The Role And Mechanism Of Chemokine Receptor 9(CCR9) In Ventricular Remodeling After Myocardial Infarction In Mice

BackgroundMyocardial infarction(MI)occurs when blood flow decreases or stops to a part of the heart,causing damage to the heart muscle,which lead to the heart cells in the territory of the blocked coronary artery.Myocardial infarction is a leading cause of morbidity and mortality in humans worldwide.Every year,millions of patients die from acute myocardial infarction in China.MI triggers an intense pathological process that includes inflammation,apoptosis,cardiomyocyte hypertrophy,and fibrosis and can lead to post-infarction remodeling,heart failure,arrhythmias and,thus,sudden cardiac death.There have been some major accomplishments in MI therapy,including electrophysiological monitoring,the routine use of beta blockers,aspirin and other medications,and reperfusion therapy.Although these improvements have reduced the acute mortality of MI,patients surviving a large MI are likely to develop ventricular remodeling,which is closely related to heart failure and arrhythmia.Therefore,it is of great importance to elucidate the exact mechanism underlying MI,and also important to point the way to slow down the progression of pathophysiology,to improve the cardiac remodeling following MI.Chemokines as well as their receptors play a important role in cardiovascular diseases:CCL2/CCR2 participate in atherosclerosis and MI.Pharmacological inhibition or genetic targeting of the CCL2/CCR2 pathway might represent an attractive approach to blunt excessive inflammation and prevent detrimental ventricular remodeling.CCL21/CCR7 play a vital role in ventricular remodeling after MI and remodeling is improved after anti-CCL21 antibody is used.CCR1 and CCR5 can recruit mononuclear macrophage.CC chemokine receptor 9(CCR9)is a G-protein coupled receptor with only one known ligand,thymus-expressed chemokine/CCL25.CCR9 is preferentially expressed on lymphocytes,dendritic cells(DCs)and monocytes/macrophages.There are papers reporting CCR9 is related to inflammatory diseases.Myocardial infarction is associated with inflammatory responses that involve a variety of chemokines.However,it is unknown whether CCR9 plays a role in the post-MI pathological processes.In this study,we used CCR9 knockout mice to determine the role of CCR9 in the mouse heart after MI.Part ?:CCR9/CCL25 is involved in ventricular remodeling following MIObjective:To explore the role of CCR9/CCL25 in ventricular remodeling following MI.Methods:C57BL/6 male mice with a body weight of 24-27g and an age of 8-10 week old were chosen to conduct MI surgery by ligating the left anterior descending coronary.The mice were divided into three groups:Sham group,MI/3d group,MI/1W group.RT-PCR and Western blot were used to measure the expression levels of CCR9 and CCL25 in heart tissue after MI.Immunohistochemistry was used to test the expression level as well as the expression position of CCR9 and CCL25 after MI.Immunofluorescence was used to figure out the cell type to express CCR9.Results:Compared with sham group,The immunohistochemistry results showed that the expression of both CCL25 and CCR9 was significantly up-regulated at 3 and 7 days after MI,whereas the sham hearts displayed low expression levels.Immunohistochemistry also indicated that CCL25 and CCR9 were mainly expressed in the infarct area and border zone,in which a large number of inflammatory cells had infiltrated,revealing that CCR9 was highly expressed in infiltrating cells.Double immunofluorescence experiments were performed using an anti-CCR9 antibody combined with anti-CD68,anti-CD3 or anti-(a-actinin)antibodies to determine which cells expressed CCR9.The results showed that CCR9 was highly expressed in CD68-positive and CD3-positive cells but not cardiomyocytes.RT-PCR and western blots indicated the same results as immunohistochemistry.Both the CCR9 RNA and protein levels were up-regulated at 3 days post-MI,and the expression levels were even higher at 1 week after MI.Conclusion:CCL25 and CCR9 were up-regulated in mouse infarcted hearts.CCL25 and CCR9 were mainly expressed in CD68-positive and CD3-positive cells in the infarct area and border zone.Part ?:The effect and mechanism of CCR9 knock out on ventricular remodeling following MIObjective:To explore the effect and mechanism of CCR9 gene knockout on ventricular remodeling following MI.Methods:CCR9 gene knockout(CCR9-/-)mice as well their littermate control(CCR9+/+)with a body weight of 24-27g and an age of 8-10 week old were chosen to conduct MI surgery by ligating the left anterior descending coronary.The mice were divided into four groups:CCR9+/+ Sham group,CCR9-/-Sham group,CCR9+/+ MI 1W group,CCR9-/-MI 1W.After surgery,mice living status were observed and death values were recorded.Standard 2D echocardiographic images of the left ventricular(LV)dimensions were collected in the short-axis view at the papillary muscle level.M-mode measurement data were obtained from an average of 4-5 cardiac cycles.The LV end-diastolic dimension(LVEDd),LV end-systolic diameter(LVESd),ejection fraction(EF)and LV fractional shortening(FS)were measured.Mice body weight,heart weight,lung weight and tibia length were measured.HE stainning was used to measure cross section area of cardiomyocytes and infracted size.Picrosirius red(PSR)was used to examine interstitial collagen deposition.RT-PCR was used to detect hypertrophy markers ANP,BNP,?