Ca2+-Sensitivity and Elementary Steps of the Cross-Bridge Cycle in Papillary Muscle Fibers from the Troponin C (TnC)-A8V Knock-In Mouse, which Exhibits Hypertrophic Cardiomyopathy (HCM)

Abstract

Mutations of TnC alter its function by two mechanisms: changing Ca2+-affinity, and/or changing the TnC-TnI interaction. A mutation, A8V, exhibits HCM and diastolic dysfunction in both mice and humans as demonstrated by echocardiography. Here we have used homozygous knock-in TnC mutant A8V mice (7-8 months-old) in which the myofilaments carry only mutant protein. These mice develop HCM at 3 months (Circ Cardiovasc Genetic 2015, in press). This mutation is located in the N-helix of TnC, a region known to affect Ca2+-affinity. Here we studied the effects of TnC-A8V mutation in small fiber bundles (90-110µm diameter) from skinned papillary muscles. These were activated at 25°C under the intracellular ionic conditions of working cardiomyocytes (5mM ATP, 1mM Mg2+, 8mM phosphate, 200mM ionic strength, and pH 7.0). With the pCa-tension study, Ca2+-sensitivity increased (3x) and cooperativity decreased (0.8x) compared to the wild-type. The maximum active tension decreased slightly (0.95x). The elementary steps of the cross-bridge cycle were investigated by small-amplitude sinusoidal analysis. With the ATP study, there was no significant change in the ATP affinity (K1) to the myosin head, but cross-bridge detachment rate (k2) and its equilibrium constant (K2) increased to 1.5x, which causes slightly fewer force-generating cross-bridges. With the phosphate study, no effect of the mutation on the rate constants of the force-generation step (k4, k-4) or Pi-release step (1/K5) was observed. When the rigor state was induced, there was no effect of the mutation on stiffness, implying that force/cross-bridge is not altered. We infer from these observations that TnC-A8V mutation recruits more cycling cross-bridges during systole as a result of increased myofilament Ca2+-sensitivity, but unable to relax adequately resulting in diastolic dysfunction, followed by HCM development.