Dominant Negative Suppression of Rad Leads to QT Prolongation and Causes Ventricular Arrhythmias via Modulation of L-type Ca ²⁺ Channels in the Heart

Abstract

Abstract-Disorders of L-type Ca 2ϩ channels can cause severe cardiac arrhythmias. A subclass of small GTP-binding proteins, the RGK family, regulates L-type Ca 2ϩ current (I Ca,L ) in heterologous expression systems. Among these proteins, Rad (Ras associated with diabetes) is highly expressed in the heart, although its role in the heart remains unknown. Here we show that overexpression of dominant negative mutant Rad (S105N) led to an increase in I Ca,L and action potential prolongation via upregulation of L-type Ca 2ϩ channel expression in the plasma membrane of guinea pig ventricular cardiomyocytes. To verify the in vivo physiological role of Rad in the heart, a mouse model of cardiac-specific Rad suppression was created by overexpressing S105N Rad, using the ␣-myosin heavy chain promoter. Microelectrode studies revealed that action potential duration was significantly prolonged with visible identification of a small plateau phase in S105N Rad transgenic mice, when compared with wild-type littermate mice. Telemetric electrocardiograms on unrestrained mice revealed that S105N Rad transgenic mice had significant QT prolongation and diverse arrhythmias such as sinus node dysfunction, atrioventricular block, and ventricular extrasystoles, whereas no arrhythmias were observed in wild-type mice. Furthermore, administration of epinephrine induced frequent ventricular extrasystoles and ventricular tachycardia in S105N Rad transgenic mice. This study provides novel evidence that the suppression of Rad activity in the heart can induce ventricular tachycardia, suggesting that the Rad-associated signaling pathway may play a role in arrhythmogenesis in diverse cardiac diseases.

Direct inhibition of P/Q-type voltage-gated Ca 2+ channels by Gem does not require a direct Gem/Ca v β interaction

“…Thus, a prevalent hypothesis is that RGK proteins need to bind Ca v β to exert their inhibitory effect (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)31). To test this hypothesis, we abolished Gem/β 3 interaction by simultaneously mutating to alanine three residues on Gem and three residues on β 3 that have been shown to be critical for this interaction (34).…”

“…Recently, members of the Rem, Rem2, Rad, and Gem/Kir (RGK) family of Ras-related monomeric small GTP-binding proteins, which are known to regulate cytoskeleton remodeling through the Rho/Rho kinase signaling cascade (review in ref. 13), have emerged as the most potent protein inhibitors of HVA Ca 2+ channels (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). RGK proteins are present in many tissues and cells where HVA Ca 2+ channels are expressed, including the brain and cardiac, skeletal, and smooth muscles (review in ref.…”

“…Accordingly, they are emerging as strong regulators of hormone secretion and cardiac and brain physiology, both in vivo and in vitro. For example, in the heart, dominant negative suppression of endogenous Rad increases L-type Ca 2+ -channel currents and action-potential duration in cardiac cells and produces longer QT intervals and arrhythmias (24). Rem2 prevents glucose-stimulated insulin secretion in pancreatic β cells (19) and regulates the development of both excitatory and inhibitory synapses, presumably through a feedback loop that controls Ca 2+ influx (32).…”

“…Two modes of action have been reported: (i) RGK proteins reduce the number of HVA Ca 2+ channels on the cell surface, either by interfering with channel trafficking to the plasma membrane or increasing endocytosis of surface channels (14,16,17,20,24,31,34,35), and (ii) RGK proteins inhibit channels already on the plasma membrane (18,19,21,25,31). The molecular mechanisms of either mode of action are unknown, but because of the central role of Ca v β in HVA Ca 2+ -channel trafficking and gating, it is widely assumed that both forms of inhibition rely on the RGK/Ca v β interaction (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)31). This key hypothesis, however, has not been tested.…”

Pregabalin-induced first degree atrioventricular block in a young patient treated for pain from extrapulmonary tuberculosis

“…For instance, non-dihydropyridine calcium blockers, such as verapamil, are selective for the blockade of L-type calcium channels and increase both functional and effective refractory period of the A-V node in a dose-dependent fashion. Pregabalin is a selective, high-affinity ligand for the α2-δ subunit of voltage-gated L-type calcium channels [3][4]. Therefore, cardiac side effects related to pregabalin use might be due to the interferences of the drug with the calcium channels of the cardiomyocytes.…”