Sarcomeric Genes Research Articles

Abstract 12408: Response to Mavacamten by Sarcomere Gene Mutation Status in EXPLORER-HCM

Background: Mavacamten is a small molecule inhibitor of cardiac myosin that was developed based on insights into the molecular basis of hypertrophic cardiomyopathy (HCM). In the phase 3 EXPLORER-HCM trial (NCT03470545), patients showed consistent benefit in the primary endpoint with mavacamten treatment vs placebo while subgroup analysis of patients with pathogenic or likely pathogenic variants in a broad panel of HCM-related genes, revealed a slightly more favorable response. However, it is unknown how patients specifically with sarcomere gene variants responded. Aims: To assess the effect of HCM-specific sarcomere gene variants on response to mavacamten. Methods: Sequencing using a 60 gene panel (Invitae) was optional in EXPLORER-HCM. In this exploratory analysis, responses to mavacamten vs placebo for the primary, secondary, and exploratory endpoints were analysed. Patients were grouped based on sarcomere gene variant status (positive for pathogenic/likely pathogenic/variants of uncertain significance [SARC+], negative [SARC–]) or no sequencing). Analyses were adjusted for clinically relevant variables. Results: Of 190/251 patients sequenced, 73 were SARC+ (mavacamten, n = 33; placebo, n = 40), 117 were SARC– (mavacamten, n = 57; placebo, n = 60) and 61 had no panel sequencing (mavacamten, n = 33; placebo, n = 28). Mavacamten treatment showed a favorable response for the primary endpoint vs placebo, for SARC+ (odds ratio [OR], 4.43 [95% CI, 1.56-12.58]), and for SARC– (OR, 2.52 [95% CI, 0.99-6.42]) ( Figure ). Improvements with mavacamten vs placebo were observed in both SARC+ and SARC– subgroups for change from baseline to week 30 in post-exercise left ventricular outflow tract gradient, and peak oxygen consumption, and in New York Heart Association class. Conclusions: In this exploratory subgroup analysis, mavacamten treatment benefit was observed both in patients with and without sarcomere gene variants.

Abstract 14463: Beyond Sarcomere Mutations Plakophilin-2 and Familial Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is a genetically heterogeneous disorder with diverse clinical presentation, course and outcomes. It is commonly caused by mutations in sarcomeric genes. Non-sarcomeric gene mutations have been associated with HCM in certain metabolic and storage diseases. Pathogenic mutations in plakophilin-2 (PKP2) gene have been associated with enhanced desmosomal protein degradation and linked to arrhythmogenic right ventricular cardiomyopathy (ARVC), Brugada syndrome and dilated cardiomyopathy. We report an association between PKP2 gene mutation and familial HCM. Case presentation: A 65-year-old man with family history of HCM (maternal grandfather, 2 aunts and an uncle) presented with dyspnea on exertion (NYHA class II-III) and pedal edema. Echo showed severe LVH, mid-cavitary obstruction and LV apical aneurysm. Cardiac MRI confirmed findings (Figure:A and B) and additionally showed significant late gadolinium enhancement (Figure:C and D, >20% of LV myocardium). Extended ambulatory heart monitor detected frequent runs on non-sustained ventricular tachycardia. A primary prevention ICD was implanted. Genetic testing identified a pathogenic mutation [c.1237C>T (p.Arg413*)] in exon 5 of PKP2 gene resulting in premature translational stop signal and disrupted desmosomal protein plakophilin-2. Family screening (Figure:E) identified a sister with positive results who also had non-obstructive HCM (Figure:E). Discussion: We present a family with typical presentations of HCM and no phenotypic evidence of ARVC. This novel observation provides evidence for pleiotropic model of complex genotype-phenotype interactions through nonlinear networks. Further evidence would be needed to understand potential molecular basis of hypertrophic signaling in PKP2 gene mutation.

Abstract 11679: The Crucial Role of Estrogen-Related Receptor in Human Cardiomyocyte Maturation

