Xu Ruodan, Srinivasan Sureshkumar Perumal, Sureshkumar Poornima, Nembo Erastus Nembu, Schäfer Christoph, Semmler Judith, Matzkies Matthias, Albrechtsen Morten, Hescheler Jürgen, Nguemo Filomain
ENKAM Pharmaceuticals A/S, Copenhagen, Denmark.
Cell Physiol Biochem. 2015;35(6):2437-50. doi: 10.1159/000374044. Epub 2015 Apr 24.
BACKGROUND/AIMS: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis.
In the present study, using a transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain (α-MHC) promoter (pαMHC-EGFP), we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGL(L), hNgf_C2, EnkaminE, Plannexin and C3) on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively.
The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF) peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor.
Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to cardiac pathologies where BDNF levels are impaired.
背景/目的：鉴于多能干细胞在合适的细胞环境中分化为心肌样细胞在再生医学中的潜在应用，已引起了广泛关注。然而，多能干细胞谱系特化的确切机制仍有待深入探索。确定参与心肌生成的各种小合成肽的作用，可能为促进心肌生成的途径提供新的见解。
在本研究中，我们使用在α-肌球蛋白重链（α-MHC）启动子（pαMHC-EGFP）控制下表达增强型绿色荧光蛋白（EGFP）的转基因小鼠胚胎干细胞（ES）系，研究了7种合成肽（Betrofin3、FGLs、FGL(L)、hNgf_C2、EnkaminE、Plannexin和C3）对心脏分化的心肌生成作用。通过RT-PCR测定几种心脏特异性标志物的表达，而通过免疫荧光和电生理学分别检查衍生心肌细胞的结构和功能特性。
结果显示，脑源性神经营养因子（BDNF）肽激动剂Betrofin3对ES细胞具有最显著的促心肌生成作用。我们发现BDNF受体TrkB在分化过程中表达上调。在第3天至第5天用Betrofin3处理分化细胞，可增强心脏特异性标志物的表达，并改善心肌细胞的分化和功能，这通过基因调控、流式细胞术和膜片钳分析得以揭示。因此，Betrofin3可能通过TrkB受体对ES细胞发挥其心肌生成作用。
综上所述，结果表明Betrofin3以阶段和剂量依赖性方式调节BDNF信号传导，具有积极的心肌生成作用，为增加基于ES细胞的心肌细胞生成提供了一种有效策略，并为BDNF水平受损的心脏疾病提供了一种新的治疗方法。
