Remedios, Cristobal G. dos

Introduction: Recently, we demonstrated transcriptional down-regulation of hypertrophy pathways in myectomy tissue derived from patients with obstructive hypertrophic cardiomyopathy (HCM) despite translational activation of associated hypertrophy pathways. Previous studies of other forms of cardiac hypertrophy demonstrate hypertrophy pathways are activated via histone acetylation and histone deacetylase (HDAC) inhibitors prevented development of hypertrophy. Whether histone modifications regulate hypertrophy pathways in HCM remains unexplored. Methods: Chromatin immunoprecipitation sequencing (ChIP-seq) was performed on myectomy tissues from 40 patients with HCM and 23 controls to characterize differential histone marks for H3K4me3 (active promoter), H3K9ac (active promoter), H3K9me3 (heterochromatin), H3K27ac (active enhancer), H3K27me3 (repression) across the genome. The differential histone marks were integrated with RNA-sequencing (RNA-seq) data derived from the same patients. Results: Overall, we identified 2,912 (7%) differential H3K4me3 peaks impacting 5 pathways, 23,339 (21%) differential H3K9ac peaks impacting 87 pathways, 33 (0.05%) differential H3K9me3 peaks, and 58,837 (42%) differential H3K27ac peaks impacting 50 pathways. There was increased H3K27ac (q-value = 0.002) and H3K9ac (q-value = 0.009) at RAS-MAPK loci. Univariate analysis of concordance between H3K9ac with RNA-seq data showed activation of cardiac hypertrophy signaling (z-score=4.5; -log [BH p-value] = 3.1), while H3K27me showed down-regulation of cardiac hypertrophy signaling (z-score=-2.4; -log [BH p-value] = 2.4). Multivariate integration of the ChIP-seq data with RNA-seq data identified 3,189 transcripts concordant with all five histone marks impacting 254 pathways, 18 (7%) of which are involved in cardiac hypertrophy. Conclusions: Myectomy tissue from patients with obstructive HCM shows enrichment of histone modifications at cardiac hypertrophy loci with activation via H3K27 and H3K9 acetylation and repression via H3K27 methylation. Further studies are needed to understand the balance and temporal relationship between these competing regulators in HCM pathobiology and the potential for epigenetic therapies.

Introduction: Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy. Approximately 40-50% of HCM is caused by pathogenic variants in genes encoding proteins of the cardiac sarcomere. Patients with sarcomeric HCM have earlier expression and more severe disease progression. Herein, we evaluated the proteomic differences between patients with sarcomeric HCM and genotype negative HCM. Methods: Mass-spectrometry was performed on cardiac tissue from genotyped patients with HCM obtained after surgical myectomy, followed by differential proteomics (log2fc ≥

Background: Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy. Currently, hypertrophy pathways responsible for HCM have not been fully elucidated. Their identification could serve as a nidus for the generation of novel therapeutics aimed at halting disease development or progression. Herein, we performed a comprehensive multi-omic characterization of hypertrophy pathways in HCM. Methods: Flash-frozen cardiac tissues were collected from genotyped HCM patients (n=97) undergoing surgical myectomy and tissue from 23 controls. RNA sequencing and mass spectrometry–enabled deep proteome and phosphoproteomic assessment were performed. Rigorous differential expression, gene set enrichment, and pathway analyses were performed to characterize HCM-mediated alterations with emphasis on hypertrophy pathways. Results: We identified transcriptional dysregulation with 1246 (8%) differentially expressed genes and elucidated downregulation of 10 hypertrophy pathways. Deep proteomic analysis identified 411 proteins (9%) that differed between HCM and controls with strong dysregulation of metabolic pathways. Seven hypertrophy pathways were upregulated with antagonistic upregulation of 5 of 10 hypertrophy pathways shown to be downregulated in the transcriptome. Most upregulated hypertrophy pathways encompassed the rat sarcoma-mitogen-activated protein kinase signaling cascade. Phosphoproteomic analysis demonstrated hyperphosphorylation of the rat sarcoma-mitogen-activated protein kinase system suggesting activation of this signaling cascade. There was a common transcriptomic and proteomic profile regardless of genotype. Conclusions: At time of surgical myectomy, the ventricular proteome, independent of genotype, reveals widespread upregulation and activation of hypertrophy pathways, mainly involving the rat sarcoma-mitogen-activated protein kinase signaling cascade. In addition, there is a counterregulatory transcriptional downregulation of the same pathways. Rat sarcoma-mitogen-activated protein kinase activation may serve a crucial role in hypertrophy observed in HCM.

Introduction: Hypertrophic cardiomyopathy (HCM) is one of the most common genetic heart diseases characterized by asymmetric left ventricular hypertrophy (LVH) and vast clinical and genetic heterogeneity. Several transcriptional studies in HCM heart tissues have shown severe alterations in gene expression. However, these earlier studies relied on microarray technology or were limited by small sample size or lack of genotype-specific analyses. Methods: We obtained flash-frozen cardiac tissue from patients with HCM undergoing surgical myectomy/septal reduction therapy for relief of left ventricular outflow tract obstruction. RNA-sequencing (RNAseq) was performed using paired-end index reading on 97 myectomy tissues (44 female [45%]; mean age at surgery of 47 ± 18 years) and 23 control tissues (10 female [43%]; mean age at death of 43 ± 12 years) to identify differentially expressed genes (DEGs) using edgeR and pathways in HCM using Ingenuity Pathway Analysis. Additionally, genome sequencing (GS) was performed and HCM patients were divided into genotype-based subgroups to compare the transcriptional profile across different genotypes. Results: Overall, 43% had a pathogenic, HCM-associated variant including 23% with MYBPC3-HCM and 15% with MYH7-HCM. Among the 15,634 genes expressed in the cardiac tissue,1,246 genes (8%) were upregulated and 738 genes (3%) were downregulated significantly in HCM vs. controls. Pathway analysis showed alterations in 126 pathways with downregulation of 44 pathways and upregulation of 1 pathway. There were notable transcriptional alterations of most canonical, pro-hypertrophic pathways, including statistically and biologically significant downregulation of five pathways [-log (BH p-value)≥ 1.3; Z-score≤ -2]: ERK/MAPK, p38 MAPK, ERK5, IL6, and NF-κB signaling. Far less DEGs were observed between genotypic subgroups, with only 37 DEGSs identified between sarcomere positive- and genotype negative-HCM. Conclusions: At the time of surgical myectomy, the ventricular myocardium’s transcriptome, independent of genotype, reveals widespread downregulation of pro-hypertrophic pathways. Whether these counter regulatory measures can be targeted therapeutically warrants further investigation.