| BackgroundCardiac remodeling after myocardial infarction(MI)is the main predictor of morbidity and mortality of complications such as heart failure and life-threatening arrhythmias.Although percutaneous coronary intervention is widely used in the initial stage of MI,which can quickly open infarction-related vessels and use the best drug treatment,the occurrence of cardiac remodeling after MI is still widespread.This phenomenon is initially caused by the increase of ventricular wall stress after large area myocardial necrosis,and the infarct size is usually an important determinant of malignant remodeling.However,more and more studies have confirmed that other factors are involved in the process of tissue repair after infarction,mainly involving fine,complex and multi-step cellular responses,including inflammation,immune response and synthesis and degradation of extracellular matrix(ECM).These factors promote the pathological process of cardiac structural and electrical remodeling after MI at the same time.In recent years,post-infarction inflammation,especially the activation and polarization of macrophages,and fibrosis have attracted wide attention in the progress of cardiac remodeling.Cardiac macrophages show different phenotypes and functions(such as M1,pro-inflammatory or M2,anti-inflammatory)to the changes of surrounding microenvironment,which aggravate or alleviate the inflammation of infarcted heart,worsen or improve cardiac function.How to promote the polarization of macrophages to M2 is related to the degree of heart healing and anti-ventricular remodeling after myocardial infarction.C1q/ tumor necrosis-associated proteins(CTRPs)is a family of 15 newly discovered adiponectin-like proteins(CTRP1-CTRP15).Among them,CTRP9 is the closest analogue of APN in the CTRP family and is highly expressed in adult heart,and its globular domain isomer is produced by proteolysis of full-length CTRP9 in myocardial tissue,which is an effective component to produce cardioprotective activity.Although previous studies have confirmed that CTRP9 has the effects of anti-inflammation and anti-ventricular remodeling in chronic phase after MI,whether it can promote the polarization of M2 macrophages in the early stage of MI(7 days)and the specific mechanism are unknown,and whether it can affect atrial structural remodeling and electrical remodeling after MI has not been studied.In this study,we used overexpressed CTRP9 adenovirus to interfere with myocardial infarction rat model established by ligation of anterior descending branch and silent CTRP9 adenovirus to interfere with normal rats,and further studied the effects of CTRP9 on atrial and ventricular macrophage polarization and atrial remodeling in the early stage of MI and its possible mechanism.Part Ⅰ: Effects of CTRP9 on Macrophage Polarization and Left Ventricular Function after Myocardial Infarction in Rats.Objective: To investigate the effects of CTRP9 on ventricular function and macrophage polarization after MI in rats.Methods: a Adenovirus carrying CTRP9 transcription factor(Ad-CTRP9-GFP)and negative control GFP airborne virus(Ad-GFP),small hairpin adenovirus silencing CTRP9 transcription factor(Ad-sh CTRP9-GFP,sh CTRP9)and negative control GFP airborne virus(scramble shRNA-GFP,shRNA)were constructed.Sixty-eight SD rats were randomly divided into sham operation group(Sham,n=12),MI group(n=21),Ad-GFP+MI group(n = 18)and Ad-CTRP9+MI group(n = 17).The MI model was established by ligating the anterior descending of coronary artery.Three or seven days after using Ad-CTRP9 by jugular vein injection in Ad-CTRP9+MI group,Ad-GFP+MI group was injected with the same dose of Ad-GFP,Sham and MI group was injected with the same dose of PBS;At the cellular level,g CTRP9 and / or shRNA interfered with lipopolysaccharide(LPS)+ interferon-γ(INF-γ)-induced M1 transformation of peritoneal macrophages or IL-4-induced M2 macrophage model,respectively,and further verified the pathway by using AMPK inhibitor Compoud C and NF-κB inhibitor PDTC.Evaluation:(1)plasma ELISA and Western blot to detect the level of CTRP9 in the periinfarct area from each group;(2)to observe the survival status of rats in each group,count the number of deaths and make survival curves;(3)to evaluate the infarct size by Masson staining;and(4)to evaluate the LVEDD,EF and FS of rats in each group by ultrasound;(5)PCR and / or Western blot were used to detect M1 polarization markers(CD86,iNOS),M2 polarization markers(Arg1,CD163),MMP2,p AMPK/AMPK and p NF-κB p65 / NF-κB p65 in peri-infarcted and/ or peritoneal macrophages;immunofluorescence double staining was used to detect the expression of CD86~+CD68~+ and CD163~+CD68~+ macrophages in myocardial tissue;(6)Flow cytometry was used to detect iNOS~+F4/80~+ and CD163~+F4/80~+ in peritoneal macrophages.(7)IL-1β,IL-6 and IL-10 in myocardial tissue and cell supernatant were detected by ELISA.Results: The expression of CTRP9 in peri-infarct area decreased after MI,and increased after jugular vein injection of Ad-CTRP9;Adenovirus-mediated CTRP9 supplementation promoted the transformation of macrophages from M1 to M2 in peri-infarct area on the 3 day after MI.g CTRP9 pretreated peritoneal macrophages promoted the polarization from M1 to M2 after LPS+INF-γ stimulation,g CTRP9 further enhanced the polarization of macrophages induced by IL-4 to M2,and partially silenced the expression of CTRP9 in macrophages(sh CTRP9)could promote the transition of macrophages to M1.CTRP9 increased the phosphorylation of AMPK after MI and inhibited the phosphorylation of NF-κB p65.The effect of CTRP9 on the transition of macrophages to M2 was partially eliminated by the use of Compoud C,while the effect of PDTC was significantly enhanced.The cardiac function of rats decreased significantly on the 7 day after MI,and CTRP9 improved the cardiac function of rats after MI.CTRP9 did not improve the survival rate and infarct size of rats in the early stage of MI.Conclusions: CTRP9 can improve cardiac function in the early stage after MI,at least partly through AMPK/NF-κB pathway to regulate the polarization transition from M1 to M2 macrophages.Part Ⅱ: Effects of CTRP9 on Atrial Inflammation and Fibrosis after Myocardial Infarction in Rats.Objective: To investigate the effect of CTRP9 on atrial inflammation and fibrosis after MI in rats.Methods: The construction of virus was the same as the first part.