Blockage Of TRPV4 Attenuates Atrial Fibrosis And Atrial Fibrillation Promotion In A Rat Sterile Pericarditis Model

Part I TRPV4 contributes to atrial fibrillation in a rat sterile pericarditis modelObjective:Establish stable rat sterile pericarditis model of atrial fibrillation and to observe whether there is any changes of TRPV4 channel’s expression in rat atrial after sterile pericarditis.Treat sterile pericarditis rats with TRPV4 channel’s oral inhibitor GSK2193874,test whether there is any improvement of AF duration and AF induction in sterile pericarditis rats.Collect atrial tissue samples from atrial fibrillation patients and sinus rhythm patients,test wether there is any difference of TRPV4channel’s expression.This study want to confirm whether TRPV4 involved in the development and progression of atrial fibrillation in rat sterile pericarditis.Method:Sterile pericarditis model were induced by generously dusted rats atrial surfaces with sterile talcum powder,and sham group only received thoracotomy operation without pericardiotomy.In different time points,heart were isolated,and atrial tissue were stored at-80 degree Centigrade for further use.The expression of TRPV4 channels in atrial and ventricle tissue was tested by western blot at different time points after sterile pericarditis operation.Immunofluorescence were used to measure the distribution of TRPV4 channels in rat atrial of different groups.Male Sprague Dawley rats were randomly assigned to 3 groups:1)sham group;2)vehicle group(sterile pericarditis model+ vehicle);3)GSK2193874 group(sterile pericarditis model+ GSK2193874).In vivo electrophysiology was performed on the third operative day in different groups of rats.The number of AF episodes,the total time of the AF episodes,and the probability of induction of AF were analyzed by esophagus.Atrial tissue from patients were tested by western blot to confirm whether there is any difference of TRPV4 expression.Result:TRPV4 channel in rat atrial increased up to the peak level on the first day after SP operation and this increase can be sustained until two weeks after sterile pericarditis operation.On the fourteenth day after operation,TRPV4 decreased compared to the first day,but still higher than sham group.We didn’t tested TRPV4 expression longer than two weeks after sterile pericarditis operation.TRPV4 expression in rat ventricle didn’t changed after sterile pericarditis operation.Immunofluorescence showed that TRPV4 expressed around nuclear in atrial tissue in sham group,few fluorescence can be seen in the membrane.Distinctively,TRPV4 transferred to the membrane of atial tissue in sterile pericarditis rats.In vivo electrophysiology detection showed that AF duration and AF induction were significantly higher in SP group compared to sham group.After the usage of TRPV4 channels,oral inhibitor GSK2193874,AF duration and induction can be reduced significantly in GSK2193874 group.Besides,TRPV4 protein level is higher in the atial of atrial fibrillation patients than sinus rhythm patients.Conclusion:These results indicated that TRPV4 channel contribute to the development and progressive of atrial fibrillation in rat SP model,blockage of TRPV4 can effectively attenuates atrial fibrillation;TRPV4 may contribute to the pathological process of atrial fibrillation patients.Blockage of TRPV4 may serve as a new therapeutic target for atrial fibrillation.Part II Blockage of TRPV4 can suppress atrial fibroblast proliferation and differentiationObjective:It has been well accepted that atrial fibrosis contribute to atrial fibrillation.But we don’t know whether TRPV4 participate to atrial fibrillation by induce atrial fibrosis.This study performed to determine whether TRPV4 dysfunction contribute to atrial fibrillation through atrial fibrosis in rat sterile pericarditis model.Method:Male SD rats were randomly assigned to 3groups:1)sham group;2)vehicle group(sterile pericarditis+vehicle);3)GSK2193874 group(sterile pericarditis+GSK2193874).To examine the role of TRPV4 in sterile pericarditis rats,TRPV4 inhibitor was administered orally once daily for four days.Rats in vehicle group were injected with the same volume of vehicle.At the third day after sterile pericarditis operation,hearts were isolated and stored under-80 degree centigrade for further use.Real time polymerase chain reaction was performed to measure mRNA level of fibrosis related indicators:TGF-β,Col－I,Col-III and a-SMA.Besides,collagen and a-SMA content were tested by masson and immunohistochemical staining.Expression of fibrosis related signaling pathway proteins STAT3,ERK,AKT and GSK-3β were measured by western blot.Besides,primary cultured of rat atrial fibrosis cells(CFs)were isolated from different groups.Then 0-1 passage CFs were used to test the TRPV4 currents by patch clamp and intracellular calcium were tested to find the reactivity