MOLECULAR PHYSIOLOGY OF MYOCARDIAL TROPONIN I VARIANTS

Johns Hopkins University, Baltimore MD

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Abstract

DESCRIPTION (the applicant's description verbatim): Contraction of the heart occurs through regulated interactions of the myofilament proteins in response to increasing intercellular calcium concentrations. Contractile dysfunction associated with myocardial disease is linked to an altered response of contractile proteins to calcium. The inhibitory subunit of troponin, troponin I, is a key regulatory protein which modulates contractility based on its phosphorylation status. Recently, modifications and mutants of troponin I have been associated with ischemic injury, heart failure and cardiomyopathy. The goal of this proposal is to understand how disease related alterations to troponin I modify its function and play a central role in myocardial disease states. A comprehensive approach is proposed to delineate the both the pathophysiology and molecular mechanisms of alteration of function produced by troponin I variants in the heart . Specific variants which will be characterized are a) truncated variant (I - 193), a loss of 16 amino acid residues from the carboxy-terminus, which recapitulates the truncated form produced by calcium dependent proteolysis in stunned myocardium, and b) troponin I variants with site-directed mutations in protein kinase A and protein kinase C phosphorylation sites to determine the role of these sites in intrinsic contractility, preconditioning, ischemia/reperfusion and heart failure and finally c) a troponin I variant with a single amino acid mutation in the inhibitory region reproducing a recently described mutant found in a familial hypertrophic cardiomyopathy. Methods include measurements of ventricular mechanics in transgenic mice using a miniaturized conductance-micromanometer catheter, studies of steady state force-calcium relationships in fura-2 loaded intact trabeculae from these mice and in vitro experiments with recombinant protein and synthesized peptides to dissect the altered biochemical properties of these troponin I variants. This work will determine a molecular mechanism of myocardial stunning, the role of troponin I phosphorylation in vivo and the pathophysiology of one form of hypertrophic cardiomyopathy. It is also anticipated that the information gained will ultimately provide a rational basis for the development of novel therapies for cardiac dysfunction.