【作者】 刘琳；

【导师】 黎莉；

【作者基本信息】 山东大学 ， 内科学， 2014， 博士

【摘要】 背景糖尿病心肌病(diabetic cardiomyopathy, DCM)是一种独立于糖尿病大血管并发症的心肌结构和功能改变的疾病,与糖尿病特有的代谢异常相关,以左室舒张和/或收缩功能障碍为主要的临床表现,是糖尿病患者高心力衰竭发生率和高死亡率的主要原因。糖尿病心肌病的主要病理变化包括细胞肥大、凋亡、变性、坏死和心肌间质的纤维化。心肌细胞凋亡是DCM发生发展的重要病理生理机制,心肌细胞凋亡在DCM进程中的作用为：1.减少心肌细胞数量,使心肌收缩单位逐步减少,导致心脏收缩功能障碍；2.由于心肌收缩单位减少,促进心肌细胞代偿性增生,引起心室肥厚及心脏重构,导致心脏舒张功能不全。Smad2/3在将TGF-p信号从细胞表面受体传导至细胞核的过程中起到关键性作用,是调控细胞增殖分化非常重要的信号分子。研究表明,多种刺激如活化蛋白-1、血管紧张素和一氧化氮均可激活Smad2/3,活化的Smad2/3与Smad4结合后形成异源寡聚物转移到细胞核内,激活凋亡相关基因如caspase-3/6/9等的转录,从而引起细胞凋亡,然而Smad2/3在高糖诱导的心肌细胞凋亡中的作用仍不清楚。ALK7是Ⅰ型活化素(Activin)受体家族新成员,它是有丝氨酸/苏氨酸激酶活性的细胞膜孤儿受体,由跨膜区、丝氨酸/苏氨酸激酶区和两者之间的GS区及胞外配体结合区组成,目前已知配体有和生长/分化因子3、Nodal、Activin AB和Activin B。在信号转导过程中上述配体首先和Ⅱ型Activin受体结合,而后ALK7和它们形成复合物,其GS区被Ⅱ型Activin受体磷酸化,激酶区则使受体激活型Smad2/3磷酸化,进而通过Smads信号通路参与细胞粘附、增殖、分化、凋亡等的调控。ALK7在人胰脏、脑组织、脂肪组织、肝脏、肠道和心脏中均有表达,现有研究证实,ALK7介导下游促凋亡信号通路参与人卵巢上皮细胞、肝癌细胞、胰岛细胞的增殖凋亡的调控,但ALK7在心肌细胞凋亡中的作用仍不清楚。结合上述研究,本课题将探讨ALK7-Smad2/3信号转导通路在高糖诱导的心肌细胞凋亡中的作用。目的1.探讨高糖对心肌细胞凋亡的影响；2.探讨Smad2/3在高糖诱导的心肌细胞凋亡中的作用；3.探讨高糖对心肌细胞ALK7表达水平的影响；4.探讨ALK7在高糖诱导的心肌细胞凋亡中的作用及其信号转导机制。材料方法本研究以H9c2大鼠心肌细胞系及原代培养的新生大鼠心肌细胞为研究对象,应用实时定量逆转录聚合酶链反应(real time RT-PCR)、蛋白免疫印迹(western blotting)、流式细胞术(flow cytometry)及细胞转染小干扰RNA(siRNA)等实验方法,分别观察：1.体外模拟糖尿病高糖状态,分为低糖组、高渗组和高糖组,分别给予葡萄糖(5.5mmol/L)、葡萄糖+甘露醇(5.5mmol/L+33mmol/L)和葡萄糖(33mmol/L)孵育H9c2细胞,检测cleaved Caspase3和Bcl2蛋白水平的表达,流式细胞术测早晚期凋亡率；2.高糖、高渗和低糖培养心肌细胞,观察ALK7mRNA.蛋白的表达水平,及Smad2/3磷酸化水平的变化；3.设计、化学合成Smad2、Smad3siRNA,采用脂质体转染大鼠H9c2心肌细胞分别抑制Smad2、Smad3的表达,观察高糖对心肌细胞凋亡的影响；4.设计、化学合成ALK7siRNA,构建质粒,采用脂质体转染大鼠H9c2心肌细胞,观察ALK7抑制后,Smad2/3磷酸化水平的变化,以及cleaved Caspase3、 Bc12蛋白水平和细胞凋亡率的变化。结果1.33mmol/L的高糖上调H9c2细胞ALK7的表达。琼脂糖凝胶电泳结果和Western blotting结果证实ALK7在H9c2细胞系及原代大鼠心肌细胞上均有表达。分别以5.5mmol/L、11mmol/L、22mmol/L、33mmol/L和38.5mmol/L的葡萄糖孵育H9c2心肌细胞24h,结果显示33mmol/L的糖浓度刺激下,ALK7的表达水平较对照组显著增加(P<0.05),因此选用33mmol/L的葡萄糖作为高糖刺激浓度；2.33mmol/L的高糖诱导H9c2细胞凋亡。分别以低糖、高渗、高糖孵育H9c2心肌细胞0h、4h、8h、12h、24h、48h,与低糖组相比,高糖刺激24h,心肌细胞cleaved Caspase3表达增加至1.84倍(P<0.001),Bcl2表达降低至51%(P<0.001),高糖刺激48h,心肌细胞cleaved Caspase3表达增加至2.41倍(P<0.001),Bcl2表达降低至43%(P<0.001),高渗组无显著变化。Annexin V/PI双染流式测凋亡结果显示,高糖刺激24h,心肌细胞出现凋亡增加,以早期凋亡为主,48h出现以晚期为主的凋亡增加；3. Smad2/3参与高糖诱导的H9c2细胞凋亡。低糖、高渗、高糖分别孵育H9c2心肌细胞0h、4h、8h、12h、24h、48h,高糖刺激12h-48h,Smad2/3的磷酸化水平呈时间依赖性增高,48h达高峰,低糖组和高渗组Smad2/3的磷酸化水平无显著变化。Smad2-siRNA与Smad3-siRNA分别转染H9c2心肌细胞,高糖培养48h,与转染无意义序列组(NS组)相比,cleaved Caspase3的表达分别降低至NS组的58%和52%(P<0.05, P<0.05), Bcl2的表达增高至NS组的2.04倍和2.01倍(P<0.01,P<0.001),流式测细胞凋亡率降低至58%和55%(P<0.01,P<0.01)。实验同样探讨了高糖对原代大鼠心肌细胞Smad2/3磷酸化水平的影响,结果显示,25mmol/L的葡萄糖浓度可显著提高原代心肌细胞Smad2/3的磷酸化水平；4.高糖培养不同时间对H9c2细胞及原代大鼠心肌细胞ALK7表达水平的影响。低糖、高渗和高糖培养H9c2细胞0h、0.5h、1h、4h、8h、12h、24h、48h, RT-PCR结果显示,高糖刺激H9c2细胞1h, ALK7mRNA表达水平增加,8h达高峰,增至低糖组的2.98倍(P<0.01),后逐渐下降,但仍高于低糖组。Western blot结果显示,高糖刺激H9c2细胞12h, ALK7表达水平开始增加,24h达高峰,增至低糖组的2.06倍(P<0.001),48h和24h无显著差异。高渗组ALK7mRNA和蛋白水平无显著变化。实验同样探讨了高糖对原代大鼠心肌细胞ALK7表达水平的影响,结果显示,25mmol/L的葡萄糖浓度可显著上调原代心肌细胞ALK7的表达水平；5.ALK7通过Smad2/3信号转导通路调控高糖诱导的H9c2细胞凋亡。应用ALK7-siRNA转染心肌细胞,与转染阴性对照质粒组(NS组)相比,cleaved Caspase3表达减少至76%(P<0.01),Bcl2表达增加至3.42倍(P<0.05),心肌细胞凋亡率减少至62%(P<0.01)；应用siRNA抑制ALK7表达后,Smad2/3的磷酸化水平分别降至65%和73%(P<0.01,P<0.05)。结论1.33mmol/L葡萄糖可以诱导H9c2细胞凋亡；2.Smad2/3参与高糖诱导的H9c2细胞凋亡的调控；3.高糖可以促进ALK7的表达；4.ALK7通过Smad2/3信号转导通路参与调控高糖诱导的H9c2细胞凋亡。背景糖尿病心肌病(DCM)是独立于糖尿病其他大血管并发症的心脏结构和功能改变的疾病,是糖尿病患者高病死率的主要原因。糖尿病心肌病的发病机制复杂,主要涉及：胰岛素抵抗、高血糖、氧化应激、炎症和脂质沉积等,左室舒张和收缩功能不全是DCM的主要临床表现。心肌细胞凋亡和间质纤维化是DCM心功能不全的重要病理机制,心肌细胞凋亡使心肌收缩单位逐步减少,间质纤维化则引起心室肥厚及心脏重构,两者最终导致心脏功能不全的发生。然而,目前关于DCM心肌细胞凋亡和间质纤维化的分子病理学机制仍不甚清楚。活化素受体样激酶7(ALK7)是Ⅰ型转化生长因子-β (TGF-β)受体家族的新成员,研究证实ALK7参与了多种细胞增殖、分化和凋亡的调控。近来在小鼠上的研究表明,ALK7的无意义突变可以改善脂肪蓄积以及肥胖所导致的糖耐量异常和胰岛素抵抗。胰岛素抵抗和脂质代谢紊乱是糖尿病发生的重要诱因,由此我们推测,ALK7在糖尿病的发生发展中起重要作用。我们第Ⅰ部分的实验结果证实,高糖可以上调H9c2细胞中ALK7的表达,但是ALK7在糖尿病心肌组织中的病理生理作用仍待进一步阐明。Smad2/3和Akt是ALK7重要的下游信号分子。