Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Tex.
Department of Genetic Medicine, Weill Cornell Medical College, New York, NY.
J Thorac Cardiovasc Surg. 2017 Feb;153(2):329-339.e3. doi: 10.1016/j.jtcvs.2016.09.041. Epub 2016 Sep 23.
The reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells improves ventricular function in myocardial infarction models. Only integrating persistent expression vectors have thus far been used to induce reprogramming, potentially limiting its clinical applicability. We therefore tested the reprogramming potential of nonintegrating, acute expression adenoviral (Ad) vectors.
Ad or lentivirus vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) were validated in vitro. Sprague-Dawley rats then underwent coronary ligation and Ad-mediated administration of vascular endothelial growth factor to generate infarct prevascularization. Three weeks later, animals received Ad or lentivirus encoding G, M, or T (AdGMT or LentiGMT) or an equivalent dose of a null vector (n = 11, 10, and 10, respectively). Outcomes were analyzed by echocardiography, magnetic resonance imaging, and histology.
Ad and lentivirus vectors provided equivalent G, M, and T expression in vitro. AdGMT and LentiGMT both likewise induced expression of the cardiomyocyte marker cardiac troponin T in approximately 6% of cardiac fibroblasts versus <1% cardiac troponin T expression in AdNull (adenoviral vector that does not encode a transgene)-treated cells. Infarcted myocardium that had been treated with AdGMT likewise demonstrated greater density of cells expressing the cardiomyocyte marker beta myosin heavy chain 7 compared with AdNull-treated animals. Echocardiography demonstrated that AdGMT and LentiGMT both increased ejection fraction compared with AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: -0.4% ± 2%; P < .05).
Ad vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into induced cardiomyocyte-like cells and improving cardiac function in postinfarct rat hearts. Short-term expression Ad vectors may represent an important means to induce cardiac cellular reprogramming in humans.
将心脏成纤维细胞重编程为诱导性心肌细胞样细胞可改善心肌梗死模型中的心室功能。迄今为止，仅整合持续表达载体被用于诱导重编程，这可能限制了其临床适用性。因此，我们测试了非整合、急性表达腺病毒（Ad）载体的重编程潜力。
在体外验证了编码 Gata4（G）、Mef2c（M）和 Tbx5（T）的 Ad 或慢病毒载体。然后，Sprague-Dawley 大鼠接受冠状动脉结扎和 Ad 介导的血管内皮生长因子给药以产生梗死前血管生成。3 周后，动物接受 Ad 或慢病毒编码 G、M 或 T（AdGMT 或 LentiGMT）或等效剂量的空载体（分别为 n=11、10 和 10）。通过超声心动图、磁共振成像和组织学分析来评估结果。
Ad 和慢病毒载体在体外提供了等效的 G、M 和 T 表达。AdGMT 和 LentiGMT 同样诱导了约 6%的心脏成纤维细胞表达心肌肌钙蛋白 T，而 AdNull（不编码转基因的腺病毒载体）处理的细胞中 <1%的心肌肌钙蛋白 T 表达。用 AdGMT 处理的梗死心肌同样表现出更高密度的表达心肌肌球蛋白重链 7 的细胞，与 AdNull 处理的动物相比。超声心动图显示，AdGMT 和 LentiGMT 均与 AdNull 相比增加了射血分数（AdGMT：21%±3%，LentiGMT：14%±5%，AdNull：-0.4%±2%；P<0.05）。
Ad 载体在诱导心脏成纤维细胞向诱导性心肌细胞样细胞转分化并改善梗死大鼠心脏功能方面至少与慢病毒载体一样有效。短期表达的 Ad 载体可能是在人类中诱导心脏细胞重编程的重要手段。
