Effects of two Gbetagamma-binding proteins--N-terminally truncated phosducin and beta-adrenergic receptor kinase C terminus (betaARKct)--in heart failure.

Myocardial overexpression of the C-terminus of beta-adrenergic receptor kinase (betaARKct) has been shown to result in a positive inotropic effect or an improvement of survival in heart failure. However, it is not clear whether this beneficial effect is mainly because of dominant-negative inhibition of betaARK1, and a consecutive resensitization of beta-adrenergic receptors (betaAR), or rather due to inhibition of other Gbetagamma-mediated effects. In this study, we tested whether overexpression of N-terminally truncated phosducin (nt-del-phosducin), another Gbetagamma-binding protein that does not resensitize betaARs owing to simultaneous inhibition of GDP release from Galpha subunits, shows the same effects as betaARKct. Adenoviral gene transfer was used to express nt-del-phosducin and betaARKct in isolated ventricular cardiomyocytes and in myocardium of rabbits, which suffered from heart failure because of rapid ventricular pacing. BetaAR-stimulated cAMP formation was increased by betaARKct, but not by nt-del-phosducin, whereas both proteins inhibited Gbetagamma-mediated effects. Both transgenes also increased contractility of normal and failing isolated cardiomyocytes and improved contractility in rabbits with heart failure after gene transfer in vivo. In conclusion, overexpression of nt-del-phosducin enhances the contractility of cardiomyocytes to the same extent as betaARKct, suggesting that the effects of betaARKct might be owing to inhibition of Gbetagamma rather than to betaAR resensitization.

Gene transfer of heterologous G protein-coupled receptors to cardiomyocytes: differential effects on contractility.

In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to beta-adrenergic stimulation. Parathyroid hormone (PTH)-related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations. To bypass the defective beta-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V(2) vasopressin receptors (rV(2)-Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant beta(2)-adrenergic receptors (rbeta(2)-ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V(2)-Rs are uniquely coupled to Gs, PTH1-Rs and beta(2)-ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or rbeta(2)-ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV(2)-R-expressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of beta(2)-ARS: In the absence of receptor agonists, rPTH1-Rs and rbeta(2)-ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV(2)-Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V(2)-Rs might be more suited to test the effects of cAMP-stimulating receptors in heart failure than that of PTH1-Rs or beta(2)-ARS: