Role And Mechanisms Of Drpl In Mitochondrial Quality Imbalance Induced By Acute Ischemic/hypoxic Injury

Mitochondria,as the main site of cellular aerobic respiration,is one of the organelles damaged earlier after ischemia and hypoxia.In the past,the treatment of mitochondrial dysfunction mainly focused on the effects of anti-oxidative stress and anti-apoptosis after the occurrence of mitochondrial dysfunction.The lack of prevention and treatment measures for the key links in mitochondrial quality control has limited the therapeutic effects of acute ischemic/hypoxic injury.Recent studies have shown that mitochondrial quality imbalance,such as excessive fission,impaired fusion,autophagy,and metabolic disorders,are closely related to the occurrence of multiple organ dysfunctions and metabolism disorders after ischemia and hypoxia.As a classical mitochondrial dynamic protein,Drp1 mainly affects the mitochondrial fission process in the regulation of mitochondrial quality.Previous studies have suggested that Drp1 could only be recruited to mitochondria to participate in mitochondrial fission only after the contact between endoplasmic reticulum and mitochondria(ER-Mito contact)caused mitochondrial precontraction.However,the recruitment mechanism of Drp1 remains to be further studied.Besides,under normal conditions,Drp1 translocated to mitochondria only accounts for 6%of the total Drp1 content,and a large amount of Drp1 exists in the cytoplasm.What are the functions of the abundant cytoplasmic Drp1?Has Drp1 been involved in the mitochondrial fission process before being recruited to mitochondria?Does Drp1 have a regulatory effect on ER-Mito contact formation and mitochondrial pre-contraction?Does Drp1 affect other key links in mitochondrial quality control,such as autophagy and mitochondrial metabolism except for mitochondrial fission?In addition,current studies suggest that Drp1 mediates mitochondrial fission mainly through its GTPase activity after its recruitment to mitochondria.However,what role does Drp1 play after the end of mitochondrial fission has always been a puzzle.In recent years,it has been found that Drp1 is involved in the regulation of mitochondrial and cellular functions in various disease models,but the specific regulatory mechanism is still unclear.Studies have shown that excessive opening of mitochondrial mPTP channel is the prerequisite for the changes of mitochondrial membrane potential and cell necrosis,and the large accumulation of ROS may be an important cause of the above-mentioned mitochondrial and cellular dysfunction.In addition to mediating mitochondrial fission,does mitochondrial Drp1 also affect the opening of mPTP channels in the outer mitochondrial membrane as well as the production and elimination of ROS after ischemia and hypoxia?In this study,we used global hemorrhagic shock in vivo and cellular glucose-oxygen deprivation in vitro to establish the model of acute ischemic/hypoxic injury.Firstly,we understand the characteristics of mitochondrial quality imbalance in multiple tissues with the extension of ischemic/hypoxic injury.Secondly,we explored the role and regulatory mechanisms of Drp1 in mitochondrial quality imbalance induced by acute ischemic/hypoxic injury.We mainly focused on the initiation mechanism of cytoplasmic Drp1 on ER-Mito contact formation and the regulatory mechanisms of mitochondrial Drp1 on mitochondrial functions such as mPTP and ROS.Finally,we observed the protective effects of Mdivi-1,a Drp1 inhibitor,or Drp1 knockout intervention on multiple organ functions after acute ischemic/hypoxic injury,which provided new ideas and new targets for clinical treatment of acute ischemic/hypoxic injury from the perspective of maintaining mitochondrial quality balance.[Research contents]Part 1:Study on the characteristics of mitochondrial quality imbalance in multiple tissues with the extension of ischemic/hypoxic injury.Hemorrhagic shock SD rat model and cellular glucose-oxygen deprivation model were used to observe the changes of mitochondrial morphology and quantity as well as mitochondrial functions including ATP production,ROS contents and mitochondrial membrane potential(??m)in vascular,intestines,and heart tissues at different time points(0.5h,1h,2h,3h and 4h)of ischemia or hypoxia,so as to understand the characteristics of mitochondrial quality imbalance in multiple tissues after acute ischemic/hypoxic injuryPart 2:Role and mechanisms of Drpl in mitochondrial quality imbalance induced by acute ischemic/hypoxic injury1.The expression,distribution,and modification of Drpl after acute ischemic/hypoxic injury.Hemorrhagic shock SD rat model and glucose-oxygen deprivation VSMC model were used to observe the expression and distribution of some mitochondrial dynamin proteins at different time points(0.5h,1h,2h,3h and 4h)of ischemia or hypoxia;to detect the modification of Drp1 after ischemic injury2.Role of Drpl in mitochondrial morphological and quantitative changes after ischemic/hypoxic injury and its regulatory mechanism on ER-Mito contacts.(1)The regulatory role of mitochondrial Drpl-mediated excessive mitochondrial fission on morphological and quantitative changes of mitochondria.VSMCs transfected with Drp1 interference/overexpression adenovirus were used to observe the effects of Drp1 intervention on mitochondrial morphology and quantity under normal and hypoxia conditions,so as to investigate the role of Drp1 in regulating the morphological and quantitative changes of mitochondria(2)The initiation mechanism of cytoplasmic Drpl on ER-Mito contacts and mitochondrial precontraction.Hemorrhagic shock SD rat and Drp1 knockout(Drp1 +/-)mouse models and glucose-oxygen deprivation VSMC model were used to observe the contact of the endoplasmic reticulum(ER)and mitochondria(Mito)at different time points(0.5h,1h,2h,3h and 4h)of ischemia or hypoxia;to observe the effects of cytoplasmic Drp1 intervention on ER-Mito contacts and mitochondrial precontraction;to explore the effects of the intervention of Drp1-Shroom4 complex on actin bundling,ER-Mito contact formation,and mitochondrial precontraction,so as to elucidate the mechanisms of cytoplasmic Drp1 initiating ER-Mito contact through shroom4-bundling F-actin after ischemic/hypoxic injury(3)The accelerating mechanism of cytoplasmic Drpl translocation to mitochondria.Hemorrhagic shock SD rat model and glucose-oxygen deprivation VSMC model were used to observe the expression and distribution of Calnexin and Fundcl at different time points(0.5h,1h,2h,3h and 4h)of ischemia or hypoxia;to observe the effects of Calnexin intervention on mitochondrial translocation of Drp1 and Fundcl,mitochondrial precontraction,and mitochondrial fission at the early stage of hypoxia,so as to investigate the accelerating mechanism of cytoplasmic Drp1 tranlocation to mitochondria under the recruitment of Calnexin-Fundcl loop at ER-Mito contact sites after ischemia and hypoxia(4)The clearance mechanism of Drpl-mediated mitophagy to damaged mitochondrial fragments.Hemorrhagic shock SD rat and Drp1 knockout(Drp1 +/-)mouse models and glucose-oxygen deprivation VSMC transfected with Drpl interference adenovirus model were used to clarify Drpl-mediated potential pathways under physiological and ischemic conditions through genomics analysis;to observe the effects of Drpl intervention on the formation of mitochondrial autophagosomes and autolysosomes after ischemic/hypoxic injury;to observe the effects of Drpl intervention on the expression and distribution of autophagy-related proteins,so as to clarify the mechanism of Drpl-mediated mitophagy through Clecl6a-Parkin pathway after ischemic/hypoxic injury3.Role and regulatory mechanisms of Drpl in the changes of mitochondrial functions such as mPTP opening and ROS accumulation after ischemic/hypoxic injury.