Abstract:
Abstract—Transgenic mouse models have been developed to manipulate β-adrenergic receptor (βAR) signal transduction. Although several of these models have altered βAR subtypes, the specific functional sequelae of βAR stimulation in murine heart, particularly those of β2-adrenergic receptor (β2AR) stimulation, have not been characterized. In the present study, we investigated effects of β2AR stimulation on contraction, [Ca2+]i transient, and L-type Ca2+ currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human β2AR (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 β2AR activation. In contrast, β2AR stimulation by zinterol or isoproterenol plus a selective β1-adrenergic receptor (β1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, β2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to β2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac β2AR 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 β2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [γ-32P]GTP azidoanilide into α subunits of Gi2 and Gi3 after β2AR stimulation by zinterol or isoproterenol plus the β1AR blocker CGP 20712A. This effect to activate Gi proteins was abolished by a selective β2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) β2ARs 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 β2AR 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 β2AR-coupled Gi proteins; and (3) spontaneous β2AR activation may differ from agonist-stimulated β2AR signaling.
