| BackgroundAtrial Fibrialltion(AF)is the most common chronic arrhythmia in the clinic,and its incidence,disability and mortality are increasing.Diabetes Mellitus(DM)is an important risk factor for the occurrence and development of atrial fibrillation.Combination with DM can increase the incidence of AF by 40%,but the pathophysiological mechanism of atrial fibrillation caused by diabetes is still unclear.Calpain-1 is a proteolytic enzyme in myocardial tissue that degrades a variety of proteins and participates in pathological and physiological processes such as cell differentiation,apoptosis and necrosis.Previous studies have found that Calpain-1 participates in atrial tissue structural remodeling and electrical remodeling,playing an important role in the occurrence and development of atrial fibrillation.Under stress,Calpain-1 degrades Junctophilin-2(JPH2),thereby reducing the stability of the dialostic closed state of the Rynedine Receptor 2(RyR2)channel,resulting in increases of calcium leakage via sarcoplasmic reticulum,which induces abnormal electrical triggering activity such as depolarization after the delay.It is reported that high-fat diet and abnormal glucose metabolism can activate Calpain-1,causing tissue fibrosis.Therefore,we speculate that the state of diabetes can cause hyperglycemia and insulin resistance,which in return activate atrial tissue Calpain-1,causing electrical remodeling and structural remodeling of atrial tissue,and thus leading to the occurrence of atrial fibrillation.ObjectivesThis study collects clinical data of diabetic patients,analyzes tissue samples of diabetic patients,constructs a mouse model of diabetes,and applies a model of diabetes in transgenic mice to:1.Identify the atrial electrical remodeling and structural remodeling of diabetic patients;2.Clarify the mechanism of action of Calpain-1 in the development of diabetes-induced atrial fibrillation;3.Verify the inhibition of Calpain-1 on the prevention of diabetic atrial fibrillation;Methods1.Clinical study of diabetes affecting atrial remodeling86 patients with coronary artery bypass grafting(CABG)who underwent coronary artery disease admitted to the Department of Cardiovascular Surgery,Changzheng Hospital,Second Military Medical University from March 2016 to June 2018.According to the medical history,there was no type 2 diabetes.Patients were divided into two groups,the diabetic group and the control group.There were no significant differences in general data such as gender,age,weight,BM,NYHA classification between the two groups.All patients received general information after admission and underwent preoperative echocardiography.During the operation,a small amount of atrial tissue was taken at the incision for experimental study.Cardiac ultrasound was used to evaluate atrial enlargement and other structural remodeling indexes.Masson staining was used to evaluate atrial fibrosis,and collagen volume fraction(CVF)was used to quantitatively assess fibrosis.Western blot was used to evaluate the expression of key proteins such as Calpain-1 and JPH2 in myocardial tissue.All data were analyzed by SPSS22.0 statistical software,P<0.05 was statistically significant.2,Construction and verification of diabetic mouse modelThe C57BL/6J experimental mice were randomly divided into the diabetic group(DM group)and the control group(Control group).The DM group was given a high fat diet(HFD)and a small dose(75 mg/Kg body weight)Streptozotocin was injected intraperitoneally(Streptozocin,STZ)to establish a diabetic model,and the Control group was given a conventional diet(Normal Diet,ND)and an equivalent dose(75 mg/kg body weight)of citrate buffer for intraperitoneal injection.The mice were tested for fasting blood glucose(FBG)before administration(12 weeks)and modeling(20 weeks),and the esophageal programmed electrical stimulation was given at 21 weeks to evaluate the recovery time of sinus node(Sinus).Node Recovery Time(SNRT)and Atrial Effective Refractory Time(AERP)were used to detect atrial fibrillation induction rate and AF duration in both groups.After the mice were sacrificed,the mouse myocardial specimens were obtained,and Calpain-1 activity detection,calcium imaging,Western blot,Hematoxylin-eosin(HE)staining,Masson staining and other experimental studies were performed.All data were analyzed by GraphPad Prsim 7.0 statistical software,P<0.05 was statistically significant.3.Experimental study on the protective effect of CalpastatinCalpastatin(CAST)is an endogenous specific inhibitory protein of Calpain-1.We used CAST overexpression(CAST OE)mice as subjects and wild type(CAST WT)mice as controls.According to the genotype,the experimental mice were divided into diabetic overexpression group(DM+OE)group,diabetic wild type group(DM+WT)group,conventional dietary overexpression group(ND+OE)group,and conventional diet wild type group(ND).