(1)The role and regulatory mechanism of Drpl in mPTP opening after ischemic/hypoxic injury.Hemorrhagic shock SD rat and Drp1 knockout(Drp1+/-)mouse models and glucose-oxygen deprivation VSMC model were used to observe the mPTP opening conditions,the expression and distribution of mPTP structural proteins at different time points(0.5h,1h,2h,3h and 4h)of ischemia or hypoxia;to clarify the effects of the intervention of HK2 expression and activity on mPTP opening after hypoxia;to clarify the specific binding site of mitochondrial Drpl and LRRK2 and its effects on HK2 activity and distribution as well as mPTP opening after hypoxia,so as to elucidate the regulatory mechanism of mitochondrial Drpl on the excessive mPTP opening induced by ischemic/hypoxic injury(2)The role and regulatory mechanism of Drpl in ROS accumulation after ischemic/hypoxic injury.Hemorrhagic shock Drp1 knockout(Drp1+/-)mouse models and glucose-oxygen deprivation VSMC model were used to observe energy metabolism changes after ischemic injury by metabolic cage monitoring;to observe the effects of the Drpl intervention on mitochondrial metabolism after ischemia through target metabolomics;to observe the effects of intervention of Drpl-mediated glutathione metabolism on mitochondrial functions,so as to elucidate the regulatory mechanism of Drpl on ROS production and clearance through mitochondrial metabolism pathwaysPart 3:The protective effects of Drpl intervention on vital organ functions after acute ischemic/hypoxic injury and its clinical significance.Mdivi-1,a Drp1 inhibitor,and Drp1 knockout intervention after acute ischemic/hypoxic injury were used to observed(1)vascular reactivity,vascular diameter change,VSMC contraction tension,and the maintenance of ambulatory blood pressure;(2)Liver and kidney blood perfusion,liver and renal functions,cardiac output,heart damage index,and acute-separated cardiomyocytes contractility;(3)intestinal epithelial tight junction,gut microbiome composition and intestinal barrier function;(4)hemodynamics(LVSP,+dp/dt max),blood gases(Lac,HCO3-,BE,pH,etc.)and 72h survival conditions,so as to elucidate the protective effect of Drpl intervention on the function of vital organs after acute ischemic/hypoxic injury and its clinical significance[Results]Part 1:Study on the characteristics of mitochondrial quality imbalance in multiple tissues with the extension of ischemic/hypoxic injury.1.The morphology and quantity of mitochondria in vascular,intestines,and heart tissues changed significantly with the extension of ischemic/hypoxic injury,but there were differences in the time,degree,and form of changes.The specific manifestations were:In vascular tissues,the changes of mitochondrial morphology and quantity started at ischemia/hypoxia 1h and gradually increased.In intestine tissues,the changes started at ischemia/hypoxia 0.5h but did not further aggravate.The changes in heart tissues were not as obvious as those in vascular and intestinal tissues.The number of myocardial mitochondria only increased slightly after ischemia/hypoxia 2h.The effects of ischemic/hypoxic injury on myocardial mitochondria were mainly reflected in the disorder of myocardial mitochondrial arrangement and the destruction of internal cristae structures,which were different from the excessive mitochondrial fission and fragmentation in vascular and intestine tissues2.The functions of mitochondria in vascular,intestines,and heart tissues changed significantly with the extension of ischemic/hypoxic injury.Consistently,the changes in mitochondrial functions were mostly later than the changes in mitochondrial morphology and quantity after ischemic/hypoxic injuryPart 2:Role and mechanisms of Drpl in mitochondrial quality imbalance induced by acute ischemic/hypoxic injury1.The expression,distribution,and modification of Drp1 after acute ischemic/hypoxic injury.