Upregulation of Paired-Related Homeobox 2 Promotes Cardiac Fibrosis in Mice Following Myocardial Infarction by Targeting of Wnt5a

Abstract

Backgrounds: Cardiac fibrosis is a key factor to determine the prognosis in patient with myocardial infarction (MI). Activation of Wnt signaling by transforming growth factor β (TGF-β) contributes to cell differentiation of fibroblasts in cardiac remodeling. However, the molecular mechanisms by how Wnt protein family 5a (Wnt5a) is upregulated in post-ischemic heart are still mostly unclear. The aim of this study is to investigate whether paired-related homeobox 2 (Prrx2), as a transcriptional factor, regulates Wnt5a gene expression in myocardial fibrosis, and, if so, to identify the role of Prrx2 in the recovery of cardiac functions in mice following MI. Methods: The MI surgery was performed by ligation of left anterior descending coronary artery. Cardiac remodeling was assessed by measuring interstitial fibrosis using Masson staining. Cell differentiation was examined by immunofluorescence analysis of alpha smooth muscle actin (α-SMA). Results: Both Prrx2 and Wnt5a gene expressions were upregulated in Apoe-/- mice following MI, accompanied with increased mRNA and protein levels of α-SMA, collagen I, and collagen III in hearts, compared to mice with sham surgery. Adenovirus-mediated gene knockdown of Prrx2 increased survival rate, alleviates cardiac fibrosis, decreased infarction sizes in hearts, and improved cardiac functions in mice with MI after 30 post-ischemia days. Importantly, inhibition of Prrx2 suppressed ischemia-induced Wnt5a gene expression and Wnt5a signaling in mice hearts. In cultured cardiac fibroblasts, TGF-β increased gene expressions of Prrx2 and Wnt5a, and induced cell differentiations, which were abolished by gene silence of either Prrx2 or Wnt5a. Further, overexpression of Prrx2 or Wnt5a mirrored the effects of TGF-β on cell differentiations of cardiac fibroblasts. Gene silence of Wnt5a also ablated cell differentiations induced by Prrx2 overexpression in cardiac fibroblasts. Mechanically, Prrx2 was able to bind with Wnt5a gene promoter to upregulate Wnt5a gene expression. Conclusion: Upregulation of Prrx2 increases Wnt5a gene expression to induce cell differentiations, which contributes to ischemia-induced cardiac fibrosis. In perspective, targeting Prrx2-Wnt5a signaling should be considered to improve cardiac remodeling in patients with ischemic heart diseases. Funding: This project was supported by National Natural Science Foundation of China (81874312, 81770576, 81770493, U1704168, 81673423, and 81570723) and the Natural Science Foundation of Shandong Province (ZR2019PH007, ZR2019QH011, ZR2016HM04). S.X.W. is an exceptional young scholar of Shandong University, a distinguished professor of Henan province, a recipient of Zhong-Yuan-Qian-Ren Program of Henan province (194200510005), an adjunct Tai-Hang Professional Scholarship of Xinxiang Medical University (505067), and an adjunct Lan-Yue Professional Scholar of Hubei University of Science and Technology (2016-18XZ07). L.Z. was supported by general program of Department of Health (B20180875) and clinical medical technology innovation guidance project from Department of Science and Technology (2017SK50504) of Hunan Province. W.D.Q. was sponsored by China Postdoctoral Science Foundation (2018M630788). Declaration of Interest: None. Ethical Approval: The animal protocol was reviewed and approved by the University of Shandong, Animal Care and Use Committee. This animal study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.