MYH7 Mutation Research Articles

Abstract EC.03: Distinct Molecular Signatures Of HCM- And DCM-causing Mutations At The Same Residue In The Transducer Of Cardiac Myosin

Mutations in cardiac myosin cause both hypertrophic (HCM) and dilated cardiomyopathy (DCM), and the clinical phenotype ultimately depends on the precise effect of the mutation on the function of myosin. Using recombinant human cardiac myosin, we studied the molecular effects of a novel Q222H mutation in MYH7 found in a patient with severe, early-onset DCM, and compared the effects to the Q222K mutation that was previously reported to cause HCM. The Q222 residue is located within the transducer of the head domain near the ATP binding pocket. The Q222H (DCM) mutant myosin exhibited significantly reduced steady-state actin-activated ATPase activity and a decreased rate of ATP binding in transient kinetic studies, while Q222K (HCM) mutant protein showed an increase in ATPase activity and ATP binding kinetics. In vitro motility assays showed 43% reduction and 32% increase in actin gliding velocity in Q222H and Q222K mutations, respectively. Finally, single nucleotide turnover assays revealed that the fraction of myosin with a slow ATP hydrolysis rate (super relaxed state, or SRX) thought to represent myosin in an autoinhibited state was significantly reduced in Q222K and increased in Q222H. Taken together, the Q222H/K mutations exerted contrasting effects on the motor properties of the cardiac myosin catalytic domain. Cellular characterization and molecular dynamic simulations of cardiac myosin to assess the structural impact of mutant residues are underway. These results highlight how subtle structural differences between cardiomyopathy-causing mutations can lead to significant differences in cardiac myosin motor activity and thus phenotypic expression of cardiomyopathy.

Abstract P1019: High-throughput Generation And Functional Characterization Of Myosin Heavy Chain 7 Variants In Genome-edited Cardiomyocytes

Hypertrophic cardiomyopathy (HCM), an inheritable cardiac condition characterized by idiopathic left ventricular hypertrophy, can cause sudden cardiac death or heart failure. Autosomal-dominant missense mutations in MYH7 (encoding the sarcomeric βMHC protein) account for 33% of genotype-positive HCM cases. While pathogenic MYH7 variants are clinically-actionable, 75% of MYH7 missense variants in ClinVar are variants of unknown significance (VUS). Gene-editing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can help determine the function of MYH7 VUS, however, the discovery of new variants by genetic testing outpaces the ability to gene-edit hiPSCs. Thus, we developed a novel method called CR ISPR a On- T arget E diting R etrieval (CRaTER) which increases the success of gene-editing by 25-fold compared to current methods. We leveraged CRaTER to enrich for hiPSCs edited with a library of MYH7 missense variants and recovered 113 different heterozygous variants. As deleterious mutations can reduce protein stability, we flow sorted MYH7 variant hiPSC-CMs into bins based on differing βMHC abundance followed by next generation sequencing of each bin to calculate variant abundance scores. This assay identified 31 MYH7 missense variants as functionally abnormal (lower abundance scores than synonymous controls), including all known pathogenic variants in the library. This βMHC depletion phenotype was validated in clonal hiPSC-CMs and supported by western blotting of myocardium from a patient with a pathogenic MYH7 missense mutation. Furthermore, we rescued βMHC protein levels by genetically ablating the gene encoding for Muscle RING-finger protein-1, an E3 protein ligase that targets βMHC for degradation, and found that the cardiomyocyte hypertrophy induced by the pathogenic MYH7 E848G HCM variant was significantly blunted. In summary, CRaTER enables the generation of gene-edited hiPSC lines at unprecedented scale. Functional analysis of MYH7 variant hiPSC-CMs reveals a novel correlation between loss of βMHC protein and clinical pathogenicity that can be leveraged for clinical variant interpretation. Interventions that prevent the loss of βMHC may potentially be therapeutic for MYH7 -associated HCM.

Long-Term Prevalence of Systolic Dysfunction in MYBPC3 Versus MYH7-Related Hypertrophic Cardiomyopathy.