of CFs from different groups to TRPV4 agonist GSK1016790A or TRPV4 inhibitor GSK2193874.Then,activation or blockage of TRPV4 to observe the influence to fibrosis related markers in CFs.After that,inhibitors of AKT or STAT3 were used to confirm wheter they participate as TRPV4,downstream in atrial fibrosis.Result:We found several fibrosis related indicators were increased in the atrial of sterile pericarditis rats.RT-PCR results showed that atrial mRNA levels of TGF-β1,Col-I,Col-III and a-SMA were significantly increased in vehicle group than sham.Immunohistochemical staining reveled that a-SMA content was significantly increased in control group.Masson result showed that the atrial from control group have more collagen element than sham group.ALL this changes can be reversed when TRPV4’s specific inhibitor was administrated.Results from western blot showed that the phosphorylation level of fibrosis related proteins STAT3,AKT and GSK-3β were significantly increased in the atrial of rats in vehicle group.When TRPV4’s inhibitor GSK2193874 was administrated,the increased phosphorylation level of STAT3,AKT and GSK-3β can be reversed.Intracellular calcium measurement showed that TRPV4 agonist GSK1016790A can mediate more calcium flux in CFs isolated from SP rats.And this effect can be effectively blocked by TRPV4’s inhibitor GSK2193874.Patch clamp results showed that GSK1016790A induced higher TRPV4 current density in CFs isolated from SP rats.Besides,blockage of TRPV4 suppressed fibrosis related markers in CFs from both of sham and SP rats.Also,AKT and STAT3 inhibtors can blocked TRPV4 activation induced CFs differentiation and collagen synthesis respectively.Conclusion:TRPV4 channel may play an important role in mediate fibrosis pathological process in the atrial of sterile pericarditis rats.TRPV4 can through the regulation of phosphorylation level of STAT3 and AKT/GSK-3P participate in CFs differentiation and collagen synthesis,then promote atial fibrosis and contribute to atrial fibrillation.Part III Blockage of TRPV4 in the process of atrial electrical remodeling in rat sterile pericarditis modelObjective:Atrial electrical remodeling contribute to atrial fibrillation.This study planned to investigate whether TRPV4 participate in atrial electrical remodeling and its potential mechanisms.Method:Male SD rats were randomly assigned to 3 groups:1)sham group;2)vehicle group;3)GSK2193874 group.On the third postoperative day,rats were anesthetized with barbiturate(40mg/kg i.p.)and injected heparin(2000IU/kg)to reduce the theoretical risk of thrombus formation.Then hearts were quickly harvested and transferred into the cold Krebs-Henseleit buffer as quickly as possible to avoid any detrimental hypoxia.After that hearts were transfected directly from the ice cold buffer onto the cannula and perfused.Atrial epicardial activation mapping in isolated Langendorff-perfused rat hearts was performed using a multielectrode array containing 64 elec-trodes(0.2-mm electrode diameter;0.43-mm interelectrode distance).Conduction velocity was detected on left appendage under 7Hz paced rhythm.Action potential duration was also measured under 7Hz stimulated.Then atrial were stored at-80 degree centigrade for further use.Expression levels of cardiac conduction related proteins connexin43 and caveolin-3 were detected through western blot technique.And the distribution of this connexin43 were measured by immunohistochemical.Result:Maps showed that control rats were characterized by inhomogeneous conduction,the index.of inhomogeneity was significantly greater in vehicle group than in sham.But there is no significant difference in the conduction velocity between the two groups.When TRPV4 inhibitor was administrated,the index of inhomogeneity can be decreased in vehicle group.Besides,APD duration(50%)was significantly prolonged in vehicle group compared to sham,and this prolongation can be reversed when TRPV4 inhibitor was used.The expression of caveolin3 was significant reduced in vehicle group compared to sham,and this reduction can be restored in GSK2193874 group.There were no significant differences in the expression of connexin43 between the two groups.Immunohistochemical staining showed that in the atrial of sham rats,connexin43 distributed in the intercalated disk,along the end-to-end direction.In the vehicle group,connexin43 distributed mainly along the side-to-side way,very little connexin43 can be seen in the intercalated disk,this change can be reversed in GSK2193 874 group.Conclusion:TRPV4 dysfunction can change the expression level and distribution mode of atrial electric conduction related proteins.Which may responsible for rat atrial APD prolongation and the increased index of conduction inhomogeneity.At last participate in the progress of atrial electric remodeling and promote the occurrence of atrial fibrillation.