Regulation of cardiac ryanodine receptors by protein kinase CK2 and small molecule kinase inhibitors

Abstract

The cardiac ryanodine receptor (RyR2) is an intracellular Ca2+ release channel located in the sarcoplasmic reticulum (SR) and is a key component in excitation-contraction coupling (the process of contraction) in cardiac muscle. However, in addition to its role in normal contraction, when the SR store Ca2+ content reaches a critical level, spontaneous Ca2+ release occurs through RyR2; a process we refer to as store-overload-induced Ca2+ release (SOICR). In turn SOICR can give rise to delayed after depolarisations (DADs) a common trigger of arrhythmia. Phosphorylation plays a vital role in the regulation of the RyR2 channel and occurs in response to physiological and disease stimuli. In this thesis, I have investigated a role for novel protein kinase CK2 (CK2) phosphorylation of RyR2 and its effect on SOICR.

I used human embryonic kidney 293 (HEK293) cells expressing RyR2 coupled with Ca2+ imaging, mass spectrometry (MS), site-directed mutagenesis (SDM) and biochemical approaches to investigate the impact of CK2 on Ca2+ release via RyR2. I found that silencing CK2 expression, using siRNA, increases SOICR due to a reduction in the release threshold for SOICR. Mass spectrometry identified three novel CK2 phosphorylation sites within RyR2- S2362, S2692 and S2693. Studying these sites directly showed that, contrary to PKA and CaMKII phosphorylation of RyR2, CK2 phosphorylation is required to prevent SOICR. In addition, I also studied the regulation of RyR2 by small molecule kinase inhibitors associated with cardiotoxic side effects in patients. I found that class I kinase inhibitors resulted in an increase in SOICR, whereas class II kinase inhibitors had no effect in either HEK cells or rat cardiomyocytes. These data provide a novel pathway through which certain anti-cancer drugs may be cardiotoxic.

Taken together, the thesis provides novel mechanisms of RyR2 regulation by protein kinase CK2 and small molecule kinase inhibitors. Importantly, the discovery of a novel negative RyR2 (anti-SOICR) signalling pathway opens up a new arena future research for controlling the function of RyR2 and reducing arrhythmias.