| BackgroundAtrial fibrillation（AF）is the most common sustained cardiac arrhythmia,also well recognized as the most severe atrial electrical activity disorder,associated with a high morbidity and mortality.AF could progress from a paroxysmal form to chronic and persistent states,result in lifestyle-limiting symptoms and remain the main risk factor of ischemic stroke,congestive heart failure and all-cause mortality.The underlying mechanism of AF initiation and progression remains elusive,which seriously restricts the effectiveness and safety of AF treatment in clinical practice.Calpain is a big family of Ca2+-dependent activated cysteine protease that translocates to the membrane and get activated under intracellular Ca2+overload,thus hydrolyzing a variety of protein substrates,causing destruction to cardiomyocytes.Recent studies have found that activatedμ-Calpain（Calpain 1）in AF patients proteolytically degrades various target proteins that are crucial for intracellular calcium homeostasis,such as L-type Ca2+channel（LTCC）,sodium-calcium exchanger（NCX）.Membrane-tethered structural protein junctophilin-2（JP2）,located between the membranes of the transverse tubules（T-tubules）and the sarcoplasmic reticulum（SR）,has recently been discovered to stabilize the SR Ca2+channel ryanodine receptor 2（RyR2）and therefore play a regulatory role in calcium homeostasis in cardiomyocytes.While calcium handling abnormality,which is reported mainly due to the increased sarcoplasmic reticulum（SR）Ca2+leak(Ca2+sparks or Ca2+waves)from“leaky”Ca2+release channel type 2ryanodine receptor（RyR2）,is one of the critical mechanisms underlying AF development.Besides,in the animal model of ischemia-reperfusion injury and heart failure,JP2 protein was reported to be degraded by activated Calpain-1,suggesting that JP2 is also one of the targeted substrate proteins of Calpain-1.Therefore,in the present study we hypothesized that,under pathological conditions such as rapid and irregular atrial beating or oxidative stress,the activation of atrial Calpain-1 may induce atrial fibrillation by degrading a series of structural protein substrates such as JP2,LTCC et al.in cardiomyocyte.While normal expression level of JP2 could play a critical protective role against atrial arrhythmia via the attenuation of the abnormal calcium leakage through RyR2,and the reduction of delayed afterdepolarization（DAD）,and potential atrial electrical and structural remodeling.Objectives1.To define the expression pattern of JP2 and Calpain-1 in patients with atrialfibrillation and some experimental atrial fibrillation models;2.To elucidate the role and mechanism of Calpain inhibitors in modifying atrialremodeling and AF susceptibility via regulating JP2;3.To explore the role and mechanism of JP2 in regulating the susceptibility of atrialfibrillation in CAPN 1-OE mice.Materials and methods1.Changes of JP2 and Calpain-1 in atrial samples of AF patients and several experimental AF models1.1 44 patients undergoing coronary artery bypass surgery or mitral valve surgery under extracorporeal circulation in our department were enrolled in our study with written consent form,among them 28 in AF and 16 in normal sinus rhythm（NSR）.Left or right atrial appendages（LAA or RAA）specimens were collected from patients before the onset of extracorporeal circulation and transferred to the biobank for further research.Western blot was used to detect the expression changes and correlation of JP2 and Calpain-1,and Calpain-1 activity was also determined.1.2 A total of 18 adult New Zealand white rabbits（2.0-2.5 kg,male）were selected and randomly divided into sham group（2 w,4 w）and RAP group（6 rabbits in each group）.Then open the chest cavity along the 3rd intercostal space on the left margin of the sternum and expose the heart.The pacing electrode was sutured and fixed on the free wall of the left atrium,which