Effects Of Intercellular Communication In H9c2 Cardiomyocytes By Rat Cardiac Fibroblasts Under Hypothermia Hypoxia/reoxygenation And Its Mechanism

Objective:The occurrence of reperfusion arrhythmias（RA）is associated with downregulation of the expression and phosphorylation of connexin43（Cx43）in cardiomyocytes.It will inhibit gap junction intercellular communication（GJIC）between cardiomyocytes,but the mechanism is unclear.This study aimed to explore that the effect on Cx43 expression and phosphorylation in H9c2 cardiomyocytes by rat cardiac fibroblasts（RCFs）under hypothermia hypoxia/reoxygenation and the possible mechanisms of action,and provides a new target for RA,thereby.Methods:（1）Rat cardiomyocyte cell line H9c2 cells and RCFs were respectively divided into two groups according to the random number table method,and recorded as normal culture H9c2 cell group（group H）,hypothermia hypoxia/reoxygenation H9c2cell group（group H/R-H）,RCFs group（group R）and hypothermia hypoxia/reoxygenation RCFs group（group H/R-R）.Group H and group R were incubated under normal conditions（37°C,95%air&5%CO₂）for 5 h.Group H/R-H and H/R-R were incubated under hypoxic conditions（4°C,95%N₂&5%CO₂）for 1 h and then reoxygenation（37°C,95%air&5%CO₂）for 4 h.The expression of Cx43 in each group were detected by Western blot.（2）RCFs were directly co-cultured with H9c2 cells in a 2:1 ratio and then randomly divided into two groups,which were recorded as the direct co-culture group（group Co）and hypothermia hypoxia/reoxygenation direct co-culture group（group H/R-Co）.Likewise,two kinds of cells were indirectly co-cultured by Transwell,and were recorded as hypothermia hypoxia/reoxygenation Transwell indirect co-culture group（group H/R-T）.Group Co was incubated under normal conditions for 5 hours,and the H/R-Co and H/R-T groups were incubated under hypothermia hypoxia/reoxygenation（same method as before）.And the expression of Cx43 in each group of cells was detected separately by Western blot.（3）H9c2 cells were randomly divided into four groups,which were recorded as H9c2 cell cultured alone under normal conditions（group H）,H9c2 cell cultured alone under hypothermia hypoxia/reoxygenation（group H/R）,co-culture cells under normal conditions（group T）and co-culture cells under hypothermia hypoxia/reoxygenation treatment（H/R-T group）.Both group T and H/R-T,H9c2 cells were co-cultured with RCFs in a 1:2 number ratio by Transwell.The vitality of H9c2 cells in each groups was examined by Trypan blue staining;GJIC was examined by Scrape loading/dye transfer（SL/DT）;the expression of Cx43 in each group of H9c2 cells was examined by immunofluorescence;and the expression and phosphorylation of Cx43 in each group of H9c2 cells was examined by Western blot.In addition,the concentration of angiotensinⅡ（AngⅡ）in the cell culture medium of each group were detected by ELISA.（4）The co-cultured cells（RCFs and H9c2 cells in a 2:1 ratio by Transwell）were randomly divided into six groups according to the random number table method and named as co-culture group（group T）,co-culture+AngⅡgroup（group T+AngⅡ）,co-culture+valsartan group（group T+ARB）,hypothermia hypoxia/reoxygenation co-culture group（group H/R-T）,hypothermia hypoxia/reoxygenation+AngⅡgroup（group H/R-T+AngⅡ）and hypothermia hypoxia/reoxygenation+valsartan group（group H/R-T+ARB）.Group T+AngⅡand H/R-T+AngⅡwere treated with the addition of AngⅡfor normal culture or hypothermia hypoxia/reoxygenation,respectively.Similarly,group T+ARB and H/R-T+ARB were treated with normal culture or hypothermia hypoxia/reoxygenation after addition of valsartan,respectively.The function of GJIC in H9c2 cells was detected by SL/DT;the expression of Cx43 and ERK1/2 in each group of H9c2 cells was detected by immunofluorescence;the expression and phosphorylation of Cx43 and ERK1/2 in each group of H9c2 cells was detected by Western blot.Results:（1）Effect of hypothermia hypoxia/reoxygenation on Cx43 expression in different cells:Compared with group H,Cx43 expression decreased in group H/R-H（P<0.05）;compared with group R,there was no significant change on Cx43expression in group H/R-R（P>0.05）.