-MHC and fibrosis markers CTGF?Collage??Collage??Western blot was used to test MAPK signal pathway.Results:All sham-operated mice survived for 1 week.The CCR9-/-MI mice displayed a trend of increased(but not significant)survival rates(61.4%)compared with the CCR9+/+ MI mice(53.7%)at 1 week after MI.Consistent with the survival rates,HE stainning showed that the CCR9-/-MI mice had a smaller infarct size than the CCR9+/+ MI mice.The echocardiographic measurements showed improved cardiac function with a decreased LVEDd and LVESd and a higher FS and EF in the CCR9-/-MI group compared with the CCR9+/+ MI group.At lweek post-MI,the CCR9-/-MI mice exhibited lower heart weight(HW)/body weight(BW),lung weight(LW)/body weight and heart weight/tibia length(TL)ratios.The cross-sectional area(CSA)of cardiomyocytes in a remote area,as analyzed by H&E staining,was much smaller in the CCR9-/-MI mice than in the CCR9+/+ MI mice;the morphologies of the cardiomyocytes were more regular and the LV collagen volume was much smaller in the CCR9-/-Ml mice.In addition,the mRNA levels for markers of hypertrophy(ANP,BNP and ?-MHC)and fibrosis(CTGF,collagen I,and collagen III)were all reduced in the CCR9-/-mice at lweek post-MI.Because the MAPK(mitogen-activated protein kinase)signaling pathway plays a vital role in cardiac hypertrophy,we further examined the levels of ERK1/2,JNK1/2 and p38 expression and phosphorylation.After MI,the levels of phosphorylated ERK1/2,JNK1/2 and p38 were all remarkably increased,but they were reduced in the CCR9-/-MI mice.Conclusion:CCR9 deletion reduced MI-induced mortality and infarct size and improved cardiac dysfunction.CCR9 deletion attenuated LV remodeling by influencing MAPK signaling pathway.Part ?:The effect and mechanism of CCR9 knock out on inflammation following MIObjective:To explore the effect and mechanism of CCR9 gene knockout on ventricular inflammation following MI.Methods:CCR9 gene knockout(CCR9-/-)mice as well their littermate control(CCR9+/+)with a body weight of 24-27g and an age of 8-10week old were chosen to conduct MI surgery by ligating the left anterior descending coronary.The mice were divided into four groups:CCR9+/+ Sham group,CCR9-/-Sham group,CCR9+/+ MI 1W group,CCR9-/-MI 1W.Tunnel staining was used to detected myocardial apoptosis.Immunohistochemistry was used to test inflammatory cell infiltration.RT-PCR was used to test the inflammatory factor expression levels.Western blot was used to measure the protein expression levels of NF-Kb signal pathway.Results:A decreased number of TUNEL-positive cardiomyocytes was observed in the CCR9-/-MI hearts compared with the CCR9+/+ MI hearts.Western blots indicated that Bc12 expression was markedly increased in the CCR9-/-MI hearts,whereas the expression of Bax and cleaved caspase 3 was decreased compared with the CCR9+/+ MI hearts.The immunohistochemistry analysis presented a decreased infiltration of these inflammatory cells in the CCR9-/-MI hearts at 1 week after MI.We also explored the expression levels of cytokines secreted by inflammatory cells.The mRNA levels of the proinflammatory cytokines IL-1?,IL-6 and TNF-a were decreased in the CCR9-/-MI LV tissue.The levels of phosphorylated p65 and IKBa were substantially reduced in the knockout MI mice,indicating that the NF-kB pathway was strongly suppressed by the CCR9 deletion.Conclusion:CCR9 deletion attenuated MI-induced cardiomyocyte death and CCR9 deficiency inhibited inflammatory responses following MI by regulating NF-kB signal pathway.Part ?:The effect of CCR9 knock out on electrical remodeling following MIObjective:To explore the effect of CCR9 gene knockout on ventricular electrical remodeling following MI.Methods:CCR9 gene knockout(CCR9-/-)mice as well their littermate control(CCR9+/+)with a body weight of 24-27g and an age of 8-10 week old were chosen to conduct MI surgery by ligating the left anterior descending coronary.The mice were divided into four groups:CCR9+/+ Sham group,CCR9-/-Sham group,CCR9+/+ MI 1W group,CCR9-/-MI 1W.We monitored the 24h ECG electric signals using implantable telemetry.Langendorff-perfused hearts were used to characterize the changes in the electrophysiological parameters(APD90,threshold of APD alternans and VA incidence).Western blot was used to detect the expression level of CX43.Results:During 24 h of continuous recording,the heart rate,RR interval,and PR interval were approximately the same among the four groups.Both the CCR9+/+ and CCR9-/-mice had a prolonged QRS interval,QT interval and QTc after MI.However,there was no difference between the CCR9+/+ MI mice and CCR9-/-MI mice.After MI,the APD90 was significantly prolonged,but it became much shorter in the CCR9-/-MI mice compared with the CCR9+/+ MI mice.The threshold of APD alternans was significantly decreased after MI but was increased in the CCR9 knockout mice.Additionally,non-sustained ventricular arrhythmias were induced in 10 of the 11 CCR9+/+ MI mice,and 7 CCR9-/-MI mice incurred non-sustained ventricular arrnytnmias,out there was no signmcant ainerence.ine raio of sustainea ventricular arrhythmias was reduced in the CCR9-/-MI mice compared with the CCR9+/+ MI mice.Compared with shams,CX43 was decreased in MI,but CX43 was increased in CCR9-/-MI mice compared with CCR9+/+ MI mice.Conclusion:CCR9 deletion can prevent the arrhythmias occurrence by inhibiting the CX43 expression decrease.