Protective Effects Of BMP-7 On Myocardial Ischemia-reperfusion Injury And Effect On Nuclear Transfer Of NF-κB P65

IntroductionBone morphogenetic protein-7 (BMP-7), also known as osteogenic proteins (OP-1), is an important member in bone morphogenetic proteins (BMPs) subfamily belonging to the transforming growth factor (TGF-β) superfamily. It is a comparatively powerful bioactivity factor. It mainly relates to maintenance tissue function, regenerate and restore. Recent researches have shown that many repair mechanisms are involved the anti-ischemia effect, which provide the experimental foundation for clinical practice. BMP-7 may play a role as cardioprotective factor in murine myocardial ischaemia reperfusion. This suggests that some mechanism exists within the cardiomyocytes that regulates BMP-7 expression. The existence of such a mechanism and its nature remains to be determined. In our experiment, by gene transfection technique recombinated rat bone morphogenetic protein-7 (rrBMP-7) was transfected into cardiomyocytes. Our aim was to make clear the effect of BMP-7 gene on ischemia-reperfusion damage of cardiomyocytes and the possible mechanisms that the effect is reacted via nuclear transfer of NF-κB p65.Material and MethodsThree-day-old neonatal Wistar rats were purchased from Animal Center of China Medical University.Cell culture: Neonatal rat hearts were collected from 3 d of age rats. The ventricles were dissected, placed in D-Hank's balanced salt solution to wash out remainting blood, and cut in small pieces. The cells were dispersed in trypase phosphate buffer solution (0.125%) at 37℃for 10 min, then centrifuged for 10 min. Subsequently, serial 20-min digestions were performed. After each digestion period the cells were collected by centrifugation and suspended in DMEM culture medium, supplemented with 15% fetal bovine serum. The cardiomyocytes were plated on 35 mm plastic culture dishes at a density of approximately 1×10⁵ cells/ml. The cells were kept at 37℃and 5%CO₂ under aerobic conditions in a carbon dioxide incubator. The supplemented DMEM culture medium was changed each day. Three groups were adopted: control group (C group), simulated ischemia/ reperfusion group (IR group) and BMP-7 gene transfecting group (BT group).Experiment one: The BMP-7 gene was cloned into the eucaryotic expression vector pcDNA3.1. The eukaryotic expression vector pcDNA containing recombinated rat BMP-7 gene was extracted and identified by sequence analysis and agarose gel electrophoresis after being cut with restricted endonuclease. Plasmid containing pcDNA3.1-rrBMP-7 was propagated in Escherichia coli competent cell JM109. E coli were grown in standard Luria Bertani medium at pH 7.0 overnight. Cells were harvested by centrifugation and the plasmid DNA was extracted and purified without endotoxin using a Qiagen Plasmid Maxi Kit. The plasmid was identified by 1% agarose gel electrophoresis after being cut with restricted endonuclease HindIII/BamH I . At the same time, myocardial cells were isolated and cultured in vitro. The plasmid carrying the BMP-7 gene was transfected into cardiomyocytes with FuGENE 6 reagent and mRNA and protein expression of rrBMP-7 were detected by RT-PCR and immunocytochemical methods and Western blot methods 24, 48 or 72 hours after transfection.Experiment two: Cells were incubated overnight in medium without serum to growth synchronization. Three groups were adopted: control group, simulated ischemia/ reperfusion group and gene transfecting group. The FuGENE 6-DNA mixture was carefully added to the cells of gene transfecting group. The cells were incubated for 72 h at 37℃, 5% CO₂. The medium of cardiomyocytes in the IR group and BT group was removed and changed to DMEM medium without glucose which had been saturated by a mixture of 95% N₂ and 5% CO₂ for 30 min before starting three experiments. After 2h, the cells were added normal medium, then the cells were returned to a standard incubator for a further 4h of reoxygenation to simulate reperfusion. Trypan blue exclusion was used to detect cell viability and the activity of LDH, CPK was assayed to evaluate cell injury. The activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) were measured by colorimetric assay to evaluate cell antioxidant ability. Apoptotic rate of cardiomyocytes were determined by flow cytometry (FCM) and TUNEL. Fluo-3 labeling method were used to observe the variation of intraceliular calcim. Immunocytochemistry method and Western blot were applied to observe the expression of NF-κB protein expression in cultured cardiacmyocytes.Experiment Three: Forty Wistar rat were randomly divided into 3 groups: ischemia-reperfusion group (group IR), rhBMP-7 group (group B) and Sham group. Group B were administered with rhBMP-7 (250μg/ kg) via the femoral vein before reperfusion. The rats of group B and group IR were made by reperfusion injury about 4 hours or 24hours after myocardial ischemia about 30 minutes. LDH, CPK, MDA and SOD were detected. Infarct size was assessed by TTC method. Histological findings were observed by electron microscope and HE. Apoptotic rate of cardiomyocytes was determined by TUNEL. Immunohistochemistry method and Western blot were applied to observe the expression of NF-κB protein expression in cell nucleus.Data were presented as the mean±standard error of the mean (SEM). Statistical significance of difference between means was evaluated by t-test and p values less than 0.05 were considered statistical significance.ResultsExperiment one: The total RNA was extracted from rat renal. cDNA library was established using RT-PCR method. PCR product was purified by gel extraction mini kit. PCR and digesting demonstrated that the eucaryotic expression plasmid pcDNA-BMP-7 was obtained. Sequence analysis of BMP gene indicated BMP-7 DNA was 1.3kb. BMP gene of pMD18-T Simple-BMP-7 were consistent with that of Genebank. RT-PCR showed that the BMP-7 mRNA was expressed in myocardial cells transfected with pcDNA3.1-BMP-7. About 18kD was achieved by SDS-PAGE and Western blot. Immunocytochemical methods showed that the expression of BMP-7 protein were positive in cardiomyocytes.Experiment two: The results showed that after 120 min of simulated ischemia followed by 240 min of reperfusion, cell pulsation decreased and trypan blue uptake and LDH, CPK increased. As compared with the normal control group, the decrease on SOD activity and increase on the formation of MDA were the most serious in Group IR, but was diminished greatly in BMP-7 gene transfecting group. FCM and TUNEL showed the increase of apoptosis rate in Group IR, while in BMP-7 gene transfecting group, apoptosis decreased. Treatment with BMP-7 gene transfecting led to decreasing of the [Ca²⁺]i content in cardiac myocytes, which prevented [Ca²⁺]i overload induced by ischemia-reperfusion (P<0.01 vs. cont to IR group ). NF-κB p65 protein- nuclear localization positive cell population and integral optical density (OD) radio increased after the reperfusion, while in BMP-7 gene transfecting group NF-κB p65 protein nuclear localization positive cell population decreased.Experiment Three: The result showed that after 30min of simulated ischemia followed by 4 and 24 hours of reperfusion, LDH, CPK increased. As compared with Sham group, the decrease on SOD activity and increase on the formation of MDA were the most serious in Group IR, but was diminished greatly in rhBMP-7 group. TTC stain showed that the infarct size in the IR24h group was the most serious. FCM and TUNEL showed the increase of apoptosis rate in Group IR, while in rhBMP-7 group, apoptosis decreased. Histological finding showed that rhBMP-7 relieved the level of injury. TTC results showed that rhBMP-7 minimize infarct size. NF-κB p65 protein nuclear localization positive cell population and integral OD radio increased 4h and 24h after the reperfusion, while rhBMP-7 inhibit NF-κB p65 protein nuclear transfer, compared with that of the same time.Conclusions1. Construction of an eucaryotic expression plasmid carrying BMP-7 gene and expression in myocardial cells provide a sound basis for gene therapy of anti-ischemia using the BMP-7 gene.2. Transfecting BMP-7 gene can significantly inhibit cardiomyocyte apoptosis and protect cardiaomyocytes against ischemia-reperfusion damage. The mechanism may partly involved suppression of [Ca²⁺]i overload, improve of oxidative stress in cardiomyocytes after ischemia-reperfusion.3. The protective effects of rhBMP-7 involved consequently downregulating nuclear translocation of NF-κB p65.