(1)After ischemia/hypoxia 1h,the expression of Drp1 decreased in the cytoplasm and increased on the mitochondria,while the expression of other mitochondrial dynamin proteins did not change significantly.Further observation revealed that the phase rule of cytoplasmic Drp1 translocation to mitochondria was positively correlated with mitochondrial fragmentation after hypoxia,suggesting that the mitochondrial translocation of Drp1 was closely related to the excessive mitochondrial fission at the early stage of ischemia/hypoxia Moreover,Drp1 translocated to mitochondria after hypoxia 1h was mainly enriched in mitochondrial precontraction sites.This distribution characteristic is of great significance for the occurrence of mitochondrial precontraction and mitochondrial fission(2)After ischemic injury,the phosphorylation,SUMOylation,and S-nitrosylation of Drp1 were significantly increased,the Ubiquitination of Drp1 was slightly reduced,and the acetylation and methylation modification of Drp1 were not significantly changed.The active modification of Drp1 found above has important regulatory significance for mitochondrial quality control after ischemic/hypoxic injury2.Role of Drpl in mitochondrial morphological and quantitative changes after ischemic/hypoxic injury and its regulatory mechanism on ER-Mito contacts.(1)The occurrence of mitochondrial fragmentation after ischemic/hypoxic injury was caused by excessive mitochondrial fission after Drp1 mitochondrial translocation.After Drp1 interference with Drp1 shRNA,a decrease in mitochondrial Drp1 was accompanied by a decrease in the number of mitochondrial fragments.Drpl overexpression under normal condition could also lead to excessive mitochondrial fission and an increase in the number of mitochondria.Mitochondrial Drpl-mediated mitochondrial fission was defined to determine the morphology and quantity of mitochondria after ischemic/hypoxic injury(2)Before a large number of cytoplasmic Drp1 translocating to mitochondria after ischemic/hypoxic injury,cytoplasmic Drp1 has already been over-activated and mainly manifested as Drp1 hyper-phosphorylation and S-nitrosylation.Activated cytoplasmic Drp1 binded to Shroom4,a cytoskeleton regulatory protein,to form a Drp1-Shroom4 complex.The Drp1-Shroom4 complex bundled the scattered F-actin around ER like a "snap",enhancing the traction and effective contact area between ER and mitochondria,and then the ER was entangled around the mitochondria under the traction of the bundled F-actin anchor end INF2,forming ER-Mito contact and further causing mitochondrial precontraction at the early stage of ischemic/hypoxic injury(3)After ER-Mito contacts-induced a large number of mitochondrial precontraction,Calnexin,an ER protein,migrated to the ER-Mito contact sites and combined with Fundcl to form a functional loop,accelerating the process of cytoplasmic Drp1 translocating along the ER trajectory to the ER-Mito contact sites after ischemic/hypoxic injury(4)Activated Drp1 after ischemic/hypoxic injury could up-regulate the expression of mitochondrial Clecl 6a.On the one hand,it inhibited the mitochondrial recruitment of Parkin,which resulted in the blockage of mitochondrial fragments to form autophagosomes;on the other hand,it also accelerated the transformation of autophagosomes to autolysosomes and promoted the process of autophagy flux after ischemic/hypoxic injury3.Role and regulatory mechanisms of Drpl in the changes of mitochondrial functions such as mPTP opening and ROS accumulation after ischemic/hypoxic injury.(1)At the late stage of ischemic/hypoxic injury,Drp1 enriched in mitochondrial contraction sites not only mediated excessive mitochondrial fission,but also recognized the mPTP channels near the contraction sites by binding to nearby BAX protein,and then recruited mitochondrial LRRK2 to mitochondrial contraction or fission sites.The formation of Drpl-LRRK2 complex on mitochondria caused the dephosphorylation of HK2,a mPTP structural protein,and its separation from mitochondria after hypoxia,destroyed the mPTP channel structure and leaded to excessive opening of mPTP channels.The Drpl-mediated mPTP opening could increase the release of mitochondrial CytC and activate Caspase-3 and Caspase-9,which further influenced mitochondrial and cellular functions after ischemic/hypoxic injury.