SD; * p < 0.05; two-tailed unpaired t-test).
FIG 8q, Relative expression of Atp5a1 mRNA in NRCs transduced with lentivirus expressing nsRNA or shAtp5a1. Data is normalized to control NRCs expressing nsRNA (set as 1.0). (n=3 biological replicates per group; results shown are the mean ± SD; *** p < 0.001 ; two-tailed unpaired t-test).
FIG 8r, NRCs transduced with nsRNA or shAtp5a1 were assessed for Atp5a1 protein levels by immunoblotting. Loading is normalized to cardiac actin.
FIG 8s, ADP/ATP ratio in NRCs transduced with lentivirus expressing nsRNA or Atp5a1 shRNA as indicated. (n=3 biological replicates per group; shown is mean ± SD; ** p < 0.01 ; two-tailed unpaired t-test).
FIG 8t,u, Quantification of ADP (t) and ATP (u) levels from NRCs transduced with lentivirus expressing nsRNA or Atp5a1 shRNA. (n=3 for both groups; shown is mean ± SD; ** p < 0.01 ; two-tailed unpaired t-test).
FIG 8v, NRCs transduced with lentivirus expressing nsRNA or Atp5a1 shRNA were stained for phalloidin and DAPI, and imaged by confocal microscopy. Representative fields from n=3 biological replicates are shown. Scale bar is 20 pm.
FIG 8w, Quantification of the percentage of multinucleated cells in NRCs treated as in (v) (n=3 biological replicates with approximately 100 cells analyzed per experiment and condition; shown is mean ± SD; * p < 0.05; two-tailed unpaired t-test).
FIG 8x, NRCs treated as in (v) were assessed for cell size using ImageJ. (n=3 independent experiments with approximately 100 cells analyzed per experiment and condition; shown is mean ± SD; * p < 0.05; two-tailed unpaired t-test).
FIG 8y, Evaluation of [3H]leucine incorporation in NRCs transduced with lentivirus expressing nsRNA or Atp5a1 shRNA. Data is represented as incorporated radioactivity relative to control NRCs expressing nsRNA (set as 1.0). (n=4 biological replicates per group; results shown are the mean ± SEM; * p < 0.05; two-tailed unpaired t-test).
FIG 9 a,b, Relative expression of Atp5a1 mRNA in ventricles of control (Hiflct fl/fl) and Hifla cKO mice subjected to sham or TAC surgery (a), or control (V hi fl/fl) and V hi cKO mice (b). Data is normalized to controls (set as 1.0). (n=5 mice per group for (a) and (b); shown is mean ± SEM; *, % p < 0.05; ** p < 0.01 ; one-way ANOVA and Bonferroni correction for (a) and two- tailed unpaired t-test for (b)).
c, Ventricular lysates of sham- or TAC-operated control ( Hifla fl/fl) and Hifla cKO mice (left panel), or control (Vhl fl/fl) and Vhl cKO mice (right panel) were processed for immunoblotting with antibodies against Hifla and Atp5a1. Loading is normalized to cardiac actin.
FIG 9 d,e, Quantification of ATP amount (d) and ejection fraction (e) in left ventricular biopsies from control ( Hifla fl/fl) and ventricle-specific Hifla conditional knockout ( Hifla cKO) mice subjected to sham or TAC surgery (d). (n=5 mice per group for (d) and (e); shown is mean ± SEM; *, % p < 0.05; one-way ANOVA and Bonferroni correction).
FIG 9 f,g, Quantification of ATP amount in left ventricular biopsies (f) and ejection fraction (g) of ventricle-specific V hi conditional knockout (V hi cKO) and respective control (V hi fl/fl) mice (g). (n=5 mice per group for (f) and (g); shown is mean ± SEM; * p < 0.05; ** p < 0.01 ; two- tailed unpaired t-test).
FIG 9 h, Expression of miRNAs containing putative HRE(s) in their promoter (p<0.0001 between technical quadruplicates, and p<0.05 of the biological triplicates with average signal intensity of 1 -fold over background) from ventricular lysates of Hifla cKO mice subjected to TAC surgery versus TAC-operated control animals, or Vhl cKO mice versus control animals.
FIG 9 i, Relative expression of mir27b, Vegfa and Ldha mRNA in ventricles of control ( Hifla fl/fl) and Hifla cKO mice subjected to sham or TAC surgery. Data is normalized to sham- operated controls (set as 1 .0). (n=5 mice per group; shown is mean ± SEM; *, % p < 0.05; one- way ANOVA and Bonferroni correction).
FIG 9 j, Relative expression of mir27b RNA in ventricles of control (Vhl fl/fl) and Vhl cKO mice. Data is normalized to control mice (set as 1 .0). (n=5 mice per group; shown is mean ± SEM; *** p < 0.001 ; two-tailed unpaired t-test).
FIG 9 k-m, Relative expression levels of AmpO (k), mature miR27b-3p, miR23b-3p and miR24- 3p (I), and miR27b-5p, miR23b-5p and miR24-5p (m) from ventricular lysates of wildtype C57BL/6J mice subjected to sham or TAC surgery. Data is normalized to sham-operated control mice (set as 1 .0). (n=5 for sham and n=8 for TAC; data shown is mean ± SEM; * p < 0.05; ** p < 0.01 ; *** p < 0.001 ; two-tailed unpaired t-test).
FIG 9 n-p, Relative expression of AmpO (n), mature miR27b-3p, miR23b-3p and miR24-3p (o), and miR27b-5p, miR23b-5p and miR24-5p (p) in NRCs ectopically expressing an empty control vector or HIFI aAODD. Data is normalized to NRCs transduced with an empty control vector (set as 1.0). (n=5 biological replicates per group; shown is mean ± SEM; ** p < 0.01 ; two-tailed unpaired t-test).
FIG 9 q, Evaluation of [3H]leucine incorporation in NRCs treated with T3 or PBS (mock). Data is represented as incorporated radioactivity relative to mock-treated NRCs (set as 1 .0). (n=4 biological replicates per group; results shown are the mean ± SEM; *** p < 0.001 ; two-tailed unpaired t-test).
FIG 9 r-t, Relative expression of AmpO (r), mature miR27b-3p, miR23b-3p and miR24-3p (s), and miR27b-5p, miR23b-5p and miR24-5p (t) in NRCs treated with PBS (mock) or T3. Data is normalized to mock-treated NRCs (set as 1 .0). (n=5 biological replicates per group; shown is mean ± SEM; *** p < 0.001 ; two-tailed unpaired t-test). FIG 9 u, Relative expression of Hifla, mir27b, Glutl and Vegfa mRNA in NRCs stimulated with T3 and transduced with non-silencing control shRNA (nsRNA) or shRNA against Hifl a (shHifl a). Data is normalized to control NRCs infected with nsRNA (set as 1 .0). (n=3 biological replicates per group; shown is mean ± SD; *, g, % p < 0.05; one-way ANOVA and Bonferroni correction).
FIG 9 v, Sequence of the human, monkey, possum, rat and mouse mir27b promoter harboring a conserved HRE located 162 bp upstream of the precursor mir27b transcription start site. HRE is shown in red, with the core HRE motif capitalized.
FIG 9 w, Co-transfection of wildtype (wt) or HRE-mutated (mut) mir27b promoters fused to luciferase with different doses of either an empty control vector or HIFI aAODD. Data is normalized to wildtype promoter transfected with empty control vector (set as 1 .0). (n=4 biological replicates per group; data shown is mean ± SEM; * p < 0.05; one-way ANOVA followed by Dunnett’s post test).
FIG 9 x, Co-transfection of mir23b and mir24-1 promoters fused to luciferase with different doses of either an empty vector control or HIFI a A ODD. Data is normalized to luciferase vector transfected with empty control vector (set as 1.0). (n=4 biological replicates per group; data shown is mean ± SEM; * p < 0.05; one-way ANOVA followed by Dunnett’s post test).
FIG 9 y, NRCs were transduced with lentivirus expressing either a nsRNA or shRNA against Vhl (shVhl) and processed for chromatin immunoprecipitation with a HIF1 a-specific antibody (IP: HIF1 a) or with a control isotype-matched antibody (IgG control). Hifl a promoter binding was analyzed by qPCR. Data shown is relative to chromatin immunoprecipitation with Ig control antibody on nuclear lysates from NRCs expressing nsRNA (set as 1 .0). (n=3 biological replicates per group; data shown is mean ± SD; *** p < 0.001 ; two-tailed unpaired t-test).
FIG 9 z, NRCs transduced with nsRNA or shVhl were assessed for Hifl a and Vhl protein levels by immunoblotting. Loading is normalized to cardiac actin.
FIG 10 a, Target gene recognition motif in the 3’UTR of human and mouse Atp5aJ The miR27b-5p seed sequence and corresponding target region on Atp5a1 are indicated by alignment.
FIG 10 b, Co-transfection of wildtype (wt) or mir27b binding site-mutated (mut) Atp5a1 3’UTR fused to luciferase with different doses of control, miR27b-3p or miR27b-5p mimics. Data is normalized to wildtype promoter transfected with control mimics (set as 1 .0). (n=4 biological replicates per group; data shown is mean ± SEM; ** p < 0.01 ; *** p < 0.001 ; one-way ANOVA followed by Dunnett’s post test).
FIG 10 c, NRCs transduced with empty control vector or ectopic mir27b were assessed for
Atp5a1 protein levels by immunoblotting. Loading is normalized to cardiac actin. FIG 10 d,e, Relative expression of mir27b precursor (d) and mature miR27b-3p and miR27b- 5p (e) in NRCs ectopically expressing an empty control vector or mir27b. Data is normalized to NRCs transduced with an empty control vector (set as 1 .0). (n=3 biological replicates per group; shown is mean ± SD; * p < 0.05; *** p < 0.001 ; two-tailed unpaired t-test).
FIG 10 f, Relative expression level of Atp5a1 mRNA from NRCs transfected with different concentrations of control, miR27b-3p and miR27b-5p mimics. Data is normalized to control mimics (set as 1 .0). (n=3 replicates per group; data shown is mean ± SD; * p < 0.05; *** p < 0.001 ; one-way ANOVA followed by Dunnett’s post test).
FIG 10 g,h, Relative expression of mature miR27b-5p (g) and miR27b-3p (h) in NRCs transduced with an empty vector control or HIFI aAODD and treated with either scrLNA or miR27b-5p LNAs. Data is normalized to NRCs transduced with an empty control vector (set as 1 .0). (n=5 biological replicates per group; shown is mean ± SEM; *, g, % p < 0.05; one-way ANOVA and Bonferroni correction).
FIG 10 i, Immunoblot for Hifl a and Atp5a1 expression in NRCs transduced with lentivirus expressing empty control vector or HIFI aAODD and treated with either scrambled control (scrLNA) or miR27b-5p LNAs. Loading is normalized to cardiac actin.
FIG 10 j,k, Relative expression of mature miR27b-5p (j) and miR27b-3p (k) in NRCs treated with PBS (mock) or T3 in the presence of a scrLNA or miR27b-5p LNA. Data is normalized to mock-treated cells (set as 1 .0). (n=5 biological replicates per group; shown is mean ± SEM; *, g, % p < 0.05; one-way ANOVA and Bonferroni correction).
FIG 10 1, NRCs treated with PBS (mock) or T3 in the presence of scrLNA or miR27b-5p LNA were assessed for Hifla, mir27b and Atp5a1 RNA levels. Data is normalized to NRCs treated with scrLNA (set as 1.0). (n=4 biological replicates per group; shown is mean ± SEM; *, g, % p < 0.05; one-way ANOVA and Bonferroni correction).
FIG 10 m, NRCs treated as in (I) were assessed for Atp5a1 protein levels by immunoblotting. Loading is normalized to cardiac actin.
FIG 10 n,o, ATP synthase enzymatic activity in NRCs transduced and treated as indicated. Data is normalized to control sets (set as 1.0). (n=5 biological replicates per group; results shown are the mean ± SEM; *, % p < 0.05; ** p < 0.01 ; *** p < 0.001 ; two-tailed unpaired t-test (n) or one-way ANOVA followed by Bonferroni correction (o).
FIG 10 p, NRCs transduced with empty control vector or ectopic ATP5A1 were assessed for Atp5a1 protein levels by immunoblotting. Loading is normalized to cardiac actin.
FIG 11 a, Relative expression of Nppa and Nppb mRNA in Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-mir27b 1 1 weeks after AAV9 injection. Data is normalized to Mlc2v- ere mice transduced with AAV9-fl/fl-mir27b (set as 1 .0). (n=6 for Mlc2v-cre and n=8 for Mlc2v- cre+ ; shown is mean ± SEM; * p < 0.05; two-tailed unpaired t-test).
FIG 11 b, LVID;s in Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-mir27b 1 1 weeks after AAV9 injection. (n=6 for Mlc2v-cre and n=8 for Mlc2v-cre+, shown is mean ± SEM; * p < 0.05; two-tailed unpaired t-test).
FIG 11 c, Relative expression of Atp5a1 mRNA in Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-mir27b 1 1 weeks after AAV9 injection by qPCR. Data is normalized to Mlc2v- cre mice transduced with AAV9-fl/fl-mir27b (set as 1 .0). (n=6 for Mlc2v-cre and n=8 for Mlc2v- cre+ mice; shown is mean ± SEM; *** p < 0.001 ; two-tailed unpaired t-test).
FIG 11 d, Left ventricular sections from Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9- fl/fl-mir27b were stained with H&E and imaged by light microscopy 1 1 weeks after AAV9 injection. (3 sections/heart with 3-5 fields/section were surveyed with representative fields shown. In total, 3 hearts were surveyed per group). Scale bar is 200 pm.
FIG 11 e, Cardiomyocytes were isolated from Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-mir27b viruses and stained for DAP I and a-actinin. Cells were imaged by confocal microscopy and a representative z-stack image from 3 mice per group is shown. Scale bar is 20 pm.
FIG 11 f, Adult cardiomyocytes stained as in (e) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes. (At least 160 cells were analyzed from n=3 mice per group).
FIG 11 g, Representative images of picrosirius red-stained histological sections of Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-mir27b viruses. Scale bar is 500 pm.
FIG 11 h, Relative expression of Col1a1, Col3a1 and TGFbl mRNA in left ventricular lysates of Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl-mir27b viruses. Data is normalized to Mlc2v-cre mice (set as 1.0). (n=6 for Mlc2v-cre and n=8 for Mlc2v-cre+ mice; results shown are the mean ± SEM; * p < 0.05; two-tailed unpaired t-test).
FIG 11 i, Longitudinal monitoring of aortic velocity of C57BL/6J mice subjected to sham or TAC surgery and treated with either scrambled (scrLNA) or miR27b-5p LNAs. Arrows in panels indicate LNA injections. (n=5 for sham scrLNA, n=5 for sham miR27b-5p LNA, n=7 for TAC scrLNA, n=8 for TAC miR27b-5p LNA; shown is mean ± SEM).
FIG 11 j, Left ventricular sections from C57BL/6J mice subjected to sham or TAC surgery and treated with either scrLNA or miR27b-5p LNAs were stained with H&E and imaged by light microscopy. (3 sections/heart with 2-4 fields/section were surveyed with representative fields shown. In total, 3 hearts were surveyed per group). Scale bar is 200 pm. FIG 11 k, Cardiomyocytes were isolated from C57BL/6J mice subjected to sham or TAC surgery and treated with either scrLNA or miR27b-5p LNAs and stained for DAPI and oactinin. Cells were imaged by confocal microscopy and a representative z-stack image from 3 mice per group is shown. Scale bar is 20 pm.
