microRNA及自噬对心肌重构的调控作用
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摘要
目的：心力衰竭是多种心血管疾病的终末期表现,是指在静脉回流正常的状态下,由于心脏的舒张和／或收缩功能发生障碍,导致心脏排血量不能满足组织器官代谢需要的一种综合症。尽管有很多心力衰竭的治疗药物和治疗方法治疗心力衰竭,但是该病的5年生存率仍然很低。各种心血管疾病首先引起的心肌代偿性肥厚,在长期的致病因素作用下,心脏功能逐渐失代偿,并逐渐发展为心力衰竭。在心力衰竭发病的过程受到多种编码及非编码基因的调控,但是至今很多基因及其调控作用机制尚不清楚。MicroRNA(miRNA)是一类细胞内有调控作用的小分子RNA,通常由21-23个核苷酸组成。近期研究表明,在多种心血管疾病中miRNA均参与心脏功能和心肌重塑的调控。我们研究组前期体外研究结果表明miR-221是一种促肥厚因子。在原代培养的新生大鼠心肌细胞中过表达miR-221诱导心肌肥厚。然而,miR-221是否能在体内条件下调控心肌重塑,以及miR-221调控心肌重塑的机制尚不清楚。因此,本研究通过构建miR-221心肌特异转基因小鼠,探讨miR-221在心肌重塑中的作用及机制,旨在为阐明心力衰竭的调控机制以及提供新的治疗靶点提供实验依据。
方法与结果：为探讨miR-221在体内条件下是否调控心脏功能及心肌重塑,本研究成功构建了心肌特异miR-221转基因小鼠模型。4周龄miR-221转基因小鼠与同窝阴性组小鼠相比心脏体积显著增加,心脏／体重比值增大。实时定量PCR结果表明,4周龄的miR-221转基因小鼠与同窝阴性对照小鼠相比,心脏胚胎基因ANP及BNP的表达显著上调。Masson三色染色显示,miR-221转基因小鼠心肌纤维化程度增加。同时,TUNEL染色结果显示,miR-221转基因小鼠心肌细胞凋亡数目增加。M型超声心动图显示,miR-221转基因小鼠表现为左室后壁增厚、左室内径增大、射血分数降低及短轴缩短率降低。上述结果表明,心肌特异过表达miR-221诱导心室肌重构、心功能衰竭。
为进一步研究miR-221诱导心力衰竭的分子机制,我们采用电子显微镜观察miR-221转基因小鼠心肌细胞超微结构的变化。结果显示,miR-221转基因小鼠心肌细胞内出现大量聚集的空泡状结构、受损线粒体数目增多。Westenn印迹检测结果证实,在miR-221转基因小鼠心肌组织以及miR-221过表达原代新生大鼠心肌细胞内自噬标记蛋白LC3脂化型LC3-Ⅱ水平均被下调,而p62蛋白则呈相反变化。将编码EGFP-LC3融合基因的质粒pEGFP-LC3与miR-221类似物共转染H9c2,采用激光共聚焦显微镜观察自噬体形成。镜下可观察到过表达miR-221的心肌细胞自噬体数目显著减少。上述结果共同表明,miR-221显著抑制心肌细胞自噬。
mTOR是自噬调控的关键分子。我们发现,4周龄的miR-221转基因小鼠的心肌组织内mTOR通路被激活。同样,在miR-221过表达的新生大鼠心肌细胞内,mTOR呈现相同趋势。为进一步验证mTOR通路的激活与miR-221诱导的心肌肥厚及自噬抑制有关,我们使用mTOR通路的特异性抑制剂雷帕霉素处理新生大鼠心肌细胞。结果表明,使用雷帕霉素(20nM)预处理的心肌细胞可以显著阻断miR-221诱导的心肌肥厚及自噬抑制。因此提示,mTOR信号通路在miR-221诱导的心力衰竭及自噬抑制中起到关键性的调控作用。
p27是miR-221的已知靶点,在miR-221转基因小鼠心肌组织内,我们观察到p27的表达被显著下调。采用siRNA靶向性干扰p27基因表达。与过表达miR-221类似,敲低p27后同样引发心肌细胞自噬抑制和mTOR通路的激活。p27可与细胞周期依赖性激酶2(cyclin dependent kinase2,CDK2)特异性结合并抑制其功能。采用CDK2的选择性抑制剂SU9516显著抑制miR-221诱导的自噬抑制和心肌肥厚。表明CDK2在miR-221诱导的心肌肥厚及自噬抑制中有关键的调控作用。
结论：综上所述,miR-221可通过调控p27/CDK2/mTOR通路介导的自噬抑制诱发心力衰竭。我们的研究结果表明,miR-221是心肌细胞内自噬调控的关键因子,miR-221可能是心力衰竭治疗的潜在靶点。
目的：各种心血管疾病如高血压、主动脉畸形等可以导致心脏的继发性肥厚。而肥厚型心肌病(Hypertrophic cardiomyopathy,HCM)是一种以左心室渐进性增厚为主要特点的原发性心肌病。其主要病理变化为左心室肥厚、心肌细胞排列紊乱和心肌间质纤维化。部分HCM患者有猝死和发展为心力衰竭的危险。因此,HCM对人类健康产生巨大威胁。HCM多是由于编码心肌细胞内肌小节蛋白的基因发生突变所引起的一种常染色体显性遗传病。尽管目前已经发现并确认十余种可以直接导致HCM的致病基因。但是,促进HCM患者心肌肥厚的分子机制并不清楚。越来越多的研究证实,微小RNA(microRNA,miRNA)参与并调控多种心血管疾病导致的心肌重塑过程。然而,miRNA在HCM中的作用并清楚。基于在其他心血管疾病中的研究结果,我们推测miRNA在HCM中也起到至关重要的调控作用。因此本研究采用miRNA微阵列筛选HCM中起主要变化的miRNA,并验证其在调控心肌细胞中的作用机制,旨在为揭示HCM发生发展过程的分子机制及治疗靶点提供实验依据。
方法与结果：为了筛选出HCM患者心肌组织中起主要调控作用的miRNA,我们随机选取了7例HCM患者和5例健康供体组织。提取总RNA后采用miRNA微阵列筛选差异表达基因。结果显示,有3个miRNA在HCM患者心肌组织中表达上调(≥2fold, P<0.01),10个miRNA在HCM患者心肌组织中表达下调(≤0.5fold,P<0.01)。其中,miR-451是下降程度最多的miRNA之一。之后,我们随机选取了16例HCM患者心肌组织和8例健康供体心肌组织,使用实时定量PCR的方法验证miR-451的表达水平。与miRNA微阵列结果一致,miR-451的表达在HCM患者心肌组织中下调4倍以上(P<0.01)。
为了验证miR-451对心肌肥厚的调控作用,我们用miR-451类似物或抑制物转染体外培养的原代新生大鼠心肌细胞。结果发现,过表达miR-451可以降低心肌细胞表面积,而用miR-451抑制物敲低内源性miR-451则导致心肌细胞表面积增大。双荧光素酶报告系统结果显示,TSC1是miR-451的直接靶点。在体外培养的原代新生大鼠心肌细胞及HeLa细胞中过表达miR-451可以显著抑制TSC1的表达水平。此外,在HCM心肌组织中TSC1处于高表达状态,这与HCM心肌组织中miR-451的下调相一致。
