Myosin-Catalyzed ATP Hydrolysis in the Presence of Disease-Causing Mutations: Mavacamten as a Way to Repair Mechanism.

Hypertrophic cardiomyopathy is one of the most common forms of genetic cardiomyopathy. Mavacamten is a first-in-class myosin modulator that was identified via activity screening on the wild type, and it is FDA-approved for the treatment of obstructive hypertrophic cardiomyopathy (HCM). The drug selectively binds to the cardiac ß-myosin, inhibiting myosin function to decrease cardiac contractility. Though the drug is thought to affect multiple steps of the myosin cross-bridge cycle, its detailed mechanism of action is still under investigation. Individual steps in the overall cross-bridge cycle must be queried to elucidate the full mechanism of action. In this study, we utilize the rare-event method of transition path sampling to generate reactive trajectories to gain insights into the action of the drug on the dynamics and rate of the ATP hydrolysis step for human cardiac ß-myosin. We study three known HCM causative myosin mutations: R453C, P710R, and R712L to observe the effect of the drug on the alterations caused by these mutations in the chemical step. Since the crystal structure of the drug-bound myosin was not available at the time of this work, we created a model of the drug-bound system utilizing a molecular docking approach. We find a significant effect of the drug in one case, where the actual mechanism of the reaction is altered from the wild type by mutation. The drug restores both the rate of hydrolysis to the wildtype level and the mechanism of the reaction. This is a way to check the effect of the drug on untested mutations.

A therapeutic leap: how myosin inhibitors moved from cardiac interventions to skeletal muscle myopathy solutions.

The myosin inhibitor mavacamten has transformed the management of obstructive hypertrophic cardiomyopathy (HCM) by targeting myosin ATPase activity to mitigate cardiac hypercontractility. This therapeutic mechanism has proven effective for patients with HCM independent of having a primary gene mutation in myosin. In this issue of the JCI, Buvoli et al. report that muscle hypercontractility is a mechanism of pathogenesis underlying muscle dysfunction in Laing distal myopathy, a disorder characterized by mutations altering the rod domain of ß myosin heavy chain. The authors performed detailed physiological, molecular, and biomechanical analyses and demonstrated that myosin ATPase inhibition can correct a large extent of muscle abnormalities. The findings offer a therapeutic avenue for Laing distal myopathy and potentially other myopathies. This Commentary underscores the importance of reevaluating myosin activity's role across myopathies in general for the potential development of targeted myosin inhibitors to treat skeletal muscle disorders.

Modeling cardiomyocyte signaling and metabolism predicts genotype-to-phenotype mechanisms in hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (HCM) is a significant precursor of heart failure and sudden cardiac death, primarily caused by mutations in sarcomeric and structural proteins. Despite the extensive research on the HCM genotype, the complex and context-specific nature of many signaling and metabolic pathways linking the HCM genotype to phenotype has hindered therapeutic advancements for patients. Here, we have developed a computational model of HCM encompassing cardiomyocyte signaling and metabolic networks and their associated interactions. Utilizing a stochastic logic-based ODE approach, we linked cardiomyocyte signaling to the metabolic network through a gene regulatory network and post-translational modifications. We validated the model against published data on activities of signaling species in the HCM context and transcriptomes of two HCM mouse models (i.e., R403Q-αMyHC and R92W-TnT). Our model predicts that HCM mutation induces changes in metabolic functions such as ATP synthase deficiency and a transition from fatty acids to carbohydrate metabolism. The model indicated major shifts in glutamine-related metabolism and increased apoptosis after HCM-induced ATP synthase deficiency. We predicted that the transcription factors STAT, SRF, GATA4, TP53, and FoxO are the key regulators of cardiomyocyte hypertrophy and apoptosis in HCM in alignment with experiments. Moreover, we identified shared (e.g., activation of PGC1α by AMPK, and FHL1 by titin) and context-specific mechanisms (e.g., regulation of Ca2+ sensitivity by titin in HCM patients) that may control genotype-to-phenotype transition in HCM across different species or mutations. We also predicted potential combination drug targets for HCM (e.g., mavacamten plus ROS inhibitors) preventing or reversing HCM phenotype (i.e., hypertrophic growth, apoptosis, and metabolic remodeling) in cardiomyocytes. This study provides new insights into mechanisms linking genotype to phenotype in familial hypertrophic cardiomyopathy and offers a framework for assessing new treatments and exploring variations in HCM experimental models.

Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration.

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the ß-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

A novel gene-trap line reveals the dynamic patterns and essential roles of cysteine and glycine-rich protein 3 in zebrafish heart development and regeneration.

Mutations in cysteine and glycine-rich protein 3 (CSRP3)/muscle LIM protein (MLP), a key regulator of striated muscle function, have been linked to hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, the roles of CSRP3 in heart development and regeneration are not completely understood. In this study, we characterized a novel zebrafish gene-trap line, gSAIzGFFM218A, which harbors an insertion in the csrp3 genomic locus, heterozygous fish served as a csrp3 expression reporter line and homozygous fish served as a csrp3 mutant line. We discovered that csrp3 is specifically expressed in larval ventricular cardiomyocytes (CMs) and that csrp3 deficiency leads to excessive trabeculation, a common feature of CSRP3-related HCM and DCM. We further revealed that csrp3 expression increased in response to different cardiac injuries and was regulated by several signaling pathways vital for heart regeneration. Csrp3 deficiency impeded zebrafish heart regeneration by impairing CM dedifferentiation, hindering sarcomere reassembly, and reducing CM proliferation while aggravating apoptosis. Csrp3 overexpression promoted CM proliferation after injury and ameliorated the impairment of ventricle regeneration caused by pharmacological inhibition of multiple signaling pathways. Our study highlights the critical role of Csrp3 in both zebrafish heart development and regeneration, and provides a valuable animal model for further functional exploration that will shed light on the molecular pathogenesis of CSRP3-related human cardiac diseases.

Myosin folding boosts solubility in cardiac muscle sarcomeres.

The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates that the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected > 5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may affect cardiac contractility.

Downregulation of salusins alleviates hypertrophic cardiomyopathy via attenuating oxidative stress and autophagy.

INTRODUCTION: Salusins, which are translated from the alternatively spliced mRNA of torsin family 2 member A (TOR2A), play a vital role in regulation of various cardiovascular diseases. However, it remains unclear precisely regarding their roles in hypertrophic cardiomyopathy (HCM). Therefore, this study was conducted to explore therapeutic effect and the underlying mechanisms of salusins on HCM. MATERIAL AND METHODS: In vivo experiments, Sprague-Dawley rats were used to induce HCM model by angiotensin (Ang) II infusion for 4 weeks. The rats were randomly divided into four groups, namely, Saline + Control shRNA (n = 7), Ang II + Control shRNA (n = 8), Saline + TOR2A shRNA (n = 7), and Ang II + TOR2A shRNA groups (n = 8). After HCM induction, doppler echocardiography is recommended to evaluate heart function. In vitro experiments, primary neonatal rat cardiomyocytes (NRCMs) and cardiac fibroblasts (NRCFs) were obtained from newborn rats, and were treated with Ang II (10-6 M) for 24 h. RESULTS: After treatment with Ang II, levels of salusin-α and salusin-ß were elevated in serum and cardiac tissues of rats and in the neonatal rat cardiomyocytes and cardiac fibroblasts. Downregulation of salusins alleviated the Ang II-induced cardiac hypertrophy by suppressing the increased atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and beta-myosin heavy chain (ß-MHC) and cardiac fibrosis by blocking collagen I, collagen III and transforming growth factor-beta (TGF-ß), and it also attenuated oxidative stress by suppressing the increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels and reversing the decreased superoxide dismutase (SOD) activity and autophagy by inhibiting the increased microtubule-associated protein light chain 3B (LC3B), Beclin1, autophagy related gene (Atg) 3 and Atg5 in the cardiac tissues of Ang II-infused rats and in the Ang II-treated NRCMs. CONCLUSIONS: All these findings suggest that the levels of salusins were elevated in the HCM, and targeting of salusins contributes to alleviation of cardiac hypertrophy and fibrosis probably via attenuating oxidative stress and autophagy. Accordingly, targeting of salusins may be a strategy for HCM therapy.

