Investigation On The Injurious Effects Of C-reactive Protein And Biological Intervention In Neonatal Rat Cardiomyocyte

Background:C-reactive protein(CRP)is an acute-phase reactant,which belongs to the highly conserved pentraxin family of plasma proteins and serves as a pattern-recognition molecule in the innate immune system. Myocardial necrosis following acute myocardial infarction is a potent acute-phase stimulus;there is a major CRP response.Furthermore,there is considerable clinical considence supporting a strong association between the peak CRP values in circulating blood after the onset and the outcome of myocardial infarction.What remains less clear is whether CRP acts simply as a marker of vascular disease burden and activity or indeed participates in the development,progression,and complications of cardiovascular disease.In past few years,an increasing number of in vitro studies have indicated that CRP had exerting adverse and ultimately harmful effects on vascular smooth muscle cells(VSMCs),aortic endothelial cells,and therefore acts as a potential initiator and mediator of atherosclerosis.CRP was also found in the area of acute myocardial infarction,and showed positive correlation between the level of CRP and the infarct size.Furthermore,recently,therapeutic inhibition of CRP has been showed a promising new approach to cardio-protection in acute myocardial infarction in rat models.These results indicate that CRP may play an important role in the progression of cardiac dysfunction.Previous studies have confirmed that cytochrome C releasing from mitochondria then activates Caspase signal pathway may played important role in the cardiac apoptosis,but whether the key proteins mentioned above take part in the progress of CRP induced cardiac damage is little known.So,it is important to illustrate the molecular mechanism involved in the effects of CRP on the myocardial infarction and the cardiac-disorder,and it is a perspective work to explore a new atoxic drug with higher efficiency. Objective:To investigate the direct effects of CRP on the cardiac myocyte and the molecular mechanism involved,and to explore a new efficient biological therapy against CRP,in this study,we used the hypoxia stimulated primary neonatal rat cardiac myocytes in vitro to simulate the cardiac myocyte in myocardial infarction in vivo.Within this system,we detected the effects of CRP on cardiac myocyte and look for the key point protein in the signal transduction pathway for intervention as the therapeutic target;the sequence of variable region was cloned from mouse hybridoma cells stably secreting anti-CRP antibody,the VH and VL were conjuncted with linker.The protein of scFv was obtained in the E.Coli,and the effects of scFv on CRP induced cardiac damage were observed in vitro.Materials and methods:1.Human CRP was isolated from malignant ascites fluid using Immobilized p-Aminophenyl Phosphoryl Choline Gel.The malignant ascites fluid was obtained from cancer patients and the investigation conforms to the principles outlined in the Declaration of Helsinki for use of human tissue or subjects.Purified human CRP was assayed by SDS-PAGE,mass-spectrum and western-blot analysis.2.The full length of human C-reactive protein gene was cloned from TNF-alpha induced hepatoma cell line(HepG2)by RT-PCR and subcloned into prokaryotic and eukaryotic expression vector plasmid, named as pET-28a-CRP and pEGFP-N1-CRP,respectively.His-CRP was induced to express and purified by Ni²⁺affinity chromatography. After transfection pEGFP-N1-CRP into HEK293T cells,the expression of CRP was observed under fluorescence microscope,and confirmed by western blotting.3.The full length of rat Bcl-2 gene was cloned into pEGFP-N1 plasmid and named as pEGFP-N1-Bcl-2(Stop codon TGC was contained in the cDNA of Bcl-2).After transfected into cultured neonatal cardiac myocytes using M-PEI,the expression of Bcl-2 was observed under fluorescence microscope,and confirmed by western blotting.4.The effects of CRP on cultured neonatal cardiac myocytes in virto were observed.(1)Myocytes were divided into three groups:A.None transfected myocytes;B.pEGFP-N1 transfected myocytes;C.pEGFP-N1-Bcl-2 transfected myocytes.48 hours after transfection,myocytes were treated with 100μg/ml CRP,hypoxia and cotreatment with them respectively. Control myocytes were incubated in DMEM containing 10%FBS under normoxia.A hypoxic condition was created by incubating the cells with serum-free DMEM in an airtight Plexiglas chamber with an atmosphere of 5%CO₂/95%N₂ at 37℃for 8h in experiment.(2)Determination of myocyte apoptosis was performed using cell apoptosis assay Kit(Hoechst 33258).(3)The localization of Cyt c was detected with immunofluorescent under confocal microscope and the quantity of translocation of cytochrome c from mitochondria to cytolist was analyzed by western blot.(4)The expressions of Bax and Bcl-2 and the ratio of Bax/Bcl-2 were analyzed by RT-PCR and western blot.Caspase-9 and Caspase-3 activities were determined with a Caspase assay kit.5.CRP-McAb variable region(VH and VL)was cloned from mouse hybridoma cells stably secreting anti-CRP antibody,the VH and VL were conjuncted with Linker.The prokaryotic and eukaryotic expression vector were constructed,His-scFv was induced to expression existed majorly in the inclusion body,after denaturation and renaturation,the protein was purified by Ni²⁺affinity chromatography and detected by SDS-PAGE and western blot.The plasmid pEGFP-N1-scFv was transfected into HEK293T cells using M-PEI,the expression of scFv was observed under fluorescence microscope and confirmed by western blotting.Cellular immunostaining was used to detect the ability of recognization between expressed scFv and human CRP.6.The anti-apoptosis effect of CRP-scFv was analyzed with in situ apoptosis detection kit—TUNEL.7.Statistics:The data mentioned above at least derived from three independent experiments.All data are presented as mean±SD.The independent samples T test,one way ANOVA and chi-square test were used