Mechanistic Study Of Mitochondrial Metabolic Dysfunction Caused By Magnesium Homeostasis Imbalance In Diesel Engine Exhaust Particulate-Induced Myocardial Hypoxic Injury

Objective:Diesel exhaust particle（DEP）is a kind of particulate matter produced by diesel combustion,which has a wide range of occupational exposure and causes serious harm to the health of related occupational population.Epidemiological studies have shown that occupational exposure to DEP is closely related to ischemic heart disease（IHD）,and DEP exposure increases the admission rate and mortality of ischemic heart disease.Elucidating the specific toxic mechanism of DEP effect on IHD is of great significance for the prevention and control of health hazards of DEP,and relevant mechanisms need to be elucidated.Studies have shown that mitochondrial energy metabolism plays a leading role in IHD ischemic injury,in which Mg²⁺and Ca²⁺are important regulatory factors of mitochondrial energy metabolism.Therefore,this study focused on the key role of mitochondrial energy metabolism and the important regulatory role of Mg²⁺and Ca²⁺to reveal the key characteristics,regulatory factors and mechanisms of DEP’s influence on ischemic heart injury,providing an important molecular mechanism explanation for ischemic heart disease caused by diesel exhaust particulate exposure.Methods:1.A mouse model of diesel exhaust particles exposed to occupational environmental concentration was established.ECG,H&E,and immunohistochemical staining were used to evaluate the changes in cardiac function,structure and hypoxia injury.After DEP exposure,hypoxia-inducible factor（HIF-1α）and hypoxia damage protein（Bax and Cleaved caspase3）expression were detected in a three-gas incubator and conditioned medium,and DEP induced hypoxia damage cell models were constructed for subsequent mechanism studies.2.Bioinformatics analysis was used to focus on mitochondrial energy metabolism,detect ATP content and expression of oxidative phosphorylated protein,and explore the disturbance effect of DEP on mitochondrial energy metabolism.Mitochondrial agonist（EGCG）was co-treated with DEP to evaluate the regulatory role of mitochondrial energy metabolism in DEP-induced myocardial hypoxia injury.3.The expression of pyruvate dehydrogenase（PDH）and the activity of isocitrate dehydrogenase（ICDH）,which are key enzymes regulated by Mg²⁺and Ca²⁺,were detected.At the same time,the changes of Mg²⁺and Ca²⁺in cells and mitochondria were analyzed by rapid ultra-high resolution confocal microscopy.The key regulatory factors mediating the perturbation of mitochondrial energy metabolism in DEP were analyzed.4.By focusing on Mg²⁺key transporters,the relationship between the uptake of Mg²⁺in cell membrane and the expression of SLC41A1 protein and the change of Mg²⁺in cell was analyzed,and the disturbance of Mg²⁺by DEP was evaluated.Further focusing on the expression of mitochondrial Mg²⁺uptake transporter Mrs2 and effector transporter SLC41A3,the mechanism of mitochondrial magnesium homeostasis induced by DEP exposure was evaluated.Results:1.Compared with the control group,exposure to occupational environmental concentration DEP resulted in arrhythmia and increased heart rate variability,right ventricular free wall thickening,cardiomyocyte hypertrophy,and HIF-1αincrease in mice.At the myocardial cell level,cardiomyocytes were cultured in low serum medium under hypoxia conditions of 2%O₂,5%CO₂ and 93%N₂,and the model of myocardial hypoxia was established by using 10μg/m L DEP.2.Bioinformatics analysis showed that mitochondrial energy metabolism pathway was an important regulatory pathway of myocardial hypoxia injury,and DEP exposure resulted in decreased ATP content and decreased expression of oxidative phosphorylated protein in myocardial tissue and cardiomyocytes of mice.In DEP-induced anoxic injury cell model,the addition of mitochondrial agonist EGCG alleviated the decrease of ATP,the decrease of oxidative phosphorylated protein expression and the increase of apoptosis caused by DEP exposure.3.DEP exposure resulted in decreased expression of PDH,a key Mg²⁺and Ca²⁺regulated enzyme,and decreased ICDH activity in mouse myocardial tissue and cardiomyocytes.In DEP-induced anoxic injury cell model,Mg²⁺and Ca²⁺supplementation alleviated the decreased expression of PDH caused by DEP exposure,and Mg²⁺accounted for a larger proportion of the effect of DEP on PDH.Meanwhile,Mg²⁺supplementation alleviated the decrease of ATP,the decrease of oxidative phosphorylated protein expression and the increase of apoptosis caused by DEP exposure.4.In DEP-induced hypoxia-damaged cell models,the expression of Mg²⁺uptake protein Mag T1 increased and the expression of efflux protein SLC41A1 decreased when DEP was exposed for 0.5 h,and the content of Mg²⁺increased when DEP was exposed for 2 h.DEP exposure resulted in decreased expression of Mg²⁺uptake protein Mrs2 and increased expression of SLC41A3 in myocardial tissue and mitochondria of myocardial cells.Conclusions:This study revealed the toxic effect of DEP exposure on cardiac hypoxia injury in mice,clarified that mitochondrial energy metabolism is an important mechanism of DEP exposion-induced hypoxia injury,and clarified for the first time the dominant role of dynamic changes of Mg²⁺rather than Ca²⁺in myocardial energy metabolism regulated by DEP exposure.It is further analyzed that the abnormal endogenous magnesium ion mediated by Mg²⁺transporter is an important mechanism of the disturbance of mitochondrial energy metabolism by DEP.The functional characteristics and regulatory mechanisms of ischemic injury induced by DEP exposure were elucidated,which provided a new perspective and thought for the study of toxicological mechanisms related to cardiovascular diseases caused by particulate matter exposure.