Loss of Myeloid-Derived Growth Factor Leads to Increased Fibrosis in Mice After Myocardial Infarction
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摘要:目的
研究心梗后骨髓源性生长因子(myeloid-derived growth factor, Mydgf)的缺失对心肌成纤维细胞活化的作用。
方法取成年野生型(wild type, WT)小鼠与Mydgf敲除(myeloid-derived growth factor knockout, Mydgf-KO)小鼠,检验这两组小鼠的心功能指标左室射血分数(left ventricular ejection fraction, LVEF)和左室短轴缩短率(left ventricular fractional shortening, LVFS)(n=10),实时定量PCR(quantitative real-time PCR, qRT-PCR)(n=3)检测肌成纤维细胞标志物α-平滑肌肌动蛋白(α-SMA)、骨膜蛋白(postn)、Ⅷ型胶原(type Ⅷ collagen, col8al)、结缔组织生长因子(connective tissue growth factor, ctgf)的mRNA表达水平,Western blot(n=3)检测肌α-SMA的蛋白表达水平;对WT与Mydgf-KO小鼠造心肌梗死(myocardial infarction, MI)模型,检测小鼠术后LVEF和LVFS(n=10),取出心脏后进行Masson染色测定梗死面积(n=10)。分别在MI后第7天、第14天收集Mydgf-KO及WT小鼠的心脏,验证肌成纤维细胞标志物(n=3)的表达情况。
结果相较于WT小鼠,Mydgf-KO成年小鼠LVEF、LVFS无明显变化(P均>0.05),但α-SMA和postn的mRNA水平上调,α-SMA蛋白表达水平增加(P均<0.05);MI后,与WT小鼠相比,Mydgf-KO小鼠LVEF、LVFS下降,且梗死面积增大(P均<0.05);进一步发现,Mydgf-KO小鼠α-SMA、col8al、postn和ctgf的mRNA水平升高,同时α-SMA蛋白表达水平上调且α-SMA阳性的成纤维细胞增加(P均<0.05)。
结论Mydgf缺失促使心脏成纤维细胞转变为肌成纤维细胞,同时也加剧心梗后的纤维化。
Abstract:ObjectiveTo investigate the effect of the loss of myeloid-derived growth factor (Mydgf) on the transformation of cardiac fibroblasts into myofibroblasts after myocardial infarction (MI).
MethodsTwo adult mouse groups, including a wild-type (WT) group and another group with Mydgf knockout (Mydgf-KO), were examined in the study. The mice in these two groups were tested for their cardiac function by measuring left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (n=10). Quantitative real-time PCR (qRT-PCR) (n=3) was performed to determine the mRNA expression levels of myofibroblast markers, including α-smooth muscle actin (α-SMA), periostin (postn), type Ⅷ collagen (col8al), and connective tissue growth factor (ctgf). Western blot (n=3) was performed to verify the protein expression levels of α-SMA. MI modeling was performed on the WT and the Mydgf-KO mice. Postoperative LVEF and LVFS (n=10) were then measured. The hearts were harvested and Masson staining was performed to determine the infarcted area (n=10). The heart samples of Mydgf-KO and WT mice were collected at d 7 and d 14 after MI, respectively, to verify the expression of myofibroblast markers (n=3).
ResultsCompared with WT mice, LVEF and LVFS in adult Mydgf-KO mice showed no significant changes (all P>0.05). However, the mRNA levels of α-SMA and postn were upregulated, and α-SMA protein expression was also increased (all P<0.05). After MI, compared with WT mice, LVEF and LVFS in Mydgf-KO mice decreased, and the infarcted area increased significantly (all P<0.05). Furthermore, mRNA levels of α-SMA, col8al, postn, and ctgf were increased in Mydgf-KO mice. In addition, the α-SMA protein expression level was upregulated and α-SMA-positive fibroblasts were increased (P<0.05).
ConclusionMydgf deletion promotes the transformation of cardiac fibroblasts into myofibroblasts and aggravates myocardial fibrosis after MI.
