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WO2014070706A1 - Régulation de croissance, de différenciation et d'hypertrophie cardiaques - Google Patents

Régulation de croissance, de différenciation et d'hypertrophie cardiaques Download PDF

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Publication number
WO2014070706A1
WO2014070706A1 PCT/US2013/067209 US2013067209W WO2014070706A1 WO 2014070706 A1 WO2014070706 A1 WO 2014070706A1 US 2013067209 W US2013067209 W US 2013067209W WO 2014070706 A1 WO2014070706 A1 WO 2014070706A1
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mhc
heart
tac
cardiomyocytes
agent
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Ching-Pin Chang
Pei Han
Wei Li
Jin Yang
Ching Shang
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • 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
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom

Definitions

  • Heart failure is the leading cause of morbidity in western cultures.
  • Congestive heart failure (CHF) develops when plasma volume increases and fluid accumulates in the lungs, abdominal organs (especially the liver), and peripheral tissues.
  • Cardiac hypertrophy is recognized as one of the independent risk factors leading to severe heart diseases such as ischemic heart diseases and heart failure.
  • cardiac hypertrophy When cardiac hypertrophy is present, there is a 2.5 to 3 fold increase in the percentage of onset of heart failure, ischemic heart diseases such as angina pectoris and myocardial infarction, and cardiovascular diseases such as arrhythmia.
  • Cardiac hypertrophy is a maladaptive mechanism made in response to an increased workload imposed on the heart. It is a specialized process reflecting a quantitative increase in cell size and mass rather than cell number, and may be the result of one or a combination of stimuli. It can be caused either by an increase of the width of myofibrils or by an increase of the length of myofibrils.
  • These contrasting hypertrophic forms are derived respectively by parallel assembly and serial assembly of the sarcomeres, and termed concentric and eccentric hypertrophy, respectively.
  • Cardiac hypertrophy can be induced by response to normal post-natal physiological adaptation or by movement, resulting in increased cardiac pump capacity corresponding to the increase in demand.
  • a pathologically generated load on the heart may also induce cardiac hypertrophy that leads to heart disease.
  • the load on the ventricles is increased by hypertension or valvular disease of the heart, or when damage to the cardiomyocytes themselves is produced by myocardial infarction or myocarditis, pathological cardiac hypertrophy can occur.
  • Cardiac hypertrophy is a compensatory mechanism of the heart to adapt to the increased mechanical load.
  • prolonged cardiac hypertrophy results in systolic and diastolic dysfunctions of the heart, and eventually heart failure.
  • hypertrophic hearts become susceptible to ischemic heart disease and prone to fatal arrhythmia.
  • a normal human left ventricle contains -10% a-MHC and -90% ⁇ -MHC; however, in the failing human heart, a-MHC is virtually eliminated from the left ventricle, causing contractile dysfunction.
  • increasing the expression of a-MHC by even a small amount can augment cardiac contraction by as much as 90%.
  • a-MHC is also reduced in the stressed hearts, and an increase of a-MHC enhances cardiac contractility and resistance to pathological stress. Therefore, regardless of the size of mammals, a-MHC reduction in the stressed heart is a critical step toward myopathy and heart failure.
  • inotropic drugs have the objective of improving systolic capacity of the heart and to increase the cardiac output.
  • inotropic drugs improved subjective symptoms and exercise tolerance, they failed to prolong life.
  • these inotropic agents increase mortality.
  • Newer therapies include inhibitors of angiotensin conversion enzyme (ACE), which suppresses the onset and development of cardiac hypertrophy in animal models, endothelin antagonists and vasopressin antagonists.
  • ACE angiotensin conversion enzyme
  • Non-pharmacological treatment is primarily used as an adjunct to pharmacological treatment.
  • One means of non-pharmacological treatment involves reducing the sodium in the diet.
  • non-pharmacological treatment may include the elimination of precipitating drugs, including negative inotropic agents, cardiotoxins and plasma volume expanders.
  • compositions and methods are provided for prevention and treatment of heart disease, including cardiac hypertrophy, myocardial infarction, and heart failure.
  • the present invention is based on the finding that G9a/Glp histone methyltransferase and DNA methyltransferase (DNMT) cooperate to assemble repressive chromatin on the key molecular motor gene a-myosin heavy chain (a-MHC) to promote heart disease, including cardiac hypertrophy and failure.
  • G9a/GLP and DNMT3 are activated in human hypertrophic hearts and in stressed human iPS-derived cardiomyocytes. Their activation is shown to be essential for H3K9/CpG methylation, which results in aberrant gene expression and hypertrophy. Identification of these pathogenic factors, and the availability of agent that inhibit G9a/GLP and DNMT activity, provide methods for the treatment and prevention of heart disease.
  • an effective dose of an inhibitor of H3K9/CpG methylation at a genetic locus that contributes to heart disease, including specifically the a-MHC locus is administered to an individual suffering from cardiac hypertrophy or myocardial infarction; or diagnosed as being at risk of cardiac hypertrophy or myocardial infarction.
  • the inhibitor is an inhibitor of one or both of G9a/GLP and DNMT activity, and in some such embodiments, the inhibitor is a direct inhibitor of the enzyme, i.e. it directly acts to inhibit the activity of the targeted enzyme.
  • the inhibitor selectively inhibits a DNMT3 enzyme, i.e. DNMT3A, DNMT3B or DNMT3L.
  • the inhibitor selectively inhibits G9a/GLP.
  • Embodiments of the present invention provide methods of reducing injury following myocardial infarction in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an inhibitor of H3K9/CpG methylation at a genetic locus that contributes to heart disease. Further embodiments of the present invention provide administering the inhibitor of H3K9/CpG methylation at a genetic locus that contributes to heart disease at the time of myocardial infarction, after myocardial infarction and/or before myocardial infarction.
  • Embodiments of the present invention also provide methods of promoting myocardial repair following myocardial injury in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an an inhibitor of H3K9/CpG methylation at a genetic locus that contributes to heart disease.
  • the an inhibitor of H3K9/CpG methylation at a genetic locus that contributes to heart disease can be administered at the time of myocardial infarction, after myocardial infarction and/or before myocardial infarction.
  • Embodiments of the present invention further provide methods of reducing myocardial injury and/or promoting mycocardial repair in reperfused or nonreperfused myocardial tissue.
  • the dose of inhibitor may be effective for increasing the expression of oc-MHC, e.g. increasing expression, relative to a control in the absence of therapy, by 10%, 20%, 50%, 100% or more.
  • the therapy may be continued for a period of time sufficient to provide such an effect, and may further be continued on a maintenance dose.
  • the therapy may be combined with conventional agents for treatment of cardiac hypertrophy, including without limitation inhibitors of angiotensin conversion enzyme (ACE); endothelin antagonists; vasopressin antagonists; and the like.
  • ACE angiotensin conversion enzyme
  • a pharmaceutical formulation comprising a unit dose of an inhibitor of H3K9/CpG methylation, e.g. at the oc-MHC locus, including an inhibitor of one or both of G9a/GLP and DNMT, to administered to an individual in need thereof.
  • the inhibitor is a direct inhibitor of the enzyme, i.e. it directly acts to inhibit the activity of the targeted enzyme.
  • the inhibitor selectively inhibits a DNMT3 enzyme, i.e. DNMT3A, DNMT3B or DNMT3L.
  • the inhibitor selectively inhibits G9a/GLP.
  • the dose of inhibitor may be effective for increasing the expression of oc-MHC, e.g. increasing expression relative to a control in the absence of therapy by 10%, 20%, 50%, 100% or more.
