The Effect Of PINK1-dependent Mitophagy On The Function Of Endothelial Cells And The Mechanism Of Mitophagy Induction Under Simulated Microgravity

BackgroundThe cardiovascular dysfunction including orthostatic intolerance and disability on physical exercise is one of the health problems for astronauts induced by longterm spaceflight.Given that the countermeasures and the effectiveness are very limited,it is of importance to detect the underlying mechanism for the future effective countermeasures.As an important part of vascular structure,the vascular endothelium,uniquely sensitive to mechanical force,plays a pivotal role in coordinating vascular functions.It is widely reported that mitophagy in endothelial cells is closely related to various cardiovascular diseases.Recently,our study have found that simulated microgravity enhanced mitophagy in endothelial cells.Based on that,we assume that mitophagy plays a vital role in functional changes in endothelial cells under simulated microgravity.Exploring the underlying mechanism can deepen the understanding of vascular functional changes under microgravity.ObjectiveBased on the previous findings,this research aimed at detecting the relationship between mitophagy and vascular endothelial functional changes and clarifying the mechanism of mitophagy induction by simulated microgravity in vitro.Methods1.HUVECs cultured in vitro were randomly divided into four groups:clinostat simulated microgravity for 12 h（12h MG）,24 h（24h MG）and 48 h（48h MG）and stationary control group（Con）.Western blot was performed to determine the expression levels of Drp1 and Mfn2 and immunofluorescence assay was performed to observe morphology of mitochondria under clinorotation.The membrane potential of mitochondria was examined using JC-1 after clinorotation for 48 h.Transmission electron microscopy was used to observe mitophagosome in HUVECs.The expression levels of COXIV,Tom20 and Tim23 were detected after clinorotation for 12 h,24 h and 48 h,and the colocalization of LC3 and Tom20 as well as the colocalization of mitochondria and lysosome were detected.Mitochondrial protein was extracted and the expression levels of PINK1,Parkin and p62on mitochondrial were detected after clinorotation for 48 h.The colocalization of PINK1and Tom20 were explored by immunofluorescence assay in HUVECs.The level of mitophagy was explored with the treatment of si RNA-PINK1 under simulated microgravity.2.The mdivi-1 was used as the specific inhibitor of the mitochondrial fission and mitophagy.NAC and Mito Tempo were used as the specific scavenger of ROS and mt ROS,respectively.HUVECs treated with DCFH-DA were visualized using a fluorescence microscope to measure ROS under simulated microgravity in the presence of NAC or not.HUVECs treated with Mitotempo were visualized to measure ROS and mt ROS.Then the protein level of the markers of inflammasome such as NLRP3,IL-1βp17 and Caspase 1p20 were determined with the treatment of NAC and Mito Tempo respectively.ELISA assay was performed to detect the levels of IL-1βin the supernatant.The effects of mdivi-1 and PINK1 knockdown on the NLRP3 inflammasome were explored by western blot and ELISA assay.The effects of PINK1 knockdown on the ROS level and inflammasome activation in the presence of Mito Tempo or not under clinorotation were determined.The expression of ZO-1,Occludin and the permeability of HUVECs were explored in the presence of NAC,si RNA-NLRP3 or si RNA-PINK1.The m RNA expression of MMP1,MMP2 and MMP9 were explored by q RT-PCR.The protein level of MMP1 was detected after PINK1 knockdown under clinorotation.The expression of MMP1,ZO-1 and Occludin in HUVECs treated with IL-1βneutralizing antibody,si RNA-NLRP3 or si RNA-MMP1 under simulated microgravity were evaluated.Transwell migration assay was performed to access cellular ability of migration in HUVECs transfected with si RNA-NLRP3,si RNA-MMP1 or si RNA-PINK1 under simulated microgravity.3.The m RNA and protein levels of Hsp70,Hsp90 and Clusterin in HUVECs under clinorotation for 12 h,24 h and 48 h were tested by q RT-PCR and western blot.The expression of Hsp70,Hsp90 and Clusterin were detected in HUVECs transfected with si RNA-HSF1 under clinorotation.The activity of proteasome was evaluated after clinorotation for 12 h,24 h and 48 h.The protein synthesis rate and the protein level of RBM3 were detected by SUn SET assay and western blot,respectively.The effect of RBM3 knockdown on protein synthesis was determined.The phosphorylation of IRE1,PERK and EIF2S1 and the expression of ATF6,CHOP and GRP78,markers of ER stress and UPR,were detected after clinorotation for 48 h.The level of autophagy was detected by examining the expression of LC3II and p62 as well as the number of LC3 puncta in HUVECs exposed to simulated microgravity.The colocalization of LC3 and ubiquitinated protein was explored by immunofluorescence assay with or without Baf A1 under simulated microgravity.The m RNA expression of Hsp60 was evaluated by q RT-PR under clinorotation