Omecamtiv mecarbil and Mavacamten target the same myosin pocket despite opposite effects in heart contraction.

Inherited cardiomyopathies are common cardiac diseases worldwide, leading in the late stage to heart failure and death. The most promising treatments against these diseases are small molecules directly modulating the force produced by ß-cardiac myosin, the molecular motor driving heart contraction. Omecamtiv mecarbil and Mavacamten are two such molecules that completed phase 3 clinical trials, and the inhibitor Mavacamten is now approved by the FDA. In contrast to Mavacamten, Omecamtiv mecarbil acts as an activator of cardiac contractility. Here, we reveal by X-ray crystallography that both drugs target the same pocket and stabilize a pre-stroke structural state, with only few local differences. All-atom molecular dynamics simulations reveal how these molecules produce distinct effects in motor allostery thus impacting force production in opposite way. Altogether, our results provide the framework for rational drug development for the purpose of personalized medicine.

Retinoic acid modulation guides human-induced pluripotent stem cell differentiation towards left or right ventricle-like cardiomyocytes.

BACKGROUND: Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other non-cardiomyocyte cells. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart. METHODS: CMs were derived from hiPSC (hi-PCS-CM) using different concentrations of RA (Control without RA, LRA with 0.05µM and HRA with 0.1 µM) between day 3-6 of the differentiation process. Engineered heart tissues (EHTs) were generated by assembling hiPSC-CM at high cell density in a low collagen hydrogel. RESULTS: In the HRA group, hiPSC-CMs exhibited highest expression of contractile proteins MYH6, MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are marker genes for left ventricular CMs, was also the highest in the HRA group. In terms of EHT, the HRA group displayed the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline, which means it was functionally more similar to the left ventricle. RNAsequencing revealed that the heightened contractility of EHT within the HRA group can be attributed to the promotion of augmented extracellular matrix strength by RA. CONCLUSION: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHTs with a phenotype similar to that of the left ventricle or right ventricle.

MYH7 R453C induced cardiac remodelling via activating TGF-ß/Smad2/3, ERK1/2 and Nox4/ROS/NF-κB signalling pathways.

Hypertrophic cardiomyopathy (HCM) is a monogenic cardiac disorder commonly induced by sarcomere gene mutations. However, the mechanism for HCM is not well defined. Here, we generated transgenic MYH7 R453C and MYH6 R453C piglets and found both developed typical cardiac hypertrophy. Unexpectedly, we found serious fibrosis and cardiomyocyte loss in the ventricular of MYH7 R453C, not MYH6 R453C piglets, similar to HCM patients. Then, RNA-seq analysis and western blotting identified the activation of ERK1/2 and PI3K-Akt pathways in MYH7 R453C. Moreover, we observed an increased expression of fetal genes and an excess of reactive oxygen species (ROS) in MYH7 R453C piglet models, which was produced by Nox4 and subsequently induced inflammatory response. Additionally, the phosphorylation levels of Smad2/3, ERK1/2 and NF-kB p65 proteins were elevated in cardiomyocytes with the MYH7 R453C mutation. Furthermore, epigallocatechin gallate, a natural bioactive compound, could be used as a drug to reduce cell death by adjusting significant downregulation of the protein expression of Bax and upregulated Bcl-2 levels in the H9C2 models with MYH7 R453C mutation. In conclusion, our study illustrated that TGF-ß/Smad2/3, ERK1/2 and Nox4/ROS pathways have synergistic effects on cardiac remodelling and inflammation in MYH7 R453C mutation.

Promising therapies for adults with symptomatic obstructive hypertrophic cardiomyopathy: 2023 and beyond.

INTRODUCTION: Hypertrophic cardiomyopathy (HCM) is a heterogeneous genetic heart disease with an estimated prevalence in the general population of 0.2% to 0.6%. Clinically, HCM can range from no symptoms to severe symptoms such as heart failure or sudden cardiac death. Currently, the management of HCM involves lifestyle modifications, familial screening, genetic counseling, pharmacotherapy to manage symptoms, sudden cardiac death risk assessment, septal reduction therapy, and heart transplantation for specific patients. Multicenter randomized controlled trials have only recently explored the potential of cardiac myosin inhibitors (CMIs) such as mavacamten as a directed pharmacological approach for managing HCM. AREAS COVERED: We will assess the existing medical treatments for HCM: beta-blockers, calcium channel blockers, disopyramide, and different CMIs. We will also discuss future HCM pharmacotherapy guidelines and underline this patient population's unfulfilled needs. EXPERT OPINION: Mavacamten is the first-in-class CMI approved by the FDA to target HCM pathophysiology specifically. Mavacamten should be incorporated into the standard therapy for oHCM in case of symptom persistence despite using maximally tolerated beta blockers and/or calcium channel blockers. Potential drug-drug interactions should be assessed before initiating this drug. More studies are needed on the use of CMIs in patients with kidney and/or liver failure and pregnant/breastfeeding patients.

Target population for a selective cardiac myosin inhibitor in hypertrophic obstructive cardiomyopathy: Real-life estimation from the French register of hypertrophic cardiomyopathy (REMY).

BACKGROUND: The efficacy of current pharmacological therapies in hypertrophic cardiomyopathy is limited. A cardiac myosin inhibitor, mavacamten, has recently been approved as a first-in-class treatment for symptomatic hypertrophic obstructive cardiomyopathy. AIMS: To assess the profile and burden of cardiac myosin inhibitor candidates in the hypertrophic cardiomyopathy prospective Register of hypertrophic cardiomyopathy (REMY) held by the French Society of Cardiology. METHODS: Data were collected at baseline and during follow-up from patients with hypertrophic cardiomyopathy enrolled in REMY by the three largest participating centres. RESULTS: Among 1059 adults with hypertrophic cardiomyopathy, 461 (43.5%) had obstruction; 325 (30.7%) of these were also symptomatic, forming the "cardiac myosin inhibitor candidates" group. Baseline features of this group were: age 58±15years; male sex (n=196; 60.3%); diagnosis-to-inclusion delay 5 (1-12)years; maximum wall thickness 20±6mm; left ventricular ejection fraction 69±6%; family history of hypertrophic cardiomyopathy or sudden cardiac death (n=133; 40.9%); presence of a pathogenic sarcomere gene mutation (n=101; 31.1%); beta-blocker or verapamil treatment (n=304; 93.8%), combined with disopyramide (n=28; 8.7%); and eligibility for septal reduction therapy (n=96; 29%). At the end of a median follow-up of 66 (34-106) months, 319 (98.2%) were treated for obstruction (n=43 [13.2%] received disopyramide), 46 (14.2%) underwent septal reduction therapy and the all-cause mortality rate was 1.9/100 person-years (95% confidence interval 1.4-2.6) (46 deaths). Moreover, 41 (8.9%) patients from the initial hypertrophic obstructive cardiomyopathy group became eligible for a cardiac myosin inhibitor. CONCLUSIONS: In this cohort of patients with hypertrophic cardiomyopathy selected from the REMY registry, one third were eligible for a cardiac myosin inhibitor.

A Laing distal myopathy-associated proline substitution in the ß-myosin rod perturbs myosin cross-bridging activity.

Proline substitutions within the coiled-coil rod region of the ß-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal dominant disorder characterized by progressive weakness of distal/proximal muscles. We report that the MDP1 mutation R1500P, studied in what we believe to be the first mouse model for the disease, adversely affected myosin motor activity despite being in the structural rod domain that directs thick filament assembly. Contractility experiments carried out on isolated mutant muscles, myofibrils, and myofibers identified muscle fatigue and weakness phenotypes, an increased rate of actin-myosin detachment, and a conformational shift of the myosin heads toward the more reactive disordered relaxed (DRX) state, causing hypercontractility and greater ATP consumption. Similarly, molecular analysis of muscle biopsies from patients with MPD1 revealed a significant increase in sarcomeric DRX content, as observed in a subset of myosin motor domain mutations causing hypertrophic cardiomyopathy. Finally, oral administration of MYK-581, a small molecule that decreases the population of heads in the DRX configuration, significantly improved the limited running capacity of the R1500P-transgenic mice and corrected the increased DRX state of the myofibrils from patients. These studies provide evidence of the molecular pathogenesis of proline rod mutations and lay the groundwork for the therapeutic advancement of myosin modulators.

Complex genotype-phenotype correlation of MYH11: new insights from monozygotic twins with highly variable expressivity and outcomes.

BACKGROUND: Thoracic aortic aneurysm/dissection (TAAD) and patent ductus arteriosus (PDA) are serious autosomal-dominant diseases affecting the cardiovascular system. They are mainly caused by variants in the MYH11 gene, which encodes the heavy chain of myosin 11. The aim of this study was to evaluate the genotype-phenotype correlation of MYH11 from a distinctive perspective based on a pair of monozygotic twins. METHODS: The detailed phenotypic characteristics of the monozygotic twins from the early fetal stage to the infancy stage were traced and compared with each other and with those of previously documented cases. Whole-exome and Sanger sequencing techniques were used to identify and validate the candidate variants, facilitating the analysis of the genotype-phenotype correlation of MYH11. RESULTS: The monozygotic twins were premature and presented with PDA, pulmonary hypoplasia, and pulmonary hypertension. The proband developed heart and brain abnormalities during the fetal stage and died at 18 days after birth, whereas his sibling was discharged after being cured and developed normally post follow-up. A novel variant c.766 A > G p. (Ile256Val) in MYH11 (NM_002474.2) was identified in the monozygotic twins and classified as a likely pathogenic variant according to the American College of Medical Genetics/Association for Molecular Pathology guidelines. Reviewing the reported cases (n = 102) showed that the penetrance of MYH11 was 82.35%, and the most common feature was TAAD (41.18%), followed by PDA (22.55%), compound TAAD and PDA (9.80%), and other vascular abnormalities (8.82%). The constituent ratios of null variants among the cases with TAAD (8.60%), PDA (43.8%), or compound TAAD and PDA (28.6%) were significantly different (P = 0.01). Further pairwise comparison of the ratios among these groups showed that there were significant differences between the TAAD and PDA groups (P = 0.006). CONCLUSION: This study expands the mutational spectrum of MYH11 and provides new insights into the genotype-phenotype correlation of MYH11 based on the monozygotic twins with variable clinical features and outcomes, indicating that cryptic modifiers and complex mechanisms beside the genetic variants may be involved in the condition.

Tail length and E525K dilated cardiomyopathy mutant alter human ß-cardiac myosin super-relaxed state.

Dilated cardiomyopathy (DCM) is a condition characterized by impaired cardiac function, due to myocardial hypo-contractility, and is associated with point mutations in ß-cardiac myosin, the molecular motor that powers cardiac contraction. Myocardial function can be modulated through sequestration of myosin motors into an auto-inhibited "super-relaxed" state (SRX), which may be further stabilized by a structural state known as the "interacting heads motif" (IHM). Here, we sought to determine whether hypo-contractility of DCM myocardium results from reduced function of individual myosin molecules or from decreased myosin availability to interact with actin due to increased IHM/SRX stabilization. We used an established DCM myosin mutation, E525K, and characterized the biochemical and mechanical activity of wild-type and mutant human ß-cardiac myosin constructs that differed in the length of their coiled-coil tail, which dictates their ability to form the IHM/SRX state. We found that short-tailed myosin constructs exhibited low IHM/SRX content, elevated actin-activated ATPase activity, and fast velocities in unloaded motility assays. Conversely, longer-tailed constructs exhibited higher IHM/SRX content and reduced actomyosin ATPase and velocity. Our modeling suggests that reduced velocities may be attributed to IHM/SRX-dependent sequestration of myosin heads. Interestingly, longer-tailed E525K mutants showed no apparent impact on velocity or actomyosin ATPase at low ionic strength but stabilized IHM/SRX state at higher ionic strength. Therefore, the hypo-contractility observed in DCM may be attributable to reduced myosin head availability caused by enhanced IHM/SRX stability in E525K mutants.

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.

Myosin inhibitor reverses hypertrophic cardiomyopathy in genotypically diverse pediatric iPSC-cardiomyocytes to mirror variant correction.