FIG 11 1, Adult cardiomyocytes stained as in (k) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes. (At least 150 cells were analyzed from n=3 mice per group).
FIG 11 m, Representative images of picrosirius red-stained histological sections of C57BL/6J mice subjected to sham or TAC surgery and treated with either scrLNA or miR27b-5p LNAs. Scale bar is 500 pm.
FIG 11 n, Relative expression of Col1a1, Col3a1 and TGFbl mRNA in left ventricular lysates of C57BL/6J mice treated with scrLNA or miR27b-5p LNAs and subjected to either sham or TAC surgery by qPCR. Data is normalized to sham-operated mice treated with scrLNA (set as 1 .0). (n=5 for sham scrLNA, n=5 for sham miR27b-5p LNA, n=7 for TAC scrLNA, n=8 for TAC miR27b-5p LNA; shown is mean ± SEM; *, % p < 0.05; one-way ANOVA and Bonferroni correction).
FIG. 12 a, Relative expression of Mthfdll mRNA in NRCs expressing empty control vector or ectopic MTHFD1 L. Data is normalized to NRCs expressing empty control vector (set as 1 .0). (n=3 biological replicates per group; results shown are the mean ± SD; *** p < 0.05; two-tailed unpaired t-test).
FIG. 12 b, Immunoblot of NRCs transduced with lentivirus expressing empty control vector or MTHFD1 L using antibodies against Mthfd 11. Loading is normalized to cardiac actin.
FIG. 12 c-h, Quantification of ATP (c,e,g) and ADP (d,e,h) levels in NRCs transduced and treated as indicated. (n=4 biological replicates per group; results shown are the mean ± SD; *, % p < 0.05; one-way ANOVA followed by Bonferroni correction).
FIG 13 a, Evaluation of [3H]leucine incorporation in NRCs stimulated with T3 and treated with scrLNA or miR27b-5p LNAs. Data is represented as incorporated radioactivity relative to control (mock) NRCs treated with scrLNAs (set as 1.0). (n=4 biological replicates per group; results shown are the mean ± SEM; *, % p < 0.05; one-way ANOVA followed by Bonferroni correction).
FIG 13 b, Relative amount of formate in NRCs stimulated with T3 and treated with scrLNA or miR27b-5p LNAs. (n=5 biological replicates per group; results shown are the mean ± SEM; *,% p < 0.05; one-way ANOVA followed by Bonferroni correction). FIG 13 c, Relative ADP/ATP ratio in NRCs stimulated with T3 and treated with scrLNA or miR27b-5p LNAs. (n=5 biological replicates per group; results shown are the mean ± SEM; *,% p < 0.05; one-way ANOVA followed by Bonferroni correction).
FIG 13 d, Heat map of relative metabolite abundance in NRCs stimulated with T3 and treated with scrLNA or miR27b-5p LNAs. Depicted are metabolites with log2(fold change)>0.4 and adjusted p value<0.01 in at least one treatment group compared to corresponding control (n=4 biological replicates per group).
FIG 13 e, Relative amount of [14C]carbon derived from [14C]glucose, [14C]serine and [14C]glycine incorporated into nucleic acids in NRCs stimulated with T3 and treated with scrLNA or miR27b-5p LNAs. (n=5 biological replicates per group; results shown are the mean ± SEM; * p < 0.05; one-way ANOVA followed by Bonferroni correction).
FIG 14 a, Differential analysis of metabolites in left ventricles of shm or TAC-operated C57BL/6J mice injected with scrLNA or mir27b-5p L C57BL/6J miceNA; circle seize reflects - logio(adj.p-value); circle color reflects log2 fold change compared to denoted control (n=6 biological replicates per group).
FIG 14 b, Hierarchical cluster analysis of metabolites in denoted samples measured in duplicates (n=6 biological replicates per group).
FIG 15 a, Principal Component Analysis (PCA) of denoted cohorts.
FIG 15 b,c. Principal Component Analysis performed separately on left ventricular samples of sham or TAC treated mice with active (scrambled, b) or LNA-mediated repressed miR27b-5p (c). Arrows represent lipid species that concur to explain 15% of the variance encompassed by PC1 (Wilcoxon non-parametric signed-rank test; n= 5 per group).
FIG 15 d, Barplot of the saturation profiles in the denoted four cohorts (mean +/- SD). Lipids were grouped according to the number of double bonds (db), and all species exceeding 6 db were gathered in a single group (6+). Analyzed lipids include the following 19 lipid classes cholesteryl esters (CE), ceramides (Cer), cholesterols (Choi), cardiolipins (CL), diacylglycerols (DAG), lyso-phosphatides (LPA), lyso-phosphatidylcholines (LPC), Lyso- phosphatidylehanolamines (LPE), ether linked LPE (LPE-O ), lyso-phosphatidylinositols (LPI), phosphatidylcholines (PC), ether linked PC (PC-O ), phosphatidylethanolamines (PE), ether linked PE (PE-O ), phosphatidylglycerols (PG), phosphatidylinositols (PI), phosphatidylserines (PS), sphingomyelins (SM) and triacylglycerols. (adj. p-value< 0.001 ***, adj. p-value< 0.01 **, adj. p-value< 0.1 *; Wilcoxon non-parametric signed-rank test; n= 5 biological samples per group)
FIG 15 e, Barplot of the total length profiles in the four denoted cohorts (mean +/- SD). Lipids (as analysed in d) were grouped according to the total length of their acyl chains, i.e. number of carbon atoms. Significant comparisons are indicated with asterisks (adj. p-value< 0.001 ***, adj. p-value< 0.01 **, adj. p-value< 0.1 *; Wilcoxon non-parametric signed-rank test; n= 5 biological samples per group).
FIG 15 f, Mean mol% abundance of lipid species in left ventricular biopsies of scrLNA treated TAC-operated mice minus mean mol% abundance of lipid species in sham-controls with.
FIG 15 g, Mean mol% abundance of lipid species in TAC-operated mice injected with miR27b- 5p LNA minus mean mol% abundance of lipid species in sham-controls with repressed (LNA) miR27b-5p.
FIG 15 h, Relative expression of indicated mRNAs in C57BL/6J mice treated with scrLNA or miR27b-5p LNAs and subjected to either sham or TAC surgery by qPCR. Data is normalized to sham-operated mice treated with scrLNA (set as 1 .0). (n=5 for sham scrLNA, n=5 for sham miR27b-5p LNA, n=7 for TAC scrLNA, n=8 for TAC miR27b-5p LNA; shown is mean ± SEM; *, % p < 0.05; one-way ANOVA and Bonferroni correction).
FIG 15 i, Oxidation of [1 -14C]-palmitate to 14C02 was measured in left ventricular biopsies from C57BL/6J mice subjected to sham or TAC surgery and treated with either scrLNA or miR27b- 5p LNAs. All values were normalised to the protein content. (n=5 for sham scrLNA, n=5 for sham miR27b-5p LNA, n=6 for TAC scrLNA, n=6 for TAC miR27b-5p LNA; shown is mean ± SD; *, % p < 0.05; one-way ANOVA and Bonferroni correction).
FIG 16 a,b, Quantification of ADP (a) and ATP (b) levels in NRCs treated with PBS or 10 mM ADP for 3 days. (n=4 biological replicates per group; results shown are the mean ± SD; *, % p < 0.05; one-way ANOVA followed by Bonferroni correction).
FIG 16 c,d, Quantification of ADP (c) and ATP (d) levels in NRCs treated with PBS or 20 nM oligomycin. (n=3 biological replicates per group; results shown are the mean ± SD; *, % p < 0.05; one-way ANOVA followed by Bonferroni correction).
FIG 16 e, NRCs treated with PBS or 10 mM ADP for 3 days were stained for DAPI and oactinin and imaged by confocal microscopy. Representative fields of three independent experiments are shown. Scale bar is 5 pm.
FIG 16 f, NRCs treated with PBS or oligomycin were stained for DAPI and oactinin and imaged by confocal microscopy. An average of 4 fields per condition and experiment were imaged and representative fields of three independent experiments are shown. Scale bar is 50 pm.
FIG 16 g,h, NRCs treated and imaged as in (e,f) were assessed for multinucleation. (At least 1 10 cells were quantified per sample from n=3 independent experiments; shown is mean ± SD; ** p < 0.01 ; two-tailed unpaired t-test). FIG 16 i,j, NRCs treated and imaged as in (e,f) were assessed for cell surface area using ImageJ. (At least 1 10 cells were quantified per sample from n=3 independent experiments; shown is mean ± SD; * p < 0.05; ** p < 0.01 ; two-tailed unpaired t-test).
FIG 17 a-d, NRCs transduced with the indicated lentiviruses were stained for the cardiac- specific marker a-actinin, DAPI and phospho-Histone H3 (p-Histone H3) for assessment of cell mitosis and imaged by confocal microscopy. p-Histone H3 positive cardiomyocytes were quantified. 3 biological replicates with 3-4 fields/replicate were analyzed per condition with representative fields shown. Scale bar is 100 pm.
FIG 17 e, Relative expression of Mthfdll mRNA in NRCs expressing nsRNA or shMthfdl l. Data is normalized to NRCs expressing nsRNA (set as 1 .0). (n=3 biological replicates per group; results shown are the mean ± SD; *** p < 0.05; two-tailed unpaired t-test).
FIG 17 f, Immunoblot of NRCs transduced with lentivirus expressing nsRNA or shMthfdl l using an antibody against Mthfdl l. Loading is normalized to cardiac actin.
FIG 17 g,h, NRCs transduced with the indicated lentiviruses were stained for the cardiac- specific marker a-actinin, DAPI and phospho-Histone H3 (p-Histone H3) for assessment of cell mitosis and imaged by confocal microscopy. p-Histone H3 positive cardiomyocytes were quantified. 3 biological replicates with 3-4 fields/replicate were analyzed per condition with representative fields shown. Scale bar is 100 pm.
FIG 18 a, Left ventricular sections from sham- or TAC-operated Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-sh Mthfd 11 were stained with H&E and imaged by light microscopy 1 1 weeks post AAV 9 injection. (3 sections/heart with 2-4 fields/section were surveyed with representative fields shown. In total, 3 hearts were surveyed per group). Scale bar is 200 pm.
FIG 18 b, Left ventricular internal dimension at systole (LVID;s) in sham- or TAC-operated M!c2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-sh Mthfd 11 1 1 weeks post AAV9 injection. (n=4 for sham Mlc2v-cre , n=5 for sham Mlc2v-cre+, n=8 for TAC Mlc2v-cre and n=7 for TAC Mlc2v-cre+ mice; shown is mean ± SEM; *, % p < 0.05; one-way ANOVA followed by Bonferroni correction).
FIG 18 c,d, Relative expression of Nppa and Nppb (c) and Mthfdll mRNA (d) from ventricular lysates obtained from sham- or TAC-operated Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-sh Mthfd 11 1 1 weeks post AAV9 injection. Data is normalized to sham-operated Mlc2v-cre mice (set as 1 .0). (n=4 for sham Mlc2v-cre , n=5 for sham Mlc2v-cre+, n=8 for TAC Mlc2v-cre and n=7 for TAC Mlc2v-cre+ mice; shown is mean ± SEM; *, % p < 0.05; one-way ANOVA followed by Bonferroni correction). FIG 18 e, Cardiomyocytes were isolated from sham- or TAC-operated Mlc2v-cre+ and Mlc2v- cre mice transduced with AAV9-fl/fl-shMthfd1 l and stained for DAPI and oactinin. Cells were imaged by confocal microscopy and a representative z-stack image from 3 mice per group is shown. Scale bar is 20 pm.
FIG 18 f, Adult cardiomyocytes stained as in (e) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes. (At least 150 cells were analyzed from n=3 mice per group).
FIG 18 g, Representative images of picrosirius red-stained histological sections of sham- or TAC-operated Mlc2v-cre+ and Mlc2v-cre mice transduced with AAV9-fl/fl-shMthfd1 l. Scale bar is 500 pm.
FIG 18 h, Relative expression of Col1a1, Col3a1 and TGFbl mRNA in left ventricular lysates of C57BL/6J in sham or TAC-operated Mlc2v-cre+ and Mlc2v-cre mice injected with AAV9-fl/fl- shMthfdl l viruses. Data is normalized to sham-operated Mlc2v-cre mice (set as 1 .0). (n=4 for sham Mlc2v-cre , n=5 for sham Mlc2v-cre+, n=8 for TAC Mlc2v-cre and n=7 for TAC Mlc2v- cre+ mice; shown is mean ± SEM; *, % p < 0.05; one-way ANOVA followed by Bonferroni correction).
FIG 18 i, Left ventricular sections from Mlc2v-cre+ and Mlc2v-cre mice co-transduced with AAV9-fl/fl-shAtp5a 1 and AAV9-fl/fl-shMthfd1 l were stained with H&E and imaged by light microscopy 1 1 weeks post AAV 9 injection. (3 sections/heart with 3-5 fields/section were surveyed with representative fields shown. In total, 3 hearts were surveyed per group). Scale bar is 200 pm.
FIG 18 j, Cardiomyocytes were isolated from Mlc2v-cre+ and Mlc2v-cre mice co-transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11 viruses and stained for DAPI and oactinin. Cells were imaged by confocal microscopy and a representative z-stack image from 3 mice per group is shown. Scale bar is 20 pm.
FIG 18 k, Adult cardiomyocytes stained as in (n) were assessed for the ratio of mononucleated to multinucleated cardiomyocytes. (At least 150 cells were analyzed from n=3 mice per group).
FIG 18 I, LVID;s in Mlc2v-cre+ and Mlc2v-cre mice co-transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11 1 1 weeks post AAV9 injection. (n=6 mice per group; shown is mean ± SEM).
FIG 18 m-o, Relative expression of Nppa and Nppb (m), Atp5a1 (n), and Mthfdll mRNA (o) from ventricular lysates obtained from Mlc2v-cre+ and Mlc2v-cre mice co-transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-sh Mthfd 11 1 1 weeks post AAV9 injection. Data is normalized to Mlc2v-cre mice (set as 1 .0). (n=6 mice per group; shown is mean ± SEM; *** p < 0.001 ; two-tailed unpaired t-test). FIG 18 p, Representative images of picrosirius red-stained histological sections of Mlc2v-cre+ and Mlc2v-cre mice co-transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-shMthfd1 l viruses. Scale bar is 500 pm.
FIG 18 q, Relative expression of Col1a1, Col3a1 and TGFbl mRNA from left ventricular lysates obtained from Mlc2v-cre+ and Mlc2v-cre mice co-transduced with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-shMthfd11 viruses. Data is normalized to Mlc2v-cre mice (set as 1 .0). (n=6 mice per group; shown is mean ± SEM; * p < 0.05; two-tailed unpaired t-test).
FIG 19 a,b, Relative expression of Ccndl (a) and Ccnel (b) mRNA in NRCs treated as indicated. Data is normalized to control infected cells transfected with non-silencing siRNA (set as 1 .0). (n=3 biological replicates per group; shown is mean ± SD; * p < 0.05; ** p < 0.01 one- way ANOVA and Bonferroni correction).
FIG 19 c, NRCs transduced with lentivirus expressing empty vector control or HIFI aAODD and transfected as indicated were stained for DAPI, Ki67 and oactinin, and imaged by confocal microscopy. An average of 4 fields per condition and experiment were imaged and representative fields of three independent experiments are shown. Scale bar is 50 pm.
