nA Current Research Articles

Increased NaV1.8 expression in patients with sleep-disordered breathing induces pro-arrhythmic activity

Abstract Background Sleep-disordered breathing (SDB) is often associated with atrial fibrillation, but detailed mechanisms remain elusive. Interestingly, late Na current (late INa) has been shown to be increased in patients with SDB, while expression of cardiac Na channel NaV1.5 and peak Na current were decreased. Indeed, recent data demonstrated that enhanced NaV1.8-dependent late INa may also induce pro-arrhythmic activity. Purpose We tested whether Na-V1.8 expression and subsequent NaV1.8-dependent pro-arrhythmic activity are increased in patients with SDB. Methods We prospectively analysed 29 right atrial appendage biopsies of patients undergoing elective coronary artery bypass grafting. SDB was assessed using polygraphy in the preoperative night and an apnoea-hypopnea index (AHI) ≥15/h defined SDB. Micro-dissected atrial trabeculae were electrically field stimulated (at 1 Hz, 5 V for 50 ms, at 37°C) to elicit regular contractions. Trabecular arrhythmias were induced using 100 nM isoproterenol at [Ca]o of 3.5 mmol/L and pro-arrhythmic activity was scored from 0 (no arrhythmias) to 5 (salve). Sarcoplasmic reticulum Ca leak was estimated by the contractility after paused stimulation (at 2 Hz, normalized to before pause). To correlate functional and expression data for each individual patient, NaV1.8 mRNA expression was quantified in each trabeculum using qPCR. Results NaV1.8 mRNA expression was increased in patients with SDB, leading to a significant positive correlation with the severity of SDB (i.e. AHI, p=0.02, r2=0.22, Fig. 1A). Multivariate regression analysis revealed that this association was independent from age, sex, atrial fibrillation, heart failure, diabetes mellitus, and renal function (p=0.03, r2=0.35). Accordingly, selective NaV1.8 blockade with PF-01247324 (PF, 1 μM, 30 min) significantly improved post-pause contractility of isolated trabeculae from 1.69±0.31 to 2.95±0.54 in patients with SDB (p=0.001), whereas no significant improvement was observed in patients without SDB. This resulted in significant positive correlations between the PF-dependent improvement of post-pause contractility and both AHI (p=0.047, r2=0.19) and NaV1.8 mRNA expression (p=0.03, r2=0.17). Most importantly, we also observed a significant increase in arrhythmia severity in patients with SDB of 2.21±0.52 (vs. 1.00±0.49, p=0.03) that could be significantly reduced by selective NaV1.8 inhibition with PF to 0.25±0.18 (p=0.0008, Fig. 1B). In accordance, there was a significant positive correlation between arrhythmia severity and AHI (p=0.01, r2=0.28) that was abolished in the presence of PF (interaction analysis: p=0.ehab724.33141, r2=0.46). Conclusion In patients with SDB, enhanced NaV1.8 expression contribute to atrial pro-arrhythmic activity independent from comorbidities. Selective NaV1.8 inhibition may have therapeutic implications for patients with SDB. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Part of the study was supported by grants from Philips Respironics (Murrysville, PA 15668) and the Medical Faculty at the University of Regensburg. Figure 1

Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

In cardiomyocytes, NaV1.5 channels mediate initiation and fast propagation of action potentials. The Ca2+-binding protein calmodulin (CaM) serves as a de facto subunit of NaV1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the NaV1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a "gain-of-function" defect, and Brugada syndrome, a "loss-of-function" phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on NaV1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca2+-free CaM preassociation with NaV1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent NaV openings. Mechanistically, these effects arise from a CaM-dependent switch in the NaV inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant NaV1.5, local Ca2+ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of NaV1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of NaV1.5 channelopathies.

