Abstract 572: Role Of Human Cardiac RLC In Modulating The Super-relaxed State Of Myosin: A Cardiomyopathy Perspective

Myosin regulatory light chain (RLC) is a major regulatory subunit of the myosin molecule, the role of which has been well-characterized in non-striated muscle. However, in striated muscle, its purpose is less well defined, and it is only in the past decade that researchers have started unraveling the function of RLC and its phosphorylation in muscle contraction. Mutations in this protein result in ~ 2% of total familial cardiomyopathy, an autosomal dominant disorder, manifested by ventricular and septal hypertrophy and myofibrillar disarray that can lead to sudden cardiac death. In this study, we report the role of human cardiac RLC in forming the super-relaxed state (SRX) of myosin in reconstituted full-length cardiac myosin thick filaments. We show that the presence of RLC fine-tunes the ability of myosin to form the SRX state and that its removal depopulates the SRX state. A similar reduction in SRX population is also achieved by phosphorylating the RLC with MLCK. The second mechanism of RLC-mediated regulation in muscle is thought to be by the binding of either Mg 2+ or Ca 2+ to the N-terminal EF-hand domain. Mg 2+ did not affect myosin SRX population, but increasing Ca 2+ enhanced the population of the myosin SRX state. Preliminary SRX studies on cardiomyopathy-causing RLC mutants R58Q (HCM), K104E (HCM) and D94A (DCM) show that none of the mutants affects the SRX population in the dephosphorylated state of the RLC; however these mutants also do not affect the SRX population when RLC is phosphorylated, unlike the wild-type. Additionally, in micro-scale thermophoresis binding experiments, only the dephosphorylated form of the DCM-causing D94A RLC mutant exhibits weaker binding to the myosin lever arm as compared to WT RLC. Binding of the mutants resembles WT upon phosphorylation. Altogether, these observations demonstrate that either RLC phosphorylation or Ca 2+ binding to RLC can alter the number of accessible myosin heads for contraction and cardiomyopathy-causing RLC mutants can modify this mechanism to varying extents.