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Nitric oxide inhibits ischemia/reperfusion-induced myocardial apoptosis by modulating cyclin A-associated kinase
Yasuhiro Maejima1, Susumu Adachi, Hiroshi Ito
1Department of Cardiovascular Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan.
Objective:
Ischemia/reperfusion in the heart causes myocardial apoptosis and increase nitric oxide (NO) production. We have reported that myocardial apoptosis is related to activation of cell cycle regulatory proteins. However, the role of nitric oxide (NO) in ischemia/reperfusion-induced apoptosis is still unclear. This study was designated to elucidate novel apoptosis mechanisms induced by ischemia/reperfusion, especially the interaction between NO and cell cycle regulators.
Methods And Results:
Neonatal cardiomyocytes from 1- or 2-day-old Wistar rats were subjected to 1-h ischemia and then to reperfusion. The rate of cardiomyocyte apoptosis increased significantly after 24 h of reperfusion as evaluated by TUNEL analysis. NO increased 1.8-fold after 15 min of reperfusion in cardiomyocytes. After 36 h of reperfusion, the apoptosis rate was greatly increased by the NO synthetase inhibitor, Nitro-L-arginine methyl ester (L-NAME), and decreased by the NO donor of S-nitroso-N-acetylpenicillamine (SNAP). Immunoblot analysis showed that the protein levels of cyclin A accumulated in a time-dependent manner in response to ischemia/reperfusion, and L-NAME inhibited this response. Ischemia/reperfusion also increased the activity of cyclin A-associated kinase, and the apoptosis was inhibited by infection of dominant-negative cdk2 adenovirus. To clarify the involvement of p21(cip1/waf1) protein, which is the suppressor of cyclin A-associated kinase, we performed immunoblot analysis and examined its kinase activity. Treatment of cardiomyocytes with L-NAME suppressed the p21(cip1/waf1) protein level and increased the cyclin A-associated kinase activity. The addition of SNAP showed inverse results.
Conclusion:
Our data indicates that NO released from cardiomyocytes under condition of ischemia/reperfusion exerts an antiapoptotic effect by modulating cyclin A-associated kinase activity via p21(cip1/waf1) accumulation.
Insights
Nitric oxide (NO) protects heart cells from apoptosis during ischemia/reperfusion by regulating cell cycle proteins. This study reveals NO
Area of Science:
- Cardiovascular Biology
- Cell Death Mechanisms
- Molecular Cardiology
Background:
- Ischemia/reperfusion (I/R) injury induces myocardial apoptosis.
- Nitric oxide (NO) production increases during cardiac I/R.
- The role of NO in I/R-induced apoptosis and its interaction with cell cycle regulators remains unclear.
Purpose of the Study:
- To elucidate novel apoptosis mechanisms induced by cardiac I/R.
- To investigate the interaction between NO and cell cycle regulators in I/R-induced apoptosis.
Main Methods:
- Neonatal rat cardiomyocytes subjected to I/R.
- TUNEL analysis for apoptosis assessment.
- Western blot for protein levels (cyclin A, p21(cip1/waf1)).
- Assay of cyclin A-associated kinase activity.
- Pharmacological modulation using NO synthase inhibitor (L-NAME) and NO donor (SNAP).
Main Results:
- I/R significantly increased cardiomyocyte apoptosis and NO production.
- L-NAME exacerbated I/R-induced apoptosis, while SNAP reduced it.
- Cyclin A accumulation and associated kinase activity increased with I/R, inhibited by L-NAME.
- L-NAME decreased p21(cip1/waf1) levels and increased kinase activity; SNAP showed opposite effects.
Conclusions:
- NO exerts an antiapoptotic effect in cardiomyocytes during I/R.
- NO modulates cyclin A-associated kinase activity through p21(cip1/waf1) accumulation.
- This highlights a novel mechanism of NO in cardioprotection against I/R injury.