A Narrative Review of the Published Pre-Clinical Evaluations: Multiple Effects of Arachidonic Acid, its Metabolic Enzymes and Metabolites in Epilepsy.

Arachidonic acid (AA), an important polyunsaturated fatty acid in the brain, is hydrolyzed by a direct action of phospholipase A2 (PLA2) or through the combined action of phospholipase C and diacylglycerol lipase, and released into the cytoplasm. Various derivatives of AA can be synthesized mainly through the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (P450) enzyme pathways. AA and its metabolic enzymes and metabolites play important roles in a variety of neurophysiological activities. The abnormal metabolites and their catalytic enzymes in the AA cascade are related to the pathogenesis of various central nervous system (CNS) diseases, including epilepsy. Here, we systematically reviewed literatures in PubMed about the latest randomized controlled trials, animal studies and clinical studies concerning the known features of AA, its metabolic enzymes and metabolites, and their roles in epilepsy. The exclusion criteria include non-original studies and articles not in English.

Transient receptor potential vanilloid-1 (TRPV1) channels act as suppressors of the growth of glioma.

The aim of this study was to investigate the expression and function of the transient receptor potential vanilloid 1 (TRPV1) in glioma. We found that the expression of TRPV1 mRNA and protein were upregulated in glioma compared with normal brain by qPCR and western blot analysis. In order to investigate the function of TRPV1 in glioma, short hairpin RNA (shRNA) and the inhibitor of TRPV1 were used. In vitro, the activation of TRPV1 induced cell apoptosis with decreased migration capability and inhibited proliferation, which was abolished upon TRPV1 pharmacological inhibition and silencing. Mechanistically, TRPV1 modulated glioma proliferation through the protein kinase B (Akt) signaling pathway. More importantly, in immunodeficient (NOD-SCID) mouse xenograft models, tumor size was significantly increased when TRPV1 expression was disrupted by a shRNA knockdown approach in vivo. Altogether, our findings indicate that TRPV1 negatively controls glioma cell proliferation in an Akt-dependent manner, which suggests that targeting TRPV1 may be a potential therapeutic strategy for glioma.

Cell-specific NFIA upregulation promotes epileptogenesis by TRPV4-mediated astrocyte reactivity.

BACKGROUND: The astrocytes in the central nervous system (CNS) exhibit morphological and functional diversity in brain region-specific pattern. Functional alterations of reactive astrocytes are commonly present in human temporal lobe epilepsy (TLE) cases, meanwhile the neuroinflammation mediated by reactive astrocytes may advance the development of hippocampal epilepsy in animal models. Nuclear factor I-A (NFIA) may regulate astrocyte diversity in the adult brain. However, whether NFIA endows the astrocytes with regional specificity to be involved in epileptogenesis remains elusive. METHODS: Here, we utilize an interference RNA targeting NFIA to explore the characteristics of NFIA expression and its role in astrocyte reactivity in a 4-aminopyridine (4-AP)-induced seizure model in vivo and in vitro. Combined with the employment of a HA-tagged plasmid overexpressing NFIA, we further investigate the precise mechanisms how NIFA facilitates epileptogenesis. RESULTS: 4-AP-induced NFIA upregulation in hippocampal region is astrocyte-specific, and primarily promotes detrimental actions of reactive astrocyte. In line with this phenomenon, both NFIA and vanilloid transient receptor potential 4 (TRPV4) are upregulated in hippocampal astrocytes in human samples from the TLE surgical patients and mouse samples with intraperitoneal 4-AP. NFIA directly regulates mouse astrocytic TRPV4 expression while the quantity and the functional activity of TRPV4 are required for 4-AP-induced astrocyte reactivity and release of proinflammatory cytokines in the charge of NFIA upregulation. NFIA deficiency efficiently inhibits 4-AP-induced TRPV4 upregulation, weakens astrocytic calcium activity and specific astrocyte reactivity, thereby mitigating aberrant neuronal discharges and neuronal damage, and suppressing epileptic seizure. CONCLUSIONS: Our results uncover the critical role of NFIA in astrocyte reactivity and illustrate how epileptogenic brain injury initiates cell-specific signaling pathway to dictate the astrocyte responses.

Transient Receptor Potential Vanilloid 4: a Double-Edged Sword in the Central Nervous System.

Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel that can be activated by diverse stimuli, such as heat, mechanical force, hypo-osmolarity, and arachidonic acid metabolites. TRPV4 is widely expressed in the central nervous system (CNS) and participates in many significant physiological processes. However, accumulative evidence has suggested that deficiency, abnormal expression or distribution, and overactivation of TRPV4 are involved in pathological processes of multiple neurological diseases. Here, we review the latest studies concerning the known features of this channel, including its expression, structure, and its physiological and pathological roles in the CNS, proposing an emerging therapeutic strategy for CNS diseases.

Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4) Mitigates Seizures.

Astrocytes are critical regulators of the immune/inflammatory response in several human central nervous system (CNS) diseases. Emerging evidence suggests that dysfunctional astrocytes are crucial players in seizures. The objective of this study was to investigate the role of transient receptor potential vanilloid 4 (TRPV4) in 4-aminopyridine (4-AP)-induced seizures and the underlying mechanism. We also provide evidence for the role of Yes-associated protein (YAP) in seizures. 4-AP was administered to mice or primary cultured astrocytes. YAP-specific small interfering RNA (siRNA) was administered to primary cultured astrocytes. Mouse brain tissue and surgical specimens from epileptic patient brains were examined, and the results showed that TRPV4 was upregulated, while astrocytes were activated and polarized to the A1 phenotype. The levels of glial fibrillary acidic protein (GFAP), cytokine production, YAP, signal transducer activator of transcription 3 (STAT3), intracellular Ca2+([Ca2+]i) and the third component of complement (C3) were increased in 4-AP-induced mice and astrocytes. Perturbations in the immune microenvironment in the brain were balanced by TRPV4 inhibition or the manipulation of [Ca2+]i in astrocytes. Knocking down YAP with siRNA significantly inhibited 4-AP-induced pathological changes in astrocytes. Our study demonstrated that astrocytic TRPV4 activation promoted neuroinflammation through the TRPV4/Ca2+/YAP/STAT3 signaling pathway in mice with seizures. Astrocyte TRPV4 inhibition attenuated neuroinflammation, reduced neuronal injury, and improved neurobehavioral function. Targeting astrocytic TRPV4 activation may provide a promising therapeutic approach for managing epilepsy.

Complement C3 Aggravates Post-epileptic Neuronal Injury Via Activation of TRPV1.

Epilepsy is a brain condition characterized by the recurrence of unprovoked seizures. Recent studies have shown that complement component 3 (C3) aggravate the neuronal injury in epilepsy. And our previous studies revealed that TRPV1 (transient receptor potential vanilloid type 1) is involved in epilepsy. Whether complement C3 regulation of neuronal injury is related to the activation of TRPV1 during epilepsy is not fully understood. We found that in a mouse model of status epilepticus (SE), complement C3 derived from astrocytes was increased and aggravated neuronal injury, and that TRPV1-knockout rescued neurons from the injury induced by complement C3. Circular RNAs are abundant in the brain, and the reduction of circRad52 caused by complement C3 promoted the expression of TRPV1 and exacerbated neuronal injury. Mechanistically, disorders of neuron-glia interaction mediated by the C3-TRPV1 signaling pathway may be important for the induction of neuronal injury. This study provides support for the hypothesis that the C3-TRPV1 pathway is involved in the prevention and treatment of neuronal injury and cognitive disorders.

Silencing of circIgf1r plays a protective role in neuronal injury via regulating astrocyte polarization during epilepsy.

Epilepsy is a common brain disorder, repeated seizures of epilepsy may lead to a series of brain pathological changes such as neuronal or glial damage. However, whether circular RNAs are involved in neuronal injury during epilepsy is not fully understood. Here, we screened circIgf1r in the status epilepticus model through circRNA sequencing, and found that it was upregulated after the status epilepticus model through QPCR analysis. Astrocytes polarizing toward neurotoxic A1 phenotype and neurons loss were observed after status epilepticus. Through injecting circIgf1r siRNA into the lateral ventricle, it was found that knocking down circIgf1r in vivo would induce the polarization of astrocytes to phenotype A2 and reduce neuronal loss. The results in vitro further confirmed that inhibiting the expression of circIgf1r in astrocytes could protect neurons by converting reactive astrocytes from A1 to the protective A2. In addition, knocking down circIgf1r in astrocytes could functionally promote astrocyte autophagy and relieve the destruction of 4-AP-induced autophagy flux. In terms of mechanism, circIgf1r promoted the polarization of astrocytes to phenotype A1 by inhibiting autophagy. Taken together, our results reveal circIgf1r may serve as a potential target for the prevention and treatment of neuron damage after epilepsy.

