Effects of the β3-Adrenergic Agonist BRL 37344 on Endothelial Nitric Oxide Synthase Phosphorylation and Force of Contraction in Human Failing Myocardium

BackgroundIn nonfailing myocardium, β3-adrenergic signaling causes a decrease in contractility via endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) release. This study investigates the hypothesis that β3-adrenergic signaling undergoes alterations in failing myocardium.MethodsWe compared eNOS- and β3-adrenoceptor expression using Western blot analysis in human nonfailing myocardium versus failing myocardium. With the use of immunohistochemistry, we investigated the distribution of the β3-adrenoceptor protein and eNOS translocation and phosphorylation under basal conditions. β3-adrenergic, eNOS activation, and inotropy were measured in failing myocardium using BRL37344 (BRL, a β3-adrenoceptor agonist).Resultsβ3-adrenoceptor expression was increased in failing myocardium. Under basal conditions, Akt- and eNOSSer1177 phosphorylation were reduced in failing myocardium. During stimulation with BRL in failing myocardium, a further dephosphorylation of eNOSSer1177 and Akt was observed, whereas eNOSSer114 phosphorylation was increased. These results suggest a deactivation of eNOS via β3-adrenergic stimulation. Nevertheless, BRL decreased contractility in failing myocardium, but this effect was not observed in the presence of the NO blocker L-NMA. In failing myocardium, the β3-adrenoceptor was predominantly expressed in endothelial cells. In the cardiomyocytes, the β3-adrenoceptor was mainly located at the intercalated disks.ConclusionIn failing cardiomyocytes, β3-adrenergic stimulation seems to deactivate rather than activate eNOS. At the same time, β3-adrenergic stimulation induced a NO-dependent negative inotropic effect. Because β3-adrenoceptors are expressed mainly in the endothelium in failing myocardium, our observations suggest a paracrine-negative inotropic effect via NO liberation from the cardiac endothelial cells.