LOCAL CONTROL OF CARDIAC EXCITATION/CONTRACTION COUPLING

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Control of the release o calcium ions (Ca2+) from sarcoplasmic reticulum (SR) is fundamental to normal cardiac function and its modulation by hormones, neurotransmitters, and electrical excitation. The overall aim of this research is to test two hypotheses on SR calcium release; 1) 'Local control': Spatially discrete groups or 'clusters" of SR release channels are first activated exclusively by Ca2+ entering via co-associated L-type. Ca2+ channels. In local control activation of a cluster occurs independently of the activation of any other such clusters. 2) Loss of 'local control" leads to certain abnormalities of cardiac function such as Ca2+ waves and triggered arrhythmias. Loss of local control means that clusters are no longer activated initially by nearby Ca2+ influx, but may be activated by Ca2+ reaching them from other clusters. It is hypothesized also that loss of local control can be antagonized by intracellular magnesium ions (Mg 2+). Local control is thought to result from a microscopic 'coupling' between individual L-type Ca2+-channels and a 'cluster' of SR release channels at the junction between SR and transverse tubule. It is hypothesized that the probability of evoking Ca2+ release there is determined by latency to first opening of the L-type Ca2+ channel, and by the magnitude of the unitary L-type current (rather than by the whole-cell Ca2+-current, Na+/Ca2+_ exchange, or by Ca2+ already released from other clusters). According to local control, the 'coupler' ([Ca2+]) in the local subcellular region of the RyR is very different from that which would be predicted from conventional measurements of whole-cell Ca2+- current and spatial-average Ca2+ transient. Accordingly, confocal laser scanning microscopy, which provides increased spatial resolution, will be used to observe local [Ca2+]i transients (LCTs) evoked by voltage clamp depolarization in single guinea-pig and rat cardiac ventricular cells perfused internally with fluorescent Ca2+ indicators. Image processing will be used to determine (spatial) characteristics of evoked local [Ca2+]i-transients and spontaneous Ca2+-sparks, as required to evaluate the hypothesis of local control rigorously. Whole-cell and patch clamp recording will be used to determine whether local [Ca2+]i-transients are evoked in a manner consistent with gating properties of L-type Ca2+ - channels. The effect of SR loading and intracellular Mg2+ on the size, probability, and location of occurrence of evoked local [Ca2+]i-transients, spontaneous Ca2+-sparks, and Ca2+ -waves will be studied. The research will provide new information on normal excitation-contraction (E-C) coupling, and it will test the hypothesis that abnormal E-C coupling arises from loss of the normal 'local control of SR Ca2+ release.