（2）Effects of different co-culture methods on Cx43 expression in H9c2 cells under hypothermia hypoxia/reoxygenation:Cx43 expression was reduced in goup H/R-Co&H/R-T compared with group Co（P<0.05）;Cx43 expression was not significantly changed in the H/R-T group compared with group H/R-Co（P>0.05）.（3）Effects of RCFs on H9c2 cell vitality,protein expression and GJIC under hypothermia hypoxia/reoxygenation:（1）Vitality of H9c2 cells in each group:compared with group H,H9c2 cell vitality was reduced in the H/R group（P<0.05）,and there was no significant difference in group T（P>0.05）;compared with the T group,H9c2 cell vitality was reduced in group H/R-T（P<0.05）;H9c2 cell vitality was reduced in group H/R-T compared with group H/R（P<0.05）.（2）GJIC of H9c2 cells in each group:compared with group H,GJIC of H9c2 cells in group H/R&T was diminished（P<0.05）;compared with T group,GJIC in group H/R-T was diminished（P<0.05）;compared with group H/R,GJIC in group H/R-T was diminished（P<0.05）.（3）Expression and phosphorylation of Cx43 in H9c2 cells:compared with group H,the expression and phosphorylation of Cx43 in group H/R&T were reduced（P<0.05）;compared with group T,the expression and phosphorylation of Cx43 in group H/R-T were reduced（P<0.05）;compared with group H/R,Cx43 expression and phosphorylation of group H/R-T were reduced（P<0.05）.（4）ERK1/2 expression and phosphorylation in H9c2 cells:compared with group H,the expression and phosphorylation of ERK1/2 were reduced in group H/R&T（P<0.05）;compared with group T,the expression and phosphorylation of ERK1/2 were reduced in group H/R-T group（P<0.05）;compared with group H/R,the expression and phosphorylation of ERK1/2 were reduced in group H/R-T（P<0.05）.（5）AngⅡconcentration in cell culture medium:AngⅡwas not detected in both group H&H/R;compared with group T,AngⅡwas increased in cell culture medium of group H/R-T（P<0.05）.（4）To verify the effects of AngⅡon protein expression and GJIC in H9c2cells:（1）Expression and phosphorylation of Cx43 and ERK1/2 in H9c2 cells:compared with group T,the expression and phosphorylation of Cx43 and ERK1/2were increased in group T+ARB（P<0.05）,and the expression and phosphorylation levels of Cx43 and ERK1/2 were decreased in group T+AngⅡand H/R-T+AngⅡ（P<0.05）;compared with the T+AngⅡgroup,the expression and phosphorylation of Cx43 and ERK1/2 were decreased in group H/R-T+AngⅡ（P<0.05）;compared with the T+AngⅡgroup,the expression and phosphorylation of Cx43 and ERK1/2 were increased in the T+ARB group（P<0.05）,and decreased in the H/R-T+AngⅡgroup（P<0.05）;compared with the H/R-T group,the expression and phosphorylation of H/R-T+AngⅡgroup showed decreased of Cx43 and ERK1/2（P<0.05）,and increased in the H/R-T+ARB group（P<0.05）;compared with the H/R-T+AngⅡgroup,increased expression and phosphorylation of Cx43 and ERK1/2 in the H/R-T+ARB group（P<0.05）.（2）GJIC of H9c2 cells:compared with T group,GJIC of H9c2 cells was increased in T+ARB group（P<0.05）,and decreased in group T+AngⅡ&H/R-T+AngⅡ（P<0.05）;compared with T+Ang II group,GJIC of H9c2 cells was increased in T+ARB group（P<0.05）,and decreased in the H/R-T+AngⅡgroup（P<0.05）;compared with the H/R-T group,the GJIC of H9c2 cells in the H/R-T+AngⅡgroup was decreased（P<0.05）,and the GJICof H9c2 cells in the H/R-T+ARB group was increased（P<0.05）;compared with the H/R-T+AngⅡgroup,the GJIC of H9c2 cells in the H/R-T+ARB group had increased（P<0.05）.Conclusions:（1）Hypothermia hypoxia/reoxygenation decreases H9c2 cells vitality,inhibits ERK1/2 expression and activation in H9c2 cells,and reduces Cx43 expression and phosphorylation,thereby downregulating GJIC;the involvement of RCFs exacerbates this change;（2）After hypothermia hypoxia/reoxygenation,the secretion of AngⅡin RCFs increases,acting on H9c2 cells and inhibiting their MAPK/ERK pathway activation,thereby downregulating their Cx43 expression and phosphorylation and inhibiting GJIC;（3）The effect of RCFs on H9c2 cells was not dependent on the direct adhesion between the two cells.