Hyperpolarization-Activated Cyclic Nucleotide-Gated Ion (HCN) Channels Regulate PC12 Cell Differentiation Toward Sympathetic Neuron.

Li-Ying Zhong,Yan Wei,Xin-Rong Fan,Na Lin,Zhang-Jing Shi,Lin Wu,Ji-Min Cao,Jian Luo,Ying-Cai Liu,Xiao-Rong Zeng,Zhong-Cai Fan

doi:10.3389/fncel.2019.00415

Abstract

Hyperpolarization-activated cyclic nucleotide-gated ion channels (HCN channels) are widely expressed in the central and peripheral nervous systems and organs, while their functions are not well elucidated especially in the sympathetic nerve. The present study aimed to investigate the roles of HCN channel isoforms in the differentiation of sympathetic neurons using PC12 cell as a model. PC12 cells derived from rat pheochromocytoma were cultured and induced by nerve growth factor (NGF) (25 ng/ml) to differentiate to sympathetic neuron-like cells. Sympathetic directional differentiation of PC12 cells were evaluated by expressions of growth-associated protein 43 (GAP-43) (a growth cone marker), tyrosine hydroxylase (TH) (a sympathetic neuron marker) and neurite outgrowth. Results show that the HCN channel isoforms (HCN1-4) were all expressed in PC12 cells; blocking HCN channels with ivabradine suppressed NGF-induced GAP-43 expression and neurite outgrowth; silencing the expression of HCN2 and HCN4 using silenced using small interfering RNAs (siRNA), rather than HCN1 and HCN3, restrained GAP-43 expression and neurite outgrowth, while overexpression of HCN2 and HCN4 channels with gene transfer promoted GAP-43 expression and neurite outgrowth. Patch clamp experiments show that PC12 cells exhibited resting potentials (RP) of about −65 to −70 mV, and also presented inward HCN channel currents and outward (K+) currents, but no inward voltage-gated Na+ current was induced; NGF did not significantly affect the RP but promoted the establishment of excitability as indicated by the increased ability to depolarize and repolarize in the evoked suspicious action potentials (AP). We conclude that HCN2 and HCN4 channel isoforms, but not HCN1 and HCN3, promote the differentiation of PC12 cells toward sympathetic neurons. NGF potentiates the establishment of excitability during PC12 cell differentiation.

Highlights

The normal structure and function of sympathetic nerve is the key to maintain the normal physiological function of the heart (Gordan et al, 2015; Meng et al, 2018)
Real-time PCR results (Figures 2B,C) show that the mRNA level of HCN1 was very low in PC12 cells, while the mRNA levels of HCN2, HCN3, and HCN4 were relatively higher compared with the reference gene GAPDH
We demonstrated here that all the four HCN channel isoforms (HCN1-4) were expressed in PC12 cells and nerve growth factor (NGF) promoted PC12 cell differentiation potentially by affecting the expression and function of certain HCN channels

Summary

Introduction

The normal structure and function of sympathetic nerve is the key to maintain the normal physiological function of the heart (Gordan et al, 2015; Meng et al, 2018). A variety of heart diseases, including myocardial infarction (MI) and cardiomyopathy, can disrupt cardiac sympathetic innervation, in which sympathetic nerves are subjected to change both in structure and function in response to injury (Gardner et al, 2016). Unbalanced cardiac sympathetic innervation, including regional denervation and nerve sprouting/hyperinnervation, is strongly linked to the occurrence of arrhythmias in the course or prognosis of heart diseases (Cao et al, 2000a,b; Triposkiadis et al, 2009; Meng et al, 2018). The process of cardiac sympathetic nerve sprouting after injury includes axonal regeneration and outgrowth and neurilemmal cell proliferation (Fallavollita et al, 2014), this could lead to sympathetic nerve remodeling (Chen et al, 2001). The underlying mechanisms of sympathetic nerve remodeling remains incompletely understood

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Conclusion