Human Hypertrophic Cardiomyopathy Research Articles

Chronic isoprenaline/phenylephrine vs. exclusive isoprenaline stimulation in mice: critical contribution of alpha1-adrenoceptors to early cardiac stress responses

Hyperactivity of the sympathetic nervous system is a major driver of cardiac remodeling, exerting its effects through both α-, and β-adrenoceptors (α-, β-ARs). As the relative contribution of subtype α1-AR to cardiac stress responses remains poorly investigated, we subjected mice to either subcutaneous perfusion with the β-AR agonist isoprenaline (ISO, 30 mg/kg × day) or to a combination of ISO and the stable α1-AR agonist phenylephrine (ISO/PE, 30 mg/kg × day each). Telemetry analysis revealed similar hemodynamic responses under both ISO and ISO/PE treatment i.e., permanently increased heart rates and only transient decreases in mean blood pressure during the first 24 h. Echocardiography and single cell analysis after 1 week of exposure showed that ISO/PE-, but not ISO-treated animals established α1-AR-mediated inotropic responsiveness to acute adrenergic stimulation. Morphologically, additional PE perfusion limited concentric cardiomyocyte growth and enhanced cardiac collagen deposition during 7 days of treatment. Time-course analysis demonstrated a diverging development in transcriptional patterns at day 4 of treatment i.e., increased expression of selected marker genes Xirp2, Nppa, Tgfb1, Col1a1, Postn under chronic ISO/PE treatment which was either less pronounced or absent in the ISO group. Transcriptome analyses at day 4 via RNA sequencing demonstrated that additional PE treatment caused a marked upregulation of genes allocated to extracellular matrix and fiber organization along with a more pronounced downregulation of genes involved in metabolic processes, muscle adaptation and cardiac electrophysiology. Consistently, transcriptome changes under ISO/PE challenge more effectively recapitulated early transcriptional alterations in pressure overload-induced experimental heart failure and in human hypertrophic cardiomyopathy.

Contraction and electrophysiological abnormalities in myofilament mutation-positive and mutation-negative human HCM myocardium

Myofilament mutation-positive and mutation-negative hypertrophic cardiomyopathy (HCM) patients show different prognosis and arrhythmic burden that are likely related to the different pathogenesis. For instance, post-translational activation of CaMKII pathway is a specific biomarker of myofilament mutation-positive HCM while depressed sarcoplasmic reticulum calcium uptake is present in both groups. However, the functional consequences of a mutation-driven pathogenesis is still not completely understood.

Allele-specific silencing by RNAi of R92Q and R173W mutations in cardiac troponin T.

Autosomal dominant mutations in sarcomere proteins such as the cardiac troponin T (TNNT2) are the main genetic causes of human hypertrophic cardiomyopathy and dilated cardiomyopathy. Allele-specific silencing by RNA interference (ASP-RNAi) holds promise as a therapeutic strategy for downregulating a single mutant allele with minimal suppression of the corresponding wild-type allele. Here, we propose ASP-RNAi as a possible strategy to specifically knockdown mutant alleles coding for R92Q and R173W mutant TNNT2 proteins, identified in hypertrophic and dilated cardiomyopathy, respectively. Different siRNAs were designed and validated by luciferase reporter assay and following analysis in HEK293T cells expressing either the wild-type or mutant TNNT2 alleles. This study is the first exploration of ASP-RNAi on TNNT2-R173W and TNNT2-R92Q mutations in vitro and gives a base for further application of allele silencing as a therapeutic treatment for TNNT2-mutation-associated cardiomyopathies.

Single Nucleus RNA-sequencing Reveals Altered Intercellular Communication and Dendritic Cell Activation in Nonobstructive Hypertrophic Cardiomyopathy.