was connected with the abdominal subcutaneous pacemaker in AOO pacing mode.The rapid atrial pacing（RAP）rabbit model of AF was established by 1000 bpm continuous stimulation for 4 w.The rabbits were divided into sham operation group（sham）and RAP group（2 w or 4 w respectively）,and western blot was used to detect the protein level of JP2 and Calpain-1,the Calpain-1 activity was also determined.2.Role and mechanism of Calpain-1 inhibitor in mouse atrial fibrillation model2.1 Selection and grouping of experimental animalsTet-Off system is employed in this experiment to establish those transgenic mouse model.The Tet-Off system makes use of the tetracycline transactivator（tTA）protein,which is able to bind to DNA at specific TetO operator sequences.The entirety of several TetO sequences with a minimal promoter is called a tetracycline response element（TRE）,expression of TRE-controlled genes can be repressed by doxycycline（Dox）.They bind tTA and render it incapable of binding to TRE sequences,thereby preventing transactivation of TRE-controlled genes.In this study,we mainly focused on the changes at 8 weeks after the removal of Dox drug feed,i.e.the effects of Calpain or Calpain/JP2 overexpression for 8 weeks（Dox withdrawal 8 weeks）.Mice were divided into three groups:matched control mice（control）,Calpain overexpression for 8 weeks（CAPN 1-OE）,and Calpain inhibitor MDL28170（10mg/kg/day,intraperitoneal injection）treatment group（CAPN 1-OE+MDL）.A total of 75 mice were included in this part.Among them,αMHC-tTA mice（Calpain/tTA-/+）without Calpain-1 overexpression were selected as control group with 25males and females in total.Cardiac-specific Calpain-1 overexpression mice（Calpain/tTA+/+）were selected accordingly and they were randomly divided into CAPN-OE group and CAPN-OE+MDL group with 25 mice in each group.2.2 The effects of Calpain inhibitor on AF susceptibility in CAPN 1-OE miceCardiac programmed electrical stimulation and recording system consisted of STG-3008 multichannel electrical stimulator（Germany）,Millar 1.1F multielectrode electrophysiological catheter（EPR-800;Millar Instruments）and Labchart Pro signal acquisition system（ADI instruments,Australia）.The cardiac surface electrical signals and body surface electrocardiograms was simultaneously recorded to determine the appropriate location in esophageal of the electrical stimulation catheter,and to carry out intra-esophageal stimulation.The incidence rate and duration of AF were detected by intra-esophageal cardiac programmed electrical stimulation.2.3 Samples collectionAfter completing the corresponding electrophysiological detection,mice were executed to collect heart samples for subsequent molecular biology,confocal imaging and other experimental studies.The expression of JP2 was detected by Western blot and the activity of Calpain 1 was evaluated by activity detection kit.2.4 The effects of Calpain inhibitor on Ca2+homeostasis in CAPN 1-OE miceAdult mouse atrial myocytes were successfully isolated by Langendorff perfusion enzymatic method.Calcium homeostasis was detected by laser confocal microscopy after 5μM Fluo-4 AM Ca2+indicator incubation.The excitation and emission wavelengths are 488nm/505-530 nm,respectively.The frequency of Ca2+sparks in each group was detected at5s after the halt of 1 Hz electrical stimulation at 15 V/cm.Furthermore,the Ca2+homeostasis abnormalities(the frequency of Ca2+waves)in atrial tissue ex vivo were observed by in situ whole heart imaging after perfusion with calcium indicator（Rhod-2）.2.5 The effects of Calpain inhibitor on atrial electrical remodeling in CAPN 1-OE miceThe atrial effective refractory period（AERP）were determined by intra-esophageal cardiac programmed electrical stimulation with S1S2 stimulation protocol.Besides,in situ whole heart imaging was observed by membrane fluorescent indicator（5μM MM 4-64）,and T-tubules structure