Elevated Wnt2 and Wnt4 Activate NF-κB Signaling to Promote Cardiac Fibrosis by Cooperation of Fzd4/2 and LRP6 Following Myocardial Infarction

Abstract

Background: Acute myocardial infarction (AMI)-induced excessive cardiac fibrosis exaggerates infarcted area and cardiac dysfunction, which is a significant clinical problem. Here, we investigated Wnt2 and Wnt4 levels in AMI patients or MI mouse model, and delineated the potential mechanism in cardiac fibrosis following MI. Methods: We examined serum Wnt2 and Wnt4 levels in AMI and non-AMI patients by ELISA analysis. MI mouse model was built by ligation of left anterior descending branch (LAD). Findings: Serum Wnt2 or Wnt4 level was increased in patients with AMI, and the high serum Wnt2 and Wnt4 levels correlated to adverse outcome of these patients. Elevated serum and cardiac Wnt2 or Wnt4 levels were observed at early-phase after experimental MI. Knockdown of Wnt2 and Wnt4 significantly attenuated myocardial fibrosis and cardiac dysfunction following MI. In vitro, hypoxia enhanced the secretion and expression of Wnt2 and Wnt4 in cardiomyocytes (CMs) or cardiac fibroblasts (CFs). Si-Wnt2 or si-Wnt4 partly abolished the pro-fibrotic effect in cardiac fibroblasts in response to hypoxia. Mechanistically, exogenous Wnt2 or Wnt4 activated β-catenin /NF-κB signaling to promote pro-fibrotic effects in cultured CFs. In cultured CFs, Wnt2 or Wnt4 upregulated Frizzled (Fzd) 2 or Fzd4 and LRP6 expression. Si-Fzd4 or si-Fzd2 significantly attenuated NF-κB activation and the pro-fibrotic effects in CFs treated with Wnt2 or Wnt4, respectively. Knockdown of LRP6 showed the similar results. However, Wnt2 or Wnt4 induced the activation of β-catenin dependently on LRP6 not Fzd4 or Fzd2. Interpretation: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 in fibroblasts, which may contribute to adverse outcome of patients with AMI, suggesting that systemic inhibition of Wnt2 and Wnt4 may improve cardiac dysfunction after MI. Clinical Trial Registration Details: ClinicalTrials.gov Identifier: NCT03342131. Funding Information: This study was supported by National Natural Science Foundation of China (No. 81770239, 81730009, 82070232). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: This study was approved by the Ethics Committee of Dalian Medical University/First Affiliated Hospital of Dalian Medical University (PJ-KS-KY-2017-104(x)). Before inclusion in the study, written informed consent was obtained from the patient's family/patient. The investigation conformed to the principles outlined in the Declaration of Helsinki. All experiment protocols about animal in this study were approved by the Animal Care and Use Committee of Zhongshan Hospital, Fudan University, and conformed to the ‘Guidelines for the Care and Use of Laboratory Animals’ published by the National Institutes of Health (No. 85-23, revised 1996).