During the developmental transition from fetal to adult, the heart undergoes dramatic mitochondrial and sarcomeric maturation. This process is partially reversed during development of heart failure. We have identified the nuclear receptors, estrogen-related receptor (ERR) α and γ, as critical transcriptional regulators of energy metabolic and sarcomeric genes during postnatal maturation of mouse heart. Here, we aimed to define the transcriptional mechanisms involved in ERR-mediated maturation in human cardiomyocytes. Genomic interrogation with published datasets revealed that approximately 50% of super-enhancer regions (SEs) overlapped with ERRγ peaks in human-induced pluripotent stem cell-derived cardiac myocytes. Intersection of the ERRγ cistrome with a published H3K27ac Hi-ChIP dataset from human hearts identified over 3,000 ERR-containing enhancer-promoter loops, which are associated with genes related to cardiac contractile function. These results highlighted the essential role of ERR in cardiomyocyte enhancer function. Among the SEs defined by mediator complex deposition, we found that both ERRα and γ occupied the enhancer region flanking the MYH6/MYH7 cluster, a well-characterized locus for fetal to adult developmental switching. Targeting ERR recruitment on the enhancer using catalytically-dead Cas9-KRAB or proteolysis targeting chimeric (PROTAC) strategies resulted in the reversal of MYH6/7 switching. RNA-sequencing analysis following PROTAC treatment revealed that deactivation of ERRα resulted in downregulation of a wide array of genes encoding cardiac structural proteins, particularly adult-type ventricular sarcomeric proteins. Additional cistrome analyses demonstrated that PROTAC treatment resulted in decreased ERRα peak signals on cardiac structural genes, including enhancer regions directing MYH6/7 transcription consistent with the observed gene expression changes. In summary, ERR signaling is essential for the transcriptional program of structural maturation in human cardiomyocytes. This process likely involves establishing proper cardiac chromatin architecture. These results support a novel therapeutic strategy of utilizing ERR activators to reverse fetal shifts in failing hearts.

Abstract 16516: CMR-ECG Imaging in Hypertrophic Cardiomyopathy Detects Electrophysiological Abnormalities Before Hypertrophy or ECG Changes

INTRODUCTION: In hypertrophic cardiomyopathy (HCM), ventricular arrhythmia associates with severity of LVH and scar, and presence vs absence of a sarcomeric gene mutation (G+LVH+ vs G-LVH+). Also, ECG changes in subclinical HCM (G+LVH-) signal increased risk of phenotype progression. HYPOTHESES: ECG Imaging (ECGI) can detect: i) subtle electrophysiological (EP) abnormalities in subclinical HCM (pre-LVH). ii) EP abnormalities related to genetic status (G+ vs G-LVH+) and structural changes (late gadolinium enhancement [LGE], max. wall thickness [MWT]) in overt disease. METHODS: 200 participant multicenter study: 70 G+LVH-, 51 G+LVH+, 53 G-LVH+ and 26 healthy volunteers (HV) underwent 12-lead ECG (to detect abnormal Q-waves, repolarization changes, LVH criteria) and CMR-ECGI computing epicardial unipolar electrograms (UEGs) to derive: activation time (AT), activation-recovery intervals (ARIc), spatial gradients (activation: G AT , repolarisation: G RT ) and fractionation. RESULTS: Compared to HV, G+LVH- had prolongation of AT (40.3±7.3 vs 35.4±6.1 ms p=0.003) and steeper G RT (mean: 1.12±0.27 vs 1.00±0.23 ms/mm p=0.042, max: 11.7±2.8 vs 10.0±1.9 ms/mm p=0.005). AT was prolonged even in G+LVH- with a normal 12-lead ECG (p<0.004). Compared to G+LVH-, G+LVH+ had similar AT but a more prolonged ARIc (275±29 vs 245±26 ms p<0.001). Compared to G-LVH+, G+LVH+ had similar MWT and LGE, but more signal fractionation (0.02±0.02 vs 0.01±0.03% UEGs with ≥2 deflections p=0.002). In overt HCM (all LVH+), MWT associated with AT ( r s 0.25 p=0.011) while LGE burden associated with G AT ( r s=0.27 p=0.006) and fractionation ( r s=0.22 p=0.025). CONCLUSION: Cardiac activation prolongation and repolarization abnormalities occur in subclinical HCM (before hypertrophy). Activation is prolonged even in subclinical HCM with normal 12-lead ECG. In overt HCM, ECGI abnormalities track adverse structural changes and reveal greater fractionation in those with a sarcomeric mutation.

Abstract 15512: Disrupting the NelfA Dependent RNA pol II Paused Complex in Heart Precipitates Dilated Cardiomyopathy

