Heterodimerization with 5-HT2BR Is Indispensable for β2AR-mediated Cardioprotection.

Rationale: The β2-adrenoceptor (β2-AR), a prototypical G protein-coupled receptor (GPCR), couples to both Gs and Giproteins. Stimulation of theβ2-AR is beneficial to humans and animals with heart failure presumably because it activates the downstream Gi-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. Objective: 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-HT2BRs). Methods and Results: 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 (Dox), hydrogen peroxide (H2O2) or ischemia/reperfusion. In 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-HT2BRs. Upregulation of the heterodimerized 5-HT2BRs and β2-ARs enhanced β2-AR-stimulated Gi-Akt signaling and cardioprotection while knockdown or pharmacological inhibition of the 5-HTattenuated β2-AR-stimulated Gi signaling and cardioprotection. Conclusions: These data demonstrate that the β2-AR-stimulated cardioprotective Gi signaling depends on the heterodimerization of β2-ARs and 5-HT2BRs.