Cdc42 Regulates Macrophage Function In Cardiac Remodeling

Objective:Cardiovascular disease is a major contributor to mortality and morbidity worldwide,and almost all cardiovascular diseases are accompanied by the development of cardiac remodeling-cardiac fibrosis.Cardiac fibrosis is mainly caused by the proliferation and activation of fibroblasts into myofibroblasts,resulting in excessive accumulation of extracellular matrix proteins in the interstitial matrix of the heart,which in turn causes cardiac systolic and diastolic dysfunction.Macrophages play a pivotal role in the development of cardiac fibrosis,and the cytokines they secrete can directly or indirectly affect cardiac remodeling.Cdc42,a member of the Rho GTPase family,is involved in various life processes such as cell growth and development,cell migration,and cytoskeleton regulation,but the function of Cdc42 on macrophage during cardiac remodeling is not clear.The results of our previous work showed that macrophage-specific knockdown of Cdc42 significantly exacerbated isoproterenol(ISO)-induced cardiac fibrosis.This work aimed to investigate the effect of Cdc42 deletion on macrophage function and its role in cardiac remodeling and its mechanisms.Experimental methods:1.Construction of macrophage-specific knockout Cdc42 mice.Macrophagespecific knockout Cdc42 mice and control mice without Cre were obtained by mating Cdc42flox/flox mice with Lyz2-Cre(myeloid lineage-specific expression of Cre recombinase)mice.2.Mice in good condition and at similar age(8-12 weeks old)were selected and randomly divided into experimental and control groups of mice.The cardiac remodeling model was constructed by subcutaneous injection of ISO(20mg/kg/day,100μL/10g)for 14 consecutive days in the experimental group;the control group was injected with an equal amount of saline.3.The migration of macrophage was examined by scratching assay(Wound-healing).The mouse peritoneal macrophages were extracted and stimulated with ISO(10n M)and H9C2 supernatants induced by ISO(10n M),respectively,their migrations were examined by scratching.4.Detection of NLRP3 pathway-related proteins.Heart tissue proteins were extracted and the protein expression levels of NLRP3,ASC,caspase1,cleavedcaspsae1,IL1β,and cleaved-IL1β were detected by Western Blot.5.Detection of cardiomyocyte apoptosis.Heart tissue proteins were extracted,and the protein expression levels of Bcl-2 and Bax were detected by Western Blot.Peritoneal macrophages were extracted,and the supernatants of ISO-induced peritoneal macrophages were collected to stimulate rat cardiomyocytes(H9C2),and cardiomyocyte apoptosis was detected by Western Blot and flow cytometry.6.The phagocytic capacity of macrophages after deletion of Cdc42 was examined by cytophagocytosis assay.Macrophage phagocytosis was observed by intraperitoneal injection of 3% thioglycolate medium into mice(FF-and FF+)and intraperitoneal injection of 0.1% chicken red blood cells on day 3,after 45 minutes,their peritoneal fluid was collected and macrophage phagocytosis was observed by Giemsa staining.7.To detect the synthesis and secretion of cytokines from macrophages or H9C2.The peritoneal macrophages were isolated,and stimulated with ISO,their supernatants were collected to stimulate H9C2,and the synthesis and secretion of inflammatory factors were detected by by Western Blot and ELISA from the peritoneal macrophages or H9C2;On the other hand,H9C2 cells were stimulated with ISO,their supernatants were collected to stimulate the peritoneal macrophages,and the synthesis and secretion of inflammatory factors were also detected by Western Blot and ELISA from the peritoneal macrophages or H9C2.8.Detection of fibroblast activation.Peritoneal macrophages were extracted,and the supernatants of ISO-induced H9C2 were used to stimulate peritoneal macrophages.In reverse,ISO-induced peritoneal macrophages were used to stimulate H9C2.The supernatants from above different treatment were collected to stimulate primary fibroblasts,respectively,and the expression of fibrosis-related genes(Collagen III,α-SMA,vimentin)in fibroblasts was detected by Western Blot.Results1.Macrophage deletion of Cdc42 significantly exacerbates inflammatory responses and apoptosis in cardiac tissues.2.Macrophage deletion of Cdc42 significantly increased cardiomyocytic apoptosis induced by ISO.3.Macrophage deletion of Cdc42 significantly increased IL-10,IL1β and decreased IL-6 in cardiac tissues induced by ISO remodeling.4.Deletion of Cdc42 significantly increased the synthesis and secretion of IL1β,IL-10 in macrophages induced by ISO.5.Deficiency of Cdc42 significantly inhibited phagocytic capacity of macrophages.6.Deletion of Cdc42 enhanced the migratory capacity of macrophage induced by the secretions from ISO-stimulated cardiomyocytes and promoted the capacity of macrophages in activating fibroblasts.Conclusion:During ISO-induced cardiac remodeling,deletion of Cdc42 in macrophages promotes infiltration of monocytes/macrophages and increases inflammatory damage in cardiac tissues induced by ISO.Deletion of Cdc42 weakened their phagocytosis and promoted the synthesis and secretion of IL10,IL1β in macrophages,which in turn aggravates cardiac fibrosis.The absence of Cdc42 may exacerbate cardiac fibrosis by regulating the synthesis and secretion of inflammatory factors such as IL-10,IL-6,and TNF-α in macrophages,promoting cardiomyocyte apoptosis and fibroblast activation,and increasing collagen synthesis.