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心肌纤维化是导致心肌梗死和心脏功能恶化的重要病理基础,同时心肌梗死往往会造成心脏重塑。在重塑过程中,肌成纤维细胞引起梗死区的心肌纤维化,之后会出现一系列的不良反应如组织结构紊乱,僵硬程度增高,细胞外基质大范围的沉积,最终形成不可逆的修复瘢痕[1-2]。虽然瘢痕可以防止心肌梗死后的心脏破裂,但它也会导致血流动力学功能障碍和心律失常,从而导致心力衰竭。在心肌梗死诱导的心肌纤维化中,其纤维化水平与心血管疾病患者致死率和不良事件频发率相关[3]。心脏组织结构纤维化的基本是转变成纤维细胞状态,在心脏损伤后首先被激活,随即高度增殖活化为肌成纤维细胞的状态,进而产生瘢痕[4]。由此得知,探究肌成纤维细胞被激活的因素至关重要。
目前发现一些神经体液介质、细胞因子等在肌成纤维细胞激活过程中发挥重要的作用,例如:血管紧张素Ⅱ[5-6]、转化生长因子-β(transforming growth factor-β, TGF-β)[7-8]等。已有研究证明盲肌样蛋白家族(musclebind-like protein, MBNL)在心肌梗死后通过调控Sox9影响心肌成纤维细胞增殖,以及参与维持成纤维细胞向肌成纤维细胞转变[4]。溴结构域和外结构域蛋白可以联合致活转录,当注射全身性溴结构域和外结构域抑制剂时可以减轻小鼠的心力衰竭[9]。溴结构域和外结构域抑制剂通过成纤维细胞中转录因子MEOX1(mesenchyme homeobox 1, MEOX1)介导心肌纤维化和心力衰竭,且其依赖性的转录开关在心力衰竭中促进成纤维细胞活化为肌成纤维细胞[10]。同源盒转录因子家族(iroquois homeobox protein, IRX)在血管紧张素Ⅱ刺激时通过作用早期生长反应因子1通路促进病理性心脏纤维化的发展[11]。19号染色体(C19orf10)编码的骨髓源性生长因子(myeloid-derived growth factor, Mydgf)又被命名为SF20或白细胞介素25,是由骨髓源性单核细胞以及巨噬细胞分泌的蛋白质[12]。前期实验发现[13],Mydgf基因敲除成年小鼠心肌梗死后表现出更大的纤维化瘢痕组织以及更严重的心脏收缩功能障碍,提示Mydgf可能与心肌纤维化相关。本研究拟探索Mydgf缺失在心肌梗死后是否促进心脏成纤维细胞活化完成纤维化修复。
1. 材料与方法
1.1 实验动物
成年(6~8周)龄野生型雄性C57BL/6J小鼠(24~26 g)(以下简称为WT小鼠),来自北京维通利华动物公司,并且其执行号SCXK(京)2021-0011。Mydgf-flox小鼠与EIIa-Cre小鼠交配产生的后代,与WT小鼠繁育后得到Mydgf-KO小鼠(敲除Mydgf基因的1、2、3号外显子)。成年SPF级Mydgf-KO雄性小鼠(6~8周龄,24~26 g)由上海南方模式实验室赠予,其后代由本实验室繁育。动物操作过程已得到北京协和医学院心血管疾病国家重点实验室实验动物管理和使用委员会的批准,批准号FW-2022-0053。
1.2 主要试剂与仪器
1.2.1 主要试剂
Masson试剂盒(Sigma,美国);免疫荧光抗体(Invitrogen,美国);兔抗Mydgf抗体(爱必信,上海);兔抗α-平滑肌肌动蛋白(α-SMA)抗体(Abcam,美国公司);DAPI细胞核染液(中杉金桥,北京);凝胶成像仪(UVP,英国);烤片机、石蜡切片机(徕卡,德国);激光共聚焦显微镜、光学体视显微镜(Zeiss,德国);iBlot 2干转印系统、−20 ℃医用低温冷冻冰箱(Thermo Fisher,美国);−80 ℃医用深低温冷冻冰箱、4 ℃低温冰箱(海尔,中国)。
1.3 实验方法
1.3.1 Mydgf敲除效果验证
选取成年Mydgf-KO小鼠10只,进行基因型鉴定:裂解鼠尾提取DNA,PCR电泳区别WT(87 bp)与Mydgf-KO(355 bp)的电泳条带。选取成年WT小鼠与Mydgf-KO小鼠,每组3只,实时定量PCR(quantitative real-time PCR, qRT-PCR)检测Mydgf的mRNA水平,Western blot检测Mydgf的蛋白表达量。qRT-PCR和Western blot检测方法具体见1.3.7和1.3.8小节。
1.3.2 成年小鼠心肌梗死模型
各选取上述Mydgf-KO成年小鼠繁育的小鼠及其对照WT同窝小鼠20只,每组留3只做术前对照,每组另外17只建立心肌梗死模型:腹腔注射三溴乙醇麻醉,气管插管,碘伏消毒后,剪开左侧第3、4肋之间的皮肤,钝性破开小鼠的肋间隙,挤出心脏,将其左心耳右下缘穿入7-0缝合线,结扎冠状动脉后见线结以下心肌组织缺血发绀发白,即为建模成功。缝合肋间隙及皮肤,待其苏醒后拔出气管插管后放回笼位。造模后各组死亡1只,故每组有19只纳入后续检测。
