Abstract 5304: Dominant Negative Suppression of Rad Leads to Intracellular Ca2+ Overload via Up-Regulation of Cardiac Ryanodine Receptor Activity
Background: We have recently reported that the ras-related small G-protein Rad plays a critical role in generating arrhythmias via regulation of L-type Ca2+ channel. However, it has remained unclear whether or not the mechanism for its arrhythmogenesis is attributed only to L-type Ca2+ channel activity. This study was designed to demonstrate the role of Rad in intracellular calcium homeostasis by cardiac-specific dominant negative suppression of Rad.
Methods and results: Transgenic mice that overexpress dominant negative mutant Rad (DN Rad TG) driven by α-myosin heavy chain promoter were generated. To measure intracellular Ca2+ concentration ([Ca2+]i), we recorded [Ca2+]i transients and Ca2+ sparks from isolated cardiomyocytes by confocal microscope. The mean amplitude of [Ca2+]i transient was significantly increased in DN Rad TG cardiomyocytes, compared with WT littermate mouse cells (F/F0 3.3 ± 0.2, n = 25, in DN Rad TG cells vs. 2.4 ± 0.1, n = 30, in WT cells, P<0.05). The frequency of Ca2+sparks was significantly higher in TG cells than in WT cells (5.2 ± 0.6 sparks•100 μm−1•s−1, n = 45 in TG cells vs. 1.9 ± 0.3 sparks•100 μm−1•s−1, n = 45, in WT controls, P<0.05), although there were no significant differences in their amplitudes. Furthermore, SR Ca2+ content was not altered in TG cells, as assessed by caffeine-induced [Ca2+]i transient. These results suggested that the properties of fundamental SR Ca2+ release units might have been affected by the inhibition of endogenous Rad activity. To elucidate the mechanism for the increased frequency of SR Ca2+ release channel, phosphorylation of Ser2809 on cardiac ryanodine receptor (RYR2) was examined. The phosphorylation of RYR2 at Ser2809 was significantly enhanced in TG mouse hearts compared with WT mice, implicating the upregulation of RYR2 activity in TG mice. Additionally, this Rad-mediated phosphorylation of RYR2 was regulated by protein kinase A (PKA) activity.
Conclusions: Our results provided the first evidence that Rad regulated RYR2 activity via PKA signaling pathway. Thus, Rad might play a critical role in cellular Ca2+ homeostasis via its inhibitory effects on RYR2 as well as L-type Ca2+ channel.