Impact of Familial Hypertrophic Cardiomyopathy-Linked Mutations in the N-Terminus of the Myosin Regulatory Light Chain on the Calcium Based Motility

Calcium binding to the Regulatory Light Chain (RLC) has been shown to alter the structure and function of isolated RLC and when it is bound to the myosin heavy chain (MHC) in muscle. Furthermore, mutations associated with hypertrophic cardiomyopathy (FHC) have been shown to modify the affinity of the RLC for calcium binding (Szczesna et al., 2001). Here we studied the effect of calcium binding to the three N-terminal RLC mutants associated with FHC on the β-myosin mechano-chemistry using in vitro motility assays. To generate mutant β-myosin, native pig RLC was depleted from porcine cardiac MHC and reconstituted with the human cardiac WT (wild-type) or mutant (A13T, F18L & E22K) RLC. We measured the actin-activated myosin ATPase, actin sliding velocity and rotational stiffness of the mutant vs. WT myosin. Our results demonstrate that as pCa levels increased from 10 to 5.5, WT sliding velocity increased ∼20% while two of the mutants (A13T and E22K) decreased ∼10% respectively. Further increase in calcium to pCa 4 restored sliding velocity of all strains of RLC toward values observed at pCa 10. In contrast the actin-activated myosin ATPase of WT was lower ∼20% as calcium was increased from pCa 10 to 5.5 while that of A13T and E22K was increased by ∼5.5%. The velocity-pCa relationship concurs with the previously reported decreased calcium affinity values of the mutant RLC vs. WT (Szczesna et al., 2001). These results suggest that calcium binding to the RLC in the β-myosin can affect its contractile activity. The function of RLC as a calcium sensor allowing the heads to respond to the initial phases of activation and a greater strong cross bridge formation is largely compromised by FHC-linked RLC mutations.