Molecular Mechanism And Protection Of Chloride Channel Blockers Target To Cardiac Cell Apoptosis With Tunicamycin Induction

Cardiomyocytes apoptosis is an important cellular basis that causes a variety of the morbidity and development of cardiovascular diseases. Such as heart failure, viral myocarditis, cardiomyocytic senescense have extensive cardiac cell apoptosis. Recent studies have found the regulation of ion channel in both inside and outside the cell membrane is closely related to cardiomyocytes apoptosis. In cardiomyocytes apoptosis model, the whole-cell patch-clamp technique recorded directly similar to volume-sensitive outwardly rectifying chloride channel VSOR currents. With related biochemical markers, we proved VSOR chloride channel activation mediated cardiomyocytic apoptosis, and blocking its channel can effectively save cell apoptosis. Moreover, there are apoptosis research reports about relationships among VSOR chloride channel and death receptor(TNFR or Fas) or mitochondrial pathway. However, the mechanism of the relevant VSOR chloride channel and apoptosis remains unclear.Endoplasmic reticulum governs protein synthesis, lipid to generate in Eukaryotic cell and calcium ion storage place.Internal environment steady-state and efficient operation, is important to maintain normal function and survival of cells. 1/3 protein synthesis within cells, correct folding and structural maturation occurs in the ER. In synthesis and a large number of folds accompanied by inside and outside the cell membrane ion channels open or close, in which involves in important VSOR chloride channel and exchange with Ca2+ in ER, and controls Material exchange balance to support Intracellular homeostasis. Eukaryotic cells through a series of adaptive way make rapid response to endoplasmic reticulum dysfunction, called the endoplasmic reticulum stress(ERS). While, Endoplasmic reticulum stress have a dual role for cell survival. Endoplasmic reticulum stress in the early, molecular chaperone GRP78 and endoplasmic reticulum stress sensitivity transmembrane protein PERK, ATF6 and IRE1α receptor protein dissociate, and activate three unfolded protein response(UPR) effector, then play UPR prosurvival function. Excessive ERS induce apoptosis through independent signaling pathways. As recent research focus, the novel independence apoptotic pathway, novel endoplasmic reticulum stress and VSOR chloride channel relationship has not been any reported.This study was based on our previous and latest preliminary experimental results(Endoplasmic reticulum stress involved in the chloride channel activation on the regulation of cell apoptosis) and we proposed two levels in vivo and in vitro, to build mouse animal heart failure induced by endoplasmic reticulum stress and cell apoptosis model, scientific problems are put forward.as following: ① Endoplasmic reticulum stress as a novel apoptotic pathway is also involved in the chloride channel and apoptosis regulation? Endoplasmic reticulum stress levels continued to increase, the chloride channel blocker can effectively save the damaged cell apoptosis? ②how both mutual regulation between VSOR chloride channels and the endoplasmic reticulum stress? The study provides the basis for a new strategy for the treatment of cardiovascular and apoptosis-related diseases.Objective:1. To establish tunicamycin-induced heart failure model and ERS related cardiomyocytic apoptosis model and make clear that dose and time-effect relationship between the presence of tunicamycin and apoptosis.2. To analysis the VSOR chloride channel function change on cell apoptosis and the effect of endoplasmic reticulum stress.3. To detect VSOR chloride channels in regulating the endoplasmic reticulum signaling molecules mechanism.Methods:1. 8-week old C57 mice received intraperitoneal injection of tunicamycin(Tm, 3 mg/kg, consecutive 2 days). DIDS, 14 mg/kg, DCPIB 5 mg/kg.2. Animals were divided into 6 groups respectively, Sham, Tm, Tm+DIDS, DIDS, Tm+DCPIB, DCPIB; Cardiac cells were divided into the following groups: A. Ctrl; B. Tm; C. Tm(100ng/ml)+DIDS(75 μM); D. DIDS(75 μM); E.Tm(100ng/ml)+DCPIB(2 μM);F. DCPIB(2 μM).3. Animal model Construction of the tunicamycin-induced heart failure model : Tm, 3 mg/kg, consecutive 2 days, DIDS, 14 mg/kg, DCPIB 5 mg/kg.to detect cardiac cell apoptosis and LVEF.4.To detect role of time-dependent tunicamycin and cardiomyocyte apoptosis rate by flow cytometry and MTT; DIDS and DCPIB effects on cardiac cell apoptosis induced by tunicamycin5. To dectect