Poster abstracts

Poster number 20 submitted by Hsiang-Ting Ho

RyR phosphorylation by oxidized CAMKII contributes to the cardiotoxic effects of cardiac glycosides

Hsiang-Ting Ho, Bin Liu, Jedidiah S. Snyder (Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio), Mark E. Anderson (Departments of Internal Medicine, Molecular Physiology and Biophysics and the Cardiovascular Center, Carver College of Medicine, University of Iowa, Iowa City, Iowa), Chandan K. Sen (Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio), Xander HT Wehrens (Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas), Thomas J. Hund, Sandor Gyrke (Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio)

Abstract:
Cardiac glycosides (CGs), important agents in the treatment of heart failure, are characterized by proarrhythmic side effects commonly attributed to ionic imbalances due to inhibition of Na+/K+- ATPase (NKA). Recent studies showed that CGs rather than acting by this orthodox mechanism, act through elevating reactive oxygen species (ROS) with subsequent redox-dependent alterations of the functional activity of RyR2s. However, the precise mechanisms of ROS generation and the following downstream molecular steps that mediate the arrhythmogenic effects of CGs remain to be defined. To address these questions, we examined the effects of glycosides on Ca handling and ROS production in cardiomyocytes using a combination of pharmacological approaches and genetic mouse models. Exposure of myocytes to digitoxin (DGT) increased the frequency of arrhythmogenic spontaneous Ca waves (SCWs) and accelerated the production of ROS monitored by confocal imaging with the Ca- and ROS- sensitive indicators Fluo-3 and DCFDA, respectively. Both DGT-induced SCWs and ROS were prevented by inhibitors of ROS release by NADPH oxidase (NOX) and mitochondria (diphenyliodonium chloride, DPI, and cyclosporin A, CsA). Myocytes isolated from mice deficient in NADPH oxidase (NOX2KO) and mice conditionally overexpressing mitochondrial SOD2 showed markedly increased tolerance to the proarrhythmic action of DGT and decrease in the rate of DGT-dependent ROS production. As determined by [Na+] measurements with sodium green, DGT caused similar [Na+] increases in wild type (WT) and NOX2KO myocytes indicating that the protective effects of NOX2 ablation were not due to changes in NKA Na+ transport capacity or responsiveness to DGT. Western blot measurements indicated increased levels of oxidized CaMKII in WT but not NOX2KO myocytes treated with DGT. DGT-induced increase in SCW frequency was abolished in mice in which the S2814 CaMKII phosphorylation site on RyR2 is constitutively inactivated. These results suggest that the arrhythmogenic adverse effects of CGs are mediated by NOX2- and mitochondria-derived ROS activating CAMKII with subsequent phosphorylation of RyR2 at Ser-2814.

Keywords: RyR, ROS, Calcium