Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells.
Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells.
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DOI：
10.2147/ijn.s75124
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发表时间：
2015
影响因子：
8
通讯作者：
Murthy N
中科院分区：
文献类型：
--
作者：
Lee K;Yu P;Lingampalli N;Kim HJ;Tang R;Murthy N
心肌梗死的治疗是医学上的一大挑战，因为心脏组织无法再生。通过转录因子mRNAs的传递将内源性心脏成纤维细胞直接重编程为功能性心肌细胞，具有再生心脏组织和治疗心力衰竭的潜力。尽管将信使核糖核酸转导到心脏成纤维细胞具有治疗的潜力，但在心脏成纤维细胞中进行信使核糖核酸的转导一直是具有挑战性的。在这里，我们开发了一种通过多聚精氨酸融合的心脏靶向多肽和脂质体复合体(称为C-Lipo)在培养的小鼠心脏成纤维细胞中高效地转导mRNA的方法，并证明了心脏成纤维细胞向心肌细胞的部分直接重编程。C-Lipo2周内可实现Gata4、MEF2C和Tbx5(GMT)mRNAs的多次转导，从而实现了mRNA诱导的心脏直接重编程。α-肌动蛋白免疫组织化学显示，诱导后的心肌样细胞有α-MHC启动子驱动的绿色荧光蛋白表达，心肌结构呈条纹状。C-Lipo基因导入培养的小鼠心脏成纤维细胞，2周后心肌细胞标志基因Actc1、Actn2、Gja1、Hand2和Tnnt2的表达显著增加。此外，这项研究首次提供了GMT重编程因子的化学计量比影响心肌细胞标记基因表达的直接证据。我们的结果表明，信使核糖核酸是一种潜在的心肌细胞生成方法。
The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation.