Effects of mouse recombinant bone morphogenetic protein-7 transfection on cell apoptosis, NF-kappaB, and downstream genes in cultured primary cardiomyocytes after simulated ischemia and reperfusion injury.
OBJECTIVE
To evaluate the acting mechanism of bone morphogenetic protein-7 in anti-ischemic protective effect, we investigate the effects of BMP-7 transfection on cell apoptosis, NF-kappaB activity, and downstream genes in neonatal rat cardiomyocytes during simulated ischemia-reperfusion.


METHODS
In vitro cultured neonatal rat cardiomyocytes were divided into four groups: normal control group (Group C), simulated ischemia-reperfusion group (Group IR: cultured cardiomyocytes were subjected to 2 hours hypoxia followed by 4 hours reoxygenation), transfected group (Group BT: after transfection with pcDNA3.1-BMP-7 plasmid, cardiomyocytes were subjected to 2 hours hypoxia/4 hours reoxygenation), and empty vector control group (Group ET: same as group BT except that cells were transfected with empty pcDNA3.1 vector). Malondialdehyde content, superoxide dismutase activity, and Ca(2+) concentration were measured. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining and fluorescence activated cell sorting assay were applied to determine the apoptotic rate of cardiomyocytes and immunocytochemistry and Western blot were used to detect nuclear expression of NF-kappaB in cardiomyocytes. Reverse transcription-polymerase chain reaction was used to detect mRNA expression of monocyte chemoattractant protein-1 and intercellular cell adhesion molecule-1.


RESULTS
Compared with Group IR, malondialdehyde content of Group BT significantly decreased, whereas superoxide dismutase activity significantly elevated. In addition, intracellular Ca(2+) concentration of Group BT significantly reduced and apoptotic cells were significantly less. Decreased optical density ratio of NF-kappaB in the nucleus and reduced monocyte chemoattractant protein-1 and intercellular cell adhesion molecule-1 mRNA expression were also detected.


CONCLUSIONS
These results suggest that rat recombinant BMP-7 transfection provides sustained support for the repair of myocardium from ischemia injury through reducing cell apoptosis, inhibiting lipid peroxidation, antagonizing intracellular Ca(2+) overload, and preventing the activation of downstream signaling factors in signal transduction pathway mediated by NF-kappaB.
Xu
Ji-Hong
JH
Department of Medical Genetics, College of Basic Medical Sciences, China Medical University, Shenyang, China.
Zhao
Yan-Yan
YY
Wang
Jun-Ke
JK
Yuan
Zhi-Gho
ZG
Zhang
Tie-Zheng
TZ
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000235:genetics; D055419:Bone Morphogenetic Protein 7 / Q000235:genetics*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D015227:Lipid Peroxidation; D008297:Male; D051379:Mice; D015428:Myocardial Reperfusion Injury / Q000503:physiopathology / Q000628:therapy*; D032383:Myocytes, Cardiac / Q000473:pathology*; D016328:NF-kappa B / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D055419:Bone Morphogenetic Protein 7; D016328:NF-kappa B; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002118:Calcium