现有研究证实,Smad2/3在血管紧张素Ⅱ和晚期糖基化终产物等纤维化因子引起的组织纤维化中起关键作用。Akt则在维持葡萄糖稳态和选择性胰岛素抵抗方面发挥重要作用,Akt活性的降低是胰岛素抵抗的潜在诱因之一。此外,ALK7-Smad2/3和ALK7-Akt信号转导通路都参与了细胞凋亡的调控。综合上述研究,我们提出如下假说：2型糖尿病上调心肌ALK7的表达,ALK7过表达引起心肌细胞凋亡和间质纤维化进而影响DCM的发生发展。在本实验中我们建立了2型糖尿病大鼠的动物模型并通过ALK7-siRNA特异性抑制大鼠体内ALK7基因的表达来探讨ALK7在DCM中的作用,实验结果将为DCM的治疗提供新的潜在靶点。目的1.观察ALK7基因沉默对2型糖尿病大鼠糖脂代谢及胰岛素敏感度的影响；2.观察ALK7基因沉默对2型糖尿病大鼠心脏整体结构和功能的影响；3.观察ALK7基因沉默对2型糖尿病大鼠心肌细胞凋亡和间质纤维化的影响；4.观察ALK7基因沉默对下游信号分子Smad2/3和Akt磷酸化水平的影响。材料方法1.2型糖尿病大鼠动物模型的建立60只体重120g±20g左右(约5周龄)的雄性SD大鼠,适应性喂养1周行腹腔葡萄糖耐量试验(IPGTT)和腹腔胰岛素耐量试验(IPITT)后随机分为4组：对照组(Control)、糖尿病组(DM)、糖尿病+ALK7-siRNA腺病毒干预组(ALK7-siRNA组)和糖尿病+腺病毒空载体组(Vehicle)。 Control组大鼠喂以基础饲料,剩余三组大鼠喂以高糖高脂高热量饲料。4周后重复IPITT和IPGTT,对于高脂喂养大鼠出现胰岛素抵抗者一次性给予腹腔注射STZ30mg/kg。STZ注射12周后,ALK7-siRNA组和Vehicle组大鼠经颈静脉分别给与2.5×1010PFU的ALK7-siRNA腺病毒或空载体,2周后重复腺病毒或空载体的注射。距第1次腺病毒注射4周后处死大鼠,留取标本。2.腹腔葡萄糖耐量试验(IPGTT)和腹腔胰岛素耐量试验(IPITT)IPGTT:大鼠禁食12h,测定空腹尾静脉血糖浓度后,给予一次性腹腔注射葡萄糖1g/kg体重,分别于葡萄糖注射15min、30min、60min和120min时从尾静脉取血测定血糖浓度,并通过个时间点血糖值计算血糖曲线下面积(AUC)。IPITT:大鼠禁食4h,测定尾静脉血糖浓度后,给予一次性腹腔注射胰岛素1unit/kg体重,血糖测定及AUC计算方法同IPGTT。3.血生化检测分离血清,应用Bayer1650血生化分析仪检测血清血糖水平(FBG)、总胆固醇水平(TC)和甘油三酯(TG)水平；应用酶联免疫法测定空腹胰岛素水平(FINS)。4.血压和心率的监测利用大鼠尾动脉血压测量仪监测大鼠尾动脉收缩压(SBP)、舒张压(DBP)、平均动脉压(MBP)和心率,每只大鼠测量3次,取3次平均值。5.血流动力学监测将充有肝素钠生理盐水的塑料导管插入大鼠右侧颈总动脉并沿主动脉插入左室(主动脉瓣开放时插入),记录左室舒张末期压力(LVEDP)和左室收缩压(LVSP)。6.腺病毒注射STZ注射12周后(第17周),经颈静脉分别给与ALK7-siRNA组和Vehicle组大鼠2.5×1010PFU的ALK7-siRNA腺病毒或空载体,2周后重复腺病毒或空载体的注射。7.心肌组织的留取制作5μm厚的组织石蜡切片以备组织病理学染色用；留取左室心肌组织冻入-80℃冰箱,以备RT-PCR及Western blotting用。8.组织和形态学分析石蜡切片H&E染色,观察左室大体形态；石蜡切片经Masson三色染色和天狼猩红染色,观察左室管周纤维化和间质纤维化情况,显微镜下拍照后应用Image-Pro Plus5.0软件对图片进行分析。9.心肌细胞凋亡的检测利用TUNEL法检测左室心肌细胞的凋亡率,凋亡率%=每个视野观察到的阳性细胞数目/该视野的细胞总数。10.免疫组织化学染色组织石蜡切片滴加兔抗大鼠Collagen Ⅱ和Collagen Ⅲ一抗,4℃孵育过夜,滴加生物素标记的山羊抗兔二抗,DAB显色,苏木素复染细胞核,显微镜下拍照后应用Image-Pro Plus5.0软件对图片进行分析。11.实时定量RT-PCR实验用Trizol提取大鼠心肌组织的总RNA,经逆转录和PCR扩增后,采用β-actin作为内参,计算目的基因的相对表达,起始模板的相对浓度＝2-△△CT(注：△ACT=(CT处理组目的基因-CT对照组目的基因)-(CT处理组内参基因-CT对照组内参基因)。12. Western blotting实验每孔加蛋白样品15μg,10%的SDS-聚丙烯酰胺凝胶电泳直至蓝色染料至分离胶底部；将待转凝胶切下,200mA电转至PVDF膜上。将PVDF膜封闭后浸入含一抗反应液的平皿中,4℃振荡孵育过夜,洗膜后浸入HRP结合的二抗,室温下孵育1-2h。 ECL发光,应用Photoshop CS4图像分析系统对Western Blotting图片进行分析,各组样品的目的蛋白条带与β-actin条带的相对光密度比值作为各目的蛋白的相对含量。13.统计分析计量资料以均数±标准差表示,两组间均数差异比较采用独立样本t检验,多组间均数差异比较采用单因素方差分析(one-way ANOVA)。所有分析均采用SPSS17.0统计软件包进行,P<0.05为差异具有统计学意义。结果1.ALK7基因沉默改善2型糖尿病大鼠胰岛素抵抗。高脂喂养大鼠4周,行IPGTT及IPITT试验,结果显示高脂喂养4周大鼠各个时间点的血糖浓度及血糖曲线下面积(AUC)均较基线水平显著升高；实验末IPGTT及IPITT试验显示,与空载体组比较,ALK7-siRNA组大鼠各时间点的血糖水平显著降低(P<0.01, P<0.01), ALK7-siRNA组大鼠的胰岛素敏感指数(ISI)较空载体组显著增加(P<0.001)。2.ALK7基因沉默改善2型糖尿病大鼠糖脂代谢紊乱。实验结果显示：DM组大鼠的总胆固醇(TC)、甘油三酯(TG)和空腹血糖(FBG)水平较正常组明显增高,TC和TG在高脂喂养4周后(4周时)开始显著升高,而FBG在STZ腹腔注射后(5周时)开始显著升高,上述糖尿病组大鼠的糖脂代谢异常一直持续到实验结束。抑制ALK7的表达后,与空载体组比较,ALK7-siRNA组大鼠的TC、TG和FBG的水平显著降低(P<0.001,P<0.01,P<0.001)。3.ALK7基因沉默改善2型糖尿病大鼠的心功能不全。DM组大鼠左室舒张及收缩功能较正常组大鼠明显降低,表现为心导管测左室舒张末期压力(LVEDP)增高(7.14±0.90vs.23.43±1.81mmHg,P<0.001)而收缩末期压力(LVSP)降低(109.29±9.78vs.88±10.6mmHg, P<0.01).抑制ALK7的表达后,ALK7-siRNA组大鼠的LVEDP较空载体组显著降低(12.43±1.62vs.22.85±2.91mmHg, P<0.001),而LVSP的改变并无统计学意义。4.ALK7基因沉默改善2型糖尿病大鼠的左室重构。DM组大鼠的心脏重量/体重(HW/BW)较正常组增加22%,抑制ALK7的表达后,ALK7-siRNA组大鼠的HW/BW较空载体组显著降低(2.82±0.33vs.3.19±0.27mg/g, P<0.05).DM组大鼠的心脏明显扩大,左室室壁增厚,镜下观察,心肌细胞肥大,扭曲,排列紊乱,间隙增大,断裂细胞增加。抑制ALK7的表达后,与空载体组比较,ALK7-siRNA组大鼠的心腔变小,室壁厚度变薄,心肌细胞的排列较整齐,断裂细胞明显减少,心肌细胞的面积也显著减小(0.35±0.03vs.0.50±0.04mm2,P<0.001)。5.ALK7基因沉默抑制2型糖尿病大鼠的心肌细胞凋亡。DM组大鼠的心肌细胞凋亡率(TUNEL法测)较正常大鼠明显增加(P<0.001), cleaved Caspase3和Bax/Bcl2的表达也明显升高(P<0.001,P<0.001)；抑制ALK7的表达后,ALK7-siRNA组大鼠的心肌细胞凋亡率及cleaved Caspase3和Bax/Bc12的表达均较空载体组显著降低(P<0.001,P<0.001,P<0.001)。6.ALK7基因沉默改善2型糖尿病大鼠的心肌纤维化。Masson及天狼猩红染色显示：与正常组比较,DM组大鼠的心肌间质及管周的胶原沉积增加,胶原纤维排列紊乱,胶原容积分数(CVF%)和血管周围胶原面积/管腔面积(PVCA/LA)均有明显增加(P<0.001,P<0.001)。抑制ALK7的表达后,大鼠的心肌间质及管周的胶原沉积减少,胶原纤维排列较为有序,CVF%和PVCA/LA均有明显降低(P<0.01,P<0.001)。免疫组织化学染色结果显示：DM组大鼠的胶原Ⅰ/Ⅲ较正常组大鼠显著升高(P<0.001),而ALK7-siRNA组大鼠胶原Ⅰ/Ⅲ较空载体组显著降低(P<0.01)。 Western blotting结果同样显示：ALK7-siRNA组大鼠的胶原Ⅰ、胶原Ⅲ和胶原Ⅰ/Ⅲ的水平较空载体组降低(P<0.001,P<0.01,P<0.01)。7.ALK7基因沉默增加Akt的磷酸化,抑制Smad2/3的磷酸化。DM组大鼠心肌组织的磷酸化Smad2(p-Smad2)和磷酸化Smad3(p-Smad3)的表达较正常组增加(P<0.001,P<0.001),而磷酸化Akt (p-Akt)的表达则较正常组明显降低(P<0.001); ALK7基因沉默则显著增加了p-Akt的水平,p-Smad2和p-Smad3的水平则分别较空载体组降低62%和37%。结论抑制ALK7的表达可以改善2型糖尿病大鼠的心肌细胞凋亡、间质纤维化和胰岛素抵抗从而减缓DCM的发生和发展。本研究为DCM的治疗提供新的潜在靶点。更多还原

