Ca-dependency Of Cardiac SR Ca Release Reveals No Sign Of Ca-dependent Inactivation And Points To Luminal Ca As A Principal Regulator Of Release

Abstract

Calcium-induced calcium release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) mediates periodic beating of cardiac myocytes and can occur spontaneously contributing to arrhythmia. Following Ca release, SR Ca signaling becomes refractory due to a transitory functional inactivation of the RyR channels. Although the precise causes continue to be debated, cytosolic Ca-dependent inactivation and luminal Ca-dependent deactivation are viewed as the most likely mechanisms responsible for this phenomenon. In order to examine the role of these mechanisms in controlling CICR, we investigated SR Ca release in a wide range of cytosolic Ca concentrations ([Ca]c; 1-100uM) in permeabilized canine ventricular myocytes by monitoring Ca concentration inside the SR ([Ca]SR) using the low affinity Ca indicator Fluo5N. Elevating Ca from 100nM to 1-50uM caused spontaneous oscillations of [Ca]SR manifested as periodic depletions followed by periods of reloading synchronized across the cell. While the duration of depletion intervals increased, the periods when the SR was reloaded shortened resulting in an overall increase in the frequency of [Ca]SR oscillations with increasing [Ca]c. At [Ca]c>50uM, [Ca]SR oscillations disappeared and the SR stayed continuously empty. Preloading the SR with low-affinity Ca chelators decreased the frequency of [Ca]SR oscillations in a concentration-dependent manner. Our results suggest that under conditions of continuous activation by cytosolic Ca, RyRs can periodically cycle between open and deactivated states due to effects of luminal Ca. Deactivation appears to involve desensitization to cytosolic Ca because it is overcome at high [Ca]c, which renders the channels continuously open. Inactivation by cytosolic Ca plays no detectable role in controlling SR Ca release.