Blockade of EP3 attenuates cardiac fibrosis by suppressing TGF-β1-Smad2/3 signaling through the Smurf2-mediated Smad7 ubiquitination.

Abstract

Background: Sustained β-adrenergic activation induces cardiac fibrosis, which is characterized by excessive deposition of extracellular matrix (ECM) secreted by activated cardiac fibroblasts (CFs). Prostaglandin E₂ (PGE₂) is a dominant cyclooxygenase metabolite of arachidonic acid and exerts important biological actions by activating four G-protein-coupled receptors, including E-type prostanoid receptors 1-4 (EP1-EP4). The present study aimed to investigate the role of the EP3 receptor in isoproterenol (ISO)-induced cardiac fibrosis.

Methods: To elucidate the role of EP3 in cardiac fibrosis, the global EP3 gene knockout mice (EP3^-/-) and the LoxP-Cre system-based CF-specific EP3 gene knockout mice (EP3^cKO) or cardiomyocyte (CM)-specific EP3 gene knockout mice (EP3^mKO) were subcutaneously injected with ISO [5 mg/(kg·d)] for 7 d to establish a cardiac fibrosis model. In parallel, the EP3 agonist sulprostone and the EP3 antagonists DG041 and L798106 were used to elucidate the underlying mechanisms. Finally, the therapeutic potential of the EP3 antagonist DG041 was evaluated in the context of cardiac fibrosis.

Results: Echocardiography assay showed that both EP3^-/- and EP3^cKO mice exhibited a significant improvement in cardiac diastolic function. Histological staining revealed that EP3^-/- and EP3^cKO, but not EP3^mKO, exhibited a significant attenuation of ISO-induced CF activation and ECM accumulation. Further investigation uncovered that EP3 was highly expressed in the CFs rather than CMs. Inhibition of EP3 in the CFs suppressed transforming growth factor-β1 (TGF-β1)-triggered phosphorylation of small mother against decapentaplegic (Smad)2/3 and the production of ECM proteins. Mechanistically, the blockade of EP3 in the CFs activated the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which promoted the degradation of Smad ubiquitination regulatory factor 2 (Smurf2) and subsequently suppressed the ubiquitination of Smad7, an inhibitory Smad, leading to an increase in the protein level of Smad7. Furthermore, the administration of an EP3 antagonist, DG041, markedly improved ISO-induced cardiac diastolic dysfunction and fibrosis.

Conclusions: This study demonstrates that CF-EP3 contributes to β-adrenergic activation-induced cardiac fibrosis and target of EP3 represents a potential therapeutic strategy for the treatment of cardiac fibrosis.