End stage, nonobstructive hypertrophic cardiomyopathy (HCM) is an intractable condition with no disease-specific therapies. To gain insights into the pathogenesis of nonobstructive HCM, we performed single nucleus RNA-sequencing (snRNA-seq) on human HCM hearts explanted at the time of cardiac transplantation and organ donor hearts serving as controls. Differential gene expression analysis revealed 64 differentially expressed genes linked to specific cell types and molecular functions. Analysis of ligand-receptor pair gene expression to delineate potential intercellular communication revealed significant reductions in expressed ligand-receptor pairs likely affecting the extracellular matrix, growth factor binding, peptidase regulator activity, platelet-derived growth factor binding and protease binding in the HCM tissue. Changes in Integrin-β1 receptor expression were responsible for many observed changes related to extracellular matrix interactions, by increasing in dendritic, smooth muscle and pericyte cells while decreasing in endothelial and fibroblast cells, suggesting potential mechanisms for fibrosis and microvascular disease in HCM and a potential role for dendritic cells. In contrast, there was an increase in ligand-receptor pair expression associated with adenylate cyclase binding, calcium channel molecular functions, channel inhibitor activity, ion channel inhibitor activity, phosphatase activator activity, protein kinase activator activity and titin binding, suggesting important shifts in various signaling cascades in nonobstructive, end stage HCM.

Stress activated signalling impaired protein quality control pathways in human hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a complex myocardial disorder with no well-established disease-modifying therapy so far. Our study aimed to investigate how autophagy, oxidative stress, inflammation, stress signalling pathways, and apoptosis are hallmark of HCM and their contribution to the cardiac dysfunction. Demembranated cardiomyocytes from patients with HCM display increased titin-based stiffness (Fpassive), which was corrected upon antioxidant treatment. Titin as a main determinant of Fpassive was S-glutathionylated and highly ubiquitinated in HCM patients. This was associated with a shift in the balance of reduced and oxidized forms of glutathione (GSH and GSSG, respectively). Both heat shock proteins (HSP27 and α-ß crystalline) were upregulated and S-glutathionylated in HCM. Administration of HSPs in vitro significantly reduced HCM cardiomyocyte stiffness. High levels of the phosphorylated monomeric superoxide anion-generating endothelial nitric oxide synthase (eNOS), decreased nitric oxide (NO) bioavailability, decreased soluble guanylyl cyclase (sGC) activity, and high levels of 3-nitrotyrosine were observed in HCM. Many regulators of signal transduction pathways that are involved in autophagy, apoptosis, cardiac contractility, and growth including the mitogen-activated protein kinase (MAPK), protein kinase B (AKT), glycogen synthase kinase 3ß (GSK-3ß), mammalian target of rapamycin (mTOR), forkhead box O transcription factor (FOXO), c-Jun N-terminal protein kinase (JNK), and extracellular-signal-regulated kinase (ERK1/2) were modified in HCM. The apoptotic factors cathepsin, procaspase 3, procaspase 9 and caspase 12, but not caspase 9, were elevated in HCM hearts and associated with increased proinflammatory cytokines (Interleukin 6 (IL-6), interleukin 18 (IL-18), intercellular cell adhesion molecule-1 (ICAM1), vascular cell adhesion molecule-1 (VCAM1), the Toll-like receptors 2 (TLR2) and the Toll-like receptors 4 (TLR4)) and oxidative stress (3-nitrotyrosine and hydrogen peroxide (H2O2)). Here we reveal stress signalling and impaired PQS as potential mechanisms underlying the HCM phenotype. Our data suggest that reducing oxidative stress can be a viable therapeutic approach to attenuating the severity of cardiac dysfunction in heart failure and potentially in HCM and prevent its progression.

Bioinformatics and Immune Infiltration Analyses Reveal the Key Pathway and Immune Cells in the Pathogenesis of Hypertrophic Cardiomyopathy.

