Enhanced Beta-Adrenergic Response, Spontaneous Calcium Release, and Arrhythmogenic Propensity in Mice with a Phosphomimetic Mutation of a PKA Site (S2030D) in the Cardiac Ryanodine Receptor

Abstract

The phosphorylation of the cardiac calcium release channel/ryanodine receptor (RyR2) by protein kinase A (PKA) has been extensively investigated, but its functional consequences remain poorly understood and controversial. We have previously shown that S2030 is a major PKA site in RyR2 responding to beta-adrenergic stimulation, and that phosphomimetic mutation of this PKA site (S2030D) enhances luminal calcium activation of single RyR2 channels and increases the propensity for spontaneous calcium release in HEK293 cells during store calcium overload. To further investigate the physiological and pathophysiological significance of PKA phosphorylation of S2030, we generated a knock-in mouse model harboring the phosphomimetic S2030D mutation in RyR2. We found that S2030D mutant mice displayed higher isoproterenol-induced heart rate increase than wt mice. ECG recordings revealed that S2030D mutant mice were more susceptible to bidirectional ventricular tachycardia induced by the injection of epinephrine plus caffeine as compared to wt mice. To determine the impact of the S2030D mutation on sarcoplasmic reticulum (SR) calcium handling, ventricular myocytes isolated from wt and mutant mice were loaded with fluo-4 and field-stimulated. Calcium imaging showed that S2030D mutant cells exhibited enhanced spontaneous calcium release activity in the presence of isoproterenol. These observations indicate that the S2030D mutation in RyR2 enhances the beta-adrenergic response, and suggest that excessive PKA phosphorylation of S2030 in RyR2 may increase the propensity for stress-induced spontaneous SR calcium release and ventricular arrhythmias (supported by CIHR).