TRPV1 translocated to astrocytic membrane to promote migration and inflammatory infiltration thus promotes epilepsy after hypoxic ischemia in immature brain.

BACKGROUND: Neonatal hypoxic-ischemic brain damage (HIBD), a leading cause of neonatal mortality, has intractable sequela such as epilepsy that seriously affected the life quality of HIBD survivors. We have previously shown that ion channel dysfunction in the central nervous system played an important role in the process of HIBD-induced epilepsy. Therefore, we continued to validate the underlying mechanisms of TRPV1 as a potential target for epilepsy. METHODS: Neonatal hypoxic ischemia and oxygen-glucose deprivation (OGD) were used to simulate HIBD in vivo and in vitro. Primarily cultured astrocytes were used to assess the expression of TRPV1, glial fibrillary acidic protein (GFAP), cytoskeletal rearrangement, and inflammatory cytokines by using Western blot, q-PCR, and immunofluorescence. Furthermore, brain electrical activity in freely moving mice was recorded by electroencephalography (EEG). TRPV1 current and neuronal excitability were detected by whole-cell patch clamp. RESULTS: Astrocytic TRPV1 translocated to the membrane after OGD. Mechanistically, astrocytic TRPV1 activation increased the inflow of Ca2+, which promoted G-actin polymerized to F-actin, thus promoted astrocyte migration after OGD. Moreover, astrocytic TRPV1 deficiency decreased the production and release of pro-inflammatory cytokines (TNF, IL-6, IL-1ß, and iNOS) after OGD. It could also dramatically attenuate neuronal excitability after OGD and brain electrical activity in HIBD mice. Behavioral testing for seizures after HIBD revealed that TRPV1 knockout mice demonstrated prolonged onset latency, shortened duration, and decreased seizure severity when compared with wild-type mice. CONCLUSIONS: Collectively, TRPV1 promoted astrocyte migration thus helped the infiltration of pro-inflammatory cytokines (TNF, IL-1ß, IL-6, and iNOS) from astrocytes into the vicinity of neurons to promote epilepsy. Our study provides a strong rationale for astrocytic TRPV1 to be a therapeutic target for anti-epileptogenesis after HIBD.

NFAT5 and HIF-1α Coordinate to Regulate NKCC1 Expression in Hippocampal Neurons After Hypoxia-Ischemia.

Hypoxic-ischemic encephalopathy (HIE) is a serious birth complication with severe long-term sequelae such as cerebral palsy, epilepsy and cognitive disabilities. Na+-K+-2Cl- cotransporters 1 (NKCC1) is dramatically upregulated after hypoxia-ischemia (HI), which aggravates brain edema and brain damage. Clinically, an NKCC1-specific inhibitor, bumetanide, is used to treat diseases related to aberrant NKCC1 expression, but the underlying mechanism of aberrant NKCC1 expression has rarely been studied in HIE. In this study, the cooperative effect of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor of activated T cells 5 (NFAT5) on NKCC1 expression was explored in hippocampal neurons under hypoxic conditions. HI increased HIF-1α nuclear localization and transcriptional activity, and pharmacological inhibition of the HIF-1α transcription activity or mutation of hypoxia responsive element (HRE) motifs recovered the hypoxia-induced aberrant expression and promoter activity of NKCC1. In contrast, oxygen-glucose deprivation (OGD)-induced downregulation of NFAT5 expression was reversed by treating with hypertonic saline, which ameliorated aberrant NKCC1 expression. More importantly, knocking down NFAT5 or mutation of the tonicity enhancer element (TonE) stimulated NKCC1 expression and promoter activity under normal physiological conditions. The positive regulation of NKCC1 by HIF-1α and the negative regulation of NKCC1 by NFAT5 may serve to maintain NKCC1 expression levels, which may shed light on the transcription regulation of NKCC1 in hippocampal neurons after hypoxia.

Barbaloin inhibits ventricular arrhythmias in rabbits by modulating voltage-gated ion channels.

