Mechanisms of Ryanodine Receptor 2 Regulation in Cardiac Pathophysiology

[摘要] The Ryanodine Receptor type 2 (RyR2) the major calcium-release channel in the heart, where it is fundamental for excitation-contraction coupling, the process transducing electrical signals into mechanical contraction. The role of RyR2 dysfunction as a trigger of cardiac arrhythmia due to inherited mutations is firmly established, but the fundamental mechanisms of RyR2 regulation in normal cardiac physiology, and dysregulation in other forms of inherited and acquired heart disease remain partially understood. In this dissertation, we took advantage of three novel genetically-engineered animal models with mutations in RyR2 to better understand of the role of this ion channel in the healthy and diseased heart.First, we derived a congenic mouse line with ablation of the S2808 phosphorylation to revisit the hypothesis that this site is critical for RyR2 regulation, and sort out one of the differences between two mouse models generated by different laboratories that have fueled a long-standing controversy: the genetic background. Consistent with previous studies performed in Sv129/C57Bl6 mice, our data demonstrate that S2808A mice in the C57Bl/6 background behave like WT when subjected to acute and chronic stress. These data support the idea that S2808 phosphorylation is unlikely fundamental for RyR2 regulation during the normal adrenergic response or heart failure progression. Furthermore, they suggest that the genetic background may not be the cause for the opposing results obtained with S2808A mice from different laboratories.Second, we used a novel RyR2 knock-out rabbit model to elucidate the effects of decreased RyR2 expression on cardiac function and the possible underlying compensatory mechanisms. We show that homozygous knock-out of RyR2 is lethal, while heterozygous knock-out decreases RyR2 expression by 60% without producing an abnormal phenotype. Our data indicate that RyR2 deficiency is likely compensated by upregulation of channel function by decreasing the phosphorylation of S2031. Hence, RyR2 function is likely backed by a protein reserve, and channel deficiency is readily compensated. Nonetheless, during acute adrenergic stimulation the contraction velocity and calcium release are slower in cardiomyocytes from mutant animals, suggesting that a 40% RyR2 level may be insufficient to maintain calcium release flux during acute stress. These results give additional significance to phosphorylation of S2031, a site often ignored in the regulatory scheme of RyR2. Third, we performed a multi-level characterization of the novel mutation P1124L, identified in a patient with hypertrophic cardiomyopathy. Since this is one of a handful of RyR2 mutations associated with structural remodeling of the heart, its study may uncover novel pathogenic mechanisms of RyR2 dysregulation. We show that P1124L induces conformational changes in the SPRY2 domain of RyR2, affecting the sensitivity of the channel to cytosolic and luminal calcium. In a mouse model, P1124L produces cardiac hypertrophy and increases the susceptibility to arrhythmia. While we have yet to fully elucidate the underlying pathogenic mechanisms, these studies suggest that specific RyR2 mutations may cause cardiac hypertrophy while at the same inducing arrhythmias that are typical of previously-described mutations.These three stories share a common aim: to obtain a better understanding of RyR2 regulation in cardiac pathophysiology. From the regulatory role of phosphorylation to the clinical relevance of mutations in the RYR2 gene and the underlying pathogenic mechanisms, each model addressed in this dissertation provides novel insights into the role of RyR2 as an essential player in excitation-contraction coupling, and the possible culprit of severe cardiac disease.

[发布机构] University of Michigan

[效力级别] excitation-contraction coupling

[关键词] ryanodine receptor;excitation-contraction coupling;heart failure;hypertrophic cardiomyopathy;arrhythmia;calcium;Physiology;Health Sciences;Molecular and Integrative Physiology