The cardiac ryanodine receptor (RyR2) is the key Ca2+ release channel in the heart. Accumulating evidence indicates that RyR2 interacts with and can be regulated by a large number of proteins, making the effect of each protein on RyR2 function difficult to discern. Dysregulation of RyR2 can lead to abnormal Ca2+ release, otherwise known as store overload-induced Ca2+ release (SOICR), which can trigger arrhythmia. The molecular mechanisms for dysregulation of RyR2 by proteins are not fully elucidated. This research aims to investigate the regulation of RyR2 by two protein families within the RyR2 complex: a recently identified RyR2 regulator called histidine-rich Ca2+ binding protein (HRC) and its arrhythmogenic HRC S96A mutant form, and a highly controversial family, FK 506 binding proteins (FKBPs) 12.0 and 12.6.
Using a single cell Ca2+ imaging approach, I found that HRC wild type (WT) significantly suppressed the propensity for SOICR by buffering store free Ca2+ and inhibiting store Ca2+ uptake. Interestingly, the S96A mutation within HRC blunted the Ca2+ buffering and Ca2+ uptake inhibitory function, leading to an enhanced SOICR. These findings not only reveal the novel regulatory mechanism by which HRC regulates RyR2, but also offer the mechanism underlying HRC S96A related arrhythmia. Additionally, I found that both FKBP 12.6 and FKBP12.0 regulated RyR2 by facilitating the termination of SOICR without changing the activation of SOICR, leading to a reduced magnitude of SOICR. Importantly, these regulatory functions of FKBPs were abolished by RyR2 catecholaminergic polymorphic ventricular tachycardia (CPVT) mutations (V4653F and R4496C), despite a conserved interaction with RyR2, suggesting that FKBPs play a critical role in regulating RyR2.
Taken together, the research findings described in this thesis improve the current understanding of regulation of RyR2 by proteins within the RyR2 complex. More importantly, it sheds light on how dysregulation of RyR2 by these proteins can give rise to SOICR and arrhythmia.
