G Protein-Coupled Receptor Internalization Signaling Is Required for Cardioprotection in Ischemic Preconditioning

The present study is designed to explore the role of G protein-coupled receptors (GPCRs) in the protection afforded by ischemic preconditioning (PC). We used TG mice with cardiac-specific overexpression of a Gβγ -sequestering peptide, βARKct (TG βARKct mice), to test whether the protection of PC is Gβγ-dependent. To test the role of G_i protein, we used wild-type mice pretreated with the G_i inhibitor pertussis toxin. Recovery of left ventricular developed pressure and infarct size were measured as indices of protection. PC induced protection in wild-type mice, but this protection was blocked by pertussis toxin treatment and was also blocked in TG βARKct mice. To determine the mechanism of Gβγ-induced protection in PC, we investigated one of the downstream targets of Gβγ, the PI3K/p70S6K pathway. PC-induced phosphorylation of p70S6K was not blocked in TG βARKct hearts; therefore, we investigated other targets of Gβγ. Recent studies suggest a role for Gβγ in GPCR internalization. We found that βARKct, a specific PI3K inhibitor wortmannin, and bafilomycin A₁, which all block receptor recycling, all blocked the protective effect of PC. To additionally test whether PI3K is involved in PC-activated receptor internalization and endosomal signaling, we used TG mice with cardiac-specific overexpression of a catalytically inactive mutant PI3Kγ, which disrupts the recruitment of functional PI3K to agonist-activated GPCRs in vivo. We found that the catalytically inactive mutant of PI3Kγ blocks the protection of PC. In summary, these data suggest the novel finding that the cardioprotective effect of PC requires receptor internalization.