1.3.3 心脏超声心动图评价
将小鼠异氟烷麻醉后,固定在操作台上。在未手术以及心肌梗死造模术后第2、7、14、21、28天,取Mydgf-KO成年小鼠及其对照WT同窝小鼠(n=10),采用Vevo 2100超声仪(VisualSonics,加拿大)取左心室长轴横切面,利用软件计算小鼠左室射血分数(left ventricular ejection fraction, LVEF)和左室短轴缩短率(left ventricular fractional shortening, LVFS)。
1.3.4 心脏组织标本的收集与处理
将19只WT小鼠与19只Mydgf-KO小鼠分别在未手术(n=3)以及心肌梗死后第7天(n=3)、第14天(n=3)、第28天(n=10)脱颈处死,采用眼科剪打开胸腔,摘取心脏,固定、脱水以及包埋。调整切片厚度为5 μm,以每层200 μm的间隔从心梗部位到心尖连续切片,每个心脏收集5~7个层面。
1.3.5 Masson染色
心肌梗死后第28天,各取10只WT与Mydgf-KO小鼠心脏组织拷片、脱蜡及水化;然后将切片放置在2.5%重铬酸钾溶液中浸泡持续12~18 h;染色后脱水:依次将切片放进体积分数70%、80%、90%三种无水乙醇溶液浸泡;再将切片浸泡在二甲苯溶液中;使用中性树胶封片。蔡司扫片机进行图像采集,利用Image J软件分析染成蓝色的心脏梗死面积占染成红色的心脏组织面积的百分比。
1.3.6 免疫荧光染色
心肌梗死后第7天,各取3只Mydgf-KO成年小鼠及其对照WT同窝小鼠的心脏组织,免疫荧光染色验证成纤维细胞状态转变情况。烤片、脱蜡、水化、EDTA修复及封闭;将一抗DAPI(1∶
1000 )、α-SMA(1∶250)、Vimentin(1∶250)稀释液滴到组织表面,4 ℃过夜孵育;加入二抗避光室温孵育1 h;避光清洗后保存,进行图像采集。1.3.7 蛋白质印迹(Western blot)
各取未手术以及心肌梗死后第7、14天的Mydgf-KO成年小鼠及其对照WT同窝小鼠(每组、每时点3只)的心脏组织,Western blot验证成纤维细胞状态转变情况。先提取组织蛋白,采用蛋白裂解试剂提取,加热变性,电泳、转膜、封闭、孵育兔抗Mydgf(1∶
1000 )、兔抗α-SMA(1∶1000 )、内参GAPDH(1∶5000 ),最终配置显影液显影。Image J软件分析每个条带的灰度值,以目的条带与内参条带灰度值的比值,为目的条带的相对表达量。1.3.8 qRT-PCR
取未手术以及心肌梗死后第7天的Mydgf-KO成年小鼠及其对照WT同窝小鼠(每组、每时点3只)的心脏组织,qRT-PCR验证成纤维细胞状态转变情况。采用Trizol法提RNA。RNA反转录cDNA,PCR反应程序为95 ℃ 10 min,95 ℃ 10 s、60 ℃ 30 s,循环40次,使用2−ΔΔCt计算各mRNA的相对表达水平。基因引物序列见表1。
表 1 实时荧光定量PCR特异性引物及序列Table 1. Specific primers and sequences for real-time PCRGene Sequence Amplified product
length/bpGAPDH F: 5′-CGTCCCGTAGACAAAATGGT-3′ 110 R: 5′-TTGATGGCAACAATCTCCAC-3′ Mydgf F: 5′-TCGTGCATTCGTTCTCCC-3′ 87 R: 5′-GCTCGTTGGTCCCTCCTT-3′ α-SMA F: 5′-GTCCCAGACATCAGGGAGTAA-3′ 102 R: 5′-TCGGATACTTCAGCGTCAGGA-3′ postn F: 5′-CCTGCCCTTATATGCTCTGCT-3′ 297 R: 5′-ACATGGTCAATAGGCATCACT-3′ ctgf F: 5′-GGGCCTCTTCTGCGATTTC-3′ 149 R: 5′-CCAGGCAAGTGCATTGGTA-3′ col8al F: 5′-ACTCTGTCAGACTCATTCAGGC-3′ 131 R: 5′-CAAAGGCATGTGAGGGACTTG-3′ GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Mydgf: myeloid-derived growth factor; α-SMA: α-smooth muscle actin; postn: periostin; ctgf: connective tissue growth factor; col8al: type Ⅷ collagen. 1.4 统计学方法
使用GraphPad Prism(8.0版,GraphPad软件)进行统计。每组实验均重复3次以上,各组间的数据表述为均值±标准误,以单因素方差分析(one-way ANOVA)比较多组间差异,选用独立样本t检验来比较两组间差异,P<0.05为差异有统计学意义。
