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

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the β-myosin heavy chain () can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant 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.

The biochemically defined super relaxed state of myosin-A paradox.

The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments.

Measuring the Contractile Kinetics of Isolated Myofibrils from Human-Induced Pluripotent Stem Cell Derived Cardiomyocyte (hiPSC-CM) Models of Cardiomyopathy.

Isolated myofibrils provide biomechanical data at the contractile organelle level that are independent of cellular calcium handling and signaling pathways. These myofibrils can be harvested from animal tissue, human muscle biopsies, or stem cell-derived striated muscle. Here we present our myofibril isolation and rapid solution switching protocols, which allow for precise measurements of activation (kinetics and tension generation) and a biphasic relaxation relationship (initial slow isometric relaxation followed by a fast exponential decay in tension).

Protocols for Myosin and Actin-Myosin Assays Using Rapid, Stopped-Flow Kinetics.

Fast transient kinetics using stopped-flow fluorimetry is now a powerful method for defining the ATPase cycle of myosin and its subfragments and has found wide use in defining the difference between myosin isoforms, myosins carrying disease linked mutations, and the effect of small molecules on the ATPase cycle. Here the protocols for completing the classical assays of myosin and actin.myosin using the stopped-flow are described.

Multi-scale models reveal hypertrophic cardiomyopathy MYH7 G256E mutation drives hypercontractility and elevated mitochondrial respiration.

Rationale: Over 200 mutations in the sarcomeric protein β-myosin heavy chain (MYH7) have been linked to hypertrophic cardiomyopathy (HCM). However, different mutations in MYH7 lead to variable penetrance and clinical severity, and alter myosin function to varying degrees, making it difficult to determine genotype-phenotype relationships, especially when caused by rare gene variants such as the G256E mutation.

Objective: This study aims to determine the effects of low penetrant MYH7 G256E mutation on myosin function.

Exploring the super-relaxed state of myosin in myofibrils from fast-twitch, slow-twitch, and cardiac muscle.

Muscle myosin heads, in the absence of actin, have been shown to exist in two states, the relaxed (turnover ∼0.05 s) and super-relaxed states (SRX, 0.005 s) using a simple fluorescent ATP chase assay (Hooijman, P.