Multiplexed Functional Assessments of MYH7 Variants in Human Cardiomyocytes.

BACKGROUND: Pathogenic autosomal-dominant missense variants in MYH7 (myosin heavy chain 7), which encodes the sarcomeric protein (ß-MHC [beta myosin heavy chain]) expressed in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically actionable. However, ≈75% of MYH7 missense variants are of unknown significance. While human-induced pluripotent stem cells (hiPSCs) can be differentiated into cardiomyocytes to enable the interrogation of MYH7 variant effect in a disease-relevant context, deep mutational scanning has not been executed using diploid hiPSC derivates due to low hiPSC gene-editing efficiency. Moreover, multiplexable phenotypes enabling deep mutational scanning of MYH7 variant hiPSC-derived cardiomyocytes are unknown. METHODS: To overcome these obstacles, we used CRISPRa On-Target Editing Retrieval enrichment to generate an hiPSC library containing 113 MYH7 codon variants suitable for deep mutational scanning. We first established that ß-MHC protein loss occurs in a hypertrophic cardiomyopathy human heart with a pathogenic MYH7 variant. We then differentiated the MYH7 missense variant hiPSC library to cardiomyocytes for multiplexed assessment of ß-MHC variant abundance by massively parallel sequencing and hiPSC-derived cardiomyocyte survival. RESULTS: Both the multiplexed assessment of ß-MHC abundance and hiPSC-derived cardiomyocyte survival accurately segregated all known pathogenic variants from synonymous variants. Functional data were generated for 4 variants of unknown significance and 58 additional MYH7 missense variants not yet detected in patients. CONCLUSIONS: This study leveraged hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments of MYH7 missense variants for the first time. Phenotyping strategies used here enable the application of deep mutational scanning to clinically actionable genes, which should reduce the burden of variants of unknown significance on patients and clinicians.

Deficiency of Transcription Factor Sp1 Contributes to Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disorder. However, the pathogenesis of HCM, especially its nongenetic mechanisms, remains largely unclear. Transcription factors are known to be involved in various biological processes including cell growth. We hypothesized that SP1 (specificity protein 1), the first purified TF in mammals, plays a role in the cardiomyocyte growth and cardiac hypertrophy of HCM. METHODS: Cardiac-specific conditional knockout of Sp1 mice were constructed to investigate the role of SP1 in the heart. The echocardiography, histochemical experiment, and transmission electron microscope were performed to analyze the cardiac phenotypes of cardiac-specific conditional knockout of Sp1 mice. RNA sequencing, chromatin immunoprecipitation sequencing, and adeno-associated virus experiments in vivo were performed to explore the downstream molecules of SP1. To examine the therapeutic effect of SP1 on HCM, an SP1 overexpression vector was constructed and injected into the mutant allele of Myh6 R404Q/+ (Myh6 c. 1211C>T) HCM mice. The human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with HCM were used to detect the potential therapeutic effects of SP1 in human HCM. RESULTS: The cardiac-specific conditional knockout of Sp1 mice developed a typical HCM phenotype, displaying overt myocardial hypertrophy, interstitial fibrosis, and disordered myofilament. In addition, Sp1 knockdown dramatically increased the cell area of hiPSC-CMs and caused intracellular myofibrillar disorganization, which was similar to the hypertrophic cardiomyocytes of HCM. Mechanistically, Tuft1 was identified as the key target gene of SP1. The hypertrophic phenotypes induced by Sp1 knockdown in both hiPSC-CMs and mice could be rescued by TUFT1 (tuftelin 1) overexpression. Furthermore, SP1 overexpression suppressed the development of HCM in the mutant allele of Myh6 R404Q/+ mice and also reversed the hypertrophic phenotype of HCM hiPSC-CMs. CONCLUSIONS: Our study demonstrates that SP1 deficiency leads to HCM. SP1 overexpression exhibits significant therapeutic effects on both HCM mice and HCM hiPSC-CMs, suggesting that SP1 could be a potential intervention target for HCM.