2. 结果
2.1 Mydgf敲除效果鉴定和造模情况
如图1显示,PCR鉴定结果显示10只Mydgf-KO小鼠组条带均在355 bp处显影(图1A)。与WT同窝小鼠相比,Mydgf-KO小鼠的Mydgf的mRNA与蛋白水平均下降(P均<0.05)(图1B~1C)。这表明引进的Mydgf-KO成年小鼠的Mydgf基因确定已被敲除,实验中均用该Mydgf-KO成年小鼠繁育的小鼠。
图 1 PCR、qRT-PCR及Western blot验证Mydgf敲除效率Figure 1. PCR, qRT-PCR, and Western blot were performed to verify the Mydgf knockdown efficiency.A, The bands of – and + are tail samples of WT mice (87 bp) and Mydgf-KO mice (355 bp) respectively, and bands 1-10 are tail samples of unknown mice (Marker: DL2000); B, the expression level of Mydgf mRNA in WT mice and Mydgf-KO adult mice ; C, protein expression level of Mydgf in WT and Mydgf-KO adult mice under physiological conditions. WT: wild type (C57BL/6J mice); Mydgf-KO: mice with Mydgf systemic gene knockout. n=3. * P<0.05, **** P<0.0001 , vs. WT group .2.2 Mydgf缺失促进成纤维细胞活化
如图2显示,超声检测心肌梗死术前小鼠的心功能,WT小鼠的LVEF为(
74.724 ±2.304)%,LVFS为(43.645 ±3.706)%;Mydgf-KO小鼠的LVEF为(64.407 ±2.103)%,LVFS为(37.326 ±2.770)%,两组间LVEF、LVFS值差异均无统计学意义(P均>0.05),表明Mydgf缺失不影响小鼠的心功能(图2A)。心肌梗死术前,与WT同窝小鼠相比,Mydgf-KO成年小鼠肌成纤维细胞标志物α-SMA和骨膜蛋白(postn) mRNA水平升高(P均<0.05)(表2),α-SMA蛋白水平增高(P<0.05)(图2B、表2)。这表明Mydgf缺失促进成纤维细胞转变为肌成纤维细胞。表 2 Mydgf敲除促进成纤维细胞转变为肌成纤维细胞Table 2. Mydgf knockdown promotes the transformation of fibroblasts into myofibroblastsIndex n WT group Mydgf-KO group Mydgf mRNA 3 1.0010 ±0.0383 0.0442 ±0.0008 #α-SMA mRNA 3 1.0080 ±0.0890 1.8620 ±0.2108 *postn mRNA 3 1.0020 ±0.0482 1.3770 ±0.1301 *α-SMA protein 3 1.0000 ±0.1655 1.8470 ±0.2418 ** P<0.05, # P< 0.000 1 , vs. WT group. The samples were all collected before MI modeling.2.3 Mydgf缺失导致成年小鼠心肌梗死后心肌纤维化加重
见图3,在术后第2、7、14、21、28天对小鼠进行了超声检测,观察小鼠心功能。与WT小鼠组相比,MI后Mydgf-KO小鼠在第21和28天LVEF和LVFS均降低(P均<0.05)(图3A)。Masson结果显示,术后第28天的WT小鼠组梗死面积占心脏组织总面积为(15.01±0.74)%,Mydgf-KO小鼠组的梗死面积占(26.57±2.04)%,与WT小鼠组相比,Mydgf-KO小鼠梗死面积显著增大(P<0.001)(图3B)。以上结果提示,缺失Mydgf促使心肌梗死后的心功能恶化及梗死面积增大。