  • Formulations may provide for systemic administration, or may provide for a localized administration to cardiac tissue.
  • Small molecule inhibitors of G9a/GLP and DNMT are known in the art and may be used in the methods of the invention, or may be modified for use by conventional medicinal chemistry modifications.
  • FIG. 1 H3K9 methylation by G9a/Glp underlies cardiac hypertrophy and failure a, b,
  • H3K9me2 ChIP Quantitation of H3K9me2 ChIP of the proximal promoters of a-MHC (a) and ⁇ -MHC (b) in fetal hearts (E12.5), sham-operated adult hearts, and TAC-treated adult hearts. Data are presented as H3K9me2 enrichment relative to IgG control.
  • P-value Student's t-test.
  • Error bar SEM, standard error of the mean
  • c mRNA expression of H3K9 methyltransferases in adult hearts after 7 days of TAC.
  • P-value Student's t-test.
  • G9a null Tnnt2-rtTA;Tre-Cre;G9af/f mice, j, k, Trichrome staining of cardiac fibrosis in littermate control and mutant mice lacking myocardial G9a 2 weeks after sham or TAC operation.
  • Ctrl control.
  • G9a null Tnnt2-rtTA;Tre-Cre;G9af/f mice.
  • Red cardiomyocytes.
  • Blue fibrosis.
  • I Echocardiographic measurement of fractional shortening of the left ventricle after 14 days of TAC.
  • Ctrl control.
  • G9a null Tnnt2-rtTA;Tre-Cre;G9at/f m ⁇ ce.
  • P-value Student's t-test. Error bar: SEM, standard error of the mean, m, n, Quantitation of ventricle-body weight ratio (m) and cardiomyocyte size (n) of PBS- and BlX-treated mice 14 days after the sham or TAC operation.
  • PBS phosphate buffered saline.
  • P value Student's t-test. Error bar: SEM, standard error of the mean, o, p, Trichrome staining of cardiac fibrosis 14 days after TAC operation. Red: cardiomyocytes. Blue: fibrosis, q, Echocardiographic measurement of fractional shortening of the left ventricle after 2 weeks of TAC.
  • P-value Student's t-test. Error bar: SEM, standard error of the mean, r, Quantitation of H3K9me2 ChIP on the a-MHC proximal promoter 2 days after sham or TAC operation. Ctrl: control heart. G9a null: Tnnt2-rtTA;Tre- Cre;G9at/f heart. BIX: BlX-treated heart. Data are presented as the enrichment relative to the sham-operated hearts. P-value: Student's t test. Error bar: SEM, standard error of the mean, s, Quantitation of a-MHC mRNA 2 days after sham or TAC operation. Ctrl: control heart.
  • G9a null Tnnt2-rtTA;Tre-Cre;G9af/f hearl
  • PBS PBS-treated heart.
  • BIX BlX-treated heart.
  • P-value Student's t-test. Error bar: SEM, standard error of the mean, t, Quantitation of H3K9me2 ChIP on the ⁇ - -ZC proximal promoter 2 days after sham or TAC operation.
  • Ctrl control heart.
  • G9a null Tnnt2-rtTA;Tre-Cre;G9af/f heart.
  • BIX BlX-treated heart. Data are presented as the enrichment relative to the sham-operated hearts.
  • P-value Student's t test. Error bar: SEM, standard error of the mean, u, Quantitation of - ⁇ mRNA 2 days after sham or TAC operation.
  • Ctrl control heart.
  • G9a null Tnnt2-rtTA;Tre-Cre;G9af/f heart.
  • PBS PBS-treated heart.
  • BIX BlX-treated heart.
  • P-value Student's t-test. Error bar: SEM, standard error of the mean.
  • CpG sites Distribution of CpG sites across the proximal promoters or 5'-untranslated regions of murine a-MHC (a) and ⁇ -MHC (b), as well as the methylation of those CpG sites in fetal heart ventricles (E12.5) and adult heart ventricles after 2 days, 7 days and 14 days of sham or TAC operation.
  • the numbers denote the position of CpG sites relative to the transcriptional start site (+1 ).
  • the CpG sites are color coded.
  • Open and closed circles represent unmethylated and methylated CpG sites, respectively. Each column of circles refers to the sequencing results (at a given CpG site) of 12 randomly selected clones of PCR products amplified from sodium bisulfite-modified genomic DNA.
  • % percentage of CpG methylation (closed circles) relative to total number of sites sequenced (closed and open circles),
  • c Quantitation of mRNA of Dnmts in adult hearts after 7 days of sham or TAC operation. P value: Student's t-test. Error bar: SEM, standard error of the mean, d, e, Immunostaining of Dnmt3a in adult hearts 7 days after sham or TAC procedure. Green:wheat germ agglutinin staining (WGA) outlining cell borders. Red: Dnmt3a immunostaining. Blue: DAPI nuclear staining. Arrows point to nuclei of cardiomyocytes.
  • f, g Quantitation of ventricle-body weight ratio (f) and cardiomyocyte size (g) of PBS- and AZA-treated mice 14 days after the sham or TAC operation.
  • P-value Student's t- test.
  • Error bar SEM, standard error of the mean, h, i, Trichrome staining of cardiac fibrosis 14 days after TAC operation. Red: cardiomyocytes. Blue: fibrosis.
  • PBS PBS-treated heart.
  • AZA AZA-treated heart
  • j Echocardiographic measurement of fractional shortening of the left ventricle after 14 days of sham or TAC operation.
  • PBS PBS-treated heart.
  • AZA AZA-treated heart
  • k Methylation of CpG sites on the proximal promoter of a-MHC in hearts with PBS or AZA treatment 2 days after sham or TAC operation. Representative sequencing results and quantitation analysis of CpG methylation of individual hearts are shown. N represents the number of different hearts used for analysis, with each heart having 12 randomly selected clones sequenced. P-value: Student's t-test. Error bar: SEM, standard error of the mean. I, m, Quantitation of a-MHC (I) and ⁇ -MHC (m) mRNA in hearts treated with PBS or AZA 2 days after sham or TAC operation. P-value: Student's t-test. Error bar: SEM.
  • FIG. 1 Sequential recruitment of chromatin regulators to build repressive chromatins a, Quantitation of G9a ChIP on the proximal promoter of a-MHC 2 days after sham or TAC operation. Data are presented as G9a enrichment relative to IgG control. P-value: Student's t- test Error bar: SEM, standard error of the mean, b, Quantitation of Dnmt3a ChIP on the proximal promoter of a-MHC 2 days after sham or TAC operation. Data are presented as Dnmt3a enrichment relative to IgG control. P-value: Student's t test.
  • Error bar SEM, standard error of the mean
  • c Co-immunoprecipitation of G9a and Dnmt3a in the left ventricles 2 days after TAC.
  • d Quantitation of G9a and H3K9me2 ChIP on the proximal promoter of a-MHC 2 days after sham or TAC operation. Data are presented as G9a or H3K9me2 enrichment relative to sham control.
  • P-value Student's t-test.
  • Error bar SEM, standard error of the mean, e, Quantitation of Dnmt3a ChIP on the proximal promoter of a-MHC 2 days in G9a-null hearts 2 days after sham or TAC operation.
  • G9a null Tnnt2-rtTA;Tre-Cre;G9af/f heart. Data are presented as Dnmt3a enrichment relative to sham control.
  • P-value Student's t-test.
  • Error bar SEM, standard error of the mean, f, Quantitation of CpG methylation of a-MHC in G9a-null hearts. Representative sequencing results and quantitation analysis of CpG methylation of individual hearts are shown.
  • G9a null Tnnt2-rtTA;Tre-Cre;G9af/f heart.
