The importance of uncoupling troponin I phosphorylation from Ca2+ sensitivity in the pathogenesis of cardiomyopathy

Abstract

Heart muscle contraction is regulated via the β adrenergic response that results to phosphorylation of Protein Kinase A (PKA), which in turn decreases the Ca2+ sensitivity of the cardiac myofilament, which is very important for the heart muscle to relax. Mutations in the thin filament that cause Dilated Cardiomyopathy (DCM) and some that cause Hypertrophic Cardiomyopathy (HCM) abolish this relationship, so that the Ca2+ sensitivity becomes independent of Troponin I (TnI) phosphorylation (uncoupling). The aim of the thesis is to unravel the molecular mechanism of the uncoupling phenomenon. It is known that there is a specific interaction between the phosphorylatable TnI N terminal peptide and the Ca2+ binding site on TnC, that is weakened by phosphorylation and we hypothesize that it is disrupted in case of a DCM or HCM mutation, giving rise to the uncoupling phenomenon. Ca2+ sensitisers and desensitisers change the Ca2+ sensitivity of the cardiac muscle like mutations do but their relationship with TnI phosphorylation has never been studied before. Using the in vitro motility assay I showed that the Ca2+ sensitisers EMD 57033 and Bepridil increased the Ca2+ sensitivity of donor thin filaments and additionally they uncoupled the Ca2+ sensitivity from the TnI phosphorylation. Epigallocatechin-3-gallate (EGCG) decreased the Ca2+ sensitivity of donor thin filaments whilst retaining the coupling. On the other hand, EGCG reduced the Ca2+ sensitivity of phosphorylated but not dephosphorylated mutant thin filaments restoring the Ca2+ sensitivity change to TnI phosphorylation. EGCG re-coupled 5 DCM (TPM1 E54K and E40K, TNNI3 K36Q, TNNC1 G159D, ACTC E361G) mutants and 3 HCM (TPM1 E180G, TNNT2 K280N, ACTC E99K) mutants which were originally uncoupled. We were given 30 analogue compounds structurally similar to EGCG and nine of them were able to re-couple uncoupled TPM1 E180G HCM mutant thin filaments. The working compounds re-coupled DCM mutation TPM1 E54K and HCM mutation ACTC E99K. I show for the first time that it is possible to mimic and reverse the effect of DCM and HCM mutations on troponin pharmacologically. EGCG and its analogue compounds might have significant implications for the effective treatment of thin filament cardiomyopathies that uncouple the Ca2+ sensitivity from TnI phosphorylation. In a separate study I investigated 11 mutations in skeletal muscle tropomyosin associated with various myopathies. I found that 7 mutations cause a gain of function that could be accounted for at the molecular level due to destabilising specific actin-tropomyosin interactions. Gain of function at the molecular level correlates with a hypercontractile phenotype in patients.Open Acces