A model of on/off transitions in neurons of the deep cerebellar nuclei: deciphering the underlying ionic mechanisms

The neurons of the deep cerebellar nuclei (DCNn) represent the main functional link between the cerebellar cortex and the rest of the central nervous system. Therefore, understanding the electrophysiological properties of DCNn is of fundamental importance to understand the overall functioning of the cerebellum. Experimental data suggest that DCNn can reversibly switch between two states: the firing of spikes (F state) and a stable depolarized state (SD state). We introduce a new biophysical model of the DCNn membrane electro-responsiveness to investigate how the interplay between the documented conductances identified in DCNn give rise to these states. In the model, the F state emerges as an isola of limit cycles, i.e. a closed loop of periodic solutions disconnected from the branch of SD fixed points. This bifurcation structure endows the model with the ability to reproduce the text{F}to text{SD} transition triggered by hyperpolarizing current pulses. The model also reproduces the text{F}to text{SD} transition induced by blocking Ca currents and ascribes this transition to the blocking of the high-threshold Ca current. The model suggests that intracellular current injections can trigger fully reversible text{F}leftrightarrow text{SD} transitions. Investigation of low-dimension reduced models suggests that the voltage-dependent Na current is prominent for these dynamical features. Finally, simulations of the model suggest that physiological synaptic inputs may trigger text{F}leftrightarrow text{SD} transitions. These transitions could explain the puzzling observation of positively correlated activities of connected Purkinje cells and DCNn despite the former inhibit the latter.

The Ketamine Antidepressant Story: New Insights.

Ketamine is a versatile agent primarily utilized as a dissociative anesthetic, which acts by blocking the excitatory receptor N-methyl-d-aspartate receptor (NMDA). It functions to inhibit the current of both Na+ and K+ voltage-gated channels, thus preventing serotonin and dopamine reuptake. Studies have indicated that administering a single subanesthetic dose of ketamine relieves depression rapidly and that the effect is sustained. For decades antidepressant agents were based on the monoamine theory. Although ketamine may not be the golden antidepressant, it has opened new avenues toward mechanisms involved in the pathology of treatment-resistant depression and achieving rapid antidepressant effects. Thus, preclinical studies focusing on deciphering the molecular mechanisms involved in the antidepressant action of ketamine will assist in the development of a new antidepressant. This review was conducted to elucidate the emerging pathways that can explain the complex dose-dependent mechanisms achieved by administering ketamine to treat major depressive disorders. Special attention was paid to reviewing the literature on hydroxynorketamines, which are ketamine metabolites that have recently attracted attention in the context of depression.

Abstract 17405: Chronic Treatment of Ibrutinib Reduces PI3K Activation and Causes Increased Mitochondrial Oxidative Stress Leading to Heightened AF in Pitx2c +/- Mice

Background: Ibrutinib, a Bruton’s tyrosine kinase inhibitor which is currently used as first line treatment to various B-cell malignancies is known to cause proarrhythmic effects in patients thus limiting its continual use. Ibrutinib treated patients have been shown to be at a 4-fold increased risk of developing atrial fibrillation (AF), but the underlying molecular mechanisms remain unclear. Studies have shown that Pitx2 , the nearest gene to the 4q25 locus has been implicated in AF pathology and thus can serve as an experimental model for clinical AF. Objective: The goal of this study is to assess the role of the late cardiac Na current (I Na,L ), PI3K activation, and oxidative stress in mediating the increased susceptibility to AF in Pitx2c +/- mice and atrial human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to ibrutinib. Method: Pitx2c +/- mice were administered ibrutinib (30mg/kg/day IP) for 23 days after which their AF burden were assessed using transesophageal rapid pacing (TErP) along with their weight, BP, and plasma glucose. Atrial hiPSC-CMs were exposed to ibrutinib for 48 hrs. ELISA, IHC, Western blotting, cellular patch clamping and, qPCR studies were performed. Results: Pitx2c +/- mice were exposed to ibrutinib showed a graded increase in AF burden. ( Fig. 1A-C ). There was a decreased activation of the PI3K pathway and pAkt leading to overall decrease in SERCA2a expression ( Fig. 1D-F ). There was a significant increase in mitochondrial fragmentation and superoxide production in ibrutinib treated atrial hiPSC-CMs ( Fig G ) and the action potential duration at 90% repolarization and the I Na-L were markedly prolonged after ibrutinib exposure( Fig H ). Conclusion: We showed in Pitx2c +/- mice and atrial hiPSC-CMs that ibrutinib-mediated AF may in part be related to enhanced I Na-L , decreased activation of PI3K and SERCA2a and increased fibrosis leading to mitochondrial fragmentation and increased oxidative stress.

