Role of runt-related transcription factor 3 in myocardial fibrosis in mice

Abstract

Objective To investigate the role of runt related transcription factor 3 (Runx3) in cardiac fibroblasts differentiation, proliferation as well as synthesis of type I collagen after myocardial infarction (MI) in mice. Methods Male C57BL/6J mice were selected and randomly divided into model group (MI group, treated with ligation of left coronary artery) and Sham group, and were sacrificed at the 7th, 14th and 28th days after modeling (n=6 for each group), respectively. In addition, Runx3 small interfering RNA (si-Runx3) was injected via the tail vein of mice before modeling to achieve Runx3 knockdown, negative siRNA (si-NC) as the matched control. According to the treatment they received, the mice were subsequently allocated into Sham+si-NC group, Sham+si-Runx3 group, MI 28 d+si-NC group and MI 28 d+si-Runx3 group (n=8), and were sacrificed in 28 d after modeling. The levels of Runx3, alpha-smooth muscle actin (α-SMA) and type I collagen in the heart were determined by immunofluorescence staining and Western blotting. The cardiac function was evaluated by echocardiography, and Ki67 positive cells were observed using immunohistochemical assay. After cardiac fibroblasts were isolated from C57BL/6 mice and cultured for 1~3 d, and the effects of TGF-β1 at different concentrations and for different stimulation times on Runx3 protein were observed in the cells. The effects of Runx3 gene knockout on type I collagen synthesis and proliferation of cardiac fibroblasts were also analyzed. Results Immunofluorescence staining and Western blotting showed that the expression levels of Runx3, α-SMA and type I collagen in the fibrosis regions were up-regulated with the extended duration of MI. As compared with the MI 28 d+si-NC group, the MI 28 d+si-Runx3 group had increased left ventricular ejection fraction (LVEF), fractional shortening (FS) and cardiac output (P < 0.05), and decreased left ventricular end-diastolic diameter (LVIDd) and decreased left ventricular (LVIDs) (P < 0.05) in 28 d after MI; the expression levels of Runx3 and α-SMA and the proliferation of Ki67 positive cells induced by MI were blocked by si-Runx3. TGF-β1 up-regulated Runx3 expression in a concentration- and time-dependent manner. Runx3 knockdown not only inhibited TGF-β1-induced differentiation of cardiac fibroblasts, reduced type I collagen synthesis, but also attenuated cardiac fibroblasts proliferation. Conclusions Runx3 is involved in the cardiac fibroblast differentiation and proliferation.

Keywords

• runt related transcription factor 3
• myocardial fibrosis
• myocardial infarction
• mice
• cardiac fibroblasts