Streptozotocin-Induced Diabetes Increases Disulfide Bond Formation on Cardiac Ryanodine Receptor (RyR2)

Abstract

In a previous study, we showed that after 6 weeks of streptozotocin-induced diabetes (6D), expression of type 2 ryanodine receptor calcium-release channels (RyR2) did not change significantly in rat hearts. However, the ability of this protein to bind [³H]ryanodine was compromised. Loss in activity therefore resulted from diabetes-induced increases in post-translational modifications on RyR2. In the present study, the effects of diabetes on one type of modification, namely, changes in oxidative state of reactive sulfhydryls was investigated. RyR2 protein from 6D bound 42.3 ± 7.6 less [³H]ryanodine than RyR2 from controls (6C). The loss in binding was minimized with 2 weeks of insulin treatment initiated after 4 weeks of diabetes (77.8 ± 5.5% of 6C). Pretreating RyR2 from 6D with 2 mM dithiothreitol in vitro increases [³H]ryanodine binding by 60.8 ± 5.3%. Dithiothreitol pretreatment of RyR2 from 6C increased [³H]ryanodine binding by 16.8 ± 4.3%. The reagent pyrocoll interacts with distinct classes of free sulfhydryl groups on 6C RyR2 to induce two major effects. At concentrations ≤10 μM, it deactivates RyR2 (decreases [³H]ryanodine binding), whereas at higher concentrations it activates them (increases [³H]ryanodine binding). This reagent was unable to activate RyR2 from 6D. Although RyR2 from insulin-treated animals was deactivated by low concentrations of pyrocoll, it was only partially activated at higher concentrations. These data suggest that the dysfunction of RyR2 induced by diabetes may be due in part to formation of disulfide bonds between adjacent sulfhydryl groups and that these changes were attenuated with insulin treatment.