The Current Practices and Gaps in Forest Accounting System of Ethiopia: A Review

The SNA provides a framework and a comprehensive set of concepts, definitions and classifications for economic accounting that allows compiling, and presenting economic data in a format designed for purposes of economic analysis, decision-making and policymaking. However, the framework is inadequate to be better indicator economic performance, since it misses most of non-marketed economic activities because of different reasons. In this paper, we assessed the gap in the current practices of SNA in general and forest resource accounting system in particular using different national data sources to review the current practice of accounting in Ethiopian Economy. We also examined structure and contents of economic values of forest products and services. In the current Ethiopian system of national accounts, there was attempt to incorporate more forestry economic activities as much as the methodological approaches permitted. Different data on gross value of outputs and cost of material inputs collected from all available sources to calculate the contribution of different economic sectors to Ethiopian GDP. However, there are limitations in full coverage of forest goods and services and up-to-date productivity indicators - calling for a robust effort by all stakeholders. SNA’s room for flexibility that allows application of satellite accounting is worthy of value addition for improving the existing SNA gaps in methodologies, data availability and economic activity coverage. Measuring forest values that is more comprehensive than the current SNA greatly contributes towards production of integrated statistical indicators for development plan & policy formulation in social, economic & environmental spheres. Keywords : SNA, GDP, Forest Goods and Services, ISIC, Gaps DOI: 10.7176/RJFA/11-19-06 Publication date: October 31 st 2020

Purification, Toxicity and Functional Characterization of a New Proteinaceous Mussel Biotoxin from Bizerte Lagoon.

The marine environment is known to be occupied by microorganisms. The potential toxicity of some of these marine microorganisms, that are capable of producing unknown biotoxins, has always been underestimated. Indeed, these biotoxins may be a threat to human health through the consumption of contaminated seafood and fish. For more than ten years, recurrent but atypical toxicity has been detected in mussels from Bizerte lagoon (North of Tunisia) during routine tests. In this study, we have isolated and characterized a new proteinaceous marine biotoxin, named Mussel Toxic Peptide (MTP). Using HPLC, electrophoresis and LC/MS studies, we showed that MTP has a protein characteristic UV-spectrum, can be visualized by protein specific reagents such as Coomassie, and has a molecular mass of 6.4 kDa. Patch-clamp experiments performed on cultured N18 neuroblastoma cells revealed that MTP (0.9–18 µM) markedly inhibited voltage-gated Na current, but was about 23 times less active in blocking voltage-gated K current at equimolar concentrations. To the best of our knowledge, this is the first time that a proteinaceous marine biotoxin with relatively high molecular mass is isolated and involved in the contamination of mussels harvested from shellfish farming areas.

Social network analysis in operations and supply chain management: a review and revised research agenda

PurposeSocial network analysis (SNA) seeks to manage the connections between entities through investigating and understanding behaviours and relationships. This study demonstrates the increasing relevance of social network approaches to solving contemporary and looming operations management (OM) and supply chain management (SCM) problems; including the coordination operations challenges raised by increased connectivity.Design/methodology/approachThe systematic literature review approach adopted here examines 63 papers in OM and SCM published between 2000 and 2019. To-date OM reviews on SNA have focussed on discussing archetypal supply chains, what differentiates this study is the focus on how value was created in other forms of chains and operations.FindingsThis study reveals that current SNA adoption in OM is dominated by a manufacturing style focus on linear, sequential value creation, with a resulting focus only on sequential interdependence. SNA studies on reciprocally co-ordinated value creation (e.g. many service and network operations) are shown to have been neglected and are linked to a new agenda on contemporary management issues.Research limitations/implicationsBeyond encouraging the use of SNA, this study seeks to re-orient SNA approaches towards how contemporary services and networks create value.Originality/valueThrough adopting a unique combination of approaches and frameworks, this study challenges extant work to offer a substantially revised agenda for SNA use in Operations and Supply Chain Management.

Xiaozhang Tie Improves Intestinal Motility in Rats With Cirrhotic Ascites by Regulating the Stem Cell Factor/c-kit Pathway in Interstitial Cells of Cajal.

