Cellular Cardiomyoplasty of Cardiac Fibroblasts by Adenoviral Delivery of MyoD Ex Vivo: An Unlimited Source of Cells for Myocardial Repair

S. Etzion, I. Barbash, M. Feinberg, Parvin Zarin, L. Miller, E. Guetta, R. Holbová, R. Kloner, L. Kedes, J. Leor
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引用次数: 45

Abstract

BackgroundThe muscle-specific MyoD family of transcription factors function as master genes that are able to prompt myogenesis in a variety of cells. The purpose of our study was to determine whether MyoD could induce primary cardiac fibroblasts, isolated from infarcted myocardium or pericardium, to undergo myogenic conversion in a clinically relevant approach. Methods and ResultsPrimary rat fibroblasts from 7-day-old infarcted myocardium or normal pericardium were transfected by an E1/E3-deleted adenoviral vector carrying both a human MyoD cDNA driven by a CMV promoter and a green fluorescent protein (GFP) reporter gene driven by a second CMV promoter. Expression of MyoD caused myogenic differentiation of cultured fibroblasts, as defined by elongation and fusion into multinucleated myotubes, typical cross striation as identified by electron microscopy, and positive immunostaining for sarcomeric actin, fast myosin heavy chain (MHC), and actinin. The myogenic cells (1.5×106) were transplanted into the infarcted myocardium 7 days after coronary artery occlusion. By 1 month after transplantation, the converted fibroblasts gave rise to a cluster of myogenic cells that in a few hearts occupied a large part of the scar with positive immunostaining for the myogenic proteins fast-MHC and sarcomeric actin. A few cells expressed the gap junction protein connexin 43 in a disorganized manner. There was no positive staining in the control hearts treated with injections of untreated fibroblasts or culture medium. ConclusionsOur work shows that it is possible to exploit the unique capacity of MyoD to activate myogenesis in fibroblasts ex vivo and to create a vast source of autologous myogenic cells for transplantation.
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体外腺病毒介导心肌成纤维细胞心肌成形术:心肌修复的无限细胞来源
肌肉特异性MyoD转录因子家族是能够促进多种细胞肌肉形成的主基因。我们研究的目的是确定MyoD是否可以诱导从梗死心肌或心包分离的原代心脏成纤维细胞在临床相关的方法中进行成肌转化。方法和结果用E1/ e3缺失腺病毒载体转染7日龄梗死心肌或正常心包原代大鼠成纤维细胞,载体携带CMV启动子驱动的人MyoD cDNA和第二CMV启动子驱动的绿色荧光蛋白(GFP)报告基因。MyoD的表达引起了培养成纤维细胞的肌源性分化,表现为延长和融合成多核肌管,电镜下发现典型的交叉条纹,肌凝蛋白、快肌球蛋白重链(MHC)和肌动蛋白免疫染色阳性。冠状动脉闭塞7天后,将成肌细胞(1.5×106)移植到梗死心肌中。移植后1个月,转化成纤维细胞产生成肌细胞簇,在少数心脏中,成肌细胞簇占据了瘢痕的大部分,成肌蛋白快速mhc和肌动蛋白免疫染色阳性。少数细胞以无组织的方式表达间隙连接蛋白连接蛋白43。注射未经处理的成纤维细胞或培养基处理的对照心脏未见阳性染色。结论我们的研究表明,利用MyoD在体外激活成纤维细胞肌生成的独特能力,为移植创造大量的自体肌生成细胞是可能的。
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