Inherited Heart Disease Research Articles

Abstract 4117767: Interplay Between ST2 Gene Downregulation and Inflammatory Processes in Hypertrophic Cardiomyopathy

Introduction: Hypertrophic Cardiomyopathy (HCM) is an inherited heart disease and the pathogenesis of HCM involves genetic mutations, hemodynamic stress, and metabolic factors, with myocardial fibrosis playing a crucial role in severe clinical events. IL-33/ST2 signaling pathway known for its roles in immune response and tissue repair, participates in cardiac protection and anti-cardiac fibrosis in heart failure. The role of ST2 in HCM remains unclear, and IL-33/ST2 pathway and broader inflammatory responses may be critical in HCM. Hypothesis: ST2 gene downregulation and dysregulation of inflammatory pathways represent a novel mechanism in HCM. Methods: We re-analyzed the raw whole transcriptome sequencing data using a comprehensive bioinformatics pipeline from 9 GEO datasets. After removing low-quality samples, myocardial tissue samples from 109 HCM patients and 210 non-HCM controls were included. HTSeq-count was used to obtain the read count of each gene. Differential expression analyses were conducted using DESeq2. Spearman correlation analysis was performed for ST2 with all other protein-coding genes. Gene set enrichment analysis (GSEA) was performed to explore the biological significance of ST2 related genes. CIBERSORTx was used to estimate the immune cell composition of heart tissues. Additionally, network analyses and statistical correlations were conducted. Results: Our analysis identified significant downregulation of the ST2 gene in HCM patients compared to controls (log2FC = -5.0, p = 2.7E-147). In total, 5,180 upregulated and 741 downregulated genes were detected. Additionally, 208 genes were significantly correlated with the expression of ST2. GSEA based on the correlation coefficients revealed a significant increase in immune response, inflammation, and IFN-γ pathway activity, along with a decrease in the expression of genes related to sarcomeric function, cardiac morphogenesis, and metabolism in HCM. Notably, the significant inverse correlation between ST2 expression and regulatory T cells (r = -0.34) and a positive correlation with neutrophils (r = 0.39) suggest that ST2 may modulate immune cell populations in HCM. Pathway analysis indicated ST2's central role in networks involving inflammatory and fibrotic responses. Conclusion: The interplay between ST2 and inflammatory pathways may drive myocardial remodeling and fibrosis in HCM. This study paves a new way for investigating pathogenic mechanisms and therapeutic strategies for HCM.

Investigating Inherited Heart Diseases Using Human Induced Pluripotent Stem Cell-Based Models

Inherited heart diseases (IHDs) are caused by genetic mutations that disrupt the physiological structure and function of the heart. Understanding the mechanisms behind these diseases is crucial for developing personalised interventions in cardiovascular medicine. Development of induced pluripotent stem cells, which can then be differentiated to any nucleated adult cell type, has enabled the creation of personalised single-cell and multicellular models, providing unprecedented insights into the pathophysiology of IHDs. This review provides a comprehensive overview of recent advancements in human iPSC models used to dissect the molecular and genetic underpinnings of common IHDs. We examine multicellular models and tissue engineering approaches, such as cardiac organoids, engineered heart tissue, and multicellular co-culture systems, which simulate complex intercellular interactions within heart tissue. Recent advancements in stem cell models offer a more physiologically relevant platform to study disease mechanisms, enabling researchers to observe cellular interactions, study disease progression, and identify therapeutic strategies. By leveraging these innovative models, we can gain deeper insights into the molecular and cellular mechanisms underlying IHDs, ultimately paving the way for more effective diagnostic and therapeutic strategies.

Novel Frameshift Variant of the MYBPC3 Gene Associated with Hypertrophic Cardiomyopathy Significantly Decreases the Level of This Gene’s Transcript in the Myocardium

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease with a prevalence of 1 : 200–1 : 500 in the general population. The majority of HCM-linked pathogenic (or likely pathogenic) variants is located in eight genes encoding proteins of sarcomere, the main contractile unit of cardiomyocytes; one of these genes, MYBPC3, is the most commonly affected and usually associated with the more benign clinical course of the disease compared to other HCM-related genes. Here, we describe a novel frame shift variant NM_000256.3:c.2781_2782insCACA of the MYBPC3 gene that causes familial HCM in the heterozygous state. The proband had a progressive heart failure despite the surgical removal of left ventricular tract obstruction. Evaluation of levels of transcripts produced from the mutant allele and wild-type allele of the MYBPC3 gene in proband myocardial tissue and comparison of their total levels with ones in the control samples from patients without HCM showed a significant allele-specific reduction of mutant transcript levels. Our results expand the spectrum of known genetic variants with a proven role in the development of HCM.

Austrian consensus statement on the diagnosis and management of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease that is characterized by left ventricular hypertrophy unexplained by secondary causes. Based on international epidemiological data, around 20,000-40,000 patients are expected to be affected in Austria. Due to the wide variety of clinical and morphological manifestations the diagnosis can be difficult and the disease therefore often goes unrecognized. HCM is associated with asubstantial reduction in quality of life and can lead to sudden cardiac death, especially in younger patients. Early and correct diagnosis, including genetic testing, is essential for comprehensive counselling of patients and their families and for effective treatment. The latter is especially true as an effective treatment of outflow tract obstruction has recently become available in the form of afirst in class cardiac myosin ATPase inhibitor, as anoninvasive alternative to established septal reduction therapies. The aim of this Austrian consensus statement is to summarize the recommendations of international guidelines with respect to the genetic background, pathophysiology, diagnostics and management in the context of the Austrian healthcare system and resources, and to present them in easy to understand algorithms.

Functional control of myosin motors in the cardiac cycle.

Contraction of the heart is driven by cyclical interactions between myosin and actin filaments powered by ATP hydrolysis. The modular structure of heart muscle and the organ-level synchrony of the heartbeat ensure tight reciprocal coupling between this myosin ATPase cycle and the macroscopic cardiac cycle. The myosin motors respond to the cyclical activation of the actin and myosin filaments to drive the pressure changes that control the inflow and outflow valves of the heart chambers. Opening and closing of the valves in turn switches the myosin motors between roughly isometric and roughly isotonic contraction modes. Peak filament stress in the heart is much smaller than in fully activated skeletal muscle, although the myosin filaments in the two muscle types have the same number of myosin motors. Calculations indicate that only ~5% of the myosin motors in the heart are needed to generate peak systolic pressure, although many more motors are needed to drive ejection. Tight regulation of the number of active motors is essential for the efficient functioning of the healthy heart - this control is commonly disrupted by gene variants associated with inherited heart disease, and its restoration might be a useful end point in the development of novel therapies.

No beneficial use of the wearable cardioverter defibrillator among patients suffering from inherited and congenital heart disease: data from a European multicenter registry.

Data on the use of the wearable cardioverter defibrillator in patients suffering from inherited and congenital heart disease are limited. Consequently, evidence for guideline recommendations in this patient population is lacking. In total 1,675 patients were included in a multicenter registry of eight European centers. In the present cohort, we included 18 patients suffering from congenital and inherited heart disease. Nine patients (50%) were male with a mean age of 41.3 ± 16.4 years. Four patients suffered from hypertrophic cardiomyopathy (HCM), four patients suffered from non-compaction cardiomyopathy (NCCM), two patients were diagnosed with arrhythmogenic right ventricular cardiomyopathy (ARVC) and one patient suffered from muscular dystrophy of the limb-girdle type with cardiac involvement, secondary cardiomyopathy. Three patients presented with Brugada syndrome (BrS). One patient suffered from long-QT syndrome type 1 (LQTS1). Furthermore, two patients had congenital heart defects and one patient suffered from cardiac sarcoidosis (CS). There were no appropriate/inappropriate shocks with the WCD in this cohort. One patient had recurrent self-limiting sustained ventricular tachycardia during the wear time, but actively inhibited a shock and was hospitalized. The compliance rate in this cohort was 77.8% with a mean wear time of 45.3 ± 26.9 days with a mean follow-up time of 570 ± 734 days. 55.6% (10/18) of the patients received an ICD after WCD wear time. This retrospective study of patients with inherited and congenital heart disease shows that WCD use is not beneficial in the majority of patients with inherited and congenital heart disease.

