The present study demonstrates a novel NO-dependent modulatory mechanism that alters the voltage-gated Na+ channel protein Nav1.5 and thereby INa in neonate cardiomyocytes. Although NO is recognized as a potent vasodilator, and its activity is highly beneficial for the ischemic heart, this investigation provide a surprising new dimension to NO signaling which is the cGMP-independent action of NO to down-regulate the Na+ channel through S-nitrosylation. The major findings of this investigation are summarized as follows: (1) Long-term but not short-term application of NO donors, NOC-18 and SNAP, reduced INa in a dose-dependent manner; (2) an NO donor NOC-18 shifted the steady-state inactivation curve, but not the activation curve, to the hyperpolarization direction; (3) a potent NO inhibitor Carboxy-PTIO masks the effect of NOC-18 on INa; (4) a GC inhibitor ODG and a PKG inhibitor KT5823 could not block the effect of NOC-18 on INa, (5) a membrane permeable cGMP (8 Br-cGMP) was without effect on INa; (6) protein thiol modulation inhibitors, NEM and DTE, suppressed the effect of NOC-18 on INa, and (7) NOC-18 decreased the protein expression of Nav1.5 channel, and increased the expression of a transcription factor FOXO1 in the nucleus. These findings suggest a novel action of NO on the voltage-gated Na+ channel through S-nitrosylation pathway, which could be adverse to cardiac function particularly in the diseased conditions of the heart.
NO is produced from virtually all cell types composing the myocardium, and regulates cardiac function through vascular-dependent and -independent manners. The role of NO in cardiac function is complex and controversial [22]. NO can bind to GC, increasing cGMP production and activate PKG. NO may also directly S-nitrosylate cysteine residues of specific proteins. In this context, it is particularly important to elucidate molecular targets of NO, or NO donors NOC-18 and SNAP in this experiment. For voltage-gated ion channels, targets of PKG include Ca2+-activated K+ channel [23], ATP-sensitive K+ channel [24], voltage-gated L, N, T-type Ca2+ channel [25, 26], and voltage-gated Na+ channels [27] with their respective impact on cellular excitability. In addition, S-nitrosylation of cysteine residues has emerged as an important feature of NO signaling. Though this post-translational modification, NO is able to regulate the function of ion channels including, Ca2+ activated K+ channel [28], voltage-gated L-type Ca2+ channel [29], cyclic nucleotide gated channel [30], and voltage-gated Na+ channels [4, 31]. In addition to these emerging evidence linking PKG/S-nitrosylation to the voltage-gated Na+ channel by regulating the state of post-translational modulation, this investigation, for the first time, shed light on the transcriptional effects of NO on the cardiac Na+ channel.
In the present study NOC-18 and SNAP were used as NO donors. It has been reported that NO released from 1 mM NOC-18 results in steady-state levels of 1–5 µM NO in medium without any cofactors [32]. This is comparable to concentrations (1–30 µM) produced by endogenous inducible NO synthase in culture media and in plasma after cytokine stimulation or lung injury [33]. For the clinical setting of NO for the treatment of pulmonary hypertension, acute lung injury, and cardiopulmonary failure, concentration of 15–30 ppm (5–10 µM) of NO has widely been applied [34]. During these clinical applications of NO, lung endothelium and cardiomyocytes are exposed to be exposed to the same concentration or slightly lower than that in the inhaled NO gas. Thus exposure of cardiomyocytes to 1–5 µM NO constitutes a pathophysiologically relevant cellular model with which to study NO-mediated modulation of ion channels in cardiomyocytes. Cultured neonatal cardiomyocytes were exposed to 1 mM NOC-18 for 24 h in this investigation. Therefore results in this study imply that NO plays an important role in regulation of myocardial Na+ channel during clinical therapeutic application of NO or NO donor in vivo.
