Abstract 880: Removal of PKA Phosphorylation Sites from the Beta2-Adrenoceptor Prevents both Protective and Cardiodepressant Effects
Dual coupling of the beta2-adrenoceptor (beta2AR) to Gi- as well as Gs-coupled pathways is thought to confer protective effects of this subtype, in comparison to the solely Gs-coupled beta1AR, in cardiac myocytes. Phosphorylation of the beta2AR itself by cyclic AMP has been hypothesised to switch the beta2AR from Gs to Gi coupling. To investigate this, we have compared the effects of overexpression of wild type (wt) human beta2AR with a mutant (PKA-KO) with S to A conversion at the PKA phosphorylation sites (261, 262, 345, 346). Overexpression was achieved in adult rat ventricular myocytes using 48 h culture with adenoviral vectors. Co-expression of GFP allowed identification of transfected myocytes: cells expressing GFP alone were used as Control. Overexpression of beta2wt produced constitutive depression of contraction at 48h, which could be reversed by pertussis toxin treatment, indicating Gi-dependence. This effect was not observed after overexpression of beta2PKA-KO: contraction amplitude (% shortening) Control (GFP-only); 8.2 ± 0.9% , beta2-wt; 4.8 ± 0.4% (P<0.01 vs Control), beta2-PKA-KO; 7.7± 0.6% (P<0.05 vs beta2-wt) n=5 preparations, 44 – 49 cells per group. Survival of myocytes was reduced by 48 h exposure to 1μM isoproterenol (Iso), and in Control cells this was completely prevented by the beta1-AR blocker CGP20712A (CGP) at 300nM (% viable cells compared to freshly isolated: untreated 59.5 ± 8.2%, Iso 14.5 ± 2.5% [P<0.01 vs untreated], Iso+CGP 56.7 ± 8.5%, n=5– 6). Overexpression of beta2-wt reduced myocyte death, and again, the residual mortality was beta1-AR dependent (untreated 56.0 ± 6.1%, Iso 25.0 ± 1.9% [P<0.05 vs control+Iso], Iso+CGP 50.6 ± 8.2%). Overexpression of beta2-PKA-KO did not have a protective effect, and death was now unaffected by beta1-AR blockade (untreated 47.6 ± 4.0%, Iso 15.7 ± 1.9% [P<0.001 vs untreated], Iso+CGP 11.3 ± 3.6% [P<0.001 vs untreated]). This indicates that stimulation of the beta2-PKA-KO produced similar cell death to that mediated by the beta1-AR. Removal of the PKA phosphorylation sites from the beta2AR prevents both the cardiodepressant and protective effects of the receptor, consistent with the hypothesis that these sites are involved in Gs to Gi switching.