Results of comprehensive genetic testing in patients presenting to a multidisciplinary inherited heart disease clinic in India

ObjectivesThis study aims to analyze the results of comprehensive genetic testing in patients presenting to a dedicated multidisciplinary inherited heart disease clinic in India. MethodsAll patients presenting to our clinic from August 2017 to October 2023 with a suspected inherited heart disease and consenting for genetic testing were included. The probands were grouped into familial cardiomyopathies namely hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmogenic cardiomyopathy (ACM) and peripartum cardiomyopathy (PPCM), channelopathies namely congenital long QT syndrome (LQTS) and Brugada syndrome (BrS), and heritable connective tissue disorder namely Marfan Syndrome (MFS). Next generation sequencing (NGS) was used, and pre-test and post-test counseling were provided to probands and cascade screening offered to relatives. ResultsMean age of the subjects (n = 77; 48 probands, 29 relatives) was 43 ± 18 years, 68 % male and 44 % symptomatic, with 36 HCM, 3 DCM, 3 ACM, 1 PPCM, 3 LQTS, 1 BrS and 1 MFS probands. The diagnostic yield of NGS-based genetic testing was 31 %; variants of uncertain significance (VUS) were identified in 54 %; and 15 % were genotype-negative. Twenty-nine relatives from 18 families with HCM (n = 12), DCM (n = 3), ACM (n = 2) and MFS (n = 1) underwent genetic testing. The genotype positive probands/relatives and VUS carriers with strong disease phenotype and/or high risk variant were advised periodic follow-up; the remaining probands/relatives were discharged from further clinical surveillance. ConclusionsGenetic testing guides treatment and follow-up of patients with inherited heart diseases and should be carried out in dedicated multidisciplinary clinics with expertise for counseling and cascade screening of family members.

Early biomarkers of disease in hypertrophic cardiomyopathy: does telomere length matter?

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Biomedical Research Institute of Murcia (IMIB-Arrixaca) Introduction Hypertrophic cardiomyopathy (HCM) is the leading cause of sudden death in people under 35 years of age. (1) The variability of the phenotype together with its incomplete penetrance has been an obstacle to a complete understanding of the clinical spectrum and consequences of the disease. In recent years, there has been increasing interest in telomeric shortening and several authors have linked it to age-related diseases and HCM. (2, 3) Our aim was to study whether telomere length could affect the age at presentation and the severity of the HCM phenotype in genetic carriers. Methods Measurement of telomere length in circulating leukocytes (LTLc) was performed in 96 patients carrying a mutation in MYBPC3 causing HCM [58 affected (24 severely affected), 38 unaffected]. The variants included were c.2308+1G>A, p.Pro108Alafs*9, p.Arg891Alafs*160 and p.Glu258Lys, all considered founder mutations in our region. Peripheral venous blood samples were obtained from patients at the Inherited Heart Diseases Unit and stored at -80 ºC after informed consent had been signed by all participants. For telomere length analysis, a telomeric restriction fragment (TRF) assay was performed using the TeloTAGGGTelomereLenghtAssay kit (Roche, 12209136001) according to the manufacturer's protocol with slight modifications. Results Our results show a lower LTLc in those affected patients with a more severe HCM phenotype compared to the affected and unaffected group (p=0.023) independently of age and sex, which are two variables that affect LTLc. Similarly, a shorter telomere length was associated with greater severity of the phenotype, with patients presenting a larger left atrial size (p=0.032), septal size (p=0.023) and greater obstruction (p=0.044). The presence of atrial fibrillation (p=0.039) and sudden death (p=0.024) was also higher in patients with shorter telomeres. Conclusions Telomere length is a variable that influences the expression of the phenotype in HCM patients carrying a MYBPC3 variant. This is an observation that supports the thesis of numerous publications linking HCM and other age-related diseases, sometimes called telomeropathies,(2) to telomere shortening. It has also been linked to non-hereditary conditions(3). This fact sheds some light on the complex phenotypic variability present in HCM, raising LTL as a promising prognostic biomarker of the disease, as has already been suggested by other authors.(4, 5, 6).Workflow in the telomere legth analysisClinical associations of LTLc

Characteristics and outcomes associated with sarcomere mutations in patients with hypertrophic cardiomyopathy: A systematic review and meta-analysis