FIG 19 d,e, Quantification of the percentage of multinucleated (d) and Ki67-positive (e) cells from NRCs treated as in (c) (n=3 independent experiments with at least 100 cells analyzed per experiment and condition; shown is mean ± SD; *, % p < 0.05; one-way ANOVA and Bonferroni correction).
FIG 19 f, NRCs treated as in (c) were assessed for cell size using ImageJ. (n=3 independent experiments with at least 100 cells analyzed per experiment and condition; shown is mean ± SD; *, % p < 0.05; one-way ANOVA and Bonferroni correction).
FIG 19 g, Relative expression of Cdknlb mRNA in NRCs treated as indicated. Data is normalized to cells transfected with non-silencing siRNA (set as 1 .0). (n=3 biological replicates per group; shown is mean ± SD; ** p < 0.01 ; two-tailed unpaired t-test).
FIG 19 h, NRCs transfected with non-silencing or Cdknl b siRNAs were stained for DAPI, Ki67 and oactinin, and imaged by confocal microscopy. An average of 4 fields per condition and experiment were imaged and representative fields of three independent experiments are shown. Scale bar is 50 pm.
FIG 19 i,j, Quantification of the percentage of multinucleated (i) and Ki67-positive (j) cells from NRCs treated as in (h) (n=3 independent experiments with at least 100 cells analyzed per experiment and condition; shown is mean ± SD; * p < 0.05; two-tailed unpaired t-test). FIG 19 k, NRCs treated as in (h) were assessed for cell size using ImageJ. (n=3 independent experiments with at least 100 cells analyzed per experiment and condition; shown is mean ± SD; * p < 0.05; two-tailed unpaired t-test).
FIG 20 a, Relative expression of AldhlH mRNA in left ventricular biopsies of C57BL/6J mice subjected to either sham or TAC surgery by qPCR. Data is shown as 2A-dCt relative to Hpr . (n=5 for sham, n=7 TAC; shown is mean ± SEM; ** p < 0.01 ; two-tailed unpaired t-test).
FIG 20 b, Relative expression of ALDH1L1 mRNA in left ventricular biopsies from hypertrophic cardiomyopathy (HCM) and aortic stenosis (AS) patients versus healthy controls . Data is shown as 2A-dCt relative to HPRT1. (n=4 for healthy controls; n=10 for AS and n=1 1 for HCM; shown is mean ± SEM).
FIG 20 c, NADPH quantification in NRCs treated as indicated. All values were normalised to the protein content. (n=3 biological replicates per group; shown is mean ± SD).
FIG 20 d, Immunoblot of left ventricular lysates from HCM and aortic stenosis patients and healthy controls using a methylated lysine antibody. Loading is normalized to cardiac actin. FIG 20 e-j, Immunoblot of left ventricular lysates from mice treated as indicated using a methylated lysine antibody. Loading is normalized to cardiac actin.
Table 1. Echocardiographic analysis of Mlc2v-cre-/-cre+ mice injected with AAV9-fl/fl- shAtp5a1 viruses
Figure imgf000031_0001
Heart rate (bpm) 472.25 ± 36.12 498 53 ± 46.53
IVS;d (mm) 0.74 ± 0.03 0.68 ± 0.05
IV5;s (mm) 1.02 ± Q.0€ 0.90 ± 0.06
LVID;d (mm) 3.68 ± 0.16 3.92 ± 0.1 !
LVID;s (mm) 2.30 ± 0.10 2.96 ± 0.08
LVPW;d (mm ) 0.72 ± 0.03 0.74 ± 0.06
LVPW;s (mm) ' .05 ± 0.03 1.06 ± 0.04
LVID Trace (CO) i ml/m in) 16.89 ± 2. !'9 16.89 ± 1.2 !
LV!D Trace CSV) (mI) 35.85 ± 4.97 34.02 ± 2.86
%FS 32.51 ± 2.92 24.55 ± 1.96"
%EF 65.45 ± 3.63 49.27 ± 337”
LV Mass (mg) 90.45 ± 8.30 88.68 ± 7.40·
LV Vol:d (pi) 57.53 ± 5.63 66.75 ± 4.49
LV Vol;s (mI) 22.42 1 1.91 33.81 ± 2.29*
LVW/BW (echo) 3.18 ± 0.26 3.57 ± 0.27
HVV/BW (post sacrifice) 3.48 ± 0.08 4.13 ± 0.12”
IVS, intraventricular septum thickness at diastole (d) and systole (s); LVID, left ventricular internal diameter at diastole (d) and systole (s); LVPW, left ventricular posterior wall thickness at diastole (d) and systole (s); FS, fractional shortening; EF, ejection fraction; LVW/BW, left ventricular weight/body weight; HW/BW, heart weight/body weight. Values shown are mean ±s.e.m.; *P<0.05; **P<0.01 ; two-tailed unpaired t-test. Table 2. Echocardiographic analysis of Mlc2v-cre-/-cre+ mice injected with AAV9-fl/fl-mir27b viruses
Figure imgf000032_0001
Heart rate (bpm) 494.81 ± 22.74 446.51 ± 1 > .06
fV5;d (mm) 0.71 ± 0.02 0.73 ± 0.01
!VS;s (mm) 1.00 ± 0 03 0.98 ± 0.01
LVID;d (mm) 3.56 ± 0.04 4.00 ± 0.08*
LVID;s (mm) 2.33 ± 0.06 2.90 ± 0.07*
LVPW;d (mm) 0.71 ± 0.02 0.71 ± 0.01
LV'PW;s (mm) 1.05 ± 0.02 0.95 ± 0.01
LV1D Trace (CO) (ml/min) 17.17 1 143 17.41 ± 0.85
LVID Trace (SV) (pi) 34.33 ± 1.40 38.99 ± 1.55
%FS 34.54 1 1.30 27.58 ± 0.62*
%EF 64.49 ± 1.72 52.59 ± 1.0 !*
LV Mass (mg) 82.39 ± 2.64 104.15 ± 3.83*
LV Vol;d (mI) 53.10 ± 1.49 72.25 ± 3.32**
LV Vol;s (mI) 18.90 1 1.21 34.39 ± 2.00**
LVW/BW (echo) 2.75 ± 0.05 3.18 ± 0.12 *
HW/BW (post sacrifice) 3.59 ± 0.07 3.9Q ± 0.13*
IVS, intraventricular septum thickness at diastole (d) and systole (s); LVID, left ventricular internal diameter at diastole (d) and systole (s); LVPW, left ventricular posterior wall thickness at diastole (d) and systole (s); FS, fractional shortening; EF, ejection fraction; LVW/BW, left ventricular weight/body weight; HW/BW, heart weight/body weight. Values shown are mean ±s.e.m.; *P<0.05; **P<0.01 ; two-tailed unpaired t-test.
Table 3. Echocardiographic analysis of sham- or TAC operated mice injected with scrLNA or miR27b-5p LNAs
Figure imgf000033_0001
HW/BW (post sacrifice) 6.06 ± 0.21 LVID, left ventricular internal diameter at diastole (d) and systole (s); LVPW, left ventricular posterior wall thickness at diastole (d) and systole (s); FS, fractional shortening; EF, ejection fraction; LVW/BW, left ventricular weight/body weight; HW/BW, heart weight/body weight. Values shown are mean ±s.e.m.; *P<0.05; **P<0.01 ; TAC scrLNAvs. sham scrLNA; miR27b- 5p LNA sham vs. miR27b-5p LNA TAC; two-tailed unpaired t-test.
Table 4. Echocardiographic analysis of sham- or TAC operated Mlc2v-cre-/-cre+ mice injected with AAV9-fl/fl-sh Mthfd 11 viruses
Figure imgf000034_0001
Heart rate (bpm) 512.91 ± 23.90 486 51 ± 8.27 583.57 ± 82.17 561.55 ± 19.39 lVS;d (mm) 0.74 ± 0.01 0.74 ± 0.01 1.11 ± 0.04 0.91 ± 0.01*
IVS;s (mm) 0.98 ± 0.02 0.96 ± 0.01 1.39 ± 0.05 1.11 ± 0.03*
LVID;d (mm) 3.84 ± 0.11 3.70 ± 0.16 426 ± 0.28 3.56 ± 0.13*
LVID;s (mm) 2.74 ± 0.13 2.57 ± 0.17 3 58 ± 0.41 2.36 ± 0.39*
LVPW;d (mm) 0.7 ± 0.01 0.75 ± 0.01 0.99 ± 0.07 0.94 ± 0.04
LVPW;s (mm) 0.96 ± 0.03 1.03 ± 0.02 1.24 ± 0.15 1.15 ± 0.10
%EF 49.64 ± 2.62 57.81 ± 3.29 37.72 ± 7.75 58.03 ± 7.27*
LV!D Trace (COj (ml'min) 17.25 ± 1.49 16.11 ± 0.47 14 98 ± 1.16 16 56 ± 4 11 LVID Trace CSV) (m|) 33.42 ± 1.74 33.25 ± 1.01 27.81 ± 1.78 31.36 ± 4.95.
IV mass (mg) 86.08 ± 5.84 95.33 ± 3.15 161 33 ± 1 1 27 82.91 ± 945***
LV Vol.d ίmί) 55.57 ± 4.43 63.98 ± 2.61 66 36 ± 10.88 48.82 ± 8.26*
LV Vol.s (mI) 23.80 ± 4.83 32.34 ± 1.32 39.39 ± 11.00 17.72 ± 2.10*
LVW/BW (echo) 3.86 ± 0.54 3.42 ± 0.19 6.32 ± 0.16 4.12 ± 0.19"
HV BW (post sacrifice] 4.07 ± 0.4 4.01 ± 0.29 6.53 ± 0.53 4.91 ± 0.03* Aortic Vel (mm/s 881 ± 68 864 ± 207 4552 ± 53 4725 ± 70
IVS, intraventricular septum thickness at diastole (d) and systole (s); LVID, left ventricular internal diameter at diastole (d) and systole (s); LVPW, left ventricular posterior wall thickness at diastole (d) and systole (s); EF, ejection fraction; LVW/BW, left ventricular weight/body weight; HW/BW, heart weight/body weight. Values shown are mean ±s.e.m.; *P<0.05; **P<0.01 TAC c2v-cre-vs. TAC Mlc2v-cre+; two-tailed unpaired t-test. Table 5. Echocardiographic analysis of Mlc2v-cre-/-cre+ mice co-injected with AAV9- I- shAtp5a1 and AAV9-fl/fl-shMthfd1l viruses
Figure imgf000035_0001
Heart rate (bpmj 499.60 ± 11.38 5 P .23 ± 9.01
IVS;d (mm) 0.72 ± 0.01 0.73 ± 0.02
lVS;s (mm) 097 ±0.03 0.99 ± 0.04
LVID;d (mm) 387 ±0.11 4.09 ± 0.12
LVID;s (mm) 2.82 ± 0.18 2.86 ± 0.17
LVPW;d (mm) 0.74 ± 0.04 1.01 ± 0.03*
LVPW;s (mm) 0.98 ± 0.04 1.06 ±0.04
L' 1D Trace (CO) fmi/mim) 18.78 ±1.36 20.03 ± 1.56
LV1D Trace (SV) (pi) 37.49 ± 2.24 39.00 ± 2.34
%FS 29.28 ±2.51 27.42 ± 2.83
%EF 56.35 ±4.04 53.20 ± 4.44
LV Mass (mg) 97.47 ± 5.73 107.89 ±4.88
LV Vol;d ίmI) 65.08 ±4.61 74.42 ±5.12
LV VoLs (mI) 29.04 ±4.41 35.68 ± 5.28
LVW/BW (echo) 366 ± 0.22 3.93 ±0.2!
HW/BW (post sacrifice) 4.14 ± 0.13 4.40 ± 0.25
IVS, intraventricular septum thickness at diastole (d) and systole (s); LVID, left ventricular internal diameter at diastole (d) and systole (s); LVPW, left ventricular posterior wall thickness at diastole (d) and systole (s); FS, fractional shortening; EF, ejection fraction; LVW/BW, left ventricular weight/body weight; HW/BW, heart weight/body weight. Values shown are mean ±s.e.m.; *P<0.05; **P<0.01; two-tailed unpaired t-test. Examples
FiFn ATP synthase repression drives cardiometabolic endoreolication and pathologic growth In order to identify mediators of ATP depletion in human and mouse pathologic cardiac hypertrophy we profiled mRNA levels of all subunits of the ATP synthase complex in patient biopsies of human HCM and AS, and in mice subjected to TAC (Fig. 1 a,b and Fig. 7a). Among the subunits profiled, ATP5A1 mRNA and protein was consistently reduced in both human and mouse cardiac hypertrophy and correlated significantly with upregulation of the hypertrophic markers natriuretic peptide A ( NPPA ), natriuretic peptide B ( NPPB ), and echocardiographically measured disease indicators of cardiac morphology and function (Fig. 1 a-d and Fig. 7a). These changes in ATP5A1 levels correlated with activation of AMP-activated protein kinase (AMPK) through increased phosphorylation at Thr172 in the diseased myocardium of both humans and mice, resulting in phosphorylation and downstream inactivation of its direct target Acetyl-CoA- Carboxylase (ACC) (Fig. 1 c,d). Critically, although historically termed as being an AMP activated kinase, AMPK is in fact more sensitive and predominantly activated by ADP. In line with repressed ATP5A1 expression and AMPK activation, ADP:ATP ratios were elevated in diseased human and mouse left ventricular biopsies (Fig. 1 e,f and Fig. 7b). In addition, an increased fraction of cardiomyocytes exhibiting multinucleation was observed in human left ventricular AS and HCM biopsies (Fig. 1 g,h and Fig. 7c). In mice, an increase in the fraction of multinucleated cells was also observed after TAC surgery (Fig. 1 i,j and Fig. 7d-f). The TAC protocol mimics human aortic stenosis through surgical constriction of the mouse aorta, resulting in increased blood velocity into the ventricle to impose a pressure overload stress leading to pathologic cardiac growth and fibrosis indicated by increased picrosirius red staining and expression of the fibrotic marker genes collagen type I alpha 1 (Col1a1), collagen type I alpha 3 (Col3a1) and transforming growth factor beta 1 (TGF1b) as encountered in human aortic stenosis (Barrick, C.J., et al., 2007, Am J Physiol Heart Circ Physiol, 292, 21 19-2130). (Fig. 7 g-j).
