Roles Of Calpain-1 In The Pathogenesis Of Atrial Fibrillation Through Endoplasmic Reticulum Stress-autophagy Pathway

BackgroundAtrial fibrillation（AF）is the most serious electrical activity disorder in the atrium of the heart and is the most common atrial arrhythmia which affects health of people and had increasing prevalence with advancing age,also seriously restricts the effectiveness and safety of clinical AF treatment.The pathogenesis of AF remains unclear.Focal electrical activation of the pulmonary veins is critical for the development of AF,but structural remodeling and electrophysiological remodeling of the atrium also play a crucial role in the AF maintenance phase which was verified in the study of persistent AF.In the development of AF,abnormal diastolic release of Ca²⁺in cardiomyocytes is considered to be the foremost mechanism in the late age of AF.A large number of studies have confirmed that autophagy plays an important role in myocardial injury.It is a non-apoptotic and programmed cell death induced by oxidative stress,injury,ischemia et al.At present,autophagy is believed to be the initial state of cell fibrosis,and a number of animal studies have also confirmed the existence of autophagic cell death in AF cells.Endoplasmic reticulum stress（ERS）-mediated autophagy has been observed in a variety of diseases and pathological processes.The ERS response can promote autophagy through the IRE1/JNK pathway.It has been confirmed that it can directly affect the phosphorylation of autophagy key proteins Beclin-1 and Bcl-2 and promote the autophagy of cells.ERS can also activated MAPKs pathways which promotes autophagy of cardiomyocytes,further atrial fibrosis,and plays an important role in the mechanism of AF.Calpain-1 belongs to the calcium-dependent family of cytosolic cysteine proteases.Studies have shown that Calpain-1 plays a crucial role in the development of AF by regulating calcium homeostasis.ERS is testified closely related to AF.The process of ERS-autophagy can promote the formation of atrial myocyte fibrosis,and atrial fibrosis is an important pathological structural remodeling of AF,which plays an important role in the mechanism of AF.Calpain-1 also plays an important regulatory role in the mechanism of ERS.Therefore,is the interaction between Calpain-1 and ERS one of the important mechanisms causing AF?This study will explore the effects of Calpain-1 on AF through the process of ERS-autophagy in from the patients,cellular and ultrastructural levels.Abnormal calcium release events and the regulation mechanism of abnormal electrical triggering activities such as DAD will be clarified in the role of the interaction between Calpain-1 and ERS-autophagy in the mechanism of AF,which can help to reveal the possible electrical remodeling of AF occurrence and development.We hope to provide new ideas and theoretical basis for the development of targeted drugs for AF treatment through our study.PartⅠExpression of Calpain-1 and ERS-autophagy related genes in atrial cadiomyocyte of patients with AFObjectiveTo investigate the clinical significance of Calpain-1 and ERS-autophagy relationship in the occurrence of AF and the correlation with AF through atrial myocardium specimens obtained from AF patients and that of sinus rhythm.MethodsAtrial cadiomyocyte tissue were obtained from consecutive surgical patients who underwent mitral valve replacement or operative surgery for mitral valve disease in the Department of Cardiac Surgery,Changcheng Hospital,Navy Military Medical University from January 2015 to December 2016.HE,MASSON staining,Western blotting and QT-PCR were used to detect the changes of Calpain-1 protein,mRNA expression and enzyme activity to detect ERS and autophagy-related proteins and mRNA,hoping to identify the role in AF pathogenesis.Results1.There were no significant differences between two groups in baseline cahracteristics including age,gender et al,as well as surgery related data such as aortic clamping time and cardiopulmonary bypass time.2.The number of cardiomyocyte in the atrial myocardium of patients with AF was significantly lower than that of the SR group.The cardiomyocyte arrangement was slightly disordered,the connective tissue hyperplasia was obvious,and the myocytes were dissolved in AF patients.3.Compared with the atrial myocytes in the SR group,the relative expression of Calpain-1 protein and enzyme activity in the AF group was significantly increased.4.The expression levels of ERS-related proteins GRP78,p-PERK,ATF6 and CHOP in atrial myocytes of AF group were significantly higher than those in SR group.5.Autophagy-specific protein LC3B-II and Beclin-1in patients in AF group expressed significantly higher than the SR group.Conclusions1.The expression of Calpain-1 protein in atrial myocytes of AF patients was significantly increased,and the activity of Calpain-1 was also significantly higher than that of SR patients.2.The expression and mRNA content of ERS-related proteins GRP78,p-PERK,ATF6and CHOP in atrial myocytes of patients with AF were significantly higher than those of SR patients.3.The expression and mRNA content of autophagy-related proteins LC3B-II and Beclin-I in atrial myocytes of patients with AF were significantly higher than those of SR patients.PartⅡEffect of Calpain-1 on ERS-autophagy related gene expressionObjectiveTo determine the changes of mRNA and protein expression of Calpain-1 in atrial tissue of AF animal model and the effects of Calpain-1 on ERS-autophagy