RNA polymerase II (pol II) pausing, a wide-spread genomic phenomenon regulates pol II dependent transcription and plays a critical role in gene expression during development and disease. Negative elongation factor (Nelf) complex consisting of five subunits, namely NelfA, NelfB, NelfC/D and NelfE, in coordination with Drb sensitive inducing factor (DISF) complex has been implicated in mediating pol II pausing. Structural studies show NelfA is indispensable for nucleation of paused complex. We have reported widespread promoter NelfA occupancy on cardiac genome, and showed that it is required for compensatory gene expression in hearts during cardiac hypertrophy. Here, we examine paused complex, with respect to its interactome, influence on gene expression and cardiac function. Immunoprecipitation of chromatin bound NelfA, followed by mass spectrometry in sham and TAC induced hypertrophied hearts identified Nelf proteins, pol II and Supt5, along with novel partners like Trim28 (chromatin remodeler), Adprhl1 (cardiac restricted, nuclear enriched, involved in DNA repair) and Numa1 (nuclear structural protein). Conditional NelfA knockout mice developed progressive dilated cardiomyopathy (LVID’d: 4.72 ±0.1, LVID;s: 3.96±0.13, %EF: 33.7±2.12, %FS: 16.15±1.12, LV vol;d: 104.5±21.9, LV vol;s: 70.26±5.5) compared to Wt-Cre (LVID’d: 4.16 ±0.09, LVID;s: 3.14±0.12, %EF: 49.1±2.3, %FS: 24.6±1.3, LV vol;d: 77.5±13.9, LV vol;s: 40.9±3.9), with sudden death by 3mths of age, in males and females KO mice compared to Wt-Cre. Interestingly, absence of NelfA resulted in decrease in cardiomyocyte size and increase in apoptosis. Single nuclei RNAseq on these hearts show decrease in cardiac-enriched sarcomeric genes, along with increase in genes involved in fibrosis and endothelial dysfunction. Further, ChIP-seq showed reduced Trim28 and NelfE promoter occupancy on essential gene, while below threshold or absent on cardiac-enriched and selected inducible promoters suggesting disruption of paused complex. Thus, we conclude that NelfA induced paused complex plays an essential role in efficient pol II dependent gene transcription and expression in heart, and deconstruction of the paused complex precipitates cardiac dysfunction and dilated cardiomyopathy

MDM2 Regulation of HIF Signaling Causes Microvascular Dysfunction in Hypertrophic Cardiomyopathy.

Microvasculature dysfunction is a common finding in pathologic remodeling of the heart and is thought to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM), a disease caused by sarcomere gene mutations. We hypothesized that microvascular dysfunction in HCM was secondary to abnormal microvascular growth and could occur independent of ventricular hypertrophy. We used multimodality imaging methods to track the temporality of microvascular dysfunction in HCM mouse models harboring mutations in the sarcomere genes Mybpc3 (cardiac myosin binding protein C3) or Myh6 (myosin heavy chain 6). We performed complementary molecular methods to assess protein quantity, interactions, and post-translational modifications to identify mechanisms regulating this response. We manipulated select molecular pathways in vivo using both genetic and pharmacological methods to validate these mechanisms. We found that microvascular dysfunction in our HCM models occurred secondary to reduced myocardial capillary growth during the early postnatal time period and could occur before the onset of myocardial hypertrophy. We discovered that the E3 ubiquitin protein ligase MDM2 (murine double minute 2) dynamically regulates the protein stability of both HIF1α (hypoxia-inducible factor 1 alpha) and HIF2α (hypoxia-inducible factor 2 alpha)/EPAS1 (endothelial PAS domain protein 1) through canonical and noncanonical mechanisms. The resulting HIF imbalance leads to reduced proangiogenic gene expression during a key period of myocardial capillary growth. Reducing MDM2 protein levels by genetic or pharmacological methods normalized HIF protein levels and prevented the development of microvascular dysfunction in both HCM models. Our results show that sarcomere mutations induce cardiomyocyte MDM2 signaling during the earliest stages of disease, and this leads to long-term changes in the myocardial microenvironment.

RBPMS regulates cardiomyocyte contraction and cardiac function through RNA alternative splicing.

RNA binding proteins play essential roles in mediating RNA splicing and are key post-transcriptional regulators in the heart. Our recent study demonstrated that RBPMS (RNA binding protein with multiple splicing) is crucial for cardiac development through modulating mRNA splicing, but little is known about its functions in the adult heart. In this study, we aim to characterize the post-natal cardiac function of Rbpms and its mechanism of action. We generated a cardiac-specific knockout mouse line and found that cardiac-specific loss of Rbpms caused severe cardiomyocyte contractile defects, leading to dilated cardiomyopathy and early lethality in adult mice. We showed by proximity-dependent biotin identification assay and mass spectrometry that RBPMS associates with spliceosome factors and other RNA binding proteins, such as RBM20, that are important in cardiac function. We performed paired-end RNA sequencing and RT-PCR and found that RBPMS regulates mRNA alternative splicing of genes associated with sarcomere structure and function, such as Ttn, Pdlim5, and Nexn, generating new protein isoforms. Using a minigene splicing reporter assay, we determined that RBPMS regulates target gene splicing through recognizing tandem intronic CAC motifs. We also showed that RBPMS knockdown in human induced pluripotent stem cell-derived cardiomyocytes impaired cardiomyocyte contraction. This study identifies RBPMS as an important regulator of cardiomyocyte contraction and cardiac function by modulating sarcomeric gene alternative splicing.