We previously discovered that Xiaozhang Tie (XZT) was helpful for cirrhotic ascites, with obvious abdominal distention relief, suggesting that it may improve gastrointestinal (GI) motility. However, the underlying mechanisms of GI motility in cirrhotic ascites are unclear. Here, we aimed to discover explored the effect of XZT on GI motility in animal cirrhotic ascites and probed the action mechanism affecting GI motility by regulating the stem cell factor (SCF)/c-kit pathway in interstitial cells of Cajal (ICCs) and GI hormones. First, rat models of cirrhotic ascites were developed and then divided randomly into the following three subgroups: model control, XZT group, and mosapride group. The efficacy of XZT on treating cirrhotic ascites was evaluated on the basis of ascites weight and volume, 24 h urine volume, and feces water content. GI motility of the cirrhotic model, intestine propulsion, and gastric residue were detected using the migration distance of ink in vivo, and the frequency of contraction and tension of isolated gastric and jejunal muscle strips were measured after incubation with XZT extracts. Serum GI hormone content, including motilin (MTL), substance P (SP), somatostatin (SS), and vasoactive intestinal polypeptide were assayed. Subsequently, ICCs were isolated from jejunum, and primarily cultured ICCs were incubated with and without XZT and SCF. The cell vitality of the ICCs was measured. A whole-cell patch recording technique was used to record the current of K+ and Na+ channels in the ICC membrane. Expressions of c-kit/p-c-kit, p-Akt, p-STAT3, and p-Erk1/2 were detected in vivo and in vitro. The results revealed that XZT significantly reduced ascites weight and increased urine volume and fecal water content in model rats. XZT promoted intestinal motility and increased MTL level but reduced SP and SS levels. It enhanced the current of Na+ and K+ in ICCs and improved c-kit expression and signaling mediator phosphorylation in SCF/c-kit, which was inhibited by imatinib in vitro and downregulated in model rats in vivo. Our study concluded that XZT reduced the amount of ascites and improved intestinal motility in cirrhotic rats, which may be associated with its effect on ascites and was involved in the mechanisms regulating the SCF/c-kit signaling pathway in ICCs and improving gastrointestinal hormone secretion.

Enhanced CaMKII-Dependent Late I Na Induces Atrial Proarrhythmic Activity in Patients With Sleep-Disordered Breathing

Sleep-disordered breathing (SDB) is frequently associated with atrial arrhythmias. Increased CaMKII (Ca/calmodulin-dependent protein kinase II) activity has been previously implicated in atrial arrhythmogenesis. We hypothesized that CaMKII-dependent dysregulation of Na current (INa) may contribute to atrial proarrhythmic activity in patients with SDB. We prospectively enrolled 113 patients undergoing elective coronary artery bypass grafting for cross-sectional study and collected right atrial appendage biopsies. The presence of SDB (defined as apnea-hypopnea index ≥15/h) was assessed with a portable SDB monitor the night before surgery. Compared with 56 patients without SDB, patients with SDB (57) showed a significantly increased level of activated CaMKII. Patch clamp was used to measure INa. There was a significantly enhanced late INa, but reduced peak INa due to enhanced steady-state inactivation in atrial myocytes of patients with SDB consistent with significantly increased CaMKII-dependent cardiac Na channel phosphorylation (NaV1.5, at serine 571, Western blotting). These gating changes could be fully reversed by acute CaMKII inhibition (AIP [autocamtide-2 related inhibitory peptide]). As a consequence, we observed significantly more cellular afterdepolarizations and more severe premature atrial contractions in atrial trabeculae of patients with SDB, which could be blocked by either AIP or KN93 (N-[2-[[[(E)-3-(4-chlorophenyl)prop-2-enyl]-methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide). In multivariable linear regression models incorporating age, sex, body mass index, existing atrial fibrillation, existing heart failure, diabetes mellitus, and creatinine levels, apnea-hypopnea index was independently associated with increased CaMKII activity, enhanced late INa and correlated with premature atrial contraction severity. In atrial myocardium of patients with SDB, increased CaMKII-dependent phosphorylation of NaV1.5 results in dysregulation of INa with proarrhythmic activity that was independent from preexisting comorbidities. Inhibition of CaMKII may be useful for prevention or treatment of arrhythmias in SDB. URL: http://www.clinicaltrials.gov. Unique identifier: NCT02877745. Visual Overview: An online visual overview is available for this article.