【Abstract】 BackgroudDiabetic cardiomyopathy (DCM) which develops independent of coronary artery disease and hypertension is a primary cause of death in patients with diabetes. DCM is characterized by early-onset diastolic dysfunction and late-onset systolic dysfunction. Many pathological changes are involved in the occurrence of DCM, including cardiomyocyte hypertrophy, apoptosis, degeneration and necrosis, and interstitial fibrosis. Cardiomyocyte apoptosis plays an important role in the development of cardiac dysfunction. It causes progressive loss of effective myocardial contractile unit, initiates cardiac remodeling and finally results in both systolic and diastolic dysfunction of the heart. However, the mechanisims underlying cardiomyocyte apoptosis in DCM remain incompletely understood.Smad2and Smad3play a crucial role in transducting the signal of TGF-β family from the receptor on the surface of membrane into the nucleus. They form a heteromeric complex with the co-Smad (Smad4) after phosphorylated by activin type I receptors and then translocate into the nucleus to regulate target gene transcription. Smad2/3are important apoptosis regulatory proteins. Previous studies have proved that nitric oxide, angiotensin II and activator protein-1could induce increased phosphorylation of Smad2/3which subsequently stimulated apoptosis-related genes such as Caspase-3/6/9. However, it is not clear whether Smad2/3participate in high glucose-induced cardiomyocyte apoptosis or not.ALK7, a new member of type Ⅰ TGF-β receptors, is firstly isolated from rat brain as an orphan receptor and consists of a transmembrane domain, a serine/threonine kinases domain and a GS domain between them. Nodal, activin AB, activin B and GDF are ligands specific for ALK7. During the signal transduction processes, the ligands firstly bound to activin type Ⅱ receptors (ActRⅡ), and once bound, ALK7is recruited and phosphorylated by ActRⅡ. The activation of ALK7then brings about a series of transformation of downstream substrates, such as Smad2/3, and participates in the regulation of cell proliferation, differentiation and apoptosis. Studies have shown the existence of ALK7in pancreas, brain tissue, adipose tissue, human liver and intestine. ALK7also participates in the proliferation and apoptosis of pancreatic beta-cell, human ovarian epithelial cells and hepatoma cell line, however, the role of ALK7in cardiomyocyte apoptosis remains unclear. In this study we would explore whether ALK7-Smad2/3signaling pathway is involved in high glucose-induced cardiomyocyte apoptosis.Objectives1. To explored the effect of high glucose on cardiomyocyte apoptosis.2. To explore the role of Smad2/3in high glucose-induced cardiomyocyte apoptosis.3. To explore the effect of high glucose on the expression level of ALK7.4. To explore whether ALK7is involved in high glucose-induced cardiomyocyte apoptosis or not.Materials and MethodsH9c2cardiomyoblasts and neonatal rat primary cardiomyocytes were cultured, and RT-PCR, western blotting, flow cytometry and transfection of siRNA were performed in this study.1. H9c2cardiomyoblasts were cultured with DMEM containing5.5mmol/L glucose (normal glucose),5.5mmol/L glucose plus27.5mmol/L mannose (osmotic control), or33mmol/L glucose (high glucose). The protein levels of cleaved Caspase3and Bcl2were measured by western blotting, and apoptosis rate was measured by flow cytometry.2. H9c2cardiomyoblasts were cultured with DMEM containing normal glucose, mannose or high glucose, and the mRNA level of ALK7was measured by RT-PCR while protein level of ALK7and phosphorylated Smad2/3were measured by western blotting.3. Smad2-siRNA and Smad3-siRNA were transfected into H9c2cardiomyoblasts respectively to explore the role of Smad2/3in high glucose-induced cardiomyocyte apoptosis. The protein level of Bcl2and cleaved Caspase3were measured by western blotting while apoptosis rate was measured by flow cytometry.4. ALK7-siRNA plasmid was transfected into H9c2cardiomyoblasts to explore the role of ALK7in high glucose-induced cardiomyocyte apoptosis. The the protein level of phosphorylated Smad2/3, Bc12and cleaved Caspase3were measured by western blotting while apoptosis rate was measured by flow cytometry.Results1. High ambient glucose at a concentration of33mmol/L elevated the expression level of ALK7in H9c2cardiomyoblasts. The results of RT-PCR and western blotting showed the expression of ALK7in both H9c2cardiomyoblasts and primary cardiomyocytes. H9c2cardiomyoblasts were maintained in DMEM with5.5mmol/L,11mmol/L,22mmol/L,33mmol/L and38.5mmol/L glucose for24h. The protein expression of ALK7increased significantly (P<0.05) after H9c2cardiomyoblasts were maintained in DMEM with33mmol/L glucose for24h, so DMEM with33mmol/L glucose was used as high glucose stimulation in subsequent experiments.2. High ambient glucose at a concentration of33mmol/L could induce the apoptosis of H9c2cardiomyoblasts. H9c2cardiomyoblasts were maintained in DMEM with5.5mmol/L glucose (Control),5.5mmol/L glucose plus27.5mmol/L mannose (OC), or33mmol/L glucose (HG) for Oh,4h,8h,12h,24h and48h. The protein expression level of cleaved Caspase3in HG group was84%higher than that in LG group (P<0.001) while the level of Bcl2in HG group was49%lower than that in LG group (P<0.001) at the time point of24h. The protein expression level of cleaved Caspase3in HG group was141%higher than that in LG group (P<0.001) while the level of Bcl2in HG group was57%lower than that in LG group (P<0.001) at the time point of48h. No significant difference of the expression levels of cleaved Caspase3and Bcl2were found between OC group and LG group at all time points.The apoptosis rate detected by Annexin V through flow cytometer showed that H9c2cardiomyoblasts underwent early stage of apoptosis after maintained in high ambient glucose condition for24h and experienced late stage of apoptosis after exposed to high glucose for48h.3. Smad2/3were involved in high glucose-induced H9c2cardiomyoblasts apoptosis.The expression levels of phosphorylated Smad2/3began increased significantly after H9c2cardiomyoblasts were maintained in DMEM with high glucose for12h and lasted to48h. The results were also confirmed in primary cardiomyocytes. The levels of phosphorylated Smad2/3began increased significantly after primary cardiomyocytes were maintained in DMEM with high glucose for12h and lasted to72h.The expressions level of phosphorylated Smad2/3decreased significantly after cells were transfected with Smad2-siRNA and Smad3-siRNA respectively for48h (P<0.001, P<0.001). The expression level of cleaved Caspase3was42%and48%lower (P<0.05, P<0.05) while the level of Bcl2was104%and101%higher (P<0.01, P<0.001) in cells transfected with Smad2-siRNA or Smad3-siRNA than those in cells transfected with nonsense-siRNA.The apoptosis rate was42%lower (P<0.01) in cells transfected with Smad2-siRNA than that in cells transfected with nonsense-siRNA while55%lower (P<0.01) in cells transfected with Smad3-siRNA than that in cells transfected with nonsense-siRNA. 