Role of protein kinase A-dependent G-protein switching of the beta2-adrenoreceptor in depression of cardiac contraction. (360G-Wellcome-077661_Z_05_Z)

Although the b2-adrenoceptor (b2AR), like the b1AR, is known to stimulate cardiac contraction through Gs/adenylyl cyclase/cAMP, we have observed two distinct cardiodepressant actions mediated through b2ARs in isolated ventricular myocytes. Overexpression of the b2AR itself produces a constitutive depression after the initial constitutive activation of contraction, and also reveals an acute negative effect of b2AR ligands (including compounds previously classed as b2AR antagonists). The second effect (and possibly the first also) is evident in myocytes from failing, but not non-failing, human hearts. We have evidence that each of these effects is due to increased b2AR-Gi coupling, with a negative inotropic mechanism mediated through activation of the Na+/Ca2+-exchanger. Phosphorylation of the b2AR by cAMP-dependent protein kinase (PKA) has been shown to switch the b2AR from Gs- to Gi-coupling in reconstituted systems. Our hypothesis is that persistent Gs activation in the animal models and failing human heart produces this switch. Our aims are 1) to manipulate PKA to produce an increased (using PKA agonists) or decreased (by overexpressing PKA inhibitor peptide using adenovirus) activity in human and b2AR-overexpressing rat ventricular myocytes, and determine the effect on cardiodepression and b2AR phosphorylation; 2) to do the same after adenoviral transfection of a mutated b2AR lacking PKA phosphorylation sites; 3) to distinguish between Gia and Gbg as the effector.