The 2 sarcomere genes most commonly associated with hypertrophic cardiomyopathy (HCM), MYBPC3 (myosin-binding protein C3) and MYH7 (β-myosin heavy chain), are indistinguishable at presentation, and genotype-phenotype correlations have been elusive. Based on molecular and pathophysiological differences, however, it is plausible to hypothesize a different behavior in myocardial performance, impacting lifetime changes in left ventricular (LV) function. We reviewed the initial and final echocardiograms of 402 consecutive HCM patients with pathogenic or likely pathogenic MYBPC3 (n=251) or MYH7 (n=151) mutations, followed over 9±8 years. At presentation, MYBPC3 patients were less frequently obstructive (15% versus 26%; P=0.005) and had lower LV ejection fraction compared with MYH7 (66±8% versus 68±8%, respectively; P=0.03). Both HCM patients harboring MYBPC3 and MYH7 mutations exhibited a small but significant decline in LV systolic function during follow-up; however, new onset of severe LV systolic dysfunction (LV ejection fraction, <50%) was greater among MYBPC3 patients (15% versus 5% among MYH7; P=0.013). Prevalence of grade II/III diastolic dysfunction at final evaluation was comparable between MYBPC3 and MYH7 patients (P=0.509). In a Cox multivariable analysis, MYBPC3-positive status (hazard ratio, 2.53 [95% CI, 1.09-5.82]; P=0.029), age (hazard ratio, 1.03 [95% CI, 1.00-1.06]; P=0.027), and atrial fibrillation (hazard ratio, 2.39 [95% CI, 1.14-5.05]; P=0.020) were independent predictors of severe systolic dysfunction. No statistically significant differences occurred with regard to incidence of atrial fibrillation, heart failure, appropriate implanted cardioverter defibrillator shock, or cardiovascular death. MYBPC3-related HCM showed increased long-term prevalence of systolic dysfunction compared with MYH7, in spite of similar outcome. Such observations suggest different pathophysiology of clinical progression in the 2 subsets and may prove relevant for understanding of genotype-phenotype correlations in HCM.

Musculoskeletal defects associated with myosin heavy chain‐embryonic loss of function are mediated by the YAP signaling pathway

Mutations in MYH3, the gene encoding the developmental myosin heavy chain‐embryonic (MyHC‐embryonic) skeletal muscle‐specific contractile protein, cause several congenital contracture syndromes. Among these, recessive loss‐of‐function MYH3 mutations lead to spondylocarpotarsal synostosis (SCTS), characterized by vertebral fusions and scoliosis. We find that Myh3 germline knockout adult mice display SCTS phenotypes such as scoliosis and vertebral fusion, in addition to reduced body weight, muscle weight, myofiber size, and grip strength. Myh3 knockout mice also exhibit changes in muscle fiber type, altered satellite cell numbers and increased muscle fibrosis. A mass spectrometric analysis of embryonic skeletal muscle from Myh3 knockouts identified integrin signaling and cytoskeletal regulation as the most affected pathways. These pathways are closely connected to the mechanosensing Yes‐associated protein (YAP) transcriptional regulator, which we found to be significantly activated in the skeletal muscle of Myh3 knockout mice. To test whether increased YAP signaling might underlie the musculoskeletal defects in Myh3 knockout mice, we treated these mice with CA3, a small molecule inhibitor of YAP signaling. This led to increased muscle fiber size, rescue of most muscle fiber type alterations, normalization of the satellite cell marker Pax7 levels, increased grip strength, reduced fibrosis, and decline in scoliosis in Myh3 knockout mice. Thus, increased YAP activation underlies the musculoskeletal defects seen in Myh3 knockout mice, indicating its significance as a key pathway to target in SCTS and other MYH3‐related congenital syndromes.

P120 NEW MALIGNANT MUTATION IN HYPERTROPHIC CARDIOMYOPATHY

Abstract A 62–year–old man diagnosed with non–obstructive hypertrophic cardiomyopathy and dual–chamber pacemaker wearer since the age of 40, for third–degree atrioventricular block, came to our attention for atrial fibrillation (AF) episodes (one complicated by cardiogenic shock and multiorgan failure) and flutter with high ventricular response. For a progressive worsening of the systolic function, and the onset of multiple episodes of sustained ventricular tachycardia, an upgrade to the defibrillator was performed, which, in the following months, intervened appropriately for recurrences of ventricular arrhythmias. The patient was admitted to our ward for exacerbation of heart failure. The echocardiography showed moderate concentric hypertrophy with mild ventricular dilatation and ejection fraction 33%, inferior and inferoseptal wall, and apex akinesia with thrombotic apposition. Due to a sudden deterioration of the clinical conditions, the patient died. After genetic counseling the patient‘s family agreed to genetic testing in the proband. This highlighted the presence of a missense mutation, i.e. the substitution of the cysteine ​​residue with a serine, in the codon 905 of the MYH7 gene, encoding the cardiac isoform of myosin heavy chain. The mutation is probably pathogenetic as it falls in the region where most of the missense variants are clustered and statistically associated with the HCM phenotype. Only in one previous case of HCM was detected a mutation of the same codon, Cys905Phe. Several studies have reported that AF tends to be more prevalent in patients carrying MYH7 mutations and is associated with a substantial risk of heart failure–related mortality, stroke, and severe functional disability. Knowledge of the entire spectrum of MYH7 mutations, especially those related to a high arrhythmic burden or SCD, combined with an accurate clinical and molecular characterization of the patient, can improve the genotype–phenotype correlation by clarifying the mechanism of HCM and allowing a better clinical and therapeutic decision–making of patients.