Profiling cardiomyocytes at single cell resolution reveals COX7B could be a potential target for attenuating heart failure in cardiac hypertrophy.

Cardiac hypertrophy can develop to end-stage heart failure (HF), which inevitably leading to heart transplantation or death. Preserving cardiac function in cardiomyocytes (CMs) is essential for improving prognosis in hypertrophic cardiomyopathy (HCM) patients. Therefore, understanding transcriptomic heterogeneity of CMs in HCM would be indispensable to aid potential therapeutic targets investigation. We isolated primary CM from HCM patients who had extended septal myectomy, and obtained transcriptomes in 338 human primary CM with single-cell tagged reverse transcription (STRT-seq) approach. Our results revealed that CMs could be categorized into three subsets in nonfailing HCM heart: high energy synthesis cluster, high cellular metabolism cluster and intermediate cluster. The expression of electron transport chain (ETC) was up-regulated in larger-sized CMs from high energy synthesis cluster. Of note, we found the expression of Cytochrome c oxidase subunit 7B (COX7B), a subunit of Complex IV in ETC had trends of positively correlation with CMs size. Further, by assessing COX7B expression in HCM patients, we speculated that COX7B was compensatory up-regulated at early-stage but down-regulated in failing HCM heart. To test the hypothesis that COX7B might participate both in hypertrophy and HF progression, we used adeno associated virus 9 (AAV9) to mediate the expression of Cox7b in pressure overload-induced mice. Mice in vivo data supported that knockdown of Cox7b would accelerate HF and Cox7b overexpression could restore partial cardiac function in hypertrophy. Our result highlights targeting COX7B and preserving energy synthesis in hypertrophic CMs could be a promising translational direction for HF therapeutic strategy.

Mitochondrial dysfunction is associated with hypertrophic cardiomyopathy in Pompe disease-specific induced pluripotent stem cell-derived cardiomyocytes.

Pompe disease (PD) is a rare autosomal recessive disorder that presents with progressive hypertrophic cardiomyopathy. However, the detailed mechanism remains clarified. Herein, PD patient-specific induced pluripotent stem cells were differentiated into cardiomyocytes (PD-iCMs) that exhibited cardiomyopathic features of PD, including decreased acid alpha-glucosidase activity, lysosomal glycogen accumulation and hypertrophy. The defective mitochondria were involved in the cardiac pathology as shown by the significantly decreased number of mitochondria and impaired respiratory function and ATP production in PD-iCMs, which was partially due to elevated levels of intracellular reactive oxygen species produced from depolarized mitochondria. Further analysis showed that impaired fusion and autophagy of mitochondria and declined expression of mitochondrial complexes underlies the mechanism of dysfunctional mitochondria. This was alleviated by supplementation with recombinant human acid alpha-glucosidase that improved the mitochondrial function and concomitantly mitigated the cardiac pathology. Therefore, this study suggests that defective mitochondria underlie the pathogenesis of cardiomyopathy in patients with PD.

Myocardial Fibrosis in Hypertrophic Cardiomyopathy: A Perspective from Fibroblasts.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease and the leading cause of sudden cardiac death in young people. Mutations in genes that encode structural proteins of the cardiac sarcomere are the more frequent genetic cause of HCM. The disease is characterized by cardiomyocyte hypertrophy and myocardial fibrosis, which is defined as the excessive deposition of extracellular matrix proteins, mainly collagen I and III, in the myocardium. The development of fibrotic tissue in the heart adversely affects cardiac function. In this review, we discuss the latest evidence on how cardiac fibrosis is promoted, the role of cardiac fibroblasts, their interaction with cardiomyocytes, and their activation via the TGF-ß pathway, the primary intracellular signalling pathway regulating extracellular matrix turnover. Finally, we summarize new findings on profibrotic genes as well as genetic and non-genetic factors involved in the pathophysiology of HCM.