Barbaloin (10-ß-D-glucopyranosyl-1,8-dihydroxy-3-(hydroxymethyl)-9(10H)-anthracenone) is extracted from the aloe plant and has been reported to have anti-inflammatory, antitumor, antibacterial, and other biological activities. Here, we investigated the effects of barbaloin on cardiac electrophysiology, which has not been reported thus far. Cardiac action potentials (APs) and ionic currents were recorded in isolated rabbit ventricular myocytes using whole-cell patch-clamp technique. Additionally, the antiarrhythmic effect of barbaloin was examined in Langendorff-perfused rabbit hearts. In current-clamp recording, application of barbaloin (100 and 200 µmol/L) dose-dependently reduced the action potential duration (APD) and the maximum depolarization velocity (Vmax), and attenuated APD reverse-rate dependence (RRD) in ventricular myocytes. Furthermore, barbaloin (100 and 200 µmol/L) effectively eliminated ATX II-induced early afterdepolarizations (EADs) and Ca2+-induced delayed afterdepolarizations (DADs) in ventricular myocytes. In voltage-clamp recording, barbaloin (10-200 µmol/L) dose-dependently inhibited L-type calcium current (ICa.L) and peak sodium current (INa.P) with IC50 values of 137.06 and 559.80 µmol/L, respectively. Application of barbaloin (100, 200 µmol/L) decreased ATX II-enhanced late sodium current (INa.L) by 36.6%±3.3% and 71.8%±6.5%, respectively. However, barbaloin up to 800 µmol/L did not affect the inward rectifier potassium current (IK1) or the rapidly activated delayed rectifier potassium current (IKr) in ventricular myocytes. In Langendorff-perfused rabbit hearts, barbaloin (200 µmol/L) significantly inhibited aconitine-induced ventricular arrhythmias. These results demonstrate that barbaloin has potential as an antiarrhythmic drug.

Andrographolide inhibits arrhythmias and is cardioprotective in rabbits.

Andrographolide has a protective effect on the cardiovascular system. To study its cardic-electrophysiological effects, action potentials and voltage-gated Na+ (INa), Ca2+ (ICaL), and K+ (IK1, IKr, Ito and IKur) currents were recorded using whole-cell patch clamp and current clamp techniques. Additionally, the effects of andrographolide on aconitine-induced arrhythmias were assessed on electrocardiograms in vivo. We found that andrographolide shortened action potential duration and reduced maximum upstroke velocity in rabbit left ventricular and left atrial myocytes. Andrographolide attenuated rate-dependence of action potential duration, and reduced or abolished delayed afterdepolarizations and triggered activities induced by isoproterenol (1 µM) and high calcium ([Ca2+]o=3.6 mM) in left ventricular myocytes. Andrographolide also concentration-dependently inhibited INa and ICaL, but had no effect on Ito, IKur, IK1, or IKr in rabbit left ventricular and left atrial myocytes. Andrographolide treatment increased the time and dosage thresholds of aconitine-induced arrhythmias, and reduced arrhythmia incidence and mortality in rabbits. Our results indicate that andrographolide inhibits cellular arrhythmias (delayed afterdepolarizations and triggered activities) and aconitine-induced arrhythmias in vivo, and these effects result from INa and ICaL inhibition. Andrographolide may be useful as a class I and IV antiarrhythmic therapeutic.

Wenxin Keli diminishes Ca2+ overload induced by hypoxia/reoxygenation in cardiomyocytes through inhibiting INaL and ICaL.