to analyze differences and statistical tests were performed with the use of SPSS 13.0 statistical software.Statistical significance was accepted at P＜0.05.Results:1.CRP purified from malignant ascites fluid using Immobilized p-Aminophenyl Phosphoryl Choline Gel was in the monomeric form (24KD)with no detection of other proteins by silver staining on SDS-PAGE(purity up to 95%),western-blot or mass-spectrum.2.The full length cDNA of human CRP was successfully cloned,DNA sequencing and BLAST algorithm revealed that the cDNA was exactly identical to the reported sequence in the Genebank.The CRP gene was then successfully subcloned into pET-28a and pEGFP-N1. After induction with 1mM IPTG at 37℃for 4h,E.coli BL21(DE3) transformed with pET28a-CRP produced a fusion protein of approximately 30 kDa,which matched well with the theoretical molecular weight of His-CRP.The expressed recombinant CRP was purified by Ni²⁺affinity chromatography and detected by anti-CRP antibody.The plasmid pEGFP-N1-Bcl-2 was successfully transfected into HEK293T cells using M-PEI which resulted in significant increased CRP protein expression.3.The full length cDNA of rat Bcl-2 was successfully cloned,DNA sequencing and BLAST algorithm revealed that the cDNA was exactly identical to the reported sequence in the Genebank.The Bcl-2 gene was then successfully subcloned into pEGFP-N1.Cultured neonatal cardiac myocytes transfected using M-PEI which resulted in 70%to 80%transfection efficiency and significant increase in Bcl-2 protein expression.4.CRP augmented hypoxia-induced cell apoptosis.The percentage of apoptotic myocytes increased significantly after 8 hours hypoxia,as compared with control.Cotreatment of hypoxia with CRP(100μg/mL) significantly increased the percentage of apoptotic cells.However, CRP did not induce myocytes apoptosis significantly under normoxia.5.CRP induced more cytochrome c release than hypoxia alone.Control cardiac myocyte demonstrated organized speckled patterns of cytochrome c that colocalized with mitochondria.In contrast,after hypoxia for 8 hours,there was little cytochrome c in cytolist but after cotreatment with hypoxia and 100μg/mL CRP,cytochrome c staining diffused throughout the cells,no longer colocalized to the mitochondria.The phenomenon was further conformed by western blot which detected the quantity of translocation of cytochrome c from mitochondria to cytolist.6.CRP increased the ratio of Bax/Bcl-2 in the hypoxia-induced cardiac myocyte.7.CRP further increased the hypoxia-induced activation of Caspase-9 and Caspase-3 significantly.However CRP showed no action on the activity Caspase-9 and Caspase-3 during normoxia.8.Bcl-2 over-expression in cultured neonatal cardiomyocytes resulted in significant inhibition of cotreatment of hypoxia with CRP-induced apoptosis,cytochrome c release,Bax/Bcl-2 ratio increase and Caspases activation.9.The cDNA of CRP-McAb variable region(VH and VL)was successfully cloned from mouse hybridoma cells stably secreting anti-CRP antibody.After DNA sequencing and read frame definition according to the V-base,the VH and VL were conjuncted with Linker using overlapping extension PCR.The scFv gene was successfully subcloned into pET-28a and pEGFP-N1.After induction with 1mM IPTG,a fusion protein of approximately 30 kDa was produced,which matched well with the theoretical molecular weight of His-scFv.The expressed recombinant scFv was purified by Ni²⁺affinity chromatography and detected by anti-His antibody.The plasmid pEGFP-N1-scFv was successfully transfected into HEK293T cells using M-PEI which resulted in significant increasing of scFv expression.Cellular immunostaining showed that the expressed scFv could be recognized by human CRP.10.Our preliminary data of myocyte apoptosis analysis indicated that anti-CRP antibody(4E8C6H6F12E7D11)and recombinant scFv protein could inhibit cardiac myocyte apoptosis induced by hypoxia cotreated with CRP.Discussion and conclusion:1.A number of recent studies have used commercial CRP preparations that remain poorly characterized and indeed contain known quantities of biologically active contaminants such as sodium azide.So,in this study,we used affinity chromatography(Immobilized p-Aminophenyl Phosphoryl Choline Gel)to purify human C-reactive protein from malignant ascites fluid of the patients.Our purified CRP is of high purity,without Na₃N contamination,and is suitable for the in vitro intervention.2.The data of this study suggest a potential mechanism that CRP could enhance apoptosis in hypoxia-stimulated neonatal rat cardiac myocytes through the mitochondrion-dependent pathway but CRP alone has no effects on neonatal rat cardiac myocytes under normoxia, which is consist with the results of Pepys MB's group that injection of human CRP to rats undergoing acute myocardial infarction increased infarct size and aggravated cardiac dysfunction.3.The present study demonstrates for the first time CRP can regulate the Bax and Bcl-2 proteins in mitochondria and decrease the ratio of Bax/Bcl-2,augment mitochondrial death pathway through provoking the mitochondrial permeability transition,therefore cytochrome c released from the mitochondria to the cytosol,activate Caspase-9 and Caspase-3 in hypoxic cardiac myocytes,a fact which indicate that therapeutic inhibition of CRP may have significant implications in the development of future therapies to combat the effects of myocardial infarction.But,further studies are needed to clarify the upstream signal pathway of the pro-apoptotic action of CRP and the receptor of CRP in the cell membrane.4.In this study,we cloned cDNA of CRP-McAb variable region(VH and VL)from mouse hybridoma cells stably secreting anti-CRP antibody and obtained scFv antibody against CRP after expression and purification.Our preliminary study suggested that recombinant scFv protein could inhibit inhibit cardiac myocyte apoptosis induced by hypoxia cotreated with CRP.These results provide exciting future for using genetically engineered antibody to biological therapy against CRP.