Nonsense mediated decay factor UPF3B is associated with cMyBP-C haploinsufficiency in hypertrophic cardiomyopathy patients.

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiac disease. Up to 40% of cases are associated with heterozygous mutations in myosin binding protein C (cMyBP-C, MYBPC3). Most of these mutations lead to premature termination codons (PTC) and patients show reduction of functional cMyBP-C. This so-called haploinsufficiency most likely contributes to disease development. We analyzed mechanisms underlying haploinsufficiency using cardiac tissue from HCM-patients with truncation mutations in MYBPC3 (MYBPC3trunc). We compared transcriptional activity, mRNA and protein expression to donor controls. To differentiate between HCM-specific and general hypertrophy-induced mechanisms we used patients with left ventricular hypertrophy due to aortic stenosis (AS) as an additional control. We show that cMyBP-C haploinsufficiency starts at the mRNA level, despite hypertrophy-induced increased transcriptional activity. Gene set enrichment analysis (GSEA) of RNA-sequencing data revealed an increased expression of NMD-components. Among them, Up-frameshift protein UPF3B, a regulator of NMD was upregulated in MYBPC3trunc patients and not in AS-patients. Strikingly, we show that in sarcomeres UPF3B but not UPF1 and UPF2 are localized to the Z-discs, the presumed location of sarcomeric protein translation. Our data suggest that cMyBP-C haploinsufficiency in HCM-patients is established by UPF3B-dependent NMD during the initial translation round at the Z-disc.

Single-nuclear transcriptome profiling identifies persistent fibroblast activation in hypertrophic and failing human hearts of patients with longstanding disease.

AIMS: Cardiac fibrosis drives the progression of heart failure in ischaemic and hypertrophic cardiomyopathy. Therefore, the development of specific anti-fibrotic treatment regimens to counteract cardiac fibrosis is of high clinical relevance. Hence, this study examined the presence of persistent fibroblast activation during longstanding human heart disease at a single-cell resolution to identify putative therapeutic targets to counteract pathological cardiac fibrosis in patients. METHODS AND RESULTS: We used single-nuclei RNA sequencing with human tissues from two samples of one healthy donor, and five hypertrophic and two failing hearts. Unsupervised sub-clustering of 7110 nuclei led to the identification of 7 distinct fibroblast clusters. De-convolution of cardiac fibroblast heterogeneity revealed a distinct population of human cardiac fibroblasts with a molecular signature of persistent fibroblast activation and a transcriptional switch towards a pro-fibrotic extra-cellular matrix composition in patients with established cardiac hypertrophy and heart failure. This sub-cluster was characterized by high expression of POSTN, RUNX1, CILP, and a target gene adipocyte enhancer-binding protein 1 (AEBP1) (all P < 0.001). Strikingly, elevated circulating AEBP1 blood level were also detected in a validation cohort of patients with confirmed cardiac fibrosis and hypertrophic cardiomyopathy by cardiac magnetic resonance imaging (P < 0.01). Since endogenous AEBP1 expression was increased in patients with established cardiac hypertrophy and heart failure, we assessed the functional consequence of siRNA-mediated AEBP1 silencing in human cardiac fibroblasts. Indeed, AEBP1 silencing reduced proliferation, migration, and fibroblast contractile capacity and α-SMA gene expression, which is a hallmark of fibroblast activation (all P < 0.05). Mechanistically, the anti-fibrotic effects of AEBP1 silencing were linked to transforming growth factor-beta pathway modulation. CONCLUSION: Together, this study identifies persistent fibroblast activation in patients with longstanding heart disease, which might be detected by circulating AEBP1 and therapeutically modulated by its targeted silencing in human cardiac fibroblasts.

Vps4a Regulates Autophagic Flux to Prevent Hypertrophic Cardiomyopathy.

