Study On The Cardioprotective Effects Of ENOS Gene Transfer After Myocardial Infarction

Part one: The effect of NO on the cultured neonatal ratcardiac myocytes (CMs) treated with hypoxia and MAPKsand TGF-β/Smad signal pathway.Objective: Observe the effect of NO on the cultured neonatal rat cardiacmyocytes (CMs) treated with hypoxia and MAPKs and TGF-β/Smadsignal pathway. To provide evidence for further study on thecardioprotective effects of eNOS gene transfer after MyocardialInfarction.Methods: Cardiac myocytes from neonatal rat were cultured. Five dayslater, cultured neonatal rat CMs was divided into two guoups, namelynormal control and hypoxia group. The hypoxia group were divided intotwo groups: in order to observe the effect of NO on MAPKs andTGF-/Smad signal pathway, the first group cultured in an incubator 95%N2 and 5%CO2 for 15 mins, 30 mins, 60 mins, 12 hours, SNP(6umol/L)were administrated thirty mins before hypoxia, in order to observe theeffect of NO on cardiac myocytes apoptosis, the second group undergohypoxia for 12 hours and 24 hours reoxygen. CMs apoptosis detected byflow cytometer (FCM). MAPKs and TGF-βwere detected by WesternBlot.Result: After treated with SNP thirty mins before hypoxia, the CMsapoptosis were markly decreased detected by FCM. The expression ofP38 ,JNK and TGF-βwere significantly inhibited compare to thehypoxia control group, but the expression of ERK have no difference inall groups. Conclusion: NO can protect CMs from hypoxia injury. NO can decreaseCMs apoptosis induced by hypoxia, the effect may have relation withinhibition of the expression of P38,JNK and TGF-β. Objective: Endothelial nitric oxide synthase (eNOS) and nitric oxide(NO) have been implicated in protection against myocardial ischemiainjury. However, the angiogenic effect of eNOS in infarcted myocardiumand the role of TGF-β1 signaling in eNOS/NO mediated cardiacremodeling have not yet been elucidated. We observed thecardioprotective effects of eNOS gene transfer after MyocardialInfarction.Methods: Wistar rats (male) were applied in our study.The study wasdivided into short-term (24-hour infarction) for apoptosis and signalinginvestigations as well as long-term (7-day infarction) for angiogenesisand ventricular remodeling studies. Human eNOS gene in an adenovirusvector was delivered locally into rat heart 4 days prior to induction ofmyocardial infarction (MI) by left anterior descending coronary arteryligation. Cardiomyocyte apoptosis was detected by TUNEL andneovascularization was identified immunohistochemically. Expression ofMAPKs and TGF-βwere detected by Western Blot.Results: eNOS gene transfer significantly reduced myocardial infarct sizeand improved cardiac contractility as well as left ventricle (LV) diastolicfunction at 24 hours after MI. In addition, eNOS significantly reducedMI-induced cardiomyocyte apoptosis. Activation of JNK,P38,TGF-β1and Smad-2 after MI were also dramatically reduced by eNOS. Moreover,the deterioration of both systolic and diastolic function in conjunctionwith thin LV remodeling at 7 days after MI were prevented by eNOS.Capillary density as identified byα-smooth muscle actin was significantly increased in the infarcted myocardium after eNOS transfercompared with MI control. All the cardioprotective effects of eNOS wereblocked by N(ω)-nitro-L-arginine methyl ester administration, indicatinga NO-mediated event.Conclusion: These results demonstrate that the eNOS/NO systemprovides cardiac protection after MI injury through inhibition of cardiacapoptosis, stimulation of neovascularization and suppression ofTGF-β/Smad-2,JNK,P38 signaling.