Effects of Nitric Oxide on Voltage-Gated K⁺ Currents in Human Cardiac Fibroblasts through the Protein Kinase G and Protein Kinase A Pathways but Not through S-Nitrosylation.

Hyemi Bae,Inja Lim,Young-Won Kim,Donghee Lee,Taeho Kim,Hyoweon Bang,Jae-Hong Ko,Jung-Ha Kim,Jeongyoon Choi

doi:10.3390/ijms19030814

Hyemi Bae, Inja Lim + Show 7 more

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https://doi.org/10.3390/ijms19030814

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Abstract

This study investigated the expression of voltage-gated K+ (KV) channels in human cardiac fibroblasts (HCFs), and the effect of nitric oxide (NO) on the KV currents, and the underlying phosphorylation mechanisms. In reverse transcription polymerase chain reaction, two types of KV channels were detected in HCFs: delayed rectifier K+ channel and transient outward K+ channel. In whole-cell patch-clamp technique, delayed rectifier K+ current (IK) exhibited fast activation and slow inactivation, while transient outward K+ current (Ito) showed fast activation and inactivation kinetics. Both currents were blocked by 4-aminopyridine. An NO donor, S-nitroso-N-acetylpenicillamine (SNAP), increased the amplitude of IK in a concentration-dependent manner with an EC50 value of 26.4 µM, but did not affect Ito. The stimulating effect of SNAP on IK was blocked by pretreatment with 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or by KT5823. 8-bromo-cyclic GMP stimulated the IK. The stimulating effect of SNAP on IK was also blocked by pretreatment with KT5720 or by SQ22536. Forskolin and 8-bromo-cyclic AMP each stimulated IK. On the other hand, the stimulating effect of SNAP on IK was not blocked by pretreatment of N-ethylmaleimide or by DL-dithiothreitol. Our data suggest that NO enhances IK, but not Ito, among KV currents of HCFs, and the stimulating effect of NO on IK is through the PKG and PKA pathways, not through S-nitrosylation.

Highlights

Human cardiac fibroblasts (HCFs) are the highest cell population in the myocardium, accounting for approximately two-thirds of the cells [1], and play a role in cardiac development, myocardial structuring, cell signaling, and electro-mechanical function in healthy and diseased myocardium [2]
We demonstrated that nitric oxide (NO) produced a concentration-dependent stimulation of IK in HCFs, and these results were consistent with other reports in guinea-pig cardiomyocytes [24,48]
The stimulating effect of NO on IK was suppressed in the presence of soluble guanylate cyclase (sGC) inhibitor (ODQ) or protein kinase G (PKG) inhibitor (KT5823), and IK was increased by 8-Br-cyclic GMP (cGMP), a cell-permeable analogue of cGMP. These results suggest that the stimulatory effects of NO are dependent on the sGC/cGMP/PKG pathway in HCFs

Summary

Introduction

Human cardiac fibroblasts (HCFs) are the highest cell population in the myocardium, accounting for approximately two-thirds of the cells [1], and play a role in cardiac development, myocardial structuring, cell signaling, and electro-mechanical function in healthy and diseased myocardium [2]. HCFs are not electrically excitable, they express a plethora of ion channels that modulate cardiac electrical function. The electrical coupling between fibroblasts/myofibroblasts and ventricular cardiomyocytes has been demonstrated in co-culture conditions [1,4,5], and in the whole heart [6]. High rate of electrical activation from tachy-paced atrial cardiomyocytes induce them to secret factors into the culture medium that in turn activate fibroblasts [8]. Cardiac fibroblasts paracrine factors alter impulse conduction and ion channel expression of cardiomyocytes [9]. These interactions are enhanced in response to cardiac injury [10]

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