ERRγ agonist under mechanical stretching manifests hypertrophic cardiomyopathy phenotypes of engineered cardiac tissue through maturation

Engineered cardiac tissue (ECT) using human induced pluripotent stem cell-derived cardiomyocytes is a promising tool for modeling heart disease. However, tissue immaturity makes robust disease modeling difficult. Here, we established a method for modeling hypertrophic cardiomyopathy (HCM) malignant (MYH7 R719Q) and nonmalignant (MYBPC3 G115∗) pathogenic sarcomere gene mutations by accelerating ECT maturation using an ERRγ agonist, T112, and mechanical stretching. ECTs treated with T112 under 10% elongation stimulation exhibited more organized and mature characteristics. Whereas matured ECTs with the MYH7 R719Q mutation showed broad HCM phenotypes, including hypertrophy, hypercontraction, diastolic dysfunction, myofibril misalignment, fibrotic change, and glycolytic activation, matured MYBPC3 G115∗ ECTs displayed limited phenotypes, which were primarily observed only under our new maturation protocol (i.e., hypertrophy). Altogether, ERRγ activation combined with mechanical stimulation enhanced ECT maturation, leading to a more accurate manifestation of HCM phenotypes, including non-cardiomyocyte activation, consistent with clinical observations.

The challenge of risk stratification in hypertrophic cardiomyopathy: Clinical, genetic and imaging insights from a quaternary referral centre

BackgroundHypertrophic cardiomyopathy (HCM) is the commonest genetic cardiomyopathy and may result in sudden cardiac death (SCD). Clinical risk stratification scores are utilised to estimate SCD risk and determine potential utility of a primary prevention implantable cardioverter defibrillator (ICD). MethodsPatients with a confirmed diagnosis of HCM from a quaternary HCM service were defined according to clinical characteristics, genetic profiles and cardiac imaging results. European Risk-SCD score and American Heart Association / American College of Cardiology (AHA/ACC) Score were calculated. The primary outcome was cardiac arrest. Results380 patients with HCM were followed up for a median of 6.4 years. 18 patients (4.7%) experienced cardiac arrest, with predictive factors being younger age (37.2 vs 54.4 years, p = 0.0041), unexplained syncope (33.3% vs 9.4%, p = 0.007), non-sustained ventricular tachycardia (50.0% vs 12.7%, p < 0.0001), increased septal thickness (21.5 vs 17.5 mm, p = 0.0003), and presence of a sarcomeric gene mutation (100.0% vs 65.8%, p = 0.038). The Risk-SCD and AHA/ACC scores had poor agreement (kappa coefficient 0.38). Risk-SCD score had poor sensitivity (44.4%), classifying 55.6% of patients with cardiac arrest as low-risk but was highly specific (93.7%). AHA/ACC risk score did not discriminate between groups significantly. 20 patients (5.3%) died, with most >60-year-olds having a non-cardiac cause of death (p = 0.0223). ConclusionThis study highlights limited (38%) agreement between the Risk-SCD and AHA/ACC scores. Most cardiac arrests occurred in ostensibly low or medium-risk patients under both scores. Appropriate ICD selection remains challenging. Incorporating newer risk markers such as HCM genotyping and myocardial fibrosis quantification by cardiac MRI may assist future risk refinement.

RAF1 mutation leading to hypertrophic cardiomyopathy in a Chinese family with a history of sudden cardiac death: A diagnostic insight into Noonan syndrome.

Hypertrophic cardiomyopathy (HCM) is predominantly caused by mutations in sarcomeric genes. However, a subset of cases is attributed to genetic disorders unrelated to sarcomeric genes, such as Noonan syndrome (NS) and other RASopathies. In this study, we present a family with a history of sudden cardiac death (SCD) and focus on two adults with syndromic left ventricular hypertrophy (LVH). Clinical evaluations, including echocardiography, were conducted to assess cardiac manifestations. Whole-exome sequencing was performed to identify potential genetic variants underlying syndromic LVH in the study participants. Whole-exome sequencing revealed a missense variant in the RAF1 gene, c.782C>T (p.Pro261Leu). This variant confirmed the diagnosis of NS in the affected individuals. The findings of this study underscore the importance of family history investigation and genetic testing in diagnosing syndromic LVH. By identifying the underlying genetic cause, clinicians can better understand the etiology of RAS-HCM and its association with SCD in young adults.

