In adult cardiac muscle, depolarization activates a small Ca2+ influx that triggers intracellular Ca2+release (i.e., Ca2+-induced Ca2+ release, CICR) resulting in contraction. This intracellular Ca2+ release is mediated by ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In neonate cardiac muscle, the RyR-mediated CICR process is absent. The Ca2+ that triggers contractile activation does not come from intracellular Ca2+stores. Instead, it comes via Ca2+ influx through voltage-dependent Ca2+ channels in the surface membrane. In both contexts (adult and neonate), there are large cytosolic Ca2+ fluctuations associated with excitation-contraction coupling (E-C coupling).All mammalian cells (including cardiac cells) contain inositol trisphosphate receptor (InsP3R) channels. The InsP3R and RyR are homologous intracellular Ca2+ release channels. There is substantial evidence that InsP3R-mediated Ca2+ signaling is important in both the development and pathophysiology of cardiac muscle. In normal adult cells, the InsP3 signaling cascade plays a role in alpha-adrenergic ionotropic actions, contractile activation, and perhaps even in the regulation of transcription.Here, we focus on defining the underlying tenets of InsP3R-medated Ca2+ signaling in neonate myocytes. Neonate ventricular muscle presents a special case because it has minimal RyR-mediated intracellular Ca2+ release and therefore represents a less complicated environment in which to study of InsP3R-mediated signals. Our long range goals (past this proposal) are (1) to establish the physiological roles of InsP3R-medated Ca2+ in heart (in neonate, health and disease), and, (2) define the mechanisms that allow high fidelity InsP3R-medated Ca2+ signaling to be maintained in the """"""""Ca2+ noise"""""""" of E-C coupling. Such physiological applications require understanding the underlying mechanisms. In this proposal, we begin by focusing on the mechanisms governing the elemental properties of InsP3R-medated Ca2+ signaling in the neonate heart. To address this goal a combination of scanning confocal imaging, single channel recording, stochastic channel gating theory and Ca2+ diffusion modeling are directed at the following two specific aims.
Specific Aim #1 is to test the hypothesis that single InsP3R channel function (permeation/gating) is impacted by the complex salt environment found in cells (neonate ventricular myocytes).
Specific Aim #2 is to test the hypothesis that InsP3R-mediated Ca2+ signaling in neonate ventricular myocytes is mediated by highly organized spatial and temporal recruitment/summation of elemental stereotypical Ca2+-release events. The goal is to provide insights into the mechanisms that govern local InsP3R-mediated Ca2+ signaling in neonate ventricular myocytes.