Objective: This study was designed to identify the key pathway and immune cells for hypertrophic cardiomyopathy (HCM) via bioinformatics analyses of public datasets and evaluate the significance of immune infiltration in the pathogenesis of HCM.Methods: Expressional profiling from two public datasets (GSE36961 and GSE141910) of human HCM and healthy control cardiac tissues was obtained from the GEO database. After data preprocessing, differentially expressed genes (DEGs) were then screened between HCM and healthy control cardiac tissues in parallel. Gene Ontology, pathway functional enrichment, and gene set enrichment analysis were performed using DAVID and GSEA application. The compositional patterns of immune and stromal cells in HCM and control cardiac tissues were estimated based on the merged data using xCell. Protein–protein interaction (PPI) network and module analyses were constructed by STRING and Cytoscape applications. Gender-based expressional differences analyses were also conducted to explore gender differences in HCM. GSE130036 and clinical samples were used for verification analyses.Results: A total of 310 DEGs were identified. Upregulated DEGs were mainly enriched in “adhesion” and “apoptotic process” in the biological process. As for the downregulated DEGs, “inflammatory response,” “innate immune response,” “phagosome,” and “JAK-STAT signaling pathway” were highly enriched. Immune infiltration analyses suggested that the scores of macrophages, monocytes, DC, Th1, Treg, and plasma cells in the HCM group were significantly decreased, while CD8+ T cells, basophils, fibroblasts, and platelets were significantly enriched. Module analyses revealed that STAT3, as the hub genes in HCM together with LYVE1+CD163+ macrophages, may play a key role in the pathogenesis of HCM while there were no obvious gender differences in the HCM samples from selected datasets. Verification analyses performed on GSE130036 and clinical samples showed a strong positive correlation (Spearman correlation = 0.7646) and a good co-localization relationship between LYVE1 and CD163, suggesting the potential function of LYVE1+CD163+ macrophages in maintaining the homeostasis of cardiac tissue.Conclusion: STAT3-related pathway and CD163+LYVE1+ macrophages were identified as the potential key pathway and immune cells in HCM and may serve as interesting targets for further in-depth research.

Differences in molecular phenotype in mouse and human hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by phenotypic heterogeneity. We investigated the molecular basis of the cardiac phenotype in two mouse models at established disease stage (mouse-HCM), and human myectomy tissue (human-HCM). We analyzed the transcriptome in 2 mouse models with non-obstructive HCM (R403Q-MyHC, R92W-TnT)/littermate-control hearts at 24 weeks of age, and in myectomy tissue of patients with obstructive HCM/control hearts (GSE36961, GSE36946). Additionally, we examined myocyte redox, cardiac mitochondrial DNA copy number (mtDNA-CN), mt-respiration, mt-ROS generation/scavenging and mt-Ca2+ handling in mice. We identified distinct allele-specific gene expression in mouse-HCM, and marked differences between mouse-HCM and human-HCM. Only two genes (CASQ1, GPT1) were similarly dysregulated in both mutant mice and human-HCM. No signaling pathway or transcription factor was predicted to be similarly dysregulated (by Ingenuity Pathway Analysis) in both mutant mice and human-HCM. Losartan was a predicted therapy only in TnT-mutant mice. KEGG pathway analysis revealed enrichment for several metabolic pathways, but only pyruvate metabolism was enriched in both mutant mice and human-HCM. Both mutant mouse myocytes demonstrated evidence of an oxidized redox environment. Mitochondrial complex I RCR was lower in both mutant mice compared to controls. MyHC-mutant mice had similar mtDNA-CN and mt-Ca2+ handling, but TnT-mutant mice exhibited lower mtDNA-CN and impaired mt-Ca2+ handling, compared to littermate-controls. Molecular profiling reveals differences in gene expression, transcriptional regulation, intracellular signaling and mt-number/function in 2 mouse models at established disease stage. Further studies are needed to confirm differences in gene expression between mouse and human-HCM, and to examine whether cardiac phenotype, genotype and/or species differences underlie the divergence in molecular profiles.

