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 β 1 -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 β 3 -adrenergic receptors (β 3 AR) in animal models. Myocardial β 3 AR signaling via neuronal nitric oxide synthase (nNOS) activation has recently emerged as a cardioprotective pathway. We tested whether chronic β 1 -adrenergic blockade with metoprolol enhances myocardial β 3 AR coupling with nitric oxide-stimulated cyclic guanosine monophosphate (β 3 AR/NO-cGMP) signaling in the MR-induced, volume-overloaded heart. We compared the expression, distribution, and inducible activation of β 3 AR/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. β 3 AR 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 β 3 AR expression was increased 1.4- and nearly twofold in MR and MR+βB, respectively, compared to Control. β 3 AR was detected within caveolae-enriched lipid rafts (Cav3 + LR) and heavy density, non-lipid raft membrane (NLR) across all groups. However, in vitro selective β 3 AR 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 β 3 AR/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 sGCβ 1 subunit away from caveolae, suggesting preserved NO-sGC-cGMP signaling, albeit without coupling to β 3 AR, within MR+βB caveolae. Chronic β 1 -blockade is associated with myocardial β 3 AR/NO-cGMP coupling in a microdomain-specific fashion. Our canine study suggests that microdomain-targeted enhancement of myocardial β 3 AR/NO-cGMP signaling may explain, in part, β 1 -adrenergic antagonist-mediated preservation of cardiac function in the volume-overloaded heart.