The 4Hz–Pulsing Magnetic Field (PMF) has Common therapeutic effects on Patients Subjected to Different Pain Stress

Previously, we have suggested a hypothesis that the metabolic net water efflux from the cells is a key mechanism for metabolic controlling semipermeable properties of the cell membrane, dysfunction of which is a common consequence of cell pathology. The existence of the electrogenic Na/K-pump in the cell membrane, beating high ATP utilizing the machine in cell, which by activation of mitochondrial function stimulate endogen water formation making it as a key mechanism for the generation of metabolic water efflux from the cells and its dysfunction as a primary step of cell pathology. It is known that the intracellular concentration Ca2+([Ca]i) which is a strong inhibitor for Na/K-pump, controlling by intracellular cyclic nucleotides dependent systems that are extra sensitive to weak chemical and physical signals, unable to activate ionic channels in the membrane. In our earlier studies, we have shown that the activation of both cGMP-activated Ca-pump in the cell membrane and Na/Ca exchange in forwarding (F) mode and cAMP-activated Na/Ca exchange in reverse (R) mode by generating water efflux from the cells, having key roles in controlling low permeability membrane for inward Na+ current (INa) controlling semipermeable properties of membrane and gradient these ions on the membrane, i.e they have a protective function of cell preceding to inward water-induced decrease Na gradient on the cell membrane. It was shown that the soluble guanylate cyclase (sGC) which is sensitive even to water structure changes of cell bathing medium, serves as a sensor for activation cGMP-dependent FNa/Ca exchange while G proteins in the membrane are sensors for activation cAMP-dependent RNa/Ca exchange. The cGMP induced activation of Ca-pump and FNa/Ca exchange by the decrease of [Ca]i–induced activation of Na/K pump activate water efflux from the cell and controlling the Na gradient on the membrane. While the same function doing the cAMP-dependent RNa/Ca exchange because of its electrogenicity and [Ca]i–induced activation of mitochondrial formation water molecules. By our previous study have shown that the activation of cGMP-dependent FNa/Ca exchange has pain relief while the activation of cAMP-dependent RNa/Ca exchange pain generation effects. It was also shown also that 4Hz pulsing magnetic fields, which activation of cGMP- dependent FNa/Ca exchange have relaxation effects on heart muscles and depressed acetylcholine (Ach) sensitivity cell membrane sensitivity, pain relief and antitumor effects. The accumulation of our and literature data on pain and stress relief, antitumor and antivirus effects of 4Hz PMF allow us to hypothesizes that 4Hz–induced activation of cGMP-dependent F Na/Ca exchange can serve as a universal therapeutic tool to increase the resistance of the cells to pathological factors directed to reverse water efflux into water influx in the cells and increases inward Na current which is the primary step of cell pathology. To check this hypothesis, in this present work the 4Hz effect on voluntary patients who have been subjected by different stress on biochemical characteristics of blood we have studied which serve as a marker for pain–the stress of organism.

Expression and Ion Transport Activity of Rice OsHKT1;1 Variants.

OsHKT1;1 in rice, belongs to the high-affinity K+ Transporter family, has been found to be involved in salt tolerance. OsHKT1;1 in japonica rice (Nipponbare) produces mRNA variants, but their functions remain elusive. In salt tolerant rice, Pokkali, eight OsHKT1;1 variants (V1-V8) were identified in addition to the full-length OsHKT1;1 (FL) cDNA. Absolute quantification by qPCR revealed that accumulation of OsHKT1;1-FL mRNA is minor in contrast to that of OsHKT1;1-V1, -V2, -V4, and -V7 mRNAs, all of which are predominant in shoots, while only V1 and V7 mRNAs are predominant in roots. Two electrode voltage clamp (TEVC) experiments using Xenopus laevis oocytes revealed that oocytes-expressing OsHKT1;1-FL from Pokkali exhibited inward-rectified currents in the presence of 96 mM Na+ as reported previously. Further TEVC analyses indicated that six of eight OsHKT1;1 variants elicited currents in a Na+ or a K+ bath solution. OsHKT1;1-V6 exhibited a similar inward rectification to the FL protein. Contrastingly, however, the rests mediated bidirectional currents in both Na+ and K+ bath solutions. These data suggest possibilities that novel mechanisms regulating the transport activity of OsHKT1;1 might exist, and that OsHKT1;1 variants might also carry out distinct physiological roles either independently or in combination with OsHKT1;1-FL.