Translational investigation of electrophysiology in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) patients are at increased risk of ventricular arrhythmias and sudden cardiac death, which can occur even in the absence of structural changes of the heart. HCM mouse models suggest mutations in myofilament components to affect Ca2+ homeostasis and thereby favor arrhythmia development. Additionally, some of them show indications of pro-arrhythmic changes in cardiac electrophysiology. In this study, we explored arrhythmia mechanisms in mice carrying a HCM mutation in Mybpc3 (Mybpc3-KI) and tested the translatability of our findings in human engineered heart tissues (EHTs) derived from CRISPR/Cas9-generated homozygous MYBPC3 mutant (MYBPC3hom) in induced pluripotent stem cells (iPSC) and to left ventricular septum samples obtained from HCM patients.We observed higher arrhythmia susceptibility in contractility measurements of field-stimulated intact cardiomyocytes and ventricular muscle strips as well as in electromyogram recordings of Langendorff-perfused hearts from adult Mybpc3-KI mice than in wild-type (WT) controls. The latter only occurred in homozygous (Hom-KI) but not in heterozygous (Het-KI) mouse hearts. Both Het- and Hom-KI are known to display pro-arrhythmic increased Ca2+ myofilament sensitivity as a direct consequence of the mutation. In the electrophysiological characterization of the model, we observed smaller repolarizing K+ currents in single cell patch clamp, longer ventricular action potentials in sharp microelectrode recordings and longer ventricular refractory periods in Langendorff-perfused hearts in Hom-KI, but not Het-KI. Interestingly, reduced K+ channel subunit transcript levels and prolonged action potentials were already detectable in newborn, pre-hypertrophic Hom-KI mice. Human iPSC-derived MYBPC3hom EHTs, which genetically mimicked the Hom-KI mice, did exhibit lower mutant mRNA and protein levels, lower force, beating frequency and relaxation time, but no significant alteration of the force-Ca2+ relation in skinned EHTs. Furthermore, MYBPC3hom EHTs did show higher spontaneous arrhythmic behavior, whereas action potentials measured by sharp microelectrode did not differ to isogenic controls. Action potentials measured in septal myectomy samples did not differ between patients with HCM and patients with aortic stenosis, except for the only sample with a MYBPC3 mutation.The data demonstrate that increased myofilament Ca2+ sensitivity is not sufficient to induce arrhythmias in the Mybpc3-KI mouse model and suggest that reduced K+ currents can be a pro-arrhythmic trigger in Hom-KI mice, probably already in early disease stages. However, neither data from EHTs nor from left ventricular samples indicate relevant reduction of K+ currents in human HCM. Therefore, our study highlights the species difference between mouse and human and emphasizes the importance of research in human samples and human-like models.

173 Investigating the structural effects of MYBPC3 variants causative for feline and human hypertrophic cardiomyopathy (HCM)

173 Investigating the structural effects of MYBPC3 variants causative for feline and human hypertrophic cardiomyopathy (HCM)

Maturation of Pluripotent Stem Cell-Derived Cardiomyocytes Enables Modeling of Human Hypertrophic Cardiomyopathy.

SummaryHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for biomedical research. However, they are immature, which is a barrier to modeling adult-onset cardiovascular disease. Here, we sought to develop a simple method that could drive cultured hiPSC-CMs toward maturity across a number of phenotypes, with the aim of utilizing mature hiPSC-CMs to model human cardiovascular disease. hiPSC-CMs were cultured in fatty acid-based medium and plated on micropatterned surfaces. These cells display many characteristics of adult human cardiomyocytes, including elongated cell morphology, sarcomeric maturity, and increased myofibril contractile force. In addition, mature hiPSC-CMs develop pathological hypertrophy, with associated myofibril relaxation defects, in response to either a pro-hypertrophic agent or genetic mutations. The more mature hiPSC-CMs produced by these methods could serve as a useful in vitro platform for characterizing cardiovascular disease.