  • N represents the number of different hearts used for analysis, with each heart having 12 randomly selected clones sequenced.
  • P-value Student's t-test.
  • Error bar SEM, standard error of the mean, g, Co-immunoprecipitation of Brg1 with G9a and Dnmt3a in the left ventricles 2 days after TAC.
  • h Quantitation of Brg1 ChIP on the proximal promoter of a-MHC in G9a-null and AZA-treated hearts 2 days after sham or TAC operation.
  • G9a null Tnnt2-rtTA;Tre-Cre;G9af/f heart.
  • P- value Student's t-test.
  • Error bar SEM, standard error of the mean
  • i Quantitation of G9a and H3K9me2 ChIP on the proximal promoter of a-MHC in Brg1-ru ⁇ hearts 2 days after sham or TAC operation.
  • Brg1 null Tnnt2-rtTA;Tre-Cre;Brg1f/f heart
  • P-value Student's t-test.
  • Error bar SEM, standard error of the mean
  • j Quantitation of Dnmt3a ChIP on the proximal promoter of a-MHC in Brg1-ru ⁇ hearts 2 days after sham or TAC operation.
  • Brg1 null Tnnt2-rtTA;Tre- Cre;Brg1f/f heart.
  • P-value Student's t test.
  • Error bar SEM, standard error of the mean
  • k Quantitation of CpG methylation of a-MHC in Brg1-ru ⁇ hearts. Representative sequencing results and quantitation analysis of CpG methylation of individual hearts are shown.
  • Brg1 null Tnnt2-rtTA;Tre-Cre;Brg1 f/f heart.
  • N represents the number of different hearts used for analysis, with each heart having 12 randomly selected clones sequenced.
  • P-value Student's t-test.
  • Error bar SEM, standard error of the mean.
  • FIG. 1 Figure 4. Activation of G9a/GLP, DNMT3, and chromatin methylation in human hypertrophic hearts and iPS-derived cardiomyocytes
  • a Quantitation of a-MHC and ⁇ -MHC mRNA in normal and hypertrophic left ventricles of human hearts.
  • Ctrl control.
  • LVH left ventricular hypertrophy.
  • P-value Student's t-test.
  • Error bar SEM, standard error of the mean
  • b Quantitation of H3K9me2 ChIP on the proximal promoters of human a-MHC and ⁇ -MHC in normal and hypertrophic left ventricles of human hearts.
  • Ctrl control.
  • LVH left ventricular hypertrophy.
  • LVH left ventricular hypertrophy. N represents the number of different hearts used for analysis, with each heart having 12 randomly selected clones sequenced. P-value: Student's t-test. Error bar: SEM, standard error of the mean, e, f, Distribution of the CpG sites across the proximal promoter of human ⁇ - -ZC (e) and quantitation of CpG methylation on ⁇ - ⁇ (f). Ctrl: control. LVH: left ventricular hypertrophy. N represents the number of different hearts used for analysis, with each heart having 12 randomly selected clones sequenced. P-value: Student's t-test.
  • Error bar SEM, standard error of the mean, g, Quantitation of human G9a and GLP. Ctrl: control. LVH: left ventricular hypertrophy. P value: Student's t-test. Error bar: SEM, standard error of the mean, h, Quantitation of human DNMT3a and DNMT3b. Ctrl: control. LVH: left ventricular hypertrophy. P-value: Student's t-test.
  • Error bar SEM, standard error of the mean, i, j, Correlation of G9a and GLP mRNA level (x axis) with -/a-MHC mRNA ratio (y axis) (i) and with H3K9 methylation of a-MHC (y axis) (j).
  • Red regression curve, e, the base of natural logarithm (-2.718).
  • Error bar SEM, standard error of the mean, n-r, Quantitation of cell size, -MHC, ⁇ -MHC, ANF and BNP mRNA in control and drug treated iCMs. Ctrl: control. ET-1 : endothelin-1 . PBS: PBS-treated cells. BIX: BlX-treated cells. AZA: AZA-treated cells. P-value: Student's t-test. Error bar: SEM, standard error of the mean, s, Working model showing that cardiac stress triggers sequential recruitment of chromatin regulators on the a-MHC locus to establish a repressive chromatin scaffold. H: histone. K9: the ninth lysine residue of histone H3 N-terminal tail. C: cytosine at the CpG site. Me: methyl group on H3K9 or CpG sites.
  • FIG. 1 G9a and GIp immunostaining and cardiomyocyte size
  • a, b Immunostaining of G9a and GIp in E12.5 left ventricle. Red: G9a or GIp immunostaining. Blue: DAPI nuclear staining. Arrows point to nuclei of cardiomyocytes.
  • c, d Immunostaining of G9a in doxycycline (DOX)-treated littermate control ⁇ Tnnt2-rtTA;Tre-Cre;G9af/+) and G9a-null ⁇ Tnnt2-rtTA;Tre- Cre;G9at/f) hearts after TAC.
  • DOX doxycycline
  • the staining shows the absence of G9a proteins in cardiomyocytes (arrows) but not endothelial cells (arrowheads) in the heart of DOX-treated Tnnt2-rtTA;Tre-Cre;G9af/f mice.
  • Green wheat germ agglutinin staining (WGA) outlining cell borders.
  • Red G9a immunostaining.
  • Blue DAPI nuclear staining, e-h, Wheat germ agglutinin (WGA) immunostaining of control and G3 ⁇ 4-null mice lacking myocardial G9a 14 days after sham or TAC operation.
  • Ctrl control mice.
  • G9a null Tnnt2-rtTA;Tre-Cre;G9af/f mice, i-l, Wheat germ agglutinin (WGA) immunostaining of PBS- and BlX-treated mice 14 days after the sham or TAC operation, m-n, Ventricle-body weight ratio of mice 2 days after TAC. P- value: Student's t-test. Error bar: SEM, standard error of the mean.
  • Vertical scale bar 2 mm; Horizontal scale bar, 200 milliseconds, a-d, Control and G9a null ( Tnnt2-rtTA;Tre-Cre;G9af/f) mice, e-h, PBS- and BlX- treated mice, i-l, PBS- and AZA-treated mice.
  • Data are presented as Dnmt3b enrichment relative to IgG control.
  • P-value Student's t-test.
  • Error bar SEM, standard error of the mean
  • j Co-immunoprecipitation of Dnmt3b with G9a and Brg1 in the left ventricle of mice 2 days after TAC operation
  • k I
  • Data are presented as Dnmt3b enrichment in TAC hearts relative to Sham control.
  • P-value Student's t-test.
  • Error bar SEM, standard error of the mean.
  • Error bar SEM, standard error of the mean, j-o Immunostaining of G9a (j, k), Dnmt3a (I, m), and Dnmt3b (n, o) in control and Brg1 null (dox-treated Tnnt2-rtTA;Tre-Cre;Brg1 f/f ) hearts 2 days after TAC.
  • Green wheat germ agglutinin staining (WGA) outlining cell borders.
  • Red G9a, Dnmt3a, or Dnmt3b.
  • Blue DAPI nuclear staining. Arrows point to nuclei of cardiomyocytes.
  • FIG. 1 Demography of heart transplantation donors.
  • the left ventricular wall thickness and function was assessed by echocardiography or cardiac magnetic resonance imaging.
  • Tissue assays performed include DNA methylation (D), RT-qPCR (q), and/or ChlP- qPCR (C). Not all assays could be performed in a given tissue sample due to the quality and amount of tissue materials available.
  • LV left ventricle.
  • EF ejection fraction (normal value is 55-65%).
  • IHC intracranial hemorrhage.
