In adult cardiac muscle, depolarization activates a small Ca 2+ influx that triggers intracellular Ca 2+ release resulting in contraction. This intracellular Ca 2+ release is mediated by type-2 ryanodine receptor (RyR2) channels in the sarcoplasmic reticulum (SR). The RyR2 channels are clustered at discrete SR Ca 2+ release sites (1). Here, our focus is on defining the local control of RyR2-mediated Ca 2+ signaling within and between release sites in adult rat myocytes. Local elemental RyR2-mediated Ca 2+ release events, called Ca 2+ sparks, occur spontaneously in heart muscle. It is thought that these elemental events temporally and spatially sum to generate the more global Ca 2+ release phenomena that governs cardiac contractility. There are several fundamental unknowns that limit our understanding of local intracellular Ca 2+ signaling in heart. For example, it is still not clear if Ca 2+ sparks arise from the opening of an individual RyR2 channel or the concerted opening of several channels. The mechanisms that modulate Ca 2+ spark properties (e.g. their amplitude, frequency, propagation, etc.) are also poorly defined. To address these (and other) unknowns a combination of single channel recording, laser flash photolysis, scanning confocal imaging, stochastic single channel theory and spatiotemporal Ca 2+ diffusion modeling will be directed to test the following hypotheses (or specific aims). Hypothesis #1: Single RyR2 channel function is governed by microscopic Ca 2+ fluctuations that are not evident in the macroscopic Ca 2+ signaling environment. Specifically, two new mechanistic concepts (e.g. stochastic and/or feed-through Ca 2+ regulation) are proposed, experimentally tested, and interpreted in a novel conceptual framework. Hypothesis #2: Inter-RyR2 channel Ca 2+ communication defines the spatiotemporal nature of local Ca 2+ signaling within and between SR Ca 2+ release sites. The mechanisms that control inter-RyR2 channel Ca2+ communication within and between release sites will be defined here at both the single channel and whole cell levels. Experimental results will be interpreted using a unique spatiotemporal model of local Ca 2+ signaling.
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