The Role Of ZFP580 In Cytoprotection By TGF-β1 Against Chemical Hypoxia-induced Injury In H9c2 Cardiac Myocytes

Objective: Hypoxia is a common clinical pathophysiology of many diseases, when the body especially heart and brain and other vital organs sustained severe hypoxia which even lead to death. Cobalt chloride(Co Cl2), a well-known hypoxia mimetic agent, which can mimic hypoxic/ischemic response by increasing reactive oxygen species(ROS) generation, decreasing cell viability, dissipating mitochondrial membrane potential(MMP), activating caspase-3, and inducing apoptosis. Transforming growth factor-β1(TGF-β1) is one of exocrine cell type signal peptide having more biological activities, which can regulate the cells proliferation, differentiation, adhesion, migration and apoptosis, and plays an important role in the biology development. Cardioprotective effects of TGF-β1 have been a hot research and many reports indicated that it usually plays an anti-apoptotic role in myocardial cell injury. ZFP580, a novel C2H2(Cys2-His2) zinc-finger transcription factor, was cloned in our laboratory. Previous studies showed that ZFP580 could be regulated by TGF-β1 and had cytoprotective effects on hypoxia/reoxygenation induced cytotoxicity and apoptosis in H9c2 cells. However, under hypoxia whether ZFP580 has a similar effect and the exact mechanism, and whether it is regulated by TGF-β1 is still unclear. The aim of this study is to determine whether ZFP580 is involved in the protective effects of TGF-β1 against chemical hypoxia-induced H9c2 cells injury.Methods: The cell viability was evaluated by MTT method; the m RNA expression level of ZFP580 and HIF-1α were detected by Real time-PCR; the protein expression levels of ZFP580, HIF-1α, Smad2, Smad3, p-Smad2, p-Smad3, Bax, Bcl-2 and Active Caspase-3 were determined by Western-blot; Annexin V-FITC/PI staining for detection of cell apoptosis; intracellular ROS generation was determined by the oxidative conversion of cell-permeable DCFH-DA to fluorescent DCF; detecting related index after using SB431542 inhibitors or Lentiviral vectors expressing RNA interfere directed against ZFP580.Results:1 The cobalt chloride decreased the cell viability and increased the expression of ZFP580 and HIF-1αCo Cl2 decreased cell viability in a time- and dose-dependent manner. The H9c2 cells were treated with different doses of Co Cl2(400, 600, 800 or 1000 μM) for 24 h, and the cell viability decreased with increasing concentrations of Co Cl2. The cell viability decreased approximately 50% using 600 μM Co Cl2 for 24 h. Then cells were treated with 600 μM Co Cl2 for 0, 4, 8, 12, 16, 20 or 24 h. The results showed that cell viability significantly decreased at 8, 12, 16, 20 and 24 h after Co Cl2 treatment, compared with the control group(P < 0.05). The results of q RT-PCR and western blot analysis showed that Co Cl2 treatment increased the ZFP580 expression at both the m RNA and protein level and its m RNA level reached a peak at 12 h prior to protein level at 16 h. The protein expression of HIF-1α accumulated rapidly during hypoxia and peaked at 12 h but without a significant increase in HIF-1α m RNA levels.2 TGF-β1 attenuated Co Cl2-induced cytotoxicity and up-regulated ZFP580 protein expressionTo analyze TGF-β1 function under hypoxic conditions, we also measured the cells viability pretreated with TGF-β1 for 30 min before exposure to Co Cl2. The MTT results showed that pretreatment with TGF-β1 dramatically attenuated Co Cl2-induced cytotoxicity and increased cell viability by approximately 10% at 16 and 24 h spots compared with that of hypoxic condition group(P < 0.05). The results of Western blot analysis indicated that ZFP580 protein expression was up-regulated by TGF-β1 stimulation for 8, 16 and 24 h under normoxic condition. Meanwhile, We noticed that ZFP580 protein expression could also be up-regulated by Co Cl2 with TGF-β1 pretreatment under hypoxic condition the expression of ZFP580 was up-regulated by TGF-β1 pretreatment at 8 and 24 h(P < 0.05). However, at the 16 h time point of Co Cl2 treatment, no difference of ZFP580 expression was observed in the TGF-β1 pretreatment group.3 Inhibition of Smad2/3 activation attenuated ZFP580 protein expression and the protective effect of TGF-β1 against Co Cl2-induced apoptosisH9c2 cells were pretreated with SB431542, a well-known selective inhibitor of TβR1-Smad2/3, and were then stimulated with TGF-β1 followed by exposure to Co Cl2. The results showed that SB431542 partially blocked the TGF-β1-induced up-regulation of ZFP580 expression. Meanwhile, flow cytometry analysis demonstrated that pretreatment with TGF-β1 could significantly decrease the proportion of apoptotic cells after Co Cl2 treatment(P < 0.05); inhibition of Smad2/3 activation lead to increase the cell apoptosis rates by almost 11%(P < 0.05). Therefore, these findings demonstrated that ZFP580, as a downstream target of the TGF-β1/smad2/3 signaling pathway, could be closely associated with the protective role mediated by TGF-β1 against Co Cl2-induced cell apoptosis.4 ZFP580 plays an important role in the protective effects of TGF-β1 against Co Cl2-induced apoptosis and ROS generationTo evaluate the role of ZFP580 in the cardioprotection mediated by the TGF-β1/smad2/3 signaling pathway against Co Cl2-induced apoptosis, H9c2 cells were transfected with RNAi directed against ZFP580(Lenti-RNAi) or a negative control(Lenti-NC) and then pretreated with or without TGF-β1 before exposure to Co Cl2. The results of the flow cytometry analysis showed that suppression of ZFP580 could significantly increase the proportion of apoptotic cells compared with the apoptosis in the negative control(Lenti-NC) group(P < 0.05); transfected cells were then pretreated with TGF-β1 before exposure to Co Cl2,the anti-apoptotic role of TGF-β1 was attenuated. The results of the confocal microscope and microplate reader analysis showed that hypoxia-induced ROS generation was significantly accumulated in Lenti-RNAi-ZFP580 cells compared with negative control(Lenti-NC) group(P < 0.05). In addition, TGF-β1 also significantly decreased the Co Cl2-induced ROS generation, the role of which can be attenuated by Lenti-RNAi transfection.5 ZFP580 is involved in the anti-apoptotic effects of TGF-β1 through inhibition of mitochondrial apoptosis pathwayWe further explored the mechanism of ZFP580 involved in the TGF-β1/smad2/3 signaling pathway during Co Cl2-induced apoptosis. As shown in the Figure 5, the results of western blot showed that TGF-β1 could significantly increase the expression of the anti-apoptotic molecular Bcl-2 and decrease expression of pro-apoptotic molecules Bax, Bax/Bcl-2 ratio and active Caspase-3 induced by Co Cl2 treatment(P < 0.05). However, suppression of ZFP580 attenuated the anti-apoptotic effects of TGF-β1 and increased the Bax/Bcl-2 ratio and Caspase-3 activation(P < 0.05)Conclusion: In conclusion, this report provided experimental evidence that ZFP580 may function as a novel regulator under hypoxia condition. We confirm that ZFP580 plays an essential anti-apoptotic role in mediating the cardioprotective effects of TGF-β1 against chemical hypoxia-induced cells injury through inhibition of mitochondrial apoptosis pathway.