  • BMI body mass index (normal value is less than 25).
  • LVH left ventricular hypertrophy.
  • HTN hypertension.
  • FIG. 10 Beating cardiomyocytes derived from human iPS cells and their hypertrophic response to ET-1 a, iPS-derived human cardiomyocytes (iCMs) form monolayer. Images were taken from bright field microscope. Lower panel shows zoom-in image of the cells, b, Homogeneous beating activity of the iCMs was recorded, c, Immunostaining of oc- Actinin and Troponin-T in iCMs. Blue: DAPI nuclear staining. Green: oc-Actinin or Troponin-T staining, d, Quantitation of cell size, -MHC, ⁇ -MHC, ANF and BNP mRNA in control and endothelin-1 treated iCMs. Ctrl: control. ET-1 : endothelin-1 . P-value: Student's t-test. Error bar: SEM, standard error of the mean.
  • FIG. 1 Expression of Class I HDACs and PARP1 in TAC-stressed mouse hearts or human myopathic hearts a, mRNA expression of Class I Hdac genes (Hdad, 2, 3) and Parpl in adult mouse hearts after sham, 2 or 7 days of TAC procedure.
  • P-value Student's t-test. Error bar: SEM, standard error of the mean
  • b mRNA expression of Class-I HDAC genes (HDAC1, 2, 3) and PARP1 in normal and hypertrophic left ventricles of human hearts.
  • Ctrl control.
  • LVH left ventricular hypertrophy.
  • P-value Student's t-test. Error bar: SEM, standard error of the mean.
  • FIG. 12 G9a inhibition reduces myocardial infarction.
  • the areas of myocardial infarction are delineated from back view (a) , side view (b) or front view (c) of the hearts, d, Myocardial sections from those hearts were also stained with TTC: white staining indicates infarction.
  • the invention is based, in part, on the evaluation of the expression and role of enzymes that are differentially expressed in the heart, including cardiomyocytes and endothelial cells, in response to pressure overload.
  • Enzymes involved in H3K9/CpG methylation are activated in stressed cardiomyocytes, resulting in the assembly of repressive chromatin on the key molecular motor gene a-myosin heavy chain (a-MHC), which promotes cardiac hypertrophy and failure.
  • Enzymes of interest include G9a/Glp histone methyltransferase and DNA methyltransferase (DNMT).
  • the findings also provides for diagnostic methods, in determining the presence of modified chromatin at the a-MHC locus, and activity of histone methyltransferase and DNA methyltransferase in cardiomyocytes.
  • the invention also provides methods for the identification of compounds that modulate cardiac hypertrophy and myopathy, e.g. through specific inhibition of G9a/Glp histone methyltransferase and/or DNA methyltransferase (DNMT) activity and/or expression, including the optimization and formulation of known inhibitors for therapeutic purposes.
  • DNMT DNA methyltransferase
  • clinical samples are assayed for the presence of repressive chromatin at the a-MHC locus; or for the presence of enzymes associated with H3K9/CpG methylation.
  • a cell sample of heart tissue is analyzed for the presence of such chromatin or enzymes.
  • H3K9 methylation The lysine at position 9 along the histone 3 tail can be modified to a mono- (H3K9me1 ), di-(H3K9me2) or tri-methylated state (H3K9me3).
  • H3K9Me2 is of particular interest because it is critical for silencing euchromatin within gene rich areas across the genome.
  • H3K9me3 on the contrary, marks heterochromatin and mainly locates to gene-poor regions of repetitive DNA.
  • H3K9me2 is demonstrated to be associated directly with gene repression. Given the myosin heavy chain genes are actively transcribed in cardiac tissue, H3K9Me2 is likely to be enriched and perform the transcriptional control.
  • H3K9 methyltransferase In mammals, the methylation of H3K9 depends on members of the histone lysine methyltransferase (HKMT) family, consisting of Ehmt1 /Glp, Ehmt2/G9a, Suv39h1 , Suv39h2, Setdbl and Setdb2, as well as the non-Suv39 enzymes Prdm2 and Ashl L.
  • HKMTs histone lysine methyltransferase
  • G9a and GLP which form a heterodimeric enzyme complex, are the major HKMTs for H3K9 methylation on the euchromatin.
  • Setdbl and 2 are also HKMTs which catalyze H3K9 methylation on the euchromatin.
  • Suv39h1 and 2 are primary HKMTs that target the pericentric heterochromatin.
  • Prdm2 functions as a tumor suppressor.
  • Ashl L is not specific for H3K9; it also methylates H3K4, H4K20, and H3K36.
  • G9a is a SET domain containing lysine-specific histone methyltransferase.
  • the human gene sequence may be referenced at Genbank accession number NM 006709.3, and is located on chromosome 6, 31 .85-31 .87 Mb.
  • G9a small molecule inhibitors of G9a are known in the art, including quinazolines, of which the class of 7-aminoalkoxy-quinazolines have been shown to be specific direct inhibitors of G9a, (see Chang et al. (2009) Nat. Struct. Bio. 16(3):312-317; Kubicek et al. (2007) Mol. Cell. 25(3):473-481 ). It has been shown that BIX-01294 (diazepin- quinazolin-amine derivative), does not compete with the cofactor S-adenosyl-methionine, and selectively impairs the G9a HMTase and the generation of H3K9me2 in vitro.
  • BIX-01294 diazepin- quinazolin-amine derivative
  • High throughput screening assays are also known in the art for screening candidate compounds for G9a inhibition, including screening of analogs and variants of known inhibitors. For example, see Dhayalan et al. (2009) J. Biomol. Screen. 14(9):1 129-1 133, herein incorporated by reference, for a continuous protein methylation assay using the G9a protein lysine methyltransferase and its substrate protein WIZ (widely interspaced zinc finger motifs).
  • the assay is based on the coupling of the biotinylated substrate protein to streptavidin-coated FlashPlates and the transfer of radioactive methyl groups from the S-adenosyl-L-methionine to the substrate.
  • the reaction progress is monitored continuously by proximity scintillation counting.
  • DNMT DNA cytosine-5 methyltransferases
  • Dnmtl methylates the hemimethylated CpG di-nucleotides in the mammalian genome during DNA replication to maintain DNA methylation during cell proliferation.
  • Dnmt3a and Dnmt3b are the methyltransferases that catalyze de novo DNA methylation. Consistent with cardiomyocytes being post-mitotic, only the de novo Dnmt3a and 3b, but not the maintenance Dnmtl , methyltransferases are induced in the cardiomyocytes after pressure overload.
  • DNMT3 is a family of DNA methyltransferases that could methylate hemimethylated and unmethylated CpG at the same rate.
  • the architecture of DNMT3 enzymes is similar to that of DNMT1 , with a regulatory region attached to a catalytic domain.
  • DNMT3a and DNMT3b can mediate methylation-independent gene repression, DNMT3L can recruit DNMT3 to target sites on the chromatin.
  • DNMT3a can co-localize HP1 and methyl-CpG-binding protein (MeCBP).
  • the reference sequence for human DNMT3A may be found in Genbank at accession number NG_029465.1 ; and the reference sequence for human DNMT3B may be found at accession number NG_007290.1 .
  • Small molecule inhibitors of DNMT are known in the art, including direct inhibitors.
  • 5- azacytidine and decitabine (5-aza-2'-deoxycytidine) are well-known inhibitors.
  • Also known in the art as inhibitors are RG108 (Savickiene et al. (2012) Cell Biol. Int. 36(1 1 ):1067-78); zebularine (You et al. (2012) Mol. Biol. Rep. 39(10):9723-9731 ); procainamide and analogs and derivatives thereof (Halby et al. (2012) Chembiochem. 13(1 ) :157-165); etc.
