Molecular Mechanism of Cardiomyopathy-Causing Mutations in Alpha-Tropomyosin

Abstract

Primary cardiomyopathy is one of the most common cardiac disorders, affecting more than 1 in 500 individuals. Primary cardiomyopathies are most frequently caused by inherited single amino acid substitutions, in a single allele encoding one of the cardiac sarcomeric proteins.Alpha-tropomyosin is a key cardiac sarcomeric protein, which interacts structurally and functionally with all other components of the sarcomeric contractile apparatus and thereby regulates cardiac muscle contraction in response to Ca2+. There are more than fifteen substitutions identified throughout the length of alpha-tropomyosin which can result in cardiomyopathy. However, the fundamental biochemical and biophysical mechanism(s) by which these single amino acid substitutions affect sarcomeric function and cause cardiomyopathy is (are) unclear. Also, there is no clear relation between the location of these substitutions in alpha-tropomyosin and the nature of the resulting cardiomyopathy. Working with a collection of less-characterised, cardiomyopathy-associated mutations in human cardiac alpha-tropomyosin, we find that even if two mutations are associated with the same cardiomyopathy, the molecular dysfunction caused by the two mutations could be different. Previously it has been characterized that most HCM mutations in alpha-tropomyosin show weaker binding to actin. However, we find that the HCM-associated alpha-tropomyosin L185R mutant binds to F-actin with a greater affinity and co-operativity. In a co-sedimentation assay to measure the binding of alpha-tropomyosin and F-actin, the Kd decreases from 200 ± 20 nM (wild-type) to 100 ± 30 nM (L185R), and the Hill's co-efficient increases. This mutation, and some others, have been characterized in further detail, within the frame-work of the three-state model of the regulated thin filament, to provide novel insights into the mechanisms underlying cardiomyopathies.