Autophagy has stabilizing functions for cardiomyocytes. Recent studies indicate that an impairment in the autophagy pathway can seriously affect morphology and function, potentially leading to heart failure. However, the role and the underlying mechanism of the endosomal sorting complex required for transport (ESCRT) family protein, in particular the AAA-ATPase vacuolar protein sorting 4a (Vps4a), in regulating myocardial autophagy remains unclear. In the present study, cardiomyocyte-specific Vps4a knockout mice were generated by crossing Vps4aflox/flox (Vps4afl/fl) with Myh6-cre transgenic mice. As a result, we observed a partially dilated left ventricular (LV) chamber, a significant increase in heart weight to body weight ratio (HW/BW), and heart weight to tibial length ratio (HW/TL), hypertrophic cardiomyopathy and early lethality starting at 3 months of age. Hematoxylin-eosin (HE), immunofluorescence assay (IFA), and Western blot (WB) revealed autophagosome accumulation in cardiomyocytes. A transcriptome-based analysis and autophagic flux tracking by AAV-RFP-GFP-LC3 showed that the autophagic flux was blocked in Vps4a knockout cardiomyocytes. In addition, we provided in vitro evidence demonstrating that Vps4a and LC3 were partially co-localized in cardiomyocytes, and the knockdown of Vps4a led to the accumulation of autophagosomes in cardiomyocytes. Similarly, the transfection of cardiomyocytes with adenovirus (Adv) mCherry-GFP-LC3 further indicated that the autophagic flux was blocked in cells with deficient levels of Vps4a. Finally, an electron microscope (EM) showed that the compromised sealing of autophagosome blocked the autophagic flux in Vps4a-depleted cardiomyocytes. These findings revealed that Vps4a contributed to the sealing of autophagosomes in cardiomyocytes. Therefore, we demonstrated that Vps4a deletion could block the autophagic flux, leading to the accumulation of degradation substances and compromised cardiac function. Overall, this study provides insights into a new theoretical basis for which autophagy may represent a therapeutic target for cardiovascular diseases.

EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy.

BACKGROUND: Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. METHODS: We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model. RESULTS: Screening of 157 kinase inhibitors in wild-type (N=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (N=24 HCM and N=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (N=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. CONCLUSIONS: We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.

iPSC-Based Modeling of Variable Clinical Presentation in Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and a frequent cause of heart failure and sudden cardiac death. Our understanding of the genetic bases and pathogenic mechanisms underlying HCM has improved significantly in the recent past, but the combined effect of various pathogenic gene variants and the influence of genetic modifiers in disease manifestation are very poorly understood. Here, we set out to investigate genotype-phenotype relationships in 2 siblings with an extensive family history of HCM, both carrying a pathogenic truncating variant in the MYBPC3 gene (p.Lys600Asnfs*2), but who exhibited highly divergent clinical manifestations. METHODS: We used a combination of induced pluripotent stem cell (iPSC)-based disease modeling and CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated genome editing to generate patient-specific cardiomyocytes (iPSC-CMs) and isogenic controls lacking the pathogenic MYBPC3 variant. RESULTS: Mutant iPSC-CMs developed impaired mitochondrial bioenergetics, which was dependent on the presence of the mutation. Moreover, we could detect altered excitation-contraction coupling in iPSC-CMs from the severely affected individual. The pathogenic MYBPC3 variant was found to be necessary, but not sufficient, to induce iPSC-CM hyperexcitability, suggesting the presence of additional genetic modifiers. Whole-exome sequencing of the mutant carriers identified a variant of unknown significance in the MYH7 gene (p.Ile1927Phe) uniquely present in the individual with severe HCM. We finally assessed the pathogenicity of this variant of unknown significance by functionally evaluating iPSC-CMs after editing the variant. CONCLUSIONS: Our results indicate that the p.Ile1927Phe variant of unknown significance in MYH7 can be considered as a modifier of HCM expressivity when found in combination with truncating variants in MYBPC3. Overall, our studies show that iPSC-based modeling of clinically discordant subjects provides a unique platform to functionally assess the effect of genetic modifiers.

Clinical Trials in Hypertrophic Cardiomyopathy Therapy: A Comprehensive Analysis of Trials Registered in Global Clinical Databases.

