Impact of familial hypertrophic cardiomyopathy-linked mutations in the NH2 terminus of the RLC on β-myosin cross-bridge mechanics

Abstract

Familial hypertrophic cardiomyopathy (HCM) is associated with mutations in sarcomeric proteins, including the myosin regulatory light chain (RLC). Here we studied the impact of three HCM mutations located in the NH2 terminus of the RLC on the molecular mechanism of β-myosin heavy chain (MHC) cross-bridge mechanics using the in vitro motility assay. To generate mutant β-myosin, native RLC was depleted from porcine cardiac MHC and reconstituted with mutant (A13T, F18L, and E22K) or wild-type (WT) human cardiac RLC. We characterized the mutant myosin force and motion generation capability in the presence of a frictional load. Compared with WT, all three mutants exhibited reductions in maximal actin filament velocity when tested under low or no frictional load. The actin-activated ATPase showed no significant difference between WT and HCM-mutant-reconstituted myosins. The decrease in velocity has been attributed to a significantly increased duty cycle, as was measured by the dependence of actin sliding velocity on myosin surface density, for all three mutant myosins. These results demonstrate a mutation-induced alteration in acto-myosin interactions that may contribute to the pathogenesis of HCM.

Keywords: cardiac ventricular myosin; hypertrophic cardiomyopathy; in vitro motility; myosin regulatory light chain.

Fig. 1.

The linear amino acid sequence of human cardiac ventricular regulatory light chain. In this figure the NH₂ and COOH termini of the molecule are shaded in gray, with the metal binding domain and the phosphorylatable serine underlined. The 11 known hypertrophic cardiomyopathy (HCM) mutations are indicated in bold and numbered. Note that 9 of the mutations reside in the gray-shaded areas with the other 2 mutations, N47K and R58Q, residing either in or near the metal binding domain. In this study we determined the mechanochemical effects of the A13T, F18L, and E22K mutant myosins, which are located near the NH₂ terminus of the molecule.

Fig. 2.

SDS-PAGE of native (lane 1), depleted (lane 2), and exchanged regulatory light chain (RLC) onto porcine cardiac beta myosin. As indicated by the gel the depletion reaction is able to remove ∼80–85% of the native RLC as indicated by densitometry traces, with the subsequent exchanges of WT, A13T, F18L, or E22K (lanes 3–6, respectively) yielding an average exchange of 85.4% ± 4.2%, 81.3% ± 0.5%, 85.7% ± 5.5%, and 81.0% ± 1.9%, respectively. ELC, essential light chain.

Fig. 3.

Impact of NH₂-terminal HCM RLC mutations on actin sliding velocity with varying amounts of frictional load. Note that the unloaded actin filament velocities driven by the mutant myosins are reduced compared with WT (P < 0.001). Similarly, actin filament velocities were significantly slower than wild type under low frictional load (up to 2 μg/ml α-actinin; solid stars; open stars indicate significance for A13T and F18L only). Yet, when the frictional load is increased beyond 3 μg/ml the actin sliding velocity for the mutants is not significantly different from the wild-type. Except for F18L at 6 μg/ml α-actinin (N = 9), all points represent the average of more than 25 movies; error bars represent SE.

Fig. 4.

Actin-activated ATPase activity of β-myosin bearing WT and HCM-exchanged RLC. The ATPase activity of all four RLC exchanged β-myosins were not significantly different (average from 3 exchanges); error bars represent SE.

Fig. 5.

Effect of HCM mutations on the duty cycle of β-myosin. As shown in A, consistent with the results from Fig. 1, the calculated V_max for the 3 HCM mutants were significantly (P < 0.0001) slower than WT-RLC exchange myosin (0.496 ± 0.009, 0.414 ± 0.008, 0.404 ± 0.004, and 0.404 ± 0.006 mm/s for WT, A13T, F18L, and E22K, respectively), as indicated by the black star. B: normalizing the sliding velocities to the calculated V_max illustrates that all 3 HCM mutants had a significantly (P < 0.01) larger duty cycle (6.3± 0.6%, 6.2± 0.3%, and 6.0± 0.4% for A13T, F18L and E22K, as shown in inset of B) than the wild-type exchanged myosin (4.4± 0.3%) (as indicated by the gray star). Data from 0-50 μg/l are plotted in the inset of B. For WT, A13T, and E22K there were 15–25 measurements per myosin concentration while for F18L the N was 25–35 movies for each point; error bars represent SE.