Integrative analysis of transcriptome, DNA methylome, and chromatin accessibility reveals candidate therapeutic targets in hypertrophic cardiomyopathy

Abstract

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease and is characterized by primary left ventricular hypertrophy usually caused by mutations in sarcomere genes. The mechanism underlying cardiac remodeling in HCM remains incompletely understood. An investigation of HCM through integrative analysis at multi-omics levels will be helpful for treating HCM. DNA methylation and chromatin accessibility, as well as gene expression, were assessed by nucleosome occupancy and methylome sequencing (NOMe-seq) and RNA-seq, respectively, using the cardiac tissues of HCM patients. Compared with those of the controls, the transcriptome, DNA methylome, and chromatin accessibility of the HCM myocardium showed multifaceted differences. At the transcriptome level, HCM hearts returned to the fetal gene program through decreased sarcomeric and metabolic gene expression and increased extracellular matrix gene expression. In the DNA methylome, hypermethylated and hypomethylated differentially methylated regions were identified in HCM. At the chromatin accessibility level, HCM hearts showed changes in different genome elements. Several transcription factors, including SP1 and EGR1, exhibited a fetal-like pattern of binding motifs in nucleosome-depleted regions in HCM. In particular, the inhibition of SP1 or EGR1 in an HCM mouse model harboring sarcomere mutations markedly alleviated the HCM phenotype of the mutant mice and reversed fetal gene reprogramming. Overall, this study not only provides a high-precision multi-omics map of HCM heart tissue but also sheds light on the therapeutic strategy by intervening in the fetal gene reprogramming in HCM.

Keywords

hypertrophic cardiomyopathy / multi-omics / chromatin accessibility / DNA methylation / fetal gene reprogramming / therapy

References

[1] Azakie A, Fineman JR, He Y. Myocardial transcription factors are modulated during pathologic cardiac hypertrophy in vivo. J Thorac Cardiovasc Surg 2006a;132:1262–1271.

[2] Azakie A, Fineman JR, He Y. Sp3 inhibits Sp1-mediated activation of the cardiac troponin T promoter and is downregulated during pathological cardiac hypertrophy in vivo. Am J Physiol Heart Circ Physiol 2006b;291:H600–H611.

[3] Baum M. A clinical trial of mithramycin in the treatment of advanced malignant disease. Br J Cancer 1968;22:176–183.

[4] Boon RA, Jae N, Holdt L et al. Long noncoding RNAs: from clinical genetics to therapeutic targets? J Am Coll Cardiol 2016;67:1214–1226.

[5] Brower GL, Gardner JD, Forman MF et al. The relationship between myocardial extracellular matrix remodeling and ventricular function. Eur J Cardiothorac Surg 2006;30:604–610.

[6] Buitrago M, Lorenz K, Maass AH et al. The transcriptional repressor Nab1 is a specific regulator of pathological cardiac hypertrophy. Nat Med 2005;11:837–844.

[7] Cao C, Xu Q, Zhu Z et al. Three-dimensional chromatin analysis reveals Sp1 as a mediator to program and reprogram HPV-host epigenetic architecture in cervical cancer. Cancer Lett 2024;588:216809.

[8] Chen J, Bardes EE, Aronow BJ et al. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res 2009;37:W305–W311.

[9] Chen Y, Lyu RT, Rong BW et al. Refined spatial temporal epigenomic profiling reveals intrinsic connection between PRDM9-mediated H3K4me3 and the fate of doublestranded breaks. Cell Res 2020;30:256–268.

[10] Chien KR, Knowlton KU, Chien S. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J 1991;5:3037–3064.

[11] Choi ES, Nam JS, Jung JY et al. Modulation of specificity protein 1 by mithramycin A as a novel therapeutic strategy for cervical cancer. Sci Rep 2014;4:7162.

[12] Coats CJ, Heywood WE, Virasami A et al. Proteomic analysis of the myocardium in hypertrophic obstructive cardiomyopathy. Circulation: Genomic Precision Med 2018;11:e001974.