因TSC1是一个已知的自噬激活蛋白,我们将编码EGFP-LC3荧光融合蛋白的质粒与miR-451类似物及其对照或miR-451抑制物及其对照共转染入HeLa细胞。在激光共聚焦显微镜下可见过表达miR-451组HeLa细胞自噬体数目减少,而抑制miR-451组自噬体数目增多。同时,Western印迹结果显示,转染了miR-451类似物的原代新生大鼠心肌细胞自噬标志蛋白LC3脂化型LC3-Ⅱ水平降低。相反,敲低内源性miR-451的原代新生大鼠心肌细胞自噬标志蛋白LC3脂化型LC3-Ⅱ水平升高。电镜下可见,HCM患者心肌细胞内自噬体数目增多。Western印迹结果显示,自噬标志蛋白LC3-Ⅱ、Beclin-1表达水平增高,而自噬抑制蛋白Bcl-2表达下调。这些结果提示,miR-451抑制心肌细胞自噬,并参与心肌重塑的调控过程。
结论：综上所述,我们的研究表明miR-451可以调控心肌肥厚,并可以通过靶向抑制TSC1的表达抑制心肌细胞自噬。HCM患者心肌组织中miR-451显著下调,这可能参与HCM患者心肌重塑的病理过程和自噬调控。因此,miR-451可能是HCM治疗的新靶点。
目的：修饰基因参与调控肥厚型心肌病(HCM)的临床表现。肌肉环指状(MuRF)蛋白是一类肌肉特异性的泛素E3连接酶。这类蛋白参与并调控心脏发育和功能。泛素-蛋白酶体系统(ubiquitin-proteasome system, UPS)是细胞内降解蛋白的途径之一。已有研究表明,HCM心脏组织中UPS功能被减弱,从而导致编码肌小节蛋白的突变基因降解减少,加快HCM疾病的进展。由MuRF1、MuRF2和MuRF3组成的环状指E3泛素连接酶亚家族特异性表达于横纹肌中。最近有研究表明,编码MuRF1的基因可以通过破坏心肌细胞蛋白质降解而导致HCM的发生,提示编码MuRF蛋白的基因发生变异可能参与HCM疾病的进展。因此,本研究通过高通量二代测序的方法,分别在HCM患者及健康对照中对MuRF亚家族三个成员,MuRF1, MuRF2和MuRF3的编码外显子进行深度测序,探讨MuRF1、MuRF2和MuRF3基因突变与HCM的关系。
方法与结果：在本研究中,包含有594个无亲缘关系的HCM患者和307个健康对照。对MuRF亚家族中三个成员MuRF1、MuRF2和MuRF3的全部外显子进行二代测序。通过Sanger毛细管测序法确认筛选出的罕见变异。结果发现,在HCM患者中MuRF1和MuRF2基因罕见变异出现的频率较对照组升高(MuRF113/594[2.2%] versus1/307[0.3%],P=0.04;MuRF222/594[3.7%] versus2/307[0.7%];P=0.007)。携带有MuRF1和MuRF2罕见变异的HCM患者年龄较无变异的HCM患者年龄更小(P=0.04),并且有更高的最大左室壁厚度(P=0.006)。相反,HCM患者和健康对照中MuRF3罕见变异携带率无统计学差异。PolyPhe2SIFT生物信息学分析结果表明,21个MuRF1和MuRF2罕见变异有潜在的致病性。这些变异中有18个存在于22个HCM患者中(3.7%),而仅有3个存在于对照组中(1%,P=0.02)。在34个携带有MuRF1和MuRF2罕见变异的HCM患者中,有19个存在编码肌小节蛋白的基因发生突变(55.9%)。这些结果提示,MuRF1和MuRF2罕见变异与HCM的高外显率和临床表现的严重程度有关。
结论：我们的研究结果证实MuRF1和MuRF2罕见变异与HCM的临床表现和严重程度有关。MuRF1和MuRF2罕见变异是HCM的修饰基因,UPS失调参与并加快HCM疾病的进展。
BACKGROUND
Heart failure is the ultimate outcome of various cardiovascular diseases and is a leading cause of morbidity and mortality worldwide. Although drugs and other therapies have been developed for the management of heart failure, its five-year mortality rate remains high. The process of heart failure involves the dysregulation of many coding and non-coding genes; however, not all of these genes have been well characterized. MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules that post-transcriptionally regulate the degradation and/or translation of their target genes. A large body of evidence indicates that miRNA-mediated gene regulation plays important roles in the control of cardiac homeostasis and pathological remodeling. We previously found that miR-221is significantly up-regulated in patients with hypertrophic cardiomyopathy and in a mouse model of cardiac hypertrophy and heart failure induced by pressure overload. The in vitro overexpression of miR-221alone is sufficient to increase the size of cardiomyocyte. However, the in vivo roles and molecular mechanisms of miR-221in the regulation of cardiac remodeling remain unclear.
