| Objective:In this study,?3-AR overexpressing lentiviruses were used to infect neonatal rat cardiomyocytes to mimic the high expression of?3-AR in heart failure,while simultaneously administering selective?3-AR agonists and antagonists and?3-AR downstream specific signaling molecule-specific inhibitors to investigate the mechanism of?3-AR mediated TGF?1 expression in cardiomyocytes of heart-failure rats during heart failure.Methods:Myocardial cells were cultured in vitro.After cardiomyocytes were completely adherent,they were transfected with multiplicity of infection?MOI?5,10,and 20 to carry the over-expression of green fluorescent protein?GFP??3-AR Lentivirus.After 72 hours of transfection of cardiomyocytes with lentivirus,the expression of GFP in the cells was observed by inverted fluorescence microscopy,and the expression of?3-AR was detected by q-PCR at the mRNA level to determine the optimal transfection complex number of lentivirus-transfected cardiomyocytes.After 48h of transfection of cardiomyocytes with the optimal MOI with the lentiviral vector carrying the?3-AR gene,and then selective?3-AR agonists and antagonists and related signal protein specific inhibitors were separately treated.The experiment was divided into 6 groups:?1?Normal control group:myocardial cells,without any intervention,normal fluid change culture for 72 hours;?2?Negative control virus group:myocardial cell+negative control virus?MOI5?culture 72h;?3?Overexpression of?3-AR group:Cardiomyocytes+MOI5 carrying?3-AR for 72h;?4?Overexpression of?3-AR+agonist group:cardiomyocytes+lentivirus?MOI5?carrying?3-AR culture48h+BRL37344(10-5mol/L)24h;?5?Overexpression of?3-AR+antagonist+agonist group:cardiomyocytes+lentivirus?MOI5?carrying?3-AR culture 48h+SR59230A(10-5mol/L)+BRL37344(10-5mol/L)for 24h;?6?Overexpression of?3-AR+antagonist group:cardiomyocytes+lentivirus carrying?3-AR?MOI5?48h+SR59230A(10-5mol/L)24h.The purity of cardiomyocytes was identified by immunofluorescence.The expression of green fluorescent protein?GFP?in the over-expression of?3-AR lentivirus transfection group was observed by inverted fluorescence microscope.q-PCR,ELISA and Western Blot were used to detect the expression of?3-AR,TGF?1,c-Jun,p-c-Jun,JNK,and p-JNK.Results:?1?Identification of rat cardiomyocytes:Identification of myocardial cells by immunofluorescence,and the purity of isolated and cultured cardiomyocytes can reach more than 90%.?2?Detection of Lentiviral Transfection Effect:Cardiomyocytes were transfected with different MOI lentiviruses,and the fluorescence abundance was observed under an inverted fluorescence microscope.Combined with q-PCR results,the virus transfection rate was found to be appropriate when MOI=5.q-PCR detection of?3-AR mRNA expression in each group of cells,compared with empty virus transfection group,overexpression of?3-AR at the MOI of 5,10,20?3-AR mRNA expression were significantly increased?5MOI vs.LV-NC,1208±58.88 vs.0.5799±0.1697,P<0.01;10MOI vs.LV-NC,1932±35.08 vs.0.5799±0.1697,P<0.001;20MOI vs.LV-NC,4290±277.5 vs.0.5799±0.1697,P<0.01?,The results showed that the constructed?3-AR lentivirus can effectively increase the expression of?3-AR in rat cardiomyocytes.?3?Effect of selective?3-AR agonists and antagonists on the expression of TGF?1 in cardiomyocytes from overexpressing?3-AR in neonatal rat cardiomyocytes:After 48 hours of virus transfection,selective?3-AR agonists and antagonists were administered for 24 hours,and the expression of TGF?1 was detected by q-PCR and ELISA.Expression of TGF?1 in the?3-AR agonist group was significantly increased by overexpressing?3-AR cells compared to the over-expressed?3-AR group?LV-Adrb3-BRL vs.LV-Adrb3,2.894±0.1086 vs.0.9945±0.0873,P<0.001?;Overexpression of?3-AR cells with?3-AR antagonist followed by?3-AR agonist resulted in marked suppression of TGF?1 expression?LV-Adrb3-SR-BRL vs.LV-Adrb3-BRL,1.21±0.1278 vs.2.894±0.1086,P<0.001?;?4?Increased expression of?3-AR can activate the PKG/JNK/c-Jun pathway:Western Blot was used to detect the phosphorylation levels of JNK and c-Jun in each group.The results showed that compared with the over-expressed?3-AR group,overexpression of?3-AR cells significantly increased JNK phosphorylation levels when administered with?3-AR agonists?LV-Adrb3-BRL vs.LV-Adrb3,3.572±0.4209 vs.1.835±0.2146,P<0.001?;the level of c-Jun phosphorylation also increased significantly?LV-Adrb3-BRL vs.LV-Adrb3,1.572±0.08953 vs.1.016±0.04305,P<0.01?.When cells were incubated with?3-AR antagonists and then given?3-AR agonists,JNK phosphorylation was significantly inhibited?LV-Adrb3-SR-BRL vs.LV-Adrb3-BRL,1.203±0.1485 vs.3.572±0.4209,P<0.001?;The c-Jun phosphorylation level was also significantly inhibited?LV-Adrb3-SR-BRL vs.LV-Adrb3-BRL,0.8117±0.02201 vs.1.572±0.08953,P<0.01?.The increase in the expression of TGF beta 1 caused by beta 3-AR agonists no longer occurs when the expression of PKG inhibitor KT5823,JNK inhibitors SP600125 and c-Jun si RNA are suppressed in the over expressed beta 3-AR cardiomyocytes?LV-Adrb3-KT-BRL vs.LV-Adrb3-BRL:TGF?1m RNA level,1.762±0.08643 vs.2.7±0.05279,P<0.001,TGF?1 protein level,555.3±27.46vs.749.4±23.97,P<0.01;LV-Adrb3-SP-BRL vs.LV-Adrb3-BRL:TGF?1 mRNA level,1.207±0.139 vs.2.118±0.153,P<0.001,TGF?1 protein level,487.1±19.94 vs.743.8±22.98,P<0.001;LV-Adrb3-siRNA-BRL vs.LV-Adrb3-BRL:TGF?1 mRNA level,1.424±0.09043vs.2.276±0.1413,P<0.001,TGF?1 protein level,512±24.92 vs.681.5±29.28,P<0.001?;It shows that?3-AR stimulation in cardiomyocytes can increase the expression of TGF?1 by activating PKG/JNK/c-Jun pathway.Conclusion:1.Increased expression of TGF?1 in?3-AR stimulation in neonatal rat cardiomyocytes;2.?3-AR stimulation mediates the increased expression of TGF?1 through PKG/JNK/c-Jun signaling pathway. |
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