Mechanism Of Apelin-13 Regulating Cx43 Remodeling And Hypertrophy In HL-1 Cells Induced By Ang?

Objiective:To investigate the inhibiting effect of Ang? on the Cx43 expression in HL-1 cells.To investigate the effect of Apelin-13 on the suppression of Cx43 expression induced by Ang? in HL-1 cells.To explore the role of Apelin-13 on the upregulation of cell hypertrophy induced by Ang? in HL-1 cells.Further explore the mechanism of the effect of Apelin-13 on the suppression of Cx43 expression and autophagy induced by Ang? in HL-1 cells.Further explore the mechanism of the effect of Apelin-13 on the regulation of cell hpertrophy induced by Ang? in HL-1 cells.Methods:Cell culture and experimental protocolsHL-1 cells were incubated in a high-glucose DMEM(Dulbecco's modified Eagle medium)in which 10%fetal bovine serum and penicillin/streptomycin(1%)solution was added.The environment in the incubator is constant 37?,5%C02 and 95%air.The cells were starved in serum-free high glucose medium for 24 hours when density reached 85%.In the treatment according to different groups,HL-1 cells were given different concentrations of drugs.All drugs were freshly dissolved and filtered before each experiment.Cell viability assayThe viability of HL-1 cells was measured by an MTT kit.HL-1 cells were seeded in 96-well plates.Following different treatments,each well was incubated with 5 mg/ml MTT for 4 h in a humidified incubator at 37?.Subsequently,the supernatant in each well was removed and only the formazan crystals formed by the MTT was left,then 150 ?l DMSO were used to dissolve the sediment for 10 min.Finally,a microplate reader was used to measure the absorbance of the sediment at a wavelength of 490 nm.At the same time,cell viability percentage was calculated.Measurement of cell sizeUpon reaching around 60%confluence,HL-1 cells on slides were washed with PBS three times.Next,4%formaldehyde was added into the 24 well plate to fix the cells for nearly 10 min at room temperature.After three times of washing,0.5%triton x-100 was added to the plate for 5 min at room temperature.After washing with PBS,the cells on slides were treated with FITC-phalloidin for 30 min.At last,the cells were lightly washed and then incubated with DAPI for about 30 s and visualized under a fluorescence microscope.Immunofluorescence stainingAfter drug treatment,all groups of HL-1 cells on slides were washed three times and fixed in 4%formaldehyde for about 15 min at room temperature.Then,5%BSA was used to block cells for 1 h.After washing,cells were incubated with a Cx43 antibody overnight at 4?.After three washes the next day,the slides were incubated with a secondary fluorescent antibody(1:500)for 1 h.Then after washing,the cells were stained with DAPI for approximately 15 min.Finally,the cell slides were visualized under a fluorescence microscope.Western blot analysisHL-1 cells in each group were treated with RIPA lysis buffer on ice.BCA assay was used to measure the protein concentration of each group.Around 30-60 ?g protein were resolved by gel electrophoresis with 8-12%separation gel and then transferred to the cut PVDF membranes.Place the membrane in a antibody incubation box with 5%non-fat dry milk to block the membranes for 1-2 h at room temperature.Then the membranes were incubated overnight in a 4? refrigerator with different dilution of primary antibodies including Cx43(1:1000),LC3B(1:1000),p-AMPK,AMPK(1:1000),mTOR(1:1000)and B-type natriuretic peptide(BNP,1:1000).GAPDH(1:3000)was incubated on 4? shaking table as a loading control.The following day,after five times washes,the washing solution was discarded,a HRP secondary antibodies(1:5000)was added in the incubation box and incubated for nearly 2 h at room temperature.After five washes,the liquid A and B in an enhanced chemiluminescence kit was mixed and prepared in a dark room for exposure.And Image J software was used to quantify all protein bands.Statistical analysisThe data are expressed as means ±standard error(SEM).Both Prism GraphPad 6.0 software and SPAA were used for the data analysis.Comparisons among groups were evaluated using one-way ANOVA followed