  • inhibitors of interest for G9a/GLP and DNMT include agents that directly inhibit expression, e.g. RNAi, antisense specific for the targeted gene; and agents that act on the protein, e.g. specific antibodies and analogs thereof, small organic molecules that block activity, etc.
  • Antisense molecules can be used to down-regulate expression in cells.
  • the antisense reagent may be antisense oligonucleotides (ODN), particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such antisense molecules as RNA.
  • ODN antisense oligonucleotides
  • the antisense sequence is complementary to the mRNA of the targeted gene, and inhibits expression of the targeted gene products.
  • Antisense molecules inhibit gene expression through various mechanisms, e.g. by reducing the amount of mRNA available for translation, through activation of RNAse H, or steric hindrance.
  • One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences.
  • RNAi technology refers to a process in which double-stranded RNA is introduced into cells expressing a candidate gene to inhibit expression of the candidate gene, i.e., to "silence" its expression.
  • the dsRNA is selected to have substantial identity with the candidate gene.
  • such methods initially involve transcribing a nucleic acids containing all or part of a candidate gene into single- or double-stranded RNA.
  • Sense and anti-sense RNA strands are allowed to anneal under appropriate conditions to form dsRNA.
  • the resulting dsRNA is introduced into cells via various methods. Usually the dsRNA consists of two separate complementary RNA strands.
  • the dsRNA may be formed by a single strand of RNA that is self-complementary, such that the strand loops back upon itself to form a hairpin loop. Regardless of form, RNA duplex formation can occur inside or outside of a cell.
  • RNA can be directly introduced intracellular ⁇ .
  • Various physical methods are generally utilized in such instances, such as administration by microinjection (see, e.g., Zernicka-Goetz, et al. (1997) Development 124:1 133-1 137; and Wianny, et al. (1998) Chromosoma 107: 430-439).
  • Other options for cellular delivery include permeabilizing the cell membrane and electroporation in the presence of the dsRNA, liposome-mediated transfection, or transfection using chemicals such as calcium phosphate.
  • a number of established gene therapy techniques can also be utilized to introduce the dsRNA into a cell. By introducing a viral construct within a viral particle, for instance, one can achieve efficient introduction of an expression construct into the cell and transcription of the RNA encoded by the construct.
  • Heart failure is a general term that describes the final common pathway of many disease processes. Heart failure is usually caused by a reduction in the efficiency of cardiac muscle contraction. However, mechanical overload with normal or elevated cardiac contraction can also cause heart failure. This mechanical overload may be due to arterial hypertension, or stenosis or leakage of the aortic, mitral, or pulmonary valves, or other causes. The initial response to overload is usually hypertrophy (cellular enlargement) of the myocardium to increase force production, returning cardiac output (CO) to normal levels. Typically, a hypertrophic heart has impaired relaxation, a syndrome referred to as diastolic dysfunction.
  • systolic dysfunction also commonly known as heart failure.
  • This natural progression typically occurs over the course of months to many years in humans, depending on the severity of the overload stimulus. Intervention at the hypertrophy stage can slow or prevent the progression to the clinically significant systolic dysfunction stage.
  • diagnosis in the early hypertrophy stage provides unique therapeutic opportunities.
  • the most common cause of congestive heart failure is coronary artery disease, which can cause a myocardial infarction (heart attack), which forces the heart to carry out the same work with fewer heart cells. The result is a pathophysiological state where the heart is unable to pump out enough blood to meet the nutrient and oxygen requirements of metabolizing tissues or cells.
  • symptoms and signs for example exertional dyspnea, orthopnea, edema, tachycardia, pulmonary rales, a third heart sound, jugular venous distention, etc. have a diagnostic specificity of 70 to 90%, the sensitivity and predictive accuracy of conventional tests are low. Elevated levels of B-type natriuretic peptide may be diagnostic. Adjunctive tests include CBC, blood creatinine, BUN, electrolytes (eg, Mg, Ca), glucose, albumin, and liver function tests. ECG may be performed in all patients with HF, although findings are not specific.
  • Patients diagnosed as being at risk for heart failure may be appropriately treated with the methods of the invention to reduce the risk of heart failure.
  • the individual may be treated with conventional therapy, including diuretics, ACE inhibitors, digitalis, and ⁇ -blockers.
  • systolic and/or diastolic BP Arterial hypertension, or the elevation of systolic and/or diastolic BP, either primary or secondary, is frequently associated with pressure overload of the heart, and is an important risk factor for heart failure.
  • Hypertensive patients may be analyzed by the diagnostic methods of the invention, in order to determine whether there is a concurrent development of hypertrophy, diastolic dysfunction, and a tendency to heart failure. Criteria for hypertension is typically over about 140 mm Hg systolic blood pressure, and/or diastolic blood pressure of greater than about 90 mm Hg.
  • Valvular disease including stenosis or insufficiency of the aortic, mitral, pulmonary, or tricuspid valves, is also frequently associated with overload of the heart, and is another important risk factor for heart failure. Patients with valvular disease may be analyzed by the diagnostic methods of the invention, in order to determine whether ther is a concurrent development of hypertrophy, diastolic dysfunction, and a tendency to heart failure. Valvular disease is typically diagnosed by echocardiographic measurement of significant valvular stenoses or insufficiencies. Valvular heart disease has many etiologies, including but not limited to rheumatic heart disease, congenital valve defects, endocarditis, aging, etc. The pathogenic mechanism whereby valvular disease leads to heart failure is the obstruction of blood outflow from various chambers of the heart, thus increasing load.
  • Cardiomyopathy refers to a structural or functional abnormality of the ventricular myocardium. Cardiomyopathy has many causes. Pathophysiologic classification (dilated congestive, hypertrophic, or restrictive cardiomyopathy) by means of history, physical examination, and invasive or noninvasive testing may be performed. If no cause can be found, cardiomyopathy is considered primary or idiopathic.
  • Pathophysiologic classification diilated congestive, hypertrophic, or restrictive cardiomyopathy
  • Hypertrophic cardiomyopathies are congenital or acquired disorders characterized by marked ventricular hypertrophy with diastolic dysfunction that may develop in the absence of increased afterload.
  • the cardiac muscle is abnormal with cellular and myofibrillar disarray, although this finding is not specific to hypertrophic cardiomyopathy.
  • the interventricular septum may be hypertrophied more than the left ventricular posterior wall (asymmetric septal hypertrophy).
  • asymmetric septal hypertrophy In the most common asymmetric form of hypertrophic cardiomyopathy, there is marked hypertrophy and thickening of the upper interventricular septum below the aortic valve.
  • Chest pain is usually typical angina related to exertion.
  • Syncope is usually exertional and due to a combination of cardiomyopathy, arrhythmia, outflow tract obstruction, and poor diastolic filling of the ventricle.
  • Dyspnea on exertion results from poor diastolic compliance of the left ventricle, which leads to a rapid rise in left ventricular end-diastolic pressure as flow increases.
  • Outflow tract obstruction by lowering cardiac output, may contribute to the dyspnea.
  • myocardial infarction refers to a rapid development of myocardial necrosis, which may be caused by the interruption of blood supply to the heart resulting in a critical imbalance between oxygen supply and demand of the myocardium This may result from plaque rupture with thrombus formation in a coronary vessel leading to an acute reduction of blood supply to a portion of the myocardium; that is, an occlusion or blockage of a coronary artery following the rupture of a susceptible atherosclerotic plaque. If untreated for a sufficient period of time, the resulting ischemia or restriction in blood supply and oxygen shortage can cause damage or death, i.e., infarction of the heart.