BackgroundHypertrophic cardiomyopathy (HCM) is an inherited heart disease that can lead to sudden cardiac death. Impact of genetic testing for the prognosis and treatment of patients with HCM needs to be improved. We conducted a systematic review and meta-analysis to investigate the characteristics and outcomes associated with sarcomere genotypes in index patients with HCM. MethodsA systematic search was conducted in Medline, Embase, and Cochrane Library up to Dec 31, 2023. Data on clinical characteristics, morphological and imaging features, outcomes and interventions were collected from published studies and pooled using a random-effects meta-analysis. ResultsA total of 30 studies with 10,825 HCM index patients were included in the pooled analyses. The frequency of sarcomere genes in HCM patients was 41%. Sarcomere mutations were more frequent in women (p < 0.00001), and were associated with lower body mass index (26.1 ± 4.7 versus 27.5 ± 4.3; p = 0.003) and left ventricular ejection fraction (65.7% ± 10.1% vs. 67.1% ± 8.6%; p = 0.03), less apical hypertrophy (6.5% vs. 20.1%; p < 0.0001) and left ventricular outflow tract obstruction (29.1% vs. 33.2%; p = 0.03), greater left atrial volume index (43.6 ± 21.1 ml/m2 vs. 37.3 ± 13.0 ml/m2; p = 0.02). Higher risks of ventricular tachycardia (23.4% vs. 14.1%; p < 0.0001), syncope (18.3% vs. 10.9%; p = 0.01) and heart failure (17.3% vs. 14.6%; p = 0.002) were also associated with sarcomere mutations. ConclusionsSarcomere mutations are more frequent in women, and are associated with worse clinical characteristics and poor outcomes.

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

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.

Autopsy of all young sudden death cases is important to increase survival in family members left behind.

Sudden cardiac death (SCD) is an important public health problem worldwide, accounting for an estimated 6-20% of total mortality. A significant proportion of SCD is caused by inherited heart disease, especially among the young. An autopsy is crucial to establish a diagnosis of inherited heart disease, allowing for subsequent identification of family members who require cardiac evaluation. Autopsy of cases of unexplained sudden death in the young is recommended by both the European Society of Cardiology and the American Heart Association. Overall autopsy rates, however, have been declining in many countries across the globe, and there is a lack of skilled trained pathologists able to carry out full autopsies. Recent studies show that not all cases of sudden death in the young are autopsied, likely due to financial, administrative, and organizational limitations as well as awareness among police, legal authorities, and physicians. Consequently, diagnoses of inherited heart disease are likely missed, along with the opportunity for treatment and prevention among surviving relatives. This article reviews the evidence for the role of autopsy in sudden death, how the cardiologist should interpret the autopsy-record, and how this can be integrated and implemented in clinical practice. Finally, we identify areas for future research along with potential for healthcare reform aimed at increasing autopsy awareness and ultimately reducing mortality from SCD.

Diagnostic yield from cardiac gene testing for inherited cardiac conditions and re-evaluation of pre-ACMG variants of uncertain significance.

Inherited cardiomyopathies (HCM, DCM, ACM) and cardiac ion channelopathies (long QT/Brugada syndromes, CPVT) are associated with significant morbidity and mortality; however, diagnosis of a familial pathogenic variant in a proband allows for subsequent cascade screening of their at-risk relatives. We investigated the diagnostic yield from cardiac gene panel testing and reviewed variants of uncertain significance from patients attending three specialist cardiogenetics services in Ireland in the years 2002 to 2020. Reviewing molecular genetic diagnostic reports of 834 patients from 820 families, the initial diagnostic yield of pathogenic/likely pathogenic variants was 237/834 patients (28.4%), increasing to 276/834 patients (33.1%) following re-evaluation of cases with variant(s) of uncertain significance. Altogether, 42/85 patients with VUS reviewed (49.4%) had a re-classification that could change their clinical management. Females were more likely to carry pathogenic/likely pathogenic variants than males (139/374, 37.2% vs 137/460, 29.8%, respectively, p = 0.03), and the diagnostic yields were highest in the 0 to < 2 years age group (6/12, 50.0%) and amongst those tested for cardiomyopathy gene panels (13/35, 37.1%). Variants in the MYBPC3/MYH7 (87/109, 79.8%) and KCNQ1/KCNH2 (91/100, 91.0%) genes were the predominant genetic causes for hypertrophic cardiomyopathy and long QT syndrome, respectively. Our study highlights the importance of collation and review of pre-ACMG genetic variants to increase diagnostic utility of genetic testing for inherited heart disease. Almost half of patients with pre-ACMG VUS reviewed had their variant re-classified to likely pathogenic/likely benign which resulted in a positive clinical impact for patients and their families.