The General Anesthetic Isoflurane Bilaterally Modulates Neuronal Excitability

SummaryVolatile anesthetics induce hyperactivity during induction while producing anesthesia at higher concentrations. They also bidirectionally modulate many neuronal functions. However, the neuronal mechanism is unclear. The effects of isoflurane on sodium channel currents were analyzed in acute mouse brain slices, including sodium leak (NALCN) currents and voltage-gated sodium channels (Nav) currents. Isoflurane at sub-anesthetic concentrations increased the spontaneous firing rate of CA3 pyramidal neurons, whereas anesthetic concentrations of isoflurane decreased the firing rate. Isoflurane at sub-anesthetic concentrations enhanced NALCN conductance but minimally inhibited Nav currents. Isoflurane at anesthetic concentrations depressed Nav currents and action potential amplitudes. Isoflurane at sub-anesthetic concentrations depolarized resting membrane potential (RMP) of neurons, whereas hyperpolarized the RMP at anesthetic concentrations. Isoflurane at low concentrations induced hyperactivity in vivo, which was diminished in NALCN knockdown mice. In conclusion, enhancement of NALCN by isoflurane contributes to its bidirectional modulation of neuronal excitability and the hyperactivity during induction.

P1596Interaction of CaMKII and NaV1.8 modulates cardiac electrophysiology in human heart failure

Abstract Introduction In human heart failure, electrical remodeling contributes to the risk of arrhythmia generation. Increased expression of Ca/Calmodulin-dependent protein kinase IIδ (CaMKIIδ) and an enhanced persistent Na current (INaL) have been linked to arrhythmogenesis. CaMKIIδ increases INaL via regulation of sodium channels thereby contributing to arrhythmias through early- and delayed-afterdepolarizations (EADs and DADs). Genome-wide association studies (GWAS) have described the implication of the neuronal sodium channel isoform NaV1.8 (SCN10A) in cardiac electrophysiology showing modulation in cardiac conduction. We showed that the expression of the isoform Nav1.8 is significantly increased in human failing cardiomyocytes and contributes substantially to the enhanced INaL. Purpose We investigated a potential interaction of CaMKIIδ and NaV1.8 and thereby its role in arrhythmia generation and electrophysiology in human and murine failing hearts. Methods Cardiomyocytes were isolated from explanted failing hearts and CaMKIIδ transgenic (TG) mice. We performed immunostainings and co-immunoprecipitation (Co-IP) to show interactions of CaMKIIδ and Nav1.8 in isolated cardiomyocytes and homogenates. Whole-cell patch clamp experiments were conducted in isolated human and murine ventricular cardiomyocytes. Additionally, Ca2+ transients were measured using epifluorescence microscopy with the Ca2+ dye fura-2 (10μmol/L) whereas Ca2+ sparks measurements were performed by using confocal microscopy with the Ca2+ dye fluo-4 (10μmol/L). PF-01247324 is a novel specific NaV1.8 inhibitor (orally bioavailable; 1 μmol/L) and autocamtide inhibitory peptide (AIP, 1 μmol/L) was used to inhibit CaMKIIδ. Results Co-immunoprecipitation experiments revealed an association of CaMKIIδ and Nav1.8 in human homogenates compared to healthy controls. Furthermore, immunohistochemistry stainings in isolated human cardiomyocytes showed a co-localization of CaMKIIδ and NaV1.8 at the intercalated disc and t-tubules. We observed a significant reduction of INaL integral and proarrhythmic SR-Ca2+ spark frequency (CaSpF) after addition of either PF-01247324 or the CaMKIIδ inhibitor AIP in failing human and murine ventricular cardiomyocytes. When PF-01247324 and AIP were added together, the decrease in INaL integral and CaSpF was comparable to PF-01247324 alone in human failing cardiomyocytes. Inhibition of NaV1.8 did not show an effect on Ca2+ transient amplitude or Ca2+ transient decay at different stimulation frequencies in CaMKIIδ TG cardiomyocytes. Conclusion Our results demonstrate the significance of both CaMKIIδ and NaV1.8 in INaL generation and their detrimental interaction. This data suggest that increased CaMKIIδ activity plays a substantial role for the activation of NaV1.8-mediated late sodium current and SR-Ca2+ leak.

Effects of FGF14 and NaVβ4 deletion on transient and resurgent Na current in cerebellar Purkinje neurons.