(1)The subjects were divided into 4 groups: sham group(n=12),MI group(n=12),Ad-GFP+MI group(n = 12)and Ad-CTRP9+MI group(n = 10).Samples were collected and tested on the 3 or 7 day after MI.(2)The subjects were divided into two groups: sh CTRP9 group and shRNA group.Sh CTRP9 and shRNA were injected into normal rats and the same operation was performed on the 5th or 10 day.Evaluation:(1)The expression of atrial inflammatory markers CD68 and the levels of CTRP9,IL-1β,IL-6 and IL-10 mRNA on the 3 or 7 day after MI were evaluated by immunofluorescence or PCR;(2)The expression of iNOS~+CD68~+ and CD163~+CD68~+macrophages in atrial muscle was detected by immunofluorescence double staining,and the mRNA levels of atrial macrophage markers iNOS,CD163,CD68 and inflammatory factors IL-1β,IL-6 and IL-10 were detected by PCR technique;(3)The degree of atrial fibrosis was evaluated by Masson staining;the expression levels of CollagenⅠ and Collagen Ⅲ were detected by PCR;the expression levels of α-SMA and TGF-β1 protein were detected by WB;(4)Cardiac function indexes of rats after sh CTRP9 intervention: LVEDD,EF and FS;(5)After partial silencing of CTRP9,the inflammation-related indexes IL-1β,IL-6,CD68,iNOS and fibrosis indexes CollagenⅠ,Collagen Ⅲ,α-SMA and TGF-β1 were detected by PCR;(6)The protein expression levels of TLR4,p NF-κB p65/NF-κB p65 and p Smad2/3 / Smad2/3 were detected by WB.Results: The infiltration of macrophages and the production of inflammatory factors in the atrium on the 3 day after MI were significantly higher than those on the7 day,and CTRP9 promoted the M2 polarization of atrial macrophages on the 3 day after myocardial infarction,thus reducing the inflammatory response.CTRP9 inhibited atrial fibrosis in the early stage of MI by reducing the production of atrial Collagen type I and Ⅲ,TGF-β1 and α-SMA.Partial silencing of CTRP9 gene in normal rats did not change the cardiac function of rats.Partial knockout of atrial CTRP9,could promote the infiltration of atrial M1 macrophages and the production of inflammatory factors,and increase the area of atrial fibrosis and the level of Collagen type I,type Ⅲ and TGF-β 1 mRNA.CTRP9 inhibited the increased expression of TLR4 and phosphorylation of NF-κB p65 and decreased the degree of phosphorylation of Smad2/3 in atrial tissue after MI.The expression of TLR4 and the degree of phosphorylation of NF-κB p65 in atrium increased after sh CTRP9 intervention,but had no significant effect on the phosphorylation of Smad2/3.Conclusions: CTRP9 may effectively reduce the degree of atrial inflammation and fibrosis in the early stage of MI by inhibiting TLR4/ NF-κB p65 and Smad2/3signal pathway.Part Ⅲ: The Effect of CTRP9 on Atrial Electrical Remodeling after Myocardial Infarction in Rats.Objective: To investigate the effect of CTRP9 on atrial electrical remodeling after MI in rats.Methods: The experimental grouping and administration regimen were the same as the second part.MI was detected after 7 days,and the detection indexes were as follows:(1)the electrocardiogram(ECG)of rats was recorded and the P wave duration and PR interval were analyzed;(2)the atrial conduction time(IACT),the effective refractory period of left and right atrium(LAERP,RAERP)and the occurrence of induced AF(AF induction rate,AF duration)were recorded by Langendorff cardiac perfusion device;(3)the expression of CollagenⅠ~+ α-actinin~+ and Collagen Ⅲ~+α-actinin~+ was detected by atrial immunofluorescence double staining,and the correlation between AF duration and CollagenⅠ/Ⅲ ratio(CVF I/Ⅲ)was evaluated;(4)The expression of IL-1β and CTRP9 in plasma was measured by ELISA to evaluate the correlation between CTRP9 and AF duration and IL-1β.Results: P wave duration did not significantly change in the early stage of MI,but PR interval was prolonged.CTRP9 had no significant effect on P wave duration after MI,but shortened PR interval after MI.MI could prolong RAERP and LAERP,but AERP was not shortened after CTRP9 intervention in MI rats.MI significantly increased the induction rate of AF and prolonged the AF duration,and CTRP9 significantly inhibited the occurrence of AF after MI.The AF duration was positively correlated with the concentration of IL-1β(r=0.93,P<0.001),negatively correlated with the concentration of CTRP9(r=0.86,P<0.001),and negatively correlated with the concentration of CTRP9 and IL-1β(r=0.79,P<0.001).AF duration was positively correlated with CVF I/Ⅲ in the early stage of MI(r=0.714,P=0.047);The CVF I/Ⅲ increased after MI,but there was no significant difference between sham groups and MI groups.CTRP9 did not significantly affect CVF I/Ⅲ after MI.Partial silencing of CTRP9 in normal rat atrium significantly increased the AF susceptibility,prolonged the duration of AF and prolonged IACT,but had no significant effect on LAERP.Conclusions: The supplement of CTRP9 can reduce the occurrence of AF after MI,which may be related to its effect of reducing atrial inflammation and fibrosis. |
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