Heterodimerization With 5-HT_2BR Is Indispensable for β₂AR-Mediated Cardioprotection.

Abstract

The β₂-adrenoceptor (β₂-AR), a prototypical GPCR (G protein-coupled receptor), couples to both G_s and G_i proteins. Stimulation of the β₂-AR is beneficial to humans and animals with heart failure presumably because it activates the downstream G_i-PI3K-Akt cell survival pathway. Cardiac β₂-AR signaling can be regulated by crosstalk or heterodimerization with other GPCRs, but the physiological and pathophysiological significance of this type of regulation has not been sufficiently demonstrated. Here, we aim to investigate the potential cardioprotective effect of β₂-adrenergic stimulation with a subtype-selective agonist, (R,R')-4-methoxy-1-naphthylfenoterol (MNF), and to decipher the underlying mechanism with a particular emphasis on the role of heterodimerization of β₂-ARs with another GPCR, 5-hydroxytryptamine receptors 2B (5-HT_2BRs). Using pharmacological, genetic and biophysical protein-protein interaction approaches, we studied the cardioprotective effect of the β₂-agonist, MNF, and explored the underlying mechanism in both in vivo in mice and cultured rodent cardiomyocytes insulted with doxorubicin, hydrogen peroxide (H₂O₂) or ischemia/reperfusion. In doxorubicin (Dox)-treated mice, MNF reduced mortality and body weight loss, while improving cardiac function and cardiomyocyte viability. MNF also alleviated myocardial ischemia/reperfusion injury. In cultured rodent cardiomyocytes, MNF inhibited DNA damage and cell death caused by Dox, H₂O₂ or hypoxia/reoxygenation. Mechanistically, we found that MNF or another β₂-agonist zinterol markedly promoted heterodimerization of β₂-ARs with 5-HT_2BRs. Upregulation of the heterodimerized 5-HT_2BRs and β₂-ARs enhanced β₂-AR-stimulated G_i-Akt signaling and cardioprotection while knockdown or pharmacological inhibition of the 5-HT_2BR attenuated β₂-AR-stimulated G_i signaling and cardioprotection. These data demonstrate that the β₂-AR-stimulated cardioprotective G_i signaling depends on the heterodimerization of β₂-ARs and 5-HT_2BRs.