Altered Intercellular Communication and Extracellular Matrix Signaling as a Disease Mechanism in Human Hypertrophic Cardiomyopathy

Altered Intercellular Communication and Extracellular Matrix Signaling as a Disease Mechanism in Human Hypertrophic Cardiomyopathy

The role and the signal pathways of Yes-associated protein 2 in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a heterogeneous myocardial disease characterized by myocardial hypertrophy, myocardial mechanical and electrical activity obstacles. This study aimed to explore the relationship between YAP2 (Yes-associated protein 2) and HCM and clarify a signaling path about the pathogenesis of HCM. Our study confirms that YAP2 can promote myocardial cell hypertrophy at the molecular level (myocardial lineage cell H9C2), organ level (clinical specimens of human HCM), and an animal model (a mouse model of HCM with cardiac-specific transgenic and knockout YAP2). The detailed molecular mechanisms linking YAP2 to cardiomyocyte hypertrophy and HCM were investigated. This study proved that YAP2, as the final reaction factor in Hippo pathways, influences Akt1 activity to affect the downstream genes, which participate in the formation of HCM by promoting myocardial cell proliferation and cardiac hypertrophy.

T-tubule remodeling in human hypertrophic cardiomyopathy

The highly organized transverse T-tubule membrane system represents the ultrastructural substrate for excitation–contraction coupling in ventricular myocytes. While the architecture and function of T-tubules have been well described in animal models, there is limited morpho-functional data on T-tubules in human myocardium. Hypertrophic cardiomyopathy (HCM) is a primary disease of the heart muscle, characterized by different clinical presentations at the various stages of its progression. Most HCM patients, indeed, show a compensated hypertrophic disease (“non-failing hypertrophic phase”), with preserved left ventricular function, and only a small subset of individuals evolves into heart failure (“end stage HCM”). In terms of T-tubule remodeling, the “end-stage” disease does not differ from other forms of heart failure. In this review we aim to recapitulate the main structural features of T-tubules during the “non-failing hypertrophic stage” of human HCM by revisiting data obtained from human myectomy samples. Moreover, by comparing pathological changes observed in myectomy samples with those introduced by acute (experimentally induced) detubulation, we discuss the role of T-tubular disruption as a part of the complex excitation–contraction coupling remodeling process that occurs during disease progression. Lastly, we highlight how T-tubule morpho-functional changes may be related to patient genotype and we discuss the possibility of a primitive remodeling of the T-tubule system in rare HCM forms associated with genes coding for proteins implicated in T-tubule structural integrity, formation and maintenance.

Abstract 14427: Differences in Molecular Phenotype in Mouse and Human Hypertrophic Cardiomyopathy Suggest Need for Precision Medicine Approach to Treatment Based on Human Studies

Background: Mouse studies at early stage of hypertrophic cardiomyopathy (HCM) indicate allele-specific differences in cardiac gene expression and mitochondrial function. But data at established disease stage in mouse and human HCM are lacking. We hypothesized that 1) allele-specific differences persist at established stage, and 2) mouse and human HCM have distinct molecular biosignatures. Methods: We analyzed the transcriptome (mRNA, miRNA) in 2 HCM mouse models (R92W-TnT, R403Q-MyHC)/littermate controls at 24weeks of age and in human myectomy samples/healthy-control hearts ( GSE36961, GSE36946 ). We examined myocyte redox, mitochondrial DNA copy number (mtDNA-CN), respiration, ROSgeneration/scavenging and Ca 2+ handling in mutant/littermate-control mice . Results: Analysis of mRNA/miRNA expression and Ingenuity Pathway Analysis (IPA) revealed distinct allele-specific gene expression in mouse HCM and marked differences from human HCM. Only CASQ1 and GPT1 were similarly regulated in both mouse HCM/human HCM. KEGG analysis revealed enrichment of several metabolic pathways, but only pyruvate metabolism was enriched in both mouse HCM/human HCM. IPA predicted upregulation of 2 pathways (inflammasome, type 2 diabetes signaling) in MyHC mutants, and upregulation of 18 pathways (including STAT3, ILK, Ca 2+ signaling) in TnT mutants; the anti-hypertrophic/anti-fibrotic LXR/RXR pathway was the most upregulated in human HCM. Losartan was a predicted therapy only in TnT mutants. Myocytes of both mutant mice exhibited an oxidized redox environment. Mitochondrial complex I respiration was lower in both mutants compared to controls. MyHC mutants had similar mtDNA-CN and mitochondrial Ca 2+ handling, but TnT mutants demonstrated lower mtDNA-CN and impaired mitochondrial Ca 2+ handling, compared to respective controls. Conclusions: Molecular profiling reveals allele-specific differences in mRNA/miRNA expression, intracellular signaling and mt-function/number in mouse HCM at established disease stage. Transcriptional differences between mouse and human HCM highlight the need for precision medicine approaches based on human studies.

