Effects Of Cdc42 On Synthesis And Secretion Of TGF-β By Cardiomyocytes And Its Molecular Mechanism

Objective:Myocardial fibrosis plays an important role in the process of myocardial hypertrophy and myocardial infarction.Cardiomyocyte hypertrophy,apoptosis,inflammation and cardiac fibroblast activation are closely related to myocardial fibrosis.Cardiac fibroblasts are the key cellular components of ventricular fibrosis.Differentiation of cardiac fibroblasts into myofibroblasts results in massive disordered extracellular matrix deposition,myocardial stiffness and impaired cardiac function.Cdc42 is a member of Rho GTPase family and plays an important role in physiological and pathological stimulation.Cdc42 is activated through the exchange of GDP for GTP and binds to PAK1 to induce PAK1 activation and regulate cell growth,shape,motility,survival and death via multiple downstream signaling pathways.Transforming growth factor-β(TGF-β)family is a multifunctional cytokine that is essential for survival.They play important roles in growth and development,inflammatory repair,and host immunity.The autocrine and paracrine effects of TGF-βcan be altered by the extracellular matrix,neighboring cells,and other cytokines.Our preliminary data showed that cardiac specific deletion of Cdc42attenuated cardiac fibrosis induced by isoprotonerol in mce.The purpose of this study was to explore the effect of Cdc42 on the synthesis and secretion of TGF-βin cardiomyocytes and its role and molecular mechanism in cardiac fibrosis.Experimental methods:1.Construction of cardiomyocyte-specific knockout mice of Cdc42.Cardiomyocyte-specific knockout Cdc42(Cre)mice(F/F+)and control group(Cre-free)mice(F/F-)were obtained by mating Cdc42 flox/flox mice with MLC-Cre tool mice.2.Mouse cardiac fibrosis model was established by subcutaneous injection of isoproterenol(ISO):F/F~+and F/F~-male mice aged 8-12 weeks were selected and divided into experimental group and control group.The experimental group was subcutaneously injected with ISO(20mg/kg/day)for 7 days to induce cardiac fibrosis in mice,while the control group was injected with the same amount of normal saline once a day at a fixed point.The protein and RNA of the heart tissue of the model mice were extracted,and the protein levels and mRNA expressions of genes related to cardiac fibrosis were detected to clarify the effect of cardiac Cdc42deletion on ISO-mediated cardiac fibrosis.3.Primary cardiomyocytes were extracted to detect fibrosis-related signaling pathways.After identifying the genotype of the neonatal mouse(0-3 days),the primary cardiomyocytes of the neonatal mouse were extracted,then stimulation with ISO(10μM)24 hours,cellular proteins were extracted to detect the expression of related proteins.4.Rat cardiomyocytes H9C2 were stimulated with different concentration gradients of ISO,and their effects on cell activity and cytoskeleton were detected to find the optimal concentration.5.At the cellular level,the synthesis and secretion of TGF-βwere detected in cardiomyocytes after stimulation of ISO.Rat cardiomyocytes H9C2 were pretreated with Cdc42-specific inhibitor ML141(10μM)for 4h,and then stimulated with ISO(10μM)for different durations.The Cells and supernatants were collected,and the contents of TGF-βwere detected by Western Blots.6.Mouse primary cardiomyocytes and primary fibroblasts were extracted and their interation were performed by treating fibroblasts with the supernatant secretions from ISO-treated cardiomytes to explore the effect of Cdc42 in activation of fibroblasts.Results:1.The activity of rat cardiomyocytes were not significantly altered by ISO or ML141 at 10μM,respectively.However,the sizes of rat cardiomyocytes were increased significantly after treating with 10μM ISO.2.The expressions of Collagen Ⅰ and Collagen Ⅲ and the phosphorylation of Smad2 and Smad3 were significantly increased in fibroblasts after treating with the secretions from ISO-stimulated cardiomyocytes and these activating forms of fibroblasts were significantly attenuated with the secretions from Cdc42 deleted cardiomyocytes.3.The transcription of TGF-βand intracellular and secrtory TGF-βs were significantly decreased in ML141 treated cardiac myocytes after ISO stimulaton.4.Deletion or inhibition of Cdc42 significantly inhibited the phosphorylation of STAT3,p38,and PI3K/Akt in cardiomyocytes or mouse heart tissue under treatment of ISO.Conclusion:Deletion or inhibition of Cdc42 significantly reduced the intracellular synthesis and extracellular secretion of TGF-βin cardiomyocyts and mouse hearts after ISO stimulation,and inhibited the differentiation of fibroblasts into myofibroblasts,and significantly alleviated cardiac fibrosis.Cardiac specific knockout of Cdc42 may alleviate cardiac fibrosis by inhibiting the transformation of fibroblasts to myofibroblasts via the classical Smads and non-classical Non-Smads signal pathway of TGF-β.