Factors Influencing Ca2+ Spark Refractoriness in Mouse

Abstract

In cardiac myocytes, Ca2+ sparks exhibit time-dependent refractoriness such that it is difficult to trigger a second spark soon after an initial spark has terminated. Recent studies in rat ventricular myocytes suggested that spark amplitude recovery is controlled by local sarcoplasmic reticulum (SR) refilling whereas refractoriness of Ca2+ spark triggering depends on both refilling and ryanodine receptor (RyR) sensitivity. Here we examined Ca2+ spark refractoriness in mouse ventricular myocytes by exposing fluo-3 loaded quiescent cells to 50 nM ryanodine, recording Ca2+ sparks with a confocal microscope, and analyzing the repeated sparks that were produced at a limited number of RyR clusters. The time constant of Ca2+ spark amplitude recovery was 70-80 ms in mouse versus 90-100 ms in rat, suggesting slightly faster SR refilling in mouse. Spark-to-spark delay histograms were similar in the two species. Depending on the conditions, incubation of mouse myocytes with Isoproterenol or the drug H89, an inhibitor of protein kinase A (PKA), led to faster or slower Ca2+ spark amplitude recovery and shorter or longer average spark-to-spark delays, respectively. Moreover, incubation with H89 caused a decrease in whole-cell Ca2+ transient amplitude, slower Ca2+ transient decay, and no apparent change in SR Ca2+ load, compared with control conditions. Together these results suggest that relatively high levels of endogenous PKA activity may act to shorten Ca2+ spark refractoriness in mouse ventricular myocytes.