+WT)group,DM+OE group,DM+WT group gave diabetes model with HFD and low dose STZ,ND+OE group,ND+WT group gave regular diet(ND)and the same dose of citrate buffer is injected intraperitoneally.Mouse FBG was detected before administration(12 weeks)and modeling was completed(20 weeks),and transesophageal electrical stimulation was performed at 21 weeks to evaluate SNRT and AERP.The atrial fibrillation rate and atrial fibrillation were detected in the two groups.duration.After the mice were sacrificed,the mouse myocardial specimens were obtained,and Calpain-1 activity assay,calcium imaging,Western blot,Masson staining and other experimental studies were performed.All data were analyzed by GraphPad Prsim 7.0 statistical software,P<0.05 was statistically significant.Results1.Clinical study of diabetes affecting atrial remodeling1.1 Baseline dataThere were no significant differences in gender,age,body mass index(BMI),smoking status,etc.between the diabetic group and the control group(P>0.05).The two groups were in NYHA classification,hypertension,atrial fibrillation,stroke,and chronic obstruction.There was no significant difference in the incidence of comorbidities such as pulmonary disease(P>0.05).The levels of FBG and glycosylated hemoglobin(HbAlc)in the diabetic group were significantly higher than those in the control group(P<0.05),while the indicators of hemoglobin(Hb),low-density lipoprotein cholesterol(LDL-C),and creatinine(Creatine)were not significant.Difference(P>0:05)1.2 Atrial structural remodelingThe left atrial diameter(LAD)and left atrial volume index(LAVI)of the diabetic group were significantly higher than those of the control group(P=0.001),while the degree of atrial fibrosis was also higher than that of the control group,and the CVF was significantly higher than that of the control group(P=0.03).Univariate linear regression analysis showed that the level of HbAlc in diabetic patients was linearly correlated with LAD(Y=1.139X+25.575,P<0.001,R2=0.291)and CVF(Y=0:444X+29.648,P=0.009,R2=0.078).1.3 Atrial electrical remodelingThe expression of Calpain-1 in myocardial tissue of diabetic patients was significantly higher than that of the control group(P<0.001),while the expression of JPH2 was significantly lower than that of the control group(P<0.001).2.Construction and yalidation of a mouse model of diabetes2.1 Model constructionThe body weight of the mice in the DM group was higher than that in the control group at the 8th week and the 12th week.After the STZ injection at the 12th week,the body weight of the DM group was significantly decreased,but the body weight of the mice increased again after the 16th week.There was no significant difference in body weight between the two groups at each time point(P>0.05).When STZ was not injected in the 12th week,the FBG of the DM group was(110.9+19.6)mg/dl,and the FBG of the Control group was(75.4+10.2)mg/dl.The difference between the two groups was statistically significant(P=0.043)..At the 20th week,the FBG of the DM group was(294.5±81.4)mg/dl,and the FBG of the control group was(80.0+13.0)mg/d1.The FBG of the DM group was significantly higher than that of the Control group(P<0.001),suggesting that the diabetic mouse model was successfully constructed.2.2 Transesophageal programmed electrical stimulationSNRT100(165.8 ± 12.43 vs 138 ± 14.25 ms,P=0.150).,SNRT90(162.1 ± 11.67 vs 148.2 ± 11.12 ms,P = 0.4042),SNRT80(158.5±12.74 vs 156.7 ± 13.83 ms.,P = 0.9262)There was no statistical difference.The AERP of the DM group was significantly lower than that of the Control group(51.48 ± 1.958 vs 60.09±1.53 ms,P=0.0023).The AF induction rate of the DM group was significantly higher than that of the Control group(52.6%vs 13.3%,P=0.0172).The duration of AF in the DM group and Control group was 23.25(16.11,61.84)s vs 18.87(7.88,30.14).s,but there was no significant difference between the two groups(P = 0.1309).2.3 Atrial