remodeling were explored.2.6 The effects of Calpain inhibitor on atrial structural remodeling in CAPN 1-OE miceAfter the electrophysiological experiments,the atrial samples were harvested and weighed,and tibia length was measured,then atrium weight-to-tibia length ratio（AW/TL）was calculated as an index of atrial enlargement.Besides,the ex vivo whole hearts were fixed by 4%PFA perfusion and atrial fibrosis was then detected by Masson trichrome staining.The expression levels of transforming growth factor（TGF）-β1,α-smooth muscle actin（SMA）and collagen I（Col I）in atrium were also measured.3.Role and mechanism of changes in JP2 protein level in regulating susceptibility to atrial fibrillation in mice3.1 Establishment of stable Calpain and/or JP2 cardiac-specific overexpression mice.In this study,2 transgenic mouse models were established using cardiac-specificα-myosin heavy chain（α-MHC）promoter vector,that is,JP2 overexpression（JP2-OE）mouse and JP2/Calpain-1 double-transgenic（CAPN 1-OE×JP2-OE）mice.In this part,the mice were divided into control group,Calpain-overexpressing mice（CAPN 1-OE）,and CAPN 1-OE×JP2-OE mice.A total of 75 mice were included in the study.Among them,25 mice with positive expression of tTA gene,that is Calpain/tTA-/+or Calpain/JP2/tTA-/-/+,were selected as control group,and Calpain-1 cardiac-specific overexpression mice（Calpain/tTA+/+or Calpain/JP2/tTA+/-/+）were selected as CAPN-OE group with 25 males and females in total,and cardiac-specific Calpain-1/JP2 overexpression mice（Calpain/tTA+/+or Calpain/JP2/tTA+/-/+）were selected as CAPN-OE×JP2-OE group with 25 males and females in total.Furthermore,according to the corresponding target-gene overexpression timepoint,above mentioned groups were then divided into 4 weeks after Dox chow withdrawal（Dox withdrawal 4w）and 8 weeks after Dox chow withdrawal（Dox withdrawal 8w）.The experimental animals in each time period were grouped as the same,so totally 150 mice were included in this part.Before the initiation of electrophysiological experiment,mice cardiac function was measured by echocardiography to detect the changes in left ventricular ejection fraction（LVEF）,end-systolic volume（ESV）,end-diastolic volume（EDV）.The changes of heart weight/body weight（HW/BW）and lung weight/body weight（LW/BW）in mice were also compared among 3 groups at each timepoint.3.2 The effects of JP2-OE on AF susceptibility in CAPN 1-OE miceThe corrected sinus node recovery time（cSNRT）were measured with different basic cycle length（BCL）of S1S1 stimulation protocol.Atrial burst pacing was performed to measure the inducibility and the duration of AF episodes.3.3 Samples collectionAfter completing the corresponding electrophysiological detection,mice were executed to collect heart samples for subsequent molecular biology,confocal imaging and other experimental studies.The expression of JP2 was detected by Western blot and the activity of Calpain 1 was evaluated by activity detection kit.3.4 The effects of JP2-OE on Ca2+homeostasis in CAPN 1-OE miceThe frequency of Ca2+sparks in atrial cardiomyocytes and the frequency of Ca2+waves in atrial tissue ex vivo in each group were observed.3.5 The effects of JP2-OE on atrial electrical remodeling in CAPN 1-OE miceThe AERP in each group was determined by intra-esophageal cardiac programmed electrical stimulation.Besides,in situ whole heart T-tubules imaging was also performed to explore the T-tubules system remodeling in both atria of mice.3.6 The effects of JP2-OE on atrial structural remodeling in CAPN 1-OE miceThe changes of AW/TL in mice were calculated as an index of atrial enlargement.Atrial fibrosis was detected by Masson trichrome staining.The typical profibrotic pathway factors levels of TGF-β1,α-SMA and Col I were also measured.Results1.The expression levels of JP2 and Calpain-1 in AF models and human patients1.1 JP2 and Calpain expression in atria