Abstract 103: Interferon Regulatory Factor-1 Is Responsible For Cardiac Protective Effect Of 5-azacytidine On Macrophages In Myocardial Infarction

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Background: During progression of cardiac injury, macrophages with specialized phenotypes are actively involved in inflammatory responses. In this study, we investigated the effect of 5-azacytidine (5AZ) on macrophages in the damaged myocardium.

Methods: A mouse macrophage cell line RAW264.7 was stimulated with lipopolysaccharide (LPS, 100 ng/mL) with or without 5AZ (10 μM). Nitric oxide was quantified by Griess’s method. Expression patterns of inducible nitric oxide synthase (iNOS) and interferon regulatory factor 1 (IRF-1) were determined by Western blot analysis. For animal study, myocardial infarction (MI) was induced by ligation of left coronary artery in mice, and divided into four groups; non MI + saline, non-MI + 5AZ , MI + saline, and MI + 5AZ. Saline or 5AZ was injected (5 mg/kg/d) every other day. Cardiac fibrosis was evaluated by Masson’s trichrome stain was performed and cardiac function was by echocardiography 2 weeks after MI.

Results: LPS-induced nitric oxide formation was reduced by 5AZ treatment in RAW264.7 cells. LPS-induced iNOS mRNA and protein inductions were blocked by 5AZ treatment. Next, the effect of 5AZ on IRF1, a regulator of iNOS, was examined. IRF-1 was dramatically increased by LPS to peak at 4 hr and then reduced to basal level. In the presence of MG132, a proteasome inhibitor, IRF-1 protein sustained maximal level without degradation. On the other hand, IRF-1 protein was significantly highly maintained by 5AZ treatment. In animal study, there were significant improvements in ejection fraction (53.25 ± 2.55 % vs. 62.50 ± 7.2 %, p < 0.05) and cardiac fibrosis (28.85 ± 5.44% vs. 16.57 ± 6.30%, p < 0.05). Collectively, 5AZ inhibited iNOS induction by modulation of IRF-1 in the activated macrophages to preserved cardiac function and fibrosis after MI.

Conclusion: 5AZ protected post-MI injury by regulation of IRF-1 kinetics to modulate macrophages in infarcted myocardium. This study suggested 5AZ as a novel therapeutic intervention for cardiac repair.

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