  • Myocardial infarction can be assessed using clinical parameters and/or assessments known to those skilled in the art of diagnosing and/or treating the same, for example, physical examinations, detection of signs and symptoms of myocardial infarction, electrocardiogram, echocardiogram, chest X-ray, blood tests to detect cardiac biomarkers including troponins, CK, and CK-MB, etc.
  • reperfusion refers to the restoration of blood flow or supply to the myocardium or myocardial tissue that has become ischemic or hypoxic.
  • Modalities for reperfusion include, but are not limited to, chemical dissolution of the occluding thrombus, i.e., thrombolysis, administration of vasodilators, angioplasty, percutaneous coronary intervention (PCI), catheterization and coronary artery bypass graft (CABG) surgery.
  • PCI percutaneous coronary intervention
  • CABG coronary artery bypass graft
  • therapeutically effective amount refers to an amount of an agent, i.e., an inhibitor of H3K9/CpG methylation at a genetic locus that contributes to heart disease, or composition that is sufficient to produce the desired therapeutic effect.
  • the therapeutically effective amount will vary with the age and physical condition of the subject, the severity of the disorder, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art.
  • An appropriate "therapeutically effective amount” in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. (See, for example, Remington, The Science and Practice of Pharmacy 20th Edition, Lippincott Williams & White, Baltimore, Md. (2000).
  • administered at the time of means that the inhibitor of H3K9/CpG methylation at a genetic locus that contributes to heart disease according to embodiments of the present invention is administered at a time sufficiently close to the onset of an impetus causing injury, a time sufficiently close to the onset of the actual injury or a time sufficiently close to the manifestation of physical symptoms characteristic of the injury. If administered at the time of injury, the inhibitor may reduce injury or prevent further injury.
  • administering after means that the inhibitor is administered after the onset of an impetus causing injury, after the onset of the actual injury or after the manifestation of physical symptoms characteristic of the injury. If administered after injury, the inhibitor may reduce injury or prevent further injury.
  • administering before means that the inhibitor is administered before the onset of an impetus causing injury, before the onset of the actual injury or before the manifestation of physical symptoms characteristic of the injury. If administered before, the inhibitor may be used as a preventive treatment.
  • treatment encompasses the improvement and/or reversal of the symptoms of heart failure (i.e., the ability of the heart to pump blood).
  • "Improvement in the physiologic function" of the heart may be assessed using, for example, measurement of ejection fraction, fractional shortening, left ventricular internal dimension, heart rate, etc., as well as any effect upon the individual's survival.
  • the response of treated transgenic animals and untreated transgenic animals may be compared. Humans and other mammals may be targeted for the methods of the invention.
  • the mammals in question are not particularly limited, and, concretely, may include rats, mice, hamsters, guinea pigs, dogs, monkeys, cows, horses, sheep, goats, and pigs, etc.
  • the term "compound” refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function. Compounds comprise both known and potential therapeutic compounds. A compound can be determined to be therapeutic by screening using the screening methods of the present invention.
  • a "known therapeutic compound” refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment.
  • agonist refers to molecules or compounds that mimic or enhance the action of a “native” or “natural” molecule.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules that interact with a molecule, receptor, and/or pathway of interest.
  • Antagonist and “inhibitor” refer to molecules or compounds that inhibit the action of a cellular factor involved in cardiac hypertrophy, usually H3K9/CpG methylation at the a-MHC locus.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules which bind or interact with a receptor, molecule, and/or pathway of interest.
  • modulate refers to a change or an alteration in the biological activity. Modulation may be an increase or a decrease in protein activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein or other structure of interest.
  • modulator refers to any molecule or compound which is capable of changing or altering biological activity as described above.
  • compositions for treating heart disease comprise as active ingredients substances that inhibit H3K9/CpG methylation at the a-MHC locus in cardiomyocytes.
  • Inhibition may be evaluated by a direct effect on histone or CpG methylation at the a-MHC locus, may be evaluated by an increase in expression of a-MHC as a result of the inhibition; or may be evaluated by determining the effect on one or more of the enzymes involved in the process, particularly G9a, and DNMT3a/b.
  • Inhibition may reduce methylation, relative to a control in the absence of treatment, by 100%, 90%, 80%, 70%, 50%, 25%, etc.
  • Inhibition may increase a-MHC expression relative to a control in the absence of therapy by 10%, 20%, 50%, 100% or more. Inhibition my inhibit activity of a targeted enzyme in cardiomyocytes by 100%, 90%, 80%, 70%, 50%, 25%, etc.
  • An inhibitor of H3K9/CpG methylation at the a-MHC locus in cardiomyocytes may be used in the form of pharmaceutical compositions at a dose effective to prevent or remedy heart diseases caused by cardiac hypertrophy together with pharmaceutically acceptable carriers and other additives.
  • the method to prevent or remedy heart disease caused by cardiac hypertrophy of the present invention may be carried out by administering to test subjects with heart diseases caused by cardiac hypertrophy or the preconditions thereof the effective amount of a substance that inhibits the functional activity or expression.
  • the method may be effectively used to prevent cardiac hypertrophy from developing into heart disease for a test subject with cardiac hypertrophy.
  • a determination of effective dose, and effective combination of agents may be determined empirically, for example using animal models as provided herein. In vitro models are also useful for the assessment of dose and selection of agent. For example, cultures are described herein where the effect on cardiomyocytes is evaluated. Such cultures may be used to assay for the effectiveness of agents alone, or in combinations.
  • a therapeutically or prophylactically effective amount of an agent composition can be done based on animal data using routine computational methods.
  • the effective dose may be measured in terms of parameters described above, in terms of hemodynamic parameters as known in the art, etc., in the individual over a suitable period of time, e.g. over 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or more.
  • the unit dose of a therapeutically or prophylactically effective amount contains between about 0.1 ⁇ g to about 100 mg/kg weight of the individual, as applicable.
  • the administration is performed over a period of time, e.g. semi-daily, daily, semi- weekly, weekly, for a period of days, weeks, months, etc.
  • administering the instant compositions can be effected or performed using any of the various methods and delivery systems known to those skilled in the art.
  • the administering can be performed, for example, intravenously, orally, via implant, transmucosally, transdermal ⁇ , intramuscularly, intrathecal ⁇ , and subcutaneously.
  • the following delivery systems, which employ a number of routinely used pharmaceutical carriers, are only representative of the many embodiments envisioned for administering the instant compositions.
  • Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA's).
  • Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone. Protein or nucleic acids of the invention can also be administered attached to particles using a gene gun.
  • Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc).
  • excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.
  • Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, xanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA).
  • suspending agents e.g., gums, xanthans, cellulosics and sugars
  • humectants e.g., sorbitol
  • solubilizers e.g., ethanol, water, PEG and propylene glycol
  • Formulations may be provided in a unit dosage form, where the term "unit dosage form,” refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active agent in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active agent in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the unit dosage forms of the present invention depend on the particular complex employed and the effect to be achieved, and the pharmacodynamics associated with each complex in the host.
  • dose levels can vary as a function of the specific agent, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the agents will be more potent than others. Preferred dosages for a given agent are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound.
  • the agent is administered at a dose that is effective to cause an improvement in hemodynamic paramaters, but which maintains the overall health of the individual.
  • Treatment regimens may utilize a short-term administration of the active agent; although typically the treatment is administered for prolonged periods, for example as a daily, semi-weekly, weekly, semimonthly, monthly dose monthly, etc.