图 3 缺失Mydgf加重心肌梗死后的心肌纤维化Figure 3. Loss of Mydgf aggravates myocardial fibrosis after MIA, LVEF and LVFS of WT and Mydgf-KO adult mice; B, Masson plot of WT and Mydgf-KO adult mice after MI. The angular black line marks the size of the infarcted area. The blue-purple color represents the infarcted area after Masson staining. The five graphs arranged from bottom to top are serial sections from the infarction site to the apex of the heart at 200 μm intervals per layer. n=10. * P<0.05, ** P<0.01, vs. Mydgf-KO group.2.4 Mydgf缺失促进心肌梗死后肌成纤维细胞的形成
见图4,与WT小鼠组相比,Mydgf-KO小鼠在心肌梗死第7天,肌成纤维细胞标志物α-SMA、Ⅷ型胶原(col8al)、postn和结缔组织生长因子(ctgf)mRNA水平增高及Mydgf mRNA水平下降(P均<0.05)(图4A、4B);在心肌梗死第7、14天,Mydgf-KO小鼠α-SMA蛋白表达水平增高(P<0.05)(图4C);免疫荧光染色结果显示在心肌梗死第7天,WT小鼠组α-SMA与Vimentin共定位占心脏组织总面积为(9.79±0.83)%,Mydgf-KO小鼠组则为(18.49±1.32)%,与WT小鼠组相比,Mydgf-KO小鼠α-SMA阳性的成纤维细胞增多(P<0.01)(图4D)。以上结果提示,缺失Mydgf在心肌梗死后促进成纤维细胞活化成肌成纤维细胞。
图 4 Mydgf缺失促进心肌梗死后成纤维细胞转变为肌成纤维细胞Figure 4. Mydgf deletion promotes the transformation of fibroblasts into myofibroblasts after MIA, The expression levels of α-SMA, postn, col8al, and ctgf mRNA in WT and Mydgf-KO adult mice 7 days after the MI; B, the mRNA expression level of Mydgf in WT and Mydgf-KO adult mice 7 days after MI; C, the protein expression levels of α-SMA in WT and Mydgf-KO adult mice 7 and 14 days after MI; D, multiplex immunofluorescence staining of α-SMA (green), Vimentin (red), and DAPI (blue) was performed for the heart tissues from the WT and the Mydgf-KO groups 7 days after MI (Scale bar: 20 μm). n=3. * P<0.05, ** P<0.01, *** P<0.001, vs. WT group.3. 讨论
2020年我国城乡居民的疾病致死率中,其中心血管疾病占首位,分别占乡村、城市死亡率的48.00%和45.86%;我国心血管病主要死因是缺血性心脏病、出血性和缺血性脑卒中[14]。而心肌纤维化是心肌梗死、心力衰竭和心律失常等多种心血管疾病中常见的病理进程,与患者的不良预后密切相关。在心脏重塑过程中,激活的成纤维细胞(肌成纤维细胞)导致心肌梗死区的心脏纤维化,以支持心脏结构[15-17]。因此,阐明心脏纤维化治疗过程中,成纤维细胞被激活这一步骤至关重要,抑制该过程的发生是治疗心肌纤维化的关键靶点。
既往研究表明,心肌梗死后Mydgf对心脏心肌细胞凋亡起负向作用、对心脏内皮细胞增殖起正向作用,来达到修补梗死心脏的目的[12]。Mydgf也可通过保护心肌细胞中肌/内质网钙泵的表达来预防压力超负荷诱导的心力衰竭[18]。由本实验室既往结果得知[13],Mydgf可通过对心肌细胞增殖起正向作用来实现新生小鼠心脏再生;与野生型小鼠相比,心肌梗死后成年和新生小鼠的Mydgf基因敲除后,瘢痕心肌组织会变得更大,心脏收缩功能障碍也会更为严重。
