Chronic β1-adrenergic blockade enhances myocardial β3-adrenergic coupling with nitric oxide-cGMP signaling in a canine model of chronic volume overload: new insight into mechanisms of cardiac benefit with selective β1-blocker therapy

The β1-adrenergic antagonist metoprolol improves cardiac function in animals and patients with chronic heart failure, isolated mitral regurgitation (MR), and ischemic heart disease, though the molecular mechanisms remain incompletely understood. Metoprolol has been reported to upregulate cardiac expression of β3-adrenergic receptors (β3AR) in animal models. Myocardial β3AR signaling via neuronal nitric oxide synthase (nNOS) activation has recently emerged as a cardioprotective pathway. We tested whether chronic β1-adrenergic blockade with metoprolol enhances myocardial β3AR coupling with nitric oxide-stimulated cyclic guanosine monophosphate (β3AR/NO-cGMP) signaling in the MR-induced, volume-overloaded heart. We compared the expression, distribution, and inducible activation of β3AR/NO-cGMP signaling proteins within myocardial membrane microdomains in dogs (canines) with surgically induced MR, those also treated with metoprolol succinate (MR+βB), and unoperated controls. β3AR mRNA transcripts, normalized to housekeeping gene RPLP1, increased 4.4 × 10(3)- and 3.2 × 10(2)-fold in MR and MR+βB hearts, respectively, compared to Control. Cardiac β3AR expression was increased 1.4- and nearly twofold in MR and MR+βB, respectively, compared to Control. β3AR was detected within caveolae-enriched lipid rafts (Cav3(+)LR) and heavy density, non-lipid raft membrane (NLR) across all groups. However, in vitro selective β3AR stimulation with BRL37344 (BRL) triggered cGMP production within only NLR of MR+βB. BRL induced Ser (1412) phosphorylation of nNOS within NLR of MR+βB, but not Control or MR, consistent with detection of NLR-specific β3AR/NO-cGMP coupling. Treatment with metoprolol prevented MR-associated oxidation of NO biosensor soluble guanylyl cyclase (sGC) within NLR. Metoprolol therapy also prevented MR-induced relocalization of sGCβ1 subunit away from caveolae, suggesting preserved NO-sGC-cGMP signaling, albeit without coupling to β3AR, within MR+βB caveolae. Chronic β1-blockade is associated with myocardial β3AR/NO-cGMP coupling in a microdomain-specific fashion. Our canine study suggests that microdomain-targeted enhancement of myocardial β3AR/NO-cGMP signaling may explain, in part, β1-adrenergic antagonist-mediated preservation of cardiac function in the volume-overloaded heart.