Although our study could not detect the acute effects of NO donor on INa, previous several studies identified NO/cGMP/PKG pathway to regulate INa as a short-term effect [16, 27, 35]. Because the life span of NO in blood milieu or in the bathing solution is less than 0.2 ms [30], biological exposure time of NO largely depends on the NO-releasing speed of NO donor. NOC-18 is a diazeniumdiolate slow-releasing NO donor with a half-life for NO release of 20 hours, where the rate of release is attributed to the structure [37]. NOC-18 is an ideal NO donor since the amine byproduct formed has no known interferences with cellular activities. Therefore, unlike experiments using gaseous NO to obtain saturated concentration of NO in the medium, experiments by use of a slow-releasing NO donor NOC-18 are suitable to assess the possible transcriptional effect of NO to cardiomyocytes. In this context, we could not observe persistent Na+ current in this experiments, presumably because increases of NO concentration by slow-releasing NO donors were neither prompt and high enough in the bath medium for the electrophysiological studies. Also as a limitation of whole-cell patch clamp experiments, in most patch clamp amplifier in the market, leak subtraction could subtract the leak current produced by single depolarizing pulse but not the leak current produced by a series of different step depolarizing pulses. Due to its assumption that leak current would be produced if only potential difference arises across membrane, recording protocols in manufactured voltage-clamp program by use of leak subtraction are not suitable to subtracting the steady-state leak current while recording voltage-gated channel currents which is also time-dependently altered.
Several studies, particularly based on plants with altered NO levels, have recently provided genetic evidence for the importance of NO in gene induction [38, 39], although little is known on the role of NO as a regulator of gene expression in mammals. Furthermore, the NO-dependent intracellular signaling pathway(s) that lead to the activation or suppression of these genes have not yet been defined. In this context, for the first time, our study has revealed a novel role of NO as a positive modulator of FOXO1 in cardiomyocytes, leading to a reduction of Nav1.5 proteins. Because FOXO1-mRNA was unchanged and phosphorylated form of FOXO1 was increased in the nucleus in response to NO (Fig. 7), it is suggested that FOXO1 translocates from the cytoplasm to the nucleus and suppresses the expression of Nav1.5. Phosphorylated Foxo1 can be dephoshosphorylated by phosphatase allowing Foxo1 to enter the nucleus [40]. Therefore functional modulation or stimulation of phosphatase by NO-mediated S-nitrosylation is possibly postulated, although we have no data to support this speculation. Obviously a complete transcriptome analysis is needed for the understanding about the mechanism of NO-mediated Nav1.5 suppression.
Derangement of NO production regulation, such as produced on excessive NO delivery from inflammatory cells (or cytokine-stimulated cardiomyocytes themselves), may result in profound cellular disturbances leading to heart failure [41]. At the same time, however, the functional consequences of altered NO synthase expression and NO bioavailability in the failing heart are poorly characterized. Namely, quite a few numbers of diverse and often contradictory effects of NO and NO donors on myocardial function have been reported. It is widely accepted that NO could modulate inotropic, chronotropic, and dromotropic response to β adrenoceptor stimulation: low dose enhances and high dose reduce β adrenergic response [42]. Interestingly, an action of NO to modulate β adrenergic inotropic responses in humans in vivo could only be demonstrated in patients with heart failure and not in “normal” subjects [43]. In relation to the action of NO to heart failure, we note the possible adverse effect of NO in patients with diseased heart. In human study, prolonged nitrate treatment was reportedly not beneficial for patients with myocardial infarction [44, 45] and heart failure [46].
Although we have successfully demonstrated that NO down-regulates INa in neonatal cardiomyocytes, and have postulated a possible FOXO1-dependent signaling pathway for the regulation of Nav1.5 (Fig. 8), we still need to identify interactor molecules between NO and FOXO1 transcription. As limitation of this investigation, it is also important to keep in mind that reduction of INa and the sodium channel protein was confirmed by NO donors in in-vitro experiments but not in in-vivo condition of the heart. Because physiological actions of NO in the heart are largely dependent on the vascular and neuronal regulation of the circulation, drug actions on cardiomyocytes without systemic circulation may not accurately represent the clinical pharmacological effects of NO. Furthermore, we are not sure whether NO donors mimic endogenous NO-related response in the heart. In addition, we could not explore the action of NO on the accessory proteins of the channel including β1 and β2 subunits, which may affect the gating properties of the channel; e.g. the shift of the steady-state inactivation curve to the hyperpolarization direction.
We conclude that NO is a negative modulator of the voltage-gated Na+ channel in cardiomyocytes. A significant reduction of INa as well as the SCN5A protein is considered to be one of mechanisms possibly related to NO-induced cardiac dysfunction particularly in heart failure. The endogenous mechanisms of transcriptional regulation of SCN5A in cardiomyocytes are largely unknown. We also demonstrate in this study that increase of a transcription factor FOXO1 in the nucleus is the trigger for the down-regulation of SCN5A in NO-treated cardiomyocytes. These findings indicate that many diverse and often contradictory effects of NO or NO donor on myocardial function could be attributed to the conduction defect and/or arrhythmias in the heart, at least in a part, caused by a reduction of the voltage-gated Na+ channel.