METHODS AND RESULTS
To investigate whether miR-221regulates cardiac remodeling in vivo, we generated transgenic mice (Tg-miR-221) with cardiac-specific overexpression of miR-221. Compared with their non-transgenic (NTG) littermates, the hearts of the Tg-miR-221mice were significantly enlarged at4weeks of age. The heart-to-body weight ratios and the expression levels of ANP and BNP were significantly higher in Tg-miR-221mice than in their NTG littermates. Increased interstitial fibrosis and apoptosis were observed in the myocardia from Tg-miR-221mice by histological examination. Cardiac function was evaluated by using high-resolution echocardiography at4and16weeks of age. Compared with their age-matched NTG controls, Tg-miR-221mice exhibited a progressive thickening of the end-diastolic left ventricular posterior wall, an increased internal dimension of the left ventricle and left ventricular mass/body weight ratio, and decreased fractional shortening. Taken together, these results indicated that the overexpression of miR-221interrupted cardiac homeostasis and induced cardiac dysfunction and heart failure in mice.
We further analyzed the ultrastructure of the myocardia by using transmission electron microscopy. In cardiomyocytes from Tg-miR-221mice, the mitochondria were disorganized and dispersed, with degraded cristae. In addition, a large amount of vacuoles with low electron densities were found in the cardiomyocytes from Tg-miR-221mice. The lipidated form of microtubule-associated protein1light chain3(LC3-Ⅱ), a marker of autophagy, were dramatically decreased in Tg-miR-221hearts. Consistently, the levels of sequestosome1(p62), an indicator of cytosolic protein clearance, were significantly increased. H9c2cells were forced to overexpress miR-221by the transient transfection of miR-221mimics. An autophagy-reporter plasmid encoding the EGFP-LC3recombinant protein was also co-transfected into H9c2cells, and the formation of autophagosomes was monitored based on the appearance of EGFP-LC3puncta. H9c2cells overexpressing miR-221had significantly lower levels of punctate EGFP-LC3compared with those of the control cells, indicating that autophagy was directly inhibited by miR-221.
As the mammalian target of rapamycin (mTOR) is a well-known negative regulator of autophagy. We first measured the activity of mTOR by detecting the phosphorylation levels of mTOR and its substrates. At4weeks of age, the mTOR signaling pathway was significantly activated in'transgenic hearts compared with those in the NTG controls. Likewise, overexpression of miR-221in cultured neonatal rat cardiomyocytes resulted in the hyper-activation of mTOR. To assess whether the cardiac remodeling induced by miR-221overexpression is dependent on the inhibition of autophagy, we used rapamycin to inhibit the activity of mTOR and re-activate autophagy in cultured neonatal rat cardiomyocytes through the transfection of miR-221mimics. Rapamycin (20nM) significantly antagonized the hypertrophic effect of miR-221overexpression. Our results indicated that the inhibition of autophagy induced by miR-221overexpression was dependent on mTOR activation and was necessary for miR-221-induced pathogenic cardiac remodeling.
Previously, we determined that p27was a direct target of miR-221in cardiomyocytes in vitro. Consistently, in the present study, we observed that the in vivo expression levels of p27were significantly down-regulated by the transgenic overexpression of miR-221in mouse heart. To determine whether the suppression of p27was responsible for the inhibition of autophagy induced by miR-221overexpression, we knocked down p27by using two different siRNAs in H9c2cells. As expected, p27knockdown showed similar effects of miR-221overexpression on autophagy and mTOR pathway. P27is a cyclin dependent kinase (CDK) inhibitor that interacts with CDK2to inhibit the cell cycle. We employed SU9516, a selective CDK2inhibitor, to suppress CDK2activity. In neonatal rat cardiomyocytes overexpressing miR-221, SU9516treatment significantly activated autophagy and attenuated cardiac hypertrophy. Thus, our results indicated that CDK2acted as a downstream mediator of p27and was required for the autophagy inhibition and pathological cardiac remodeling induced by miR-221overexpression in cardiomyocytes.
CONCLUSIONS
We found that miR-221promoted heart failure through mTOR-mediated autophagy inhibition. A previously unknown signaling pathway, the p27/CDK2/mTOR, governed the regulation of autophagy by miR-221in cardiomyocytes. Our study provides insights into the regulation of cardiomyocyte autophagy by miRNAs and implicates miR-221as a potential therapeutic target for heart failure.
BACKGROUND
Hypertrophic cardiomyopathy (HCM) is a common monogenetic disease of myocardium. HCM is characterized by unexplained cardiac hypertrophy, myocyte disarray and interstitial fibrosis, and is an important cause of sudden cardiac death and heart failure. This disease is mostly caused by mutations in genes encoding sarcomere proteins. However, the molecular mechanisms that drive the expression of HCM phenotypes are still poorly understood.
MicroRNAs (miRs) are endogenous non-coding small RNAs that usually act as repressors of target genes by either inhibiting translation and/or by promoting degradation of the target mRNAs. Previous studies have shown that a number of microRNAs play critical roles in controlling cardiac homeostasis and pathological remodeling, and suggest that microRNAs may serve as potential therapeutic targets for cardiac disease.
METHODS AND RESULTS
To identify differently expressed microRNAs between NCM and HCM hearts, a microarray assay was performed on specimens from5NCM donors and7HCM patients. In total,3microRNAs were significantly up-regulated (>2fold, P<0.01), whereas10down-regulated (≤0.5fold, P<0.05) in hearts from HCM patients. Among these differently expressed microRNAs, miR-451ranked as the most down-regulated. To validate the under-expression of miR-451, a qRT-PCR assay was performed with expanded tissue samples (NCM, n=8and HCM, n=16). As expected, the expression of miR-451had decreased more than four fold P<0.01).
To analyze the effects of miR-451on cardiac hypertrophy, we transfected primary neonatal rat cardiomyocytes with miR-451mimics or miR-451antagomir for48h after which the cell surface areas were analyzed. The surface area of miR-451overexpressed cardiomyocytes was significantly decreased. Conversely, knockdown of miR-451with antagomir increased surface area. The results from dual luciferase assays suggested that TSC1was a direct target of miR-451. Overexpression of miR-451in Primary neonatal rat cardiomyocytes and HeLa cells induced the down-regulation of TSC1expression. As the expression of miR-451was down-regulated in the hearts from HCM patients, we observed that the expression of TSC1was consistently up-regulated in HCM hearts.
As TSC1positively regulates autophagy, we assessed whether autophagy was regulated by miR-451. An autophagy-reporter plasmid encoding EGFP-LC3recombinant protein was co-transfected with miR-451mimics or antagomir into HeLa cells. The formation of autophagosomes was observed based on the appearance of EGFP-LC3punctae. We found that HeLa cells transfected with miR-451mimics had a significantly lower number of EGFP-LC3punctae compared with those transfected with scrambled microRNAs. Conversely, the suppression of miR-451in HeLa cells resulted in larger amounts of EGFP-LC3punctae.
We transfected miR-451mimics or miR-451antagomir into primary neonatal rat cardiomyocytes and HeLa cells. We observed that the LC3-Ⅱ in cells with miR-451overexpression was significantly decreased, whereas cells transfected with miR-451antagomir showed increased LC3-Ⅱ.
Transmission electron microscopy showed that large quantities of early and late autophagic vacuoles with cytoplasmic remnants and mitochondria had accumulated in the cytosol of cardiomyocytes from HCM hearts. The levels of LC3-Ⅱ were dramatically increased in hearts from HCM patients. The expression of Beclin-1, a required protein in membrane nucleation during the early stage of autophagy, was also significantly up-regulated in HCM hearts, whereas Bcl-2protein, an inhibitor of autophagy, is down-regulated. Taken together, these results indicated that autophagy was increased in HCM hearts.
CONCLUSIONS
In summary, our study reveals that miR-451is one of the most down-regulated microRNAs in HCM and regulates cardiac hypertrophy and cardiac autophagy by targeting TSC1. The down-regulation of miR-451may contribute to the development of HCM and may be a potential therapeutic target for the disease.
BACKGROUND
Modifier genes contribute to the diverse clinical manifestations of hypertrophic cardiomyopathy (HCM), but are still largely unknown. Muscle ring finger (MuRF) proteins are a class of muscle-specific ubiquitin E3-ligases that appear to modulate cardiac mass and function by regulating the ubiquitin-proteasome system. MuRF1,2and3comprise a subfamily of the RING-finger E3ubiquitin ligases that are specifically expressed in striated muscles. Recently, mutations in the gene encoding MuRF1were reported to cause HCM by impairing protein degradation in cardiomyocytes, suggesting that genetic variants in genes encoding MuRF proteins might be involved in the pathogenesis of HCM. In present study, we evaluated the association between genetic variants of MuRF genes and HCM phenotype by screening all the three members of MuRF subfamily in a large HCM cohort and matched healthy controls.
METHODS AND RESULTS
In this study we screened all the three members of the MuRF family, MuRF1, MuRF2and MuRF3, in594unrelated HCM patients and307healthy controls by targeted resequencing. Identified rare variants were confirmed by capillary Sanger sequencing. The prevalence of rare variants in both MuRF1and MuRF2in HCM patients was higher than that in control subjects (MuRFl13/594[2.2%] versus1/307[0.3%], P=0.04; MuRF222/594[3.7%] versus2/307[0.7%];P=0.007). Patients with rare variants in MuRF1or MuRF2were younger (P=0.04) and had greater maximum left ventricular wall thickness (P=0.006) than those without such variants. In contrast, rare variants in MuRF1were detected with equal frequency in patients and control individuals. PolyPhen2and SIFT analysis showed21rare variants of MuRFl and MuRF2were potentially pathogenic, including18variants among22(3.7%) HCM patients and3variants among3(1%) control individuals (P=0.02). Mutations in genes encoding sarcomere proteins were present in19(55.9%) of the34HCM patients with rare variants in MuRF1and MuRF2. These data strongly supported that rare variants in MuRF1and MuRF2are associated with higher penetrance and more severe clinical manifestations of HCM.
