Abstract
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.