Effects Of Histone Demethylase KDM3A On Myocardial Injury After Infarction By Regulating Macrophage Polarization

Aims: Recently,emerging studies have verified that KDM3 A involved in an important epigenetic mechanism in cardiovascular diseases,such as diabetic cardiomyopathy,myocardial infarction and cardiac hypertrophy.our previous studies confirmed that KDM3 A regulated the function of smooth muscle cells in high glucose environment and vascular remodeling in diabetes mellitus,the underlying mechanism was that KDM3 A could regulate pathophysiological responses such as inflammation,apoptosis,and oxidative stress.According to the above findings,we arrive the conclusion that KDM3 A may play a central role in the inflammatory response of the cardiovascular system.Meanwhile,owing to macrophages play a key role in inflammatory repair after myocardial infarction,we attempt to explore whether KDM3 A could regulate the polarization of macrophages to affect the inflammatory response after myocardial infarction and targeting KDM3 A could influence the prognosis of myocardial infarction and adverse left ventricular(LV)remodeling.Methods: In order to explore the biological function of KDM3 A and its potential mechanism,we designed the experiments by down-regulating the expression of KDM3 A into three groups randomly in vitro: a)control group: The BMDMs without any treatment;b)Adsh RNA group: The BMDMs were infected by Adsh RNA;c)Adsh KDM3 A group: The BMDMs were infected by Adsh KDM3 A.Four groups were divided randomly in vivo: a)wild-type rat sham operation group(WT-SO);b)KDM3A knockout rat sham operation group(KO-SO);c)WT-MI operation group;d)KO-MI operation group.We analyzed the function of macrophages by phagocytosis and migration assay,and explored the polarization of macrophages.The expression of macrophage inflammation related genes in acute inflammatory phase and surface makers were detected by western blotting and immunofluorescence assay.Echocardiography,Masson’s trichrome staining and Hematoxylin & Eosin(H&E)staining were detected the cardiac ventricular function.Results: we confirmed that KDM3 A affected the process of inflammation by regulating the major function and phenotypic polarization of macrophages,thus affecting the prognosis of myocardial infarction and adverse left ventricular(LV)remodeling.In vitro,inhibition of the expression of KDM3 A in BMDMs led to the decrease of phagocytosis and attenuated the migration ability respond to chemokines of myocardial cell necrosis,which suggested that KDM3 A inhibition might contribute the loss of macrophages homing to injured tissues and the inefficient clearance of cell debris.KDM3 A deficiency led to increase in secretion of pro-inflammatory cytokines and more differentiation of macrophages into M1 phenotype under the LPS stimulation,but the ability of macrophages differentiated into M2 phenotype were inhibited under the IL-4stimulation.Furthermore,our results showed that there was a significantly difference in the infiltration of M1 and M2 macrophages between KO-MI and WT-MI groups on the7 th day post-MI.Remarkably,we observed that more M1 macrophages(CD86+)infiltration and higher M1/M2(CD86+/CD206+)ratio in the myocardial infarction area of KO-MI rats.WT-MI rats,on the other hand,were more infiltrated of M2 macrophages(CD206+).Moreover,the secretion trend of M1 or M2 related phenotypic markers and cytokines in the two groups were consistent in vitro.These observations consistently clarify that inhibition of KDM3 A could lead to the polarization of macrophages toward pro-inflammatory M1 and not be converted into repaired M2 in time during the inflammation-reparative transition phase.Importantly,in the post-MI both left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)at seven days and one month,fibrosis and cardiac function at 7 days in KO-MI group were worse than those in WT-MI group.Western blot and Ch IP-PCR analysis confirmed that IRF4 acts as a downstream effector of KDM3 A to regulate the polarization of macrophages.KDM3 A affects the transcription of IRF4 by enhancing the level of H3K9me2 in the IRF4 promoter region through epigenetic effects,thereby regulating the polarization of macrophages.Conclusion: our results revealed that KDM3 A were critical for mediating macrophage polarization,phagocytosis and migration to participate in cardiac repair progress and left ventricular remodeling in post-MI.We also found that IRF4 was downstream effector of KDM3A-dependent pathway.These findings not only indicated that targeting KDM3 A to modulate macrophage polarization via IRF4 signal pathway in the infarcted heart might be a promising novel therapeutic strategy for cardiac repair and function,but also suggested that skewing macrophage polarization towards a reparative phenotype might be a pivotal way to treat post-MI.