AMPK Attenuation of β-Adrenergic Receptor-Induced Cardiac Injury via Phosphorylation of β-Arrestin-1-ser330.

Abstract

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.