Nonmuscle Myosin IIA Regulates the Precise Alignment of Hexagonal Eye Lens Epithelial Cells During Fiber Cell Formation and Differentiation.

Epithelial cells in the equatorial region of the ocular lens undergo a remarkable transition from randomly packed cells into precisely aligned and hexagon-shaped cells organized into meridional rows. We investigated the function of nonmuscle myosin IIA (encoded by Myh9) in regulating equatorial epithelial cell alignment to form meridional rows during secondary fiber cell morphogenesis. We used genetic knock-in mice to study a common human Myh9 mutation, E1841K, in the rod domain. The E1841K mutation disrupts bipolar filament assembly. Lens shape, clarity, and stiffness were evaluated, and Western blots were used to determine the level of normal and mutant myosins. Cryosections and lens whole mounts were stained and imaged by confocal microscopy to investigate cell shape and organization. We observed no obvious changes in lens size, shape, and biomechanical properties (stiffness and resilience) between the control and nonmuscle myosin IIA-E1841K mutant mice at 2 months of age. Surprisingly, we found misalignment and disorder of fiber cells in heterozygous and homozygous mutant lenses. Further analysis revealed misshapen equatorial epithelial cells that cause disorientation of the meridional rows before fiber cell differentiation in homozygous mutant lenses. Our data indicate that nonmuscle myosin IIA bipolar filament assembly is required for the precise alignment of the meridional rows at the lens equator and that the organization of lens fiber cells depends on the proper patterning of meridional row epithelial cells. These data also suggest that lens fiber cell organization and a hexagonal shape are not required for normal lens size, shape transparency, or biomechanical properties.

Myosin post-translational modifications and function in the presence of myopathy-linked truncating MYH2 mutations.

Congenital myopathies are a vast group of genetic muscle diseases. Among the causes are mutations in the MYH2 gene resulting in truncated type IIa myosin heavy chains (MyHCs). The precise cellular and molecular mechanisms by which these mutations induce skeletal muscle symptoms remain obscure. Hence, in the present study, we aimed to explore whether such genetic defects would alter the presence as well as the post-translational modifications of MyHCs and the functionality of myosin molecules. For this, we dissected muscle fibers from four myopathic patients with MYH2 truncating mutations and from five human healthy controls. We then assessed 1) MyHCs presence/post-translational modifications using LC/MS; 2) relaxed myosin conformation and concomitant ATP consumption with a loaded Mant-ATP chase setup; 3) myosin activation with an unloaded in vitro motility assay; and 4) cellular force production with a myofiber mechanical setup. Interestingly, the type IIa MyHC with one additional acetylated lysine (Lys35-Ac) was present in the patients. This was accompanied by 1) a higher ATP demand of myosin heads in the disordered-relaxed conformation; 2) faster actomyosin kinetics; and 3) reduced muscle fiber force. Overall, our findings indicate that MYH2 truncating mutations impact myosin presence/functionality in human adult mature myofibers by disrupting the ATPase activity and actomyosin complex. These are likely important molecular pathological disturbances leading to the myopathic phenotype in patients.

Embryonic myosin mutations alter the mechanics and maturation of HiPSC derived skeletal muscle.

