Endogenous RGS Proteins and Gα Subtypes Differentially Control Muscarinic and Adenosine-Mediated Chronotropic Effects

Abstract

Abstract-Cardiac automaticity is controlled by G protein-coupled receptors, such as adrenergic, muscarinic, and adenosine receptors. The strength and duration of G protein signaling is attenuated by regulator of G protein signaling (RGS) proteins acting as GTPase-activating proteins for G␣ subunits; however, little is known about the role of endogenous RGS proteins in cardiac function. We created point mutations in G␣ subunits that disrupt G␣-RGS binding and introduced them into embryonic stem (ES) cells by homologous recombination. Spontaneously contacting cardiocytes derived from the ES cells were used to evaluate the role of endogenous RGS proteins in chronotropic regulation. The RGS-insensitive G␣ o G184S homozygous knock-in (G␣ o GS/GS) cells demonstrated enhanced adenosine A 1 and muscarinic M 2 receptor-mediated bradycardic responses. In contrast, G␣ i2 GS/GS cells showed enhanced responses to M 2 but not A 1 receptors. Similarly M 2 but not A 1 bradycardic responses were dramatically enhanced in G␣ i2 GS/GS mice. Blocking G protein-coupled inward rectifying K ϩ (GIRK) channels largely abolished the mutation-induced enhancement of the M 2 receptor-mediated response but had a minimal effect on A 1 responses. The G␣ s -dependent stimulation of beating rate by the ␤ 2 adrenergic receptor agonist procaterol was significantly attenuated in G␣ o GS/GS and nearly abolished in G␣ i2 GS/GS cells because of enhanced signaling via a pertussis toxin sensitive mechanism. Thus, endogenous RGS proteins potently reduce the actions of G␣ i/o -linked receptors on cardiac automaticity. Key Words: RGS Ⅲ automaticity Ⅲ adenosine receptor Ⅲ ␤ 2 adrenergic receptor Ⅲ muscarinic receptor G protein-coupled receptors (GPCRs) control many cardiovascular functions, such as automaticity and contractility and cell growth and survival. 1 Cardiac ion channels are key effectors, including activation of the G protein-coupled inward rectifying potassium (GIRK) channels by ␤␥ subunits released from G␣ i and inhibition of I f and I Ca,L by G␣ o , whereas I Ca,L is also regulated by G␣ i and nitric oxide. [2][3][4][5] GIRK channels contribute approximately 50% of the in vivo bradycardic response to vagal stimulation, 6 but the relative contribution of different G␣ subunits to in vivo cardiac automaticity is less clear.The recently identified regulators of G protein signaling (RGS) proteins reduce the strength and duration of G protein signaling by acting as GTPase accelerating proteins (GAPs) through their conserved RGS domain. 7 Mammalian myocardium expresses more than 10 different RGS proteins (2, 3, 4, 5, 6, 7, 12, 14, 16, and 19), which have GAP activity toward G␣ i/o and G␣ q/11 family G proteins. 8 Indeed, the first reported mammalian RGS effect was the rapid deactivation of GIRK currents in atrium 9 and RGS proteins also contribute to agonist-, calcium-, and voltage-dependent relaxation of GIRK currents. 10 -12 These results suggest an important role for RGS proteins in sinoatrial (SA) node function and potentially in atrial arrhyth...

Pleiotropic Phenotype of a Genomic Knock-In of an RGS-Insensitive G184S Gnai2 Allele

“…Maximum inhibition of forskolinstimulated AC by LPA was 37% for wild-type MEFs and 52% and 54% for G␣ i2 ϩ/G184S and G␣ i2 G184S/G184S , respectively (n ϭ 6 to 9; P Ͻ 0.05, t test). In contrast to results for adenosine and carbachol signaling in ES-derived cardiocytes with this mutation (24), there was no significant shift in the 50% effective concentration for LPA-mediated AC inhibition (71, 95, and 80 nM for ϩ/ϩ, ϩ/G184S, and G184S/G184S, respectively), and so the only change was in the maximum response.…”

“…All protocols and procedures were approved by the University Committee on Use and Care of Animals, and animal care was overseen by the Unit for Laboratory Animal Medicine (University of Michigan). The production of the G␣ i2 G184S/G184S mice has been described in detail in the supporting online material of Fu et al (24). Briefly, the targeting construct pTKLNL/G␣ i2 G184S harbors a 5Ј sequence encoding thymidine kinase followed by a 13.8-kb G␣ i2 genomic sequence that is divided into two regions of G␣ i2 homology (6.7 and 7.1 kb in size) surrounding a loxP-flanked neo cassette.…”

The role of inhibitory heterotrimeric G proteins in the control of in vivo heart rate dynamics

“…A similar experiment was performed using the selective A1-receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1 mg/kg) over a 10-min time course. Our cardioinhibition protocols are based on previous reports specifically investigating parasympathetic heart rate modulation (10,11,30). Similarly, CCPA is a pure A1 receptor agonist (1,000-fold A1 to A2 selectivity), which results in similar chronotropic effects (10,48).…”

“…Our cardioinhibition protocols are based on previous reports specifically investigating parasympathetic heart rate modulation (10,11,30). Similarly, CCPA is a pure A1 receptor agonist (1,000-fold A1 to A2 selectivity), which results in similar chronotropic effects (10,48). Similar experiments were performed after pretreatment with tertiapinQ (0.05 mg/kg) to determine the effects of GIRK channel blockade on receptor-mediated heart rate slowing (5, 23).…”

Ventricular Tachycardia

“…In addition, in supraventricular tissue, adenosine attenuates cAMP-stimulated increases of the L-type calcium current I CaL through activation of Gα i2 /Gα o . 10 In contrast to its effects on supraventricular tissue, adenosine has little or no direct effect on ventricular resting membrane potential, amplitude, or action potential duration ( Figure 1A). This is due to the absence of G-protein-coupled inward rectifying K + channel expression in ventricular tissue.…”