Variants in non-coding DNA in inherited heart disease

Variants in non-coding DNA in inherited heart disease

Circular RNAs: Biogenesis, Functions, and Role in Myocardial Hypertrophy.

Circular RNAs (circRNAs) are a large class of endogenous single-stranded covalently closed RNA molecules. High-throughput RNA sequencing and bioinformatic algorithms have identified thousands of eukaryotic circRNAs characterized by high stability and tissue-specific expression pattern. Recent studies have shown that circRNAs play an important role in the regulation of physiological processes in the norm and in various diseases, including cardiovascular disorders. The review presents current concepts of circRNA biogenesis, structural features, and biological functions, describes the methods of circRNA analysis, and summarizes the results of studies on the role of circRNAs in the pathogenesis of hypertrophic cardiomyopathy, the most common inherited heart disease.

In silico analysis of TRPM4 variants of unknown clinical significance

Background TRPM4 is a calcium-activated channel that selectively permeates monovalent cations. Genetic variants of the channel in cardiomyocytes are associated with various heart disorders, such as progressive familial heart block and Brugada syndrome. About97% of all known TRPM4 missense variants are classified as variants of unknown clinical significance (VUSs). The very large number of VUSs is a serious problem in diagnostics and treatment of inherited heart diseases. Methods and results We collected 233 benign or pathogenic missense variants in the superfamily of TRP channels from databases ClinVar, Humsavar and Ensembl Variation to compare performance of 22 algorithms that predict damaging variants. We found that ClinPred is the best-performing tool for TRP channels. We also used the paralogue annotation method to identify disease variants across the TRP family. In the set of 565 VUSs of hTRPM4, ClinPred predicted pathogenicity of 299 variants. Among these, 12 variants are also categorized as LP/P variants in at least one paralogue of hTRPM4. We further used the cryo-EM structure of hTRPM4 to find scores of contact pairs between parental (wild type) residues of VUSs for which ClinPred predicts a high probability of pathogenicity of variants for both contact partners. We propose that 68 respective missense VUSs are also likely pathogenic variants. Conclusions ClinPred outperformed other in-silico tools in predicting damaging variants of TRP channels. ClinPred, the paralogue annotation method, and analysis of residue contacts the hTRPM4 cryo-EM structure collectively suggest pathogenicity of 80 TRPM4 VUSs.

Titin domains with reduced core hydrophobicity cause dilated cardiomyopathy

The underlying genetic defect in most cases of dilated cardiomyopathy (DCM), a common inherited heart disease, remains unknown. Intriguingly, many patients carry single missense variants of uncertain pathogenicity targeting the giant protein titin, a fundamental sarcomere component. To explore the deleterious potential of these variants, we first solved the wild-type and mutant crystal structures of I21, the titin domain targeted by pathogenic variant p.C3575S. Although both structures are remarkably similar, the reduced hydrophobicity of deeply buried position 3575 strongly destabilizes the mutant domain, a scenario supported by molecular dynamics simulations and by biochemical assays that show no disulfide involving C3575. Prompted by these observations, we have found that thousands of similar hydrophobicity-reducing variants associate specifically with DCM. Hence, our results imply that titin domain destabilization causes DCM, a conceptual framework that not only informs pathogenicity assessment of gene variants but also points to therapeutic strategies counterbalancing protein destabilization.

