Effect Of Calpain-I Overactivation On Atrial Fibrillation Via Degradation Of Junctophilin-2

BackgroundAtrial fibrillation（AF）is the most common atrial arrhythmia in clinics,with a severe atrial electrical activity disorder.The specific mechanism of AF is not completely clear,which severely restricts the efficacy and safety of AF treatment in clinics.A large number of studies have shown that abnormal intracellular calcium homeostasis in atrial myocytes,especially calcium leakage,is one of the important mechanisms in the initiation and progression of AF.Ryanodine receptor 2（RyR2）,the specific calcium channel mainly located at sarcoplasmic reticulum（SR）,plays an important role in cardiomyocyte calcium homeostasis.If the RyR2 channel is abnormally leaking during diastole,the SR Ca²⁺leakage will result in spontaneous calcium release events.In this case,the extra cytosolic Ca²⁺will be pumped out through the sodium calcium exchange（NCX）on the cell membrane,which may cause cardiomyocyte delayed afterdepolarization（DAD）meanwhile.The spontaneous action potential（AP）may be induced once DAD reaches the threshold,acting as trigger activities or“ectopic activities”.Thus inducing on the basis of atrial structural remodeling including atrial enlargement or atrial fibrosis and so on.Recent studies have found that decreased stability of RyR2 and the Junctophilin（JPH）-2（JPH2）down-regulation were important reasons that affect the Ca²⁺leak of atrial myocytes.We have documented that rapid atrial pacing leads to increased intracellular Ca²⁺concentration and activation of calpain-I,and its activation increases as AF progressed.Therefore,we hypothesized that high-frequency atrial rhythm could lead to atrial myocytes intracellular Ca²⁺overload,thereafter might activate calpain-Ⅰ,JPH2 protein degradation,leading to increased Ca²⁺leak,and progressing to AF.In this study,the correlation between calpain-Ⅰand JPH2 expression level was determined in AF patients and animal model.Objectives1.To explore the correlation between Calpain-I/JPH2 and AF in humans.2.To clarify whether rapid atrial pacing（RAP）could affect calcium homeostasis via inducing the changes in Calpain-I/JPH2 expression,and result in atrial fibrillation.Methods1.The expression changes of Calpain-I/JPH2 in AF patients1.1 The object of researchA total of 64 Patients who were hospitalized in Changzheng hospital affiliated to Naval Medical University to undergo cardiac surgery,were enrolled in this study.1.2 The groupsAccording to the patients’history and preoperative electrocardiogram,64 patients were divided into two groups:The atrial fibrillation group（AF group,n=32）and the sinus rhythm group（SR group,n=32）.1.3 The collection of specimen and clinical dataThe Left atrial appendage（LAA）for the study were removed in surgery and All the general information of patients admitted were collected.1.4 Biochemical study（1）Tissue explant-collagenase digestion and fluorescent staining method were used to evaluate the intracellular Ca²⁺concentration in atrial myocytes.（2）The kit was used to detect the activity of Calpain-Ⅰin different groups.（3）Q-PCR was used to detect the mRNA levels of JPH2 in different groups.（4）Western blot was used to detect the protein levels of JPH2 and Calpain-Ⅰin different groups.2.The Correlation of Calpain-I/JPH2 Protein Levels in left atrium and its relationship with Atrial Fibrillation in rabbit model2.1 The establishment and evaluation of rabbit AF model（1）Adult New Zealand rabbits weighing from 2.0 to 2.5 kg.（2）The preparation of model and the groups of research:Suture the stimulating electrodes to the surface of LAA by thoracotomy,start continuous upper-threshold stimulation and close the chest while the effective stimulation is observed.According to the experimental requirements,The operation group was divided into the RAP group and the RAP+Calpain-Ⅰinhibitor group.One week after operation,The RAP group started continuous RAP and intraperitoneal injection of Dimethyl sulfoxide（DMSO,solvent of Calpain-Ⅰinhibitor）for 4 weeks,The RAP+calpain-Ⅰinhibitor group began continuous RAP and injection of MDL28170（specific Calpain-I inhibitor）for 4 weeks and The sham-operated group which only performed chest operation was with continuous administration of DMSO for 4 weeks.（3）The telemetry system was used to record the duration of AF in AF（>30min for persistent AF<5min and able to self-terminator for non-persistent AF or paroxysmal AF）.2.2 The collection of specimenThe experimental rabbits were sacrificed at 5 weeks after operation,partial rabbits for isolating cardiomyocytes and partial for biochemical study.2.3 Biochemical study（1）The kit was used to detect the activity of Calpain-Ⅰin different groups.（2）Q-PCR was used to detect the mRNA levels of JPH2 in different groups.（3）Western blot was used to detect the protein levels of JPH2 and Calpain-Ⅰin different groups.（4）Langendorff perfusion and fluorescent staining method were used to evaluate the intracellular Ca²⁺concentration in atrial myocytes.3.AnalysisIBM SPSS statistics 21.0 statistical software was used for data processing and statistical analysis.A P value<0.05 was considered significant.Results1.The expression changes of Calpain-I/JPH2 in AF patients1.1 There was no significant difference in the Baseline characteristics of patients between two groups（P>0.05）.but the LAD in the AF patients was significantly higher than that in the SR patients（53.97±8.37 vs 41.81±8.18,P<0.05）.1.2 The intensity of intracellular Ca²⁺-related fluorescence in the AF group was significantly higher than that in SR group,and the mean fluorescence intensity of the AF group was significantly higher than that of the SR group（20.56±1.98 vs 8.92±1.26,P<0.01）.1.3 Based on the OD value of the SR group,the activity of Calpain-Ⅰin the AF group was significantly higher（P<0.05）.1.4 The JPH2 mRNA level in the AF group was not significantly different from that in the SR group（P>0.05）.1.5 The expression of JPH2 protein in AF group was significantly lower than that in the SR group（0.53±0.14 vs 0.82±0.27,P<0.01）.However,the expression of Calpain-Ⅰin the AF group was significantly higher than that in the SR group（0.89±0.09 vs 0.46±0.09,P=0.015）.1.6 There was a significant negative correlation between JPH2 protein expression and Calpain-Ⅰactivity in the AF group（P<0.01）.2.The Correlation of Calpain-I/JPH2 Protein Levels in left atrium and its relationship with Atrial Fibrillation in rabbit model2.1 Calpain-I inhibitor can reduce the incidence of atrial fibrillation caused by RAP（P<0.01）.2.2 Calpain-I inhibitor can reduce the increase of RAP-induced enzyme activity（P<0.01）.2.3 Calpain-I inhibitor had no effect on the expression of JPH2 mRNA（P>0.05）2.4 Calpain-I inhibitors inhibited the degradation of JPH2 protein induced by RAP（P<0.05）.2.5 There was a negative correlation between JPH2 protein expression and Calpain-Ⅰactivity in AF rabbits（P<0.01）.2.6 Calpain-I inhibitor can reduce the RAP-induced increase in intracellular calcium concentration（P<0.01）.Conclusions1.Calpain-I/JPH2 is closely related to the occurrence of atrial fibrillation in human.2.Calpain-Ⅰcan affect the intracellular calcium homeostasis of atrial myocardium by degrading JPH2 protein and induce the occurrence of atrial fibrillation.