DIDS and DCPIB effects on tunicamycin-induced cardiac cell apoptosis rate by FCM, TUNEL with a laser confocal microscope.6. GRP78, CHOP, caspase-3, β-catenin protein levels were detected by Western blotting;to detect ATF 4,pe IF2α,e IF2α protein levels and effect by DIDS and DCPIB; caspase-12, p-JNK、JNK、caspase-3、Bcl-2、Bax protein levels and Bax mitochondrial transfer level by Western blotting.7. Using of caspase-3 activity assay kit detected caspase-3 activity.8. Using MQAE test chloride ion concentration of cardiomyocytes by Tunicamycin.9. Using double immunofluorescent labeling with DAPI and GRP78 and CHOP expression were observed with a laser confocal microscope.10. Real-time quantitative PCR test XBP 1S m RNA level.11. Using si RNA CHOP to test VSOR channel function and endoplasmic reticulum stress.12. Using Topflash luciferase report system to detect β-catenin activity. Results:1. To build the ideal tunicamycin-induced cardiac apoptosis model, in vitro experiments suggest that tunicamycin plays a dose and time-dependent increase cytotoxic and cardiomyocytes viability decreases, finally 100ng/ml, 72 h as best model experimental conditions. GRP78, CHOP protein expression was significantly up-regulated in the induction of endoplasmic reticulum stress,Tunicamycin can increase GRP78, CHOP transcription level, within the induction of endoplasmic reticulum stress, GRP78, m RNA expression peaked at 24 h. Tunicamycin lead to cells apoptosis through endoplasmic reticulum stress pathway.2. Construction of the ideal tunicamycin-induced heart failure model. In vivo experiments show that tunicamycin aggravated left ventricular dysfunction due to myocardial injury. Left ventricular ejection fraction about 66.5 ± 3.6%, after tunicamycin treatment, dropped to 32.7 ± 1.3%, ejection fraction was signifincantly decreased(P < 0.05,n=5). Chloride channel blockers reduced the apoptotic rate of myocardial tissue, and lead to myocardial protection.3. The effects of tunicamycin on cardiomyocyte viability: MTT assay showed tunicamycin group, DIDS and DCPIB group were 57.4 ± 3.18%, 80.4 ± 1.2% and 78.4 ± 0.8%, respectively; cardiomyocyte intracellular caspase-3 activity in tunicamycin group, DIDS and DCPIB group respectively were 88.4 ± 3.4%, 45.6 ± 1.2% and 42.1 ± 2.6%; compared with tunicamycin group, DIDS and DCPIB group was statistically significant(P<0.05, n = 5). All evidence indicated that DIDS and DCPIB group could significantly inhibit caspase-3 activity and cell apoptosis.4. Intracellular chloride concentration in control group, tunicamycin group and DIDS groups were 0.9 ± 0.18, 11.3 ± 0.7 and 3.6 ± 0.4,respectively; tunicamycin group vs the control group, MQAE significantly fluorescence intensity was higher, two groups was statistically significant(P<0.05, n=5); compared with tunicamycin group, DIDS group MQAE fluorescence intensity decreased significantly(P <0.05, n = 5). The results suggest that the fluorescence intensity can reflect intracellular chloride ion concentration level, the intensity and the chloride ion concentration was negatively correlated, DIDS could significantly inhibit chloride ion outflow.5. Signaling molecules ATF4, CHOP and pe IF2α / e IF2α protein expression in the control group, tunicamycin group, DIDS group and DCPIB group are about 1,2.5,1.5 and 1.25 times respectively, compared with the control group, ATF4, CHOP and pe IF2α / e IF2α are a significant increase in tunicamycin group,(P <0.05, n = 5); compared with tunicamycin group, DIDS and DCPIB group, these indexes significantly decreased(P<0.05, n = 5); DIDS and DCPIB could significantly inhibit ATF 4, CHOP and pe IF2α / e IF2α protein expression(P <0.05, n = 5).6. The signal molecule p-JNK, JNK pathway protein expression in control group, tunicamycin group, DIDS group were no significant difference(P> 0.05, n = 5), showed that the endoplasmic reticulum stress signaling pathway activation of JNK protein were not affected by tunicamycin.7. XBP 1S m RNA transcript levels in the control group, tunicamycin group, DIDS groups and DCPIB groups were about 1,0.5,0.85 and 0.88 times respectively,compared with the control group, tunicamycin group XBP 1S m RNA decreased significantly(P<0.05, n = 5); compared with tunicamycin, DIDS and DCPIB group XBP 1S m RNA were significantly increased(P <0.05, n=5); showed DIDS and DCPIB could significantly increase myocardial cell XBP 1S m RNA transcript levels.8. Apoptotic gene Bcl-2 / Bax protein ratio in the control group, tunicamycin group, DIDS groups were 1,0.6,1.25 