Sarcoplasmic reticulum (SR) Ca2+ leak is an important component of cardiac Ca2+ signalling. Together with the SR Ca2+-ATPase (SERCA)-mediated Ca2+ uptake, diastolic Ca2+ leak determines SR Ca2+ load and, therefore, the amplitude of Ca2+ transients that initiate contraction. Spontaneous Ca2+ sparks are thought to play a major role in SR Ca2+ leak. In this study, we determined the quantitative contribution of sparks to SR Ca2+ leak and tested the hypothesis that non-spark mediated Ca2+ release also contributes to SR Ca2+ leak. We simultaneously measured spark properties and intra-SR free Ca2+ ([Ca2+]SR) after complete inhibition of SERCA with thapsigargin in permeabilized rabbit ventricular myocytes. When [Ca2+]SR declined to 279 ± 10 μm, spark activity ceased completely; however SR Ca2+ leak continued, albeit at a slower rate. Analysis of sparks and [Ca2+]SR revealed, that SR Ca2+ leak increased as a function of [Ca2+]SR, with a particularly steep increase at higher [Ca2+]SR (>600 μm) where sparks become a major pathway of SR Ca2+ leak. At low [Ca2+]SR (<300 μm), however, Ca2+ leak occurred mostly as non-spark-mediated leak. Sensitization of ryanodine receptors (RyRs) with low doses of caffeine increased spark frequency and SR Ca2+ leak. Complete inhibition of RyR abolished sparks and significantly decreased SR Ca2+ leak, but did not prevent it entirely, suggesting the existence of RyR-independent Ca2+ leak. Finally, we found that RyR-mediated Ca2+ leak was enhanced in myocytes from failing rabbit hearts. These results show that RyRs are the main, but not sole contributor to SR Ca2+ leak. RyR-mediated leak occurs in part as Ca2+ sparks, but there is clearly RyR-mediated but Ca2+ sparks independent leak.Contraction of the heart relies on calcium stored in the sarcoplasmic reticulum (SR) that is released and subsequently taken up again with every heart beat. Calcium content of the SR is determined by the balance between calcium pump activity and passive calcium leak. SR calcium leak plays an important role in the normal and diseased heart. We found that the ryanodine receptor, a calcium release channel in the SR, is the main source of calcium leak of ventricular myocytes. In the SR ryanodine receptors are organized in individual clusters. Depending on the amount of calcium stored, calcium leak can occur as openings of a single ryanodine receptor, as simultaneous activation of multiple channels in a cluster or through pathways that do not involve the ryanodine receptor. We also found that calcium leak through ryanodine receptors is significantly increased in myocytes from failing hearts leading to impaired cardiac function.
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