Stromal cell models of arrhythmogenic cardiomyopathy

Abstract Background Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease mainly associated with mutations in genes encoding cardiac desmosomes, most frequently Plakophilin-2 (PKP2). Primary cardiac mesenchymal stromal cells (CMSCs) were shown to contribute to the aberrant ACM cardiac remodeling based on their ability to differentiate into adipocytes and myofibroblasts, thus representing a useful in vitro model for the disease. The limitations associated with access to biopsies and finite passages in culture have been overcome by the advancement of stem cell technology, especially human induced pluripotent stem cells (hiPSCs) and their ability to differentiate towards cardiac stromal population. Specifically, CMSCs from hiPSCs provide a virtually unlimited cell sources that are genetically identical to patient cells of origin. Purpose The study compares hiPSC-derived stromal cells (hiPSC-D) and primary stromal cells (PRs), to explore the feasibility to use iPSC-D as an alternative in vitro model to study ACM. Methods PRs were isolated from ACM patients and healthy donors (HC). hiPSCs from a PKP2 mutated ACM patient (c.2013delC) and a sex/aged matched HC were used to produce iPSC-D. Flow cytometry analysis and specific differentiation induction (osteogenic, chondrogenic, adipogenic and pro-fibrotic) were performed to confirm mesenchymal lineage and multipotency. qRT-PCR and western blot analysis were performed to evaluate the expression of desmosomes, both gene and protein levels. Illumina transcriptomic profiling of both PRs and iPSC-D were compared and enriched pathways were identified by GSEA. Results Both PR and iPSC-D cells expressed mesenchymal surface markers and were able to differentiate towards osteogenic, chondrogenic, adipogenic and myofibroblast lineages. Desmosomal genes and proteins were expressed by both populations, showing a general trend toward lower expression of desmosomal markers in ACM compared to HC cells. Overall, higher expression of desmosomes was observed in iPSC-D stromal cells compared to their primary counterparts, independently on the genotype. As occurs for PRs, ACM iPSC-D exhibited an increased propensity to accumulate lipid droplets and collagen compared to HC cells. Moreover, both cell populations shared a highly similar transcriptomic profile, showing a significant linear correlation (R=0.85). Interestingly, PRs were enriched for genes associated with the immune response and differentiation processes, hinting to the hypothesis that an inflammatory stimulus received from the cardiac tissue might activate the innate immunity and drive the cell differentiation fates in the PRs. Conclusion(s) Both PR and iPSC-D cardiac stromal cells obtained from ACM patients can be used to model the disease, where the choice for the most suitable cell populations can be determined by the experimental strategy and/or the availability of human primary material.

Enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction in patient-specific induced pluripotent stem cell-derived cardiomyocytes with MYH7 mutation

Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is frequently caused by mutations in the β-cardiac myosin heavy chain gene (MYH7). Abnormal calcium handling and diastolic dysfunction are archetypical features of HCM caused by MYH7 gene mutations. However, the mechanism of how MYH7 mutations leads to these features remains unclear, which inhibits the development of effective therapies. Initially, cardiomyocytes were generated from induced pluripotent stem cells from an eight-year-old girl diagnosed with HCM carrying a MYH7(C.1063 G>A) heterozygous mutation(mutant-iPSC-CMs) and mutation-corrected isogenic iPSCs(control-iPSC-CMs) in the present study. Next, we compared phenotype of mutant-iPSC-CMs to that of control-iPSC-CMs, by assessing their morphology, hypertrophy-related genes expression, calcium handling, diastolic function and myofilament calcium sensitivity at days 15 and 40 respectively. Finally, to better understand increased myofilament Ca2+ sensitivity as a central mechanism of central pathogenicity in HCM, inhibition of calcium sensitivity with mavacamten can improveed cardiomyocyte hypertrophy. Mutant-iPSC-CMs exhibited enlarged areas, increased sarcomere disarray, enhanced expression of hypertrophy-related genes proteins, abnormal calcium handling, diastolic dysfunction and increased myofilament calcium sensitivity at day 40, but only significant increase in calcium sensitivity and mild diastolic dysfunction at day 15. Increased calcium sensitivity by levosimendan aggravates cardiomyocyte hypertrophy phenotypes such as expression of hypertrophy-related genes, abnormal calcium handling and diastolic dysfunction, while inhibition of calcium sensitivity significantly improves cardiomyocyte hypertrophy phenotypes in mutant-iPSC-CMs, suggesting increased myofilament calcium sensitivity is the primary mechanisms for MYH7 mutations pathogenesis. Our studies have uncovered a pathogenic mechanism of HCM caused by MYH7 gene mutations through which enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction. Correction of the myofilament calcium sensitivity was found to be an effective method for treating the development of HCM phenotype in vitro.