times, compared with the control group, tunicamycin group Bcl-2/Bax protein ratio decreased significantly(P<0.05, n=5); compared with tunicamycin group, DIDS group Bcl-2 / Bax protein ratio increased, the difference was statistically significant(P <0.05, n = 5). Compared with tunicamycin group, DIDS group cleaved caspase-12 protein expression decreased nearly doubled, with statistical significance(P<0.05, n = 5). Data indicated DIDS can significantly increase intracellular Bcl-2 / Bax protein expression and pro-apoptotic protein expression of cleaved caspase-12 can be reduced.9. Wnt pathway-associated protein β-catenin in the nucleus under normal circumstances, shows a certain level of expression functions, and tunicamycin treatment after 24 h, the nucleus of β-catenin levels were significantly decreased, the difference was statistical difference(P <0.05, n = 5), prompt tunicamycin-induced nuclear translocation of β-catenin significantly reduced; interference by blocking CHOP pathway, β-catenin activity levels was observed, Topflash fluorescence reading of si CHOP group is 3 times control group,with statistical difference(P <0.05, n = 5). Data showed si CHOP gene pathway, β-catenin activity was significantly increased, indicating that CHOP upregulates to Wnt proteins.10. Topflash luciferase reporter system to observe the level of β-catenin activity, fluorescence readings in the control group, tunicamycin group, si CHOP, DIDS and DCPIB were 1,0.5,0.8,0.9 and 0.85, respectively, compared with the control group, tunicamycin group β-catenin activity was significantly reduced, with statistical difference(P <0.05, n = 5); compared with tunicamycin group, si CHOP, DIDS, DCPIB group β-catenin activity was significantly increased, with statistical difference(P <0.05, n = 5). Data showed DCPIB or DIDS can inhibit tunicamycin induced Wnt-associated protein β-catenin downregulation.11. After si CHOP transfection and tunicamycin treatment,β-catenin activity was detect in the control group, tunicamycin group, DIDS groups and DCPIB groups, showed that β-catenin activity reduction was not increased after DIDS and DCPIB treatment, there was no significant improvement(P > 0.05, n = 5), on the other hand, blocking CHOP signaling pathway, DIDS or DCPIB lost helpful recovery on β-catenin activity, and further reflected that Wnt and CHOP signaling pathway regulation relationship, CHOP located upstream of Wnt molecule. Particularly after the use of the Wnt signaling pathway inhibitor s FRP, DIDS or DCPIB equally lost to the regulation of β-catenin activity, and the difference was statistically significant(P <0.05, n=5). Data showed silence after CHOP signaling pathway or Wnt signaling pathway directly block, chloride channel blockers can regulate the expression of β-catenin activity.12. Cell viability in tunicamycin group, si CHOP, DIDS and DCPIB group were 56.8 ± 7.23%, 74.4 ± 5.2%, 76.6 ± 3.1% and 77.8 ± 5.8%; compared with tunicamycin group, si CHOP, DIDS and DCPIB were significantly increased cell survival, the difference was statistically significant(P <0.05, n = 12). s FRP made si CHOP, DIDS or DCPIB lost cytoprotective effect. si CHOP, DIDS and DCPIB significantly inhibit apoptosis caused by tunicamycin, its protective effect was blocked by s FRP, showed Wnt signaling pathway is CHOP downstream regulatory molecules. Conclusion:1. Our current study demonstrated that Tunicamycin-induced ER stress can lead to cardiomyocyte apoptosis; animal experiments confirmed that ER stress inducer Tunicamycin can lead to increased apoptosis in cardiac tissue damage systolic function; chloride channel blockers reduced cardiac apoptosis related to ERS, then improved cardiac function;2. Tunicamycin could regulate the p-e IF2α / ATF4 and XBP1 protein pathway, increasing the generation of pro-apoptotic gene CHOP rather than induced JNK pathway to trigger ER stress response. DIDS and DCPIB reduced the generation of CHOP by inhibiting the above pathway; and DIDS can significantly increase cadiac intracellular Bcl-2 / Bax protein expression and reduced ERS sepecial pro-apoptotic protein caspase-12 expression to protect cardiomyocytes.3. CHOP signaling has significant regulatory role in the Wnt signaling pathway, then Wnt pathway is downstream molecule. DIDS or DCPIB could significantly inhibit tunicamycin downregulation effect on Wnt-associated protein β-catenin, which effect could be blocked by s FRP. Chloride channel blockers exert a protective effect of anti-apoptosis through CHOP / Wnt signaling pathway.