Voltage-gated Na channels of Purkinje cells are specialized to maintain high availability during high-frequency repetitive firing. They enter fast-inactivated states relatively slowly and undergo a voltage-dependent open-channel block by an intracellular protein (or proteins) that prevents stable fast inactivation and generates resurgent Na current. These properties depend on the pore-forming α subunits, as well as modulatory subunits within the Na channel complex. The identity of the factors responsible for open-channel block remains a question. Here we investigate the effects of genetic mutation of two Na channel auxiliary subunits highly expressed in Purkinje cells, NaVβ4 and FGF14, on modulating Na channel blocked as well as inactivated states. We find that although both NaVβ4 and the FGF14 splice variant FGF14-1a contain sequences that can generate resurgent-like currents when applied to Na channels in peptide form, deletion of either protein, or both proteins simultaneously, does not eliminate resurgent current in acutely dissociated Purkinje cell bodies. Loss of FGF14 expression does, however, reduce resurgent current amplitude and leads to an acceleration and stabilization of inactivation that is not reversed by application of the site-3 toxin, anemone toxin II (ATX). Tetrodotoxin (TTX) sensitivity is higher for resurgent than transient components of Na current, and loss of FGF14 preferentially affects a highly TTX-sensitive subset of Purkinje α subunits. The data suggest that NaV1.6 channels, which are known to generate the majority of Purkinje cell resurgent current, bind TTX with high affinity and are modulated by FGF14 to facilitate open-channel block.

Effects of ketamine on voltage-gated sodium channels in the barrel cortex and the ventral posteromedial nucleus slices of rats.

Ketamine is commonly used as a dissociative anesthetic with unique actions in the central nervous system. Previous studies have found that the thalamocortical systems play an important role in general anesthetics induced unconsciousness. Whether the voltage-gated sodium channels in the thalamocortical systems are the target of ketamine remain unclear. The present study used a whole-cell patch-clamp technique to observe the effects of ketamine on voltage-gated Na channels in thalamocortical pyramidal neurons. We found that IC50 of ketamine on Na currents in the primary somatosensory barrel cortex pyramidal neurons and the thalamus ventral posteromedial nucleus pyramidal neurons was 686.72 ± 39.92 and 842.65 ± 87.28 μM, respectively. Ketamine accelerated the Na channels inactivation and slowed inactivation of Na channels after recovery but did not affect the activation. We demonstrated the detailed suppression process of neural voltage-gated Na channels by ketamine on thalamocortical slice. This may provide a new insight into the mechanical explanation for the ketamine anesthesia.

Nuclear Factor-kappaB Gates Nav1.7 Channels in DRG Neurons via Protein-Protein Interaction.

SummaryIt is well known that nuclear factor-kappaB (NF-κB) regulates neuronal structures and functions by nuclear transcription. Here, we showed that phospho-p65 (p-p65), an active form of NF-κB subunit, reversibly interacted with Nav1.7 channels in the membrane of dorsal root ganglion (DRG) neurons of rats. The interaction increased Nav1.7 currents by slowing inactivation of Nav1.7 channels and facilitating their recovery from inactivation, which may increase the resting state of the channels ready for activation. In cultured DRG neurons TNF-α upregulated the membrane p-p65 and enhanced Nav1.7 currents within 5 min but did not affect nuclear NF-κB within 40 min. This non-transcriptional effect on Nav1.7 may underlie a rapid regulation of the sensibility of the somatosensory system. Both NF-κB and Nav1.7 channels are critically implicated in many physiological functions and diseases. Our finding may shed new light on the investigation into the underlying mechanisms.

Abstract 102: Molecular, Cellular and Systemic Mechanism of Nonlinear Dynamic Patterns of Ventricular Repolarization and Spontaneous Arrhythmic Sudden Death in Non-ischemic Heart Failure