specimens of patients with AFIn AF patients,the expression level of JP2 protein in atrial myocardium decreased significantly,while Calpain protein level and enzyme activity greatly increased.The level of JP2 protein in atrial myocardium was negatively correlated with the activity of Calpain-1protease.1.2 The expression of JP2 and Calpain in rabbits AF model under rapid atrial pacingIn RAP rabbit AF model,with the prolongation of atrial pacing time,the expression level of JP2 protein in atrial samples decreased gradually,while the Calpain expression level and activity increased gradually.The level of JP2 protein in atrial myocardium was negatively correlated with the activity of Calpain-1 protease.2.The effects and the underlying mechanism of Calpain inhibitor on AF susceptibility2.1 Calpain inhibitor significantly ameliorated AF susceptibility in CAPN 1-OE miceCompared with control mice,CAPN 1-OE mice at 8 weeks after Dox withdrawal had significantly higher AF incidence rate,longer AF events duration,while the increased AF susceptibility was remarkably restored in CAPN 1-OE+MDL group.2.2 Calpain inhibitor MDL28170 significantly reduced the overactivation of Calpain-1in atria of CAPN-1-OE miceCompared with the control group,the atrial Calpain-1activity in CAPN 1-OE mice was significantly increased,accompanied by a significant decrease in the JP2 expression,while MDL28170 treatment greatly reduced the atrial Calpain-1activity,and the expression of JP2protein was markedly restored.2.3 Calpain inhibitor greatly attenuated abnormality in calcium homeostasis in CAPN1-OE miceCompared with control mice,CAPN 1-OE mice at 8 weeks after Dox withdrawal had significantly higher calcium sparks frequency in atrial myocytes,and obviously abnormal calcium waves in atria tissue detected by in situ whole heart imaging.In the CAPN 1-OE+MDL treatment group,the abnormal calcium homeostasis in vitro and ex vivo were totally greatly improved.2.4 Calpain inhibitor markedly improved atrial electrical remodeling in CAPN 1-OE miceCompared with control mice,AERP in CAPN 1-OE mice at 8 weeks after Dox withdrawal was significantly shortened,while AERP in CAPN 1-OE+MDL mice was notably improved.Obviously abnormal T-tubules system in atria of CAPN 1-OE mice at 8weeks after Dox withdrawal was detected by in situ whole heart imaging,and MDL28170treatment greatly improved the intactness and regularity of T-tubules system.2.5 Calpain inhibitor markedly improved atrial structural remodeling in CAPN 1-OE miceCAPN 1-OE mice at 8 weeks after Dox withdrawal showed significantly bilateral atrial cavity enlargement,higher AW/TL.Besides,enhanced atrial fibrosis and increased level of TGF-β1,α-SMA and Col I were also detected.However,after Calpain inhibitor MDL28170treatment,atrial dilatation was significantly improved,and atrial fibrosis levels and the activation of profibrotic pathways were also attenuated in CAPN 1-OE+MDL mice.3.The effect and mechanism of JP2-OE in regulating the susceptibility to AF in CAPN 1-OE mice.3.1 Successful establishment of CAPN-1-OE×JP2-OE miceIn this part,the cardiac-specific JP2 overexpression mice（JP2-OE）were first established.Compared with control mice,the expression level of JP2 protein in atrial myocardium of cardiac-specific JP2-OE mice increased significantly at 4 weeks after withdrawal of Dox chow.Calpain-1/JP2 co-overexpression mouse model（CAPN-1-OE×JP2-OE）was established by crossing JP2-OE mice with CAPN-1-OE mice.3.2 No significant abnormality of left ventricular function was observed in all groups of mice at 4 weeks after Dox withdrawal.Echocardiographic results showed that there were no significant differences in LVEF,ESV and EDV among control mice,CAPN-1-OE mice and CAPN-1-OE×JP2-OE mice.Heart weight/body weight（HW/BW）,lung weight/body weight（LW/BW）among three groups were also similar.3.3 CAPN-1-OE and CAPN-1-OE×JP2-OE