Ang-?1-7? Attenuates Ang? Induced Fibroblast-myofibroblast Transition Via Inhibiting The NCX1/Ca²⁺/CaMKII Pathway

Background:Fibroblasts are an important components of the heart,and the function of fibroblasts are closely related to cardiomyocytes,directly affecting the development,hypertrophy,mechanical and electrical activity of heart.Moreover,the fibroblast myofibroblast transition?FMT?of cardiac fibroblasts is closely related to myocardial inflammation,cardiac hypertrophy and myocardial infarction.In the external pathological stimulation,fibroblasts undergo proliferation,differentiation,and secrete a large amount of extracellular matrix proteins?ECM?,causing collagen deposition and fibrotic remodeling,resulting in arrhythmia and cardiac dysfunction.In recent years,it has been found that sodium/calcium exchangers?NCX?and calcium/calmodulin-dependent protein kinase II?CaMKII?are important signalling molecules for FMT,in which CaMKII directly mediates angiotensin II-induced FMT.Recently,we have demonstrated that high expression of angiotensin-converting enzyme 2?ACE2?in the epicardium can increase angiotensin-?1-7??Ang-?1-7??expression in atrial myocytes,thereby reversing atrial fibrotic remodeling in the atrial rapid pacing model,and subsequently decreasing the inducibility and duration of AF.Ang-?1-7?is an important end-product of biological activity in the renin-angiotensin-aldosterone system?RAAS?that antagonizes Ang?.Furthermore,the physiological function of Ang-?1-7?is often associated with the activity of endothelial nitric oxide synthase?eNOS?and nitric oxide?NO?,which have the effects of inhibiting fibroblasts proliferation and reducing heart fibrosis.Objective:To investigate the effect of Ang-?1-7?on the FMT and the relationship with Ang?-induced Ca²⁺/CaMKII activation,and further clarify the role of NCX in Ang?-induced Ca²⁺ dysregulation.MethodsThe experiment was divided into three parts: Part one: Primary cardiac fibroblasts were cultured in vitro,cells were divided into 6 groups: Control group,Ang? group,Ang? + KB-R7943?NCX1 inhibitor?group,Ang? + autocamtide 2-related inhibitory peptide?AIP??CaMKII inhibitor?Group,Ang? + Ang-?1-7?group and Ang? + Ang-?1-7?+ L-NAME?eNOS inhibitor?group.Cellular immunofluorescence was used to detect the proliferation and phenotypic transformation of fibroblasts.Western blot was used to detect the expression of NCX1,CaMKII,p-CaMKII?Autophosphorylated Thr-287?,ox-CaMKII?Oxidized Met-281/282?,Transforming Growth Factor-?1?TGF-?1?and phenotypic conversion related proteins.Fluo 4-AM fluorescence probe was used to monitor the changes of Ca²⁺ in fibroblasts.Part two: Animal experiments.Adult Sprague-Dawley?SD?rats were divided into 5 groups: Control group,Ang? group,Ang? + AIP?CaMKII inhibitor?group,Ang? + Ang-?1-7?group and Ang? + Ang-?1-7?+ LNAME group.Hematoxylin/eosin?HE?and sirius red staining were used to observe the structure and fibrosis of the heart;Dihydroethidium?DHE?probe was used to detect myocardial reactive oxygen species?ROS?level;Western blot was used to detect the expression of CaMKII,p-CaMKII,ox-CaMKII,alpha smooth muscle Actin??-SMA?,endothelial nitric oxide synthase?eNOS?,p-eNOS?Ser 1177?;Immunohistochemistry was used to evaluate the expression of ?-SMA,TGF-?1,ox-CaMKII and Collagen I?Col I?in paraffin sections;To evaluate the expression of ox-CaMKII in fibroblasts,double immunofluorescence was used to detected the expressionof ox-CaMKII and ?-SMA in cryosections simultaneously.Part three: Cell and animal experiments: To investigate the effect of Cx43 on FMT in Ang? activated CaMKII pathway,Fibroblasts and rats were divided into 4 groups: Control group,Ang? group,Ang? + AIP?CaMKII inhibitor?group and Ang? + Ang-?1-7?group.In vivo,HE and sirius red staining were used to observe the structure and fibrosis of the heart;DHE probe was used to detect myocardial ROS level;Western blot was used to detect the expression of CaMKII,p-CaMKII,ox-CaMKII,?-SMA,Connexin 43?Cx43?and TGF-?1;To evaluate the expression of Cx43 in fibroblasts,double immunofluorescence was used to detected the expressionof Cx43 and ?-SMA in cryosections simultaneously.In vitro,double immunofluorescence for Cx43?green?and ?