The Inactivation Of SERCA2 C674 Accelerates Angiotensin Ii-induced Aortic Aneurysm And The Mechanism Involved

Aortic aneurysm is the crucial macrovascular disease with high mortality and lethality.It is a serious threat to human health and there are currently no effective drugs for its treatment.Sarcoplasmic/Endoplasmic Reticulum Ca²⁺ATPase（SERCA）,located in Sarcoplasmic/Endoplasmic Reticulum（SR/ER）,is vital to maintain cellular calcium homeostasis.SERCA2 is the main subtype in cardiovascular system,its Cysteine-674（C674）is surrounded by positively charged arginine.In physiology,C674 is glutathiolated by nitric oxide while in many pathological conditions,such as restenosis,atherosclerosis,etc.,the significant irreversible oxidation（SERCA2 C674-SO₃H）occured.However,the causative link between the irreversible oxidation of SERCA2C674 and aortic aneurysm remain enigmatic.We therefore sought to elucidate the relationship of SERCA2 C674 with aortic aneurysm.By using SERCA2 C674S knock-in（SKI）mouse line,in which half of C674 was substituted by serine 674（S674）to represent partial irreversible oxidation of C674（refer to as C674 inactivation）,then the SKI mice was cross-bred with low density lipoprotein deficiency(LDLR^-/-)mice to construct the Ang II infusion aortic aneurysm model.The cell biology,molecular biology and omics technologies were used to investigate the contribution of inactivation of C674 to the development of aortic aneurysm and the mechanism involved.The new finding and novel insights of this study are presented as follows,（1）Protein sequence comparison and analysis showed that the conserved cysteine site near the 674 site existed ranging from birds to mammals which demonstrated the conservation of this site in different species.In addition,the conserved cysteine（in some subtype,675）was existed in all subtypes of SERCA in mice which highlighted the importance of C674.（2）The WT(LDLR^-/-)male mice were infused with saline or Ang II（1000ng/kg/day）for 28 days to induce experimental aortic aneurysm.We found that Ang II induced the aortic aneurysm formation,mainly in abdominal aneurysm.The C674-SO₃H was up-regulated in both human and mouse aortic aneurysm samples by immunohistochemistry and immunofluorescence,mainly in the SMC,indicating that irreversible oxidation of C674 was associated with aneurysm formation.（3）The aortic smooth muscle cells（SMC）in C57/B6J background showed that C674inactivation increased intracellular calcium.The calcineurin（Ca N）activity and the nuclear translocation of NFAT4/NFκB were significantly increased in SKI SMC.Overexpression of human SERCA2a/b S674 in WT SMC using adenoviral system activated the NFAT4 nucleation.The Ca N inhibitor cyclosporine A（Cs A）significantly inhibited the nucleation of NFAT4/NFκB in SKI SMC,implying that inactivation of C674 activated the Ca²⁺dependent Ca N-NFAT4/NFκB signaling pathway.（4）In aorta and aortic SMC,the inactivation of C674 up-regulated the SMC dedifferentiated proteins Col I,Col III,MMP2,OPN,VCAM1,ICAM1 and p-p65NFκB while decreased the differentiated protein MYOCD expression;increased the proliferation,migration and the adhesion of macrophages to SMC.However,those changes were all reversed after the treatment of Cs A or PDTC.In addition,the intracellular Ca²⁺chelator BAPTA-AM reversed part of proteins expression of SKI SMC.These results suggested that inactivation of SERCA2 C674 promoted SMC phenotypic modulation by activating the Ca²⁺dependent Ca N-NFAT4/NFκB signaling pathway.（5）Gene expression profiles showed that 40 DEGs were differentially expressed between the SKI/LDLR^-/-and LDLR^-/-.Those DEGs enriched in 20 pathways（p<0.05）,mainly in pyruvate metabolism,PPAR signaling,oxidative phosphorylation and myocardial contraction.Both the q PCR and western blot confirmed that the inactivation of C674 inhibited the PPARγ2 m RNA expression and PPARγ（PPARγ1 and PPARγ2）protein expression.PPARγactivator PIOG or PPARγ2 overexpression reversed the expression of SMC phenotypic modulation related proteins;inhibited SMC proliferation,migration and macrophage adhesion in SKI SMC.Those results indicated that PPARγmediated the inactivation of C674 promoted phenotypic modulation.（6）By constructing the mice PPARγ2-Luciferase promoter,we found that the inactivation of SERCA2 C674 obviously inhibited the PPARγ2 promoter activity,while both the Cs A and PDTC reversed the reduced PPARγ2 promoter activity,suggesting that NFAT4/NFκB enable to bind to PPARγ2 promoter to suppress its transcription.Similarly,we found that Cs A and PDTC significantly inhibited the protein expression of PPARγ（PPARγ1 and PPARγ2）in SKI SMC while PIOG inhibited the nuclear translocation of NFAT4/NFκB in SKI SMC,indicating that NFAT4/NFκB and PPARγregulated mutually to maintain cellular homeostasis and the inactivation of C674 broke this vicious circle thus promoted SMC phenotypic modulation.（7）In vivo,the WT(LDLR^-/-)and SKI(SKI/LDLR^-/-)male mice were infused with Ang II（1000 ng/kg/min）for 28 days to induce experimental aortic aneurysm and then the SKI was divided into two groups.After one-week,sterilized water（SKI/Ctrl）and PIOG（SKI/PIOG）were given to the stomach respectively.Meanwhile,the WT was also given the same volume of sterilized water（WT/Ctrl）as the control.We found that the survival rate of the SKI/Ctrl was significantly lower than that of the WT/Ctrl control.While the incidence rate,the number of aortic aneurysms,the aneurysm rupture,elastin degradation ratio of grade IV,aneurysm expansion and collagen deposition were increased,and these indexes were ameliorated to WT/Ctrl level in SKI/PIOG.Those results demonstrated that the inactivation of C674 significantly promoted the aortic aneurysms formation,while PPARγabrogated the inactivation of C674 accelerated aortic aneurysm formation.