The Overexpression of S105N Mutant Rad Leads Intracellular Ca2+ Overload Via Up-Regulation of Cardiac Ryanodine Receptor Activity

Background: The ras-related small G-protein Rad was originally identified from skeletal muscle of patients with type 2 diabetes mellitus. We have recently reported that Rad plays a critical role in generating arrhythmias. The study was aimed to elucidate the role of Rad in intracellular calcium homeostasis. Methods and Reslts: We developed the transgenic mice that overexpress S105N mutant Rad driven by α-myosin heavy chain promoter. We mesure intracellular Ca concentration ([Ca²⁺]_I) from isolated cardiomyocytes by confocal microscopy. The amplitude of [Ca²⁺]_I transient was significantly increased in S105N-Rad-TG cardiomyocytes, compared with littermate. Furthermore, the Ca²⁺ sparks and the spontaneous Ca²⁺ waves occurred with greater frequency in S105N-Rad-TG cells, implicating the enhanced activity of SERCA. We recorded L-type calcium currents (I_Ca-L) and action potentials (APs) from isolated cardiomyocytes using whole cell patch-clamp technique. The peak I_Ca-L was dramatically larger in the S105N-Rad-TG cells than in littermate. Early afterdepolarization (EAD) and delayed afterdepolarization (DAD) were frequently observed in S105N-Rad-TG. Then, the phosphorylation of RYR2 at Ser²⁸⁰⁹ and PKA was significantly enhanced in S105N-Rad-TG by Western blot. Conclusions: Our results provided the first evidence that Rad might regulate RYR2 activity possibly via downstream of PKA signaling pathway.