Mutations in myosin S2 alter cardiac myosin-binding protein-C interaction in hypertrophic cardiomyopathy in a phosphorylation-dependent manner

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disorder primarily caused by mutations in the beta-myosin heavy-chain gene. The proximal subfragment 2 region (S2), 126 amino acids of myosin, binds with the C0-C2 region of cardiac myosin-binding protein-C to regulate cardiac muscle contractility in a manner dependent on PKA-mediated phosphorylation. However, it is unknown if HCM-associated mutations within S2 dysregulate actomyosin dynamics by disrupting its interaction with C0-C2, ultimately leading to HCM. Herein, we study three S2 mutations known to cause HCM: R870H, E924K, and E930 Delta. First, experiments using recombinant proteins, solid-phase binding, and isothermal titrating calorimetry assays independently revealed that mutant S2 proteins displayed significantly reduced binding with C0C2. In addition, CD revealed greater instability of the coiledcoil structure in mutant S2 proteins compared with S2(Wt) proteins. Second, mutant S2 exhibited 5-fold greater affinity for PKA-treated C0-C2 proteins. Third, skinned papillary muscle fibers treated with mutant S2 proteins showed no change in the rate of force redevelopment as a measure of actin-myosin cross-bridge kinetics, whereas S2(Wt) showed increased the rate of force redevelopment. In summary, S2 and C0-C2 interaction mediated by phosphorylation is altered by mutations in S2, which augment the speed and force of contraction observed in HCM. Modulating this interaction could be a potential strategy to treat HCM in the future.

Automated summary

This study investigates how mutations in S2 related to Hypertrophic Cardiomyopathy (HCM) disrupt the interaction with C0-C2, leading to the disease. Results show that mutant S2 proteins had reduced binding with C0C2 and exhibited higher affinity for PKA-treated proteins. Additionally, mutant S2 did not affect the rate of force redevelopment in skinned papillary muscle fibers, indicating alteration in actomyosin cross-bridge kinetics.