To understand the physiologic implication of ATP5A1 depletion, we utilized a novel adeno- associated virus (AAV)9-based system for in vivo ventricular-specific transgenesis (Mirtschink, P., et al., 2015, Nature, 522, 444-449). Briefly, delivery of an AAV containing TAT A-lox flanked genes or short-hairpin RNAs (shRNA) imparts Cre recombinase ( Cre ) dependency, which upon delivery to tissue-specific Cre transgenic mice results in tissue-specific ectopic gene or shRNA expression (Sauer, B., et al., 1988, Proc Natl Acad Sci U S A, 85, 5166-5170). AAV9 harboring shRNAs targeting Atp5a1 (AAV9-fl/fl-shAtp5a1 ) was delivered to Mlc2v-Cre transgenic mice (Chen, J., et al., 1998, Development, 125, 1943-1949). that express Cre recombinase specifically in the ventricular myocardium (Fig. 1 k). AAV9-fl/fl-shAtp5a1 delivery led to repressed Atp5a1 mRNA and protein expression (Fig. 11 and Fig. 8a), with concomitant activation of Ampk and augmented phosphorylation of its downstream target Acc (Fig. 11), increased ADP:ATP levels (Fig. 1 m and Fig. 8b), cardiomyocyte multinucleation and overgrowth combined with significantly reduced cardiac contractility and fibrosis (Fig. 1 n-s, Fig. 8c-i and Table 1 ). Next, we confirmed these findings in induced pluripotent stem cell (iPSC)- derived human cardiomyocytes and primary neonatal rat cardiomyocytes (NRC). Human and rat cardiomyocytes were stained with DAPI to visualize nuclei, and phalloidin to label filamentous actin and outline the cell surface. As noted in Fig. 8j-y, knockdown of ATP5A1 with shRNAs in human and rat heart cells resulted in increased ADP:ATP ratio (Fig. 8k-m, s-u), confirmed by elevated levels of ADP and ATP depletion (Fig. 8t, u), and cardiomyocyte multinucleation, concomitant to cell overgrowth as quantified by 2D cell size analysis and leucine incorporation, a readout for protein synthesis as an indirect measure of growth (Fig. 8n-p, v-y) (Fukuzawa, J., et al., 2000. Hypertension, 35, 1 191 -1 196). Thus, ATP5A1 depletion is sufficient to induce endoreplication and multinucleation to drive pathologic cardiac growth and compromise cardiac function.
HIF1a regulates FiFn ATP synthase activity via mir27b-5o
We detected inverse co-regulation of cardiac Hifl a activation with Atp5a1 mRNA and protein expression in ventricular-specific Hifla conditional knockout (cKO) mice subjected to TAC, and in mice deficient for the von Hippel Lindau protein (Vhl) in the ventricular myocardium, leading to myocardial Hifl a accumulation, due to inhibited HIF1 a degradation via its oxygen dependent subunit (Fig. 9a-c). As a consequence of the inverse relationship between HIF1 a accumulation and ATP5A1 repression, ventricular-specific Hifla cKO mice were protected from the ATP deficiency and contractile dysfunction observed in control Hifla fl/fl littermates subjected to TAC (Fig. 9d,e), whereas Vih/ cKO mice displayed low ventricular ATP levels associated with decreased systolic function (Fig. 9f,g). However, in silico and expression studies repeatedly failed to pinpoint direct Hifl a target genes capable of mediating the observed effects on Atp5a1 mRNA and ATP levels (data not shown). This led us to explore the role of Hifl a-regulated miRNAs in mediating these effects. We performed expression profiling of miRNAs from left ventricles of control and ventricular-specific Hifla cKO mice subjected to TAC, and on ventricle-specific Vhl cKO mice. By comparing expression data within the respective groups and filtering for miRNAs containing cross-species conserved hypoxia response element (HRE) motifs, we identified several potential Hifl a-regulated miRNAs (Fig. 9h). A subset of these miRNAs have been previously linked to hypoxia and/or Hifl a dependence. Of these miRNAs, mir27b was unique by its repression in Hifla cKO mice subjected to TAC (compared to littermate control Hifla fl/fl TAC mice) in line with the expression pattern of established HIF1 a target genes, and its upregulation in ventricular-specific Vhl cKO mice (Fig. 9i,j), suggesting potential regulation by Hifl a. mir27b is contained within the mir23b-27b-24 cluster in intron 15 of the aminopeptidase O {AmpO) gene in humans and mice (Zhou, Q., et at. , 201 1 , Proc Natl Acad Sci U S A ,108, 8287-8292). To determine the stress- and Hifl odependence of mir27b transcription, we assayed expression of AmpO and all miRNAs contained within the cluster in mice subjected to TAC, and in vitro in response to expression of a constitutively active Hifl a mutant lacking the oxygen dependent degradation domain (ODD) (referred to as HIFI aAODD) or by stimulation with the hypertrophy-inducing b-adrenergic receptor agonist triiodothyronine (T3) (Fig. 9k-t). Application of TAC in mice resulted in a slight induction of AmpO, largely unchanged expression of mature miR23b-3p/5p and miR24-3p/5p, and a -2.5-fold induction of miR27b- 3p/5p (Fig. 9k-m).
In vitro ectopic HIFI aAODD expression led to AmpO downregulation and unchanged levels of miR23b-3p/5p and miR24-3p/5p (Fig. 9n-p), while T3 stimulation induced AmpO expression but not that of miR23b-3p/5p or miR24-3p/5p (Fig. 9r-t). In contrast, mature miR27b-3p/5p was consistently upregulated in both in vitro models (Fig. 9p,t). These results suggest that miR27b- 3p/5p is regulated independently of its host gene AmpO and its neighboring miRNAs in response to stress and Hifl a. To confirm MIR27B as a direct HIF1 a target, we stimulated control non-silencing (ns) and shHifl a transduced NRCs with T3 and analyzed the expression of mir27b and established Hifl a target genes as controls (Fig. 9u). In silico analysis for cross- species conserved HREs in the mir27b promoter, corresponding to -1 kb upstream of the pre- mir27b transcriptional start site (TSS) revealed a conserved HRE motif close to the TSS in humans and mice (Fig. 9v). To assess functionality of this HRE, mir27b promoter-luciferase assays with the wildtype and HRE-mutated promoter were performed. Hifl a increased luciferase reporter expression only of the wildtype promoter but not in the HRE mutant promoter or in control cells, respectively (Fig. 9w). Promoters of the neighboring miRNAs mir23b and mir24 did not show increased promoter activity in the presence of ectopic expression of HIFI aAODD (Fig. 9x). Moreover, Hifla specifically associated with the mir27b promoter in native chromatin of NRC transduced with shVhl as revealed by Hifl a chromatin- immunoprecipitation (ChIP) from nuclear extracts (Fig. 9y). The efficiency of pVhl depletion by shVhl and consequent Hifla accumulation was confirmed by immunoblotting (Fig. 9z). Taken together, these data define MIR27B as a bona fide HIF1 a target miRNA.
Atp5a1 harbors a miR27b-binding site in the 3’UTR, conserved between human and mice (Fig. 10a), which upon disruption by site-directed mutagenesis, results in de-repression of mir27b- mediated inhibition (Fig. 10b). In accord, ectopic mir27b expression in NRC resulted in pronounced repression of Atp5a1 mRNA and protein (Fig. 10c-e). Despite upregulation of both miR27b-3p and miR27b-5p RNA by HIF1 a, miR27b-5p was unique by its capacity to inhibit wildtype Atp5a1 UTR-reporter expression (Fig. 10b). Treatment of cardiomyocytes with miR27b-5p mimics led to the dose-dependent downregulation of Atp5a1 mRNA but miR27b- 3p mimics did not (Fig. 10f). These observations were recapitulated with locked nucleic acid (LNA)-mediated miR27b-5p inhibition in cardiomyocytes expressing ectopic HIFI aAODD ( Fig. 10g-i). LNAs specifically targeting miR27b-5p led to de-repression of HIFI aAODD induced Atp5a1 downregulation (Fig. 10g-i). Similar results were observed upon T3 stimulation where Atp5a1 repression was rescued upon simultaneous treatment with miR27b-5p LNAs (Fig. 10j- m). Thus, miR27b inhibition of Atp5a1 is mediated specifically by the miR27b-5p species.
In order to determine if downregulation of Atp5a1 under these settings affected ATP synthase activity, we measured the ATP regenerating capacity of immunoprecipitated ATP synthase isolated from rat cardiomyocytes ectopically expressing mir27b or shAtp5a1 ; or upon ectopic HIFI aAODD expression in combination with miR27b-5p LNAs (Fig. 10n,o). As noted, ectopic mir27b and HIFI aAODD expression led to significant repression of ATP synthase activity, with activity rescued upon parallel treatment with miR27b-5p LNAs (Fig. 10n,o). Atp5a1 inactivation also mimicked the effects of ectopic mir27b expression, while concomitant ATP5A1 overexpression with a flag-tagged construct (Fig. 10p) rescued the diminished ATP synthase activity upon ectopic HIFI aAODD expression (Fig. 10o). Thus, ATP synthase activity is highly dependent on Atp5a1 expression levels. miR27b-5o inhibition atenuates severe cardiac hypertrophy and dysfunction
In order to assess if MIR27B expression correlates with human cardiac pathology, we profiled its expression in healthy and diseased human cardiac biopsies. As noted in Fig. 2a, MIR27B expression was elevated in left ventricular biopsies of HCM and AS patients compared to healthy subjects, thus inversely correlating with ATP5A1 expression (Fig. 1 a). We examined the causal association between mir27b and Atp5a1 in vivo through delivery of AAV9 carrying pre-mir27b (AAV9-fl/fl-mir27b) to Mlc2v-Cre transgenic mice to achieve ectopic mir27b expression specifically in the ventricular myocardium. As noted ectopic mir27b expression was sufficient to drive cardiac overgrowth, contractile dysfunction and Atp5a1 mRNA and protein repression at 1 1 weeks after transduction (Fig. 2b-h,). Furthermore, an increase in the fraction of multinucleated cardiomyocytes, as a readout of elevated endoreplication, was observed in left ventricles of Mlc2v-Cre+ mice compared to Cre littermates (Fig. 2i,j and Fig. 1 1 d-f), to a comparable extent as observed upon TAC surgery (Fig. 1 i,j). Cardiac fibrosis was similarly elevated (Fig. 1 1 g,h). These observations and our identification of miR27b-5p as the principal regulator of Atp5a1 expression (Fig. 10b,f-p) led us to interrogate miR27b-5p function in mice exhibiting severe heart failure - a disease state commonly associated with ATP synthase repression and ATP depletion. C57BL/6J mice were randomly assigned to two groups, with the groups subjected to either sham or TAC surgery and further subdivided into treatment groups with scrambled LNA (scrLNA) or miR27b-5p LNA (Fig. 2k). In order to mimic late-stage human heart failure, TAC surgery was performed and hypertrophy allowed to progress till severe cardiac dysfunction was detected by echocardiography. At which time LNA treatment was initiated and echocardiography performed to monitor disease progression, as indicated in the respective figure panels (Fig. 2k-p). TAC surgery led to an increase in aortic flow velocity, a control for the degree of overload applied on the myocardium, while sham treated mice displayed normal aortic flow (Fig. 1 1 i). Concomitant to severe cardiac hypertrophy, as demonstrated by the increased ventricular weight: body weight ratio and ventricular dilatation, systolic cardiac dysfunction was evident by a dramatically decreased ejection fraction (EF) (Fig. 2l-p and Table 3) in TAC mice at 42-days post-surgery. Following this observation, mice from the respective groups were subdivided for scrLNA or miR27b-5p LNA therapy. Longitudinal monitoring of disease progression by echocardiography revealed an absence of pathology in sham operated mice. TAC operated mice treated with scrLNA displayed a further decline in cardiac function and heart failure as evidenced by pronounced hypertrophy development, ventricular dilatation and reduced cardiac ejection fraction (Fig. 2l-p and Table 3). In contrast, TAC mice treated with miR27b-5p LNA exhibited improved cardiac function and partial reversion of hypertrophic growth (Fig. 2l-p and Table 3). Analysis of miR27b-3p and miR27b-5p expression confirmed specific inactivation of the miR27b-5p species (Fig. 2q,r), correlating with reduced hypertrophic marker gene expression (Fig. 2s) and increased Atp5a1 mRNA and protein expression compared to scrambled LNA treated TAC operated mice, mimicking expression levels in sham-treated mice (Fig. 2t,u). Strikingly, the stress-induced increase in multinucleation detected by immunofluorescent staining of left ventricular sections from TAC operated mice injected with scrambled LNAs was markedly reduced in TAC operated mice receiving miR27b-5p LNAs (Fig. 2v,w and Fig. 1 1 j-l). Likewise, miR27b-5p LNA therapy significantly reduced cardiac fibrosis and mortality in mice subjected to TAC surgery compared to mice that were injected with scrambled LNAs (Fig. 2x and Fig 1 1 m,n). Thus, pathologic- stress induced MIR27B-5p expression and ATP5A1 repression is required for maintenance of key aspects of pathologic cardiac growth in humans and mice.
Mitochondrial ADP drives purine biosynthesis
Given that ATP synthase inactivation elevates intra-mitochondrial ADP levels, we explored if ADP in the absence of substrate competition from ATP synthase could be rechanneled to MTHFD1 L, an intra-mitochondrial rate-limiting enzyme of the 1 -carbon pathway that accounts for 70-99% of formate produced in cells. MTHFD1 L utilizes ADP as a rate-limiting cofactor for the hydrolysis of 10-formyltetrahydrofolate (CHO-THF) to formate (Fig. 3a). To test this we measured formate and mitochondrial ATP and ADP levels in genetic Hifl a, miR27b, Atp5a1 and Mthfd 11 gain- and loss-of-function settings (Fig. 3b, c, Fig. 12a-h). Increased formate generation and ADP:ATP ratios correlated directly with ectopic Hifl a or miR27b expression, and shRNA-mediated Atp5a1 knockdown (Fig. 3b, c). Next, we performed metabolomics in similar settings as above to investigate the metabolic link between activation of the HIF1 o MIR27B-ATP5A1 axis and elevated levels of multinucelation and cardiomyocyte growth (Fig. 3d). Strikingly ectopic Hifl a, miR27b or shAtp5a1 expression resulted in increased levels of glucose and glycolytic intermediates (Dihydroxyacetonephosphate, 3-Phosphoglycerate) pointing to increased glucose uptake and glycolysis. Moreover, elevated levels of serine and glycine, intermediates of the purine biosynthesis pathway (Ribose-5-phosphate, FGAR) as well as purine and pyrimidine derivatives (Inosine, Xanthine, dUTP, GTP, Uridine) were observed. In contrast, concomitant miR27b-5p inactivation, depletion of Mthfd 11 or ectopic ATP5A1 expression partially reversed these effects (Fig. 3d). Moreover, ectopic expression of MTHFD1 L alone was not sufficient to induce the de novo purine biosynthesis pathway, supporting the view that mitochondrial ADP serves preferably as a substrate for ATP Synthase and that the induction of mitochondrial formate biosynthesis is highly dependent on repression of ATP5A1 (Fig. 3d). Similar results were observed with T3, resulting in highly miR27b- dependent growth (Fig. 13a), formate production (Fig. 13b) and ADP/ATP increase (Fig. 13c). LC-MS based analysis of the metabolome revealed increased production of purine and pyrimidine derivatives as a function of miR27b expression (Fig. 13d).
Thus, ADP rechanneling connects energetic compromise to stress-induced biosynthesis pathways.
Purines are essential for nucleic acid synthesis, endoreplication and cell growth. Its de novo synthesis can be traced to the glucose that enters the cell and undergoes glycolysis to form 3- phosphoglycerate (3-PG), and further metabolized to generate serine and (indirectly) glycine via the serine biosynthesis pathway, which then serve as key 1 -carbon donors through incorporation of its carbon into the purine ring (Fig. 3a). As the carbon atom incorporated into the purine ring essentially traces back to glucose carbons, we followed the flux of carbon into the purine ring of nucleic acids through labeling of glucose, serine and glycine, respectively (Fig. 3e). As shown, increased incorporation of carbon atoms derived from glucose, serine and glycine was observed in the nucleic acid fraction of cells ectopically expressing HIFI aAODD, miR27b or shAtp5a1 (Fig. 3e), while simultaneous ectopic MTHFD1 L expression did not appreciably increase contribution of labeled carbons to the nucleic acid fraction (Fig. 3e), in line with the obtained metabolome profile (Fig. 3d). Similar effects were observed in settings of T3-induced cell growth and miR27b-5p inactivation (Fig. 13e). These data suggest that the entire upstream network impinges on the crosstalk between ATP5A1 and MTHFD1 L in regulating pathology-induced de novo nucleic acid synthesis.
