Up to 40% of heart failure patients have impaired relaxation, known as diastolic dysfunction. Cardiac muscle relaxation is not merely a passive reversal of contraction, but a complex process requiring a series of events: 1) decline in intracellular Ca2+, 2) dissociation of Ca2+ from troponin C (TnC), and 3) cross-bridge detachment. There is controversy regarding the relative importance of each of these events in controlling relaxation. Despite the fact that Ca2+ must dissociate from the regulatory domain of TnC to allow relaxation, contribution of TnC to the rate of relaxation is in dispute. The objective of this project is to test the overriding hypothesis that the rate of Ca2+ dissociation from the regulatory domain of TnC is a major determinant of cardiac relaxation. To test this hypothesis, the proposed study will utilize the following AIMs:
AIM 1 : Elucidate and manipulate factors controlling Ca2+ binding and exchange with TnC in increasingly structured systems, ranging from isolated TnC to reconstituted thin filaments. The goal of this aim is to determine how Tnl, thin and thick filament proteins and key residues in TnC affect Ca2+ affinity and Ca2+ dissociation rate from the regulatory domain of TnC. TnC proteins, generated in this AIM by mutagenesis of key residues in TnC, will be used in AIMs 2 and 3 to test the overriding hypothesis.
AIM 2 : Determine the contribution of Ca2+ dissociation from TnC to the rate of relaxation in skinned rabbit trabeculae. This study will probe the contribution of Ca2+ dissociation from TnC to the rate of relaxation at the level of the myofilaments. Relaxation of skinned trabeculae reconstituted with TnC mutants, possessing dramatically faster or slower Ca2+ dissociation rates, will be induced by rapid laser photolysis of the caged Ca2+ chelator diazo-2.
AIM 3 : Determine the contribution of Ca2+ dissociation from TnC to the rate of relaxation in intact, continuously contracting rabbit trabeculae in culture. These studies will probe the contribution of TnC to the rate of relaxation, under the conditions where Ca2+ sequestration can be a determinant of relaxation. TnC mutants with dramatically faster or slower Ca2+ dissociation rates will be introduced into intact cultured trabeculae by adenoviral gene transfer. These experiments should clarify whether TnC should be a target for the treatment of diastolic dysfunction.
