Effects of Cytosolic Ca Buffering on Sarcoplasmic Reticulum Ca Leak in Permeabilized Rabbit Ventricular Myocytes

Abstract

In this study we tested the hypothesis that local Ca-induced Ca release (CICR) within ryanodine receptor (RyR2) release clusters plays an important role in defining spark-mediated sarcoplasmic reticulum (SR) Ca leak. We studied to what degree changes of cytosolic Ca buffer capacity affect single RyR activity in lipid bilayers, Ca spark properties, and SR Ca leak in permeabilized ventricular myocytes. No substantial changes in single RyR2 function were observed if BAPTA (a fast Ca buffer) was added (holding free Ca constant) to the EGTA-buffered cytosolic solution. However, the same cytosolic BAPTA addition almost completely eliminated Ca sparks in cells. The elimination of Ca sparks by BAPTA was not due to a decrease in SR Ca load. The addition of the fast cytosolic Ca buffer significantly decreased SR Ca leak, particularly at high [Ca]SR (>400 μM) where Ca sparks significantly contribute to SR Ca leak. In contrast, lowering cytosolic Ca buffer capacity (by decreasing EGTA from 0.36 to 0.1 mM; keeping free Ca constant) drastically increased Ca spark frequency and width, leading to generation of multifocal (or propagating) Ca sparks. This decrease in cytosolic Ca buffer capacity also significantly accelerated SR Ca leak as a result of augmentation of Ca spark-mediated component of SR Ca leak. In conclusion, by changing cytosolic Ca buffering (capacity or affinity) we were able to experimentally separate Ca spark- and non-spark- mediated component of the overall SR Ca leak in ventricular myocytes. These data also suggest that changes in the dynamics of local cytosolic Ca handling (buffering, extrusion, etc.) are physiologically important in determining the degree of spark and non-spark mediated SR Ca leak.