[13] Cook T, Gebelein B, Urrutia R. Sp1 and its likes: biochemical and functional predictions for a growing family of zinc finger transcription factors. Ann N Y Acad Sci 1999;880:94–102.

[14] De Bold A, Borenstein HB, Veress AT et al. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci 1981;28:89–94.

[15] Deng J, Tian AL, Pan H et al. Everolimus and plicamycin specifically target chemoresistant colorectal cancer cells of the CMS4 subtype. Cell Death Dis 2021;12:978.

[16] Depre C, Shipley GL, Chen W et al. Unloaded heart in vivo replicates fetal gene expression of cardiac hypertrophy. Nat Med 1998;4:1269–1275.

[17] Dirkx E, da Costa Martins PA, De Windt LJ. Regulation of fetal gene expression in heart failure. Biochim Biophys Acta 2013;1832:2414–2424.

[18] Dutcher JP, Coletti D, Paietta E et al. A pilot study of alpha-interferon and plicamycin for accelerated phase of chronic myeloid leukemia. Leuk Res 1997;21:375–380.

[19] England J, Granados-Riveron J, Polo-Parada L et al. Tropomyosin 1: multiple roles in the developing heart and in the formation of congenital heart defects. J Mol Cell Cardiol 2017;106:1–13.

[20] Eto K, Kaur V, Thomas MK. Regulation of insulin gene transcription by the immediate-early growth response gene Egr-1. Endocrinology 2006;147:2923–2935.

[21] Fajardo OA, Thompson K, Parapuram SK et al. Mithramycin reduces expression of fibro-proliferative mRNAs in human gingival fibroblasts. Cell Prolif 2011;44:166–173.

[22] Federico A, Steinfass T, Larribère L et al. Mithramycin A and Mithralog EC-8042 Inhibit SETDB1 expression and its oncogenic activity in malignant melanoma. Mol Ther Oncolytics 2020;18:83–99.

[23] Feng P, Che Y, Gao C et al. Profibrotic role of transcription factor SP1 in cross-talk between fibroblasts and M2 macrophages. iScience 2023;26:108484.

[24] Gao J, Collyer J, Wang M et al. Genetic dissection of hypertrophic cardiomyopathy with myocardial RNA-Seq. Int J Mol Sci 2020;21:3040.

[25] Gao J, Zheng Y, Li L et al. Integrated transcriptomics and epigenomics reveal chamber-specific and species-specific characteristics of human and mouse hearts. PLoS Biol 2021;19:e3001229.

[26] Garmany R, Bos JM, Tester DJ et al. Multi-omic architecture of obstructive hypertrophic cardiomyopathy. Circ Genom Precis Med 2023;16:e003756.

[27] Gashler A, Sukhatme VP. Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog Nucleic Acid Res Mol Biol 1995;50:191–224.

[28] Geisterfer-Lowrance AA, Kass S, Tanigawa G et al. A molecular basis for familial hypertrophic cardiomyopathy: a β cardiac myosin heavy chain gene missense mutation. Cell 1990;62:999–1006.

[29] Geng H, Su Y, Huang R et al. Specific protein 1 inhibitor mithramycin A protects cardiomyocytes from myocardial infarction via interacting with PARP. In Vitro Cell Dev Biol Anim 2021;57:315–323.

[30] Gentleman RC, Carey VJ, Bates DM et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004;5:R80.

[31] Gersh BJ, Maron BJ, Bonow RO et al. 2011 ACCF/AHA guideline for the diagnosis and treatment of hypertrophic cardiomyopathy: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation 2011;124:2761–2796.

[32] Goetze JP, Bruneau BG, Ramos HR et al. Cardiac natriuretic peptides. Nat Rev Cardiol 2020;17:698–717.