by the Student-Newman-Keuls test.P value<0.05 was considered to represent statistical significance.Results:Apelin-13 restored the Cx43 downregulation induced by Ang? in HL-1 cellsCells were separately treated with Ang? and apelin-13 for 48 h.Treatment with 10?M Ang? alone caused significant downregulation of Cx43 expression(P<0.05).But treatment with apelin-13 alone resulted in no change in Cx43 expression.However,when co-treated with 10 ?M Ang? for 48 h,100 nM apelin-13 reversed the decrease in Cx43 expression induced by Ang? in HL-1 cells(P<0.05).Apelin-13 reversed the inhibition of AMPK activity induced by Ang? in HL-1 cellsCells were treated with Ang?(10 ?M)and Ang?(10 ?M)+apelin-13(100 nM)for 15,30,60,or 120 min,and expression of p-AMPK and AMPK were evaluated by Western blot.Ang? alone decreased the p-AMPK/AMPK ratio at 60 min.Compared with the control,the p-AMPK/AMPK ratio significantly improved with a peak at 30 min and slightly fluctuated at 60min and 120 min when co-treated with Ang? and apelin-13.Compound C and rapamycin inhibited AMPK expression and abolished the protective effect of apelin-13AMPK expression was measured in cells treated with compound C,an AMPK inhibitor,as a negative control.Rapamycin,a common activator of autophagy and inhibitor of mTOR activity.HL-1 cells were treated in five groups:control group,Ang? group,AngII+Apelin-13 group,AngII+Apelin-13+CompoundC group,AngII+Apelin-13+Rapamycin group.Compared with control group,the level of AMPK decreased in Ang?group while increased in AngII+Apelin-13 group.The results showed that both compound C and rapamycin significantly decreased AMPK expression,which was prevented by apelin-13(P<0.05).Apelin-13 promoted autophagy via the AMPK/mTOR signaling pathwayHL-1 cells were treated in five groups and the expression of mTOR and the autophagic marker LC3 were examined.Compared with Ang? group,the expression of mTOR was decreased and that of LC3 increased in cells treated with both Ang? and apelin-13(P<0.05).And AngII+Apelin-13+Rapamycin group was characterized by decreased mTOR expression and increased LC3II expression(P<0.05).However,AngII+Apelin-13+CompoundC group was characterized by increased mTOR expression and decreased LC3II expression(P<0.05).Apelin-13 decreased Ang?-induced proliferation of HL-1 cellsCells were incubated in 96-well plates and treated with apelin-13(10,100,1000 nM)alone or in combination with Ang?(10 ?M).MTT assays indicated that apelin-13 alone had no significant effect on cell survival.However,1000 nM of apelin-13 was obviously reduced the increase of cell survival rate induced by Ang?(10 ?M)(P<0.05).Apelin-13 reversed Ang?-induced downregulation of Cx43 expression and distribution via the AMPK/mTOR signaling pathwayWestern blot and cell immunofluorescence was also used to visualize the distribution and expression of Cx43.The results indicated that Apelin-13 inhibited the downregulation of Cx43 expression and distribution induced by Ang? in HL-1 cells(P<0.05).Compared with Ang?+apelin-13 group,adding additional compound C(10 ?M)or rapamycin(10 nM)decreased the expression and distribution of Cx43(P<0.05).These data supported that the AMPK/mTOR signaling pathway may involved in the effect of Apelin-13 on Cx43.Rapamycin reversed the downregulation of BNP and cell hypertrophy induced by apelin-13The protein level of BNP was measured by western blot and cell immunofluorescence using FITC-phalloidin was performed to assess cytoskeletal structure.The results showed that apelin-13 decreased the expression of BNP and reduced cell hypertrophy,which was highly increased by rapamycin(P<0.05).However,compound C had no apparent effect.Conclusion:Ang? decreased Cx43 expression and distribution,stimulated autophagic flux and inreased the hypertrophy of HL-1 cells.However,apelin-13 via AMPK/mTOR pathway reversed the Ang?-induced effects on Cx43 downregulation and cell hypertrophy in HL-1 cells and further increased autophagy,which in turn inhibited Cx43 expression and promoted cell hypertrophy.Thus,it' s promising that apelin-13 may be a potential agent in prevention or treatment of AF in the futhure.