Effect Of Macrophage-specific Deletion Of Cdc42 On Isoproterenol-induced Cardiac Fibrosis And Its Molecular Mechanism

Objective:Heart failure is strongly associated with cardiac fibrosis.Adult myocardial cells do not have the ability to regenerate,they will be continuously reduced and replaced by myofibroblasts when mechanically or chemically damaged,eventually it will lead to cardiac fibrosis.Cardiac fibrosis manily manifests as decreasing cardiomyocytes,infiltration of macrophages,conversion of fibroblasts into myofibroblasts,increasing of collagen content,etc..Although cardiac fibrosis is a repair mechanism to maintain the integrity of the heart after heart injury,it will further decline the cardiac contractile ability,weaken blood pumping function and even heart failure.Cdc42,a small molecule switch protein,belongs to the Rho family of small GTPase and has GTPase activity.It is reported that CDC42 is involved in the regulation of cell migration,polarization,vesicle transport,cell cycle and cytoskeleton,and its role in cardiac fibrosis has not been revealed.The purpose of this study was to explore the effect of macrophage specific deletion of Cdc42 on isoproterenol-induced cardiac fibrosis and its molecular mechanism.Experimental methods:1.The macrophage-specific knockout CDC42 mice(FF+)were obtained by crossbreeding CDC42FL/FL mouse with Ly Z2 Cre mouse in which Cre recombinase is specifically expressed in bone marrow derived macrophages.2.Cardiac fibrosis model was established by subcutaneously injecting ISO into mice.Echocardiography and ECG were operated to detect the cardiac function,and Routine blood tests were performed to analyze the blood parameters.3.The hearts of mice were isolated,fixed,dehydrated,embedded,and sectioned after the disease fibrosis model was established.The structure and morphology were examined with HE staining and Masson staining.Macrophage infiltration was detected by immunofluorescence.4.Total proteins and RNAs were extracted from mice hearts,Western Blots and RT-PCR were operated to detect the expressions of genes related to cardiac fibrosis.5.Primary bone marrow macrophages and fibroblasts were isolated to explore the interactions between macrophages and fibroblasts.Results1.The macrophage-specific knockout CDC42 mice were successfully obtained,and the m RNA and protein levels of CDC42 were reduced significantly in macrophages compared with control group.2.Cardiac fibrosis model was successfully established after the mice were subcutaneously injected with ISO.Echocardiography and electrocardiogram results showed that cardiac function,the length of QRS were impaired,and the numbers of WBCs and median cells were remarkably decreased in ISO-induced CKO mice.3.HE staining,Masson staining and immunofluorescence revealed that cardiac fibrosis and macrophage infiltration were aggravated in ISO-induced CKO mice.4.Western Blots showed that the expressions of matrix metalloproteinase MMP9 and cardiac TGF-β were notably decreased while the expressions of interleukin-10(IL-10)of FF+ group were significantly up-regulated in ISO-induced CKO mice.5.The secretions from ISO-induced FF+ macrophages significantly enhanced the expressions of fibrotic markers—Col III and SMA,but phosphorylated Smad3 was not simultaneously enhanced in cardiac fibroblasts.The contents of TGF-β were not elevated in the secretions from ISO-induced FF+ macrophages.Our results suggested the fibrotic role of ISO-induced FF+ macrophages might not be TGF-βdependent differentiation of fibroblasts into myofibroblasts.Conclusion:Macrophage-specific knockout of CDC42 exacerbated ISO-induced myocardial injury,cardiac function and aggravate cardiac fibrosis.It increased the infiltration of macrophages and elevated Col III,decreased the expressions of MMP9,IL6 and TGFβ.The fibrotic role of deficiency of Cdc42 in macrophages is not TGF-βdependent in cardiac fibrosis,and might accelerate fibrosis via reducing MMP9.