The planned work will investigate a new phenomenon in heart muscle that we have recently discovered: """"""""Calcium sparking"""""""". Calcium-sparking can be observed when imaging the distribution of calcium in normal heart cells with very high temporal and spatial resolution. The calcium spark is a highly localized (microns) elevation of intracellular calcium that arises spontaneously and lasts only briefly (msec). The """"""""calcium spark"""""""" occurs largely randomly in time and space within a heart muscle cell. In normal, healthy cardiac myocyte the calcium spark event is rare. However, it produces a significant elevation of intracellular calcium (near-micromolar) that, surprisingly, does not activate additional calcium release from the sarcoplasmic reticulum (SR). Our preliminary investigation of calcium sparks in isolated rat heart muscle cells suggests that the calcium spark could result from the opening of a single SR calcium release channel, but this finding will be examined during the planned work. The calcium spark may activate propagated waves of elevated calcium when the heart cells are """"""""overloaded"""""""" with calcium. Over the next five years we plan to examine the cellular, biophysical and molecular basis of [Ca2+]i- sparking and explore its cellular consequences. Four experimental series are planned that will address the following broad questions: (1) What controls the [Ca2+]i sparking process? (2) What is the relationship between [Ca2+]i sparks and the normal [Ca2+]i transient? (3) What is the relationship between [Ca2+]i sparks and propagated waves of elevated [Ca2+]i? (4) Are the kinetic and functional properties of the SR Ca-release channel appropriate to explain calcium sparking? We expect this planned investigation to resolve issues that have been raised by our preliminary results: (a) What activates the calcium-spark? (b). How many SR Ca-release channels are involved in contributing calcium to a spark? (c) Why doesn't a spark usually give rise to a propagated wave of elevated calcium? (d) Is the frequency of calcium sparking a direct function of the open probability (P) of the SR- Ca-release channel? We will use isolated guinea pig and rat heart cells examined by high-speed confocal fluorescence microscopy in combination with a patch-clamp method in whole-cell mode. Additionally, the novel planar lipid bilayer method of Gyorke & Fill (1993) will be used to examine the SR Ca-release channels from rats and guinea-pig hearts. Both bilayer and imaging methods will exploit flash photolysis of caged calcium in the planned work. Fundamental new information on the cellular processes that control SR Ca- release channels and EC coupling in the heart muscle should result from the propose experiments. The work should thus broaden our understanding of calcium-dependent arrhythmias and diverse forms of heart failure.
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