Regulation of sarcoplasmic reticulum Ca2+ leak by cytosolic Ca2+ in rabbit ventricular myocytes

Sarcoplasmic reticulum (SR) Ca2+ leak determines SR Ca2+ content and, therefore, the amplitude of global Ca2+ transients in ventricular myocytes. However, it remains unresolved to what extent Ca2+ leak can be modulated by cytosolic [Ca2+] ([Ca2+](i)). Here, we studied the effects of [Ca2+](i) on SR Ca2+ leak in permeabilized rabbit ventricular myocytes. Using confocal microscopy we monitored SR Ca2+ leak as the change in [Ca2+](SR) (with Fluo-5N) after complete SERCA inhibition with thapsigargin (10 mu m). Increasing [Ca2+](i) from 150 to 250 nM significantly increased SR Ca2+ leak over the entire range of [Ca2+](SR). This increase was associated with an augmentation of both Ca2+ spark-and non-spark-mediated Ca2+ leak. Further increasing [Ca2+](i) to 350 nMled to rapid [Ca](2+)](SR) depletion due to the occurrence of Ca2+ waves. The augmentation of SR Ca2+ leak by high [Ca2+](i) was insensitive to inhibition of Ca2+-calmodulin-dependent protein kinase II. In contrast, lowering [Ca2+](i) to 50 nM markedly decreased SR Ca2+ leak rate and nearly abolished Ca2+ sparks. When the ryanodine receptor (RyR) was completely inhibited with ruthenium red (50 mu M), changes in [Ca2+](i) between 50 and 350 nM did not produce any significant effect on SR Ca2+ leak, indicating that [Ca2+](i) alters SR Ca2+ leak solely by regulating RyR activity. In summary, [Ca2+](i) in the range of 50-350 nM has a significant effect on SR Ca2+ leak rate mainly via direct regulation of RyR activity. As RyR activity depends highly on [Ca2+](i) and [Ca2+](SR), SR Ca2+ leak remains relatively constant during the declining phase of the Ca2+ transient when [Ca2+](SR) and [Ca2+](i) change in opposite directions.