263Through differential targeting of cardiac myocytes and fibroblasts, miR-221 promotes myocardial recovery and averts adverse remodeling after infarction
Authors: Zhou Y Z , Richards A M R , Wang P P W 
Published: October 2019

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.