Study On The Relationship And Mechanism Between The Inactivation Of 674 Cysteine In SERCA2 And Mitochondrial Dysfunction Of Aortic Smooth Muscle Cells

Mitochondria are the centers of aerobic respiration release of eukaryotic cells.They are closely related to the maintenance of calcium homeostasis,the generation of free radicals,the regulation of signal transduction and growth cycle,and the autophagy and apoptosis.Mitochondrial osmotic transformation,abnormal levels of intracellular calcium ion(intracellular Ca²⁺,[Ca²⁺]_I),electron transport chain damage,abnormal mitochondrial enzyme activity,mitochondrial DNA damage,and excessive generation and accumulation of mitochondrial free radicals can all cause disturbance of energy metabolism in cells.Causes mitochondrial dysfunction.Abnormal mitochondrial function directly affects the normal function of cells,leading to the occurrence of various metabolic diseases.Therefore,clarifying the molecular mechanism of mitochondrial dysfunction is particularly critical for the prevention and treatment of related diseases.The main role of sarcoplasmic/endoplasmic reticulum calcium ATPase（SERCA）is to actively transport[Ca²⁺]_I to sarcoplasmic reticulum and endoplasmic reticulum,ER)is a key enzyme that regulates Ca²⁺in cells,and is essential for the maintenance of intracellular calcium homeostasis and ER homeostasis.SERCA2 is the main subtype of SERCA in the cardiovascular system.Its 674th cysteine（C674）is a major redox site that regulates SERCA2 function.Through analysis of tissue samples such as atherosclerosis and abdominal aortic aneurysm,we found that the pathological conditions leading to oxidative stress all cause the irreversible oxidation of SERCA2C674（C674-SO₃H）inactivation.In order to simulate the irreversible oxidative inactivation of this site under pathological conditions,we mutated the cysteine at position 674 of SERCA2 to serine（C674→S674）,constructed the SERCA2 C674S gene knock-in mouse（SKI）,wild type mice（WT）from the same litter（without S674）were used as controls.Homozygous SKI is embryonic lethal.Heterozygous SKI（hereinafter referred to as SKI）contains half C674 and half S674,simulating the partial inactivation of C674 under pathological conditions（hereinafter referred to as C674inactivation）.In aortic smooth muscle cells（SMC）,C674 inactivation significantly increased[Ca²⁺]_I and mitochondrial calcium ion(mitochondrial Ca²⁺,[Ca²⁺]_M),inhibiting the synthesis of mitochondrial adenosine triphosphate（ATP）,increasing the accumulation of reactive oxygen species（ROS）.It indicated that C674 inactivation causes mitochondrial dysfunction.This topic mainly analyzes the relationship between SERCA2 C674 inactivation and mitochondrial dysfunction and related regulatory mechanisms from the perspective of aortic SMC,providing new theoretical basis and intervention targets for the prevention and treatment of clinically related diseases,and has good application value.The main research contents are as follows:（1）C674 inactivation induces endoplasmic reticulum stress by interfering with the intracellular calcium homeostasis,affecting the energy metabolism of cellsSERCA is a key enzyme for ingesting Ca²⁺from the cytoplasm to the sarcoplasmic reticulum/ER.ER is the largest and most important calcium store in the cell and is essential for maintaining the normal function of the cell.In the aortic SMC,we found that C674 inactivation significantly increased[Ca²⁺]_I,activated IRE1α,PERK and ATF6signaling pathways,and induced ER stress.Further research found that C674inactivation leads to[Ca²⁺]_M overload,reduces the mitochondrial transmembrane potential（Δψm）,inhibits the phosphorylation of AMP-activated protein kinase（AMPK）,which in turn affects fatty acidβoxidation pathway related protein acetyl-Co A carboxylase 1（ACC1）phosphorylation downregulates the protein expression of carnitine palmitoyl transferase 1A（CPT-1A）.At the same time,C674 inactivation downregulates the expression of oxidative phosphorylation（OXPHOS）pathway related proteins,including ATP synthase F0 subunit 6（ATP6）and cytochrome c oxidase Ⅱ（MT-CO2）and cytochrome c oxidase Ⅳ（COXIV）and NADH dehydrogenase subunit1（ND1）.It indicated that C674 inactivation caused disturbance of cell energy metabolism and induced mitochondrial dysfunction by interfering with intracellular calcium homeostasis.Inhibition of ER stress（4-PBA and 4μ8c）and activation of AMPK（AICAR and metformin）can significantly alleviate the effect of C674inactivation on fatty acidβoxidation and OXPHOS pathway related protein expression,indicating that C674 inactivation can be induced by ER stress Stimulates and inhibits AMPK activity to block intracellular energy metabolism.（2）C674 inactivation inhibits mitochondrial ATP productionFluorescent carbon quantum dots,as a new type of fluorescent carbon nanomaterials,are widely used in the field of biomedicine due to their advantages such as good light stability,non-flickering light,and no bleaching.We used polyethyleneimine and polybutenetetracarboxylic anhydride as raw materials,ethanol as a solvent,and hydrothermally prepared single-layered graphene quantum dots（s-GQDs）with large conjugate planes and positive charge.Because ATP has a negatively charged triphosphate moiety and aromatic conjugated adenine,the s-GQDs specifically bind to ATP throughπ-πstacking and electrostatic interaction,and the aggregation of s-GQDs causes fluorescence quenching.At the same time,s-GQDs are mainly enriched in mitochondria due to a certain degree of hydrophobicity and positive charge,which can be used for real-time analysis of ATP in mitochondria.To further investigate the effect of SERCA2 C674 inactivation on the energy metabolism of aortic SMC,we used this s-GQDs to perform real-time imaging analysis of ATP in WT and SKI aortic SMC.The results showed that compared with WT,the fluorescence intensity of s-GQDs in SKI aortic SMC was significantly increased,indicating that C674 inactivation inhibited the generation of ATP;and inhibition of ER stress could significantly alleviate the effect of C674 inactivation on cellular ATP production,indicating that C674 Inactivation-induced ER stress is involved in the regulation of ATP production.（3）C674 inactivation promotes the generation of ROS and induces apoptosisMitochondria are the main sites of ROS production in cells,and also the main organelles attacked by ROS.We found that C674 inactivation significantly increased the level of ROS in aortic SMC,inhibited mitochondrial antioxidant enzyme activity,and induced apoptosis.In SKI aortic SMC,the superoxide scavenger Tempol reduces the level of ROS,inhibits cell apoptosis,alleviates the inhibition of OXPHOS and fatty acidβoxidation by C674 inactivation,and increases the production of ATP.These results indicate that C674 inactivation participates in mitochondrial energy metabolism abnormalities by increasing ROS production,while inhibiting excessive ROS production improves C674 inactivation-mediated energy metabolism disorders.In addition,we found that activating AMPK can alleviate the oxidative stress caused by C674 inactivation and promote mitochondrial energy metabolism.In summary,the inactivation of C674 in SERCA2 leads to disordered calcium regulation,induces ER stress and oxidative stress,interferes with mitochondrial energy metabolism,and causes mitochondrial dysfunction.