BACKGROUND: An increase in the late sodium current (INaL ) causes intracellular Na+ overload and subsequently intracellular Ca2+ ([Ca2+ ]i ) overload via the stimulated reverse Na+ -Ca2+ exchange (NCX). Wenxin Keli (WXKL) is an effective antiarrhythmic Chinese herb extract, but the underlying mechanisms are unclear. METHODS AND RESULTS: The INaL , NCX current (INCX ), L-type Ca2+ current (ICaL ), and action potentials were recorded using the whole-cell patch-clamp technique in rabbit ventricular myocytes. Myocyte [Ca2+ ]i transients were measured using a dual excitation fluorescence photomultiplier system. WXKL decreased the enhanced INaL , reverse INCX , diastolic [Ca2+ ]i , and the amplitude of Ca2+ transients induced by sea anemone toxin II (ATX II, a specific INaL channel opener) in a concentration-dependent manner. Hypoxia increased INaL , INCX , and diastolic [Ca2+ ]i , and decreased amplitude of [Ca2+ ]i transients. Hypoxia-reoxygenation aggravated these changes and induced spontaneous [Ca2+ ]i transients and hypercontraction in 86% cells (6/7). The application of WXKL during hypoxia or reoxygenation periods decreased the increased INaL , INCX , and diastolic [Ca2+ ]i , and prevented those events in 82% cells (9/11) under hypoxia-reoxygenation conditions. WXKL also inhibited the ICaL in a dose-dependent manner. Furthermore, WXKL shortened the action potential duration and completely abolished ATX II-induced early afterdepolarizations from 9/9 to /9. In isolated heart electrocardiogram recordings, WXKL inhibited ischemia-reperfusion induced ventricular premature beats and tachycardia. CONCLUSIONS: WXKL attenuated [Ca2+ ]i overload induced by hypoxia-reoxygenation in ventricular myocytes through inhibiting INaL and ICaL and prevents arrhythmias. This could, at least partly, contribute to the antiarrhythmic effects of WXKL.

Icariin, a Novel Blocker of Sodium and Calcium Channels, Eliminates Early and Delayed Afterdepolarizations, As Well As Triggered Activity, in Rabbit Cardiomyocytes.

Icariin, a flavonoid monomer from Herba Epimedii, has confirmed pharmacological and biological effects. However, its effects on arrhythmias and cardiac electrophysiology remain unclear. Here we investigate the effects of icariin on ion currents and action potentials (APs) in the rabbit myocardium. Furthermore, the effects of icariin on aconitine-induced arrhythmias were assessed in whole rabbits. Ion currents and APs were recorded in voltage-clamp and current-clamp mode in rabbit left ventricular myocytes (LVMs) and left atrial myocytes (LAMs), respectively. Icariin significantly shortened action potential durations (APDs) at 50 and 90% repolarization (APD50 and APD90) and reduced AP amplitude (APA) and the maximum upstroke velocity (Vmax) of APs in LAMs and LVMs; however, icariin had no effect on resting membrane potential (RMP) in these cells. Icariin decreased the rate-dependence of the APD and completely abolished anemonia toxin II (ATX-II)-induced early afterdepolarizations (EADs). Moreover, icariin significantly suppressed delayed afterdepolarizations (DADs) and triggered activities (TAs) elicited by isoproterenol (ISO, 1 µM) and high extracellular calcium concentrations ([Ca2+]o, 3.6 mM) in LVMs. Icariin also decreased INaT in a concentration-dependent manner in LAMs and LVMs, with IC50 values of 12.28 ± 0.29 µM (n = 8 cells/4 rabbits) and 11.83 ± 0.92 µM (n = 10 cells/6 rabbits; p > 0.05 vs. LAMs), respectively, and reversed ATX-II-induced INaL in a concentration-dependent manner in LVMs. Furthermore, icariin attenuated ICaL in a dose-dependent manner in LVMs. The corresponding IC50 value was 4.78 ± 0.89 µM (n = 8 cells/4 rabbits), indicating that the aforementioned current in LVMs was 2.8-fold more sensitive to icariin than ICaL in LAMs (13.43 ± 2.73 µM; n = 9 cells/5 rabbits). Icariin induced leftward shifts in the steady-state inactivation curves of INaT and ICaL in LAMs and LVMs but did not have a significant effect on their activation processes. Moreover, icariin had no effects on IK1 and IKr in LVMs or Ito and IKur in LAMs. These results revealed for the first time that icariin is a multichannel blocker that affects INaT, INaL and ICaL in the myocardium and that the drug had significant inhibitory effects on aconitine-induced arrhythmias in whole rabbits. Therefore, icariin has potential as a class I and IV antiarrhythmic drug.

Modulation of late sodium current by Ca2+ -calmodulin-dependent protein kinase II, protein kinase C and Ca2+ during hypoxia in rabbit ventricular myocytes.

NEW FINDINGS: What is the central question of this study? Hypoxia-induced increase in late sodium current (INa,L ) is associated with conditions causing cellular Ca2+ overload and contributes to arrhythmogenesis in the ventricular myocardium. The INa,L is an important drug target. We investigated intracellular signal transduction pathways involved in modulation of INa,L during hypoxia. What is the main finding and its importance? Hypoxia caused increases in INa,L , reverse Na+ -Ca2+ exchange current and diastolic [Ca2+ ], which were attenuated by inhibitors of Ca2+ -calmodulin-dependent protein kinase II (CaMKII) and protein kinase C and by a Ca2+ chelator. The findings suggest that CaMKII, protein kinase C and Ca2+ all participate in mediation of the effect of hypoxia to increase INa,L . Hypoxia leads to augmentation of the late sodium current (INa,L ) and cellular Na+ loading, increased reverse Na+ -Ca2+ exchange current (reverse INCX ) and intracellular Ca2+ loading in rabbit ventricular myocytes. The purpose of this study was to determine the intracellular signal transduction pathways involved in the modulation of INa,L during hypoxia in ventricular myocytes. Whole-cell and cell-attached patch-clamp techniques were used to record INa,L , and the whole-cell mode was also used to record reverse INCX and to study intercellular signal transduction mechanisms that mediate the increased INa,L . Dual excitation fluorescence photomultiplier systems were used to record the calcium transient in ventricular myocytes. Hypoxia caused increases of INa,L and reverse INCX . These increases were attenuated by KN-93 (an inhibitor of Ca2+ -calmodulin-dependent protein kinase II), bisindolylmaleimide VI (BIM; an inhibitor of protein kinase C) and BAPTA AM (a Ca2+ chelator). KN-93, BIM and BAPTA AM had no effect on INa,L in normoxia. In studies of KN-93, hypoxia alone increased the density of INa,L from -0.31 ± 0.02 to -0.66 ± 0.03 pA pF-1 (n = 6, P < 0.01 versus control) and the density of reverse INCX from 1.02 ± 0.06 to 1.91 ± 0.20 pA pF-1 (n = 7, P < 0.01 versus control) in rabbit ventricular myocytes. In the presence of 1 µm KN-93, the densities of INa,L and reverse INCX during hypoxia were significantly attenuated to -0.44 ± 0.03 (n = 6, P < 0.01 versus hypoxia) and 1.36 ± 0.15 pA pF-1 (n = 7, P < 0.01 versus hypoxia), respectively. In studies of BIM, hypoxia increased INa,L from -0.30 ± 0.03 to -0.60 ± 0.03 pA pF-1 (n = 6, P < 0.01 versus control) and reverse INCX from 0.91 ± 0.10 to 1.71 ± 0.27 pA pF-1 (n = 6, P < 0.01 versus control). In the presence of 1 µm BIM, the densities of INa,L and reverse INCX during hypoxia were significantly attenuated to -0.48 ± 0.02 (n = 6, P < 0.01 versus hypoxia) and 1.33 ± 0.21 pA pF-1 (n = 6, P < 0.01 versus hypoxia), respectively. In studies of BAPTA AM, hypoxia increased INa,L from -0.26 ± 0.04 to -0.63 ± 0.05 pA pF-1 (n = 6, P < 0.01 versus control) and reverse INCX from 0.86 ± 0.09 to 1.68 ± 0.35 pA pF-1 (n = 6, P < 0.01 versus control). The effects of hypoxia on INa,L and reverse INCX were significantly attenuated in the presence of 1 mm BAPTA AM to -0.39 ± 0.02 (n = 6, P < 0.01 versus hypoxia) and 1.12 ± 0.27 pA pF-1 (n = 6, P < 0.01 versus hypoxia), respectively. Results of single-channel studies showed that hypoxia apparently increased the mean open probability and mean open time of sodium channels. These effects were inhibited by either 1 µm KN-93 or 1 mm BAPTA AM. The suppressant effects of drug interventions were reversed upon washout. In addition, KN-93, BIM and BAPTA AM also reversed the hypoxia-enhanced diastolic Ca2+ concentration and the attenuated amplitude of the [Ca2+ ]i transient, maximal velocities of Ca2+ increase and Ca2+ decay. In summary, the findings suggest that Ca2+ -calmodulin-dependent protein kinase II, protein kinase C and Ca2+ all participate in mediation of the effect of hypoxia to increase INa,L .