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

Ras-related small G-protein Rad plays a critical role in generating arrhythmias via regulation of the L-type Ca²⁺ 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 Ca²⁺ concentration ([Ca²⁺]_i), we recorded [Ca²⁺]_i transients and Ca²⁺ sparks from isolated cardiomyocytes using confocal microscopy. The mean [Ca²⁺]_i transient amplitude was significantly increased in S105N Rad TG cardiomyocytes, compared with control littermate mouse cells. The frequency of Ca²⁺ sparks was also significantly higher in TG cells than in control cells, although there were no significant differences in amplitude. The sarcoplasmic reticulum Ca²⁺ content was not altered in the S105N Rad TG cells, as assessed by measuring caffeine-induced [Ca²⁺]_i transient. In contrast, phosphorylation of Ser²⁸⁰⁹ 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).