electrical remodelingThe results of Western blotting showed that the expression of JPH2 protein in DM group was significantly lower than that in Control group(P=0.026),while the expression of Calpaim-1 was significantly higher than that in Control group(P=0.007).The Calpain-1 activity in the DM group was also significantly higher than that in the Control group(P<0.001).The results of laser confocal calcium imaging showed that the calcium transient amplitude(F/FO)of DM group was significantly lower than that of Control group(P<0.001).There was no significant difference in calcium transient time Tpeak,T50 and T75 between the two groups(P>>0.05),the T90 of the DM group was significantly lower than that of the Control group(P<0.001),while the calcium spark frequency of the DM group was significantly higher than that of the Control group(P<0.001),and the calcium spark amplitude(F/F0)was significantly lower than that of the Control group(P= 0.0143).Immunofluorescence results showed that the expression of JPH2 in the DM group was significantly lower(P<0.001),and the transverse tube structure was destroyed.2.4 Atrial structural remodelingThere was no significant difference in the gross appearance and HE staining between the two groups.There was no significant difference in atrnal weightibody weight ratio(AW/BW)and atrial weight/tibia length(AW/TL)between the two groups(P>values>0.05).Masson staining showed that the degree of atrial fibrosis in the DM group was more obvious than that in the Control group,and the CVF%was significantly higher than that in the Control group(P=0.0023).3.Experimental study on the protective effect of Calpastatin3.1 Overexpression gene identificationWe used PCR and agarose gel electrophoresis to identify genotypes of male mice in the same litter.The samples of atrial myocardium were collected from the experimental mice.The expression of CAST mRNA was identified by RT-PCR.The expression of CAST protein was determined by Western blot.The results showed that the mRNA expression and protein expression of CAST OE mice were significantly higher than those of CAST WT mice(P>0.05).3.2 Model buildingThe results showed that there was a significant difference in FBG between the 4 groups at week 12(F=5.636,P=0.0018).FBG in the DM+OE group was significantly higher than that in the ND+OE group(85.5+32.7 mg/).dL vs 63.3+15.6 mg/dL,P=0.0469),FBG in DM+WT group was slightly higher than ND+WT group,but there was no statistical difference(94.1+30.9 mg/dL vs 71.4+9.8 mg/dL,P=0.0775).There was no significant difference between the DM+OE group and the DM+WT group,the ND+OE group and the ND+WT group(P=0.7588,P=0.7970).At the 4th week and the 8th week,between the groups There was no significant difference in body weight(P>0.05).At 12th,16th and 20th week,at week 12,DM+OE group weight was significantly lower than DM+WT group(P=0.0025),ND The weight of the +OE group was significantly lower than that of the ND+WT group(P=0.0273).There was no significant difference between the DM+OE group and the ND+OE group,the DM+WT group and the ND+WT group(P>0.05);the 16th week The body weight of DM+OE group was significantly lower than that of DM+WT group(P=0.0434).There was no significant difference between DM+OE group and ND+OE group,DM+WT group and ND+WT group(P>0.05).At week 20,DM+OE group weight was significantly lower than DM+WT group(P=0.0016),ND+OE group weight was significantly lower than ND+WT group(P=0.0409),DM+OE group was significantly lower than ND+ OE group(P=0.0 101)There was no significant difference between the DM+WT group and the ND+WT group(P=0.2369).At week 12,there was a significant difference in FBG between the 4 groups(F=5.636,P=0.0018).FBG in the DM+OE group was significantly higher than that in the ND+OE group(85.5+32.7 mg/dL vs.63.3+15.6 mg/dL,P=0.0469),FBG in DM+WT group was slightly higher than ND+WT group,but there was no statistical difference(94.1±30.9 mg/dL vs 71.4±9.8 mg/dL,P=0.0775).There was no significant difference between DM+OE group and DM+WT group,ND+OE group and ND+WT group(P=0.7588.,P=0.7970).At the 20th week,there were significant differences in FBG between the 4 groups.