The cardioprotective potential of melatonin on cardiac hypertrophy: A mechanistic overview

Cardiac hypertrophy (CH) is an increment of muscle mass to maintain the heart regular operations. A physiological cardiac hypertrophy due to exercise or other normal physiological process is characterized by normal contractile function and structural framework of heart tissue. In contrast, pathological hypertrophy occurs in response to increased pressure or volume overload from several cardiovascular diseases including hypertension, valvular diseases, cardiac infarction and heart failure. It is of major concern as it is one of the leading causes of death worldwide. Despite much progress in this field there is a scope for understanding of the molecular mechanisms of this condition. In this review, various types of cardiac hypertrophy and their intricate physio-pathological mechanisms have been discussed. In addition, the genetic mutations in sarcomere genes and oxidative stress are also closely linked to hypertrophic cardiomyopathy. Although several drugs against cardiac hypertrophy have been used, it appears that melatonin, due to its high bioavailability and low side effects, is a better candidate than the conventional medicine for treatment of hypertrophic cardiomyopathy. Melatonin, a hormone and a potent antioxidant, is secreted mainly from the pineal gland, but it is also synthesized from different peripheral tissues including the heart. This molecule can regulate a myriad of cellular functions. It can protect against cardiac hypertrophy via reducing oxidative stress, elevating Cu-Mn SOD via controlling several cell signalling pathways of Akt/mTOR, ROR-α and NLRP3 cascades. Melatonin also mitigates cardiac hypertrophy by suppressing pro-inflammatory cytokines including TNF-α and TGF-β and cardiac hypertrophy markers like β-MHC, ANP, BNP, LDH. This review focuses on the molecular mechanisms of cardiac hypertrophy and the defensive role of melatonin on it. We propose melatoninas a propitious adjunct for the treatment of cardiac hypertrophy.

Mature human induced pluripotent stem cell-derived cardiomyocytes promote angiogenesis through alpha-B crystallin

BackgroundHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used to treat heart diseases; however, the optimal maturity of hiPSC-CMs for effective regenerative medicine remains unclear. We aimed to investigate the benefits of long-term cultured mature hiPSC-CMs in injured rat hearts.MethodsCardiomyocytes were differentiated from hiPSCs via monolayer culturing, and the cells were harvested on day 28 or 56 (D28-CMs or D56-CMs, respectively) after differentiation. We transplanted D28-CMs or D56-CMs into the hearts of rat myocardial infarction models and examined cell retention and engraftment via in vivo bioluminescence imaging and histological analysis. We performed transcriptomic sequencing analysis to elucidate the genetic profiles before and after hiPSC-CM transplantation.ResultsUpregulated expression of mature sarcomere genes in vitro was observed in D56-CMs compared with D28-CMs. In vivo bioluminescence imaging studies revealed increased bioluminescence intensity of D56-CMs at 8 and 12 weeks post-transplantation. Histological and immunohistochemical analyses showed that D56-CMs promoted engraftment and maturation in the graft area at 12 weeks post-transplantation. Notably, D56-CMs consistently promoted microvessel formation in the graft area from 1 to 12 weeks post-transplantation. Transcriptomic sequencing analysis revealed that compared with the engrafted D28-CMs, the engrafted D56-CMs enriched genes related to blood vessel regulation at 12 weeks post-transplantation. As shown by transcriptomic and western blot analyses, the expression of a small heat shock protein, alpha-B crystallin (CRYAB), was significantly upregulated in D56-CMs compared with D28-CMs. Endothelial cell migration was inhibited by small interfering RNA-mediated knockdown of CRYAB when co-cultured with D56-CMs in vitro. Furthermore, CRYAB overexpression enhanced angiogenesis in the D28-CM grafts at 4 weeks post-transplantation.ConclusionsLong-term cultured mature hiPSC-CMs promoted engraftment, maturation and angiogenesis post-transplantation in infarcted rat hearts. CRYAB, which was highly expressed in D56-CMs, was identified as an angiogenic factor from mature hiPSC-CMs. This study revealed the benefits of long-term culture, which may enhance the therapeutic potential of hiPSC-CMs.

Early-life exposure to lead changes cardiac development and compromises long-term cardiac function

Lead (Pb) is widely used in industrial and daily-use consumer products. Early-life exposure may increase the risk of lead-related heart problems in childhood. However, the effects of early-life lead exposure on fetal heart development and long-term cardiac outcomes are unknown. In this study, pregnant ICR mice were exposed to lead acetate trihydrate (50 mg/kg/d) via oral gavage from gestation day 1.5 until offspring weaning. Thereafter, the second hit model was established, two groups of offspring (4 weeks old) were either administered sterile saline or Angiotensin II (Ang II) for 4 weeks until euthanasia. We investigated lead-induced offspring heart damage from embryonic period to adulthood by echocardiographic analysis, pathological H&E staining, and ultrastructural examination, as well as mitochondrial function detection. The results showed early-life lead exposure predisposed offspring mice to decreased ejection fraction, increased left ventricular volume, accompanied by hypertrophy and dilation, cardiomyocyte sarcomere dysplasia, abnormal mitochondrial structure, mitochondrial dysfunction, and decreased expression of key sarcomeric and mitochondrial genes, rendering them more susceptible to cardiac hypertrophy, vascular wall thickening, cardiac fibrosis, apoptosis, and heart failure induced by Ang II infusion. This study elucidates early-life low dose lead exposure compromises cardiac development and exacerbates second hit-induced cardiac pathological responses in adulthood, which furnishes crucial scientific evidence pertaining to the cardiac toxicity and risk evaluation associated with early-life exposure to lead.