Distal arthrogryposis (DA) is a skeletal muscle disorder characterized by joint contractures predominantly localized in the distal extremities. DA associated syndromes like Freeman-Sheldon syndrome (FSS) are linked to mutations in the MYH3 gene that encodes the embryonic skeletal muscle myosin. To study the mutation and its effect on developing muscle, we generated human induced pluripotent stem cell (hiPSC) lines bearing T178I or R672C MYH3 mutations. hiPSC were differentiated into skeletal muscle and evaluated for differences in the maturation of the contractile unit and its functional performance. Preliminary myofibril mechanics measurements showed T178I myotubes generated significantly greater specific force compared to control myofibrils. Diseased myofibrils also expressed significantly slower rates of relaxation and prolonged thin filament deactivation compared to control myofibrils. Myofibril preparations were also used to compare ATP binding rates across conditions. Preliminary results showed homozygous R672C myofibrils have faster ATP binding rates compared to heterozygous and control preparations. Mutations in MYH3 were also associated with alterations in myosin isoform content, with homozygous R672C having reduced MYH3 levels in day 7 cells. Molecular dynamics simulations of R672C and T178I mutations in the post rigor state showed greater separation between the beta-sheet and SH-helix than in controls. Mutation also disrupted local salt bridges and hydrogen bond formation in some residues that help stabilize the nucleotide binding pocket and converter-connected helix. Interrupted structural communication between the nucleotide binding pocket and surrounding functional regions may underly the impaired relaxation phenotype. Ongoing studies will further characterize the effect of mutation on actin-myosin binding, crossbridge cycling kinetics, the maturation of skeletal myotubes over time, and myosin isoform switching dynamics.

MYH7 mutations induce changes in single cell mechanobiology.

Hypertrophic Cardiomyopathy (HCM) is characterized by thickening of the left ventricular wall and hypercontractility and has been linked to mutations in the sarcomere motor protein β-myosin (MYH7). Using single cell mechanobiology studies, we examined how the effects of single point mutations propagate to change the contractile dynamics and cellular morphology (sarcomere spacing, spread area, myofibril alignment) of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). We micropattern islands of adhesive protein to constraining the spreading and alignment of hiPSC-CM on hydrogel substrates containing fluorescent microbeads as fiducial markers for traction force microscopy (TFM). We deployed substrate stiffnesses ranging from physiological (10 kPa) to heavily diseased/fibrotic (100 kPa) to test the role of increased “afterload” in functional phenotypes. We use image and video analysis to assess the contractile dynamics of the hiPSC-CM in terms of force, power, and velocities of relaxation and contraction. For example, we assessed multiple MYH7 mutations edited into the WTC line along with isogenic controls. Some lines carried an endogenously labeled alpha-actinin GFP reporter of sarcomere structure to enable visualization of sarcomere structure and dynamics. We assessed the magnitude and dynamics of contractile force output from TFM video analysis and observed increased the contractile force when compared to the control hiPSC-CMs. We also measured significantly different dynamics in the relaxation or contraction velocities compared to control hiPSC-CMs. Interestingly, not all HCM mutant lines presented a significant increase in cell spread area, a proxy for hypertrophy, and this correlated with culture conditions, such as the size of the protein pattern constraining the cells or stiffness of the substrate. Taken together, these results suggest a role for MYH7 mutations driving remodeling of structure and function at a cell-intrinsic level via changes in mechanosignaling.

Hypercontractility by hypertrophic cardiomyopathy mutant iPSC-CMs induces hippo-yap activation through enhanced nuclear deformation.

Hypertrophy Cardiomyopathy (HCM) is the most prevalent hereditary cardiovascular disease - affecting >1:500 individuals. Advanced forms of HCM clinically present with hypercontractility, hypertrophy and fibrosis. Several single-point mutations in β-myosin heavy chain (MYH7) have been associated with HCM and increased contractility at the organ level. Different MYH7 mutations have resulted in increased, decreased, or unchanged force production at the molecular level. Yet how these molecular kinetics link to cell and tissue pathogenesis remains unclear. The Hippo Pathway has been demonstrated to be reactivated in pathological hypertrophic growth, intriguing because it signals proliferative growth during developmental growth and is quiescent during cardiac homeostasis. We hypothesized that changes in force production (intrinsically or extrinsically) directly alter the homeostatic mechano-signaling of the Hippo pathway through changes in stresses on the nucleus. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we asked whether homeostatic mechanical signaling through the canonical growth regulator, Hippo-YAP, is altered 1) by changes in the biomechanics of single HCM mutant cardiomyocytes and 2) by alterations in the mechanical environment. We use genetically edited hiPSC-CM with point mutations in MYH7 associated with HCM, and their matched controls, combined with micropatterned traction force microscopy substrates of different stiffness to measure changes in contractility and cell size. Mutant hiPSC-CMs display hypercontractile phenotypes and activated Hippo-YAP (% of hiPSC-CMs with nuclear localized YAP, mutants: 56.4% +/-6.85%, control: 32.1% +/-4.67%). The modulation of contractility in healthy hiPSC-CMs by treatment with inotropic drugs or culture in fibrotic-like stiff conditions also activated YAP compared to untreated controls. We hypothesize the activation of YAP in both HCM mutants and healthy hiPSC-CMs treated with contractility modulators is through enhanced nuclear deformation (nuclear aspect ratio amplitude, mutants: 0.063 +/-0.016, control: 0.027 +/-0.005).

