Effect Of Cardiomyocyte-Specific Knockout Cdc42 On Isoproterenol-Induced Cardiac Fibrosis And Its Molecular Mechanism

ObjectiveCardiac fibrosis is a common pathological feature of cardiovascular disease at advanced stage.Loss of cardiomyocytes,increased cardiac hardness,transformation of fibroblasts into myofibroblasts,and accumulation of interstitial ECM were present in cardial fibrous hearts.Small GTPase Cdc42 is a small molecule switch protein belonging to the Rho GTPase family.There are two forms of Cdc42,including an inactive form that binds to GDP and an active form that binds to GTP.Currently,the studies of Cdc42 involve cell migration,polarization,and cytoskeletal regulation,but little is known about its role in the cardiac fibrosis.The aim of this study is to investigate the effect of Cdc42 in isoproterenol-induced myocardial fibrosis and its molecular mechanism.Experimental methods:1.Myocardial deletion of Cdc42 mice were obtained by crossing MLC-2v Cre recombinase mice with Cdc42 flox mice(F/F+ knockout mice,F/F-control mice).2.Cardial fibrosis was induced by Isoproterol(ISO,20 mg/day)subcutaneously for 7 days.The male mice,8-10 weeks old,were divided into four groups:(1)F/F-group with saline treatment;(2)F /F+ group with saline treatment;(3)F/F-group with ISO treatment;(4)F/F+ group with ISO treatment.3.Echocardiography and electrocardiogram were performed to record cardiac function.4.The hearts were isolated,fixed,paraffin-embedded,and cut into 4?m thick sections.The sections were stained for histological structures and collagen fibers,respectively.5.RT-q PCR and Western blots were performed to analyze the expressions of collagen type I and III collage,TGF-?,Erk,Gsk-3? etc.6.Primary mouse or rat cardiomyocytes and fibroblasts were isolated and cultured to explore their interaction under isoproterenol stimulation.Experimental results:1.Cdc42 is widely expressed in various tissues and moderately expressed in the hearts of adult mice.2.Cdc42 expressions were down-regulated in cardiac tissues in myocardial specific deletion of Cdc42 mice about 50% in m RNA and 40% in protein,respectively.3.Deletion of Cdc42 significantly attenuated cardiac alterations induced by ISO administration in left ventricular mass,left ventricular end-systolic wall thickness,the left ventricular end-systolic diameter and R wave in ECG in myocardial specific deletion of Cdc42 mice mice.The heart-body weight ratios were increased 30% and 20% in F/F-group and F/F+ group,respectively.4.There were much less cardiac atrophy,myocardial mesenchymal hyperplasia and interstitial collagen deposition in F/F+ group under ISO administration.5.Deletion or inactivation of Cdc42 significantly inhibited the expression of TGF-?,and the phosphorylation of GSK-3?,MAPKs including ERK and P38 in mouse hearts or isolated cardiac myocytes.Phosphorylation of PI3K/Akt upstream of MAPKs were not alterred by ISO stimulation.6.Deletion or inactivation of Cdc42 significantly significantly decreased the expression of CD11 b and IL-6 in mouse hearts under ISO stimulation.Conclusion:Myocardial specific deletion of Cdc42 improves cardiac function and attenuates the deposition of ECM and myocardial damage via MAPKs/GSK-3? signaling pathway in ISO-induced cardiac fibrosis mouse models.