1.Increased Expression Of Integrin-linked Kinase Promotes Proliferation Of Cardiomyocytes 2.Metformin Inhibits Angiotensin ?-Induced Differentiation Of Cardiac Fibroblasts Into Myofibroblasts

Background and Obective-Loss of cardiomyocytes is a main pathology of many heart diseases such as myocardial infarction and heart failure.However,the mammalian heart can not regenerate effectively after cardiac injuries due to the restricted proliferative potential of cardiomyocytes.Hence,stimulating proliferation of endogenous cardiomyocytes may provide a potential approach to repair the injuried heart.Here we investigated whether integrin-linked kinase(ILK)could promote the proliferation of mammalian cardiomyocytes and the underlying mechanisms of the beneficial effects.Methods-The expression of ILK in rat cardiac tissue during myocardial development were determined by western blotting and real-time PCR.Primary cultured neonatal rat cardiomyocytes were isolated from 2-day-old SD rat and infected with adeno-ILK or adeno-GFP viruses 48 hours after isolation.The infection efficiency was assessed by confocal microscopy and western blotting 48h after infection.Cardiomyocyte number was evaluated by MTT assay and direct cell counting,the DNA synthesis,karyokinesis and cytokinesis,were detected by immunofluorescence staining,and the number of cardiac stem cells were also detected by flow cytometry.To further investigate the underlying mechanisms,we also detected the phosphorylation of PKB/Akt on serine 473 and GSK-3p on serine 9,as well as the expression of cyclin D1 using western blotting.Results-The expression levels of ILK were high in the fatal hearts and were markedly decreased after birth.Overexpression of ILK was associated with increased DNA synthesis,karyokinesis and cytokinesis,as well as an increase in cardiomyocyte number.FACS analysis of alpha sacromeric actinin positive cells revealed that infection with adeno-ILK dramatically increased the percentage of cells in S/G2/M phase compared with controls.Activation of PKB/Akt kinase was required for ILK-induced cardiomyocyte proliferation,whereas the ERK1/2 signaling pathway was not associated with ILK-induced cardiomyocyte proliferation.Moreover,ILK can activate PKB/AKT by direct phosphorylation at serine 473,inhibit GSK-3? activity by indirect phosphorylation at serine 9 and increase the protein level of cyclin D1.Additionally,overexpression of ILK did not affect the proliferation of c-kit+ cardiac stem cells in our culture condition.Conclusions-Our findings indicate that ILK promotes proliferation of mammalian cardiomyocytes through the PKB/Akt/GSK-3? signaling pathway in vitro.Thus,stimulating proliferation of endogenous cardiomyocytes with ILK may provide an innovative approach to induce myocardial repair.Background and Objective-Cardiac fibrosis is a critical aspect of cardiac remodeling following myocardial infarction,hypertension,and other cardiovascular diseases.Differentiation of cardiac fibroblasts into myofibroblasts,characterized by expression of ?-smooth muscle actin(?-SMA)and production of extracellular matrix(ECM)components such as collagen types ? and ?,is a critical event in cardiac fibrosis.Metformin,an antidiabetic agent which improves insulin sensitivity and decreases hepatic glucose output,exhibits a number of cardioprotective effects beyond its antihyperglycemic properties.However,whether metformin affects myofibroblast differentiation remains unknown.Here we investigated whether metformin could inhibit angiotensin ?(Ang ?)-induced myofibroblast differentiation and the underlying mechanisms of the beneficial effects.Methods-Adult rat cardiac fibroblasts were isolated from 8-to 10-week-old male Sprague-Dawley rats,and myofibroblast differentiation was induced by exposure to Ang ?(100nM).To evaluate myofibroblast differentiation,?-smooth muscle actin(?-SMA)expression was detected by western blotting and immunofluorescence staining,and the mRNA levels of collagen ? and collagen ? were assessed by real-time PCR.To determine whether the PKC-NADPH oxidase-ROS pathway is necessary for Ang ?-induced cardiac myofibroblast differentiation,we exposed cardiac fibroblasts to N-acetyl cysteine(NAC,a glutathione precursor and scavenger of H2O2),calphostin C(an inhibitor of PKC)or apocynin(an inhibitor of NADPH oxidase)and then examined the expression of a-SMA,collagen I and collagen III.To further investigate the effects of metformin on the PKC-NADPH oxidase-ROS pathway,the membrane translocation of PKC isoforms ?,?,? and ? were detected by western blotting,the intracellular ROS generation was measured with a DCFH-DA method and analyzed by flow cytometry and confocal microscopy,and the NADPH oxidase activity was measured with cell NADPH oxidase colorimetric assay kit.Results-Ang ? stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts,as indicated by increased expression of a-smooth muscle actin(a-SMA)and collagen types ? and ?.This effect of Ang ? was inhibited by pretreatment of cardiac fibroblasts with metformin in a concentration-dependent manner.Furthermore,pretreatment of cardiac fibroblasts with NAC,PKC inhibitor calphostin C or NADPH oxidase inhibitor apocynin markedly inhibited Ang?-induced ROS generation and myofibroblast differentiation.In addition,incubation with Ang ? induced a significant translocation of PKC s from the cytosol to the membrane,whereas the other isoforms did not change significantly.Metformin pretreatment decreased Ang ?-induced reactive oxygen species(ROS)generation in cardiac fibroblasts via inhibiting the activation of the PKC-NADPH oxidase pathway.Conclusions-Our findings indicate that metformin inhibits Ang ?-induced myofibroblast differentiation by blocking ROS generation via the inhibition of the PKC-NADPH oxidase pathway in adult rat cardiac fibroblasts.This may provide new mechanistic insights regarding the cardioprotective effects of metformin and provide an efficient therapeutic strategy to attenuate or prevent cardiac fibrosis.