Rad as a novel regulator of excitation-contraction coupling and beta-adrenergic signaling in heart.
ABSTRACT Rad (Ras associated with diabetes) GTPase, a monomeric small G protein, binds to Ca(v)beta 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.
This study aimed to investigate possible role of Rad in cardiac EC coupling and beta-adrenergic receptor (betaAR) inotropic mechanism.
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, approximately 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 I(Ca). 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(2+) sparks and propensity for Ca(2+) waves all remained unperturbed regardless of Rad manipulation. Rad overexpression, but not knockdown, negated betaAR effects on I(Ca) and Ca(2+) transients.
These results establish Rad as a novel endogenous regulator of cardiac EC coupling and betaAR signaling and support a parsimonious model in which Rad buffers Ca(v)beta to modulate LCC activity, EC coupling, and betaAR responsiveness.
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ABSTRACT: Ca(V)1/Ca(V)2 channels, comprised of pore-forming α(1) and auxiliary (β,α(2)δ) subunits, control diverse biological responses in excitable cells. Molecules blocking Ca(V)1/Ca(V)2 channel currents (I(Ca)) profoundly regulate physiology and have many therapeutic applications. Rad/Rem/Rem2/Gem GTPases (RGKs) strongly inhibit Ca(V)1/Ca(V)2 channels. Understanding how RGKs block I(Ca) is critical for insights into their physiological function, and may provide design principles for developing novel Ca(V)1/Ca(V)2 channel inhibitors. The RGK binding sites within Ca(V)1/Ca(V)2 channel complexes responsible for I(Ca) inhibition are ambiguous, and it is unclear whether there are mechanistic differences among distinct RGKs. All RGKs bind β subunits, but it is unknown if and how this interaction contributes to I(Ca) inhibition. We investigated the role of RGK/β interaction in Rem inhibition of recombinant Ca(V)1.2 channels, using a mutated β (β(2aTM)) selectively lacking RGK binding. Rem blocked β(2aTM)-reconstituted channels (74% inhibition) less potently than channels containing wild-type β(2a) (96% inhibition), suggesting the prevalence of both β-binding-dependent and independent modes of inhibition. Two mechanistic signatures of Rem inhibition of Ca(V)1.2 channels (decreased channel surface density and open probability), but not a third (reduced maximal gating charge), depended on Rem binding to β. We identified a novel Rem binding site in Ca(V)1.2 α(1C) N-terminus that mediated β-binding-independent inhibition. The Ca(V)2.2 α(1B) subunit lacks the Rem binding site in the N-terminus and displays a solely β-binding-dependent form of channel inhibition. Finally, we discovered an unexpected functional dichotomy amongst distinct RGKs- while Rem and Rad use both β-binding-dependent and independent mechanisms, Gem and Rem2 use only a β-binding-dependent method to inhibit Ca(V)1.2 channels. The results provide new mechanistic perspectives, and reveal unexpected variations in determinants, underlying inhibition of Ca(V)1.2/Ca(V)2.2 channels by distinct RGK GTPases.PLoS ONE 01/2012; 7(5):e37079. · 3.73 Impact Factor
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ABSTRACT: The L-type calcium channels (LTCC) are critical for maintaining Ca(2+)-homeostasis. In heterologous expression studies, the RGK-class of Ras-related G-proteins regulates LTCC function; however, the physiological relevance of RGK-LTCC interactions is untested. In this report we test the hypothesis that the RGK protein, Rem, modulates native Ca(2+) current (I(Ca,L)) via LTCC in murine cardiomyocytes. Rem knockout mice (Rem(-/-)) were engineered, and I(Ca,L) and Ca(2+) -handling properties were assessed. Rem(-/-) ventricular cardiomyocytes displayed increased I(Ca,L) density. I(Ca,L) activation was shifted positive on the voltage axis, and β-adrenergic stimulation normalized this shift compared with wild-type I(Ca,L). Current kinetics, steady-state inactivation, and facilitation was unaffected by Rem(-/-) . Cell shortening was not significantly different. Increased I(Ca,L) density in the absence of frank phenotypic differences motivated us to explore putative compensatory mechanisms. Despite the larger I(Ca,L) density, Rem(-/-) cardiomyocyte Ca(2+) twitch transient amplitude was significantly less than that compared with wild type. Computer simulations and immunoblot analysis suggests that relative dephosphorylation of Rem(-/-) LTCC can account for the paradoxical decrease of Ca(2+) transients. This is the first demonstration that loss of an RGK protein influences I(Ca,L) in vivo in cardiac myocytes.Channels (Austin, Tex.) 05/2012; 6(3):166-73. · 1.91 Impact Factor
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ABSTRACT: Despite our cognizance that diabetes can enhance the chances of heart failure, causes multiorgan failure,and contributes to morbidity and mortality, it is rapidly increasing menace worldwide. Less attention has been paid to alert prediabetics through determining the comprehensive predictors of diabetic cardiomyopathy (DCM) and ameliorating DCM using novel approaches. DCM is recognized as asymptomatic progressing structural and functional remodeling in the heart of diabetics, in the absence of coronary atherosclerosis and hypertension. The three major stages of DCM are: (1) early stage, where cellular and metabolic changes occur without obvious systolic dysfunction; (2) middle stage, which is characterized by increased apoptosis, a slight increase in left ventricular size, and diastolic dysfunction and where ejection fraction (EF) is <50%; and (3) late stage, which is characterized by alteration in microvasculature compliance, an increase in left ventricular size, and a decrease in cardiac performance leading to heart failure. Recent investigations have revealed that DCM is multifactorial in nature and cellular, molecular, and metabolic perturbations predisposed and contributed to DCM. Differential expression of microRNA (miRNA), signaling molecules involved in glucose metabolism, hyperlipidemia, advanced glycogen end products, cardiac extracellular matrix remodeling, and alteration in survival and differentiation of resident cardiac stem cells are manifested in DCM. A sedentary lifestyle and high fat diet causes obesity and this leads to type 2 diabetes and DCM. However, exercise training improves insulin sensitivity, contractility of cardiomyocytes, and cardiac performance in type 2 diabetes. These findings provide new clues to diagnose and mitigate DCM. This review embodies developments in the field of DCM with the aim of elucidating the future perspectives of predictors and prevention of DCM.Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 01/2013; 6:151-60.