Oxidation of RyR2 Has a Biphasic Effect on the Threshold for Store Overload-Induced Calcium Release

Abstract

At the single-channel level, oxidation of the cardiac ryanodine receptor (RyR2) is known to activate and inhibit the channel depending on the level of oxidation. However, the mechanisms through which these changes alter the activity of RyR2 in a cellular setting are poorly understood. In this study, we determined the effect of oxidation on a common form of RyR2 regulation; store overload-induced Ca(2+) release (SOICR). We found that oxidation resulted in concentration and time-dependent changes in the activation threshold for SOICR. Low concentrations of the oxidant H2O2 resulted in a decrease in the threshold for SOICR, which led to an increase in SOICR events. However, higher concentrations of H2O2, or prolonged exposure, reversed these changes and led to an increase in the threshold for SOICR. This increase in the threshold for SOICR in most cells was to such an extent that it led to the complete inhibition of SOICR. Acute exposure to high concentrations of H2O2 led to an initial decrease and then increase in the threshold for SOICR. In the majority of cells the increased threshold could not be reversed by the application of the reducing agent dithiothreitol. Therefore, our data suggest that low levels of RyR2 oxidation increase the channel activity by decreasing the threshold for SOICR, whereas high levels of RyR2 oxidation irreversibly increase the threshold for SOICR leading to an inhibition of RyR2. Combined, this indicates that oxidation regulates RyR2 by the same mechanism as phosphorylation, methylxanthines, and mutations, via changes in the threshold for SOICR.

Activation of RyR2 by class I kinase inhibitors

“…Our previous work studying the effect of caffeine and oxidation has shown that a common mechanism by which the propensity of SOICR is increased is due to a reduction in the F SOICR (Kong et al ., ; Waddell et al ., ). Therefore, we next explored the effect of class I and class II kinase inhibitors on F SOICR (which is expressed as a percentage of the total store) using the luminally expressed Ca 2+ sensor D1ER as previously described (Jones et al ., ; Zhang et al ., ).…”

“…Class I kinase inhibitors increase the propensity for SOICR HEK293 cells expressing RyR2 have been well established to display SOICR with very similar characteristics to cardiac cells (Zhang et al, 2014;Waddell et al, 2016). In this study, HEK293 cells expressing RyR2 were used to investigate the impact of class I and II kinase inhibitors on the occurrence of SOICR.…”

“…It is well established that a reduction in the threshold for SOICR, combined with an increase in SR load, enhances the propensity for SOICR which is commonly linked to arrhythmias (Jones et al ., ; Chen et al ., ; Jones et al ., ). We have found this to be a common mechanism contributing to how mutations within RyR2, oxidation, doxorubicin and methylxanthine derivatives such as aminophylline and theophylline all lead to arrhythmia (Jones et al ., ; Kong et al ., ; Hanna et al ., ; Waddell et al ., ). Although class I kinase inhibitors appear to have a strong agonist effect at the cell level, at the single channel, the effect is less clear cut.…”

“…Measurements of cytosolic Ca 2+ were conducted in stable, inducible HEK293 cells (RRID:CVCL_U427) expressing RyR2, generated as previously described (Waddell et al ., ). RyR2 expression was induced ~16–18 h before the experiment via the addition of tetracycline (0.1 μg·mL −1 ).…”

“…Two images were collected by a CoolSNAP HQ2 CCD camera (Photometrics, AZ, USA) using a dual‐channel imaging system (DV2). The amount of FRET was determined from the ratio of the emissions at 535 and 480 nm (YFP : CFP) as previously described (Waddell et al ., ; Zhang et al ., ). From each trace, several parameters were determined.…”