| Objective: In this study, we investigated whether lentivirus-mediated genetransduction improves the cardiac function in rats with myocardial infarction and themechanisms involved.Methods: hNRG-1genomic sequence was amplified by PCR and cloned intopGC-FU-GFP lentivirus vector, hNRG-1eukaryotic expression vector wasconstructed. Rats were randomly assigned to4groups: Sham, intramyocardialinjection with PBS (PBS), lentivirus-GFP (GFP) and lentivirus-hNRG-1(NRG) intoacute myocardial infarction. Acute myocardial infarction (AMI) models were madeby ligation of the left anterior descending (LAD) coronary artery. Lentivirus carryinghNRG-1gene or GFP alone, or equivalent volume of PBS alone was injected at foursites into the infarcted border zone. The sham group received the same surgicalprocedure except the LAD ligation. Four weeks later, the survived rats (n=8in eachgroup) received echocardiographic studies to measure the heart function. Terminaldeoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) stainingwas performed to detect the apoptotic cells. The total blood vessels weredemonstrated by immunohistochemical staining using the von Willebrand-factor(vWF) antibody. Real-time PCR was performed to detect the mRNA expression levelof hNRG-1, VEGF, bcl-2and bax genes. Western blot was performed to measure theprotein expression levels of phospho-Akt, total Akt, Phospho-eNOS or total eNOS.Results:(1) PCR identification and DNA sequencing showed that the eukaryoticexpression vector for hNRG-1constructed successfully.(2) Four weeks after transduction, echocardiographic studies were performed.The cardiac function in AMI rats was remarkably deteriorated compared tosham-operated rats. Compared to the PBS and GFP group, the NRG group exhibited asignificantly improved myocardial function, which was verified by increased LVEFand FS, the difference was significant (P<0.05).(3) The expression of hNRG-1mRNA in NRG group was confirmed byqRT-PCR, however, other groups were not detected the hNRG-1mRNA expression. The difference was significant (P<0.05).(4) vWF immunohistochemical staining showed that the microvessel density inthe NRG group was higher than that of either the PBS or GFP group, the differencewas significant (P<0.05). The mRNA expression level of VEGF in the NRG, PBS andGFP group was increased compared to the sham group. However, we found that theNRG group presented the highest mRNA expression level of VEGF, the differencewas significant (P<0.05).(5) The number of TUNEL-positive nucleus in the infarct border zonesignificantly increased in the AMI groups compared to the sham group, whereas theinjection of lentivirus-hNRG-1alleviated this apoptotic change. Quantitativereal-time PCR showed that the expression level of the proapoptosis gene (bax) wasup-regulated while the anti-apoptosis gene (bcl-2) was down-regulated in the infarctborder area in all three AMI groups compared to the sham group. However, the NRGgroup exhibited the mildest changes in the expression level of these apoptotic genesamong three groups, the difference was significant (P<0.05). The phospho-Akt andphospho-eNOS protein expression level in the infarct border zone was significantlyup-regulated in the NRG group compared to the Sham, PBS and GFP groups. Whenthe phospho-Akt and phospho-eNOS level was normalized to their total protein level,the ratios were increased significantly in the NRG group compared to PBS, GFP andsham groups, the difference was significant (P<0.05).Conclution: In summary, we showed that the hNRG-1gene transduction canimprove the mRNA expression of hNRG-1in myocardial infarcted heart. NRG-1activates the VEGF expression and PI3K/Akt/eNOS pathway to promoteneovascularization and angiogenesis. By manipulating the bax/bcl-2signaling,NRG-1protects cells in the infarcted border zone from apoptosis. All of thesechanges improve the ventricular function after myocardial infarction in rats. |
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