4. High ambient glucose elevated the expression level of ALK7in both H9c2cardiomyoblasts and primary cardiomyocytes.The mRNA expression of ALK7began to increase after H9c2cardiomyoblasts were exposed to high glucose for1h (P<0.05) and reached the peak after cultured for8h (P<0.01). The protein level of ALK7elevated significantly after H9c2cells were exposed to high ambient glucose for12h (P<0.001) and lasted to48h (P<0.001).The results were also confirmed in primary cardiomyocytes. The protein level of ALK7began increased significantly after primary cardiomyocytes were maintained in DMEM with25mmol/L glucose for8h (P<0.01) and lasted to72h (P<0.01).5. ALK7participated in high glucose-induced H9c2cardiomyoblasts apoptosis through activating Smad2/3signaling pathway.The expression level of cleaved Caspase3was24%lower (P<0.01), apoptosis rate was38%lower (P<0.01) while the level of Bcl2was242%higher (P<0.05) in cells transfected with ALK7-siRNA plasmid than those in cells transfected with nonsense-siRNA plasmid.Both the expression levels of phosphorylated Smad2and phosphorylated Smad3decreased significantly after the expression of ALK7was inhibited (P<0.01,P<0.05).Conclusion1. High ambient glucose at a concentration of33mmol/L could induce the apoptosis of H9c2cardiomyoblasts.2. Smad2/3participate in high ambient glucose-induced H9c2cardiomyoblasts apoptosis.3. High glucose could elevate the expression level of ALK7in both H9c2cardiomyoblasts and rat cardiomyocytes.4. ALK7is involved in high glucose-induced H9c2cardiomyoblasts apoptosis through activating Smad2/3signaling pathway. BackgroudDiabetic cardiomyopathy (DCM), an independent diabetes-associated cardiovascular complication, is one of the leading causes of increased morbidity and mortality in diabetic patients. Previou studies showed that multifaceted stimuli such as hyperglycemia, insulin resistance, oxidative stress, inflammation and lipid accumulation were involved in the onset and progression of DCM. Both cardiomyocytes apoptosis and interstitial fibrosis are important pathological mechanisms for the development of cardiac dysfunction in DCM. The apoptosis and fibrosis could cause persistent loss of effective contractile tissue and increased myocardial stiffness both of which inevitably result in early-onset diastolic dysfunction and late-onset systolic dysfunction. However, the underlying mechanisms by which diabetes leads to apoptosis and fibrosis remain incompletely elucidated.Activin receptor-like kinase7(ALK7), a new member of type I TGF-β receptors, has been demonstrated to be associated with cell proliferation, differentiation and apoptosis. Recent studies in rodents showed that the nonsense mutation of ALK7ameliorates fat accumulation and obesity-induced glucose intolerance and insulin resistance. The observations suggested that the activation of ALK7contributed to the abnormalities of lipometabolism and insulin sensitivity which are important triggers of DCM. Our previous study in H9c2cardiomyoblasts has demonstrated that the inhibition of ALK7attenuated high ambient glucose-induced cardiomyocytes apoptosis. However, its pathophysiological significance in diabetic heart is incompletely understood.Both Smad2/3and Akt are crucial downstream mediators of ALK7signaling pathway. Previous findings showed that Smad2/3were critical for the fibrosis induced by profibrotic