Abstract
Diastolic sarcoplasmic reticulum (SR) Ca leak determines Ca content and, therefore, the amplitude of action potential-induced global Ca transients in ventricular myocytes. However, the pathways and properties of SR Ca leak have been poorly described. Here, we studied the effects of cytosolic [Ca] ([Ca]i) on SR Ca leak in permeabilized rabbit ventricular myocytes. Using confocal microscopy we simultaneously measured intra-SR free Ca ([Ca]SR) with fluo-5N and cytosolic Ca sparks with rhod-2, and monitored SR Ca leak as the change in [Ca]SR over time after complete SERCA inhibition with thapsigargin (10 μM). Increasing [Ca]i from 150 to 250 nM significantly increased SR Ca leak (by ∼30%) over the entire range of [Ca]SR. This increase in SR Ca leak associated with an increase in Ca spark frequency. Further increasing [Ca]i to 350 nM led to rapid [Ca]SR depletion due to the occurrence of spontaneous Ca waves. In contrast, lowering [Ca]i to 50 nM markedly decreased SR Ca leak rate (by ∼60%) and nearly abolished Ca spark activity. When the ryanodine receptor (RyR) was completely inhibited with ruthenium red (50 μM), changes in [Ca]i between 50 and 350 nM did not produce any significant effect on SR Ca leak, showing that changes of [Ca]i over a physiological range alter SR Ca leak solely by regulating RyR activity. However, decreasing [Ca]i to a lower, non-physiologically level (5 nM) activated additional SR Ca leak pathway(s) that were insensitive to RyR or SR Ca ATPase inhibition. In summary, [Ca]i plays an important role in regulating SR Ca leak by activating RyR and preventing Ca leak through unspecified pathways.
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