-SMA?red?in fibroblasts was used to evaluated the Ang?-induced FMT under different treatment time;Western blot was used to detect the expression of ?-SMA,TGF-?1 and Cx43,CaMKII,p-CaMKII and ox-CaMKII;TGF-?1 si-RNA transfection was used to evaluate the role of TGF-?1 on Cx43 expression.ResultsPart one:?1?Compared with the Control group,Ang? could promote the proliferation of fibroblasts and ROS production and phenotypic transformation,Western blot suggested that Ang? upegulated the expression of ?-SMA,NCX1,TGF-?1,CaMKII,p-CaMKII and ox-CaMKII.Intracellular calcium detection suggested that Ang? promoted storeoperated calcium entry?SOCE?mediated Ca²⁺ influx,and that NCX was involved in this process.?2?Compared with Ang? group,AIP and Ang-?1-7?pretreatment effectively attenuated the changes of the above results and the phenotypic conversion of fibroblasts induced by Ang?.Contrary to Ang?,intracellular calcium detection suggested that Ang-?1-7?pretreatment could reduce the Ca²⁺ influx and the expression of NCX1,therefore,the intracellular Ca²⁺ concentration was decreased.?3?Compared with the Ang? + Ang-?1-7?group,L-NAME addition promoted FMT and its effect was similar to that of Ang?Part two:?1?Compared with the Control group,Ang? infusion promoted myocardial hypertrophy,interstitial fibrosis,ROS production,and upregulated the expression of ?-SMA,Col I,ox-CAMKII,p-CAMKII,TGF-?1 and NCX1.Double immunofluorescence for ?-SMA and ox-CAMKII showed that Ang? promoted the ox-CAMKII expression in ?-SMA positive areas.?2?Compared with Ang? group,AIP and Ang-?1-7?treatments effectively attenuated the changes of the above results.?3?Compared with Ang? + Ang-?1-7?group,eNOS inhibition reversed the inhibitory effect of Ang-?1-7?on Ang?-induced FMT.Similar to Ang?,the expression of oxCAMKII was increased in ?-SMA positive areas.Part three:?1?The results of animal experiments showed that: Ang? could reduce the expression of Cx43 in myocardium,with CaMKII activation and high expression of TGF-?1.Furthermore,double immunofluorescence for ?-SMA and Cx43 indicated that Ang? decreased the Cx43 expression in ?-SMA positive areas.?2?Compared with Ang? group,AIP and Ang-?1-7?restored the expression of Cx43 in myocardium,with downregulation of CaMKII,p-CaMKII,ox-CaMKII and TGF-?1.?3?In in vitro cultured fibroblasts,Ang? elevated the expression of Cx43 in a time-dependent manner,and the time for upregulation of Cx43 was earlier than that of ?-SMA.Once fibroblasts totally transformed into myofibroblasts,Cx43 sharply reduced to basal level.?4?Compared with Ang? group,TGF-?1 si-RNA transfection could inhibit the upregulaton of Cx43 and ?-SMA induced by Ang?,and furthermore,AIP and Ang-?1-7?pretreatment reduced the expression of Cx43,TGF-?1 and CaMKII activation,and effectively attenuated the Ang?-induced FMT.ConclusionThe main findings of this study were as follows:?1?Ang? can increase Ca²⁺ in fibroblasts through NCX,thereby activating CaMKII,causing upregulationof TGF-?1,and promoting fibroblasts proliferation and phenotypic conversion.?2?At the same time,Ang? can induce ROS production.ROS derived from the NADPH oxidase 2?NOX2?,NADPH oxidase 4?NOX4?and eNOS uncoupling.Enhanced ROS production promotes ox-CaMKII expression and further activates CaMKII.?3?The antifibrotic effect of Ang-?1-7?is associated with its antioxidative capacity and its ability to inhibit the reverse transport of Ca²⁺ by NCX,reducing the intracellular Ca²⁺ concentration,both of which simultaneously decrease CaMKII activation and downregulate the expression of TGF-?1,therefore,attenuates cardiac fibrosis remodeling;?4?Cx43 is also involved in the process of FMT induced by CaMKII/TGF-?1 pathway;?5?Ang-?1-7?reduces the expression of TGF-?1 and Cx43 through inhibition of CaMKII activity,thereby reducing cardiac fibrosis remodeling.In conclusion,Ang-?1-7?can alleviate Ang?-induced fibroblast Ca²⁺ dysregulation by promoting the reverse transport mode of NCX,thereby inhibiting CaMKII activation,reducing the expression of TGF-?1 and Cx43,and finally inhibiting FMT.Decreased deposition of collagen in the heart effectively maintains normal cardiac mechanical and electrical activity.