  • the size of the dose administered must be determined by a physician and will depend on a number of factors, such as the nature and gravity of the disease, the age and state of health of the patient and the patient's tolerance to the agent itself.
  • myocardial repair can be an improvement or a decrease in infarct size, necrosis, apoptosis, autophagy, angiogenesis, remodeling, chamber dilation, wall thinning, inflammation, reduction in serum cardiac troponin I and/or other markers of cardiomyocyte degradation or a combination thereof.
  • the pharmaceutical composition of the present invention may further contain well- known therapeutic drugs for heart disease as necessary.
  • the therapeutic drugs for heart disease are not particularly limited, but ⁇ -blockers, anti-hypertensive agents, cardiotonic agents, anti-thrombosis agents, vasodilators, endothelial receptor blockers, calcium channel blockers, phosphodiesterase inhibitors, Angll receptor blockers, cytokine receptor blockers, gp130 receptor inhibitors, and the like.
  • the change of H3K9/CpG methylation at the oc-MHC locus in cardiomyocytes in hypertrophic cardiomyocytes provides for its use as a marker for diagnosis, and in prognostic evaluations to detect individuals at risk for cardiac pathologies, including atrial enlargement, ventricular hypertrophy, heart failure, etc.
  • Prognostic methods can also be utilized to monitor an individual's health status prior to and after an episode, as well as in the assessment of the severity of the episode and the likelihood and extent of recovery.
  • diagnostic and prognostic methods involve detecting an altered level of repressive chromatin structures at the oc-MHC locus in the cells or tissue of an individual or a sample therefrom. Usually this determined value or test value is compared against some type of reference or baseline value.
  • Samples can be obtained from the tissues or fluids of an individual, as well as from cell cultures or tissue homogenates. For example, samples can be obtained from heart tissue biopsy of cardiomyocytes, etc. Also included in the term are derivatives and fractions of such cells and fluids. Where cells are analyzed, the number of cells in a sample will often be at least about 10 2 , usually at least 10 3 , and may be about 10 4 or more. The cells may be dissociated, in the case of solid tissues, or tissue sections may be analyzed. Alternatively a lysate of the cells may be prepared.
  • Diagnostic samples are collected any time after an individual is suspected to have cardiomyopathy, atrial enlargement, ventricular hypertrophy, etc. or has exhibited symptoms that predict such pathologies.
  • samples can be obtained from an individual who present with risk factors that indicate a susceptibility to heart failure, which risk factors include high blood pressure, obesity, diabetes, etc. as part of a routine assessment of the individual's health status.
  • the various test values determined for a sample from an individual believed to suffer pressure overload, cardiac hypertrophy, diastolic dysfunction, and/or a tendency to heart failure typically are compared against a baseline value to assess the extent of increased or decreased expression, if any.
  • This baseline value can be any of a number of different values.
  • the baseline value is a value established in a trial using a healthy cell or tissue sample that is run in parallel with the test sample.
  • the baseline value can be a statistical value (e.g., a mean or average) established from a population of control cells or individuals.
  • the baseline value can be a value or range that is characteristic of a control individual or control population.
  • the baseline value can be a statistical value or range that is reflective of expression levels for the general population, or more specifically, healthy individuals not susceptible to stroke.
  • Individuals not susceptible to stroke generally refer to those having no apparent risk factors correlated with heart failure, such as high blood pressure, high cholesterol levels, diabetes, smoking and high salt diet, for example.
  • -MHC dynamically changes its expression: it has low expression in fetal hearts, is upregulated in adult hearts, but repressed in hypertrophic and failing hearts.
  • H3K9 histone 3 lysine 9
  • H3K9me2 on the oc- MHC promoter was reactivated and increased 3.6-fold on the a-MHC promoter, comparable to the fetal level of H3K9 methylation (Fig. 1 a).
  • very low H3K9me2 was found on the proximal -MHC promoter (approximately -322 to +278) relative to the IgG control, and H3K9me2 showed no significant changes on the -MHC promoter during heart development and hypertrophy (Fig.
  • H3K9MTs histone H3K9 methyltransferases
  • mice treated with BIX- 01294 a potent and direct specific inhibitor of the G9a/Glp methyltransferase activity, exhibited cardiac resistance to TAC-induced hypertrophy, fibrosis, and failure (Fig. 1 m-q and Fig. 5i-l, 2e-h).
  • BIX BIX- 01294
  • Another potent marker for gene repression is DNA methylation at the CpG dinucleotides.
  • DNA methylation at the CpG dinucleotides To examine the role of DNA methylation in heart failure, we identified five CpG sites on the a- MHC proximal promoter and eight CpG sites on the proximal promoter/5'- untranslated region of -MHC (Fig. 2a, b). Bisulfite genomic sequencing showed that the oc- MHC CpG sites were highly methylated in E12.5 fetal heart ventricles, and the methylation decreased by 44% in healthy adult heart ventricles (from 50% to 28%, Fig. 2a).
  • AZA 5- Azacytidine
  • the human and mouse studies show that the activation of G9a/GLP, DNMT3, and H3K9/CpG methylation is causal to human cardiac hypertrophy.
  • iCMs human iPS-derived cardiomyocytes
  • iCMs iPS-derived cardiomyocytes
  • Fig. 10a, b cardiac-specific proteins a-Actinin and Troponin-T
  • iCMs were treated with endothelin-1 (ET-1 ) to induce cell hypertrophy and gene reprogramming.
  • mice Brg1f/f, G9at/f and Tnnt2-rtTA;Tre-Cre mice have been described previously.
  • the mouse embryonic date was determined by the conventional method, in which the date of observing a vaginal plug was set as embryonic day E0.5.
  • the use of mice for studies is in compliance with the regulations of Stanford University and National Institute of Health.
  • Histology, immunostaining, and trichrome Staining were performed as described.
  • the following primary antibodies were used for immunostaining: anti-G9a (Cat* PP-A8620A-00, R&D Systems), anti-GLP (Cat* PP- B0422-00, R&D Systems), anti-DNMT3a (H-295, Cat* sc20703, Santa Cruz Biotechnology), anti-DNMT3b (Cat* ab16049, Abeam), anti-oc-Actinin (EA-53, Cat* A781 1 , Sigma) and Troponin-T (CT-3, DSHB).
  • TAC Transaortic constriction
  • mice were then tied around the aorta and needle, and secured with a second knot.
  • the needle was immediately removed to create a lumen with a fixed stenotic diameter.
  • the chest cavity was closed by 6-0 silk suture. Sham-operated mice underwent similar surgical procedures, including isolation of the aorta, looping of aorta, but without tying of the suture.
  • the pressure load caused by TAC was verified by the pressure gradient across the aortic constriction measured by echocardiography. Only mice with a pressure gradient >30 mmHg were analyzed for cardiac hypertrophy and gene expression.
  • Echocardiography The echocardiographer was blinded to the genotypes, surgical, or pharmacological treatment of the mice tested. Transthoracic ultrasonography with a GE Vivid 7 ultrasound platform (GE Health Care, Milwaukee, Wl) and a 1 3 MHz transducer was used to measure aortic pressure gradient and left ventricular function. Echocardiography was performed on control and Tnnt2-rtTA;Tre-Cre;G9af/f mice, as well as on mice treated with PBS (phosphate buffered saline), BIX, and AZA at 8 to 1 2 weeks of age.
  • PBS phosphate buffered saline
  • BIX phosphate buffered saline
  • the flow of isoflurane (inhalational) was adjusted to anesthetize the mice while maintaining their heart rates at 450-550 beats per minute.
  • the peak aortic pressure gradient was measured by continuous wave Doppler across the aortic constriction.
