β-Arrestin–Biased Allosteric Modulator Potentiates Carvedilol-Stimulated β Adrenergic Receptor Cardioprotection

β(1) adrenergic receptors (β(1)ARs) are central regulators of cardiac function and a drug target for cardiac disease. As a member of the G protein–coupled receptor family, β(1)ARs activate cellular signaling by primarily coupling to Gs proteins to activate adenylyl cyclase, cAMP-dependent pathways, and the multifunctional adaptor-transducer protein β-arrestin. Carvedilol, a traditional β-blocker widely used in treating high blood pressure and heart failure by blocking β adrenergic receptor–mediated G protein activation, can selectively stimulate Gs-independent β-arrestin signaling of β adrenergic receptors, a process known as β-arrestin–biased agonism. Recently, a DNA-encoded small-molecule library screen against agonist-occupied β(2) adrenergic receptors (β(2)ARs) identified Compound-6 (Cmpd-6) to be a positive allosteric modulator for agonists on β(2)ARs. Intriguingly, it was further discovered that Cmpd-6 is positively cooperative with the β-arrestin–biased ligand carvedilol at β(2)ARs. Here we describe the surprising finding that at β(1)ARs unlike β(2)ARs, Cmpd-6 is cooperative only with carvedilol and not agonists. Cmpd-6 increases the binding affinity of carvedilol for β(1)ARs and potentiates carvedilol-stimulated, β-arrestin–dependent β(1)AR signaling, such as epidermal growth factor receptor transactivation and extracellular signal-regulated kinase activation, whereas it does not have an effect on Gs-mediated cAMP generation. In vivo, Cmpd-6 enhances the antiapoptotic, cardioprotective effect of carvedilol in response to myocardial ischemia/reperfusion injury. This antiapoptotic role of carvedilol is dependent on β-arrestins since it is lost in mice with myocyte-specific deletion of β-arrestins. Our findings demonstrate that Cmpd-6 is a selective β-arrestin–biased allosteric modulator of β(1)ARs and highlight its potential clinical utility in enhancing carvedilol-mediated cardioprotection against ischemic injury. SIGNIFICANCE STATEMENT: This study demonstrates the positive cooperativity of Cmpd-6 on β(1)ARs as a β-arrestin–biased positive allosteric modulator. Cmpd-6 selectively enhances the affinity and cellular signaling of carvedilol, a known β-arrestin–biased β-blocker for β(1)ARs, whereas it has minimal effect on other ligands tested. Importantly, Cmpd-6 enhances the β-arrestin–dependent in vivo cardioprotective effect of carvedilol during ischemia/reperfusion injury–induced apoptosis. The data support the potential therapeutic application of Cmpd-6 to enhance the clinical benefits of carvedilol in the treatment of cardiac disease.