The Effect of Hypertrophic Cardiomyopathy (HCM) Mutations of Tropomyosin on Force Generation and Cross-Bridge Kinetics in Thin-Filament Reconstituted Bovine Cardiac Muscle Fibers

Abstract

Three HCM mutations (V95A, D175N and E180G) of Tropomyosin (Tm) were examined using the thin-filament extraction and reconstitution technique. Effects of Ca2+, ATP, phosphate and ADP concentrations on force and its transients were studied at 25°C and compared to WT. E180G showed larger isomeric tension (1.89±0.11) than WT (1.59±0.08). Tension of two other mutants (V95A 1.53±0.09, D175N 1.47±0.07) was not different from WT. pCa50 (Ca2+ sensitivity) of V95A (6.20±0.06) and E180G (6.51±0.02) was larger than WT (5.85±0.03), while that of D175N (5.88±0.05) remained the same. The cooperativity was reduced in all three mutants (V95A 1.70±0.11, D175N 1.87±0.09, E180G 1.91±0.14) compared to WT (2.79±0.25). Four equilibrium constants were deduced using sinusoidal analysis. The largest effect was on K5 (Pi association constant) which was reduced to ∼0.5X in all mutants, while K4 (force generation step) was unchanged. V95A showed significantly lower K2 (cross-bridge detachment step: 0.93±0.06) than WT (1.37±0.13). D175N and V95A showed significantly lower K1 (ATP association constant, 0.91±0.13 and 0.86±0.16, respectively) than E180G (1.84±0.33) and WT (1.60±0.35). However, the cross-bridge distribution was not significantly different among 4 Tms, indicating that force/cross-bridge in E180G is larger than WT, but it is unchanged in V95A and D175N. In conclusion, all three mutants showed significant deviations in force/cross-bridge, pCa50, cooperativity or cross-bridge kinetics; in particular, E180G had the largest effect. Because E180G and D175N are located in the Tm-Troponin (Tn) interaction region and result in the net charge increase, and E180G causes the largest hydropathy change, we infer that both electrostatic and hydrophobic interactions between Tm and Tn play vital roles in maintaining normal muscle functions.