Hypertrophic cardiomyopathy mutation R58Q in the myosin regulatory light chain perturbs thick filament-based regulation in cardiac muscle

Abstract

Hypertrophic cardiomyopathy (HCM) is frequently linked to mutations in the protein components of the myosin-containing thick filaments leading to contractile dysfunction and ultimately heart failure. However, the molecular structure-function relationships that underlie these pathological effects remain largely obscure. Here we chose an example mutation (R58Q) in the myosin regulatory light chain (RLC) that is associated with a severe HCM phenotype and combined the results from a wide range of in vitro and in situ structural and functional studies on isolated protein components, myofibrils and ventricular trabeculae to create an extensive map of structure-function relationships. The results can be understood in terms of a unifying hypothesis that illuminates both the effects of the mutation and physiological signaling pathways. R58Q promotes an OFF state of the thick filaments that reduces the number of myosin head domains that are available for actin interaction and ATP utilization. Moreover this mutation uncouples two aspects of length-dependent activation (LDA), the cellular basis of the Frank-Starling relation that couples cardiac output to venous return; R58Q reduces maximum calcium-activated force with no significant effect on myofilament calcium sensitivity. Finally, phosphorylation of R58Q-RLC to levels that may be relevant both physiologically and pathologically restores the regulatory state of the thick filament and the effect of sarcomere length on maximum calcium-activated force and thick filament structure, as well as increasing calcium sensitivity. We conclude that perturbation of thick filament-based regulation may be a common mechanism in the etiology of missense mutation-associated HCM, and that this signaling pathway offers a promising target for the development of novel therapeutics.

Phosphomimetic‐mediated in vitro rescue of hypertrophic cardiomyopathy linked to R58Q mutation in myosin regulatory light chain

“…They also showed that MLCK‐dependent phosphorylation of R58Q RLC‐exchanged trabeculae led to an increase by about ~ 30% of maximum active isometric force. While this supports the notion that R58Q promotes an OFF state of the thick filaments, it does not provide information about the effect of R58Q mutation on the number of functional myosin heads that are available for actin interaction and ATP utilization and on the population of myosin head existing in SRX versus DRX state . Our mant‐ATP chase experiments in both reconstituted porcine and Tg fibers indicate that R58Q mutation stabilizes the slow SRX phase of myosin compared to WT, and thereby decreases the number of ‘working' myosin heads that are able to participate in the force‐generating state, without significantly affecting the turnover rate of slow/fast proportion compared to WT.…”

“…Study from M. Irving laboratory using polarized fluorescence experiments in rat ventricular trabeculae has shown that RLC phosphorylation can shift the equilibrium between perpendicular ON and parallel OFF states toward more ON state of myosin [39]. Using bifunctional sulforhodamine labeled RLCs exchanged into demembranated rat right ventricular trabeculae, the authors further showed that order fluorescence polarization parameter <P 2 > was significantly higher for R58Q under both relaxing (pCa 9) and full calcium activation (pCa 4.3) conditions, suggesting that R58Q mutation induces a more parallel OFF orientation of the myosin heads [40]. They also showed that MLCK-dependent phosphorylation of R58Q RLC-exchanged trabeculae led to an increase by about~30% of maximum active isometric force.…”

Slow‐twitch skeletal muscle defects accompany cardiac dysfunction in transgenic mice with a mutation in the myosin regulatory light chain

“…P10916; National Center for Biotechnology Information (NCBI), Bethesda, MD, USA]. The molecular basis of heart pathology associated with the R58Q mutation has been studied with a wide range of research tools, including investigations of structure-function effects in R58Q-mutant reconstituted cardiac muscle preparations (22)(23)(24)(25)(26) and transgenic hearts of R58Q mice (15,16,(27)(28)(29). Combined results suggest that the mutation imposes significant perturbations in the motor function of myosin head, leading to its reduced ability to interact with actin and generate force.…”

Mavacamten stabilizes the auto-inhibited state of two-headed cardiac myosin

“…Regulation of the cardiac myosin thick filament-independent of the actomyosin interaction-is hypothesized to be a critical determinant of contraction and contribute to the Frank-Starling relationship, a fundamental regulator of cardiac performance [26], and to cardiac dysfunction in inherited cardiomyopathies [18,27]. The structural state of cardiac thick-filaments is hypothesized to be controlled by an evolutionarily-conserved mechanism where the heads of each myosin dimer fold back and interact with their S2 coiled-coil domain and with the thick-filament backbone [13].…”