Hypoxia Sensing of beta-Adrenergic Receptor Is Regulated by Endosomal PI3K gamma

Background:Impaired beta-adrenergic receptor (beta 1 and beta 2AR) function following hypoxia underlies ischemic heart failure/stroke. Activation of PI3K gamma (phosphoinositide 3-kinase gamma) by beta-adrenergic receptor leads to feedback regulation of the receptor by hindering beta-adrenergic receptor dephosphorylation through inhibition of PP2A (protein phosphatase 2A). However, little is known about PI3K gamma feedback mechanism in regulating hypoxia-mediated beta 1 and beta 2AR dysfunction and cardiac remodeling. Methods:Human embryonic kidney 293 cells or mouse adult cardiomyocytes and C57BL/6 (WT) or PI3K gamma knockout (KO) mice were subjected to hypoxia. Cardiac plasma membranes and endosomes were isolated and evaluated for beta 1 and beta 2AR density and function, PI3K gamma activity and beta 1 and beta 2AR-associated PP2A activity. Metabolic labeling was performed to assess beta 1 and beta 2AR phosphorylation and epinephrine/norepinephrine levels measured post-hypoxia. Results:Hypoxia increased beta 1 and beta 2AR phosphorylation, reduced cAMP, and led to endosomal accumulation of phosphorylated beta 2ARs in human embryonic kidney 293 cells and WT cardiomyocytes. Acute hypoxia in WT mice resulted in cardiac remodeling and loss of adenylyl cyclase activity associated with increased beta 1 and beta 2AR phosphorylation. This was agonist-independent as plasma and cardiac epinephrine and norepinephrine levels were unaltered. Unexpectedly, PI3K gamma activity was selectively increased in the endosomes of human embryonic kidney 293 cells and WT hearts post-hypoxia. Endosomal beta 1- and beta 2AR-associated PP2A activity was inhibited upon hypoxia in human embryonic kidney 293 cells and WT hearts showing regulation of beta-adrenergic receptors by PI3K gamma. This was accompanied with phosphorylation of endogenous inhibitor of protein phosphatase 2A whose phosphorylation by PI3K gamma inhibits PP2A. Increased beta 1 and beta 2AR-associated PP2A activity, decreased beta-adrenergic receptor phosphorylation, and normalized cardiac function was observed in PI3K gamma KO mice despite hypoxia. Compared to WT, PI3K gamma KO mice had preserved cardiac response to challenge with beta 1AR-selective agonist dobutamine post-hypoxia. Conclusions:Agonist-independent activation of PI3K gamma underlies hypoxia sensing as its ablation leads to reduction in beta 1- and beta 2AR phosphorylation and amelioration of cardiac dysfunction.