factors, such as angiotensin II and advanced glycation end products. Akt plays a key role in the maintenance of normal glucose homeostasis and selective insulin resistance, and depressed activity of Akt is considered to be a potential inducer of insulin resistance. Besides, both ALK7-Smad2/3and ALK7-Akt signaling pathway are involved in the regulation of cell apoptosis.On the basis of the above considerations, we hypothesized that ALK7plays a critical role in myocardial remodeling during the development of DCM, and this action of ALK7may be associated with cardiomyocyte apoptosis and cardiac fibrosis in diabetic hearts. In the present study, we established the rat model of type2DCM and used ALK7gene silencing in vivo to elucidate the role of ALK7in the pathogenesis of DCM. We try to look forward for a new potential target for the treatment of human DCM.Objectives1. To investigate the effect of ALK7gene silencing on glucose and lipid metabolism and insulin sensitivity in type2diabetic rats.2. To investigate the effect of ALK7gene silencing on cardiac structure and cardiac function in type2diabetic rats.3. To investigate the effect of ALK7gene silencing on cardiomyocyte apoptosis and interstitial fibrosis in type2diabetic rats.4. To investigate the effect of ALK7gene silencing on the expression level of phosphorylated Smad2/3and Akt in type2diabetic hearts.Materials and Methods1. Induction of type2diabetes in rats. Sixty male Sprague-Dawley rats (120-140g) were randomized into four groups:control, DM, DM+ALK7-siRNA, and DM+vehicle after intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed. The control group received normal chow and the other three groups were fed a high-fat (HF) diet. Four weeks later, IPGTT and IPITT were repeated. Rats with insulin resistance were intraperitoneally injected with streptozotocin (STZ) at the dose of30mg/kg for only one time. After12weeks of diabetes, rats in DCM+ALK7-siRNA group and DCM+vehicle group received adenovious injection. After16weeks of diabetes, the rats were killed.2. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT).IPGTT was performed after rats fasted for12h. The rats were intraperitoneally injected with the20%glucose solution (1g/kg) and blood samples were collected sequentially from the tail vein at the time point of0,15,30,60, and120min. IPITT was performed after rats fasted for4h. The rats were intraperitoneally injected with insulin solution (1unit/kg), and the blood was sampled and serum glucose was measured as described above.3. Blood serum analysis.Serum levels of total cholesterol (TC), triglyceride levels (TG), and fasting blood glucose (FBG) were determined using the Bayer1650blood chemistry analyzer and fasting insulin level (FINS) was analyzed using enzyme-linked immunosorbent assay.4. Measurement of blood pressure and heart rate.The heart rate, systolic blood pressure, diastolic blood pressure, and mean arterial pressure were measured by a noninvasive tail-cuff method.5. Evaluation of left ventricular function by cardiac catheterization.Left ventricular (LV) function was assessed by invasive hemodynamic measurement at the end of the experiment. A pressure tip-catheter filled by fluid was advanced from the right carotid artery into the left ventricle, and LV end-diastolic pressure (LVEDP) and LV end-systolic pressure (LVSP) were measured.6. Gene silencing of ALK7.After12weeks of diabetes, rats in DCM+ALK7-siRNA group and DCM+vehicle group received an adenovirus harboring ALK7gene with green fluorescent protein (GFP) or a control empty virus with GFP at a dose of2.5×1010plaque-forming units via the jugular vein. Adenovirus