Functional analysis of a unique troponin c mutation, gly159asp, that causes Familial dilated cardiomyopathy, studied in explanted heart muscle

Abstract

Background-Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca2+ sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca 2+-regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation. Methods and Results-Mass spectroscopy showed that the mutant cTnC was expressed approximately equimolar with wild-type cTnC. Contraction was compared in skinned ventricular myocytes from the cTnC G159D patient and nonfailing donor heart. Maximal Ca2+-activated force was similar in cTnC G159D and donor myocytes, but the Ca2+ sensitivity of cTnC G159D myocytes was higher (EC50 G159D/donor=0.60). Thin filaments reconstituted with skeletal muscle actin and human cardiac tropomyosin and troponin were studied by in vitro motility assay. Thin filaments containing the mutation had a higher Ca2+ sensitivity (EC50 G159D/donor=0.55±0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca 2+ sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca2+ sensitivity was increased (EC50 P/unP=4.7±1.9) but not with cTnC G159D troponin (EC50 P/unP=1.2±0.1). Conclusions-We propose that uncoupling of the relationship between phosphorylation and Ca2+ sensitivity could be the cause of the dilated cardiomyopathy phenotype. The differences between these data and previous in vitro results show that native phosphorylation of troponin I and troponin T and other posttranslational modifications of sarcomeric proteins strongly influence the functional effects of a mutation. © 2009 American Heart Association, Inc.