Pathogenic variants in MYH7 and MYBPC3 account for the majority of hypertrophic cardiomyopathy (HCM). Targeted drugs like myosin ATPase inhibitors have not been evaluated in children. We generate patient and variant-corrected iPSC-cardiomyocytes (CMs) from pediatric HCM patients harboring single variants in MYH7 (V606M; R453C), MYBPC3 (G148R) or digenic variants (MYBPC3 P955fs, TNNI3 A157V). We also generate CMs harboring MYBPC3 mono- and biallelic variants using CRISPR editing of a healthy control. Compared with isogenic and healthy controls, variant-positive CMs show sarcomere disorganization, higher contractility, calcium transients, and ATPase activity. However, only MYH7 and biallelic MYBPC3 variant-positive CMs show stronger myosin-actin binding. Targeted myosin ATPase inhibitors show complete rescue of the phenotype in variant-positive CMs and in cardiac Biowires to mirror isogenic controls. The response is superior to verapamil or metoprolol. Myosin inhibitors can be effective in genotypically diverse HCM highlighting the need for myosin inhibitor drug trials in pediatric HCM.

Novel MYH7 Variant in the Neonate of a Mother with Gestational Diabetes Mellitus Showing Left Ventricular Hypertrophy and Noncompaction.

BACKGROUND: Left ventricular hypertrophy (LVH) is a well-recognized cardiac dysfunction in infants of mothers with gestational diabetes mellitus (GDM). Left ventricular noncompaction (LVNC) is a cardiomyopathy that is morphologically characterized by numerous prominent trabeculations and deep intertrabecular recesses on cardiovascular imaging. However, there have been no case reports on neonates of mothers with GDM showing LVH and LVNC. CASE PRESENTATION: A patient, with LVH of a mother with GDM, was delivered at 36 weeks of gestation. Prominent trabeculations in the LV, suggesting LVNC, instead of LVH, were apparent 1 week after birth. A heterozygous deletion variant in the MYH7 gene (NM_000257.4: c.1090T>C, p.Phe364Leu) was discovered through genetic testing using a cardiomyopathy-associated gene panel in the patient and his father and the older brother who had LVNC. The patient is now 5 years old and does not have major cardiac events, although LVNC persisted. This is the first case of LVH secondary to a mother with GDM and LVNC with a novel variant in the MYH7 gene. CONCLUSION: Genetic testing should be conducted to obtain an accurate outcome and medical care in a patient with LVH and subsequently prominent hypertrabeculation in the LV.

Exploring the Super-Relaxed State of Human Cardiac ß-Myosin by Molecular Dynamics Simulations.

Human ß-cardiac myosin plays a critical role in generating the mechanical forces necessary for cardiac muscle contraction. This process relies on a delicate dynamic equilibrium between the disordered relaxed state (DRX) and the super-relaxed state (SRX) of myosin. Disruptions in this equilibrium due to mutations can lead to heart diseases. However, the structural characteristics of SRX and the molecular mechanisms underlying pathogenic mutations have remained elusive. To bridge this gap, we conducted molecular dynamics simulations and free energy calculations to explore the conformational changes in myosin. Our findings indicate that the size of the phosphate-binding pocket can serve as a valuable metric for characterizing the transition from the DRX to SRX state. Importantly, we established a global dynamic coupling network within the myosin motor head at the residue level, elucidating how the pathogenic mutation E483K impacts the equilibrium between SRX and DRX through allosteric effects. Our work illuminates molecular details of SRX and offers valuable insights into disease treatment through the regulation of SRX.

A New Leu714Arg Variant in the Converter Domain of MYH7 is Associated with a Severe Form of Familial Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy is the most frequent autosomal dominant disease, yet due to genetic heterogeneity, incomplete penetrance, and phenotype variability, the prognosis of the disease course in pathogenic variant carriers remains an issue. Identifying common patterns among the effects of different genetic variants is important. METHODS: We investigated the cause of familial hypertrophic cardiomyopathy (HCM) in a family with two patients suffering from a particularly severe disease. Searching for the genetic variants in HCM genes was performed using different sequencing methods. RESULTS: A new missense variant, p.Leu714Arg, was identified in exon 19 of the beta-myosin heavy chain gene (MYH7). The mutation was found in a region that encodes the 'converter domain' in the globular myosin head. This domain is essential for the conformational change of myosin during ATP cleavage and contraction cycle. Most reports on different mutations in this region describe severe phenotypic consequences. The two patients with the p.Leu714Arg mutation had heart failure early in life and died from HCM complications. CONCLUSIONS: This case presents a new likely pathogenic variant in MYH7 and supports the hypothesis that myosin converter mutations constitute a subclass of HCM mutations with a poor prognosis for the patient.

