Department of Cardiology, University RWTH Aachen, Aachen, Germany.
J Mol Cell Cardiol. 2010 Jul;49(1):79-87. doi: 10.1016/j.yjmcc.2010.01.019. Epub 2010 Feb 2.
Neuronal remodeling with increased sympathetic innervation density has been implicated in the pathogenesis of atrial fibrillation (AF). Recently, increased transcardiac nerve growth factor (NGF) levels were observed in a canine model of AF. Whether atrial myocytes or cardiac sympathetic neurons are the source of neurotrophins, and whether NGF is the main neurotrophic factor contributing to sympathetic nerve sprouting (SNS) in AF still remains unclear. Therefore, neonatal rat atrial myocytes were cultured under conditions of high frequency electrical field stimulation (HFES) to mimic rapid atrial depolarization. Likewise, sympathetic neurons from the superior cervical ganglia of neonatal rats were exposed to HFES to simulate the physiological effect of sympathetic stimulation. Real-time PCR, ELISA and Western blots were performed to analyze the expression pattern of NGF and neurotrophin-3 (NT-3). Baseline NGF and NT-3 content was 3-fold higher in sympathetic neurons than in atrial myocytes (relative NGF protein expression: 1+/-0.0 vs. 0.37+/-0.11, all n=5, p<0.05). HFES of sympathetic neurons induced a frequency dependent NGF and NT-3 gene and protein up-regulation (relative NGF protein expression: 0Hz=1+/-0.0 vs. 5Hz=1.13+/-0.19 vs. 50Hz=1.77+/-0.08, all n=5, 0Hz/5Hz vs. 50Hz p<0.05), with a subsequent increase of growth associated protein 43 (GAP-43) expression and morphological SNS. Moreover, HFES of sympathetic neurons increased the tyrosine kinase A (TrkA) receptor expression. HFES induced neurotrophic effects could be abolished by lidocaine, TrkA blockade or NGF neutralizing antibodies, while NT-3 neutralizing antibodies had no significant effect on SNS. In neonatal rat atrial myocytes, HFES resulted in myocyte hypertrophy accompanied by an increase in NT-3 and a decrease in NGF expression. In summary, this study provides evidence that high-rate electrical stimulation of sympathetic neurons mediates nerve sprouting by an increase in NGF expression that targets the TrkA receptor in an autocrine/paracrine manner.
交感神经支配密度增加导致的神经元重塑与心房颤动（AF）的发病机制有关。最近，在犬 AF 模型中观察到心脏神经生长因子（NGF）水平升高。然而，尚不清楚神经营养因子的来源是心房肌细胞还是心脏交感神经元，以及 NGF 是否是导致 AF 中交感神经发芽（SNS）的主要神经营养因子。因此，本研究采用高频电场刺激（HFES）模拟快速心房去极化来培养新生大鼠心房肌细胞。同样，新生大鼠颈上神经节的交感神经元暴露于 HFES 以模拟交感刺激的生理效应。实时 PCR、ELISA 和 Western blot 用于分析 NGF 和神经营养因子-3（NT-3）的表达模式。基础状态下，交感神经元中的 NGF 和 NT-3 含量比心房肌细胞高 3 倍（相对 NGF 蛋白表达：1+/-0.0 比 0.37+/-0.11，所有 n=5，p<0.05）。HFES 诱导交感神经元产生频率依赖性 NGF 和 NT-3 基因和蛋白上调（相对 NGF 蛋白表达：0Hz=1+/-0.0 比 5Hz=1.13+/-0.19 比 50Hz=1.77+/-0.08，所有 n=5，0Hz/5Hz 比 50Hz p<0.05），随后生长相关蛋白 43（GAP-43）表达和形态学 SNS 增加。此外，HFES 还增加了酪氨酸激酶 A（TrkA）受体的表达。利多卡因、TrkA 阻断或 NGF 中和抗体可阻断 HFES 诱导的神经营养作用，而 NT-3 中和抗体对 SNS 无明显影响。在新生大鼠心房肌细胞中，HFES 导致心肌细胞肥大，同时 NT-3 增加，NGF 表达减少。总之，这项研究提供了证据表明，交感神经元的高频电刺激通过增加 NGF 表达介导神经发芽，而 NGF 表达通过自分泌/旁分泌方式靶向 TrkA 受体。
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