(F=57.19,P<0.001),FBG in DM+OE group was significantly higher than ND+OE group(283.7±87.6 mg/dL vs 65.2±16.1 mg/dL.,P<0.001),DM The FBG of the WT group was significantly higher than that of the ND+WT group(271.8±94.4 mg/dL vs 59.8±13.8 mg/dL,P<0.001),while the DM+OE group and the DM+WT group,the ND+OE group and the ND+ group.There was no significant difference between the WT groups(P=0.9562,P=0.9960).3.3 Transesophageal programmed electrical stimulationThere were no significant differences in SNRT 100ms,90ms,and 80ms between the 4 groups(P>0.05).The AERP of DM+OE group,DM+WT group,ND+OE group and ND+WT group were(53.78+7.765)ms,(46.59+8.007)ms,(56.37+4.982)ms,(59.64+5.441).There was no significant difference in AERP levels between the 4 and 4 groups of mice(P<0.001).The AF induction rates of mice in DM+OE group,DM+WT group,ND+OE group and ND+WT group were 17.7%,53.3%,25.0%and 18.8%,respectively.The AF induction rate of DM+WT mice was significantly higher than that of DM+WT group.There was no significant difference in AF induction rate between the DM+OE group and the ND+OE group(P=0.5882).The AF durations of DM+OE group,DM+WT group,ND+OE group and ND+WT group were 12.1(7.9,27.6)s,18.1(8.4,49.7)s,8.0(5.2,11.4)s,respectively.7.9(5.2,10.7)s,DM+WT group had higher duration of AF,but there was no statistical difference in 4 groups of mice(P>0.05).3.4 Atrial electrical remodelingWestern blot analysis showed that the expression of JPH2 protein in DM+WT group was significantly lower than that in other three groups(P<0.001).The expression of JPH2 mRNA in DM+WT group and DM+OE group was significantly higher than that in ND+WT group.ND+OE group(P<0.001.,P=0.012).Western blot analysis showed that the expression of Calpain-1 in DM+WT group and DM+OE group was significantly higher than that in ND+WT group and ND+OE group(P<0.001.,P=0.0487).,DM+WT group,DM+.The expression of Calpain-1 mRNA in OE group was significantly higher than that in ND+WT group and ND+OE group(P<0.001,P=0.0092),while the activity of Calpain-1 in DM+WT group was significantly higher than that in other three groups(P=0.001).The results of laser confocal calcium imaging showed that the calcium transient amplitude(F/FO)of DM+WT group was significantly lower than that of DM+OE group(P<0.001)and ND+WT group(P=0.0279),while DM+OE group and There was no significant difference between the ND+OE group(P=0.2235).Compared with calcium transient time,there were no significant differences in Tpeak,T50 and T75 between the four groups(P>0.05),and T90 in DM+OE group was significantly higher than DM+WT(P<0.001),significantly lower.In the ND+OE group(P=0.0076),the T90 in the DM+WT group was significantly lower than that in the ND+WT group(P<0.001),but there was no significant difference between the ND+OE group and the ND+WT group(P=0.1686)..The calcium wave frequency in the DM+WT group was significantly higher than the other three groups(P<0.001).T-Tubule imaging results showed that the transverse tube structure of the DM+WT group was disordered.The TT-Power value of the DM+WT group was significantly lower than that of the ND+WT group(P<0.001)and the DM+OE group(P<0.001).There was no significant difference between the +OE group and the ND+OE group(P=0.0956).3.5 Atrial structural remodelingThere were no significant differences in atrial weight(AW/BW,AW/TL)and cardiac weight(HW/BW,HW/TL)between DM+OE group,DM+WT group,ND+OE group and ND+WT group.P values were al>0.05).Masson staining showed that the CVFo-/%of DM+OE group,DM+WT group,ND+OE group and ND+WT group were(6.328 ± 3.229),(14.39±7.327),(8.536±2.999),(7.016±2.392),respectively.The CVF%was significantly higher than the other 3 groups(F=4.403,P=0.0149).The comparison between the two groups showed that there was no significant difference in CVF%between the other three groups(P>0.05).Conclusions1.Diabetic patients have atrial electrical remodeling and structural remodeling,and are closely related to glycemic control,which may be the main pathophysiological process of diabetes to promote the occurrence and development of atrial fibrillation.2.In the state of hyperglycemia and insulin resistance,Calpain-1 can atrial electrical remodeling by degrading JPH2,affecting calcium homeostasis,and atrial structural remodeling induced by atrial fibrosis,ultimately promoting the occurrence and development of atrial fibrillation.3.Overexpression of Calpain-1 endogenous inhibitor Calpastatin,can reduce the atrial structural remodeling and electrical remodeling caused by diabetes,thereby slowing the occurrence and development of atrial fibrillation. |
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