transfer was repeated in two weeks.7. Tissue preparation.Histological paraffin-embedded sections (5μm) were prepared for histology staining. The remaining portion of the left ventricular tissue was stored at-80℃for RT-PCR and western blotting analysis.8. Histology and morphometric analysis.The LV paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E), masson’s trichrome and0.5%sirius red to assess LV architecture and perivascular and interstitial fibrosis.9. In situ detection of apoptotic cells.In situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed to detect apoptotic cells. Percent TUNEL stained positive nuclei=number of TUNEL positive nuclei/total nuclei×100.10. Immunohistochemical staining.Tissue sections were incubated with polyclonal rabbit anti-rat collagen I and collagen III primary antibodies at4℃overnight, and then incubated with biotinylated goat anti-rabbit secondary antibody. The reaction was visualized with3,3’-diaminobenzidine (DAB) solution and stained sections were then counterstained with hematoxylin. The positive staining was analyzed with Image-Pro Plus5.0analysis software.11. Real-time quantitative RT-PCR.Total RNA was preparing with trizol agent. The reverse transcription and PCR reactions were performed using standard methods. The relative expression of the genes was analyzed with the2(-Delta Delta C (T)) method with β-actin as endogenous reference gene.12. Western blotting analysis.A total of15μg protein from each sample was separated by10%SDS—polyacrylamide gels and electrophoretically transferred to polyvinylidene fluoride membranes. The membranes were incubated with different primary antibodies overnight at4℃, and then treated with horseradish peroxidase-conjugated secondary antibodies for2h. β-actin was used as a loading control. The ECL kit was used to visualize the blots, and Photoshop CS4software was used to quantify the bands.13. Statistical analysis.Values are presented as mean mean±SD. Intergroup comparisons involved one-way ANOVA, followed by Tukey’s post hoc test and independent samples t-test. SPSS17.0was used for statistical analysis. A value of P<0.05was considered statistically significant.Results1. ALK7gene silencing ameliorated insulin resistance in type2diabetic rats.The4-week HF diet induced insulin resisitance in rat. Both the results of IPGTT and IPITT showed that levels of blood glucose in rats fed with HF diet were significantly higher at week4than at baseline at all of the time points tested. The area under the curve (AUC) of IPGTT and IPITT across the time for glucose level was also significantly higher at week4than at baseline.Both IPGTT and IPITT were repeated at the end of the experiment, and the results showed significantly higher glucose levels in DM group than control group (P<0.001, P<0.001). With ALK7gene silencing, the results of IPGTT and IPITT showed significant decreases of glucose levels in ALK7-siRNA group compared with vehicle group (P<0.01, P<0.01). The AUC for IPGTT and IPITT was also lower in ALK7-siRNA group than vehicle group (P<0.001, P<0.001). Besides, the decreased ISI in DCM group was restored after ALK7silencing (P<0.001). 