CONCLUSIONS
In conclusion, rare variants in the genes encoding MuRFl and MuRF2act as modifiers that increase the risk of development of HCM and lead to more severe disease phenotypes. Our study is consistent with the hypothesis that impaired ubiquitin-proteasome system contributes to the pathogenesis of HCM.
方法与结果：为探讨miR-221在体内条件下是否调控心脏功能及心肌重塑,本研究成功构建了心肌特异miR-221转基因小鼠模型。4周龄miR-221转基因小鼠与同窝阴性组小鼠相比心脏体积显著增加,心脏／体重比值增大。实时定量PCR结果表明,4周龄的miR-221转基因小鼠与同窝阴性对照小鼠相比,心脏胚胎基因ANP及BNP的表达显著上调。Masson三色染色显示,miR-221转基因小鼠心肌纤维化程度增加。同时,TUNEL染色结果显示,miR-221转基因小鼠心肌细胞凋亡数目增加。M型超声心动图显示,miR-221转基因小鼠表现为左室后壁增厚、左室内径增大、射血分数降低及短轴缩短率降低。上述结果表明,心肌特异过表达miR-221诱导心室肌重构、心功能衰竭。
为进一步研究miR-221诱导心力衰竭的分子机制,我们采用电子显微镜观察miR-221转基因小鼠心肌细胞超微结构的变化。结果显示,miR-221转基因小鼠心肌细胞内出现大量聚集的空泡状结构、受损线粒体数目增多。Westenn印迹检测结果证实,在miR-221转基因小鼠心肌组织以及miR-221过表达原代新生大鼠心肌细胞内自噬标记蛋白LC3脂化型LC3-Ⅱ水平均被下调,而p62蛋白则呈相反变化。将编码EGFP-LC3融合基因的质粒pEGFP-LC3与miR-221类似物共转染H9c2,采用激光共聚焦显微镜观察自噬体形成。镜下可观察到过表达miR-221的心肌细胞自噬体数目显著减少。上述结果共同表明,miR-221显著抑制心肌细胞自噬。
mTOR是自噬调控的关键分子。我们发现,4周龄的miR-221转基因小鼠的心肌组织内mTOR通路被激活。同样,在miR-221过表达的新生大鼠心肌细胞内,mTOR呈现相同趋势。为进一步验证mTOR通路的激活与miR-221诱导的心肌肥厚及自噬抑制有关,我们使用mTOR通路的特异性抑制剂雷帕霉素处理新生大鼠心肌细胞。结果表明,使用雷帕霉素(20nM)预处理的心肌细胞可以显著阻断miR-221诱导的心肌肥厚及自噬抑制。因此提示,mTOR信号通路在miR-221诱导的心力衰竭及自噬抑制中起到关键性的调控作用。
p27是miR-221的已知靶点,在miR-221转基因小鼠心肌组织内,我们观察到p27的表达被显著下调。采用siRNA靶向性干扰p27基因表达。与过表达miR-221类似,敲低p27后同样引发心肌细胞自噬抑制和mTOR通路的激活。p27可与细胞周期依赖性激酶2(cyclin dependent kinase2,CDK2)特异性结合并抑制其功能。采用CDK2的选择性抑制剂SU9516显著抑制miR-221诱导的自噬抑制和心肌肥厚。表明CDK2在miR-221诱导的心肌肥厚及自噬抑制中有关键的调控作用。
结论：综上所述,miR-221可通过调控p27/CDK2/mTOR通路介导的自噬抑制诱发心力衰竭。我们的研究结果表明,miR-221是心肌细胞内自噬调控的关键因子,miR-221可能是心力衰竭治疗的潜在靶点。
目的：各种心血管疾病如高血压、主动脉畸形等可以导致心脏的继发性肥厚。而肥厚型心肌病(Hypertrophic cardiomyopathy,HCM)是一种以左心室渐进性增厚为主要特点的原发性心肌病。其主要病理变化为左心室肥厚、心肌细胞排列紊乱和心肌间质纤维化。部分HCM患者有猝死和发展为心力衰竭的危险。因此,HCM对人类健康产生巨大威胁。HCM多是由于编码心肌细胞内肌小节蛋白的基因发生突变所引起的一种常染色体显性遗传病。尽管目前已经发现并确认十余种可以直接导致HCM的致病基因。但是,促进HCM患者心肌肥厚的分子机制并不清楚。越来越多的研究证实,微小RNA(microRNA,miRNA)参与并调控多种心血管疾病导致的心肌重塑过程。然而,miRNA在HCM中的作用并清楚。基于在其他心血管疾病中的研究结果,我们推测miRNA在HCM中也起到至关重要的调控作用。因此本研究采用miRNA微阵列筛选HCM中起主要变化的miRNA,并验证其在调控心肌细胞中的作用机制,旨在为揭示HCM发生发展过程的分子机制及治疗靶点提供实验依据。
方法与结果：为了筛选出HCM患者心肌组织中起主要调控作用的miRNA,我们随机选取了7例HCM患者和5例健康供体组织。提取总RNA后采用miRNA微阵列筛选差异表达基因。结果显示,有3个miRNA在HCM患者心肌组织中表达上调(≥2fold, P<0.01),10个miRNA在HCM患者心肌组织中表达下调(≤0.5fold,P<0.01)。其中,miR-451是下降程度最多的miRNA之一。之后,我们随机选取了16例HCM患者心肌组织和8例健康供体心肌组织,使用实时定量PCR的方法验证miR-451的表达水平。与miRNA微阵列结果一致,miR-451的表达在HCM患者心肌组织中下调4倍以上(P<0.01)。
为了验证miR-451对心肌肥厚的调控作用,我们用miR-451类似物或抑制物转染体外培养的原代新生大鼠心肌细胞。结果发现,过表达miR-451可以降低心肌细胞表面积,而用miR-451抑制物敲低内源性miR-451则导致心肌细胞表面积增大。双荧光素酶报告系统结果显示,TSC1是miR-451的直接靶点。在体外培养的原代新生大鼠心肌细胞及HeLa细胞中过表达miR-451可以显著抑制TSC1的表达水平。此外,在HCM心肌组织中TSC1处于高表达状态,这与HCM心肌组织中miR-451的下调相一致。
