Ca²+ spark-dependent and -independent sarcoplasmic reticulum Ca²+ leak in normal and failing rabbit ventricular myocytes.

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(+)) after complete inhibition of SERCA with thapsigargin in permeabilized rabbit ventricular myocytes. When Ca²(+) declined to 279 ± 10 μm, spark activity ceased completely; however SR Ca²(+) leak continued, albeit at a slower rate. Analysis of sparks and Ca²(+) revealed, that SR Ca²(+) leak increased as a function of Ca²(+), with a particularly steep increase at higher Ca²(+) ( >600 μm) where sparks become a major pathway of SR Ca²(+) leak. At low Ca²(+) (< 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.