Inactivation Of SERCA2 Cysteine 674 Accelerates Atherosclerosis And The Mechanism Involved

Atherosclerosis is the main cause of cardio-cerebrovascular diseases such as coronary heart disease and stroke.Atherosclerosis possesses extremely high morbidity and fatalities,which is a serious threat to human health.The number of deaths caused by cardiovascular diseases ranks the first among all deaths in the world.Atherogenesis is complex,and the control rate of atherosclerosis is still at a low level.It is particularly important to clarify the occurrence and development mechanism of atherosclerosis.Sarcoendoplasmic reticulum Ca²⁺ATPase（SERCA）is the only protein that transports Ca²⁺in the cytoplasm to the sarcoplasmic reticulum/endoplasmic reticulum,which is essential for maintaining intracellular calcium homeostasis.SERCA2 is the main subtype of SERCA in cardiovascular system.The glutathionylation of the sulfhydryl group of cysteine 674（C674）is the active regulation form of SERCA2.Excessive reactive oxygen species causes the irreversibly oxidization of C674 sulfhydryl groupand interferes with the function of SERCA.SERCA2 C674 is found irreversible oxidated in the plaques of atherosclerotic patients and atherosclerosis animal models.In the pathological conditions susceptible to atherosclerosis,whether C674 inactivation is directly involved in the development of atherosclerosis and related regulatory mechanisms is still unclear.This article is mainly discusse the relationship between C674inactivation in SERCA2 and atherosclerosis and the possible molecular mechanisms.Conducts drug interventions against the relevant targets of these molecular mechanisms,and explores their role in the treatment of atherosclerosis.The main research contents are as follows:1.Inactivation of C674 in SERCA2 induces endoplasmic reticulum stress and inflammation to exacerbate atherosclerosisWe constructed SERCA2 C674S mutant knock-in mice（SKI）to study the role of SERCA2 C674 inactivation in atherogenesis.Compared with the littermate of wild-type control（WT）mice,SKI mice demonstrated significantly increased aortic and aortic root plaque deposition,aortic root necrotic nucleus ratio and macrophage infiltration;The expression of ICAM-1 and VCAM-1 in the aortic endothelium were significantly up-regulated in SKI mice.In macrophages and endothelial cells,inactivation of C674increases cytoplasmic calcium ion concentration,up-regulates the protein expression of endoplasmic reticulum stress and inflammatory response markers,and promotes the interaction between macrophages and endothelial cells;The endoplasmic reticulum stress inhibitor 4-PBA reduces the protein level of endoplasmic reticulum stress and inflammatory response markers,and inhibits the interaction between macrophages and endothelial cells.Compared with the solvent control,oral administered 4-PBA reduced the deposition of atherosclerotic plaque and macrophage infiltration in the aorta and aortic root in SKI mice,which proves that the inactivation of C674 in SERCA2 aggravates atherosclerosis by inducing endoplasmic reticulum stress and inflammation.2.Macrophage C674 inactivation induces oxidative stress,interferes cholesterol metabolism and promotes foam cell formationIn pathological conditions such as diabetes,hypertension and atherosclerosis,the massive production of ROS causes oxidative stress interferes cholesterol metabolism in macrophages and promotes the transform to foam cells therefore intensifies atherosclerosis.We found that compared with WT macrophages,ROS level and the protein expression of NADPH oxidase 4（Nox4）,the main enzymes catalyzing ROS production,are both increased in SKI macrophages.In addition,C674 inactivation increases lipid deposition and cholesterol intake in macrophages,the expression of CD36,SRA-1,and ACAT-2 are upregularted,which promote cholesterol intake and esterification,the expression of n-CEH-1,ABCA-1,and ABCG-1 are downregularted,which inhibited lipid hydrolysis and cholesterol efflux.In SKI macrophages,compared with the solvent control,administration of the antioxidant-Tempol reduces the Nox4expression and ROS production as well as lipid deposits and cholesterol intake,and increases the expression of n-CEH-1,ABCA-1 and ABCG-1.These results indicate that the inactivation of C674 in SERCA2 induces oxidative stress,which leads to abnormal cholesterol metabolism in macrophages,therefore promotes the conversion of macrophages to foam cells.3.Construction of a novel nanoprobe for real-time monitoring of intracellular Ca²⁺The main function of SERCA2 is to regulate Ca²⁺,so it is very important to efficiently and sensitively detect the intracellular calcium content and calcium distribution.In view of the shortcomings of traditional calcium measurement reagents,we synthesized CPM NPs（CPM nanoparticles）by precipitation.The CPM NPs can specifically detect Ca²⁺,the degree of fluorescence enhancement of which is depending on the icreasing concetration of Ca²⁺,with the lowest detection limit of 0.04μM.CPM NPs have good biocompatibility,photobleaching resistance,and can be used for intracellular Ca²⁺detection.CPM@Cur NPs were prepared by co-precipitation and encapsulation of CPM and curcumin.Fluorescence energy resonance transfer between CPM and curcumin makes CPM@Cur NPs possess stronger fluorescence output and lower background interference than CPM NPs used for the intracellular Ca²⁺determination.Using CPM NPs and CPM@Cur NPs to detect the cytoplasmic Ca²⁺concentration of aortic smooth muscle cells,we confirmed that the inactivation of C674significantly increased the cytoplasmic Ca²⁺level.In summary,we are the first time found that the inactivation of C674 in SERCA2aggravate the process of atherosclerosis by interfering intracellular calcium homeostasis,activating the endoplasmic reticulum stress and inflammatory response in both macrophages and endothelial cells.Moreover,in macrophages,C674 inactivation induces oxidative stress,causes abnormal cholesterol metabolism and promotes the conversion of macrophages to foam cells.In addition,we synthesized CPM NPs and CPM@Cur NPs for intracellular Ca²⁺detection,further proved that the redox state of C674 is critical for maintaining intracellular calcium homeostasis.