Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physiology

Abstract

The aim of my dissertation is to understand the regulation of RyR2. The whole dissertation is composed of two parts. The first part focused on RyR2-CaM interaction. The second focused on synthetic RyR2 domain peptide (DPc10), which worked as a powerful molecular tool for RyR2 functional and structural studies.

CaM has been long identified as an important cardiac RyR regulator. Broad studies suggest CaM is a critical RyR2 stabilizer and CaM-RyR2 interaction is a critical molecular substrate for arrhythmias and HF pathogenesis, but the in situ binding properties for CaM-RyR2 are still unknown. Here we, Using FRET detection and permeabilized myocytes, identified RyR2-bound CaM from other potential targets and revealed that CaM binds to RyR2 with high affinity (Kd =10-20 nM) in myocytes. Therefore physiologically CaM is bound to > 70% of RyR2 monomers and inhibits SR Ca release. Using RyR2ADA/+ knock-in mice in which half of the RyR2-CaM binding is suppressed, we estimated that >90% of Z-line CaM is RyR2-bound and identify RyR2 as one of main CaM cellular binding sites. In consistence with binding properties studies, functional tests indicated a higher propensity for Ca waves in RyR2ADA/+ mice upon ISO challenge. In a post MI rat HF model, I detected a decrease binding affinity for CaM-RyR2 (Kd = ~51nM, ~3 fold increase) and unchanged binding affinity for FKBP12.6-RyR2 (Kd = ~0.8nM).

The defective interaction between interdomains (the N-terminal and the central domains) is a leading hypothesis to explain the RyR2 dysfunction in HF and lethal arrhythmias, such as CPVT. And DPc10 can bind to RyR2 and recapitulate this arrhythmogenic RyR2 leakiness by unzipping N- and central-domains. In this study fluorescently-labeled DPc10, FKBP12.6, and CaM were used to characterize the binding properties of DPc10 to RyR2 in permeabilized rat ventricular myocytes. DPc10 access to its binding site is extremely slow in resting RyR2, but accelerated by promoting RyR2 opening or unzipping (by unlabeled DPc10). RyR2-bound CaM (but not FKBP12.6) drastically slowed DPc10 binding. Conversely, DPc10 binding significantly reduced CaM (but not FKBP12.6) binding to the RyR2. FRET measurements indicate that DPc10 and CaM binding sites are separate.

The accomplishment of this study enriched our understanding about RyR2-CaM interaction and functional effects on RyR-mediated Ca release in cardiac myocytes. It also expanded our knowledge of molecular mechanisms that underlie arrhythmogenesis and HF. Importantly, it pinpointed a potential molecular target responsible for Ca triggered arrhythmias and may translate into the development of novel therapeutic strategies

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