Background: With the disappointing results associated with the use of cardiac myosin inhibitors in the treatment of hypertrophic cardiomyopathy (HCM), the development of new therapies in clinical trials for HCM has rapidly increased. We assessed the characteristics of therapeutic intervention in HCM registered on ClinicalTrials.gov and the International Clinical Trials Registry Platform (ICTRP). Methods: We conducted a cross-sectional, descriptive study of clinical trials for therapeutic intervention in HCM registered on ClinicalTrials.gov and ICTRP. Results: This study analyzed 137 registered trials. Regarding study designs of these trials, 77.37% were purpose of treatment, 59.12% were randomized, 50.36% were parallel assignment, 45.26% were performed with masking, 48.18% recruited less than 50 participants, and 27.74% were Phase 2 trials. In total, 67 trials were new drug trials, of which 35 drugs were tested in these trials, and 13 trials involved treatment with mavacamten. Of these 67 clinical drug trials, 44.78% of trials involved the study of amines, and 16.42% involved 1-ring heterocyclic compounds. Regarding the NCI Thesaurus Tree, 23.81% of trials involved myosin inhibitors, 23.81% of trials involved drugs belonging to agents affecting the cardiovascular system, and 20.63% were involved in testing cation channel blockers. The drug-target network showed that myosin-7, potassium voltage-gated channel subfamily h member 2, beta-1 adrenergic receptor, carnitine o-palmitoyltransferase 1, and liver isoform were the most targeted pathways of the clinical trials analyzed in the drug-target network. Conclusion: The number of clinical trials investigating therapeutic interventions for HCM has increased in recent years. Ultimately, recent HCM therapeutic clinical trials generally did not incorporate either randomized controlled trials or masking and were small studies recruiting fewer than 50 participants. Although recent research has focused on targeting myosin-7, the molecular signaling mechanisms involved in the pathogenesis of HCM have the potential to elucidate novel target pathways.

Molecular and cellular evidence for the impact of a hypertrophic cardiomyopathy-associated RAF1 variant on the structure and function of contractile machinery in bioartificial cardiac tissues.

Noonan syndrome (NS), the most common among RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770 C > T missense change. We characterize the molecular, structural, and functional consequences of aberrant RAF1-associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes and myocardial tissue biopsies. The aforementioned changes correlated with the isoform shift of titin from a longer (N2BA) to a shorter isoform (N2B) that also affected the active force generation and contractile tensions. The genotype-phenotype correlation was confirmed using cardiomyocyte progeny of an isogenic gene-corrected RAF1S257L-iPSC line and was mainly reversed by MEK inhibition. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease.

1-Deoxynojirimycin promotes cardiac function and rescues mitochondrial cristae in mitochondrial hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most prominent cause of sudden cardiac death in young people. Due to heterogeneity in clinical manifestations, conventional HCM drugs have limitations for mitochondrial hypertrophic cardiomyopathy. Discovering more effective compounds would be of substantial benefit for further elucidating the pathogenic mechanisms of HCM and treating patients with this condition. We previously reported the MT-RNR2 variant associated with HCM that results in mitochondrial dysfunction. Here, we screened a mitochondria-associated compound library by quantifying the mitochondrial membrane potential of HCM cybrids and the survival rate of HCM-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in galactose media. 1-Deoxynojirimycin (DNJ) was identified to rescue mitochondrial function by targeting optic atrophy protein 1 (OPA1) to promote its oligomerization, leading to reconstruction of the mitochondrial cristae. DNJ treatment further recovered the physiological properties of HCM iPSC-CMs by improving Ca2+ homeostasis and electrophysiological properties. An angiotensin II-induced cardiac hypertrophy mouse model further verified the efficacy of DNJ in promoting cardiac mitochondrial function and alleviating cardiac hypertrophy in vivo. These results demonstrated that DNJ could be a potential mitochondrial rescue agent for mitochondrial hypertrophic cardiomyopathy. Our findings will help elucidate the mechanism of HCM and provide a potential therapeutic strategy.