Functional Effects of Two Troponin I Mutations Linked to Restrictive Cardiomyopathy

Abstract

Mutations in cardiac troponin, a protein complex that regulates muscle contraction, have been shown to be linked to cardiomyopathies, which commonly lead to chest pains, myocardial infarction, or sudden cardiac death. The troponin complex consists of three proteins: Troponin T, Troponin I (TnI), and Troponin C (TnC). In recent clinical studies, two novel mutations in cardiac TnI were discovered co-segregated with cardiomyopathy, but their specific functional effects remain unknown. These mutations are the first frameshift mutations in cTnI known to be linked to restrictive cardiomyopathy (RCM). The deletion of two adenines at codon 177 (Delbp529AA) in cardiac TnI, was discovered in a six year old female RCM patient. The second cTnI mutation included in this investigation was the result of a deleted guanine in codon 168 which caused a frame shift and premature stop codon at 176 (DelG502). cTnI DelG502 was associated with sudden cardiac death. It was found during column purification that the 34 residue truncation of cTnI removed or greatly decreased its binding affinity for TnC. However, the Delbp529AA mutant protein, containing 32 alternate C-terminal residues, was successfully purified and formed a functional troponin complex. Actomyosin ATPase assays demonstrated similar maximal ATPase activity for complexes containing TNNI3 Delbp529AA compared to wild type complexes. cTnI Delbp529AA showed increased calcium sensitivity of ATPase and less inhibitory function compared to wild-type cTnI. Calpain digestion studies indicate that cTnI Delbp529AA is degraded at a faster rate than wild-type cTnI. These results suggest that the poor prognosis of patients carrying these RCM-linked mutants is due to protein dysfunction at multiple levels and suggest possible mechanisms of RCM pathology.