mice showed decreased left ventricular function at 8 weeks after withdrawal of Dox chow.Compared with control mice,the LVEF,ESV and EDV of CAPN 1-OE mice and CAPN1-OE×JP2-OE mice were impaired significantly,while the HW/BW increased significantly.LW/BW showed no significant difference among three groups.3.4 No significant abnormality in sinoatrial node conduction function in three groupsAt 4 weeks after withdrawal of Dox diet,the corrected recovery time of sinus node（cSNRT）in three groups were measured,and the results showed that there were no abnormal changes in sinoatrial node（SAN）function in control mice,CAPN 1-OE mice and CAPN 1-OE×JP2-OE mice,suggesting that CAPN 1-OE and/or JP2-OE did not affect SAN function in mice.At 8 weeks after withdrawal of Dox diet,the changes of SNRT and cSNRT in control mice,CAPN 1-OE mice and CAPN 1-OE×JP2-OE mice did not reach statistical difference,suggesting that the function of sinoatrial node remained normal at this indicated timepoint.3.5 Changes of atrial calpain-1 activity in three groupsCalpain-1 activity in atrial myocardium of CAPN-1-OE mice and CAPN-1-OE×JP2-OE mice increased significantly after 4 weeks of overexpression of JP2.Atrial Calpain-1activity in CAPN-1-OE mice and CAPN-1-OE×JP2-OE mice increased further after 8 weeks of overexpression of JP2,suggesting that JP2-OE could not interfere with the activity of Calpain-1.3.6 Changes in AF inducibility and duration time in CAPN-OE mice at 4 weeks after Dox withdrawal.Compared with control group,AF inducibility and duration time in CAPN 1-OE mice significantly increased,which could be attenuated by JP2-OE.3.7 Changes in AF inducibility and duration time in CAPN-OE mice at 8 weeks after Dox withdrawal.AF inducibility in CAPN 1-OE×JP2-OE mice remained increased compared to control mice,while the AF duration in CAPN 1-OE×JP2-OE mice at this timepoint was attenuated.3.8 Changes in atrial JP2 expression in CAPN 1-OE×JP2-OE mice.JP2-OE significantly improved the expression of JP2 protein in atrium of CAPN 1-OE mice after 4 weeks of withdrawal of Dox chow;however,the normal level of atrial JP2 could not be maintained after 8 weeks of withdrawal of Dox chow,the JP2 protein in the atrium of CAPN 1-OE×JP2-OE mice also decreased significantly.It is suggested that maintaining normal levels of JP2 protein is the structural basis for JP2-OE to improve the enhanced atrial fibrillation susceptibility.3.9 The regulatory effects of JP2-OE on Ca2+homeostasis in CAPN 1-OE mice at 4weeks after Dox withdrawal.Compared with control group,spontaneous calcium sparks were significantly increased in CAPN 1-OE mice while JP2-OE could decreased the high frequency of calcium sparks in CAPN 1-OE×JP2-OE mice,suggesting that JP2-OE was involved in calcium homeostasis in mouse atrial myocytes.In addition,the results of in situ calcium imaging in isolated hearts showed that abnormal calcium leaking events such as spontaneous calcium wave in the atrial tissues ex vivo of CAPN 1-OE mice increased significantly compared with the control group,while JP2-OE could significantly reduce the frequency of calcium wave in the atrial tissues of CAPN 1-OE×JP2-OE mice ex vivo.The results of in situ calcium imaging in isolated hearts were consistent with those of isolated single atrial myocytes.3.10 The regulatory effects of JP2-OE on Ca2+homeostasis in CAPN 1-OE mice at 8weeks after Dox withdrawal.Compared with the control group,the frequency of spontaneous calcium sparks was significantly increased in the atrial myocytes of CAPN 1-OE and CAPN 1-OE×JP2-OE mice.In addition,the results of in situ calcium imaging in isolated hearts showed that compared with CAPN 1-OE mice,the frequency of spontaneous calcium waves in atrial tissues of CAPN 1-OE×JP2-OE mice did not decrease significantly.Again,the results of in situ calcium imaging in isolated hearts ex vivo were consistent with