为探究Mydgf缺失在心肌梗死后心肌成纤维细胞激活过程中的潜在作用,本研究利用Mydgf-KO小鼠,结合心肌梗死模型[19],检测纤维化过程中成纤维细胞状态转变。通过M型超声和Masson染色检验小鼠心功能和瘢痕面积,结果发现,与WT组相比,Mydgf-KO鼠心肌梗死第21天LVEF和LVFS下降,表现出更明显的收缩功能障碍;Mydgf-KO小鼠出现了更大的瘢痕面积。鉴于Mydgf在心肌纤维化中的关键作用[13],本研究收集Mydgf-KO成年小鼠及其对照WT同窝小鼠的心脏组织,通过Western blot和qRT-PCR验证成纤维细胞状态转变情况。进一步发现Mydgf-KO小鼠心肌梗死后,心肌组织中肌成纤维细胞标志物α-SMA、postn表达水平增高,提示缺失Mydgf促进心脏成纤维细胞转变成肌成纤维细胞。虽然生理情况下Mydgf-KO小鼠心功能没有恶化,但是α-SMA、postn表达水平依然升高,猜测在生理情况下Mydgf缺失生成的肌成纤维细胞活化的数目不足以生成富含胶原的瘢痕,从而影响心功能的变化。与WT鼠相比,Mydgf-KO小鼠心肌梗死后的心肌组织中肌成纤维细胞标志物col8al、ctgf mRNA表达水平增高,提示缺失Mydgf心肌梗死后促进心脏成纤维细胞转变成肌成纤维细胞。通过本实验室质谱数据发现与对照WT同窝小鼠相比,Mydgf-KO小鼠心肌梗死后成纤维细胞生长因子1(fibroblast growth factor 1, FGF1)的表达水平下降(数据待发表)。并且已有研究证明FGF1亚家族(FGF1、FGF2)可以促进心肌细胞存活,并减少心肌梗死时的梗死面积,并且也能减弱主动脉弓缩窄模型(transverse aortic constriction, TAC)和血管紧张素Ⅱ(angiotensin Ⅱ, Ang Ⅱ)诱导的病理性心脏重构[20]。由此提示Mydgf可能通过分泌FGF1抑制成纤维细胞活化。本研究将继续探讨Mydgf缺失可能加剧心肌纤维化的分子生物学机制。
综上,本实验结果显示缺失Mydgf能够促进成纤维细胞转变为肌成纤维细胞,促使心肌梗死后心肌纤维化加剧。但本研究缺乏Mydgf特异性过表达对心脏成纤维细胞的影响,课题组将继续探索Mydgf是否特异性作用心脏成纤维细胞,进一步将其转变为肌成纤维细胞;以及Mydgf缺失加剧心肌纤维化作用机制是否与FGF1的表达有关,或是否存在其他相关机制仍需后续实验进行深入探究。应用药物学等手段来抑制肌成纤维细胞活性,可以很大程度上防止心脏及其邻近组织损失后的组织重构,是解决过度纤维化的问题的潜在方向。
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作者贡献声明 韩国玲负责论文构思、正式分析、调查研究、研究方法、验证、可视化和初稿写作,郝琰琰负责数据审编、研究方法和审读与编辑写作,李若朴负责研究项目管理、软件和监督指导,刘维静负责研究方法和监督指导,刘俊负责正式分析和监督指导,聂宇负责经费获取和提供资源,白丽娜负责研究项目管理、监督指导、可视化和审读与编辑写作,王玉瑶负责研究项目管理、提供资源、监督指导和审读与编辑写作。所有作者已经同意将文章提交给本刊,且对将要发表的版本进行最终定稿,并同意对工作的所有方面负责。
Author Contribution HAN Guoling is responsible for conceptualization, formal analysis, investigation, methodology, validation, visualization, and writing--original draft. HAO Yanyan is responsible for data curation, methodology, and writing--review and editing. LI Ruopu is responsible for project administration, software, and supervision. LIU Weijing is responsible for methodology and supervision. LIU jun is responsible for formal analysis and supervision. NIE Yu is responsible for funding acquisition and resources. BAI Lina is responsible for project administration, supervision, visualization, and writing--review and editing. WANG Yuyao is responsible for project administration, resources, supervision, and writing--review and editing. All authors consented to the submission of the article to the Journal. All authors approved the final version to be published and agreed to take responsibility for all aspects of the work.