Abstract 12927: Impact of AAV- MYBPC3 Gene Transfer on Heart Structure and Function in Human and Mouse Models of Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is a life-threatening inherited heart disease characterized by left ventricular hypertrophy and diastolic dysfunction. The most common cause of HCM is genetic variants in MYBPC3 , encoding cardiac myosin binding protein C (cMyBP-C), a sarcomeric protein with structural and regulatory roles. The majority of MYBPC3 gene variants are truncating leading to protein haploinsufficiency. Hypothesis: Transfer of a functional copy of MYBPC3 to heart muscle deficient in cMyBP-C will lead to sustained improvements in cardiac function. Aims: To determine whether BMN 293 (AAV-hMYBPC3) can restore cMyBP-C levels in the sarcomere and halt and/or reverse disease progression in non-clinical models of genetic HCM due to MYPBC3 deficiency. Methods: BMN 293 is an adeno-associated virus (AAV) vector that encodes wild-type human MYBPC3 under the control of a cardiomyocyte-selective promoter. We transduced human iPSC-derived cardiomyocytes and engineered heart tissues (EHTs) carrying a compound heterozygous truncating MYBPC3 mutation (MYBPC3 -/- ) with BMN 293 and assessed cMyBP-C levels and contractile parameters. We also systemically administered BMN 293 to MYBPC3 -/- mice and assessed cardiac distribution of human cMyBP-C by molecular and histological methods and determined its impact on left ventricular hypertrophy and function by echocardiography and other imaging techniques. Results: BMN 293 transduction of human iPSC MYBPC3 -/- cardiomyocytes and EHTs resulted in high levels of human MYBPC3 mRNA and cMyBP-C protein, correct incorporation of cMyBP-C into the sarcomere, and complete normalization of contractile kinetics. BMN 293 was well tolerated in MYBPC3 -/- mice and resulted in uniform restoration of cMyBP-C expression throughout the heart and significant correction of structural and functional cardiac abnormalities. Conclusions: A single IV infusion of BMN 293 to MYBPC3 -/- mice resulted in early and sustained reduction in left ventricular hypertrophy and durable improvements in diastolic function.

Improved Cardiac Performance and Decreased Arrhythmia in Hypertrophic Cardiomyopathy With Non-β-Blocking R-Enantiomer Carvedilol.

Hypercontractility and arrhythmia are key pathophysiologic features of hypertrophic cardiomyopathy (HCM), the most common inherited heart disease. β-Adrenergic receptor antagonists (β-blockers) are the first-line therapy for HCM. However, β-blockers commonly selected for this disease are often poorly tolerated in patients, where heart-rate reduction and noncardiac effects can lead to reduced cardiac output and fatigue. Mavacamten, myosin ATPase inhibitor recently approved by the US Food and Drug Administration, has demonstrated the ability to ameliorate hypercontractility without lowering heart rate, but its benefits are so far limited to patients with left ventricular (LV) outflow tract obstruction, and its effect on arrhythmia is unknown. We screened 21 β-blockers for their impact on myocyte contractility and evaluated the antiarrhythmic properties of the most promising drug in a ventricular myocyte arrhythmia model. We then examined its in vivo effect on LV function by hemodynamic pressure-volume loop analysis. The efficacy of the drug was tested in vitro and in vivo compared with current therapeutic options (metoprolol, verapamil, and mavacamten) for HCM in an established mouse model of HCM (Myh6R403Q/+ and induced pluripotent stem cell (iPSC)-derived cardiomyocytes from patients with HCM (MYH7R403Q/+). We identified that carvedilol, a β-blocker not commonly used in HCM, suppresses contractile function and arrhythmia by inhibiting RyR2 (ryanodine receptor type 2). Unlike metoprolol (a β1-blocker), carvedilol markedly reduced LV contractility through RyR2 inhibition, while maintaining stroke volume through α1-adrenergic receptor inhibition in vivo. Clinically available carvedilol is a racemic mixture, and the R-enantiomer, devoid of β-blocking effect, retains the ability to inhibit both α1-receptor and RyR2, thereby suppressing contractile function and arrhythmias without lowering heart rate and cardiac output. In Myh6R403Q/+ mice, R-carvedilol normalized hyperdynamic contraction, suppressed arrhythmia, and increased cardiac output better than metoprolol, verapamil, and mavacamten. The ability of R-carvedilol to suppress contractile function was well retained in MYH7R403Q/+ iPSC-derived cardiomyocytes. R-enantiomer carvedilol attenuates hyperdynamic contraction, suppresses arrhythmia, and at the same time, improves cardiac output without lowering heart rate by dual blockade of α1-adrenergic receptor and RyR2 in mouse and human models of HCM. This combination of therapeutic effects is unique among current therapeutic options for HCM and may particularly benefit patients without LV outflow tract obstruction.