263Through differential targeting of cardiac myocytes and fibroblasts, miR-221 promotes myocardial recovery and averts adverse remodeling after infarction

Abstract

Abstract Background Reducing cardiac myocyte (CM) death has been the major focus of cardioprotection in the treatment of myocardial infarction (MI). The post-MI role of cardiac fibroblasts (cFB) has received less attention compared to CM. miRNAs can multi-target in condition-dependent and/or cell type-oriented manner. Reduced miR-211 expression in myocardium is associated with severe cardiac fibrosis in patients with end stage heart failure. We previously reported that miR-221 mimics protect CM in vitro against hypoxic injury. Purpose Taking comprehensive in vivo and in vitro approaches, we tested our hypothesis that miR-221 regulate CM and cFB differently to reduce CM death and inhibit adverse fibrosis following MI. Methods In vitro, H9c2 and rat cFB were transfected with miR-221 mimics (miR-221) and mimic control (MC) and subjected to hypoxia/reperfusion (H/R). Apoptosis (Annexin V and 7-AAD), cell injury (LDH release), and autophagy LC3 II/I and p62 by Western blot (WB). Myofibroblast (myoFB) activation (α-SMA and gel contraction), and collagen synthesis (Sircol assay) were measured. In vivo, following left coronary artery ligation (MI), rats were treated with miR-221 mimics (i.v. 1mg/kg) immediately and 3-days post-MI. Hearts were collected at 2-, 7- and 30-days post-MI. Infarction and fibrosis were determined by Masson trichrome staining and myoFBs identified by α-SMA immunofluorescence. Cardiac function was assessed by echocardiography and LV catheterization. WB, qPCR and Luciferase reporter assays were applied. Results The novel findings of this study are: (1) miR-221 protects CM and cFB through different mechanisms, namely combined anti-apoptotic and anti-autophagic effects vs. anti-autophagic alone, respectively. (2) For the first time we demonstrated that p53 is a direct target of miR-221; downregulation of total and phosphorylated p53 is associated with reduced apoptosis in CM while this effect is completely missing in cFB. Direct targeting of Ddit4 is responsible for anti-autophagy effects in both cell types. (3) miR-221 increases cFB in number but inhibits α-SMA activation and collagen synthesis. (4) Multiple predicted and previously reported targets of miR-221, e.g. Bmf, Puma, p27 and Tp53inp1, are down-regulated in cultured cells but are not affected in the heart in vivo. (5) Working through CM and cFB, miR-221 reduces infarct size, post-MI fibrosis and α-SMA+ cells in both infarct and remote myocardium, and improves LV function (as indicated by preserved ejection fraction, LV developed pressure, +/− dP/dt and end diastolic pressure). Conclusion miR-221 prevents CM and cFB death without extension of injury-stimulated cardiac fibrosis in the infarct zone or adverse fibrosis in the peri-infarct zone. The integrated effects of miR-221 ameliorate adverse post-ischemic LV remodeling and augment cardiac functional recovery. Therefore miR-221 is a unique therapeutic target in the treatment of cardiac infarction.