Gene-echocardiography: refining genotype-phenotype correlations in hypertrophic cardiomyopathy.

This study aims to clarify the association between hypertrophic patterns and genetic variants in hypertrophic cardiomyopathy (HCM) patients, contributing to the advancement of personalized management strategies for HCM. A comprehensive evaluation of genetic mutations was conducted in 392 HCM-affected families using Whole Exome Sequencing. Concurrently, relevant echocardiographic data from these individuals were collected. Our study revealed an increased susceptibility to enhanced septal and interventricular septal thickness in HCM patients harbouring gene mutations compared with those without. Mid-septal hypertrophy was found to be associated predominantly with myosin binding protein C3 (MYBPC3) variants, while a higher septum-to-posterior wall ratio correlated with myosin heavy chain 7 (MYH7) variants. Mutations in MYH7, MYBPC3, and other sarcomeric or myofilament genes (troponin I3 [TNNI3], tropomyosin 1 [TPM1], and troponin T2 [TNNT2]) showed a relationship with increased hypertrophy in the anterior wall, interventricular septum, and lateral wall of the left ventricle. In contrast, alpha kinase 3 (ALPK3)-associated hypertrophy chiefly presented in the apical region, while hypertrophy related to titin (TTN) and obscurin (OBSCN) mutations exhibited a uniform distribution across the myocardium. Hypertrophic patterns varied with the type and category of gene mutations, offering valuable diagnostic insights. Our findings underscore a strong link between hypertrophic patterns and genetic variants in HCM, providing a foundation for more accurate genetic testing and personalized management of HCM patients. The novel concept of 'gene-echocardiography' may enhance the precision and efficiency of genetic counselling and testing in HCM.

Abstract P2046: The Role Of A Novel Alpha-crystallin B Chain Variant In Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disorder affecting 1 in 500 people in the general population. Characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray and cardiac fibrosis, HCM is a highly complex disease with heterogenous clinical presentation. While mutations in sarcomere genes can account for a substantial proportion of familial cases, 40-50% of HCM patients do not carry such sarcomere variants and the causal mutations for their diseases remain elusive. Recently, we identified a novel variant of the alpha-crystallin B chain (CRYAB R123W ) in a pair of homozygotic twins who developed concordant HCM phenotypes that manifested over a nearly identical time course. Yet, how CRYAB R123W promotes HCM phenotype remains unclear. Here, we generated mice carrying the Cryab R123W -knockin allele and demonstrated that hearts from these animals exhibit increased maximal elastance, reduced diastolic function and are more susceptible to ventricular tachycardia with programmed stimulation. Upon transverse aortic constriction, mice carrying the Cryab R123W allele developed pathogenic left ventricular hypertrophy with substantial cardiac fibrosis and progressively decreased ejection fraction. In contrast to another well-characterized CRYAB variant (R120G) which induced Desmin aggregation, no evidence of protein aggregation was observed in hearts expressing CRYAB R123W despite its potent effect on driving cellular hypertrophy. Unexpectedly, CRYAB R123W appears to enhance calcium signaling by promoting nuclear localization of NFAT through direct interaction with calcineurin. Studies on isolated adult cardiomyocytes reveal altered fractional shortening, calcium dyshomeostasis, prolonged action potential duration, and T-wave alternans. Crossing of CRYAB R123W mice with a mybpc3 knock-in model of HCM did not potentiate pathological hypertrophy in compound heterozygotes, indicating that the pathological mechanisms in this model are independent of the sarcomere. Thus, our data establish the Cryab R123W allele as a novel genetic model of HCM that potentially unveils additional sarcomere-independent mechanisms of cardiac pathological hypertrophy and sudden cardiac death.

Abstract P3052: RNA Binding Protein Csdc2 Is Required For Metabolic And Cardiac Stress Responses

