The Effect of HCM and RCM Causing Mutations of Troponin T on Force Generation and Cross-Bridge Kinetics in Thin-Filament Reconstituted Bovine Cardiac Muscle Fibers

Abstract

Two troponin T mutations (I79N and ΔE96), which cause hypertrophic cardiomyopathy and restrictive cardiomyopathy respectively, 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. I79N and ΔE96 showed no significant change in maximum tension generated compared to WT, but both of them showed significantly increased Ca2+ sensitivity (pCa50: 5.65±0.04 and 5.62±0.04, respectively) than WT (5.44±0.04). The cooperativity increased in I79N (2.53±0.18) but remained the same in ΔE96 (1.75±0.19) compared to WT (1.71±0.18). Both I79N and ΔE96 showed lowered low-Ca (pCa 8.0) stiffness (YLC, 14.52±2.67 and 23.93±2.73) than WT (30.21±4.36). High-Ca (pCa 4.66) stiffness remained similar among all three Tns. However, The Ca2+ activatable stiffness of I79N and ΔE96 (Yact=YHC-YLC, 55.43±4.92 and 48.49±6.91) was significantly higher than WT (Yact=33.54±4.22) (±sem, N=9-20 for all studies). The All tensions were normalized to Ta, the tension of actin-filament reconstituted myocardium at the standard activating condition. Five equilibrium constants were deduced using sinusoidal analysis. I79N and ΔE96 showed significantly decreased K0 (ADP dissociation constant, 30.20±2.43 mM−1 and 34.61±3.89 mM−1, respectively) than WT (46.8±1.24 mM−1). I79N showed significantly lower K2 (cross-bridge detachment step: 0.80±0.11) than ΔE96 (1.74±0.24) and WT (1.41±0.19). I79N and ΔE96 also showed significantly higher K4 (force generation step: 0.70±0.06 and 0.28±0.01, respectively) than WT (0.21±0.03). The cross-bridge distribution of the mutants and WT was calculated from the equilibrium constants. I79N showed ∼33% more force generating cross-bridges than WT, but ΔE96 remained similar to WT. These results indicate that, under pCa4.66, force/cross-bridge is ∼25% less in I79N than WT, but remains similar in ΔE96.