Departments of Cardiovascular and Metabolic Sciences (Y.S., K.S., M.L.M., M.K.G., S.V., N.P.), Lerner Research Institute, Cleveland Clinic, OH.
Inflammation and Immunity (N.W., K.A., S.E.), Lerner Research Institute, Cleveland Clinic, OH.
Circ Res. 2023 Mar 17;132(6):690-703. doi: 10.1161/CIRCRESAHA.122.321735. Epub 2023 Feb 13.
Impaired beta-adrenergic receptor (β1 and β2AR) function following hypoxia underlies ischemic heart failure/stroke. Activation of PI3Kγ (phosphoinositide 3-kinase γ) 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 PI3Kγ feedback mechanism in regulating hypoxia-mediated β1 and β2AR dysfunction and cardiac remodeling.
Human embryonic kidney 293 cells or mouse adult cardiomyocytes and C57BL/6 (WT) or PI3Kγ knockout (KO) mice were subjected to hypoxia. Cardiac plasma membranes and endosomes were isolated and evaluated for β1 and β2AR density and function, PI3Kγ activity and β1 and β2AR-associated PP2A activity. Metabolic labeling was performed to assess β1 and β2AR phosphorylation and epinephrine/norepinephrine levels measured post-hypoxia.
Hypoxia increased β1 and β2AR phosphorylation, reduced cAMP, and led to endosomal accumulation of phosphorylated β2ARs 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 β1 and β2AR phosphorylation. This was agonist-independent as plasma and cardiac epinephrine and norepinephrine levels were unaltered. Unexpectedly, PI3Kγ activity was selectively increased in the endosomes of human embryonic kidney 293 cells and WT hearts post-hypoxia. Endosomal β1- and β2AR-associated PP2A activity was inhibited upon hypoxia in human embryonic kidney 293 cells and WT hearts showing regulation of beta-adrenergic receptors by PI3Kγ. This was accompanied with phosphorylation of endogenous inhibitor of protein phosphatase 2A whose phosphorylation by PI3Kγ inhibits PP2A. Increased β1 and β2AR-associated PP2A activity, decreased beta-adrenergic receptor phosphorylation, and normalized cardiac function was observed in PI3Kγ KO mice despite hypoxia. Compared to WT, PI3Kγ KO mice had preserved cardiac response to challenge with β1AR-selective agonist dobutamine post-hypoxia.
Agonist-independent activation of PI3Kγ underlies hypoxia sensing as its ablation leads to reduction in β1- and β2AR phosphorylation and amelioration of cardiac dysfunction.
缺氧后β-肾上腺素能受体（β1 和 β2AR）功能受损是缺血性心力衰竭/中风的基础。β-肾上腺素能受体激活 PI3Kγ（磷酸肌醇 3-激酶 γ）可通过抑制 PP2A（蛋白磷酸酶 2A）来阻碍β-肾上腺素能受体去磷酸化，从而对受体进行反馈调节。然而，关于 PI3Kγ 反馈机制在调节缺氧介导的β1 和 β2AR 功能障碍和心脏重塑中的作用知之甚少。
将人胚肾 293 细胞或成年小鼠心肌细胞和 C57BL/6（WT）或 PI3Kγ 敲除（KO）小鼠置于缺氧环境中。分离心脏质膜和内体，评估β1 和 β2AR 密度和功能、PI3Kγ 活性以及β1 和 β2AR 相关的 PP2A 活性。进行代谢标记以评估β1 和 β2AR 磷酸化，检测缺氧后去甲肾上腺素/肾上腺素水平。
缺氧可增加β1 和 β2AR 磷酸化，降低 cAMP，并导致人胚肾 293 细胞和 WT 心肌细胞中磷酸化的β2AR 在内体中的积累。WT 小鼠的急性缺氧导致心脏重塑和腺苷酸环化酶活性丧失，同时伴有β1 和 β2AR 磷酸化增加。这与激动剂无关，因为血浆和心脏去甲肾上腺素和肾上腺素水平没有改变。出乎意料的是，PI3Kγ 活性在人胚肾 293 细胞和 WT 心脏缺氧后选择性增加。在人胚肾 293 细胞和 WT 心脏中，β1 和 β2AR 相关的内体 PP2A 活性在缺氧时受到抑制，表明 PI3Kγ 对β-肾上腺素能受体进行调节。这伴随着内源性蛋白磷酸酶 2A 抑制剂的磷酸化，PI3Kγ 对其磷酸化可抑制 PP2A。尽管缺氧，PI3Kγ KO 小鼠仍表现出β1 和 β2AR 相关的 PP2A 活性增加、β-肾上腺素能受体磷酸化减少和心脏功能正常化。与 WT 相比，PI3Kγ KO 小鼠在缺氧后对β1AR 选择性激动剂多巴酚丁胺的心脏反应得到保留。
PI3Kγ 的非激动剂依赖性激活是缺氧感应的基础，因为其缺失可导致β1 和β2AR 磷酸化减少和心脏功能障碍改善。