Abstract 16683: Profiling Cardiac Inflammation and Molecular Changes During the Transition of Hypertrophic to Dilated Cardiomyopathy

Introduction: Hypertrophic cardiomyopathy (HCM) is a disease caused primarily by sarcomere gene mutations [like myosin-binding protein C3 ( MYBPC3 )], with several distinct phenotypes. Tissue-level inflammation has been reported in human HCM, tissue-specific neutrophil subsets, and their dysregulation cause inflammatory myopathies, and neutrophil extracellular traps (NETs) are associated with cardiac fibrosis in aged rodents. However, the mechanism(s) responsible for the broad phenotypic variability and changes observed over time transitioning from HCM to dilated cardiomyopathy (DCM) has not yet been elucidated. Objective: To determine the cardiac inflammatory signature of HCM and transition from HCM to DCM and its relationship to fibrosis and ventricular remodeling in an established MYBPC3 ( cMyBP-C t/t ) mouse model. Method and Results: Mixed-gender cMyBP-C +/+ (WT) and mutant mice ( cMyBP-C t/t ) underwent echocardiography and were euthanized at one, two and three months of age (n=8) and blood and hearts were harvested. Echocardiography and histopathology examination confirmed significant heart dysfunction and tissue remodeling in cMyBP-C t/t mice showing cardiac hypertrophy at one month of age (Left ventricular (LV) posterior wall systole 1.20 ±0.39 vs 1.51±0.49 and LV posterior wall diastole 0.96 ±0.22 vs 1.38 ±0.36 mm; * p<0.05 ), and DCM phenotype (LV internal diameter systole 2.40 ±0.41 vs 3.67 ±0.25 and LV internal diameter diastole 3.44 ±0.29 vs 4.30 ±0.39 mm; * p<0.05 ) along with myofibrillar loss and increased cardiac fibrosis at three months. Interestingly, LV dysfunction and hypertrophy resulted in a time-dependent increase in the activation of coagulation, as measured by plasma microvesicle tissue factor activity, thrombin antithrombin, and D-dimer. In silico analysis of RNA-Seq data from 3-months old cMyBP-C t/t mice heart revealed key genes and pathways associated with HCM, DCM, tissue remodeling, renin-angiotensin-aldosterone signaling, fibrosis, apoptosis, NETosis, and inflammation. Conclusions: The transition from HCM to a DCM phenotype involves proinflammatory and profibrotic signaling. Therapies directed at tissue-specific inflammation and NETs may be a novel and impactful strategy for HCM.