2. ALK7gene silencing ameliorated metabolism abnormalities in type2diabetic rats. The diabetic rats got increased serum levels of TC, TG and FBG compared with control group. The elevated TC and TG appeared at week4while the FBG increased markedly after the onset of diabetes (at week5). With ALK7silencing, the elevated serum levels of TC, TG and FBG decreased significantly in ALK7-siRNA group compared with vehicle group (P<0.001, P<0.01, P<0.001).3. ALK7gene silencing improved cardiac function in DCM. Rats in DM group exhibited increased LVEDP (7.14±0.90vs.23.43±1.81mmHg, respectively; P<0.001) and decreased LVSP (109.29±9.78vs.88±10.6mmHg, respectively; P<0.01) compared with control group. This impaired cardiac function was partially restored by ALK7-siRNA treatment. Rats in ALK7-siRNA group showed significantly lower LVEDP compared with the vehicle group (12.43±1.62vs.22.85±2.91mmHg, respectively; P<0.001).4. ALK7gene silencing ameliorated cardiac remodeling in DCM. Rats of DM group showed a significantly larger heart compared with the control group, and the heart weight to body weight ratio (HW/BW) was22%higher in DM group than the control group. With ALK7silencing, HW/BW was significantly decreased in DM group compared with vehicle group (2.82±0.33vs.3.19±0.27mg/g, respectively; P<0.05).The cross-section staining of H&E at papillary muscle level showed that rats of DM group had dilated ventricle and thickened ventricular wall compared with control group. The cardiomyocytes were compact and well-organized in control group while hypertrophic, disorganized and twisty in DM group. With ALK7-siRNA treatment, the myocyte hypertrophy, rupture, disorganization as well as LV dilation and wall thickening were ameliorated. The myocyte size were significantly decreased in ALK7-siRNA group compared with vehicle group (0.35±0.03vs.0.50±0.04mm2, respectively; P<0.001).5. ALK7gene silencing prevented cardiomyocyte apoptosis in DCM.Rats in DM group showed significantly increased cardiomyocyte apoptosis rate (P<0.001) and elevated levels of cleaved Caspase3and Bax/Bcl2(P<0.001, P<0.001, respectively) compared with rats in control group. After a4-week ALK7-siRNA treatment, the cardiomyocyte apoptsis rate as well as protein level of cleaved Caspase3and Bax/Bcl2was significantly decreased in ALK7-siRNA group compared with vehicle group (P<0.001, P<0.001, P<0.001, respectively).6. ALK7gene silencing attenuated diabetes-induced cardiac fibrosis.Rats of DM group showed a diffuse, reticular, pockety and disorganized collagen network structure in the interstitial and perivascular areas. The collagen volume fraction (CVF) and perivascular collagen area/luminal area (PVCA/LA) were higher in the DM group than control group (P<0.001; P<0.001). With ALK7-siRNA treatment, the collagen deposition was significantly ameliorated in both the interstitial and perivascular areas, and the CVF and PVCA/LA were reduced significantly (P<0.01; P<0.001) compared with vehicle group.The immunohistochemistry analysis showed decreased level of collagen Ⅰ-to-Ⅲ ratio in ALK7-siRNA group compared with vehicle group (P<0.001). Western blotting analysis also showed decreased protein level of collagen Ⅰ and Ⅲ, and collagen Ⅰ-to-Ⅲ ratio in ALK7-siRNA group compared with vehicle group (P<0.001; P<0.01; P<0.01).7. ALK7regulated Akt and Smad2/3signaling pathways in DCM.The expression of phosphorylated Akt decreased markedly (P<0.001) while phosphorylated Smad2/3increased significantly in DM group (P<0.001, P<0.001). With ALK7silencing, the depressed phosphorylation of Akt was restored, and the phosphorylation of Smad2and Smad3was abolished by62%and37%, respectively.ConclusionThe inhibition of ALK7significantly attenuates cardiomyocyte apoptosis, cardiac fibrosis as well as insulin resistance in type2diabetic rats. The protective effects of ALK7gene silencing may be mediated through the suppression of Smad2/3signaling pathway and restoration of Akt signaling pathway. The cardioprotective effects with ALK7gene silencing suggest a potential therapeutic approach for the treatment of DCM in type2diabetes.更多还原