20-HETE induces apoptosis in H9c2 cardiomyocytes via GPR75 receptor coupled Gαq signaling pathway
Objective To investigate the role and mechanism of GPR75 receptor-coupled Gαq signaling pathway in 20-HETE-induced apoptosis of H9c2 cardiomyocytes.Methods Lentiviral vector was employed to interfere GPR75 expression in H9c2 cardiomyocytes,and RT-qPCR and Western blot was utilized to assess the knockdown efficiency.The levels of IP3 and cAMP were quantified using ELISA.Apoptosis rate was evaluated by TUNEL assay.The intracellular Ca2+concentration,ROS content and mitochondrial membrane potential(△Ψm)were detected by Fluo-4/AM,DHE and JC-1 fluorescent probes,respectively.Protein expressions of EGFR,ERK1/2,AKT,GSK3β,Bax,Bcl-2,Cyt C,and Caspase-3 were analyzed via Western blot.Results After transfection of H9c2 cardiomyocytes with lentiviral vector shGPR75,the mRNA and protein expression of GPR75 receptor de-creased by 67.16％and 63.99％,respectively(P＜0.05).Knockdown of GPR75 expression or blockade of its effect by AAA significantly attenuated 20-HETE-induced apoptosis of H9c2 cardiomyocytes,reversed the reduc-tion in mitochondrial membrane potential(△Ψm)as well as the upregulation of apoptosis-related proteins Bax,Cyt C,and Caspase-3(P＜0.05).After treatment with 20-HETE,the intracellular IP3 content was significantly increased(P＜0.05),but there was no significant effect on cAMP generation(P＞0.05).Knockdown of GPR75 expression,pretreatment with AAA,or inhibition of the PLC signaling pathway using U73122 effectively abrogated 20-HETE-induced IP3 production(P＜0.05).Knockdown of GPR75 expression and pretreatment with AAA or U73122 effectively inhibited the increase of Ca2+concentration and ROS generation induced by 20-HETE(P＜0.05).After treatment with 20-HETE,the phosphorylation levels of EGFR,ERK1/2,AKT,and GSK3β exhibited a significant increase(P＜0.05).Importantly,these effects induced by 20-HETE could be effectively attenuated either by knocking down GPR75 expression or by applying AAA(P＜0.05).Finally,blocking PLC or PI3K signaling pathway significantly inhibited 20-HETE-induced cardiomyocyte apoptosis(P＜0.05).Conclusion 20-HETE induces apoptosis of H9c2 cardiomyocytes through GPR75 receptor-coupled Gαq protein and its mediated PLC and EGFR/ERKl/2/AKT/GSK3β signaling pathways.
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