Finally, we determined if similar changes in metabolite distribution could be detected in left ventricular biopsies of mice subjected to either sham or TAC surgery with scrambled LNA (scrLNA) or miR27b-5p LNA treatment (Fig. 2k-p). As noted in Fig. 14a, the metabolite changes observed in cardiac left ventricles closely paralleled changes observed in vitro above, including the elevation of purine precursors such as N-Formylglycinamide ribonucleotide (FGAR) and 5- Formamidoimidazole-4-carboxamide ribotide (FAICAR) in scrLNA treated TAC mice compared to TAC-operated animals treated with miR27b-5p LNA or scrLNA treated mice subjected to sham surgery. Furthermore, the purines xanthine and inosine were significantly lower abundant in miR27b-5p treated TAC mice compared to TAC-operated mice treated with scrLNA (Fig. 14a). Hierarchical clustering revealed that the metabolic signature of left ventricular biopsies from TAC-treated mice injected with miR27b-5p LNAs was altered compared to scrLNA injected TAC-mice, but comparable to that of of sham-operated scrLNA and miR27b-5p LNA treated mice (Fig. 14b). In particular, TAC-operated left ventricular biopsies of miR27b-5p LNA injected mice revealed lower levels of free fatty acids and other lipid species compared to TAC scrLNA controls (Fig. 14a). Thus, we have in addition investigated the lipidome in the above-named in vivo samples (Fig. 15a-g). Principal component analyses (PCA) indicated a unique lipidomic signature in left ventricles of TAC- operated scrLNA vs. miR27b-5p LNA injected mice (Fig. 15a). Lipidomes for sham- or TAC- operated scrLNA treated hearts partially overlap, but significantly separate along the first Principal Component (PC1 ) whereas lipidomes of sham-or TAC treated hearts with diminished miR27b-5p expression levels significantly separate along the second principal component (PC2) (Fig. 15b, c). A closer look into the lipid composition revealed that left ventricular biopsies of TAC-operated/miR27b-5p LNA injected mice show indeed the highest amount of lipids containing 6 double-bonds (db), while TAC scrLNA samples significantly stand out for 2db, 4db and for lipids with 34 and 36 carbon atoms (Fig. 15d,e).
Analyzing the abundance of individual lipid species in TAC-operated mice minus mean mol% abundance of lipid species in corresponding controls revealed an enrichment of arachidonic (20:4) and linoleic (18:2) acids in TAC samples (Fig. 15f). Strikingly, lipids containing docosahexaenoic acid (22:6) accumulate in the left ventricle of TAC-operated mice with repressed miR27b-5p expression (Fig. 15g). In line with this result, free docosahexanoic acid was also increased in the untargeted metabolic analysis (Fig. 14a). Taken together the comprehensive analysis of the cardiac lipidome revealed a correlation between the prevention of left ventricular growth in response to pressure overload and an enrichment of polyunsaturated lipids, especially lipids containing docosahexanoic acid, which has been shown to be cardioprotective. Increased hypertrophy on the other hand is linked with a significant upregulation of lipids containing fatty acids with 34 and 36 carbons, pointing to decreased fatty acid oxidation. In accord with the greater oxidative phenotype of hearts derived from TAC mice treated with miR27b-5p LNA (compared to mice subjected to TAC and treated with scrLNA) (Fig. 14a), lipid catabolism was markedly elevated in miR27b-5p LNA-treated mice, as evidenced by enrichment of long chain fatty acids in TAC-operated hearts and treated with scrLNA compared to miR27b-5p LNA-treated TAC hearts (Fig. 15a-g). Furthermore, an increased expression of lipid catabolism mediators was observed in miR27b-5p LNA-treated TAC hearts (compared to mice subjected to TAC and treated with scrLNA), corresponding to an increased palmitate oxidation capacity in miR27b-5p LNA-treated TAC hearts (Fig. 15h,i). In sum, these analyses revealed a unique lipid signature for miR27b-5p LNA-treated TAC hearts characterized by a significant upregulation of lipids bearing the cardioprotective docosahexanoic acid and an increased fatty acid oxidation capacity.
ATP synthase controls cardiomvocvte endoreplication and pathologic growth
Cardiomyocyte endoreplication precedes pathologic cardiac overgrowth(Ahuja, P., et al., 2007, Physiol Rev, 87, 521 -544). To understand if the axis identified indeed contributes to cardiomyocyte multinucleation and growth through ADP, the inventors mimicked miR27b- mediated ATP5A1 repression through treatment of primary cardiomyocytes with oligomycin (an Fo subunit ATP synthase inhibitor (Shchepina, L.A., et al., 2002, Oncogene, 21 , 8149- 8157).) or stimulated primary cardiomyocytes with ADP (Hu, J., et al., 2014, Cell Death Dis, 5, 1424). As noted in Fig. 16a-d, ADP levels were indeed elevated in cells treated with oligomycin or ADP. Next, the inventors assessed the sufficiency for oligomycin or ADP to induce endoreplication and multinucleation, as visualized by immunofluorescent staining with DAPI and skeletal a-Actinin to quantify the fraction of multinucleated cells (Fig. 16e-h), and cardiomyocyte hypertrophy by cell size quantification (Fig. 16i,j).
To better define the underlying mechanism, the inventors analyzed cardiomyocyte ploidy and cell size in Hifl a, miR27b, Atp5a1 and Mthfdl l gain- and loss of function settings (Fig. 4a-i and Fig. 17a-h). As shown by imaging coupled flow cytometry and flow cytometry of propidium iodide (PI) stained DNA, ectopic HIF1 a, mir27b or shAtp5a1 expression was sufficient to increase the population of multinucleated polyploid cells (Fig. 4a-f), an effect reverted upon simultaneous miR27b-5p LNA-mediated inactivation (Fig. 4a, b), ectopic ATP5A1 expression (Fig. 4c, d) or depletion of Mthfdl l by co-transduction of shMthfdl l expressing lentiviruses (4e,f). In order to directly visualize endoreplicated multinucleated cells, cardiomyocytes were stained for phosphorylated histone 3 at serine 10 (p-Histone H3) and imaged by confocal microscopy (Fig. 17a-h). Phosphorylated histone H3 marks condensed chromosomes that are characteristic of karyokinetic cells. Consistent with the flow cytometry data, ectopic HIFI aAODD expression led to increased phosphorylated histone H3 stained nuclei which was rescued upon parallel miR27b-5p inhibition with LNAs (Fig. 17a,b). Furthermore, mir27b overexpression or Atp5a1 inhibition similarly led to increased numbers of karyokinetic cells compared to corresponding controls (Fig. 17c,d). shRNA-mediated Mthfdl l inhibition led to efficient depletion of its mRNA and protein (Fig. 17e,f) and resulted in comparable phospho- Histone H3 staining as control cells (Fig. 17g,h). Remarkably, simultaneous expression of ectopic ATP5A1 and mir27b, or co-transduction of NRC with shMthfdl l and shAtp5a1 lentiviruses likewise reduced the extensive pattern of phospho-Histone H3 staining as observed upon ectopic expression of mir27b or shAtp5a1 alone (Fig. 17c-h). To verify that the increase in de novo purine biosynthesis and multinucleation links directly to cell growth, the inventors performed leucine-incorporation assays under identical treatment conditions. As shown in Fig. 4g-i ectopic HIFI aAODD, mir27b expression, or Atp5a1 knockdown led to increased leucine-incorporation (indicative of increased protein synthesis and cell growth), which was negated upon simultaneous miR27b-5p inactivation (Fig. 4g), or ectopic expression of ATP5A1 (Fig. 4h). In line with the critical requirement for MTHFD1 L in purine biosynthesis, the fraction of multinucleated cells was reduced upon simultaneous depletion of MTHFD1 L and ATP5A1 (Fig. 4e,f) as was pathologic cell growth (Fig. 4i). Thus, our data supports the view that re-programming of the metabolic environment is sufficient to drive cardiomyocyte endoreplication, resulting in multinucleation and cell growth.
MTHFD1L is a key modulator of pathologic cardiac growth
To confirm that MTHFD1 L impacts cardiac pathologic hypertrophy, the inventors injected Mlc2v-cre~ and cre+ mice subjected to sham or TAC surgeries with AAV9 harboring short- hairpin RNAs targeting Mthfdl l (AAV9-fl/fl-shMthfd1 l) (Fig. 5a). Consistent with the previous TAC data (Fig. 1 i,j), multinucleation was significantly increased in Mlc2v-cre~ mice, as quantified from immunofluorescent staining (Fig. 5b, c and Fig. 18a). Concomitant with stress- induced endoreplication, Mlc2v-cre~ mice developed pathologic cardiac hypertrophy and systolic dysfunction after TAC surgery, evident by increased left ventricular weighbbody weight ratio, increased left ventricular internal diameter in diastole (LVId) and eleveated hypertrophic marker genes and reduced ejection fraction compared to sham-operated Mlc2v-cre~ mice. (Fig. 5d-g, Fig. 18b-d and Table 4). Strikingly, the percentage of multinucleated cells was significantly lower in TAC operated Mlc2v-cre+ mice compared to similarly treated Mlc2v-cre~ littermates (Fig. 5b, c and Fig. 18a,e,f). In accord, cardiac hypertrophy, fibrosis and systolic dysfunction was significantly reduced (Fig. 5d-g, Fig. 18b,g,h and Table 4). qRT-PCR analysis and immunoblotting for Mthfdl l expression revealed sufficient repression of Mthf 11 on both the RNA and protein level by AAV9-fl/fl-sh Mthfdl l transduction (Fig. 5h and Fig. 18d) coincident with resistance to TAC-induced cardiac overgrowth and contractile dysfunction.
In line with the accumulation of HI F1 a in TAC treated Mlc2v-cre~ and Mlc2v-cre+ hearts, Atp5a1 expression was repressed resulting in AMPK phosphorylation at Thr172 and hyperphosphorylation of ACC (Fig. 5h). In line with the fact that Mthfdl l repression was sufficient to prevent increased multinucleation in response to cardiometabolic endoreplication driven by TAC mediated pressure-overload, multinucleation, overgrowth, fibrosis and dysfunction induced by Atp5a1 downregulation (Fig. 1 k-s and Fig. 8a-i) was similarly prevented by the parallel inactivation of Mthfdl I in vivo (Fig. 5i-o, Fig. 18i-q and Table 5). Mlc2v-cre+ mice co-injected with AAV9-fl/fl-shAtp5a1 and AAV9-fl/fl-shMthfd11 showed no increase in multinucleated cells (Fig. 5j,k and Fig. 18i-k), normal ventricular size (Fig. 5l,m and Fig. 181), left ventricular weight:body weight ratio (Fig. 5n), hypertrophic marker gene expression (Fig. 18m) and systolic left ventricular function (Fig. 5o and Table 5), despite efficient inhibition of Atp5a1 (Fig. 5p and Fig. 18n) when Mthfd 11 was inhibited simultaneously (Fig. 18o) indicating that Mthfdl l functions downstream of Atp5a1 . Levels of cardiac fibrosis were similarly comparable between groups (Fig. 18p,q). Thus, ATP5A1 repression and the resulting rechanneling of ADP to MTHFD1 L and the consequent increase in purine biosynthesis, drives cardiometabolic endoreplication and multinucleation to enforce pathologic cardiac growth.
ATP5A1 integrates metabolic and growth signaling to drive pathologic growth
AMPK activation has been shown to drive inhibition of the tumor suppressor Retinoblastoma protein (Rb) via phosphorylation at Ser804. Rb restricts DNA replication and cell cycle progression from G1 to S through its sequestration and inhibition of E2F transcription factors, which are composed of dimers E2F protein and a dimerization partner (DP) protein (E2F-DP). Rb phosphorylation results in the release and de-repression of E2F transcription factors from the E2F-DP complex, culminating in E2F target gene induction and cell cycle re-entry. Given that the HIF1 omiR27b-ATP5A1 -MTHFD1 L axis creates a metabolic context permissive for cell cycle re-entry and endomitosis in post-mitotic adult cardiomyocytes, the inventors interrogated possible cooperation of metabolic and growth factor signaling in cardiac pathologic hypertrophy.
First the inventors confirmed direct interaction of AMPK with Rb in our setting through co- immunoprecipitation of AMPK and Rb. To that end, the AMPKa subunit was ectopically expressed in cardiomyocytes and subsequently immunoprecipitated. Pull-down products revealed co-precipitation of AMPKa with phosphorylated-Rb (Fig, 6a, b). The inventors confirmed this interaction through pull-down of endogenous AMPK in cells expressing ectopic HIFI aAODD. As noted, AMPK immunoprecipitated with phosphorylated Rb in cardiomyocytes ectopically expressing HIFI aAODD, but not upon simultaneous inhibition of AMPK with compound C (CC), a potent AMPK inhibitor (Fig, 6c, d).
Having confirmed the direct interaction between AMPK and phospho-Rb, the inventors next assessed if Rb was hyperphosphorylated in our human biopsies and various mouse models (Fig. 6e-j). Indeed, hyperphosphorylation of Rb was detected in lysates of left ventricular biopsies of TAC operated mice injected with scrLNA (Fig. 6e), ectopic mir27b expression (Fig. 6f) and depletion of Atp5a1 (Fig. 6g) in line with the phosphorylation of AMPK at Thr172 under these conditions as demonstrated before (Fig. 11 and Fig. 2h,u). Left ventricles of mice injected with LNAs targeting miR27b-5p (Fig. 6e) or simultaneously transduced with AAV9 bearing shRNAs against Mthfdl l alone or Mthfdl l and Atp5a1 were protected from pRb induction after TAC surgery (Fig. 6h,i). In accord with experiments in mice and the observed Thr172 phosphorylation of AMPK (Fig. 1 c) immunoblotting of lysates from ventricular biopsies of AS and HCM patients revealed likewise increased phospho-Rb expression (Fig. 6j). Taken together, pRb is a downstream target of phosphorylated AMPK in the presence of the cardiometabolic endoreplication driving HIF1 oMIR27B-ATP5A1 axis.
To investigate whether the predicted release of E2F1 upon Rb hyperphosphorylation leads to increased transcription activity of E2F1 , the inventors performed qRT-PCRs to detect changes in RNA levels of E2F1 -target genes (Cyclin A1 , A2, D1 , E1 ( Ccnal , Ccna2, Ccndl, Ccnel ) and Cyclin-dependent kinase \ (Cdk1)) on left ventricular samples of mice treated with the above mentioned conditions and on human heart biopsies (Fig. 6k-n). These gene expression analysis could confirm the activation of the E2F pathway in vivo, downstream of phospho- AMPK mediated Rb inhibition in mice and AS and HCM patients (Fig. 6k-n). E2F luciferase reporter assays performed in NRC further confirmed E2F transcriptional activity under ectopic expression of HIFI aAODD, mir27b or Atp5a1 depletion (Fig. 6o,p). Finally, to functionally assess the impact of cell cycle regulators on cardiomyocyte endomitosis and multinucleation the inventors inactivated Ccndl (cyclin D1 ) and Ccnel (cyclin E1 ), two key downstream components of Rb activation in cardiomyocytes expressing ectopic HIFI aAODD. As evidenced, inactivation of Ccndl and Ccnel inhibited HIFI omediated endomitosis, multinucleation and hypertrophy (Fig. 19a-f). In contrast, inactivation of the cell cycle inhibitor Cdknl b (p27/Kip1 ) was sufficient to induce cardiomyocyte endomitosis, multinucleation and pathologic growth (Fig. 19g-k). Taken together, the inventors have demonstrated that cardiac endoreplication and multinucleation requires simultaneous activation of de novo purine biosynthesis (as facilitated by the identified HIF1 oMIR27B-ATP5A1 -MTHFD1 L axis) as well as activation of cell cycle by E2F1 . This is reflected by the fact that depletion of Mthfd 11 in combination with Atp5a1 inhibition was sufficient to induce AMPK activation and E2F transcription (Fig. 6h,i,l,m) but failed to induce cardiometabolic endoreplication (Fig. 5b,c,j,k).