those of isolated single atrial myocytes.3.11 The regulatory effects of JP2-OE on atrial electrical remodeling in CAPN 1-OE mice at 4 weeks after Dox withdrawal.Compared with the control group,CAPN 1-OE mice showed a significant shortening of AERP while JP2-OE improved the AERP in CAPN 1-OE×JP2-OE mice.Furthermore,In situ observation of atrial T-tubules system showed that the T-tubules in both left and right atria of CAPN-1-OE mice were obviously disordered and impaired at 4 weeks after Dox withdrawal,while the integrity and intactness of T-tubules system in CAPN 1-OE×JP2-OE was significantly improved,indicating JP2-OE was helpful to maintain the dedicated junctional membrane complex and to improve the electrical signaling in atrial myocytes.3.12 The regulatory effects of JP2-OE on atrial electrical remodeling in CAPN 1-OE mice at 8 weeks after Dox withdrawal.Compared with the control group,AERP in CAPN 1-OE and CAPN 1-OE×JP2-OE mice were significantly shortened detected by S1S2 intra-esophageal electrical stimulation.In situ observation of atrial T-tubules system indicated that the integrity of atrial T-tube system in CAPN 1-OE×JP2-OE mice was not significantly improved after 8 weeks of overexpression of JP2,suggesting that overexpression of JP2 could not effectively improve the electrical remodeling of CAPN 1-OE×JP2-OE mice at this timepoint.3.13 The regulatory effects of JP2-OE on atrial structural remodeling in CAPN 1-OE mice at 4 weeks after Dox withdrawal.The AW/TL and the right/left atrium of CAPN 1-OE mice increased significantly compared to control mice,while JP2-OE greatly improved the atrial enlargement.Besides,the atrial fibrosis levels in CAPN 1-OE and CAPN 1-OE×JP2-OE mice were both significantly increased.WB results show that TGF-β1,α-SMA and Col I from atrial samples in CAPN 1-OE and CAPN 1-OE×JP2-OE mice were notably activated.These results suggested that JP2-OE is involved in regulating the enlargement of atrial cavity in mice,but JP2-OE could not inhibit the overactivation of profibrotic signaling pathway in CAPN 1-OE×JP2-OE mice.3.14 The regulatory effects of JP2-OE on atrial structural remodeling in CAPN 1-OE mice at 8 weeks after Dox withdrawal.Compared with CAPN 1-OE mice,the enlargement of atrial cavity in CAPN 1-OE×JP2-OE mice was not effectively attenuated after 8 weeks of overexpression of JP2.Besides,Masson staining results showed that the degree of atrial fibrosis in CAPN 1-OE and CAPN 1-OE×JP2-OE mice was further increased compared with control group.Conclusion1.In atrial fibrillation patients and a variety of atrial fibrillation experimental models,with the persistence of rapid atrial stimulation,the expression of JP2 protein in atrial myocytes gradually decreased,while the expression of Calpain-1 was significantly up-regulated and its enzyme activity was significantly activated;while the level of atrial JP2level was significantly negatively correlated with the activity of Calpain-1.2.In CAPN-1-OE mouse,JP2 protein was proved to be one of the critical substrates of Calpain-1.At 8 weeks after cardiac-specific Calpain-1 overexpression,MDL28170treatment mitigated the susceptibility to atrial fibrillation by inhibiting the activity of Calpain-1 and reducing the degradation of substrate protein like JP2.3.The changes of JP2 protein in atrial myocytes can affect the susceptibility of atrial fibrillation（the incidence and duration of atrial fibrillation）by regulating the calcium homeostasis（abnormal calcium leaking events like calcium spark and wave）,thereby affecting the electrical remodeling（AERP changes,T-tubules remodeling）and structural remodeling（atrial chamber enlargement,atrial fibrosis）.As a potential therapeutic approach to atrial fibrillation,its role in regulating the susceptibility of atrial fibrillation has a certain time-course. |
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