(2)Activated Drp1 could lead to excessive accumulation of ROS,which further damaged mitochondrial functions and caused abnormal cellular energy metabolism after ischemic/hypoxic injury.The underlying mechanisms included:(a)activated Drp1 destroyed the mitochondrial respiratory chains by inhibiting the biosynthesis of coenzyme Q(Quinone),which increased mitochondrial ROS production;(b)activated Drp1 reduced ROS clearance by inhibiting mitochondrial glutamate-glutathione metabolism;(c)activated Drp1 further reduced ROS clearance by blocking neurotransmitter pathways(including tyrosine-dopamine metabolism and vitamin E related pathways).Part 3:The protective effects of Drpl intervention on vital organ functions after acute ischemic/hypoxic injury and its clinical significance.1.The combined fluid resuscitation with Mdivi-1,a Drp1 inhibitor,in acute ischemic/hypoxic injury could significantly improve vascular reactivity and vasoconstriction tension The intervention of Drp1 could well-maintain ambulatory blood pressure after ischemic/hypoxic injury.2.The intervention of Drp1 could improve the blood perfusion and functions of the liver and kidney after ischemic injury.The intervention of Drp1 also had a certain impact on the contractility of acute-separated cardiomyocytes after ischemic injury.3.The intervention of Drp1 could protect the intestinal barrier after ischemic injury by influencing the gut microbiome and restoring the production of short-chain fatty acids(SCFA).4.The intervention of Drp1-mediated mitochondrial quality imbalance could significantly improve hemodynamics,blood gases,and survival conditions after acute ischemic/hypoxic injury.[Conclusions]1.Drp1 plays an important role in mitochondrial quality imbalance induced by acute ischemic/hypoxic injury."Drp1 overactivation(phosphorylation,SUMOylation,and S-nitrosylation)" and "cytoplasmic Drp1 accelerates mitochondrial translocation" after ischemia and hypoxia are important reasons for the changes of mitochondrial morphology and functions after ischemia and hypoxia.2.At the early stage of ischemic/hypoxic injury,a large number of cytoplasmic Drp1 has already participated in the preprocess of mitochondrial fission before being recruited to mitochondria.Abnormal activation(phosphorylation and S-nitrosylation)of cytoplasmic Drp1 bundle scattered F-actin through Shroom4,enhance the traction between the ER and mitochondria,initiate a large number of ER-Mito contacts,and cause a large number of mitochondrial precontraction,which lay the structural foundation for the subsequent translocation of cytoplasmic Drp1 to the mitochondrial precontraction sites to mediate excessive mitochondrial fission after ischemic/hypoxic injury.3.After ER-Mito contacts-induced a large number of mitochondrial precontraction,Calnexin-Fundcl loops accelerate mitochondrial translocation of cytoplasmic Drpl via ER trajectory,which laid the material foundation for mitochondrial Drpl-mediated excessive mitochondrial fission after ischemic/hypoxic injury4.At the late stage of ischemic/hypoxic injury,mitochondrial Drpl may further affect the mitochondrial functions after mediating mitochondrial fission at the ER-Mito contact sites On the one hand,mitochondrial Drpl can recognize and destroy the mitochondrial mPTP channel structure at the mitochondrial contraction or fission sites,resulting in the excessive opening of mPTP and the increase of CytC release;on the other hand,Drpl may also affect the clearance and production of ROS through a variety of mitochondrial metabolic pathways,resulting in excessive accumulation of ROS and ultimately aggravate the damage of mitochondrial and cellular functions after ischemia and hypoxia5.The intervention of Drp1 with Mdivi-1 or Drp1 knockout can protect vasoconstriction tension,improve liver and kidney blood perfusion,and intestinal barrier after ischemic/hypoxic injury.The combined fluid resuscitation with Mdivi-1 makes up for the shortcomings of conventional resuscitation that "although it can expand volume quickly,it is difficult to maintain blood pressure for a long time".It is of great clinical significance for prolonging the treatment time and improving the survival conditions of patients with acute ischemic/hypoxic injury.