Abstract 17402: Single Nuclei RNA-sequencing of Human Hypertrophic Cardiomyopathy Myectomy Samples Reveals Common Novel Mechanisms of Pathogenesis and Potential Therapeutic Targets Regardless of Genotype

Introduction: Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disease, often resulting in left ventricular outflow tract obstruction, relieved by surgical myectomy. Current treatments are largely palliative and do not target the root causes. Understanding the molecular drivers of the disease could lead to alternative treatment strategies through identification of novel therapeutic targets. Methods: We performed single nuclei RNA-sequencing (snRNA-seq) on thousands of nuclei from 9 patient myectomy samples and septal tissue from 4 unused donor hearts selected randomly without regard to genotype to identify the cell populations and determine the gene expression patterns in individual cells. Each sample was processed individually using Seurat v3 for quality control and normalization. Next, all 13 samples were integrated into a combined dataset for clustering and differential gene expression analysis to identify markers specific to each cluster and to assign cell identities. Results: Our results revealed several clusters of cardiomyocytes with differences in sarcomeric and metabolic gene expression. Several fibroblast populations were also observed. Numerous genes were differentially expressed between the HCM and normal samples. For example, RARRES1 expression was observed in many of the fibroblast populations in the normal samples, but was absent in the HCM samples. RARRES1 is involved in regulating fatty acid metabolism and autophagy, both of which are altered in HCM. Additionally, expression of PLA2G2A was absent in the HCM samples but was present in almost every cell type in the normal controls. PLA2G2A is involved in suppression of RTK mediated hypertrophic signaling, impacts lipid signaling, and has tumor suppressor properties. Thus, both RARRES1 and PLA2G2A may represent novel targets in HCM. Conclusions: This approach reveals novel potential therapeutic targets within common final HCM pathological pathways independent of genotype that have the potential to guide development of alternative treatment strategies. Further analysis of larger datasets using this approach will likely identify even more common pathway targets and identify additional common mechanisms in the pathogenesis of obstructive HCM.

MYBPC3 Haplotype Linked to Hypertrophic Cardiomyopathy in Rhesus Macaques (Macaca mulatta).

In humans, abnormal thickening of the left ventricle of the heart clinically defines hypertrophic cardiomyopathy (HCM), a common inherited cardiovascular disorder that can precede a sudden cardiac death event. The wide range of clinical presentations in HCM obscures genetic variants that may influence an individual's susceptibility to sudden cardiac death. Although exon sequencing of major sarcomere genes can be used to detect high-impact causal mutations, this strategy is successful in only half of patient cases. The incidence of left ventricular hypertrophy (LVH) in a managed research colony of rhesus macaques provides an excellent comparative model in which to explore the genomic etiology of severe HCM and sudden cardiac death. Because no rhesus HCM-associated mutations have been reported, we used a next-generation genotyping assay that targets 7 sarcomeric rhesus genes within 63 genomic sites that are orthologous to human genomic regions known to harbor HCM disease variants. Amplicon sequencing was performed on 52 macaques with confirmed LVH and 42 unrelated, unaffected animals representing both the Indian and Chinese rhesus macaque subspecies. Bias-reduced logistic regression uncovered a risk haplotype in the rhesus MYBPC3 gene, which is frequently disrupted in both human and feline HCM; this haplotype implicates an intronic variant strongly associated with disease in either homozygous or carrier form. Our results highlight that leveraging evolutionary genomic data provides a unique, practical strategy for minimizing population bias in complex disease studies.

Abstract 415: Asporin Contributes to Reactive Interstitial Cardiac Fibrosis by Regulating Cardiac Fibroblast Activation