Post transcriptional gene regulation (PTGR) mediated by networks of microRNAs and RNA binding proteins (RBP's) controls mRNA processing, localization, and expression. Hundreds of microRNAs and RBPs are expressed in heart, and much remains unknown regarding PTGR’s influence on cardiovascular health and disease. Through bioinformatic analyses, we identified Csdc2 as a cardiac-enriched RBP that is consistently downregulated in failing hearts. Csdc2’s paralog, Carshp1, controls metabolic gene expression in liver through PPARα. We hypothesize that Csdc2 plays key roles in cardio-metabolic stress. To address this, we first assessed mRNA-seq changes in neonatal rat cardiomyocytes after overexpression and knockdown of Csdc2. Pathway analyses revealed reciprocal dysregulation of fatty acid metabolism, ion channels, and cytoskeletal and sarcomere genes. We generated constitutive Csdc2-KO mice, which are viable with no overt baseline phenotypes. Cardiac phenotyping revealed that Csdc2-KO mice have slightly decreased PR interval (~10%, p=1.3e-4, n=16-22) but maintain normal cardiac structure/function. To assess Csdc2’s potential role in metabolic stress, mice were fed a high-fat-diet for 10 weeks and monitored for lean/fat mass and insulin/glucose tolerances. Csdc2-KO mice gained 20% more weight than wildtype littermates (p=0.04, n=13-18) and developed insulin resistance (p&lt;0.01). To test if Csdc2 loss influences cardiac stress, normal chow cohorts were subjected to microsurgery-induced myocardial infarction (MI); Csdc2-KO mice showed ~2-fold increase in mortality within two weeks (70% versus 40% for wildtype). Echocardiographic measures support that Csdc2-KO hearts have larger infarcts, dilation without compensatory remodeling and worse systolic function. Together, these data highlight Csdc2 as a disease-relevant RBP that controls cardio-metabolic stress responses.

Abstract P2049: Myosin Heavy Chain-7 E848G Mutation-induced Sarcopenia And Hypocontractility In Induced Pluripotent Stem Cell Model Of Hypertrophic Cardiomyopathy Are Rescued By Muscle Ring Finger 1 Ablation

Hypertrophic cardiomyopathy (HCM) presents with idiopathic left ventricular hypertrophy and occasionally systolic dysfunction. In a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of HCM, we previously reported the pathogenic MYH7 E848G variant increased hiPSC-CM size and impaired contractility. To elucidate the mechanisms by which the E848G variant causes HCM, we now examined cardiac tissue from a MYH7 wt/E848G patient. We found reduced expression of the following sarcomeric proteins: β-myosin heavy chain (βMHC) (97.8 ± 1.1% reduction), α-sarcomeric actinin (αSA) (84.8 ± 7.3%), and ventricular myosin light chain (MLC-1v) (45.9 ± 13.9%) relative to healthy patient tissue. Isogenic MYH7 wt/E848G hiPSC-CMs also had reduced protein expression of βMHC (22.8%) and αSA (24.5%) relative to MYH7 wt/wt hiPSC-CMs and unchanged mRNA expression. These results suggest MYH7 E848G HCM induces broad sarcomere component deficiency. We tested hiPSC-CM contractility using single cell traction force microscopy (TFM) and engineered heart tissues (EHT). Single isogenic MYH7 wt/E848G hiPSC-CMs had lower maximum twitch force (80 ± 12 nN) than MYH7 wt/wt hiPSC-CMs (150 ± 20 nN) by TFM. Isogenic MYH7 wt/E848G EHTs had lower maximum twitch force (118.9 ± 23.7 μN) than MYH7 wt/wt EHTs (287.4 ± 27.7 μN). Because MuRF1, an E3 ubiquitin-protein ligase, is known to target βMHC for proteasomal degradation, we hypothesized MuRF1 mediates the sarcopenic and hypocontractile phenotype. Genetic ablation of TRIM63 (encoding MuRF1) significantly reduced MYH7 wt/E848G hiPSC-CM cell size by flow cytometry (6.8 ± 1.7%). MYH7 wt/E848G TRIM63 -/- hiPSC-CMs increased protein expression of βMHC (154.7 ± 51.9%), αSA (44.9 ± 25.6%), and MLC-1v (246.9 ± 57.3%) relative to MYH7 wt/E848G TRIM63 +/+ hiPSC-CMs. MYH7 wt/E848G TRIM63 -/- had significantly higher maximum twitch force by TFM (133 ± 14 nN) and EHTs (178.2 ± 9.8 μN) than MYH7 wt/E848G TRIM63 +/+ by TFM (67 ± 8.0 nN) and EHTs (118.9 ± 23.7 μN). Our results implicate sarcopenia in the pathogenesis of MYH7 E848G HCM and identify MuRF1 as a potential novel therapeutic target for HCM. Further study will determine if these findings are generalizable to other pathogenic HCM variants in MYH7 and other sarcomeric genes.

Hypertrophic Cardiomyopathy: Genetic Foundations, Outcomes, Interconnections, and Their Modifiers.

