Excitation contraction (EC) coupling in the heart depends upon the process of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum (SR), which occurs via calcium release channels commonly referred to as ryanodine receptors (RyR). It is believed physiological CICR is mediated by the stochastic interaction between calcium microdomains and RyR's at the dyad junction between the t-tubule and the SR release terminal (the local control hypothesis). Stochastic calcium release events, known as calcium sparks are believed to be elementary events of EC coupling, but their exact nature is controversial. We sought to determine whether calcium sparks arise from a single or multiple RyR's by examining the effect of SR calcium depletion, which should decrease the coupling between nearby RyR's. Spontaneous calcium sparks were studied in rat ventricular myocytes using confocal microscopy and the fluorescent calcium probe fluo-3. A computer algorithm was developed to count and measure objectively calcium sparks in linescan images. Thapsigargin (25-150 nM) depleted caffeine-releasable SR calcium by up to 64%. During SR depletion, calcium sparks were robustly observed albeit at reduced frequency (?30% of control) and amplitude (greater than otr equal to 60% of control). After correction for bias due to the reduced detectability of smaller sparks, we found that the spark frequency was independent of SR load whereas the amplitude was proportional to load. We conclude that, although spark amplitude depends on SR filling status, the frequency of spark generation is independent of SR calcium load and therefore of the local calcium release rate. This implies that sparks are single channel events, or collective events that are well above threshold for local regeneration.
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