Therapeutic silencing miR-146b-5p improves cardiac remodeling in a porcine model of myocardial infarction by modulating the wound reparative phenotype

Protein Cell 2021, 12(3):194–212 https://doi.org/10.1007/s13238-020-00750-6 Protein & Cell RESEARCH ARTICLE Protein & Cell Therapeutic silencing miR-146b-5p improves cardiac remodeling in a porcine model of myocardial infarction by modulating the wound reparative phenotype Yiteng Liao1,2 , Hao Li1,2 Hongming Zhu1& , Hao Cao3, Yun Dong4, Lei Gao1, Zhongmin Liu1,3&, Junbo Ge5& 1 Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China 2 Department of Cardiology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China 3 Department of Cardiovascular Surgery, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China 4 Department of Ultrasound in Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China 5 Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital of Fudan University, Shanghai 200032, China & Correspondence: (Z. Liu), (J. Ge), (H. Zhu) Received March 13, 2020 Accepted May 29, 2020 ABSTRACT Fibrotic remodeling is an adverse consequence of immune response-driven phenotypic modulation of cardiac cells following myocardial infarction (MI). MicroRNA146b (miR-146b) is an active regulator of immunomodulation, but its function in the cardiac inflammatory cascade and its clinical implication in fibrotic remodeling following MI remain largely unknown. Herein, miR-146b5p was found to be upregulated in the infarcted myocardium of mice and the serum of myocardial ischemia patients. Gain- and loss-of-function experiments demonstrated that miR-146b-5p was a hypoxia-induced regulator that governed the pro-fibrotic phenotype transition of cardiac cells. Overexpression of miR-146b-5p activated fibroblast proliferation, migration, and fibroblast-to-myofibroblast transition, impaired endothelial cell function and stress survival, and disturbed macrophage paracrine Yiteng Liao, Hao Li, and Hao Cao have contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13238-020-00750-6) contains supplementary material, which is available to authorized users. signaling. Interestingly, the opposite effects were observed when miR-146b-5p expression was inhibited. Luciferase assays and rescue studies demonstrated that the miR-146b-5p target genes mediating the above phenotypic modulations included interleukin 1 receptor associated kinase 1 (IRAK1) and carcinoembryonic antigen related cell adhesion molecule 1 (CEACAM1). Local delivery of a miR-146b-5p antagomir significantly reduced fibrosis and cell death, and upregulated capillary and reparative macrophages in the infarcted myocardium to restore cardiac remodeling and function in both mouse and porcine MI models. Local inhibition of miR-146b-5p may represent a novel therapeutic approach to treat cardiac fibrotic remodeling and dysfunction following MI. KEYWORDS cardiac fibrosis, microRNA, porcine model, myocardial infarction INTRODUCTION Myocardial infarction is the leading cause of mortality and morbidity worldwide (Nagpal et al., 2016), indicating an © The Author(s) 2020 urgent need for studies investigating the underlying mechanisms of MI to identify innovative therapeutic strategies. Pathological cardiac remodeling is characterized by complex multicellular alterations, such as cardiomyocyte death, immune cell activation, and excessive deposition of the extracellular matrix, that exacerbate cardiac dysfunction, and often progresses to heart failure (Sutton and Sharpe, 2000; Prabhu and Frangogiannis, 2016a, b; Shiraishi et al., 2016). Cells are well known to sense their surrounding physical and signaling environments and respond accordingly by altering their functions and fates (Zhu et al., 2014a, 4b). Pioneering studies have demonstrated that adverse remodeling following MI is caused by the phenotypic modulation of cardiac cells, with the inappropriate and untimely activation and resolution of inflammation being a crucial driving factor (Epelman et al., 2015; Westman et al., 2016; Meyer et al., 2017; Huang and Frangogiannis, 2018). Thus, therapeutic modulation of immunoregulatory factors and reparative phenotypes of resident cells may be a promising approach for preventing post-infarction remodeling (Prabhu and Frangogiannis, 2016a, b). MicroRNAs are functional, single-stranded, short noncoding RNAs that regulate a large array of biological processes through the degradation or inhibition of target messenger RNAs (mRNAs). The miR-146 family was the first family of microRNAs reported to be involved in mammalian immunomodulation (Taganov et al., 2006; Baltimore et al., 2008). However, the functions of these microRNAs in the cardiac inflammatory cascade and adverse remodeling remain unclear. To address this, we generated two animal models (mouse and pig) to demonstrate the benefits of inhibiting miR-146b5p post-MI. The results indicated that hypoxia induced miR146b-5p expression through the NF-κB signaling pathway. miR-146b-5p gain-of-function was found to activate fibroblast proliferation, migration, and fibroblast to myofibroblast transition (FMT), impair endothelial cell function and stress survival, and disrupt macrophage paracrine signaling. The opposite effects were observed upon miR-146b-5p loss-offunction. IRAK1 and CEACAM1 were found to be the targets of miR-146b-5p in the above phenotypic modulations. The local delivery of a miR-146b-5p antagomir significantly reduced fibrosis and cell death, and upregulated capillary and reparative macrophages in infarcted myocardium to restore cardiac remodeling and function in both mice and porcine MI models. RESULTS miR-146b-5p was upregulated in patients with chronic total occlusion (CTO) and in a murine MI model CTO consists of the complete obstruction of a coronary artery, resulting in severe myocardial ischemia. Using quantitative real-time polymerase chain reaction (qPCR), we found that miR-146b-5p expression was significantly © The Author(s) 2020 RESEARCH ARTICLE increased in the plasma of CTO patients compared to noischemia volunteers (n = 8 per group, P = 0.02; Fig. 1A). Clinical parameters and medical histories were available for all patients (Table S1). To elucidate the connection between plasma miR-146b-5p expression and myocardial ischemia, a mouse MI model was constructed, and qPCR was performed. Morphological observation and expression of the fibrotic genes COL1A1 (Col1) and ACTA2 (αSMA) were used to validate myocardial ischemia (Fig. 1B–D). We found that miR-146b-5p expression increased continuously in the infarct zone (reaching approximately 5-fold above the baseline), but not in the remote myocardium, over 14 days following the induction of MI (Fig. 1E). Hypoxia upregulated miR-146b-5p in fibroblasts, endothelial cells, and macrophages may through NF-κB signaling Given that miR-146b-5p is upregulated in t (...truncated)