因TSC1是一个已知的自噬激活蛋白,我们将编码EGFP-LC3荧光融合蛋白的质粒与miR-451类似物及其对照或miR-451抑制物及其对照共转染入HeLa细胞。在激光共聚焦显微镜下可见过表达miR-451组HeLa细胞自噬体数目减少,而抑制miR-451组自噬体数目增多。同时,Western印迹结果显示,转染了miR-451类似物的原代新生大鼠心肌细胞自噬标志蛋白LC3脂化型LC3-Ⅱ水平降低。相反,敲低内源性miR-451的原代新生大鼠心肌细胞自噬标志蛋白LC3脂化型LC3-Ⅱ水平升高。电镜下可见,HCM患者心肌细胞内自噬体数目增多。Western印迹结果显示,自噬标志蛋白LC3-Ⅱ、Beclin-1表达水平增高,而自噬抑制蛋白Bcl-2表达下调。这些结果提示,miR-451抑制心肌细胞自噬,并参与心肌重塑的调控过程。
结论：综上所述,我们的研究表明miR-451可以调控心肌肥厚,并可以通过靶向抑制TSC1的表达抑制心肌细胞自噬。HCM患者心肌组织中miR-451显著下调,这可能参与HCM患者心肌重塑的病理过程和自噬调控。因此,miR-451可能是HCM治疗的新靶点。
目的：修饰基因参与调控肥厚型心肌病(HCM)的临床表现。肌肉环指状(MuRF)蛋白是一类肌肉特异性的泛素E3连接酶。这类蛋白参与并调控心脏发育和功能。泛素-蛋白酶体系统(ubiquitin-proteasome system, UPS)是细胞内降解蛋白的途径之一。已有研究表明,HCM心脏组织中UPS功能被减弱,从而导致编码肌小节蛋白的突变基因降解减少,加快HCM疾病的进展。由MuRF1、MuRF2和MuRF3组成的环状指E3泛素连接酶亚家族特异性表达于横纹肌中。最近有研究表明,编码MuRF1的基因可以通过破坏心肌细胞蛋白质降解而导致HCM的发生,提示编码MuRF蛋白的基因发生变异可能参与HCM疾病的进展。因此,本研究通过高通量二代测序的方法,分别在HCM患者及健康对照中对MuRF亚家族三个成员,MuRF1, MuRF2和MuRF3的编码外显子进行深度测序,探讨MuRF1、MuRF2和MuRF3基因突变与HCM的关系。
方法与结果：在本研究中,包含有594个无亲缘关系的HCM患者和307个健康对照。对MuRF亚家族中三个成员MuRF1、MuRF2和MuRF3的全部外显子进行二代测序。通过Sanger毛细管测序法确认筛选出的罕见变异。结果发现,在HCM患者中MuRF1和MuRF2基因罕见变异出现的频率较对照组升高(MuRF113/594[2.2%] versus1/307[0.3%],P=0.04;MuRF222/594[3.7%] versus2/307[0.7%];P=0.007)。携带有MuRF1和MuRF2罕见变异的HCM患者年龄较无变异的HCM患者年龄更小(P=0.04),并且有更高的最大左室壁厚度(P=0.006)。相反,HCM患者和健康对照中MuRF3罕见变异携带率无统计学差异。PolyPhe2SIFT生物信息学分析结果表明,21个MuRF1和MuRF2罕见变异有潜在的致病性。这些变异中有18个存在于22个HCM患者中(3.7%),而仅有3个存在于对照组中(1%,P=0.02)。在34个携带有MuRF1和MuRF2罕见变异的HCM患者中,有19个存在编码肌小节蛋白的基因发生突变(55.9%)。这些结果提示,MuRF1和MuRF2罕见变异与HCM的高外显率和临床表现的严重程度有关。
结论：我们的研究结果证实MuRF1和MuRF2罕见变异与HCM的临床表现和严重程度有关。MuRF1和MuRF2罕见变异是HCM的修饰基因,UPS失调参与并加快HCM疾病的进展。
BACKGROUND
Heart failure is the ultimate outcome of various cardiovascular diseases and is a leading cause of morbidity and mortality worldwide. Although drugs and other therapies have been developed for the management of heart failure, its five-year mortality rate remains high. The process of heart failure involves the dysregulation of many coding and non-coding genes; however, not all of these genes have been well characterized. MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules that post-transcriptionally regulate the degradation and/or translation of their target genes. A large body of evidence indicates that miRNA-mediated gene regulation plays important roles in the control of cardiac homeostasis and pathological remodeling. We previously found that miR-221is significantly up-regulated in patients with hypertrophic cardiomyopathy and in a mouse model of cardiac hypertrophy and heart failure induced by pressure overload. The in vitro overexpression of miR-221alone is sufficient to increase the size of cardiomyocyte. However, the in vivo roles and molecular mechanisms of miR-221in the regulation of cardiac remodeling remain unclear.