利益冲突 上海南方模式实验室赠送了SPF级Mydgf-KO雄性小鼠。除此之外,所有作者均声明不存在利益冲突。
Declaration of Conflicting Interests The Shanghai Southern Model Laboratory donated male mice with SPF grade Mydgf-KO. Other than this, all authors declare no competing interests.
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图 1 PCR、qRT-PCR及Western blot验证Mydgf敲除效率
Figure 1. PCR, qRT-PCR, and Western blot were performed to verify the Mydgf knockdown efficiency.
A, The bands of – and + are tail samples of WT mice (87 bp) and Mydgf-KO mice (355 bp) respectively, and bands 1-10 are tail samples of unknown mice (Marker: DL2000); B, the expression level of Mydgf mRNA in WT mice and Mydgf-KO adult mice ; C, protein expression level of Mydgf in WT and Mydgf-KO adult mice under physiological conditions. WT: wild type (C57BL/6J mice); Mydgf-KO: mice with Mydgf systemic gene knockout. n=3. * P<0.05, **** P<
0.0001 , vs. WT group .图 3 缺失Mydgf加重心肌梗死后的心肌纤维化
Figure 3. Loss of Mydgf aggravates myocardial fibrosis after MI
A, LVEF and LVFS of WT and Mydgf-KO adult mice; B, Masson plot of WT and Mydgf-KO adult mice after MI. The angular black line marks the size of the infarcted area. The blue-purple color represents the infarcted area after Masson staining. The five graphs arranged from bottom to top are serial sections from the infarction site to the apex of the heart at 200 μm intervals per layer. n=10. * P<0.05, ** P<0.01, vs. Mydgf-KO group.
图 4 Mydgf缺失促进心肌梗死后成纤维细胞转变为肌成纤维细胞
Figure 4. Mydgf deletion promotes the transformation of fibroblasts into myofibroblasts after MI
A, The expression levels of α-SMA, postn, col8al, and ctgf mRNA in WT and Mydgf-KO adult mice 7 days after the MI; B, the mRNA expression level of Mydgf in WT and Mydgf-KO adult mice 7 days after MI; C, the protein expression levels of α-SMA in WT and Mydgf-KO adult mice 7 and 14 days after MI; D, multiplex immunofluorescence staining of α-SMA (green), Vimentin (red), and DAPI (blue) was performed for the heart tissues from the WT and the Mydgf-KO groups 7 days after MI (Scale bar: 20 μm). n=3. * P<0.05, ** P<0.01, *** P<0.001, vs. WT group.
表 1 实时荧光定量PCR特异性引物及序列
Table 1 Specific primers and sequences for real-time PCR
Gene Sequence Amplified product
length/bpGAPDH F: 5′-CGTCCCGTAGACAAAATGGT-3′ 110 R: 5′-TTGATGGCAACAATCTCCAC-3′ Mydgf F: 5′-TCGTGCATTCGTTCTCCC-3′ 87 R: 5′-GCTCGTTGGTCCCTCCTT-3′ α-SMA F: 5′-GTCCCAGACATCAGGGAGTAA-3′ 102 R: 5′-TCGGATACTTCAGCGTCAGGA-3′ postn F: 5′-CCTGCCCTTATATGCTCTGCT-3′ 297 R: 5′-ACATGGTCAATAGGCATCACT-3′ ctgf F: 5′-GGGCCTCTTCTGCGATTTC-3′ 149 R: 5′-CCAGGCAAGTGCATTGGTA-3′ col8al F: 5′-ACTCTGTCAGACTCATTCAGGC-3′ 131 R: 5′-CAAAGGCATGTGAGGGACTTG-3′ GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Mydgf: myeloid-derived growth factor; α-SMA: α-smooth muscle actin; postn: periostin; ctgf: connective tissue growth factor; col8al: type Ⅷ collagen. 表 2 Mydgf敲除促进成纤维细胞转变为肌成纤维细胞
Table 2 Mydgf knockdown promotes the transformation of fibroblasts into myofibroblasts
Index n WT group Mydgf-KO group Mydgf mRNA 3 1.0010 ±0.0383 0.0442 ±0.0008 #α-SMA mRNA 3 1.0080 ±0.0890 1.8620 ±0.2108 *postn mRNA 3 1.0020 ±0.0482 1.3770 ±0.1301 *α-SMA protein 3 1.0000 ±0.1655 1.8470 ±0.2418 ** P<0.05, # P< 0.000 1 , vs. WT group. The samples were all collected before MI modeling. -
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