Mechanisms Of Inactivation The Redox Cysteine 674 Of SERCA2 To Promote Pulmonary Artery Remodeling

Pulmonary arterial hypertension?PAH?is a progressive cardiopulmonary disorder and clinical hemodynamic syndrome characterized by elevated pulmonary arterial pressure and pulmonary vascular resistance,leading to right heart failure and death.The incidence and cure rate of PAH are low,but its mortality is high.The main reasons of increased pulmonary arterial pressure include vasoconstriction,vascular remodeling,and formation of in situ thrombosis.Pulmonary arterial remodeling is the most prominent and irreversible histopathological feature in PAH.Pulmonary arterial remodeling in PAH has been draw more and more attention,involving the thickening of the adventitia,the media and the intima.The media of the pulmonary arterial composed of smooth muscle cells has been the focus of PAH research.Phenotype switching from contractile to synthetic type,increased proliferation and migration,collagen deposition,decreased apoptosis and autophagy are all involved in pulmonary arterial remodeling.Sarcoplasmic/endoplasmic reticulum calcium ATPase?SERCA?plays an important role in transporting cytosolic Ca²⁺into the sarcoplasmic reticulum and endoplasmic reticulum to regulate calcium homeostasis and calcium signaling.SERCA2 is the major subtype of SERCA in blood vessels.Its sulfhydryl group at cysteine 674?C674?is an important redox regulation site,which could be glutathiolated by the vasodilator nitric oxide?NO?to increase SERCA2 activity.The irreversible oxidation of C674?C674-SO₃H?of SERCA2,which interferes with its glutathionylation by NO,was significantly increased in pathological conditions such as diabetes and atherosclerosis.We found that C674-SO₃H was significantly increased in lung tissue of hypoxia-induced PAH mice,and we hypothesized that the irreversible oxidation of C674 of SERCA2 under pathological conditions might be involved in pulmonary arterial remodeling and PAH.We constructed a SERCA2 C674S knock-in?SERCA2C674S knock-in,SKI?in C57BL/6J background to mimic the inactivation of the C674redox site of SERCA2 under pathological conditions.The homozygous SKI mice were embryonic lethal,indicating that redox status of C674 of SERCA2 is key to maintain cell homeostasis.We found that heterozygous SKI mice developed significant pulmonary arterial remodeling,confirmed the direct contribution of the inactivation of redox C674.Our study was to investigate the regulatory mechanisms of the inactivation of C674 redox site of SERCA2 that promoted pulmonary arterial remodeling,aiming to provide a new theoretical basis and intervention target for pulmonary arterial remodeling and PAH prevention and treatment.We found significant pulmonary arterial remodeling in SKI mice.Compared to PASMC from wild type?WT?,PASMC from SKI mice has decreased expression of SERCA2 and increased expression of C674-SO₃H,decreased expression of peroxisome proliferator-activated receptor gamma?PPAR??,increased expression of endoplasmic reticulum stress-related protein ATF-6,BIP and IRE1?,decreased expression of SMC contractile-associated proteins?SMA and myocardin,decreased expression of apoptosis-related protein?cl-caspase3?and autophagy-related protein?the ratio of LC3BII/LC3BI?,increased expression of collagen deposition related protein MMP2 and collagen I and increased proliferation and migration.It was speculated that the inactivation of the redox site of C674 of SERCA2 under pathological conditions promotes the remodeling of the pulmonary arterial by regulating the above protains.In PASMC from SKI,PPAR?agonist pioglizatone increased the expression of cl-caspase3and LC3BII and the ratio of LC3BII/LC3BI,inhibited SMC phenotypic switching by increasing the expression of?SMA and myocardin,inhibited the expression of MMP2and collagen I,had no effect on the expression of endoplasmic reticulum stress-related proteins and inhibited proliferation and migration.Endoplasmic reticulum stress inhibitor 4-phenylbutyric acid?4-PBA?in PASMC from SKI mice inhibited endoplasmic reticulum stress-related protein expression,up-regulated the expression of cl-caspase3,inhibited the expression of MMP2 and collagen I,had no effect on the expression of PPAR?and inhibited cell proliferation and migration.In addition,PASMC from WT with overexpressing SERCA2b C674S?including SERCA2a C674S and SERCA2b C674S?by defective adenovirus,compared to control-deficient adenovirus increased expression of endoplasmic reticulum stress-related protein,decreased the expression of PPAR?and increased proliferative ability.Overexpression of SERCA2?including SERCA2a and SERCA2b?in PASMC from SKI mice increased expression of PPAR?and inhibitd proliferative ability.Our results indicated that inactivation of C674 redox site of SERCA2 involved in pulmonary arterial remodeling by?1?reducing the expression of PPAR?to reduce autophagy and apoptosis of PASMC,promote phenotypic switching and collagen deposition of PASMC;and?2?activating endoplasmic reticulum stress to reduce the apoptosis and promote collagen deposition of PASMC.