Cytosolic Ca2+ buffering determines the intra-SR Ca2+ concentration at which cardiac Ca2+ sparks terminate

Abstract

Single ryanodine receptor (RyR) Ca2+ flux amplitude (iCa-RyR) decreases as intra-sarcoplasmic reticulum (SR) Ca2+ levels fall during a cardiac Ca2+ spark. Since iCa-RyR drives the inter-RyR Ca2+-induced Ca2+ release (CICR) that underlies the spark, decreasing iCa-RyR may contribute to spark termination because RyRs that spontaneously close may stay closed. To test this possibility, we simultaneously measured local cytosolic and intra-SR ([Ca2+]cyto and [Ca2+]SR) during Ca2+ sparks in permeabilized rabbit ventricular myocytes. Local cytosolic or intra-SR Ca2+ dynamics were manipulated using Ca2+ buffers. Buffer manipulations applied in cells had no effect on individual RyR channels reconstituted in planar lipid bilayers. Presence of a fast cytosolic Ca2+ buffer (BAPTA) significantly suppressed Ca2+ spark activity and sparks terminated earlier at a higher than usual [Ca2+]SR level (∼80% vs. ∼62%). When cytosolic Ca2+ buffer power was reduced (i.e. cytosolic EGTA level decreased), sparks terminated later and at a lower than usual [Ca2+]SR level (∼45% vs. ∼62%). When intra-SR Ca2+ buffer power was increased, sparks also terminated later and at a lower than usual [Ca2+]SR (∼48% vs. ∼62%). These results suggest that cytosolic local control of inter-RyR CICR by iCa-RyR plays a substantial role during the spark termination process. Thus, alterations in local cytosolic Ca2+ handling dynamics in the dyadic cleft (Ca2+ buffering, extrusion, etc.) likely influence Ca2+ spark termination.