New mechanisms of cardiac ryanodine receptor dysfunction during oxidative stress: the role of intersubunit cross-linking - PROJECT SUMMARY/ABSTRACT Calcium (Ca) release through the ryanodine receptor (RyR) Ca channel is essential for regular heart contraction. Defects in RyR regulation cause an imbalance in intracellular Ca homeostasis in a variety of cardiac diseases. The most common cardiac pathology, such as myocardial infarction, is associated with oxidative stress. RyR contains a large number of cysteine residues that link oxidative stress and Ca homeostasis. However, the pathologically relevant cysteines within RyR remain largely unknown. As a result, the mechanisms of RyR dysfunction during oxidative stress and myocardial infarction are not fully understood. This delays our progress in designing effective therapeutic interventions that can improve Ca homeostasis during ischemic heart diseases. The main goal of this proposal is to define the molecular mechanisms of RyR dysfunction during oxidative stress and myocardial infarction. We have recently discovered that oxidative stress activates RyR by forming disulfide bonds between two neighboring subunits: the intersubunit cross-linking. It appears that only two cross-linking cysteines play a critical role in the RyR response to oxidative stress. In this proposal we will test the hypothesis: the intersubunit cross-linking is the principal redox modification of RyR responsible for Ca dysregulation during oxidative stress and myocardial infarction. To test this hypothesis, we have developed a transgenic mouse model with a unique RyR2 knock-in mutation that protects RyR from the intersubunit cross-linking. In Aim 1, we will define molecular mechanisms of RyR dysfunction induced by the intersubunit crosslinking. We expect to show that the cross-linking activates RyR2 by promoting longer channel openings. Such alteration of RyR function increases Ca leak and triggers pro-arrhythmogenic Ca waves. We expect to show that RyR mutation that protects the channel from the cross-linking will normalize RyR activity, reduce Ca leak, and prevent Ca waves during oxidative stress. In Aim 2, we will define the critical role of the RyR intersubunit cross-linking in Ca dysregulation and contractile dysfunction during myocardial infarction. We expect to show that RyR mutation that protects the channel from the crosslinking will normalize cardiac Ca regulation and improve myocardial contraction in the infarcted heart. We will also test whether selective pharmacological interventions that stabilize RyR at the intersubunit cross-linking region can reduce Ca leak and prevent Ca waves during oxidative stress and myocardial infarction.
