Role Of GPR75-mediated PLC/PKC Signaling Pathway In 20-HETE-induced Cardiomyocyte Apoptosis

Objective:To observe the role of GPR75 receptor-regulated PLC/PKC signaling pathway on 20-HETE-induced cardiomyocyte apoptosis in neonatal rats,and to investigate the molecular and cellular mechanisms of GPR75 receptor in 20-HETE-induced cardiac injury.Methods:Neonatal SD rat ventricular cardiomyocytes were cultured,and knockdown of GPR75 gene expression was done by lentiviral transfection in neonatal rat cardiomyocytes（NRCMs）.Cultured cardiomyocytes were randomly divided into the following groups:Control group,sh GPR75 group and 20-HETE group（100 nmol/L）.The other groups of cells,according to the aims of different experiments,were treated with different concentrations of pretreatment drugs for 1 h before administration of20-HETE,and cells were then collected after 24 h of culture for analyses.ELISA method was performed to measure the content of inositol triphosphate（IP₃）and the activities of PKC and NADPH oxidase.The Ca²⁺-sensitive fluorescent probe Fluo-3/AM was applied to measure the intracellular concentration of Ca²⁺.DHE staining was performed to determine the ROS levels.The mitochondrial membrane potential was detected by JC-1 fluorescent probe.Protein levels of Cyt C and Caspase-3 were determined by Western-blot assays.TUNEL assay was used to evaluate cardiomyocytes apoptosis.Results:1.Knockdown of GPR75 or treatment with U73122 decreased IP3 production induced by 20-HETE in cardiomyocytesTreatment of cultured NRCMs with 20-HETE induced a significant increase in IP₃ production from 2.78±0.11 ng/ml to 5.14±0.32 ng/ml when compared with control group（P<0.05）.Compared with 20-HETE group,knockdown of GPR75 gene expression or pre-treatment with PLC inhibitor U73122 markedly reduced the IP₃production to 3.26±0.14 ng/ml and 3.65±0.18 ng/ml,respectively（P<0.05）.2.Knockdown of GPR75,treatment with U73122 or 2-APB reduced20-HETE-induced intracellular Ca²⁺concentrationCompared with control group,treatment of NRCMs with 20-HETE showed2.7-fold increases in intracellular Ca²⁺concentration([Ca²⁺]_i)（P<0.05）,whereas knockdown of GPR75 gene expression,pre-treatment with PLC inhibitor U73122 or IP₃ receptor inhibitor 2-APB,blocked the effects of 20-HETE-induced increase in[Ca²⁺]_i（P<0.05）.3.Knockdown of GPR75 inhibited the effect of 20-HETE-induced increase in PKC activityTreatment of NRCMs with 20-HETE induced a remarkable enhancement in PCK activity by 1.78-fold when compared with control group（P<0.05）.While knockdown of GPR75 gene expression significantly inhibited the effect of 20-HETE-induced increase in PKC activity（P<0.05）.4.Knockdown of GPR75 or treatment with GF109203X inhibited the effect of20-HETE-induced increase in NADPH oxidase activityThe activity of NADPH oxidase in NRCMs was significantly enhanced by1.64-fold in 20-HETE group as compared with control group（P<0.05）.While knockdown of GPR75 gene expression or pre-treatment with PKC inhibitor GF109203X significantly blocked the effects of 20-HETE mentioned above（P<0.05）.5.Knockdown of GPR75,treatment with GF109203X or Apocynin attenuated ROS generation induced by 20-HETEThe production of ROS was significantly increased by 3.74-fold in cardiomyocytes stimulated with 20-HETE as compared to the control cells（P<0.05）.While knockdown of GPR75 gene expression,pre-treatment with GF109203X or Apocynin significantly attenuated the effect of 20-HETE-induced increase in the generation of ROS（P<0.05）.6.Knockdown of GPR75,treatment with U73122 or GF109203X reversed the decrease in the mitochondrial membrane potentials（ΔYm）induced by20-HETEΔYm was significantly decreased by 84.63%in 20-HETE group as compared to the control cells（P<0.05）,whereas knockdown of GPR75 gene expression,pre-treatment with PLC inhibitor U73122 or PKC inhibitor GF109203X significantly reversed the effect of 20-HETE induced decrease onΔYm in NRCMs（P<0.05）.7.Knockdown of GPR75,treatment with U73122 or GF109203X reduced the increase of Cyt C protein level induced by 20-HETECompared with control group,treatment of NRCMs with 20-HETE induced a significant increase in Cyt C protein level（P<0.05）.While knockdown of GPR75gene expression,pre-treatment with PLC inhibitor U73122 or PKC inhibitor GF109203X significantly reduced the increase in Cyt C protein level induced by20-HETE（P<0.05）.8.Knockdown of GPR75,treatment with U73122 or GF109203X reduced the increase of Caspase-3 protein level induced by 20-HETECompared with control group,treatment of NRCMs with 20-HETE induced a significant increase in Caspase-3 protein level（P<0.05）.While knockdown of GPR75gene expression,pre-treatment with PLC inhibitor U73122 or PKC inhibitor GF109203X significantly reduced the increase in Caspase-3 protein level induced by20-HETE（P<0.05）.9.Knockdown of GPR75,treatment with U73122 or GF109203X blocked the rate of 20-HETE-induced apoptosis in cardiomyocytesThe percentage of apoptotic cells was significantly increased to 54.5%in20-HETE group as compared to the control group（3.33%,P<0.05）.Compared with20-HETE group,knockdown of GPR75 gene expression,pre-treatment with U73122or GF109203X effectively blocked the apoptotic effect of 20-HETE in NRCMs（P<0.05）.Conclusion:20-HETE-induced increase in intracellular Ca²⁺concentration and excessive ROS production is mediated by GPR75 receptor via PLC/PKC signal pathway,which results in the dysfunction of mitochondria and finally induce cardiomyocyte apoptosis.