The Role And Mechanism Of G Protein-coupled Receptor 75 On 20-HETE-Induced Cardiomyocyte Apoptosis

Objective:1.To observe the expression of GPR75 receptor and the role of 20-HETE on activation of GPR75 in cardiomyocytes;2.To investigate the role and the potential mechanisms of GPR75 receptor on20-HETE-induced cardiomyocyte apoptosis;Methods:Neonatal rat cardiomyocytes and H9c2 cardiomyocytes were cultured,and knockdown of GPR75 gene expression by using lentiviral RNA interference in cardiomyocytes.The cultured cells were randomly divided into 6 groups（n=6）:Control group,20-HETE group（100nmol/L）,shCtrl+20-HETE group（100nmol/L）,shGPR75+20-HETE group（100nmol/L）,shCtrl group and shGPR75 group.After 24 hours of treatment with the drugs indicated,cells were harvested and TUNEL assay was performed to assess the ratio of apoptosis;The mRNA expression of GPR75 and apoptosis-related protein,Bcl-2 and Bax were measured by using qRT-PCR;DHE staining was utilized to measured the intracellular ROS level;the mitochondrial membrane potential was detected by JC-1 fluorescent probe;Western blot method was applied to evaluate the protein expression of GPR75,Bcl-2,Bax and Cytochrome C（CytC）;Cysteinyl aspartate specific proteinase-3（Caspase-3）activity was determined by the method of chromogenic substrate assay;Production of inositol triphosphate（IP₃）in myocardial cells was measured by the method of ELISA.Results:1.The expression of GPR75 receptor in the neonatal rat cardiomyocytes and H9c2cardiomyocytes.Western blot and qRT-PCR method were applied to detect the mRNA and protein expression of GPR75 receptor in the cultured neonatal rat cardiomyocytes and H9c2 rat cardiomyocytes,respectively.The results showed that expression of GPR75 receptor was confirmed both in the neonatal rat cardiomyocytes and H9c2 rat cardiomyocytes at mRNA and protein level.In H9c2 cardiomyocytes transfected with shRNA against GPR75（shGPR75）,the mRNA and protein expression levels of GPR75 were decreased by 71.24%and 64.95%compared to control group（P<0.05）.Consistent with the results in H9c2cardiomyocytes,the mRNA and protein expression levels of GPR75 receptor in the neonatal rat cardiomyocytes transfected with shGPR75 were also decreased by 70.98%and75.25%（P<0.05）.However,cardiomyocytes transfected with a non-targeting shRNA（shCtrl）did not alter the GPR75 expression compared to control groups（P>0.05）.2.Role of 20-HETE promotes GPR75 receptor expression and IP₃ production in the neonatal rat cardiomyocytes and H9c2 cardiomyocytes.Compared with control group,treatment of H9c2 cardiomyocytes with 20-HETE showed 1.53-fold increases in protein expression levels of GPR75 receptor,and 2.09-fold increases in IP₃ production,respectively（P<0.05）.Consistent with the results in H9c2cardiomyocytes,GPR75 receptor protein levels and IP₃ production in neonatal rat cardiomyocytes treated with 20-HETE were increased by 1.51-and 3.23-fold respectively（P<0.05）,whereas knockdown of GPR75 receptor significantly blocked the stimulatory effect of 20-HETE on GPR75 receptor protein expression and IP₃ production both in H9c2cardiomyocytes and neonatal rat cardiomyocytes（P<0.05）.3.Effects of knockdown of GPR75 receptor in 20-HETE-induced cardiomyocyte apoptosisThe percentage of apoptotic cells was 2.29±0.88%in control group and significantly increased to 51.98±5.58%（P<0.05）by treatment of H9c2 cardiomyocytes with 20-HETE,whereas knockdown of GPR75 receptor by shGPR75 significantly inhibited20-HETE-induced apoptosis（P<0.05）.As a positive control,20-HETE treatment significantly increased apoptosis in in H9c2 cardiomyocytes transfected with shCtrl（P<0.05）.There was no significant difference between shCtrl,shGPR75 groups and Control group（P>0.05）.4.Knockdown of GPR75 receptor inhibits 20-HETE-induced ROS overproductionCompared with control group,treatment of H9c2 cardiomyocytes with 20-HETE significantly increased the intracellular ROS production by 3.9-fold（P<0.05）,whereas knockdown of GPR75 receptor significantly reduced the increasing of ROS production induced by 20-HETE（P<0.05）.In addition,ROS production also increased by 3.7-fold in H9c2 cardiomyocytes transfected with shCtrl by treatment with 20-HETE（P<0.05）.5.Knockdown of GPR75 receptor blocks 20-HETE-induced the decline of mitochondrial membrane potential（Δ?m）Compared with control group,Δ?m significantly decreased by 78.83%in H9c2cardiomyocytes treatment with 20-HETE（P<0.05）,whereas 20-HETE-induced decline ofΔ?m was markedly suppressed by knockdown of GPR75 receptor（P<0.05）.In addition,Δ?m also significantly decreased by 80.98%with 20-HETE treatment in cardiomyocytes transfected with shCtrl（P<0.05）.6.Effect of knockdown of GPR75 receptor on 20-HETE-induced alteration of Bax and Bcl-2 expression.The Bax mRNA expression was significantly up-regulated by 1.91-fold in H9c2cardiomyocytes treated with 20-HETE as compared with control group（P<0.05）.In contrast,the Bcl-2 mRNA expression was significantly down-regulated by 63.52%after treatment with 20-HETE as compared with control group（P<0.05）.Similar to the results of mRNA analysis,the protein expression levels of Bax was up-regulated by 1.60-fold,while that of Bcl-2 was down-regulated by 53.07%in H9c2 cardiomyocytes（P<0.05）.Whereas knockdown of GPR75 receptor by shGPR75 markedly attenuated the20-HETE-induced alteration of Bax and Bcl-2 expression（P<0.05）.7.Knockdown of GPR75 receptor inhibits 20-HETE-induced increase of CytC expressionCompared with control group,treatment of H9c2 cardiomyocytes with 20-HETE significantly increased the protein expression levels of CytC by 1.83-fold（P<0.05）,whereas knockdown of GPR75 receptor significantly inhibited 20-HETE-induced increases in CytC protein levels（P<0.05）.As a positive control,treatment with 20-HETE also increased CytC protein expression in cardiomyocytes transfected with shCtrl（P<0.05）.8.Knockdown of GPR75 receptor blocks 20-HETE-induced the enhancement of Caspase-3 activity20-HETE treatment significant increased Caspase-3 activity by 2.68-fold as compared with control group in H9c2 cardiomyocytes（P<0.05）,however,knockdown of GPR75receptor significantly attenuated the 20-HETE-induced promotion in Caspase-3 activity（P<0.05）.In addition,20-HETE treatment similarly increased Caspase-3 activity by2.67-fold in cardiomyocytes transfected with shCtrl（P<0.05）.There was no significant difference between shCtrl,shGPR75 groups and the Control group（P>0.05）.Conclusion:1.In neonatal rat cardiomyocytes and H9c2 cardiomyocytes,GPR75 receptor is expressed at gene and protein level and can be activated by 20-HETE both in cultured neonatal rat cardiomyocytes and H9c2 cardiomyocytes.2.In neonatal rat cardiomyocytes,GPR75 receptor participates in 20-HETE-induced cardiomyocytes apoptosis.3.GPR75 mediates 20-HETE-induced apoptosis via the mechanisms of initiating the mitochondrial-dependent apoptotic pathway by inducing ROS overproduction and mitochondrial dysfunction.