Structural changes in myosin affect chemo-mechanical properties of the myosin-actin interaction.

Mutations in the genes encoding striated muscle myosins lead to a variety of diseases including hypertrophic (HCM) and dilated (DCM) cardiomyopathies (MYH7) and distal arthrogryposis syndromes (MYH3 and MYH8). We have used a muscle myoblast cell line to express and purify recombinant human beta-cardiac myosin and human embryonic skeletal muscle myosins, comparing the WT versions of these proteins with an array of disease-causing variants. HCM-causing mutations in the beta-cardiac motor domain often destabilize a folded back, autoinhibited state of myosin, leading to an increase in the number of heads available within the sarcomere to interact with actin and produce force, which in turn results in the hypercontractility characteristic of HCM. Destabilization is seen for mutations that alter residues involved in the regions of myosin important for forming the intramolecular contacts in the folded back state. However, mutations affecting residues remote from these interfaces can also increase the number of available heads by destabilizing the pre-power stroke state of myosin that enables formation of the folded back state. HCM mutations that affect only myosin motor function without affecting the stability of the folded back state are less common. In contrast, we have studied a variety of DCM-causing mutations and found that most DCM-mutant myosins exhibit decreases in one or more aspects of motor function, including actin-activated ATPase activity and in vitro gliding velocity. Current efforts focus on correlating the effects of distal arthrogryposis syndrome-causing mutations on MYH3 biomechanical function and contrasting the effects of these mutations with paralogous mutations in MYH7.

The Z-disc: Mechanosensor at the interface between myosin biomechanics and hypertrophic signaling.

Despite over two decades of research on the etiology of hypertrophic cardiomyopathy (HCM) and the identification of >1000 mutations in sarcomeric proteins such as β-cardiac myosin (MYH7), the mechanism by which altered force at the level of the sarcomere is transduced into cellular hypertrophy is still incompletely understood. We have previously described a striking widening of the Z-disc in septal myectomy samples from patients with HCM, as well as in human induced pluripotent stem cell derived cardiomyocyte (hiPSC-CM) lines in which we CRISPR-edited two different HCM mutations in MYH7. We propose that the Z-disc is the mechano-transduction link at the interface of myosin biomechanics and hypertrophy. We performed a comparative genome and mutation-wide molecular key driver analysis. Genome-wide analysis (RNAseq) of 26 HCM myectomy samples revealed altered expression of 52 Z-disc-related genes vs. donor heart controls. In HCM hiPSC-CMs, 24 Z-disc genes were altered (single-cell RNA-seq). 14 altered Z-disc genes were shared between human samples and hiPSC-CMs. Pro-hypertrophic signaling was activated through differential expression of five modulators (GLRX3, LMCD1, MYOZ2, PAK1IP1, C10ORF7) of calcineurin. We hypothesized that widening of the Z-disc, differential expression of Z-disc genes and downstream hypertrophic signaling were due to an increase in active tension on Z-disc anchored actin by adjacent myosin molecules, which was confirmed using traction force microscopy. Our findings suggest that the Z-disc acts as a mechano-sensor at the interface between myosin biomechanics and hypertrophic signaling. To further test this hypothesis, we used a vinculin FRET-tension sensor to directly measure intracellular force at the Z-disc with different myosin perturbations. This unique combination of patient samples and CRISPR-edited hiPSC-CMs confirms the fidelity of our in vitro model in recapitulating human disease and increases the potential translation of our findings to develop new therapies.

Molecular characterization of a novel MYH7 mutation Q222H in a patient with severe dilated cardiomyopathy.