METHODS AND RESULTS
To investigate whether miR-221regulates cardiac remodeling in vivo, we generated transgenic mice (Tg-miR-221) with cardiac-specific overexpression of miR-221. Compared with their non-transgenic (NTG) littermates, the hearts of the Tg-miR-221mice were significantly enlarged at4weeks of age. The heart-to-body weight ratios and the expression levels of ANP and BNP were significantly higher in Tg-miR-221mice than in their NTG littermates. Increased interstitial fibrosis and apoptosis were observed in the myocardia from Tg-miR-221mice by histological examination. Cardiac function was evaluated by using high-resolution echocardiography at4and16weeks of age. Compared with their age-matched NTG controls, Tg-miR-221mice exhibited a progressive thickening of the end-diastolic left ventricular posterior wall, an increased internal dimension of the left ventricle and left ventricular mass/body weight ratio, and decreased fractional shortening. Taken together, these results indicated that the overexpression of miR-221interrupted cardiac homeostasis and induced cardiac dysfunction and heart failure in mice.
We further analyzed the ultrastructure of the myocardia by using transmission electron microscopy. In cardiomyocytes from Tg-miR-221mice, the mitochondria were disorganized and dispersed, with degraded cristae. In addition, a large amount of vacuoles with low electron densities were found in the cardiomyocytes from Tg-miR-221mice. The lipidated form of microtubule-associated protein1light chain3(LC3-Ⅱ), a marker of autophagy, were dramatically decreased in Tg-miR-221hearts. Consistently, the levels of sequestosome1(p62), an indicator of cytosolic protein clearance, were significantly increased. H9c2cells were forced to overexpress miR-221by the transient transfection of miR-221mimics. An autophagy-reporter plasmid encoding the EGFP-LC3recombinant protein was also co-transfected into H9c2cells, and the formation of autophagosomes was monitored based on the appearance of EGFP-LC3puncta. H9c2cells overexpressing miR-221had significantly lower levels of punctate EGFP-LC3compared with those of the control cells, indicating that autophagy was directly inhibited by miR-221.
As the mammalian target of rapamycin (mTOR) is a well-known negative regulator of autophagy. We first measured the activity of mTOR by detecting the phosphorylation levels of mTOR and its substrates. At4weeks of age, the mTOR signaling pathway was significantly activated in'transgenic hearts compared with those in the NTG controls. Likewise, overexpression of miR-221in cultured neonatal rat cardiomyocytes resulted in the hyper-activation of mTOR. To assess whether the cardiac remodeling induced by miR-221overexpression is dependent on the inhibition of autophagy, we used rapamycin to inhibit the activity of mTOR and re-activate autophagy in cultured neonatal rat cardiomyocytes through the transfection of miR-221mimics. Rapamycin (20nM) significantly antagonized the hypertrophic effect of miR-221overexpression. Our results indicated that the inhibition of autophagy induced by miR-221overexpression was dependent on mTOR activation and was necessary for miR-221-induced pathogenic cardiac remodeling.
Previously, we determined that p27was a direct target of miR-221in cardiomyocytes in vitro. Consistently, in the present study, we observed that the in vivo expression levels of p27were significantly down-regulated by the transgenic overexpression of miR-221in mouse heart. To determine whether the suppression of p27was responsible for the inhibition of autophagy induced by miR-221overexpression, we knocked down p27by using two different siRNAs in H9c2cells. As expected, p27knockdown showed similar effects of miR-221overexpression on autophagy and mTOR pathway. P27is a cyclin dependent kinase (CDK) inhibitor that interacts with CDK2to inhibit the cell cycle. We employed SU9516, a selective CDK2inhibitor, to suppress CDK2activity. In neonatal rat cardiomyocytes overexpressing miR-221, SU9516treatment significantly activated autophagy and attenuated cardiac hypertrophy. Thus, our results indicated that CDK2acted as a downstream mediator of p27and was required for the autophagy inhibition and pathological cardiac remodeling induced by miR-221overexpression in cardiomyocytes.
CONCLUSIONS
We found that miR-221promoted heart failure through mTOR-mediated autophagy inhibition. A previously unknown signaling pathway, the p27/CDK2/mTOR, governed the regulation of autophagy by miR-221in cardiomyocytes. Our study provides insights into the regulation of cardiomyocyte autophagy by miRNAs and implicates miR-221as a potential therapeutic target for heart failure.
BACKGROUND
Hypertrophic cardiomyopathy (HCM) is a common monogenetic disease of myocardium. HCM is characterized by unexplained cardiac hypertrophy, myocyte disarray and interstitial fibrosis, and is an important cause of sudden cardiac death and heart failure. This disease is mostly caused by mutations in genes encoding sarcomere proteins. However, the molecular mechanisms that drive the expression of HCM phenotypes are still poorly understood.
