Stopped-Flow Calcium Kinetics of Hypertrophic Cardiomyopathy-Associated Troponin T Mutations

Abstract

Hypertrophic cardiomyopathy (HCM) is a complex genetic disorder of the cardiac sarcomere affecting 1/500 individuals worldwide. The cardiac thin filament (CTF) protein, cardiac troponin T (cTnT), is known to play a critical role in the regulation of contraction, and mutations in cTnT often cause HCM. This study focuses on 4 HCM-causing, clinically relevant point mutations located within the cTnT N-terminus (R92L/Q/W and I79N) which are each associated with distinct phenotypes and disease severity. Despite this, they exhibit comparable changes in steady-state calcium sensitivity of both force generation and myosin ATPase activity. Thus, we monitored the rates of calcium association to and dissociation from IAANS-labeled, Cys-substituted cardiac troponin C in CTFs to explain the comparable changes in steady-state calcium sensitivity. We and others previously characterized mutation-specific differences in calcium dissociation rates for the R92 mutations in CTFs. I79N CTFs significantly increased the rate of calcium dissociation from 116.0±3.2s−1 measured in WT CTFs to 136.1±6.1s−1. The observed calcium association rates to WT, R92L, R92W, and I79N CTFs were measured over a range of calcium concentrations (2.5µM-20µM) and were found to be 9.88±2.051 x106 M−1s−1, 9.141±1.94 x106 M−1s−1, 31.37±2.948 x106 M−1s−1, and 28.2±2.736 x106 M−1s−1, respectively. Both I79N and R92W, but not R92L, significantly increased the rate of calcium association compared to WT CTFs. Furthermore, both I79N and R92W, but not R92L, trend with increased responsiveness to artificially evoked calcium transients compared to WT. These data show that in addition to changing the rate of calcium dissociation, mutations in cTnT can significantly alter the rate of calcium association to affect CTF calcium sensitivity. Together this suggests that alterations in CTF calcium kinetics may represent a highly mutation-specific mechanism of disease initiation and are thus a target for potential therapeutic intervention.