for 48 h.The level of Ca²⁺in the mitochondria was evaluated with or without IP₃R inhibitor 2-APB under clinorotation using Rhod-2 AM,a mitochondrion specific chemical Ca²⁺indicator.The morphology and the membrane potential of mitochondria and the level of mitophagy were determined under clinorotation with or without 2-APB.Results1.The expression of Drp1 was increased and the expression of Mfn2 was decreased in HUVECs exposed to clinorotation for 24 h and 48 h.After exposure to simulated microgravity for 48 h,the membrane potential of mitochondria was decreased and the mitochondrial fission was increased.The expression of Tom20,Tim23 and COXIV decreased after 24 h and 48 h clinorotation.Exposure to simulated microgravity for 12 h,24 h and 48 h led to increased expression of PINK1.The colocalization of LC3 and Tom20 and the colocalization of Mito Tracker and Lyso Tracker increased in HUVECs under clinorotation for 48 h.TEM showed the typical autophagosome and mitophagosome in HUVECs after clinorotation for 48 h.Simulated microgravity for 48 h increased the expression of p62,PINK1 and Parkin in mitochondrial extracts and the colocalization of Tom20 and PINK1 in HUVECs.PINK1 knockdown inhibited the induction of mitophagy under clinorotation.2.The ROS level and mt ROS level were increased after clinorotation for 48 h.Compared to Con group,the expression of NLRP3,IL-1βp17 and Caspase 1 p20 were increased and NAC and Mito Tempo inhibited NLRP3 activation.The levels of IL-1βin the supernatant in each group were consistent to the results of western blot.The presence of mdivi-1 under clinorotation made the expression of NLRP3,IL-1βp17 and Caspase 1p20 higher than MG group.PINK1 knockdown under clinorotation increased ROS level and expression of NLRP3,IL-1βp17 and Caspase 1 p20 compared to MG group whereas Mito Tempo inhibited the activation of inflammasome.Simulated microgravity decreased the expression of ZO-1 and Occludin and increased cell permeability.NLRP3 knockdown or NAC reversed these changes whereas PINK1 knockdown aggravated these changes.Clinorotation increased the m RNA and protein expression of MMP1 but had no effect on m RNA level of MMP2 and MMP9.PINK1 knockdown under clinorotation made the protein level of MMP1 higher than MG group.IL-1βneutralizing antibody or NLRP3knockdown inhibited the changes of protein levels of MMP1,ZO-1 and Occludin induced by clinorotation.MMP1 knockdown under clinorotation inhibited the decreased expression of Occludin but had no effect on ZO-1.Clinorotation enhanced cellular migration.MMP1 knockdown or NLRP3 knockdown inhibited the cellular migration whereas PINK1 knockdown under clinorotation enhanced the cellular migration compared to MG group.3.Exposure to clinorotation for 12 h,24 h and 48 h increased the expression of Hsp70,Hsp90 and Clusterin at both m RNA and protein levels in HUVECs.Knockdown of HSF1under clinorotation decreased the expression of Hsp70,Hsp90 and Clusterin compared to MG group.Clinorotation for 12 h,24 h and 48 h increased the proteasome activity and decreased the expression of RBM3 as well as the protein synthesis rate.RBM3knockdown decreased the protein synthesis rate.Clinorotation for 48 h increased the phosphorylation of PERK and EIF2S1 and the expression of CHOP,GRP78 and ATF6.The expression of LC3II,the number of LC3 puncta and the colocalization of LC3 and ubiquitinated protein were increased and the expression of p62 was decreased by clinorotation.The treatment with Baf A1 increased the colocalization of LC3 and ubiquitinated protein.Ca²⁺level in mitochondria was elevated by clinorotation.2-APB could reverse the decreased mitochodrial membrane potential and the increased Ca²⁺level and fission of mitochondria in HUVECs under simulated microgravity.Conclusion1.Simulated microgravity induces the PINK1-dependent mitophagy in HUVECs.2.Mitophagy inhibits activation of NLRP3 inflammasome induced by microgravity,thus inhibiting increased permeability of HUVEC monolayer and enhanced cellular migration.3.Clinorotation induces ER stress and UPR,upregulates the transfer of Ca²⁺from the ER to mitochondria through the IP₃R enriched in the MAM.The transfer of Ca²⁺causes the collapse in mitochondrial membrane potential,fission of mitochondria and mitophagy.Taken together,this study found out the enhanced PINK1-dependent mitophagy in HUVECs after exposure to simulated microgravity.The increased level of mitophagy inhibits activation of NLRP3 inflammasome,increased permeability of endothelial cell monolayer and enhanced cell migration under clinorotation.The mechanism is that ER stress and UPR under clinorotation causes exaggerated ER-mitochondria Ca²⁺transfer through MAMs,leading to Ca²⁺accumulation in mitochondria which results in collapse of mitochondrial membrane potential,mitochondrial fission and mitophagy.This study deepens the understanding of mechanism of cardiovascular dysfunction under microgravity and may help to provide potential therapeutic actions to protect cardiovascular system.