MicroRNAs (miRs) are endogenous non-coding small RNAs that usually act as repressors of target genes by either inhibiting translation and/or by promoting degradation of the target mRNAs. Previous studies have shown that a number of microRNAs play critical roles in controlling cardiac homeostasis and pathological remodeling, and suggest that microRNAs may serve as potential therapeutic targets for cardiac disease.
METHODS AND RESULTS
To identify differently expressed microRNAs between NCM and HCM hearts, a microarray assay was performed on specimens from5NCM donors and7HCM patients. In total,3microRNAs were significantly up-regulated (>2fold, P<0.01), whereas10down-regulated (≤0.5fold, P<0.05) in hearts from HCM patients. Among these differently expressed microRNAs, miR-451ranked as the most down-regulated. To validate the under-expression of miR-451, a qRT-PCR assay was performed with expanded tissue samples (NCM, n=8and HCM, n=16). As expected, the expression of miR-451had decreased more than four fold P<0.01).
To analyze the effects of miR-451on cardiac hypertrophy, we transfected primary neonatal rat cardiomyocytes with miR-451mimics or miR-451antagomir for48h after which the cell surface areas were analyzed. The surface area of miR-451overexpressed cardiomyocytes was significantly decreased. Conversely, knockdown of miR-451with antagomir increased surface area. The results from dual luciferase assays suggested that TSC1was a direct target of miR-451. Overexpression of miR-451in Primary neonatal rat cardiomyocytes and HeLa cells induced the down-regulation of TSC1expression. As the expression of miR-451was down-regulated in the hearts from HCM patients, we observed that the expression of TSC1was consistently up-regulated in HCM hearts.
As TSC1positively regulates autophagy, we assessed whether autophagy was regulated by miR-451. An autophagy-reporter plasmid encoding EGFP-LC3recombinant protein was co-transfected with miR-451mimics or antagomir into HeLa cells. The formation of autophagosomes was observed based on the appearance of EGFP-LC3punctae. We found that HeLa cells transfected with miR-451mimics had a significantly lower number of EGFP-LC3punctae compared with those transfected with scrambled microRNAs. Conversely, the suppression of miR-451in HeLa cells resulted in larger amounts of EGFP-LC3punctae.
We transfected miR-451mimics or miR-451antagomir into primary neonatal rat cardiomyocytes and HeLa cells. We observed that the LC3-Ⅱ in cells with miR-451overexpression was significantly decreased, whereas cells transfected with miR-451antagomir showed increased LC3-Ⅱ.
Transmission electron microscopy showed that large quantities of early and late autophagic vacuoles with cytoplasmic remnants and mitochondria had accumulated in the cytosol of cardiomyocytes from HCM hearts. The levels of LC3-Ⅱ were dramatically increased in hearts from HCM patients. The expression of Beclin-1, a required protein in membrane nucleation during the early stage of autophagy, was also significantly up-regulated in HCM hearts, whereas Bcl-2protein, an inhibitor of autophagy, is down-regulated. Taken together, these results indicated that autophagy was increased in HCM hearts.
CONCLUSIONS
In summary, our study reveals that miR-451is one of the most down-regulated microRNAs in HCM and regulates cardiac hypertrophy and cardiac autophagy by targeting TSC1. The down-regulation of miR-451may contribute to the development of HCM and may be a potential therapeutic target for the disease.
BACKGROUND
Modifier genes contribute to the diverse clinical manifestations of hypertrophic cardiomyopathy (HCM), but are still largely unknown. Muscle ring finger (MuRF) proteins are a class of muscle-specific ubiquitin E3-ligases that appear to modulate cardiac mass and function by regulating the ubiquitin-proteasome system. MuRF1,2and3comprise a subfamily of the RING-finger E3ubiquitin ligases that are specifically expressed in striated muscles. Recently, mutations in the gene encoding MuRF1were reported to cause HCM by impairing protein degradation in cardiomyocytes, suggesting that genetic variants in genes encoding MuRF proteins might be involved in the pathogenesis of HCM. In present study, we evaluated the association between genetic variants of MuRF genes and HCM phenotype by screening all the three members of MuRF subfamily in a large HCM cohort and matched healthy controls.
METHODS AND RESULTS
In this study we screened all the three members of the MuRF family, MuRF1, MuRF2and MuRF3, in594unrelated HCM patients and307healthy controls by targeted resequencing. Identified rare variants were confirmed by capillary Sanger sequencing. The prevalence of rare variants in both MuRF1and MuRF2in HCM patients was higher than that in control subjects (MuRFl13/594[2.2%] versus1/307[0.3%], P=0.04; MuRF222/594[3.7%] versus2/307[0.7%];P=0.007). Patients with rare variants in MuRF1or MuRF2were younger (P=0.04) and had greater maximum left ventricular wall thickness (P=0.006) than those without such variants. In contrast, rare variants in MuRF1were detected with equal frequency in patients and control individuals. PolyPhen2and SIFT analysis showed21rare variants of MuRFl and MuRF2were potentially pathogenic, including18variants among22(3.7%) HCM patients and3variants among3(1%) control individuals (P=0.02). Mutations in genes encoding sarcomere proteins were present in19(55.9%) of the34HCM patients with rare variants in MuRF1and MuRF2. These data strongly supported that rare variants in MuRF1and MuRF2are associated with higher penetrance and more severe clinical manifestations of HCM.
CONCLUSIONS
In conclusion, rare variants in the genes encoding MuRFl and MuRF2act as modifiers that increase the risk of development of HCM and lead to more severe disease phenotypes. Our study is consistent with the hypothesis that impaired ubiquitin-proteasome system contributes to the pathogenesis of HCM.
引文
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