Refractoriness of Calcium Release Units in Rat Cardiac Myocytes

Abstract

Calcium release units (CRUs) in cardiac myocytes contain a large number of ryanodine receptors (RyRs), but it seems that only a small fraction of RyRs is being activated during a single calcium release event. Repetitive activity of CRUs is not well understood from the point of refractoriness. We used direct measurement of Ca-spikes activated by calcium current (ICa) to unveil the refractoriness of CRU firing from the properties and occurrence of calcium spikes under control conditions and in the presence of the DHPR activator FPL.ICa was activated by voltage pulses from 50 to 0 mV under whole-cell patch-clamp. Ca-spikes were measured by confocal microscopy using Ca2+ indicators (Fluo-3 and Oregon Green BAPTA-5N) in the presence of EGTA. A large majority of CRUs responded to stimulation by a single Ca-spike, while 13.4 % produced two subsequent (twin) Ca-spikes. Early Ca-spikes (single spikes and the first of twin Ca-spikes) had similar latencies, while the second Ca-spikes were significantly delayed. Amplitude distribution of the early Ca-spikes consisted of four quantal levels with equal amplitudes and binomially distributed frequency of occurrence, supporting the hypothesis that different Ca-spike amplitudes are due to a different number of independent quantal levels, likely individual RYR openings. The probability of occurrence of second Ca-spikes was inversely proportional to the quantal size of the early Ca-spike. In the presence of FPL, the probability of occurrence of twin spikes was decreased relative to control, despite persistent activation of DHPR receptors.We conclude that refractoriness of local calcium release does not result from Ca-dependent DHPR inactivation, but rather it is caused by inactivation of the release unit due to local depletion of the SR.Supported by APVV-0721-10, APVV-0441-09, VEGA 2/0203/11, VEGA 2/0917/11, and VEGA 2/0190/10.