Cardiomyocytes Initiate Studies On The Mechanism Of Anti-hypoxia Injury By TRPV4 Localized To Primary Cilia

With the gradual improvement of China’s military and economic capabilities,more and more people go to plateau areas with an altitude of more than 3000 m for work,study,tourism and other reasons.Highland due to low pressure hypoxia and other reasons will cause acute altitude sickness,even life-threatening cases.At present,the process of human body sensing hypoxia environment and activating anti-hypoxia mechanism is not completely clear,and there are no effective prevention and treatment methods.Primary cilia are axon-like structures located on the cell surface,which have been found to have the function of sensing chemical,physical,mechanical and other external stimuli and transmitting signals into the cell to trigger relevant reactions.So do primary cilia have the function of sensing hypoxia environment and initiating anti-hypoxia injury mechanism in human body?In this study,we investigated the role of primary cilia as oxygen receptors in cultured cardiomyocytes and the mechanism of intracellular anti-hypoxia injury.In this paper,H9C2 myocardial cell line was used to carry out the research according to the following six steps:1.After the cells were placed in anoxic environment for 0,12h,24h and 36h,the cell survival rate,intracellular ROS content,LDH release rate,MDA content,SOD and CAT enzyme activities were detected by CCK-8 method,respectively,and the optimal anoxic duration was determined according to the detection results.2.The length and incidence of primary cilia after 24 h hypoxia were observed by immunofluorescence staining.Intracellular ROS content,LDH,SOD and CAT enzyme activities before and after hypoxia were detected by RNA interference method after primary cilia.3.Intracellular Ca²⁺level and TRPV4 protein and gene expression levels were detected before and after hypoxia.Cells were pretreated 2 h in advance with TRPV4 inhibitor HC-067047,and intracellular Ca²⁺level,mitochondrial membrane potential,ROS content,LDH release rate and cell survival rate were detected before and after inhibition of TRPV4 and hypoxia for 24 h.4.Immunofluorescence was used to explore the position relationship between primary cilia and TRPV4,and to detect the influence of interfering primary cilia on TRPV4 protein and gene expression.Interference primary cilia and HC-067047 acted together.The intracellular Ca²⁺level,mitochondrial membrane potential,ROS content,LDH release rate,MDA content,SOD and CAT enzyme activities were detected before and after 24 h of hypoxia.In this paper,the main experimental results are as follows:1.With the prolongation of hypoxia time,the survival rate of H9C2 cells gradually decreases,only 72%in 36 h hypoxia.The enzyme activities of LDH,ROS,MDA,SOD and CAT gradually increased with the hypoxia time,peaked at 24 h,and then gradually decreased.2.The ciliary length of H9C2cells was 1.35±0.42μm under normoxia,and the ciliary incidence was 51%.After 24 h of hypoxia,the ciliary length was 2.30±0.67μm,and the ciliary incidence was 71%,and the results showed significant statistical difference（P<0.01）.The protein and gene levels of IFT88 were effectively interfered by sh RNA,and the ROS level in H9C2 cells under normoxia condition increased 4.5 times compared with NC group after primary cilia interference（SH group）at 0 h.After 24 h hypoxia,ROS levels in both groups increased,but the SH group was still much higher than NC group.The trend of LDH and ROS was basically consistent,that is,the SH group was significantly increased at 0 h,and the SH group was significantly higher than the NC group after 24 h hypoxia.There was no significant difference in SOD enzyme activity between NC group and SH group at 0 h.SOD enzyme activity in NC group increased significantly after 24 h hypoxia,while SOD activity in SH group also increased,but decreased significantly compared with NC group.There was no significant difference in the enzyme activity of CAT between NC and SH groups at 0 h.After hypoxia for 24 h,the level of CAT in NC group was much higher than that in 0 h,and the increase rate in SH group was much lower than that in NC group.3.Fluo-4/AM calcium probe showed calcium overload after hypoxia.WB and RT-PCR results showed that TRPV4protein and gene were upregulated after 24 h hypoxia.After pretreatment with HC-067047,the protein and gene of TRPV4 were inhibited.Hc-067047 was combined with hypoxia,and it was found that after 24 h of hypoxia,Ca²⁺level increased significantly,mitochondrial membrane potential decreased,ROS content increased significantly,LDH release rate increased significantly,and cell survival rate decreased significantly,indicating obvious oxidative damage occurred in H9C2 cells after 24 h of hypoxia.However,the above indexes were significantly reversed after the addition of HC-067047.4.Immunofluorescence results showed that TRPV4 was located above the primary cilia.The protein and gene expression of TRPV4 were both high after interference with primary cilia.The interference of primary cilia and HC-067047 combined with H9C2 cells.After the interference of primary cilia and hypoxia for 24 h,the increase of Ca²⁺level,the decrease of mitochondrial membrane potential,the increase of ROS content,the increase of LDH release rate,the increase of MDA content,the decrease of CAT and SOD activity were all reversed by the pretreatment of HC-067047.These results suggest that primary cilia in H9C2 cells,as oxygen receptors,can sense the extracellular hypoxia environment,mediate Ca²⁺influx through the TRPV4 channel located on the primary cilia,affect mitochondrial membrane potential,lead to changes in ROS content,and then regulate the activity of antioxidant enzymes to maintain intracellular oxidation and antioxidant balance.This study provided a theoretical basis for illustrating the mechanism of cardiomyocytes sensing hypoxia environment through primary cilia and responding to oxidative stress,and provided a potential drug target for the research and development of drugs for the treatment of altitude heart disease.