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.

Variants of the promoter of MYH6 gene in congenital isolated and sporadic patent ductus arteriosus: case-control study and cellular functional analyses.

Patent ductus arteriosus (PDA) is a common form of congenital heart disease. The MYH6 gene has important effects on cardiovascular growth and development, but the effect of variants in the MYH6 gene promoter on ductus arteriosus is unknown. DNA was extracted from blood samples of 721 subjects (428 patients with isolated and sporadic PDA and 293 healthy controls) and analyzed by sequencing for MYH6 gene promoter region variants. Cellular function experiments with three cell lines (HEK-293, HL-1, and H9C2 cells) and bioinformatics analyses were performed to verify their effects on gene expression. In the MYH6 gene promoter, 11 variants were identified. Four variants were found only in patients with PDA and 2 of them (g.3434G>C and g.4524C>T) were novel. Electrophoretic mobility shift assay showed that the transcription factors bound by the promoter variants were significantly altered in comparison to the wild-type in all three cell lines. Dual luciferase reporter showed that all the 4 variants reduced the transcriptional activity of the MYH6 gene promoter (P < 0.05). Prediction of transcription factors bound by the variants indicated that these variants alter the transcription factor binding sites. These pathological alterations most likely affect the contraction of the smooth muscle of ductus arteriosus, leading to PDA. This study is the first to focus on variants at the promoter region of the MYH6 gene in PDA patients with cellular function tests. Therefore, this study provides new insights to understand the genetic basis and facilitates further studies on the mechanism of PDA formation.

Missense variants in phospholamban and cardiac myosin binding protein identified in patients with a family history and clinical diagnosis of dilated cardiomyopathy.

As the genetic landscape of cardiomyopathies continues to expand, the identification of missense variants in disease-associated genes frequently leads to a classification of variant of uncertain significance (VUS). For the proper reclassification of such variants, functional characterization is an important contributor to the proper assessment of pathogenic potential. Several missense variants in the calcium transport regulatory protein phospholamban have been associated with dilated cardiomyopathy. However, >40 missense variants in this transmembrane peptide are currently known and most remain classified as VUS with little clinical information. Similarly, missense variants in cardiac myosin binding protein have been associated with hypertrophic cardiomyopathy. However, hundreds of variants are known and many have low penetrance and are often found in control populations. Herein, we focused on novel missense variants in phospholamban, an Ala15-Thr variant found in a 4-year-old female and a Pro21-Thr variant found in a 60-year-old female, both with a family history and clinical diagnosis of dilated cardiomyopathy. The patients also harbored a Val896-Met variant in cardiac myosin binding protein. The phospholamban variants caused defects in the function, phosphorylation, and dephosphorylation of this calcium transport regulatory peptide, and we classified these variants as potentially pathogenic. The variant in cardiac myosin binding protein alters the structure of the protein. While this variant has been classified as benign, it has the potential to be a low-risk susceptibility variant because of the structural change in cardiac myosin binding protein. Our studies provide new biochemical evidence for missense variants previously classified as benign or VUS.

The Revolution of Cardiac Myosin Inhibitors in Patients With Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiomyopathy worldwide and causes significant morbidity and mortality. For decades, medical treatment options have been limited and untargeted, with frequent need for invasive interventions not readily accessible to many HCM patients. More recently, our understanding of the genetic basis and pathophysiologic mechanism of HCM has grown significantly, leading to the discovery of a new class of medications, cardiac myosin inhibitors (CMIs), that shift myosin into the super-relaxed state to counteract the hypercontractility in HCM. Subsequent clinical trials have proven the mechanism and efficacy of CMIs in humans with obstructive HCM, and additional trials are under way in patients with nonobstructive HCM. With favourable results in the completed clinical trials and ongoing research on the horizon, CMIs represent a bright new era in the targeted management of HCM. This review is focused on the discovery of CMIs, provides a summary of the results of clinical trials to date, provides clinicians with a roadmap for implementing CMIs into practice, and identifies gaps in our current understanding as well as areas of ongoing investigation.