[33] Green L, Donehower RC. Hepatic toxicity of low doses of mithramycin in hypercalcemia. Cancer Treat Rep 1984;68:1379–1381.

[34] Green EM, Wakimoto H, Anderson RL et al. A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice. Science 2016;351:617–621.

[35] Guo B, Tian XC, Li DD et al. Expression, regulation and function of Egr1 during implantation and decidualization in mice. Cell Cycle 2014;13:2626–2640.

[36] Guo H, Hu B, Yan L et al. DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells. Cell Res 2017;27:165–183.

[37] Hayashi T, Arimura T, Itoh-Satoh M et al. Tcap gene mutations in hypertrophic cardiomyopathy and dilated cardiomyopathy. J Am Coll Cardiol 2004;44:2192–2201.

[38] Heinz S, Benner C, Spann N et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell 2010;38:576–589.

[39] Hershberger RE, Parks SB, Kushner JD et al. Coding sequence mutations identified in MYH7, TNNT2, SCN5A, CSRP3, LBD3, and TCAP from 313 patients with familial or idiopathic dilated cardiomyopathy. Clin Transl Sci 2008;1:21–26.

[40] Ho CY, Olivotto I, Jacoby D et al. Study design and rationale of EXPLORER-HCM: evaluation of mavacamten in adults with symptomatic obstructive hypertrophic cardiomyopathy. Circ Heart Fail 2020;13:e006853.

[41] Hoffman M, Palioura D, Kyriazis ID et al. Cardiomyocyte Kruppel-like factor 5 promotes De Novo ceramide biosynthesis and contributes to eccentric remodeling in ischemic cardiomyopathy. Circulation 2021;143:1139–1156.

[42] Hojayev B, Rothermel BA, Gillette TG et al. FHL2 binds calcineurin and represses pathological cardiac growth. Mol Cell Biol 2012;32:4025–4034.

[43] Houweling AC, van Borren MM, Moorman AF et al. Expression and regulation of the atrial natriuretic factor encoding gene Nppa during development and disease. Cardiovasc Res 2005;67:583–593.

[44] Huang H, Han Y, Chen Z et al. ML264 inhibits osteosarcoma growth and metastasis via inhibition of JAK2/STAT3 and WNT/beta-catenin signalling pathways. J Cell Mol Med 2020;24:5652–5664.

[45] Huber W, Carey VJ, Gentleman R et al. Orchestrating high-throughput genomic analysis with bioconductor. Nat Methods 2015;12:115–121.

[46] Izumo S, Nadal-Ginard B, Mahdavi V. Protooncogene induction and reprogramming of cardiac gene expression produced by pressure overload. Proc Natl Acad Sci U S A 1988;85:339–343.

[47] Jaffre F, Miller CL, Schanzer A et al. Inducible pluripotent stem cell-derived cardiomyocytes reveal Aberrant extracellular regulated kinase 5 and mitogen-activated protein kinase kinase 1/2 signaling concomitantly promote hypertrophic cardiomyopathy in RAF1-associated Noonan syndrome. Circulation 2019;140:207–224.

[48] Jones PA. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 2012;13:484–492.

[49] Kelly TK, Liu Y, Lay FD et al. Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules. Genome Res 2012;22:2497–2506.

[50] Kennedy BJ, Torkelson JL. Long-term follow-up of stage III testicular carcinoma treated with mithramycin (plicamycin). Med Pediatr Oncol 1995;24:327–328.

[51] Klemm SL, Shipony Z, Greenleaf WJ. Chromatin accessibility and the regulatory epigenome. Nat Rev Genet 2019;20:207–220.

[52] Krueger F, Andrews SR. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 2011;27:1571–1572.

[53] Ku HJ, Ahn Y, Lee JH et al. IDH2 deficiency promotes mitochondrial dysfunction and cardiac hypertrophy in mice. Free Radic Biol Med 2015;80:84–92.