  • the left ventricular function was assessed by the M-mode scanning of the left ventricular chamber, standardized by two-dimensional, short-axis views of the left ventricle at the mid papillary muscle level.
  • the fractional shortening (FS) of the left ventricle was defined as 100% x (1 - end systolic/end diastolic diameter), representing the relative change of left ventricular diameters during the cardiac cycle.
  • the mean FS of the left ventricle was determined by the average of FS measurement of the left ventricular contraction over 5 beats. P-values were calculated by the Student-t test. Error bars indicate standard error of mean.
  • RT-qPCR Reverse transcription-quantitative PCR analysis
  • Mouse Dnmt3b-F ( ACC AAATCC AG GG CCTTCTTT) , Mouse Dnmt3b-R (GATAATGCACTCCTCATACCCGC), Mouse Suv39hl-F (CTGTGCCGACTAGCCAAGC), Mouse Suv39hl-R ( ATACCC ACG CC ACTTAACC AG ) , Mouse Suv39h2-F (GCTGTGGTTGGGGTGTAAAA), Mouse Suv39h2-F ⁇ (GCTGCATCCACTGTG AACTC) , Mouse Setdbl-F (GATTCTGGGCAAGAAGAGGA), Mouse Setdbl-R (GTACTTGGCCACCACTCGAC), Mouse Setdb2-F (TCAGTCGCGTTTCCCCCACC), Mouse Setdb2-F ⁇ (CAAGGCCAGGTTGAAAGCCGGA), Mouse Hdacl-F (GCGAGACGGCATTGACGACGA), Mouse Hdacl-H (GTCCAGGGCCACCGCTGTTT), Mouse Hdac2-
  • Human H3F3A-R (TTGTT AC ACG TTTG G C ATG G ) .
  • E12.5 mouse embryos, adult mice, and human subjects were used for ChIP assay as described previously. Chromatin was sonicated to generate average fragment sizes of 200- 600 bp, and immunoprecipitated using anti-BRG1 J1 antibody, anti-G9a antibody (Cat# G6919, Sigma), anti-H3K9Me2 antibody (Cat* 17-648, Millipore), anti-DNMT3a antibody (H- 295, Cat# sc20703, Santa Cruz Biotechnology), anti-DNMT3b (Cat* ab16049, Abeam), or normal control IgG.
  • PCR primers (listed below) were designed to amplify the proximal promoter regions of mouse -MHC (-426, -320), mouse ⁇ - HC (-102, +58), human -MHC (-169, -5) and human ⁇ - HC (-343, -189). The DNA positions are denoted relative to the transcriptional start site (+1 ).
  • Immuunoprecipitation with antibody and following western blotting were performed as described above and previously. 2 ⁇ g primary antibodies of anti-BRG1 (G-7, Cat# sc17796, Santa Cruz Biotechnology), anti-G9a (Cat* PP-A8620A-00, R&D Systems), anti- DNMT3a antibody (H-295, Cat* sc20703, Santa Cruz Biotechnology) or normal control IgG, were used.
  • mice were implanted with subcutaneous micro- osmotic pumps (Alzet Model 1002, DURECT, Cupertino, CA) to infuse BIX01294 (BIX, 25 mg/ml, Cat* 3364, Tocris Bioscience). Micro-pumps were activated prior to implantation to initiate continuous delivery (0.25 ⁇ /hr. of vehicle, PBS) or BIX. Sham or TAC procedures were performed 12 hours post implantation of pumps. After 14 days of sham or TAC operation, mice were evaluated by echocardiography for heart function and harvested for cardiac hypertrophy and fibrosis studies.
  • mice were injected intraperitoneally and daily with vehicle (PBS) or 5- Azacytidine (AZA, 2.5 mg/kg/day) (Cat* A2385, Sigma). Sham or TAC procedures were performed 12 hours after the first injection. After 14 days of the sham or TAC operation, mice were evaluated by echocardiography for heart function and harvested for cardiac hypertrophy and fibrosis studies.
  • vehicle PBS
  • 5- Azacytidine AZA, 2.5 mg/kg/day
  • mice were evaluated by echocardiography for heart function and harvested for cardiac hypertrophy and fibrosis studies.
  • mice were injected intraperitoneally and daily with vehicle (PBS), BIX (1 mg/kg/day), or AZA (2.5 mg/kg/day). Sham or TAC operation was performed 12 hours after the first injection. Two days after the operation, hearts were harvested for bisulfate genomic sequencing, RT-qPCR, immunostaining, or ChIP analysis.
  • vehicle PBS
  • BIX 1 mg/kg/day
  • AZA 2.5 mg/kg/day
  • iPS-derived human cardiomyocytes culture, stress and analysis.
  • the iPS-derived human cardiomyocytes (Cellular Dynamics, Madison, Wl) were cultured according to the manufacture's recommendations. Briefly, cells were cultured in the Plating Medium (Cellular Dynamics, Madison, Wl) for 48 hours and then cultured in William's E Medium with Cocktail B (Life Technology). After another 48 hours, the cells were stressed with 10 nM of endothelin-1 (Sigma) freshly reconstituted in William's E Medium. For BIX and AZA treatment, 100 nM of each chemical was applied together with the vehicle (William's E Medium) or endothelin-1 .
  • the total cell RNA was extracted using the Quick-RNA Microprep kit (Zymo Research) for gene expression analysis.
  • Zymo Research Quick-RNA Microprep kit
  • cells were plated on the coverslip and processed after 48 hours of culture. Cell borders were outlined by WGA staining, and the areas were determined by the ImageJ (NCBI) software. The examiner was blinded to the information of drug treatment. 100 to 300 cells were measured for each control or experimental group.
  • the human tissues were processed for RT-qPCR, ChlP-qPCR, and bisulfite genomic sequencing analysis as described in above sections.
  • the use of human tissues is in compliance with the regulation of Sanford/Burnham Medical Research Institute, Intermountain Medical Center, and Stanford University.

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Abstract

L'invention concerne des procédés et des compositions pour le diagnostic et le traitement de maladies cardiaques associées à l'hypertrophie cardiaque. L'inhibition de l'activité de méthylation fournit une intervention thérapeutique lors du développement de l'hypertrophie cardiaque.
PCT/US2013/067209 2012-11-02 2013-10-29 Régulation de croissance, de différenciation et d'hypertrophie cardiaques Ceased WO2014070706A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008125846A2 (fr) * 2007-04-12 2008-10-23 The Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Suppression et remplacement génétique
US20090162329A1 (en) * 2007-11-30 2009-06-25 Piero Anversa Compositions comprising hdac inhibitors and methods of their use in restoring stem cell function and preventing heart failure
WO2011082038A2 (fr) * 2009-12-31 2011-07-07 Fate Therapeutics, Inc. Compositions de reprogrammation améliorées
WO2011106442A1 (fr) * 2010-02-24 2011-09-01 The Board Of Trustees Of The Leland Stanford Junior University Contrôle de croissance, différenciation et hypertrophie cardiaques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008125846A2 (fr) * 2007-04-12 2008-10-23 The Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Suppression et remplacement génétique
US20090162329A1 (en) * 2007-11-30 2009-06-25 Piero Anversa Compositions comprising hdac inhibitors and methods of their use in restoring stem cell function and preventing heart failure
WO2011082038A2 (fr) * 2009-12-31 2011-07-07 Fate Therapeutics, Inc. Compositions de reprogrammation améliorées
WO2011106442A1 (fr) * 2010-02-24 2011-09-01 The Board Of Trustees Of The Leland Stanford Junior University Contrôle de croissance, différenciation et hypertrophie cardiaques

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