Intra-sarcoplasmic reticulum Ca2+ oscillations are driven by dynamic regulation of ryanodine receptor function by luminal Ca2+ in cardiomyocytes

摘要

During the cardiac cycle, the release of Ca²⁺ from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR2) channel complex is controlled by the levels of cytosolic and luminal Ca²⁺ and alterations in these regulatory processes have been implicated in cardiac disease including arrhythmia. To better understand the mechanisms of regulation of SR Ca²⁺ release by Ca²⁺ on both sides of the SR membrane, we investigated SR Ca²⁺ release in a wide range of cytosolic Ca²⁺ concentrations ([Ca²⁺]cyt; 1–100 μm) in permeabilized canine ventricular myocytes by monitoring [Ca²⁺] inside the SR ([Ca²⁺]SR). Exposing myocytes to activating [Ca²⁺]cyt resulted in spontaneous oscillations of [Ca²⁺]SR due to periodic opening and closing of the RyR2s. Elevating [Ca²⁺]cyt (up to 10 μm) increased the frequency of [Ca²⁺]SR oscillations; however at higher [Ca²⁺]cyt (>50 μm) the oscillations diminished due to RyR2s staying perpetually open, resulting in depleted SR. Ablation of cardiac calsequestrin (CASQ2) altered the [Ca²⁺]cyt dependence of Ca²⁺ release oscillations such that oscillations were highly frequent at low [Ca²⁺]cyt (100 nm) but became diminished at moderate [Ca²⁺]cyt (10 μm), as determined in myocytes from calsequestrin-null versus wild-type mice. Our results suggest that under conditions of continuous activation by cytosolic Ca²⁺, RyR2s can periodically cycle between open and deactivated states due to effects of luminal Ca²⁺. Deactivation at reduced [Ca²⁺]SR appears to involve reduction of sensitivity to cytosolic Ca²⁺ and might be mediated by CASQ2. Inactivation by cytosolic Ca²⁺ plays no detectable role in controlling SR Ca²⁺ release.