Effects of Exogenous Nitric Oxide on the Ischemic Damage of H9c2 Cardiac Myoblast Cells

Jung, Sung Koo; Jang, Hyun Yong; Kim, Myung Chun; Ko, Young Gwan; Chung, Joo Ho; Bae, Young Mee; Park, Weon Seo; Kim, Dae Joong; Yoo, Young Min; Kim, Sung Soo; Yim, Sung Vin

Abstract

BACKGROUND: Nitric oxide(NO) is known to have protective effects on an ischemic heart and to exert triggering effects on ischemic preconditioning. However, the effects of NO during the ischemic period have not been investigated. To investigate the role of exogenous nitric oxide in a model of ischemic heart cell death, we studied the effects of ischemic preconditioning and ischemia in a normal and an ischemic buffer.
METHODS
Rat cardiac myoblast cells(H9c2) were cultured in a normal and an ischemic buffered medium. For the ischemic culture of heart cells, the cells were cultured in a dessicator with GasPak for 5 hrs. In ischemic preconditioning, the cells were pretreated with ischemic buffer for 5 min and then perfused with normal medium for 30 min. For the measurement of the cytotoxicity, a MTT(3-4-5dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) assay was performed. A DAPI(4',6-diamidino-2-phenylindole dihydrochloride) staining procedure and a flow cytometry analysis were performed to confirm apoptotic cell death by ischemia.
RESULTS
Cell viability, as determined by using a MTT assay, showed that the preconditioned group treated with NO showed more cell death than with the not-preconditioned groups in both normal and ischemic buffers. But, In normal medium and not-preconditioned groups, NO showed protective effect according to the concentrations(100, 1000 microM) . No treatment with NO produced the different results. In normal medium, the protective effect of ischemic preconditioning was demonstrated, but no protective effect of ischemic preconditioning could be seen in the case of the ischemic buffer. The DAPI staining and flow cytometry analysis of heart cells showed characteristic apoptotic features.
CONCLUSION
NO added in the ischemic phase had deterious effects on heart cells. Ischemic preconditioning was more harmful than ischemia alone. The toxicity of the cells was characteristic apoptosis.