Alterations in excitation-contraction (EC) coupling are a central finding in animal models of CHF and failing human myocardium. In this application, the investigators show preliminary data demonstrating that t-tubules are severely depleted or totally absent in failing canine cardiomyocytes. This observation has important functional implications as normal EC coupling requires the close apposition of L-type Ca2+ channels (DHPRs) in t-tubules with Ca release channels (RyR) in the sarcoplasmic reticulum forming junctional domains (dyads). Given the role of t-tubules in forming junctional domains and the localization of key proteins in the b-adrenergic receptor (bAR) signaling cascade there, the investigators'central hypothesis is that sub-cellular remodeling of the t-tubule system and junctional domains results in contractile failure and abnormal b-adrenergic regulation in failing ventricular myocytes. This application brings together a strong interactive group of investigators from basic and clinical disciplines who will collaborate in two R01 applications and a myocyte core project to examine remodeling of the dyad cleft in failing cardiomyocytes.
Specific aims will focus on the fact that t-tubular remodeling will result in significant functional alterations of EC coupling and its regulation as a result of changes in: 1) the number or at least location of junctional domains; 2) the composition of junctional domains (local density of DHPRs, RyRs, bARs), 3) the geometry of junctional domains; and/or 4) the functional properties of proteins in the junctional domain. To rigorously address these subhypotheses, a complementary array of experiments are proposed employing 2-photon microscopy, confocal microscopy, electron microscopy, whole-cell patch clamp techniques, lipid bilayer reconstitution studies, and biochemical methods to study t-tubular structure and function in failing and nonfailing canine and human ventricular myocytes. The mechanism of this subcellular remodeling will be examined in temporal studies during the onset and recovery from CHF in the canine model as well as utilizing human tissue from patients undergoing left ventricular assist device implantation followed by cardiac transplantation including correlation of t-tubular loss with apoptosis.
