Cardiomyopathy-Related Mutations (E244D, K247R, D270N, and K273E) in the H2-Helix of Cardiac Troponin T Have Varied Effects on Myofilament Responsiveness to Calcium and Crossbridge Recruitment Dynamics

Abstract

Two hypertrophic cardiomyopathy (HCM)-related mutations, E244D and K247R, and two dilated cardiomyopathy (DCM)-related mutations, D270N and K273E, have been identified along a centralized helix of cardiac troponin T (cTnT). This helix, termed H2(T), is centrally located in the core domain of cardiac troponin, interacting with cardiac troponin I and troponin C. This indicates a functional role of H2(T) in translating conformational changes sensed in troponin C and troponin I to the rest of the thin filament, but this structure-function relationship is not well understood. To determine the significance that disease-related alterations in the structure of H2(T) have in altering contractile function, we measured the contractile properties of rat cardiac muscle fibers containing rat cTnT variants cTnTE245D, cTnTK248R, cTnTD271N, or cTnTK274E corresponding to human E244D, K247R, D270N, or K273E mutations, respectively. We measured simultaneous force production and ATPase activity, as well as force responses (F(t)) to step-length perturbations in demembranated cardiac muscle fibers activated at various [Ca2+], at both sarcomere lengths 2.0 and 2.3 μm. Fibers containing cTnTK274E exhibited an increase in myofilament Ca2+ sensitivity when compared to those containing wild-type (WT)-cTnT. In addition, crossbridge recruitment dynamics, as estimated by model-predictions of F(t) and ktr measurements, were slower in fibers containing cTnTK274E, a trend that was also seen in fibers containing cTnTD271N. Ca2+ sensitivity of fibers containing cTnTK248R or cTnTD271N was less than that of fibers containing WT-cTnT. Furthermore, maximal ATPase activity was slightly but significantly increased in fibers containing cTnTE245D or cTnTK248R. These findings suggest that mutations along H2(T) influence the cTnT-modulated mechanisms of myofilament activation and crossbridge recruitment dynamics, and may contribute in part to cardiac dysfunction associated with HCM and DCM diseases.