Study On The Mechanism Of SERCA2 Redox Site C674 Inactivation Aggravating Cardiac Fibrosis

BACKGROUND:Cardiac fibrosis is involved in the development and progression of many heart diseases.And it is the compensatory response of the heart to external harmful stimuli.The pathological is featured with deposition of myocardial extracellular matrix with collagen as the main component.Accumulation of these matrices can cause tissue stiffness,decreased heart compliance,and diastolic dysfunction,which can eventually lead to ventricular dilatation,cardiac hypertrophy,and congestive heart failure.The pathogenesis of cardiac fibrosis mainly includes phenotypic transformation,inflammatory reaction and oxidative stress of cardiac fibroblasts.The mechanisms promote each other.The transformation of fibroblasts into myofibroblasts is the key factor to cardiac fibrosis.Sarcoplasmic/endoplasmic reticulum calcium ATPase（SERCA）is a key protein transporting Ca²⁺on the endoplasmic reticulum membrane and is essential for maintaining calcium homeostasis in the cytoplasm and endoplasmic reticulum.SERCA2 is the major subtype in the heart,and its sulfhydryl group on cysteine 674（C674）is an important redox site that regulates cardiovascular homeostasis.A commonly used model of cardiac fibrosis is induced by angiotensinⅡ（AngⅡ）perfused mice.We found that the AngⅡ-treated mouse heart was significantly fibrotic,and the production of reactive oxygen species（ROS）increased in both cardiac and cardiac fibroblasts,further increasing the irreversible oxidation of C674 on SERCA2.It is speculated that the irreversible oxidation of C674 is involved in the progression of cardiac fibrosis.METHOD AND RESULTS:To demonstrate that irreversible oxidation of C674 on SERCA2 is not only a marker of increased ROS,but may also be directly involved in the progression of cardiac fibrosis,we mutated C674 to serine（serine 674,S674）to mimic the inactivation of redox modification caused by irreversible oxidation of this site under pathological conditions,and SERCA2C674S knockin（SKI）mice were constructed.We found that in the AngⅡ-induced cardiac fibrosis model,the collagen deposition in the cardiac interstitial of SKI mice was significantly higher than that in the control wild type（WT）mice,indicating that the inactivation of C674 on SERCA2 was directly involved in cardiac fibrosis process.Compared with WT mice:1)The expression of fibrosis-associated factors（α-smooth muscle actin,collagenⅠ&Ⅲ）and inflammatory factors（interleukin6,vascular adhesion factor1,phosphorylated p65nuclear transcription factor）in SKI mouse hearts was significantly upregulated.2)The expression of proteins involved in cell transformation and fibrosis in heart fibroblasts of SKI mice（α-smooth muscle actin,collagenⅠ&Ⅲ,matrix metalloproteinase）and inflammatory factors（interleukin6,blood vessels adhesion factor1,phosphorylated p65nuclear transcription factor）was also significantly up-regulated.3)Ca²⁺concentration in cardiac fibroblasts of SKI mice was increased,and calcineurin-activated T cell nuclear factor pathway was actived.Cyclosporin A inhibiting this pathway reverses up-regulated fibrosis and inflammatory-associated proteins expression.4)In cardiac fibroblasts of SKI mice,activation of activated T cell nuclear factor can induce transdifferentiation of cardiac fibroblasts into myofibroblasts by up-regulating the expression of gap junction protein 43 and transforming growth factor beta,thereby promoting cardiac fibrosis.After treatment with SERCA activator CDN1163 in cardiac fibroblasts of SKI mice,the elevated expression of fibroblast transition-related proteins（α-smooth muscle actin,collagenⅠ&Ⅲ,matrix metalloproteinase）,inflammatory factors（interleukin6,blood vessels adhesion factor1,phosphorylated p65nuclear transcription factor）,connexin 43 and transforming growth factorβ1 was significantly down-regulated,and fibrosis reaction was alleviated.CONCLUSION:Irreversible oxidation of cysteine at position 674 on SERCA2promotes cardiac fibrosis by inducing the conversion of cardiac fibroblasts into myofibroblasts and inflammatory responses.