Cardiac fibrosis critically contributes to heart failure progression. Depending on the pathological insult, cardiac fibrosis either replaces necrotic cardiomyocytes or is reactive to cardiac fibroblast (CF) activation. The extracellular matrix (ECM) consists of various proteins and the role of fibrillar collagen has been well studied. However, the role of non-fibrillar ECM proteins in cardiac fibrosis is less clear. To explore the role of ECM in reactive cardiac fibrosis, we performed bioinformatic analysis on online available microarray GEO datasets from hearts of human hypertrophic cardiomyopathy patients and two mouse models of transverse aortic constriction and Angiotensin II (AngII) infusion. We found that 27 differentially expressed genes were common between the three datasets. Among these genes was the small leucine-rich proteoglycan Asporin (ASPN). ASPN was previously shown to be upregulated in the ECM of replacement fibrosis in porcine ischemia/reperfusion injury. However, not much is known about the role of ASPN in reactive interstitial fibrosis. We show that cardiac ASPN expression is enhanced in mice after short- and long-term AngII infusion compared to saline infusion. In resident CF isolated from adult mice, ASPN expression is upregulated by both AngII and TGF-β stimulation. Here, ASPN expression correlates with a gene signature of activated CFs including periostin ( postn ), α-smooth muscle actin ( acta2 ) and collagens I and III ( col1a1, col3a1 ), and with functional characteristics of activated CFs including proliferation, migration and collagen production. Modulating ASPN via siRNA in mouse resident CFs inhibits postn, acta2, col1a1 and col3a1 expression and total collagen production, indicating repressed CF activation upon ASPN knockdown. Taken together, ASPN may be an attractive novel target against reactive interstitial fibrosis.

Silencing of MYH7 ameliorates disease phenotypes in human iPSC-cardiomyocytes.

Allele-specific RNA silencing has been shown to be an effective therapeutic treatment in a number of diseases, including neurodegenerative disorders. Studies of allele-specific silencing in hypertrophic cardiomyopathy (HCM) to date have focused on mouse models of disease. We here examine allele-specific silencing in a human-cell model of HCM. We investigate two methods of silencing, short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) silencing, using a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model. We used cellular micropatterning devices with traction force microscopy and automated video analysis to examine each strategy's effects on contractile defects underlying disease. We find that shRNA silencing ameliorates contractile phenotypes of disease, reducing disease-associated increases in cardiomyocyte velocity, force, and power. We find that ASO silencing, while better able to target and knockdown a specific disease-associated allele, showed more modest improvements in contractile phenotypes. These findings are the first exploration of allele-specific silencing in a human HCM model and provide a foundation for further exploration of silencing as a therapeutic treatment for MYH7-mutation-associated cardiomyopathy.

Functional characterization of human myosin-binding protein C3 variants associated with hypertrophic cardiomyopathy reveals exon-specific cardiac phenotypes in zebrafish model.

Myosin-binding protein C 3 (MYBPC3) variants are the most common cause of hypertrophic cardiomyopathy (HCM). HCM is a complex cardiac disorder due to its significant genetic and clinical heterogeneity. MYBPC3 variants genotype-phenotype associations remain poorly understood. We investigated the impact of two novel human MYBPC3 splice-site variants: V1: c.654+2_654+4dupTGG targeting exon 5 using morpholino MOe5i5; and V2: c.772+1G>A targeting exon 6 using MOe6i6; located within C1 domain of cMyBP-C protein, known to be critical in regulating sarcomere structure and contractility. Zebrafish MOe5i5 and MOe6i6 morphants recapitulated typical characteristics of human HCM with cardiac phenotypes of varying severity, including reduced cardiomyocyte count, thickened ventricular myocardial wall, a drastic reduction in heart rate, stroke volume, and cardiac output. Analysis of all cardiac morphological and functional parameters demonstrated that V2 cardiac phenotype was more severe than V1. Coinjection with synthetic human MYBPC3 messenger RNA (mRNA) partially rescued disparate cardiac phenotypes in each zebrafish morphant. While human MYBPC3 mRNA partially restored the decreased heart rate in V1 morphants and displayed increased percentages of ejection fraction, fractional shortening, and area change, it failed to revert the V1 ventricular myocardial thickness. These results suggest a possible V1 impact on cardiac contractility. In contrast, attempts to rescue V2 morphants only restored the ventricular myocardial wall hypertrophy phenotype but had no significant effect on impaired heart rate, suggesting a potential V2 impact on the cardiac structure. Our study provides evidence of an association between MYBPC3 exon-specific cardiac phenotypes in the zebrafish model providing important insights into how these genetic variants contribute to HCM disease.