Hypertrophic cardiomyopathy (HCM) is the most prevalent heritable cardiomyopathy. HCM is considered to be caused by mutations in cardiac sarcomeric protein genes. Recent research suggests that the genetic foundation of HCM is much more complex than originally postulated. The clinical presentations of HCM are very variable. Some mutation carriers remain asymptomatic, while others develop severe HCM, terminal heart failure, or sudden cardiac death. Heterogeneity regarding both genetic mutations and the clinical course of HCM hinders the establishment of universal genotype-phenotype correlations. However, some trends have been identified. The presence of a mutation in some genes encoding sarcomeric proteins is associated with earlier HCM onset, more severe left ventricular hypertrophy, and worse clinical outcomes. There is a diversity in the mechanisms implicated in the pathogenesis of HCM. They may be classified into groups, but they are interrelated. The lack of known supplementary elements that control the progression of HCM indicates that molecular mechanisms that exist between genotype and clinical presentations may be crucial. Secondary molecular changes in pathways implicated in HCM pathogenesis, post-translational protein modifications, and epigenetic factors affect HCM phenotypes. Cardiac loading conditions, exercise, hypertension, diet, alcohol consumption, microbial infection, obstructive sleep apnea, obesity, and environmental factors are non-molecular aspects that change the HCM phenotype. Many mechanisms are implicated in the course of HCM. They are mostly interconnected and contribute to some extent to final outcomes.

Abstract P2068: β Iv -spectrin/stat3 Are Responsible For Concentric Versus Eccentric Cardiac Hypertrophy

Hypertrophy is critical to development of heart failure following cardiac stress. In pressure overload, hypertrophy is divided by early concentric and chronic eccentric orientations, however, the mechanisms regulating this transition and the impact on heart function are largely unknown. Previous work identified the structural protein β IV -spectrin and its interaction with the transcription factor, STAT3, in coordinating hypertrophic profiles. Specifically, mice expressing truncated β IV -spectrin lacking interaction with the kinase CaMKII ( qv 3J mice) resisted β IV -spectrin degradation and maintained concentric hypertrophy and systolic function compared to WT following 6 weeks transaortic constriction (TAC). Alternatively, mutant β IV -spectrin mice lacking the β IV -spectrin/STAT3 binding domain ( qv 4J ) expressed reduced EF and LV dilation, consistent with eccentric remodeling, suggesting β IV -spectrin/STAT3 is a regulator of cardiac profiles. This relationship was examined by adenoviral expression of the hypertrophic driver MEK1/2 on isolated myocytes, where MEK increased the width of WT cells but length in qv 4J . Notably, application of STAT3 inhibitor S3I-201 on qv 4J myocytes restored growth in width. Additionally, in vivo delivery of S3I-201 to WT mice after 6 weeks TAC reduced LV dilation and recovered systolic function. To enable hypertrophy, recent work identified the need for microtubules to traffic and locally translate sarcomeric transcripts. Therefore, we tested the hypothesis that β IV -spectrin/STAT3 direct orientation by altering microtubule stability and sarcomeric transcript trafficking. Isolated myocytes from WT and qv 4J mice were treated with the microtubule depolymerizing drug Nocodazole and then washed to evaluate microtubule repolymerization. Microtubules restored faster in qv 4J myocytes but could be reduced to WT levels by treating with S3I-201. Furthermore, localization of the sarcomeric gene, actc1 (α-actinin) by RNAScope showed more peripheral localization in qv 4J myocytes, compared to WT, and was normalized by S3I-201. Together, these data identify β IV -spectrin/STAT3 as modulators of microtubule dynamics, sarcomeric transcript distribution, hypertrophic orientation, and systolic function.

Abstract P3041: Stage-specific Alternative Rna Splicing Attunes Cardiomyocyte Proliferation And Contraction

During heart development, cardiomyocytes display reduced proliferation and enhanced contractility to support cardiac functional maturation, but the mechanistic basis is not fully understood. Alternative RNA splicing is an important mechanism of RNA posttranscriptional modification and gene regulation in the heart. In studying a cardiac RNA binding protein, RBPMS (RNA-binding protein with multiple splicing), we found that RBPMS mediates distinct RNA splicing profiles in adult hearts versus neonatal hearts, indicating its stage-specific modulation of alternative RNA splicing. We showed that RBPMS regulates the splicing of cytoskeletal genes to enable proliferation of neonatal cardiomyocytes. In adult hearts, RBPMS regulates the splicing of sarcomere genes to support cardiac contraction. Cardiac-specific loss of RBPMS causes severe dilated cardiomyopathy and early lethality in adult mice. Using both in vivo and in vitro approaches, we found that the absence of RBPMS caused aberrant splicing of some key sarcomeric components, including Titin and Nexilin, causing cardiomyocyte contractile defects. Intriguingly, we also showed that RBPMS interacts with other cardiac RNA binding proteins, such as RBM20, suggesting their synergistic effects on cardiomyocyte physiology. Our work provides novel insights into how RNA binding proteins regulate cardiomyocyte proliferation and contraction by mediating alternative RNA splicing.