in AF animal model.MethodsThe animal AF model of rabbit was established by rapid atrial pacing（RAP）method,and the experimental animals were divided into Sham,AF and AF+MD28170 groups by the intervention of Calpain-1 specific inhibitor MD28170.After the experimental treatment,the rabbit atrial myocytes were obtained to laboratory tests.HE,MASSON staining,Western blotting,QT-PCR,electron transmission electron microscopy and immunofluorescence confocal microscopy were used to detect the expression of Calpain-1,enzyme activity,ERS and autophage from the atrial myocytes of each group.The expression of autophage-associated protein and mRNA,morphological changes of autophagy in atrial myocytes and abnormal calcium release events were also detected to determine the effect of Calpain-1 on the ERS and autophagy in AF.Results1.The AF maintenance rate in each group was significantly different,while no significant difference was detected between AF and AF+MDL28170 AF group,also no significant difference in AF duration time was obtained between three groups.2.In the AF group,the myocardial cell interstitial showed obvious increase of blue-stained collagen fibers,collagen fiber proliferation and collagen fibers intertwined.The content of interstitial collagen fibers in AF+MDL28170 group was significantly lower than that in AF group with myocardial arrangement slightly ordered.3.The expression of Calpain-1 protein and enzyme activity in AF group was significantly up-regulated,but the expression of Calpain-1 protein and enzyme activity in AF+MDL28170group was significantly lower than that in AF group and Sham group.4.The expression of GRP78 and CHOP protein and mRNA in AF group was significantly higher than that in AF+MDL28170 group,but the expression of p-PERK and ATF6 was not significantly different.5.Autophagy-specific protein LC3B-II and Beclin-1 expression were significantly higher in AF group and AF+MDL28170 groups than Sham group.6.The autophagosomes and LC3B-II fluorescence spots of atrial myocytes in AF group were significantly increased.The autophagosomes and LC3B-II fluorescence points of AF+MDL28170 group were significantly reduced,but still increased compared with Sham group.Conclusions1.In the RAP AF model of rabbit,the expression of ERS-related proteins and mRNA of GRP78,p-PERK,ATF6 and CHOP in the AF group and the Calpain-1-inhibitor group were significantly increased compared with the Sham group.2.The expression of CHOP in cardiomyocytes of AF group were significantly higher than those of Calpain-1-inhibitor group.3.The autophagy-associated protein LC3B-II and autophagic flow expression in AF atrial myocytes were significantly increased.PartⅢRoles of Calpain-1 in the pathogenesis of AF through ERS-autophagy pathwayObjectiveTo clarify the expression of Calpain-1 through the ERS-autophagy pathway in AF animal models and their possible mechanism of AF development.MethodsOn the basis of the part II,the experimental animals were divided into 6 groups by intervention with ERS activators,inhibitors and Calpain-1 specific inhibitors.HE,MASSON staining,Western blotting,QT-PCR,electron transmission electron microscopy and immunofluorescence confocal microscopy were used to detect the changes of Calpain-1 activity,ERS and autophagy-related proteins and mRNA in atrial myocytes of experimental animals.The expression,autophagy morphological changes of atrial myocytes and abnormal calcium release events were detected to determine the effect of Calpain-1 on AF through ERS and autophagy of atrial myocytes.Results1.Atrial myocardial interstitial in AF and AF+MT group showed blue staining of collagen fibers,collagen fibrosis increased,myocardial arrangement disorder and a little nuclear lysis;2.The activity of Calpain-1 protein and enzyme in AF,AF+4PBA and AF+MT groups increased significantly.In addition,the expression of Calpain-1 protein in Sham group was higher than that in AF+MDL28170+4PBA and AF+MDL28170+MT group.3.The expressions of ERS-related proteins GRP78,p-PERK,ATF6 and CHOP were significantly higher in AF+MT group.The expression of ERS-related protein in AF and AF+MDL28170+MT group was lower than that in AF+MT group,but higher than Sham,AF+4-PBA and AF+MDL28170+4-PBA groups.4.The expressions of autophagy-specific proteins LC3B-II and Beclin-1 in AF group and AF+MDL28170 group were significantly higher than those in Sham group.Compared with AF group,autophagy-related protein in AF+MT group was significantly increased,while there was no significant difference between AF group and AF+MDL28170+MT group.5.Compared with Sham group and with other groups,autophagosome and LC3B-II fluorescence point in AF group and AF+MT group significantly increased,but more obvious in AF+MT group;in atrial myocytes from There were no significant differences in autophagosome and LC3B-II fluorescence spots between AF+MDL28170+MT and AF group.Conclusions1.In the RAP AF model of rabbit,ERS activator can significantly increase the expression of autophagy-related proteins and mRNA Benlin-1 and LC3II-B of atrial myocytes.2.The extent to which Calpain-1 activates autophagy in atrial myocytes in atrial myocytes of AF animals can be reduced by ERS inhibitors.3.Calpain-1 inhibitor combined with ERS inhibitor can significantly reduce the degree and occurrence of autophagy in AF atrial myocytes.In summary,Calpain-1 may activate the autophagy level of atrial myocytes through the CHOP signaling pathway in ERS,and thus play an important role in the mechanism of AF.