Mutations in the cardiac myosin heavy chain gene (MYH7) are known to cause both hypertrophic and dilated cardiomyopathy (HCM and DCM), and the use of recombinant human cardiac myosin has enabled the study of the precise biochemical and biophysical effects of these mutations on myosin function at the molecular level. Here we report a case of a patient with severe, early-onset DCM who carries a previously unreported Q222H mutation in MYH7. This mutation affects a residue at the distal end of the ATP binding pocket of cardiac myosin. The Q222H mutant myosin exhibited significantly reduced steady-state actin-activated ATPase activity and a decreased rate of ATP binding in transient kinetic studies. It also showed a ∼50% reduction in actin gliding velocity in an in vitro motility assay. Single-nucleotide turnover assays were performed comparing Q222H mutant myosin 2-headed constructs with either a long S2 tail capable of forming a folded-back auto-inhibited state or a short S2 tail that cannot stably fold back and sequester heads. These studies revealed that the fraction of myosin with a slow ATP hydrolysis rate (super relaxed state, or SRX) thought to represent myosin in an autoinhibited state did not differ significantly between WT and the Q222H mutant myosins. Taken together, the Q222H mutation exerts substantial effects on the motor properties of the cardiac myosin catalytic domain, rather than affecting the availability of myosin heads in the sarcomere. Further transient kinetic assays are underway to delineate precise steps in the actin-myosin ATPase cycle affected by the mutation. Future work also includes comparing these effects with the Q222K mutation that has been reported to cause HCM, to gain further insights into the molecular basis for phenotypic determination of cardiomyopathy.

Changes in myosin biomechanics influence growth and maturation of iPSC-cardiomyocytes.

Hypertrophic cardiomyopathy (HCM) is characterized by hypercontractility, cardiomyocyte hypertrophy, myofibril disarray, and altered energetics. Despite the identification of >1000 mutations in sarcomeric proteins, one-third of which are in β-cardiac myosin (MYH7), the mechanism by which altered force at the level of the sarcomere is transduced into cellular hypertrophy and other phenotypes is still incompletely understood. Our biochemical studies reveal surprising heterogeneity in the impact of different MYH7 mutations on myosin force generation and ATPase activity with some mutations reducing activity at the molecular level. Using CRISPR-edited hiPSC-cardiomyocytes (hiPSC-CMs), we studied the impact of myosin HCM mutations on the generation of contractile forces, hypertrophy, myofibril organization, and mitochondrial function, and compared cellular phenotypes with the effects of the mutations on myosin biomechanics. Our studies reveal a critical role for alterations in the myosin super-relaxed state (SRX) as a mechanism for cellular hypercontractility. To validate the fidelity of our findings to human disease, we compared hiPSC-CM results to those in 26 septal myectomy samples from HCM patients and 13 donor heart controls. In both hiPSC-CMs and myectomy samples we observed fiber disarray, altered mitochondrial metabolism, and widening of the Z-disc. A genome-wide key driver analysis revealed altered expression of 52 Z-disc-related genes in patient samples; 24 Z-disc genes in hiPSC-CMs; with 14 Z-disc genes shared between the two platforms. Activation of calcineurin pro-hypertrophic signaling provides a potential link between Z-disc mechano-sensation of sarcomeric forces and downstream hypertrophic signaling, which we explore using a vinculin FRET-tension sensor to directly measure intracellular force at the Z-disc. Our multi-scale approach, combined with both patient samples and CRISPR-edited hiPSC-CMs, confirms the fidelity of our in vitro model in recapitulating human disease and increases the potential translation of our findings to develop new precision-based HCM therapies.

The β-myosin G256E mutation impairs relaxation by altering transducer stiffness.

Associations between mutations in sarcomeric proteins and familial cardiomyopathies are myriad. However, detailed structural and functional studies are required to describe the mechanistic relationship between a mutation and its pathology. To accelerate such studies, we utilize a combined computational and experimental platform to probe how mutations to b-myosin lead to familial cardiomyopathies. Here, we report results on the HCN-associated MYH7 mutation G256E, which is located in the myosin transducer. Experimentally, the specific force generation of mutant G256E myofibrils was ∼2x greater than isogenic control myofibrils. The activation rate was similarly increased. The initial slow phase relaxation kinetics of G256E myosin were significantly reduced relative to isogenic controls, indicating a slower cross-bridge detachment rate. Using stopped flow kinetics, the ATP binding rate was reduced in G256E relative to WT. Molecular dynamics simulations of the rigor and myosin.ATP states showed that G256E led to reorganization of hydrogen bonding networks within the transducer region. Enhanced sampling MD simulations modeled the rigor (nucleotide free) to post-rigor (ATP-bound) structural transition along multiple reaction coordinates. The transition required that G256E myosins surmount a greater energetic barrier that describes the binding of switch 1 to ATP, rotation of the transducer backbone, and opening of the actin binding cleft. This increased barrier is attributed to salt bridges formed by the mutant Glu residue. Based on our combined computational and experimental results, we hypothesize that G256E leads to a hypercontractile and delayed relaxation phenotype via modulation of the transducer structure. This alters communication between the nucleotide binding pocket and the actin binding surface during steps in the chemo-mechanical cycle that involve transducer motion during nucleotide release-induced strong actin binding and ATP binding-induced release from actin. Other effects on myosin structure and its activation during contraction are being investigated.

