Rad as a novel regulator of excitation-contraction coupling and β-adrenergic signaling in heart

Abstract

Rationale: Rad (Ras associated with diabetes) GTPase, a monomeric small G protein, binds to Ca vβ subunit of the L-type Ca 2+ channel (LCC) and thereby regulates LCC trafficking and activity. Emerging evidence suggests that Rad is an important player in cardiac arrhythmogenesis and hypertrophic remodeling. However, whether and how Rad involves in the regulation of excitation-contraction (EC) coupling is unknown. Objective: This study aimed to investigate possible role of Rad in cardiac EC coupling and β-adrenergic receptor (βAR) inotropic mechanism. Methods and Results: Adenoviral overexpression of Rad by 3-fold in rat cardiomyocytes suppressed LCC current (I Ca), [Ca 2+]i transients, and contractility by 60%, 42%, and 38%, respectively, whereas the "gain" function of EC coupling was significantly increased, due perhaps to reduced "redundancy" of LCC in triggering sarcoplasmic reticulum release. Conversely, ≈70% Rad knockdown by RNA interference increased I Ca (50%), [Ca 2+] i transients (52%) and contractility (58%) without altering EC coupling efficiency; and the dominant negative mutant RadS105N exerted a similar effect on ICa. Rad upregulation caused depolarizing shift of LCC activation and hastened time-dependent LCC inactivation; Rad downregulation, however, failed to alter these attributes. The Na +/Ca 2+ exchange activity, sarcoplasmic reticulum Ca 2+ content, properties of Ca sparks and propensity for Ca waves all remained unperturbed regardless of Rad manipulation. Rad overexpression, but not knockdown, negated βAR effects on I Ca and Ca 2+ transients. Conclusion: These results establish Rad as a novel endogenous regulator of cardiac EC coupling and βAR signaling and support a parsimonious model in which Rad buffers Ca vβ to modulate LCC activity, EC coupling, and βAR responsiveness. © 2010 American Heart Association, Inc.