Materials and Methods
Animal breeding and maintenance
Hifla fl/fl mice were obtained from Randall S. Johnson (University of California, San Diego, USA) and V hi fl/fl mice were kindly provided by Rudolf Jaenisch (Massachusetts Institute of Technology, USA). The myosin light-chain ( Mlc)2v-Cre (Chen, J., ef a/., 1998, Development 125, 1943-1949) line was from Ju Chen (University of California, San Diego, USA). The respective ventricular-specific mouse lines described in this manuscript were generated by crossing loxP-flanked Hifla (Hifla fl/fl) (Ryan, H.E., ef a/., 2000, Cancer Res, 60, 4010-4015) or Vhl (Vhl fl/fl) (Haase, V.H., ef a/., 2001 , Proc Natl Acad Sci U S A, 98, 1583-1588) mice to myosin light-chain Mlc2v-Cre transgenic mice. The data presented represent studies with male mice aged 3-20 weeks of the C57BL/6J background. By 3-20 weeks the inventors were referring to the whole duration of the animal experiments. The AAV9 viruses were administered to 3-week old mice. As theAAV9 vectors have a lag phase of approximately 6-10 weeks until it reaches maximal expression, surgeries were performed at 9-1 1 weeks and hearts harvested at least 1 1 weeks after AAV9 administration as shown in the experimental outlines in Fig. 1 k, Fig. 2b, k and Fig. 5a, i. In TAC experiments, mice were 10-12 weeks old at the beginning of the experiment. Only mice of a similar age (+/- 1 week) were used in the corresponding experiments. In experiments with the Mlc2v-cre mice, littermates and mice of a similar age (+/- 1week) were used. After baseline echocardiography mice were randomly assigned to groups, AAV injections, LNA delivery and echocardiography was performed blinded. In experiments including TAC surgery experiments were kept blinded over the baseline echocardiography until operation. All mice were maintained at the MRC Clinical Sciences Centre (Imperial College London), Institute of Molecular Health Sciences (ETH Zurich) and/or the Cardiovascular Assessment Facility (CAF), Department of Medicine, Department of Medicine, University of Lausanne in a specific pathogen-free facility. Maintenance and animal experimentation were in accordance with the Swiss Federal Veterinary Office (BVET) guidelines.
Human and mouse ventricular biopsies
Human heart biopsies and clinical data were generously provided by Samuel Sossalla ( Georg - August-University Goettingen and DZHK, Goettingen, Germany) and Sebastian Stehr (University Hospital Leipzig, Germany). Human HCM and aortic stenosis biopsies were conducted in compliance with the local ethics committee, and written informed consent was received from all subjects prior to inclusion. Myocardial samples were obtained from patients with severe aortic stenosis undergoing aortic valve replacement and a Morrow resection from the hypertrophied left ventricular septum. Only patients without significant aortic valvular regurgitation and with preserved contractile function were included. Human left ventricular biopsies of HCM patients were obtained from left ventricular papillary muscle of explanted hearts. The myocardial samples were acquired directly in the operating room during the surgery and immediately washed in precooled cardioplegic solution (1 10 mM NaCI, 16mM KCI, 16mM MgCh, 16mM NaHCOs, 1 .2 mM CaCh, 1 1 mM glucose) followed by rapid snap-freezing in liquid nitrogen. Healthy heart samples were obtained from left ventricles of donor hearts. Sample weight was approximately 20-150 mg. Clinical data pertaining to these subjects are shown in a previous publication 16.
Transaortic Banding
9-14 week old mice were subjected to transaortic banding (TAC) through constriction of the descending aorta as described (Kassiri, Z., et al., 2005, Circ Res, 97, 380-390).. The mice were monitored up to 9 weeks after surgery and their heart dimensions and functions were determined by echocardiography. In vivo miRCURY LNA Power Inhibitors were injected intraperitoneally (i.p.) into C57BL/6J mice at a dose of 10 mg/kg for 4 consecutive days at 49 days post surgery as described in Fig. 2k. The following In vivo miRCURY LNA Power Inhibitors were purchased from Exiqon: i-mmu-miR-27b-5p (199900). i-Cel-controIJnh (199900) was used as scrambled control LNA.
In vivo transthoracic ultrasound imaging
Transthoracic echocardiography was performed using the MS400 (18-38MHz) probe from Vevo 2100 color doppler ultrasound machine (VisualSonics). Mice were lightly anesthetized with 1-1.5% isoflurane, maintaining heart rate at 400-550 beats per minute. The mice were placed in decubitus dorsal on a heated 37°C platform to maintain body temperature. A topical depilatory agent is used to remove the hair and ultrasound gel is used as a coupling medium between the transducer and the skin. Hearts were imaged in the 2D mode in the parasternal long-axis view. From this view, an M-mode cursor was positioned perpendicular to the inter- ventricular septum and the posterior wall of the left ventricle at the level of the papillary muscles. Diastolic and systolic interventricular septum diameter (IVS;d and IVS;s), diastolic and systolic left ventricular posterior wall diameter (LVPW;d and LVPW;s), and left ventricular internal end-diastolic and end-systolic chamber (LVID;d and LVID;s) dimensions were measured. The measurements were taken in three separate M-mode images and averaged. Left ventricular fractional shortening (%FS) and ejection fraction (%EF) was also calculated. Fractional shortening was assessed from M-mode based on the percentage changes of left ventricular end-diastolic and end-systolic diameters. %EF is derived from the formula of (LV vol;d - LV vol;s)/ LV vol;dx100. At the end of the duration of the experiment, the animals were sacrificed and the heart weight-to-body weight ratio was measured.
Isolation and maintenance of primary neonatal rat cardiomyocytes
Isolation of primary neonatal rat cardiomyocytes (NRC) was performed using the neonatal heart dissociation kit (130-098-373, Miltenyi Biotec) as recommended by the manufacturer. Isolated cells were pre-plated with plating medium (65% DMEM, 16% M199, 10% fetal calf serum (FCS), 5% horse serum (HS), 2% glutamine and 1 % penicillin/streptomycin (P/S)) for 1.5 h to deplete of the fibroblasts. NRC were plated on Type-I Collagen-coated (Advanced Biomatrix) 3 cm dishes (Nunc) in plating medium. The plating medium was changed to maintenance medium (88% DMEM, 9% M199, 1% HS, 2% glutamine and 1% P/S) 24 h after isolation of NRCs. Cardiomyocytes were treated with 3,3',5-Triiodo-L-thyronine (T3, T5516, Sigma Aldrich) for 6 days at a concentration of 15 nM. NRCs were stimulated with exogenous ADP (A2754, Sigma Aldrich) at a concentration of 10 mM for 3 days. Oligomycin A (75351 , Sigma Aldrich) and the AMPK inhibitor Compound C (CC; 171260, Sigma Aldrich) were applied at concentrations of 20 nM for 2 days and 5 mM for 24 h, respectively. NRC were randomly chosen for treatment the day after isolation.
Isolation of adult mouse cardiomyocytes
The left ventricle of adult mouse hearts was cut into pieces of 1 mm3 and fixed for 2 h with 4% paraformaldehyde (PFA)/PBS. The biopsies were digested with 1000 U collagenase type II (17101 -015, Gibco) in HBSS for approximately 42 h at 37°C while rotating. Appropriate concentrations of isolated adult cardiomyocytes were centrifuged at 600 rpm for 1 min onto gelatin-coated microscope slides using a cytospin (SCA-0030, Shandon Southern).
Human cardiomyocyte culture
Human induced pluripotent stem cell-derived human cardiomyocytes (iPSC-CM) were purchased from Cellular Dynamics International (CMC-100-010-001 ) and cultured as recommended by the manufacturer. These cells show a clear sarcomeric organization as evidenced by striations. Furthermore, the cells are terminally differentiated as they do not proliferate and cannot be passaged. Gene expression analysis showed that iPSC-CM express more than 200 cardiac genes, among them oactinin and cardiac troponin T, whose expression profiles are similar to adult human cardiac tissue. Cells started to beat spontaneously after 24- 48 h post thawing and formed electrically connected syncytial layers that beat in synchrony. Cells were deemed suitable for studies when they formed a synchronically beating syncytium. Cells were randomly chosen for transduction with lentiviruses after 6-7 days in culture and experiments were performed on day 10.
Isolation of mitochondria
Mitochondria were isolated using the Mitochondria Isolation Kit for Cultured Cells (ab1 10171 , Abeam). Briefly, 1 .2x106 NRCs were seeded per 6 cm dish and cells collected by scraping. After a freeze-thaw step to weaken the cell membrane, the cells were resuspended to a protein concentration of 5 mg/ml_ in Reagent A and incubated for 10 min on ice. The cells were homogenized with 30 strokes in a Dounce Homogenizer and centrifuged at 1000 g for 10 min at 4°C. The pellet was resuspended in the same volume of Reagent B than was used for Reagent A and rupturing and centrifugation was repeated. The supernatants after the two centrifugation steps were combined and centrifuged at 12000 g for 15 min at 4°C. Pellet was resuspended in 80 pl_ Reagent C supplemented with protease inhibitors and stored at -80°C. Mitochondrial ADP:ATP quantification was performed using the EnzyLight ADP/ATP Ratio Assay Kit (ELDT-100, Bioassay Systems) as recommended by the manufacturer.
Lentivirus production and infection
HEK-293T cells were transfected at 80-90% confluence with polyethylenimine (PEI) transfection reagent. 10 pg transgene, 7.5 pg pMD2.G and 6.5 pg psPAX2 were mixed with 2 ml serum-free DMEM and 45 pg PEI per 10 cm dish. After 10 min incubation at room temperature (RT) the DNA/PEI-complexes were added slowly to the cells cultured in DMEM containing 0.5% FCS and L-glutamine. Medium was changed to NRC maintenance medium 4 h after transfection. Lentiviruses were harvested 48 h after transfection and stored at -80°C. NRC were infected 20 h after isolation and incubated at 37°C/5% CO2 overnight.
AA V9 constructs
For the generation of the adeno-associated viral constructs, an shRNA targeting Mthfdl l was cloned into a pSico vector where the U6 promoter-driven shRNA expression is controlled in a Cre dependent manner. The shRNAs targeting Mthfdl l were designed using the online software pSico Oligomaker (MIT, version 1 .5). The following shRNA sequence was used: AAV9-fl/fl-sh Mthfdl l,
Sense (SEQ ID NO 003):
T G AAT G GTGT CAG AG AATTTTT CAAG AG AAAATT CT CT G ACACCATT CTTTTTT C , antisense (SEQ ID NO 004):
T CGAGAAAAAAGAAT GGT GT CAGAGAATTTT CT CTT GAAAAATT CT CT GACACCATT CA. After in vitro validation of the knockdown efficiency of the shRNA using Adeno-Cre (Cat No ADV-005, Cell Biolabs) expression to induce Cre-mediated recombination and activation of shMthfdl l expression, the region bearing the U6 promoter, the shRNA and the TATA-lox sites of the pSico construct was amplified and ligated into Nhel/Xhol linearized AAV-bGH(+) vector. All other viruses were designed and engineered by Targeted T ransgenesis. AAV9 viruses were injected at a concentration of 1 .2x1013 genome copies (GC) per kg body weight into tail veins of 3-week-old Mic2v-cre and Mlc2v-cre+ mice.
In vitro administration of locked nucleic acids (LNA), miRNA mimics and siRNAs
miRCURY LNA Power Inhibitors were added directly to the cell culture medium at a final concentration of 50 nM and fresh LNAs added every second day. The following miRCURY LNA Power Inhibitors were purchased from Exiqon: i-mmu-miR27b-5p (4101712-101 ). Negative Control A (199006-101 ) was used as non-targeting LNA.
miRIDIAN microRNA Mimics were transfected into NRCs using Lipofectamine 2000 (Invitrogen). Per well in a 96-well plate, 0.4 pL Lipofectamine 2000 was mixed with 25 pL OptiMEM (Invitrogen). After incubation of 5 min at RT, the mixture was added to an Eppendorf tube containing 6.25 nM or 12.5 nM miRNA mimics in 50 pL OptiMEM and incubated for 20 min at RT to form the complexes. 50 pL of the complexes was added to the cells cultured in 100 pL medium without antibiotics. The medium was replaced 4 h after transfection and cells were harvested 48 h after transfection. The following miRIDIAN microRNA Mimics were purchased from Dharmacon: mmu-miR27b-3p (C-310380-05-0005), mmu-miR27b-5p (C- 310810-01 -0005). miRIDIAN microRNA Mimic Negative Control #1 (CN-001000-01 ) was used as negative control.
siRNAs were transfected at a final concentration of 50 nM into NRCs using the DharmaFECT 1 transfection reagent (Dharmacon) as recommended by the manufacturer. Gene expression was analysed 72 h after transfection. The following ON-TARGETplus SMARTpool siRNAs from Dharmacon were used: Ccndl (L-089285-02-0005), Ccnel (L-101575-02-0005) and Cdknl b (L-090938-02-0005). Non-targeting siRNA pool (D-001810-10-05) was used as control.
Transient transfections
pCMV6 MTHFD1 L (RC223034, Origene) or pEBG-AMPKi-3i2 (#27632, addgene) were transiently transfected into primary neonatal rat cardiomyocytes 2 days after isolation using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were harvested and analyzed 48 h after transfection.
Plasmid constructions
The pLenti-HIFI aAODD puro lentiviral expression vector was generated by subcloning the HIFI aAODD fragment from a pcDNA3-HA-HIF1 aAODD (401-603) plasmid (Huang, L.E., et al., Proc Natl Acad Sci U S A, 95, 7987-7992). into pLenti-pgk puro vector as described previously (Troilo, A., et al. ,2014, EMBO Rep, 15, 77-85). As a corresponding control the pLenti-pgk puro empty vector was used. The miR27b overexpression construct was generated by amplification of the precursor mir27b sequence and the flanking sequence of 158 bp from either end of the mir27b precursor transcript from mouse genomic DNA. The sequence was cloned into the pLKO.1 CMV puro construct provided by A. Ittner (ETH Zurich), by using EcoRI and Sail restriction sites. As a control vector the empty pLKO.1 CMV puro construct was used.
