Suppression of Rad leads to arrhythmogenesis via PKA-mediated phosphorylation of ryanodine receptor activity in the heart

Abstract Ras-related small G-protein Rad plays a critical role in generating arrhythmias via regulation of the L-type Ca 2+ channel (LTCC). The aim was to demonstrate the role of Rad in intracellular calcium homeostasis by cardiac-Specific dominant-negative suppression of Rad. Transgenic (TG) mice overexpressing dominant-negative mutant Rad (S105N Rad TG) were generated. To measure intracellular Ca 2+ concentration ([Ca 2+ ] i ), we recorded [Ca 2+ ] i transients and Ca 2+ sparks from isolated cardiomyocytes using confocal microscopy. The mean [Ca 2+ ] i transient amplitude was significantly increased in S105N Rad TG cardiomyocytes, compared with control littermate mouse cells. The frequency of Ca 2+ sparks was also significantly higher in TG cells than in control cells, although there were no significant differences in amplitude. The sarcoplasmic reticulum Ca 2+ content was not altered in the S105N Rad TG cells, as assessed by measuring caffeine-induced [Ca 2+ ] i transient. In contrast, phosphorylation of Ser 2809 on the cardiac ryanodine receptor (RyR2) was significantly enhanced in TG mouse hearts compared with controls. Additionally, the Rad-mediated RyR2 phosphorylation was regulated via a direct interaction of Rad with protein kinase A (PKA).