903 NOVEL MALIGNANT MUTATION IN HYPERTROPHIC CARDIOMYOPATHY

Abstract Background Hypertrophic cardiomyopathy (HCM) is a common inherited disease almost invariably caused by mutations in sarcomeric genes. The HCM phenotype is clinically heterogeneous with myocyte hypertrophy, disarray, and myocardial fibrosis as histological hallmarks. This condition is recognized as an important cause of sudden cardiac death (SCD) in the youth and of heart failure (HF) in the elderly. Current guidelines mandate genetic analysis as a class I indication in HCM. Indeed, the advent of the next generation sequencing in the medical practice has led to decipher the molecular etiology of HCM and to assess the disease risk in family members with relevant insights into the clinical course. Clinical case A 62-year-old man followed-up at another Hospital with a non-obstructive HCM diagnosis, was referred to our Hospital for therapy optimization and clinical follow-up. Past medical history included third degree atrioventricular block at age 40 treated with dual chamber pacemaker (PM) and, at age of 60, atrial flutter with high ventricular response complicated with cardiogenic shock and stroke. Due to worsening systolic function and ventricular sustained tachycardia he was upgraded to implantable cardioverter defibrillator which delivered appropriate shocks. Echocardiography performed during current hospitalization showed a moderate concentric hypertrophy (intraventricular septum 17 mm, cardiac mass index 258 g/m2), dilated ventricle with reduced ejection fraction (33%) and akinesis of the mid inferior and infero-septal walls of the left ventricle and of the apex, with a stratified thrombotic apposition. Magnetic resonance was contraindicated because of non-compatible PM. Due to alteration in serum proteins and free light chain, to rule out systemic disease, extensive imaging diagnosis was performed including bone scintigraphy and abdominal ultrasonography which resulted negative. While waiting for bone marrow aspiration and biopsy the patient rapidly deteriorated with worsening renal failure, ensuing proteinuria until exitus. After an appropriate pre-test counseling the family consented to genetic test in the proband. Clinical exome sequencing revealed the presence of a missense mutation in MYH7 gene. This new mutation is characterized by the substitution of cysteine residue by a serine in 905 codon of MYH7 gene. Cys905Ser results in a semi-conservative amino acid substitution which may impact disulfide bond formation in the MYH7 protein. To date no study has described this mutation as a cause of HCM. A mutation affecting this same codon, Cys905Phe was reported in only one patient with HCM. From a biological point of view, this variant lies in the head region of the protein where the majority of the missense variants are grouped and statistically associated to HCM phenotype. Several studies reported that the occurrence of atrial fibrillation (AF) tends to be more prevalent in patients carrying the MYH7 mutation. Furthermore, AF is associated with substantial risk for HF-related mortality, stroke, and severe functional disability. Awareness regarding the spectrum of MYH7 mutations probably related to SCD or HF combined with a thorough patient characterization for molecular and clinical features, may help to improve the genotype and phenotype correlation. This important challenge could elucidate better the mechanism of HCM enabling cardiologists to a better clinical decision-making and patients’ care.

Heterozygous MYH9 Mutations in 2 Children With Cochlear Nerve Canal Stenosis.

MYH9 is a gene that encodes for a subunit of the myosin heavy chain IIA protein. Mutations in MYH9 are associated with hematologic abnormalities, renal dysfunction, and hearing loss. Bony cochlear nerve canal stenosis (CNCS), which is diagnosed on computed tomography (CT) imaging, has been associated with congenital deafness, cochlear nerve aplasia/hypoplasia, and inner ear malformations. We report two cases of CNCS presenting with profound congenital hearing loss whom we diagnosed with mutations in MYH9 and discuss the genotype-phenotype association and implications for management.

