Effects Of Nerve Growth Factor (NGF) RNA Interference On Post-infarcted Hearts

BackgroundDespite of developing of medical science and improving of the people healthy concept, myocardial infarction (MI) is still one of the main reasons of death and disability. MI has become a serious public health issue in China because of aging population and unhealthy life style. The prevalence of arrhythmia and heart failure complicated infarction remains unacceptably high. Malignant ventricular arrhythmia is the main cause of sudden cardiac death. Ventricular arrhythmias and sudden cardiac death is co-related with increased sympathetic nerve density. Series of studies found that directly or indirectly increasing the expression of nerve growth factor could increase the density of sympathetic nerve at infarcted border and no-infarcted left ventricle, and then increase the incidence or susceptibility of ventricular arrhythmias in post-infarcted hearts. MI results in upregulated NGF expression, and induces sympathetic nerve regeneration, sprouting and then remodeling. However, NGF has protetive function due to its angiogenic and anti-apoptosis effects in post-infarcted hearts. Upregulation of NGF benefits infarcted repair. NGF exerts both harmful (proarrhythmia) and protective effects (myocardial and vascular repair). Whether modulating NGF via molecular approach could affect the prognosis was still unknown and should be focused.Part1Construction and identification of recombinant lentivirus expressing short hairpin RNA (shRNA) against nerve growth factor gene ObjectiveRecombinant lentivirus vector expressing short hairpin RNA (shRNA) was designed and constructed against NGF gene targeted three different sites. Then lentiviral vector was dentified, purified, packaged, screened and titer determinated step by step. Moreover, the selected small interfering RNA (siRNA) was used to evaluate the effect of silencing.MethodThree pairs for NGF gene-specific siRNA fragments, were designed and synthesized. After annealing and double enzyme digested with Hpa I and Xho1,they were connected to the vector and amplified. Then plasmids were extracted, double enzyme digested and sequenced. Vectors which had the correct sequence and the lentiviral packaging plasmid were co-transfected293T cells. Cell supernatant including lentiviral particles was collected, purified and concentrated. Lentivirus suspensions including three candidate shRNA were randomly divided into three groups (interfererce group1, interference group2and interference group3) and then were transfected primary rat glial cells. In the meantime, the control group was setted. The morphology,fluorescence and NGF mRNA of cell were observed in each group after transfection to select the most valid siRNA. To test silencing effects of the selected siRNA, the lentivirus carrying shRNA was transfected into rat myocardial cell. After96h transfection, Western blot and RT-PCR were used to evaluate the expression of NGF protein and mRNA respectively.ResultsResults of double enzyme digestion and sequencing showed that parts of each recombinant lentiviral vector of shRNA have entirely consistent with our design targeted synthesis chain in three groups. RT-PCR showed that the largest decline in the levels of NGF mRNA expression. It also had a good silencing effect via transfected rat myocardial cells in the following vitro experiments and then largely packaged for late experiments.ConclusionThe study successfully synthesized three targeted NGF gene interference oligonucleotide sequences and constructed carrying shRNA-NGF gene expression lentiviral vector plasmid.High titer recombinant lentivirus was produced through293T cells packaged. Then ideal interference shRNA was determinated and successfully performed interference experiments in myocardial cells. Part2. Effects of NGF silencing on cardiac pathophysiology in post-infarcted rat heartsObjectiveTo explore local NGF silencing on cardiac sympathetic nerve, vessel, infarcted size and cardiac function in an infarcted model of rats.MethodsMI was modeled in the rat using left anterior descending coronary artery ligation. Rats were randomly grouped to receive intramyocardial injection of lentiviral solution containing NGF-shRNA (n=19, MI-SiNGF group), lentiviral solution containing empty vector (n=18,MI-GFP group) or0.9%NaCl solution (n=18, MI-control group), or to receive thoracotomy and pericardiotomy (n=17, sham-operated group). At the endpoint of the study time8weeks, ultrasound and hemodynamic detection were used to evaluate cardiac function. Moreover, density of capillary, arteriole and sympathetic nerve were detected and activity of nerve and vessel sprouting were assessed.ResultsAt1,2,4, and8wk after transduction, rats in the MI-control group had higher levels of NGF mRNA and protein than those in the sham-operated group. Rats in the MI-GFP group showed similar levels as well as the MI-control group, and rats in the MI-SiNGF group had lower levels compared to the MI-GFP group, indicating that MI model was successfully established and NGF silencing effectively inhibited the expression of NGF. At8wk, echocardiographic and hemodynamic studies revealed a more severe cardiac dysfunction in the MI-siNGF group compared to the MI-GFP group. Moreover, rats in the MI-siNGF group had lower mRNA and protein expression levels of tyrosine hydroxylase (TH) and growth-associated protein43-positive nerve fibers (GAP-43) at both the infarcted border and within the non-infarcted left ventricles (LV). NGF silencing also reduced the vascular endothelial growth factor (VEGF) expression and decreased the arteriolar and capillary densities at the infarcted border compared to the MI-GFP group. Histological analysis indicated a large infarcted size in the MI-SiNGF group.Conclusion These findings suggested that endogenous NGF silencing attenuated sympathetic nerve sprouting and angiogenesis, enlarged the infarct size, aggravated cardiac dysfunction, and potentially contributed to an unfavorable prognosis after MI.