[54] Lay FD, Liu Y, Kelly TK et al. The role of DNA methylation in directing the functional organization of the cancer epigenome. Genome Res 2015;25:467–477.

[55] Li H, Handsaker B, Wysoker A et al. The sequence alignment/map format and SAMtools. Bioinformatics 2009;25:2078–2079.

[56] Li L, Guo F, Gao Y et al. Single-cell multi-omics sequencing of human early embryos. Nat Cell Biol 2018;20:847–858.

[57] Lim D-S, Roberts R, Marian AJ. Expression profiling of cardiac genes in human hypertrophic cardiomyopathy: insight into the pathogenesis of phenotypes. J Am Coll Cardiol 2001;38:1175–1180.

[58] Lin H, Xiao J, Luo X et al. Transcriptional control of pacemaker channel genes HCN2 and HCN4 by Sp1 and implications in re-expression of these genes in hypertrophied myocytes. Cell Physiol Biochem 2009;23:317–326.

[59] Liu X, Ma Y, Yin K et al. Long non-coding and coding RNA profiling using strand-specific RNA-seq in human hypertrophic cardiomyopathy. Sci Data 2019;6:90.

[60] Liu X, Yin K, Chen L et al. Lineage-specific regulatory changes in hypertrophic cardiomyopathy unraveled by singlenucleus RNA-seq and spatial transcriptomics. Cell Discov 2023;9:6.

[61] Lombardi R, Betocchi S, Losi MA et al. Myocardial collagen turnover in hypertrophic cardiomyopathy. Circulation 2003;108:1455–1460.

[62] Long Y, Wang L, Li Z. SP1-induced SNHG14 aggravates hypertrophic response in in vitro model of cardiac hypertrophy via up-regulation of PCDH17. J Cell Mol Med 2020;24:7115–7126.

[63] Lorenzen JM, Thum T. Long noncoding RNAs in kidney and cardiovascular diseases. Nat Rev Nephrol 2016;12:360–373.

[64] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014;15:550.

[65] Luo X, He S, Hu Y et al. Sp1-induced LncRNA CTBP1-AS2 is a novel regulator in cardiomyocyte hypertrophy by interacting with FUS to stabilize TLR4. Cardiovascular Pathol 2019;42:21–29.

[66] Marian AJ, Braunwald E. Hypertrophic cardiomyopathy: genetics, pathogenesis, clinical manifestations, diagnosis, and therapy. Circ Res 2017;121:749–770.

[67] Maron BJ. Hypertrophic cardiomyopathy: a systematic review. JAMA 2002;287:1308–1320.

[68] Maron BJ. Hypertrophic cardiomyopathy: an important global disease. Am J Med 2004;116:63–65.

[69] Maron BJ. Contemporary insights and strategies for risk stratification and prevention of sudden death in hypertrophic cardiomyopathy. Circulation 2010;121:445–456.

[70] Maron BJ. Recognition of hypertrophic cardiomyopathy as a contemporary, relatively common, and treatable disease (from the International Summit V). Am J Cardiol 2014;113:739–744.

[71] Maron BJ, Maron MS. Hypertrophic cardiomyopathy. Lancet 2013;381:242–255.

[72] Maron BJ, Gardin JM, Flack JM et al. Prevalence of hypertrophic cardiomyopathy in a general population of young adults. Echocardiographic analysis of 4111 subjects in the CARDIA Study. Coronary Artery Risk Development in (Young) Adults. Circulation 1995;92:785–789.

[73] Mercadier J-J, Lompré A-M, Wisnewsky C et al. Myosin isoenzyme changes in several models of rat cardiac hypertrophy. Circ Res 1981;49:525–532.

[74] Meriin AB, Zaarur N, Roy D et al. Egr1 plays a major role in the transcriptional response of white adipocytes to insulin and environmental cues. Front Cell Dev Biol 2022;10:1003030.

[75] Müller I, Rössler OG, Wittig C et al. Critical role of Egr transcription factors in regulating insulin biosynthesis, blood glucose homeostasis, and islet size. Endocrinology 2012;153:3040–3053.

