Coupling of β ₂ -Adrenoceptor to G _i Proteins and Its Physiological Relevance in Murine Cardiac Myocytes

Abstract

-Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (betaAR) signal transduction. Although several of these models have altered betaAR subtypes, the specific functional sequelae of betaAR stimulation in murine heart, particularly those of beta2-adrenergic receptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta2AR stimulation on contraction, [Ca2+]i transient, and L-type Ca2+ currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergic receptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-32P]GTP azidoanilide into alpha subunits of Gi2 and Gi3 after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Gi proteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gs and Gi signaling, resulting in null inotropic response, unless the Gi signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of beta2AR-coupled Gi proteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling.

Phosphodiesterase-5 Inhibitor Sildenafil Preconditions Adult Cardiac Myocytes against Necrosis and Apoptosis

“…Effect of Sildenafil on Cardiomyocyte Necrosis-Our method for cell preparations yielded at least 70% of the cardiomyocytes with rod shape morphology, which was similar to previously reported studies (24,25). Fig.…”

“…The animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. The ventricular cardiomyocytes were isolated using an enzymatic technique modified from the previously reported method (24,25). In brief, the mouse was anesthetized with pentobarbital sodium (100 mg/kg intraperitoneally), and the heart was quickly removed from the chest.…”

A Simple Technique for Isolating Healthy Heart Cells from Mouse Models

“…In these cells, one can take advantage of the versatility of recent genetic engineering technology [1] and also of the accuracy of single-cell measurement techniques such as patch-clamp and Ca 2+ imaging. So far, molecular mechanisms of fundamental heart functions, such as excitation-contraction coupling, action potential shaping, and β-adrenergic signaling, have been successfully unveiled by using single heart cells of mouse models [2][3][4][5][6][7]. Moreover, these cells also provide a convenient platform for analyzing pathogenic mechanisms and the pathophysiology of hereditary heart diseases in molecular detail [8,9].…”

Age Increases Cardiac Gαi2 Expression, Resulting in Enhanced Coupling to G Protein-coupled Receptors

“…Activation of AC increases the formation of cAMP, which activates cAMP-dependent protein kinase A resulting in the phosphorylation of proteins controlling cardiac excitationcontraction (1). An important recent discovery is that ␤ 2 ARs (but not ␤ 1 ARs) in both rat (2,3) and human heart (4) also activate G␣ i , a G␣-subunit that inhibits AC (5). We also demonstrated that G␣ i couples to several other G␣ s -coupled receptors in human heart, including receptors for histamine, glucagon, and serotonin (4).…”

“…Determination of the effects of age on G␣ i has become increasingly important in light of the recent demonstrations that many cardiac G␣ s -coupled receptors, including ␤ 2 AR, also couple to G␣ i (2)(3)(4). The data in the present study clearly indicate an increased level of G␣ i2 in old Fisher 344 rats, and importantly, also demonstrate an elevation in the receptorstimulated activation of G␣ i2 .…”