Myosin Regulatory Light Chain: A Major Player in Defining the 'OFF' State of Cardiac Myosin

Myosin regulatory light chain (RLC) is a regulatory subunit of the myosin molecule, which plays a major role in stabilizing the Interacting Head Motif (IHM) of myosin 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. 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 and show that removal of RLC destabilizes the ability of myosin to form the SRX state. Phosphorylation of the RLC with MLCK has the same effect, which renders the myosin to the ‘OFF’ state as confirmed by its ATPase activity. Interestingly we found that Ca 2+ and not Mg 2+ binding to synthetic myosin filaments had differential effects on the population of the myosin SRX state. At systolic Ca 2+ myosin exhibits lower SRX state than at diastolic Ca 2+ . This effect is obliterated in RLC-lacking myosin constructs such as sS1, or RLC stripped myosin filaments suggesting that Ca 2+ binding to RLC can activate the myosin from the ‘OFF’ state to a more active state. Naturally occurring cardiomyopathy-causing RLC mutants R58Q, K104E and D94A did not alter this property, but two of them, K104E and D94A abolished the phosphorylation regulation of the SRX state of myosin. Altogether, these observations demonstrate that either RLC phosphorylation or binding of systolic Ca 2+ can reduce the number of accessible myosin heads for contraction and could form the basis of the Ca 2+ -mediated activation of the sarcomeric thick filament and mutations in RLC can alter these abilities differentially.