Generation of miRNA Promoter-Luciferase Constructs
A 1 .0 kilobyte (kb) fragment of the mir23b, mir24-1 and mir27b promoter was amplified from mouse genomic DNA and cloned into the pGL3 luciferase reporter vector (Stratagene) between the Xhol and Hindlll restriction sites. Mutation of the HRE in the mir27b promoter was generated by recombinant PCR (Elion, E.A., et al., 2007, CurrProtoc Mol Biol, Chapter 3, Unit 3 17). Sense and Antisense primers were designed bearing HRE mutations, which were used to amplify the mutant 5’ and 3’ regions of the mir27b promoter, respectively. Afterwards, the 5’ and 3’ products generated from the respective PCR reactions were mixed at a 1 :1 ratio and the entire fragment was amplified using primers targeting the 5’ and 3’ ends of the promoters. The wildtype constructs and the mutation of the HRE in the mir27b promoter were confirmed by DNA sequencing (Microsynth).
Generation ofAtp5a1 3’ UTR Luciferase Construct 3’ UTR of Atp5a1 was amplified from mouse cDNA and cloned into the pmirGLO Dual- Luciferase miRNA Target Expression Vector (Promega). Mutation of mir27b binding site on the 3’ UTR of Atp5a1 was generated by recombinant PCR. Sense and antisense primers were generated bearing mir27b binding site mutations, which were used to amplify the mutant 5’ and 3’ regions of the 3’UTR, respectively. Afterwards, the 5’ and 3’ products generated from the respective PCR reactions were mixed at a 1 :1 ratio and the entire sequence was amplified using primers targeting the 5’ and 3’ ends of the Atp5a1 3’UTR. The mutation of the mir27b binding site in the 3’ UTR of Atp5a1 was confirmed by sequencing (Microsynth).
shRNA knockdown of Mthfdll
shRNAs targeting Mthfdl l were designed using the online software BLOCK-iT RNAi Designer (Life Technologies) and compared against the rat genome using BLAST. The shRNA was cloned into the pLKO.1 vector. Sense and antisense oligomers were resuspended to a concentration of 20 mM in Annealing Buffer (100 mM NaCI, 50 mM HEPES, pH 7.4). 5 pL of sense and antisense oligomers were mixed and annealed by incubating them in a beaker containing boiling water and letting it cool to room temperature. The annealed oligomers were ligated into the pLKO.1 vector between the Agel and EcoRI restriction sites. 50 ng vector, 200 ng annealed oligomers, 1 pL T4 DNA ligase were mixed withl pL 10x ligation buffer and brought to a final volume of 10 pL with PCR-grade water. The ligation reaction was incubated overnight at 16°C and 2 pL of the ligation reaction was transformed into competent Stbl3 bacteria by heat shock transformation. Colonies were screened for insertion of shRNA by restriction digest using Ndel and BamHI and positive clones were confirmed by DNA sequencing (Microsynth).
Lentiviral and expression constructs
For knockdowns with lentiviral shRNAs the following TRC pLKO.1 shRNA vectors were used: Atp5a1 (shAtp5a1 , TRCN0000076239), Hifl a (shHifl a, TRCN0000232220) and Vhl (shVhl, TRCN0000436052). pLKO.1 vector containing non-silencing shRNA (nsRNA; SHC002, Sigma) was used as a control. pLenti ATP5A1 (RC214840L1 ) and pCMV6 MTHFD1 L (RC223034) plasmids were from Origene. pEBG-AMPKcd (1 -312) was a gift from Reuben Shaw (Addgene plasmid # 27632).
RNA-isolation, reverse transcription and qRT-PCR
Samples were harvested in Trizol (Invitrogen) and total RNA isolated as recommended by the manufacturer. 750 ng RNA were reverse transcribed into cDNA using RNA to cDNA EcoDry Premix (random hexamers) kit (Clontech, Cat No 639545) following the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) reactions were set up using iTaq Universal SYBR Green Supermix (Biorad, Cat No 1725121 ) according to manufacturer’s recommendations and run on a PikoReal Real-Time PCR machine (Thermo Scientific). Ct values were normalized against the housekeeping gene HprU. The qRT-PCR primer sequences are shown in Table 6.
To assess mature miRNA levels, 10 ng total RNA was transcribed into cDNA using TaqMan MicroRNA Reverse Transcription Kit (4366596, Applied Biosystems) as recommended by the manufacturer. qRT-PCR was performed using TaqMan 2x Universal PCR Master Mix (4304437, Applied Biosystems) following the manufacturers instructions and run on a DNA Engine Opticon 2 (Bio-Rad). Ct values were normalized against the housekeeping genes snoRNA (rat) or snoRNA202 (mouse). The following primers from Thermo Fisher Scientific were used: miR23b-3p (ID000400), miR23b-5p (ID243680_mat) miR24-3p (ID000402), miR24-5p (ID 000488), miR27b-3p (ID000409), miR27b-5p (ID002174), snoRNA (ID001718) and snoRNA202 (ID001232).
PCR
To assess the integrity of the Nnt transcript in BL/6N and BL/6J mice, the PCR was performed using cardiac cDNA as described previously (Huang, T.T., et al., 2006, Hum Mol Genet, 15, 1 187-1 194).
Chromatin immunoprecipitation
ChIP assays were performed with material from NRCs and the assays carried out using the ChIP-IT Kit (Active Motif) according to the manufacturer’s instructions and analyzed by qRT- PCR. ChIP was performed with a ChIP-grade antibody against Hifl a (mouse, ab1 , Abeam). In silico promoter analyses and alignments were performed using Matlnspector and DiAlignTF (Genomatix). Primer sequences used for mir27b in the ChIP were 5'- G CAT G CT GATTT GT G ACTT GAG-3’ (SEQ ID NO 006) and 5'-
CCT CT GTT CT CCAAACTGCAG-3' (SEQ ID NO 007).
MicroRNA microarray
The microRNA microarrays were performed on 3 biological replicates of V hi cKO mice and three control mice ( 7?/ fl/fl), and on 3 biological replicates of Hifla cKO mice subjected to TAC and three controls subjected to TAC surgery (TAC Hif1a M\), respectively. Cardiac dimensions and function were confirmed in all mice by echocardiography. Total RNA was isolated from left ventricle and miRNAs were labelled using the miRCURY LNA microRNA Power Labelling Kit (Exiqon) and hybridized on miRNA arrays (miRXplore) that carry 1 194 DNA oligonucleotides with the reverse-complementary sequence of the mature miRNAs. These arrays cover 728 human, 584 mouse, 426 rat and 122 viral miRNAs, each spotted on the arrays in quadruplicate. The Cy5-labelled miRNAs were normalized to a reference pool of miRNAs that were simultaneously labeled with Cy3. All the data are represented as ratios of logarithmic values between the diseased and control animals and deposited under GSE62418.
NADPH quantification
4x105 NRCs were seeded per well in a 3cm dish and the assay was performed using the NADP/NADPH Quantitation Kit (MAK038, Sigma) as recommended by the manufacturer. Data was normalized to protein amount in the cell lysate by the Bradford assay.
ATP quantification in vivo
ATP was separated and quantified on an anion exchange column (Nucleosil 4000-7 PEI, 50/4 from Macherey-Nagel) with a linear gradient (0-1 .5 M NaCI in 10 mM Tris-HCI, pH 8.0) using an HPLC system equipped with two independent UV-visible spectrometers (Shimadzu,). Elution of samples was monitored at 259 and 220 nm. The 220 nm wavelength was used to detect possible traces of contaminants.
ADP and A TP assays
For the ADP/ATP ratio analysis, 4x105 NRCs were seeded per well in a 3cm dish and the assay was performed using the EnzyLight ADP/ATP Ratio Assay Kit (ELDT-100, Bioassay Systems) as recommended by the manufacturer. For ADP and ATP quantifications the EnzyLight™ ADP Assay Kit (EADP-100, BioAssay Systems) and EnzyLight ATP Assay Kit (EATP-100, BioAssay Systems) were used as recommended by the manufacturers, respectively. The signal was measured on a FLUOstar Omega plate reader (BMG). For ADP/ATP quantification in human and mouse heart tissue, the assay was performed using the ADP/ATP Ratio Assay Kit (ab65313, Abeam) as recommended by the manufacturer with small modifications. Briefly, the heart tissue was frozen and stored in liquid nitrogen immediately after the harvest. The tissue was powdered with a mortar and suspended in lysis buffer (10 mI/mg of tissue powder) for 5 min at room temperature. After the centrifugation at 10000 g for 1 min, the supernatant was used for the assay. Data was normalized to protein amount in the supernatant by the Bradford assay.
ATP synthase Enzyme Activity Assay
4x105 cells were seeded per 3 cm dish. Cells were harvested by trypsinization, followed by centrifugation at 1 .2 krpm for 5 min. Pellet was washed 1 x with PBS (Invitrogen) and centrifuged at 1.2 krpm for 5 min. Pellet was resuspended in 100 mI PBS and frozen at -80°C and sample preparation was continued using the ATP synthase Enzyme Activity Assay Kit (ab109714, Abeam) as recommended by the manufacturer. Absorbance was measured at 340 nm and 30°C for 3 h at 1 min intervals using a FLUOstar Omega plate reader (BMG Labtech). ATP synthase activity was normalized to protein concentration. Luciferase assay
4x104 NRCs were plated in a white 96-well plate and cultured for 3 days. 40 ng of wildtype or mutant pmirGLO Atp5a1 3’UTR construct was co-transfected with 6.25, 12.5 or 25 nM control or miR27b mimics using Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. Luciferase activity was measured 24 h after transfection using the Dual Luciferase Reporter Assay System (Promega) as recommended by the manufacturer on a FLUOstar Omega Microplate Reader (BMG Labtech).
To assess promoter activity, 20 ng pGL3 vector was co-transfected with 1 .25 ng Renilla and 0-260 ng HIFI aAODD. Luciferase activity was measured 36 h after transfection using the Dual Luciferase Reporter Assay System (Promega) as recommended by the manufacturer. E2F transactivation activities were measured using the Cignal Reporter Assay Kit (CCS-003L, Qiagen) according to the manufacturer’s instructions using the Dual Luciferase Reporter Assay System (Promega).
Immunoblotting
Heart tissue was solubilized in blue wonder sample buffer (3.7 M urea, 134.6 mM Tris pH 6.8, 5.4% (v/v) sodium dodecyl sulphate (SDS), 2.3% (v/v) NP-40, 4.45% (v/v) b-mercapto-ethanol, 4% (v/v) glycerol, 60 mg/L bromphenol blue) and proteins denatured for 5 min at 95°C after homogenization using an Ultra-Turrax T10 tissue homogeniser (IKA). NRCs were washed twice with ice-cold PBS and harvested in blue wonder sample buffer. Samples were sonicated to reduce viscosity using a Bransonic 5510 Ultrasonic water bath (Branson) and boiled for 5 min. After brief centrifugation, protein lysates were loaded into 8% or 10% polyacrylamide minigels (Biorad) and transferred to nitrocellulose membrane (GE Healthcare) by wet transfer. Membranes were blocked in 5% (w/v) milk powder (Biorad) in TBST before incubation with primary antibody diluted in 5% (w/v) bovine serum albumin (BSA) in TBST for 2 hours at room temperature or overnight at 4°C. Following three washes in 5% (w/v) milk powder in TBST, membranes were incubated for 1 -2 hours with the appropriate HRP conjugated secondary antibody (anti-Goat IgG HRP, 61 -1620; anti-Mouse IgG HRP, 62-6520; anti-Rabbit IgG HRP, 65-6120, Invitrogen) at a dilution of 1 :5000. Membranes were then washed three times with TBST before detection with ECL (Amersham) on X-ray RX NIF films (Fisher Scientific) to detect the chemiluminescence. Signal intensities were quantified by densitometry using Image J (version 1 .47) (Schneider, C.A., et al., 2012, Nat Methods, 9, 671 -675). The following antibodies were used for immunoblotting: anti-oactinin (A781 1 , Sigma), anti-ACC (3676, Cell Signaling), anti-phospho-ACC Ser79 (1 1818, Cell Signaling), anti-AMPKa (5832, Cell Signaling), anti-phospho-AMRKa Thr172 (2535, Cell Signaling), anti-Atp5a1 (ab14748, Abeam), anti-cardiac actin (61075, Progen Biotechnik), anti-Hif1 a (NB100-479, Novus Biologicals), methylated Lysine (di methyl) (ab23366, Abeam), anti-Mthfd1 l (ab1 16615, Abeam), anti-Rb (9309, Cell Signaling), anti-phospho-Rb Ser807/81 1 (8516, Cell Signaling), anti-Vhl (2738, Cell Signaling) and IgG isotype-control antibody (ab171870).
Co-immunopprecipitation.
0.5x107 transfected (pEBG-AMPK-1-312) or transduced (HIFI aAODD-lentiviruses) NRC per condition were lysed in TNN cell lysis buffer (50 mM Tris-HCI pH 7.5, 250 mM NaCI, 5 mM EdTA, 0.5% NP-40, 50 mM NaF, 0.5 mM EGTA, 1 mM PMSF, 1 mMDTT, and protease inhibitor cocktail) for 30 min. Magnetic dynabeads (Invitrogen) were incubated with anti- AMPKa antibody or IgG control antibody (ab171870) for 10 min at room temperature. AMPK was then immunoprecipitated from cell lysate by incubation for 3 hours at 4°C, IgG control antibody (ab171870). Finally, reaction was terminated by boiling beads in 6x Laemmli buffer for 5 min. Samples were resolved in 10% SDS-PAGE, and Western blot analysis was performed with phospho-RbSer800/804 (Cell Signaling Technology) and phospho-AMPKa Thr172 (Cell Signaling).
Immunofluorescent stainings
Immunofluorescent stainings were performed as described previously (Krishnan, J., et al., 2009, Cell Metab, 9, 512-524). After fixation of NRCs with 4% PFA/PBS, the cells were permeabilized and incubated with primary antibodies diluted in 2% (v/v) HS for 1 h at RT. After 3 washes with PBS for 5 min, cells were incubated with 4',6-diamidino-2-phenylindole (DAPI; D1306, Thermo Fisher Scientific, 0.1 pg/ml), Phalloidin 555 (A34055, Molecular Probes), AlexaFluor 647 anti-mouse (A-1 1001 , Thermo Fisher Scientific) and AlexaFluor 488 anti- mouse (A-21235, Thermo Fisher Scientific) secondary antibody for 1 h at room temperature. For adult cardiomyocytes, the slides were incubated with the primary and secondary antibodies overnight at 4°C in a humidified chamber. Slides and dishes were mounted onto glass slides (Fisher Scientific) with a drop of ProLong Antifade (Thermo Fisher Scientific). The slides/dishes were fixed with clear nail polish and left to dry. The following primary antibodies were used: sarcomeric oactinin (A781 1 , Sigma Aldrich), Ki67 (ab15580, Abeam) and phospho-Histone H3 (Ser10) (05-817, Cell Signaling). Fluorescent images were acquired with the SP5 confocal microscopy (Leica) using a 20x magnification. Cell size was quantified blinded using the software Image J (version 1 .47).