Representativeness of EXPLORER-HCM trial and prevalence of eligibility criteria for Mavacamten in a nationwide cohort

Abstract Introduction The EXPLORER-HCM trial evaluated the safety and efficacy of Mavacamten, a cardiac myosin inhibitor, in symptomatic obstructive hypertrophic cardiomyopathy (HCM). Mavacamten improved symptoms, exercise capacity and left ventricular outflow tract (LVOT) obstruction. However, the proportion of patients eligible for this therapy in real practice remains unclear. Purpose To determine the prevalence of eligibility criteria for Mavacamten and to describe the profile of patients complying with EXPLORER-HCM criteria in a nationwide cohort. Methods Cross-sectional study including all HCM patients enrolled in a European country-based nationwide registry. Presence of EXPLORER-HCM inclusion [≥18 years, obstructive HCM (LVOT gradient ≥50 mmHg), left ventricular ejection fraction (LVEF) ≥55%, New York Heart Association (NYHA) class II–III] and exclusion criteria [syncope or sustained ventricular tachycardia with exercise] were analyzed. Results Of 1042 patients included in this analysis (57.8% male, mean age at diagnosis 52 years, 39.6% obstructive HCM), 45 met the EXPLORER-HCM inclusion criteria and, after applying exclusion criteria, 30 patients were eligible for therapy with Mavacamten. Eligible patients were all Caucasian, mostly female (63.3%), with a mean age at diagnosis of 58±14 years. All patients presented with heart failure symptoms, and were mostly in NYHA class II (83.3%); 4 patients (13.3%) presented with atrial fibrillation. In the baseline echocardiogram, mean LVEF was 68±9%; mean interventricular septum thickness was 20±4 mm; most patients presented with systolic anterior motion of the mitral valve (96.7%) and mitral regurgitation (96.7%). In cardiac magnetic resonance imaging evaluation, maximal wall thickness was 22±4 mm and 76.9% had late gadolinium enhancement. A pathogenic or likely pathogenic HCM gene variant was identified in 5 patients (MYH7 and MYBPC3 gene mutations). In this group, 86.2% of patients were treated with beta-blockers and 34.5% with calcium channel antagonists. Only 1 patient was on Disopyramide. Septal reduction techniques were used in 43.3% of patients: alcohol septal ablation and septal myectomy were performed in 7 (23.3%) and 6 (20.0%) patients, respectively. Cardioverter-defibrillators were implanted in 2 patients for primary prevention of sudden cardiac death, and 6 patients received a pacemaker for bradycardia indications. Conclusion Specific therapies remain an unmet need in HCM. In this large nationwide cohort, only 2.9% of patients were eligible for therapy with Mavacamten according to EXPLORER-HCM criteria. Among patients complying with the study criteria, invasive septal reduction therapies were offered to a high proportion of patients. Further real-world data are warranted to estimate the proportion of HCM patients that will benefit from Mavacamten. Funding Acknowledgement Type of funding sources: None.

Arrhythmogenic right ventricular cardiomyopathy presenting as heart failure in a child.

Arrhythmogenic right ventricular cardiomyopathy is an uncommon diagnosis in the paediatric population, most commonly presenting with arrhythmia. We report an 11-year-old male presenting with right heart failure due to biventricular systolic dysfunction found to have arrhythmogenic right ventricular cardiomyopathy with de novo Desmin and MYH7 mutations.

Potential of eight mutations for marker-assisted breeding in Chinese Lulai black pigs

Molecular marker-assisted selection (MAS) provides an efficient tool for pig breeding. In this study, according to the literature, we selected eight effective or causal mutations from eight functional genes, including five causal mutations in PHKG1 ( rs330928088), MUC13 ( rs319699771), IGF2 ( g.3072 G&gt; A), VRTN ( g.20311_20312ins291), and MYH3 ( XM_013981330.2:g.-1805_-1810del) genes, and three effective mutations in LIPE ( rs328830166), LEPR ( rs45435518), and MC4R ( rs81219178) genes, to investigate their potential breeding effect in 418 Lulai pigs. The linear model was used to analyse the association between mutations and intramuscular fat (IMF) content, average backfat thickness, and muscle moisture %. The results revealed that amongst the three effective mutations, only the mutation in the LEPR gene, which affects IMF deposition, was significantly associated with IMF content. However, the other molecular markers were not significantly associated with the affected traits reported in previous studies, and these mutations are ineffective for MAS in the Lulai black pig population. Therefore, causal mutations in PHKG1, IGF2, and VRTN genes, and an effective mutation in LEPR gene could be used as effective breeding makers for MAS in Lulai pigs. These results can provide helpful information for further breeding in Lulai black pigs.