Pathophysiologic Changes Induced by Mutations in the TNT1 Domain of cTnT that Cause FHC

Abstract

Familial Hypertrophic Cardiomyopathy (FHC) is a primary cardiac muscle disorder and one of the most common causes of sudden cardiac death in young people. A majority of Cardiac Troponin T (cTnT) mutations are located in the TNT1 domain and cluster at its N- and C-termini. We are investigating the cTnT deletion of glutamine 160 (delta-160E) that is known to be a severe mutation located in a predicted hinge region at the C-terminal end of TNT1. Previous in vitro motility studies in our laboratory showed that mutations in this region disrupt weak electrostatic interactions between the thin filament and myosin necessary for strong crossbridge formation. In the current study, we aim to examine the downstream pathophysioloic consequences of this mutation. Cardiac myocytes isolated from hearts of transgenic mice expressing delta-160E cTnT with 35% and 70% replacement and non-transgenic siblings were used to study mechanical function and calcium transients. Our study shows impairments in myocellular mechanics during contraction and relaxation and in the rise and decline of the calcium transient. Furthermore, the alterations in calcium kinetics were dose-dependent. These results support the progressive nature of delta-160E FHC suggested by electron micrographs that demonstrate ultrastructural sarcomeric disarray that increase with transgene expression. In addition, we determined downstream effects of the mutation on expression and function of calcium handling proteins in transgenic mouse hearts using functional assays and immunoblotting. We found that the delta-160E cTnT mutation causes secondary alterations in calcium handling, leading to decreased SR calcium uptake, increased NCX expression, and increased diastolic leak through RyR2. Collectively, these novel findings indicate a phenotype that is distinct from other cTnT mutations and support the need to establish genotype-phenotype links in order to better design molecular therapies to treat FHC.