Immunohistochemistry
Hearts were embedded in optimal cutting temperature (OCT) compound and sectioned at 10 pm. Sections were fixed for 10 min with 4% PFA/PBS and after 2 washes with PBS for 2 min blocked for 1 h with 2% HS/PBS for 1 h at room temperature. After permeabilisation for 10 min with 0.2% Triton X-100/PBS, the sections were washed 3 times with PBS for 5 min and incubated with primary antibodies against sarcomeric oactinin (A781 1 , Sigma Aldrich, 1 :800) and Laminin (ab1 1575, Abeam, 1 :300) diluted in 2% (v/v) HS overnight in a humidified chamber at 4°C. After 3 washes with PBS for 10 min, sections were incubated with 4',6-diamidino-2- phenylindole (DAPI; D1306, Thermo Fisher Scientific, 0.1 pg/ml), AlexaFluor 555 anti-mouse (A-21422, Thermo Fisher Scientific, 1 :500) and AlexaFluor 488 anti-rabbit (A-1 1034, Thermo Fisher Scientific, 1 :500) secondary antibody for 2 h at room temperature. Sections mounted with a drop of ProLong Antifade (Thermo Fisher Scientific) and fixed with clear nail polish and left to dry. Fluorescent images were acquired with the SP8 confocal microscopy (Leica) using a 20x magnification. The entire z-axis was imaged and z-stack image generated from approximately 0.2 pm steps. To determine the number of nuclei per myocyte the inventors included only myocytes that had been cut along their longitudinal axis. Nuclei that were surrounded by the extracellular matrix stain laminin were excluded from the analysis. Quantification of nuclei was performed blinded.
Cryosections were stained with hematoxylin and eosin (H&E) or picrosirius red (Sigma). Slides were visualized using a Motic AE2000 with a Moticam 3 or the Axioscan.ZI
slide scanner (Carl Zeiss).
[3H]leucine incorporation assay
[3H]leucine incorporation assay was used to measure de novo protein synthesis as an indirect readout for cell growth(Fukuzawa, J., et at. , 2000, Hypertension, 35, 1 191 -1 196).. 4x105 cells were seeded per 3 cm dish. After 3 days, cells were serum-starved overnight. Next day, cells were cultured in leucine-free medium for 4h, followed by culturing in maintenance medium containing labeled L-[4,5-3H(N)]isoleucine (specific activity 30-60 Ci/mmol, ART0233, American Radiolabelled Chemicals) at a concentration of 0.5 pCi/mL for 20 h. The next day, cells were washed with PBS, trypsinized and the radioactivity was measured for 5 min in in the liquid scintillation analyzer Tri-Carb 2800TR (Perkin Elmer). Cells were counted blinded with a Neubauer chamber after trypsinization and scintillation counts were normalized to absolute cell number.
Nucleic acid incorporation assays
4x105 cells were seeded per 3 cm dish. After 4 days, cells were starved of glucose for 1 .5 h and serine or glycine for 3 h. For radiolabelled glucose incorporation, cells were incubated for 4 h in glucose-free DMEM (Invitrogen) containing 1 pCi/mL uniformly labeled [U-14C]glucose (specific activity 250-360 mCi/mmol, NEC042V250UC, Perkin Elmer). For radiolabelled serine incorporation, cardiomyocytes were incubated overnight in MEM (Invitrogen) supplemented with MEM Vitamin Solution (Invitrogen) and containing 0.6 pCi/mL radiolabelled serine at carbon 3 ([3-14C]serine, specific activity 50-62 mCi/mmol, NEC827050UC, Perkin Elmer). For radiolabelled glycine incorporation, cells were incubated in BME (Invitrogen) containing 1 pCi/mL uniformly radiolabelled glycine ([14C(U)]glycine, specific activity >100mCi/mmol, NEC276E250UC, Perkin Elmer) for 4 h. After washing the cells three times with PBS, cells were harvested in Trizol (Invitrogen) and nucleic acids isolated as recommended by the manufacturer. Isolated nucleic acids were transferred to a scintillation vial containing 4 ml. of the liquid scintillation cocktail Ultima Gold (Perkin Elmer) and radioactivity was measured for 5 min in the Liquid Scintillation Analyzer Tri-Carb 2800TR (Perkin Elmer). Scintillation counts were normalized to absolute nucleic acid quantity.
Fatty acid oxidation
Fatty acid oxidation rate in animal tissues using [1 -14C]-palmitic acid (NEC075H, Perkin Elmer) was determined as described previously (Huynh, F.K., et al., 2014, Methods Enzymol, 542, 391 -405). with the exception that 0.7% BSA/500 mM palmitate/2 pCi 14C-palmitate was used per reaction. Radioactivity was measured for 5 min in the Liquid Scintillation Analyzer Tri-Carb 2800TR (Perkin Elmer). Scintillation counts were normalized to protein amount.
Flow cytometry
1 x106 NRCs were plated on a 6 cm dish (Nunc) and after 4-6 days cells were harvested by trypsinization. Cells were washed 1 x with PBS and centrifuged at 80 g for 5 min. Cells were resuspended in 500 pL PBS and 5 mL cold 70% (v/v) ethanol (kept at -20°C) was added immediately. The fixed cells were kept at 4°C for up to 1 week. Prior to flow cytometry analysis, cells were centrifuged and washed with PBS. After centrifugation at 80 g for 5 min the cells were resuspended in 500 pL propidium iodide solution (69 pM propidium iodide in 38 mM sodium citrate, pH 7.4) containing 40 pg/mL RNase and incubated for 1 h at 37°C. Samples were run on the ImageStream (Amnis), a flow cytometer coupled to fluorescence image acquisition to obtain representative images of the flow cytometry data. The multinucleation was quantified from the ImageStream images.
Formate assay
4x105 cells were seeded per 3 cm dish and the assay was performed using the Formate Assay Kit (Sigma). Cells were washed twice with cold PBS and collected in 25 pL Formate Assay Buffer by scraping. After centrifugation at 15000 g at 4°C for 5 min, 20 pL lysate was mixed with 5 pL Formate Assay Buffer and added to a 96-well plate. 25 pL of the Reaction Mix consisting of 23 pL Formate Assay Buffer, 1 pL Formate Enzyme Mix and 1 pL Formate Substrate Mix was added to the plate containing the lysate. The samples were incubated for 1 h at 37°C protected from light and the absorbance was measured at 450 nm on a FLUOstar Omega plate reader (BMG Labtech). The data was normalized to protein concentration. Metabolomics
4x105 NRCs were cultured per 3 cm dish. The whole cell culture plates were snap frozen in liquid nitrogen after the cells were washed with 75 mM Ammonium carbonate (Sigma), adjusted to pH 7.4 with acetic acid. The metabolites were extracted with cold extraction buffer (-20°C) containing acetonitrile:methanol:water in a 40:40:20 ratio. Untargeted analysis of metabolites by flow injection-time-of-flight mass spectrometry as previously described (Fuhrer, T., et ai, 201 1 , Anal Chem, 83, 7074-7080). Data was processed and analyzed with Matlab. Metabolomics analysis performed by Metabolon (Fig. 12d) was performed as previously described (Cimen, I., et al., 2016,. Sci Trans! Med, 8, 358ra126).
Lipidomics
Mouse heart samples were homogenized on ice in ammonium-bicarbonate buffer (150 mM ammonium bicarbonate, pH 7) with ultra-turax homogenizer. Protein content was assessed using BCA Protein Assay Kit (Thermo Fisher). Equivalents of 20 pg of protein were taken for mass spectrometry analysis. Mass spectrometry-based lipid analysis was performed by Lipotype GmbH (Dresden, Germany) as described (Sampaio, J.L., et al., 201 1 , Proc Natl Acad Sci U S A, 108, 1903-1907). Lipids were extracted using a two-step chloroform/methanol procedure(Ejsing, C.S., et ai, 2009, Proc Natl Acad Sci U S A, 106, 2136-2141 ). Samples were spiked with internal lipid standard mixture containing: cardiolipin 16:1/15:0/15:0/15:0 (CL), ceramide 18:1 ;2/17:0 (Cer),, hexosylceramide 18:1 ;2/12:0 (HexCer), lyso-phosphatidate 17:0 (LPA), lyso-phosphatidylcholine 12:0 (LPC), lyso-phosphatidylethanolamine 17:1 (LPE), lyso-phosphatidylglycerol 17:1 (LPG), lyso-phosphatidylinositol 17:1 (LPI), lyso- phosphatidylserine 17:1 (LPS), phosphatidate 17:0/17:0 (PA), phosphatidylcholine 17:0/17:0 (PC), phosphatidylethanolamine 17:0/17:0 (PE), phosphatidylglycerol 17:0/17:0 (PG), phosphatidylinositol 16:0/16:0 (PI), phosphatidylserine 17:0/17:0 (PS), cholesterol ester 20:0 (CE), sphingomyelin 18:1 ;2/12:0;0 (SM), cholesterol D6 (Choi) (all Avanti Polar Lipids), triacylglycerol 17:0/17:0/17:0 (TAG) and diacylglycerol 17:0/17:0 (DAG) (both Larodan Fine Chemicals). After extraction, the organic phase was transferred to an infusion plate and dried in a speed vacuum concentrator. 1 st step dry extract was re-suspended in 7.5 mM ammonium acetate (Sigma) in chloroform/methanol/propanol (1 :2:4, V:V:V) and 2nd step dry extract in 33% ethanol solution of methylamine in chloroform/methanol (0.003:5:1 ; V:V:V) (all liquids were LC grade obtained from VWR). All liquid handling steps were performed using Hamilton Robotics STARIet robotic platform with the Anti Droplet Control feature for organic solvents pipetting.
Samples were analyzed by direct infusion on a QExactive mass spectrometer (Thermo Scientific) equipped with a T riVersa NanoMate ion source (Advion Biosciences). Samples were analyzed in both positive and negative ion modes with a resolution of Rm/z=200=280000 for MS and Rm/z=200=17500 for MSMS experiments, in a single acquisition. MSMS was triggered by an inclusion list encompassing corresponding MS mass ranges scanned in 1 Da increments 119. Both MS and MSMS data were combined to monitor CE, DAG and TAG ions as ammonium adducts; PC, PC 0-, as acetate adducts; and CL, PA, PE, PE 0-, PG, PI and PS as deprotonated anions. MS only was used to monitor LPA, LPE, LPE 0-, LPI and LPS as deprotonated anions; Cer, HexCer, SM, LPC and LPC O- as acetate adduct and cholesterol as ammonium adduct of an acetylated derivative.
Data were analyzed with a lipid identification software based on LipidXplorer (Herzog, R., et al., 2012, PLoS One, 7, e29851 ) (Herzog, R., et ai, 2011 , Genome Biol, 12, R8). Data post- processing and normalization were performed using an in-house developed data management system. Only lipid identifications with a signal-to-noise ratio >5, and a signal intensity 5-fold higher than in corresponding blank samples were considered for further data analysis.
All downstream analyses were performed in R 123on the mol%-transformed dataset, i.e., after transforming raw data (picomol) to mole percent (each quantity was divided by the sum of the lipids detected in its respective sample and multiplied by 100). Principal Component Analysis (PCA) was computed using the Singular Value Decomposition function. Total Carbon chain length and saturation plots result from grouping together all the lipids that present the same number of carbon atoms (total length) or the same number of double bonds (saturation) and calculating their mean and standard deviation in each cohort of samples. The difference between the means was calculated for each species by subtracting the mean of the controls from the mean of the treated samples (Treated minus Reference), but only the lipids with a delta exceeding |0.3| are shown in the supplemental material. Significance was calculated by means of the non-parametric test Wilcoxon and, in case of multiple comparisons, p-values were adjusted after the Benjamini-Hochberg correction. Significance was set at p<0.05. Alongside R-base functions the following packages were used: factoextra (Kassambara, A. & Mundt, F., 2017, factoextra: Extract and Visualize the Results of Multivariate Data Analyses.)., reshape2 (Wickham, H., 2017, Journal of Statistical Software, 21 , 1-20). and ggplot2 (Wickham, H., 2009, ggplot2: Elegant Graphics for Data Analysis).
Bioinformatic analysis
In silico promoter analyses were performed using Matlnspector (Genomatix). Sequence alignments were performed with BLAST alignment (http://www.ncbi.nlm.nih.gov/blast). shRNAs were designed by proprietary algorithm (Targeted Transgenesis), or computationally predicted using pSico Oligomaker (MIT, version 1.5), BLOCK-iT RNAi Designer (Life Technologies). To predict miR27b targets the miRWalk 2.0 database (http://mirwalk.uni-hd.de) which includes many databases such as TargetScan, miRanda and RNA22 was used (Dweep, et at., 2011 , J Biomed Inform, 44, 839-847). Confocal images were quantified using Image J (version 1.47) (Schneider, C.A., et al., 2012, Nat Methods 9, 671-675).

Claims

Claims
1. An oligonucleic acid agent directed at and capable of specifically inhibiting and/or degrading the microRNA miR27b-5p, for use in a method of treatment or prevention of heart disease.
2. The oligonucleic acid agent for use in a method of treatment or prevention of heart disease of claim 1 , wherein the oligonucleic acid agent comprises, or essentially consists of a hybridizing sequence of nucleotides, which is capable of forming a hybrid with the microRNA miR27b-5p.
3. The oligonucleic acid agent for use in a method of treatment or prevention of heart disease of claim 1 or 3, wherein the oligonucleic acid agent comprises, or essentially consists of, the sequence ACC AAT CAG CTA AGC T (SEQ ID NO 001 ).
4. The oligonucleic acid agent for use in a method of treatment or prevention of heart disease according to any one of the preceding claims, wherein the oligonucleic acid agent is an antisense oligonucleotide.
5. The oligonucleic acid agent for use in a method of treatment or prevention of heart disease of any one of the preceding claims, wherein the oligonucleic acid agent comprises one or several, or essentially consists of, locked nucleic acid (LNA) and/or peptide nucleic acid (PNA) moieties, particularly wherein the oligonucleic acid agent essentially consists of locked nucleic acid (LNA) moieties.
6. The oligonucleic acid agent for use in a method of treatment or prevention of heart disease according to claim 5, wherein the LNA moieties are connected by thiophosphate bonds.
7. The oligonucleic acid agent for use in a method of treatment or prevention of heart disease according to any of the above claims, wherein the oligonucleic acid agent comprises 12 - 20 nucleotides, particularly 14-16 nucleotides.
8. The oligonucleic acid agent for use in a method of treatment or prevention of heart disease according to any one of the above claims, wherein the oligonucleic acid agent comprises or essentially consists of a central block of 5 to 10 deoxyribonucleotides flanked on either side by 2’-0 modified ribonucleotides or PNA oligomers, more particularly a central block of 5 to 10 deoxyribonucleosides flanked by LNA nucleoside analogues, even more particularly wherein said LNA nucleoside analogues are linked by phosphothioate moieties.
9. The oligonucleic acid agent for use in a method of treatment or prevention of heart disease according to any one of the above claims, wherein the oligonucleic acid agent comprises or essentially consists of the sequence ACCA-atcagcta-AGCT (SEQ ID NO 005), wherein
the capital letters signify nucleoside analogues, particularly LNA, more particularly LNA linked by phosphothioate esters, and
the lower case letters signify DNA nucleosides linked by phosphate esters, and the link between a nucleoside analogue and a DNA nucleoside is selected from phosphate ester and thiophosphate.
10. The oligonucleic acid agent for use in a method of treatment or prevention of heart disease according to any one of the above claims, wherein the oligonucleic acid agent is linked to a nanoparticle, or encapsulated in a virus or a lipid complex.
1 1. The oligonucleic acid agent for use in a method of treatment or prevention of heart disease according to any one of the above claims, wherein the heart disease is selected from cardiomyopathy, hypertrophic cardiomyopathy, aortic stenosis, hypertension and heart failure.
12. The oligonucleic acid agent for use in a method of treatment or prevention of heart disease according to any one of the above claims, wherein the heart disease is associated with reduced or absent mitochondrial Complex V (ATP Synthase) activity.
13. A method for the prevention or treatment of heart disease, comprising administering to a patient in need thereof an oligonucleic acid agent as specified in any one of the above claims.
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