BACKGROUND β-adrenergic receptor (β-AR) overactivation is a major pathological cue associated with cardiac injury and diseases. AMPK (AMP-activated protein kinase), a conserved energy sensor, regulates energy metabolism and is cardioprotective. However, whether AMPK exerts cardioprotective effects via regulating the signaling pathway downstream of β-AR remains unclear. METHODS Using immunoprecipitation, mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we determined whether AMPK phosphorylates β-arrestin-1 at serine (Ser) 330. Wild-type mice and mice with site-specific mutagenesis (S330A knock-in [KI]/S330D KI) were subcutaneously injected with the β-AR agonist isoproterenol (5 mg/kg) to evaluate the causality between β-adrenergic insult and β-arrestin-1 Ser330 phosphorylation. Cardiac transcriptomics was used to identify changes in gene expression from β-arrestin-1-S330A/S330D mutation and β-adrenergic insult. RESULTS Metformin could decrease cAMP/PKA (protein kinase A) signaling induced by isoproterenol. AMPK bound to β-arrestin-1 and phosphorylated Ser330 with the highest phosphorylated mass spectrometry score. AMPK activation promoted β-arrestin-1 Ser330 phosphorylation in vitro and in vivo. Neonatal mouse cardiomyocytes overexpressing β-arrestin-1-S330D (active form) inhibited the β-AR/cAMP/PKA axis by increasing PDE (phosphodiesterase) 4 expression and activity. Cardiac transcriptomics revealed that the differentially expressed genes between isoproterenol-treated S330A KI and S330D KI mice were mainly involved in immune processes and inflammatory response. β-arrestin-1 Ser330 phosphorylation inhibited isoproterenol-induced reactive oxygen species production and NLRP3 (NOD-like receptor protein 3) inflammasome activation in neonatal mouse cardiomyocytes. In S330D KI mice, the β-AR-activated cAMP/PKA pathways were attenuated, leading to repressed inflammasome activation, reduced expression of proinflammatory cytokines, and mitigated macrophage infiltration. Compared with S330A KI mice, S330D KI mice showed diminished cardiac fibrosis and improved cardiac function upon isoproterenol exposure. However, the cardiac protection exerted by AMPK was abolished in S330A KI mice. CONCLUSIONS AMPK phosphorylation of β-arrestin-1 Ser330 potentiated PDE4 expression and activity, thereby inhibiting β-AR/cAMP/PKA activation. Subsequently, β-arrestin-1 Ser330 phosphorylation blocks β-AR-induced cardiac inflammasome activation and remodeling.

中文翻译：

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通过 β-arrestin-1-ser330 磷酸化 β-肾上腺素能受体诱导的心脏损伤的 AMPK 衰减。


背景 β-肾上腺素能受体 （β-AR） 过度激活是与心脏损伤和疾病相关的主要病理线索。AMPK（AMP 活化蛋白激酶）是一种保守的能量传感器，可调节能量代谢并具有心脏保护作用。然而，AMPK 是否通过调节 β-AR 下游的信号通路发挥心脏保护作用仍不清楚。方法 使用免疫沉淀、质谱、位点特异性突变、体外激酶测定和体内动物研究，我们确定了 AMPK 是否在丝氨酸 （Ser） 330 位点磷酸化 β-arrestin-1。野生型小鼠和位点特异性诱变小鼠 （S330A 敲入 [KI]/S330D KI） 皮下注射 β-AR 激动剂异丙肾上腺素醇 （5 mg/kg） 以评估 β-肾上腺素能损伤与 β-arrestin-1 Ser330 磷酸化之间的因果关系。心脏转录组学用于鉴定 β-arrestin-1-S330A/S330D 突变和 β-肾上腺素能损伤引起的基因表达变化。结果 二甲双胍可降低异丙肾上腺素诱导的 cAMP/PKA （蛋白激酶 A） 信号传导。AMPK 与 β-arrestin-1 结合并磷酸化 Ser330，磷酸化质谱评分最高。AMPK 激活在体外和体内促进 β-arrestin-1 Ser330 磷酸化。过表达 β-arrestin-1-S330D （活性形式） 的新生小鼠心肌细胞通过增加 PDE （磷酸二酯酶） 4 的表达和活性来抑制 β-AR/cAMP/PKA 轴。心脏转录组学显示，异丙肾上腺素处理的 S330A KI 和 S330D KI 小鼠的差异表达基因主要参与免疫过程和炎症反应。 β-arrestin-1 Ser330 磷酸化抑制异丙肾上腺素诱导的新生小鼠心肌细胞活性氧产生和 NLRP3 （NOD 样受体蛋白 3） 炎性小体活化。在 S330D KI 小鼠中，β-AR 激活的 cAMP/PKA 通路减弱，导致炎性小体激活受到抑制，促炎细胞因子的表达降低，巨噬细胞浸润减轻。与 S330A KI 小鼠相比，S330D KI 小鼠在异丙肾上腺素暴露后表现出心脏纤维化减少和心脏功能改善。然而，AMPK 施加的心脏保护在 S330A KI 小鼠中被消除。结论 β-arrestin-1 Ser330 的 AMPK 磷酸化增强了 PDE4 的表达和活性，从而抑制了 β-AR/cAMP/PKA 的激活。随后，β-arrestin-1 Ser330 磷酸化阻断 β-AR 诱导的心脏炎性小体激活和重塑。