Cardiac ryanodine receptor activation by high Ca2+ store load is reversed in a reducing cytoplasmic redox environment

Abstract

We report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca2+]. Basal leak from the sarcoplasmic reticulum (SR) is required for normal Ca2+ handling, but excess diastolic Ca2+ leak attributed to oxidative stress is thought to lower RyR2 threshold for spontaneous SR Ca2+ release to induce arrhythmia in pathological situations. Therefore we examined RyR2 response to luminal [Ca2+] under reducing or oxidising cytoplasmic redox conditions. Unexpectedly as luminal [Ca2+] increased from 0.1–1.5 mM RyR2 activity declined when pretreated with cytoplasmic 1 mM DTT, or GSH∶GSSG buffered to a “healthy” reduced cytoplasmic redox potential (−220 mV). Conversely, with 20 µM cytoplasmic 4,4′-DTDP, or redox buffered to an oxidising −180 mV, RyR2 activity increased with increasing luminal [Ca2+]. The luminal redox potential was constant at −180 mV in each case. These responses to luminal Ca2+ were maintained with 2 mM Na2ATP or 5 mM MgATP (1 mM free Mg2+). Overall the results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than the bulk cytoplasm.