[76] Nadal-Ginard B, Mahdavi V. Molecular basis of cardiac performance. Plasticity of the myocardium generated through protein isoform switches. J Clin Invest 1989;84:1693–1700.

[77] Newman MS, Nguyen T, Watson MJ et al. Transcriptome profiling reveals novel BMI-and sex-specific gene expression signatures for human cardiac hypertrophy. Physiol Genomics 2017;49:355–367.

[78] Nunez L, Gimeno-Blanes JR, Rodriguez-Garcia MI et al. Somatic MYH7, MYBPC3, TPM1, TNNT2 and TNNI3 mutations in sporadic hypertrophic cardiomyopathy. Circ J 2013;77:2358–2365.

[79] Osgood CL, Maloney N, Kidd CG et al. Identification of Mithramycin analogues with improved targeting of the EWS-FLI1 transcription factor. Clin Cancer Res 2016;22:4105–4118.

[80] Parker TG, Schneider MD. Growth factors, proto-oncogenes, and plasticity of the cardiac phenotype. Annu Rev Physiol 1991;53:179–200.

[81] Pei J, Schuldt M, Nagyova E et al. Multi-omics integration identifies key upstream regulators of pathomechanisms in hypertrophic cardiomyopathy due to truncating MYBPC3 mutations. Clin Epigenetics 2021;13:61.

[82] Previs MJ, O’Leary TS, Morley MP et al. Defects in the proteome and metabolome in human hypertrophic cardiomyopathy. Circ Heart Fail 2022;15:e009521.

[83] Quarni W, Dutta R, Green R et al. Mithramycin A inhibits colorectal cancer growth by targeting cancer stem cells. Sci Rep 2019;9:15202.

[84] Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 2010;26:841–842.

[85] Rajabi M, Kassiotis C, Razeghi P et al. Return to the fetal gene program protects the stressed heart: a strong hypothesis. Heart Fail Rev 2007;12:331–343.

[86] Razeghi P, Young ME, Alcorn JL et al. Metabolic gene expression in fetal and failing human heart. Circulation 2001;104:2923–2931.

[87] Ream NW, Perlia CP, Wolter J et al. Mithramycin therapy in disseminated germinal testicular cancer. JAMA 1968;204:1030–1036.

[88] Ren CW, Liu JJ, Li JH et al. RNAseq profiling of mRNA associated with hypertrophic cardiomyopathy. Mol Med Rep 2016;14:5573–5586.

[89] Robertson KD. DNA methylation and human disease. Nat Rev Genet 2005;6:597–610.

[90] Ruiz de Sabando A, Wang C, He Y et al. ML264, a novel small-molecule compound that potently inhibits growth of colorectal cancer. Mol Cancer Ther 2016;15:72–83.

[91] Schuldt M, Pei J, Harakalova M et al. Proteomic and functional studies reveal detyrosinated tubulin as treatment target in sarcomere mutation-induced hypertrophic cardiomyopathy. Circ Heart Fail 2021;14:e007022.

[92] Semsarian C, Ingles J, Maron MS et al. New perspectives on the prevalence of hypertrophic cardiomyopathy. J Am Coll Cardiol 2015;65:1249–1254.

[93] Sewanan LR, Park J, Rynkiewicz MJ et al. Loss of crossbridge inhibition drives pathological cardiac hypertrophy in patients harboring the TPM1 E192K mutation. J Gen Physiol 2021;153:e202012640.

[94] Sharma V, Goessling LS, Brar AK et al. Coxsackievirus B3 infection early in pregnancy induces congenital heart defects through suppression of fetal cardiomyocyte proliferation. J Am Heart Assoc 2021;10:e017995.

[95] Stacklies W, Redestig H, Scholz M et al. pcaMethods—a bioconductor package providing PCA methods for incomplete data. Bioinformatics 2007;23:1164–1167.

[96] Sun X, Huang H, Pan X et al. EGR1 promotes the cartilage degeneration and hypertrophy by activating the Kruppel-like factor 5 and beta-catenin signaling. Biochim Biophys Acta Mol Basis Dis 2019;1865:2490–2503.

[97] Taberlay PC, Statham AL, Kelly TK et al. Reconfiguration of nucleosome-depleted regions at distal regulatory elements accompanies DNA methylation of enhancers and insulators in cancer. Genome Res 2014;24:1421–1432.

[98] Teekakirikul P, Eminaga S, Toka O et al. Cardiac fibrosis in mice with hypertrophic cardiomyopathy is mediated by non-myocyte proliferation and requires Tgf-beta. J Clin Invest 2010;120:3520–3529.

[99] Thum T, Galuppo P, Wolf C et al. MicroRNAs in the human heart: a clue to fetal gene reprogramming in heart failure. Circulation 2007;116:258–267.

[100] Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 2009;25:1105–1111.

[101] Tsoutsman T, Wang X, Garchow K et al. CCN2 plays a key role in extracellular matrix gene expression in severe hypertrophic cardiomyopathy and heart failure. J Mol Cell Cardiol 2013;62:164–178.

[102] Tsybouleva N, Zhang L, Chen S et al. Aldosterone, through novel signaling proteins, is a fundamental molecular bridge between the genetic defect and the cardiac phenotype of hypertrophic cardiomyopathy. Circulation 2004;109:1284–1291.

[103] Tucholski T, Cai W, Gregorich ZR et al. Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics. Proc Natl Acad Sci U S A 2020;117:24691–24700.

[104] Tuohy CV, Kaul S, Song HK et al. Hypertrophic cardiomyopathy: the future of treatment. Eur J Heart Fail 2020;22:228–240.

[105] Uchida S, Dimmeler S. Long noncoding RNAs in cardiovascular diseases. Circ Res 2015;116:737–750.

[106] Verrecchia F, Mauviel A, Rossert J. Blocking Sp1 transcription factor broadly inhibits extracellular matrix gene expression in vitro and in vivo: implications for the treatment of tissue fibrosis. J Investig Dermatol 2001;116:755–763.

[107] Vizcaíno C, Mansilla S, Portugal J. Sp1 transcription factor: a long-standing target in cancer chemotherapy. Pharmacol Ther 2015;152:111–124.

[108] Walsh R, Buchan R, Wilk A et al. Defining the genetic architecture of hypertrophic cardiomyopathy: re-evaluating the role of non-sarcomeric genes. Eur Heart J 2017;38:3461–3468.

[109] Wang B, Guo H, Yu H et al. The role of the transcription factor EGR1 in cancer. Front Oncol 2021;11:642547.

[110] Wehrens M, de Leeuw AE, Wright-Clark M et al. Single-cell transcriptomics provides insights into hypertrophic cardiomyopathy. Cell Rep 2022;39:110809.

[111] Wong TC, Martinez M. Novel pharmacotherapy for hypertrophic cardiomyopathy. Cardiol Clin 2019;37:113–117.

[112] Xiao S, Peng K, Li C et al. The role of sphingosine-1-phosphate in autophagy and related disorders. Cell Death Discov 2023;9:380.

[113] Yuan P, Wang L, Wei D et al. Therapeutic inhibition of Sp1 expression in growing tumors by mithramycin a correlates directly with potent antiangiogenic effects on human pancreatic cancer. Cancer 2007;110:2682–2690.

[114] Zhang F, Zhou H, Xue J et al. Deficiency of transcription factor SP1 contributes to hypertrophic cardiomyopathy. Circ Res 2024;134:290–306.

[115] Zou C, Li W, Zhang Y et al. Identification of an anaplastic subtype of prostate cancer amenable to therapies targeting SP1 or translation elongation. Sci Adv 2024;10:eadm7098.

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