Introduction: Sudden cardiac death (SCD) is the leading cause of death in USA. Heart failure (HF) confers high SCD risk, but most SCD victims do not have HF or well-defined genetic abnormalities. The underlying mechanisms are poorly understood, precluding the design of more effective strategies for risk stratification and therapy. We previously showed that distinct non-linear patterns of cardiac repolarization strongly and independently predict SCD in HF patients [PMID: 27044982]. We also showed in a unique guinea pig HF model that increased levels of mitochondrial reactive oxygen species (mROS) drive spontaneous SCD, even before HF onset [PMID: 29898892]. Herein, we further dissect the underlying mechanisms. Hypothesis: Increased mROS levels in pressure overloaded hearts reduce K+ currents, destabilize the resting membrane potential, increase repolarization lability and cause SCD. Methods: Guinea pigs were randomized to Sham, aortic banding (AC), or AC with daily brief low-dose β-adrenergic stress (ACi) with and without in vivo mROS scavenger MitoTEMPO (ACi+MT). We performed time series analyses on recordings of: (1) 24-hour ECG QT interval in freely ambulating animals before sacrifice at 4 weeks; (2) pressure-volume and electrophysiology (EP) in excised perfused hearts; and (3) duration of action potential (APd), calcium transient (CaTd) and sarcomere shortening (SSd) in isolated LV myocytes. Results: About 50% of ACi animals had SCD by 4 weeks. Increased QT entropy in ACi (at 1 week) predicted SCD over followup, similar to HF patients. Compared to Sham and ACi+MT, LV myocytes isolated from AC and ACi had reduced inward (IK1) and delayed slow (IKs) and rapid (IKr) rectifier currents; and increased beat-to-beat lability in APd, CaTd and SSd. The late Na and L-type Ca currents were similar between models. Acute exposure to isoproterenol (Iso) and carbachol (CCh) increased lability in LV myocytes compared to Iso or CCh alone. Conclusions: High mROS levels reduce repolarization reserve in LV myocytes and contribute, at least in part, to the non-linear dynamics of ventricular repolarization (reflected by high QT entropy), leading to spontaneous arrhythmic SCD. These findings provide important new mechanistic insight with direct clinical implications.

Abstract 811: The Role of Mitochondrial Stress and the Late Sodium Current in Ibrutinib-Mediated Atrial Fibrillation

Background: Ibrutinib (a Bruton’s tyrosine kinase inhibitor, BTKI) is rapidly becoming the treatment of choice for many B-cell malignancies, but its proarrhythmic effects may limit its widespread use. GWAS have consistently identified an AF risk locus on chromosome (chr) 4q25, and the nearest gene Pitx2c has been implicated in AF development as evidenced by increased AF in Pitx2c +/- mice which serve as an experimental model for clinical AF. Objective: To assess the role of the late cardiac Na current (I Na,L ) and oxidative stress in mediating increased susceptibility to AF in Pitx2c +/- mice which are genetically prone to developing AF as well as in atrial human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to ibrutinib. Method: Pitx2c +/- mice were administered ibrutinib (30mg/kg/day IP) for 14 days while atrial hiPSC-CMs were exposed to ibrutinib for 48 hrs. Results: There was a graded increase in AF episodes and burden with the number of days the Pitx2c +/- mice were exposed to ibrutinib versus controls ( Fig. 1A ). The action potential duration at 90% repolarization and the I Na-L were markedly prolonged ( Fig. 1B-C ). Furthermore, we observed a significant increase in mitochondrial fragmentation ( Fig. 1D-E ) and superoxide production ( Fig. 1F-G ) in ibrutinib treated atrial hiPSC-CMs versus controls. Conclusion: We showed in Pitx2c +/- mice and atrial hiPSC-CMs that ibrutinib-mediated AF may in part be related to enhanced I Na-L and mitochondrial dysfunction leading to increased oxidative stress. Patients undergoing ibrutinib treatment may benefit from novel therapies which target mitochondrial dysfunction or the late cardiac sodium current.

High-Throughput Ca2+ Flux Assay To Monitor Cyclic Nucleotide-Gated Channel Activity and Characterize Achromatopsia Mutant Channel Function.

Cone photoreceptor cyclic-nucleotide gated channels (CNG) are tetrameric proteins composed of subunits from CNGA3 and CNGB3. These channels transduce light information into electrical signals carried by both Na+ and Ca2+ ions. More than 100 mutations in the CNGA3 gene are associated with the inherited retinal disorder, achromatopsia 2 (ACHM2), which results in attenuation or loss of color vision, daylight blindness, and reduced visual acuity. Classical techniques to measure CNG channel function utilize patch clamp electrophysiology measuring Na currents in the absence of divalent cations, yet intracellular Ca2+ regulates both light and dark adaptation in photoreceptors. We developed a fluorescence-based, high-throughput Ca2+ flux assay using yellow fluorescent protein (YFP) tagged CNGA3 channels expressed in HEK293 cells which allow monitoring for folding defects in mutant channels. The cell permeant cGMP analog, 8-(4-chlorophenylthio)-cGMP (CPT-cGMP), was used to activate Ca2+ flux. The assay was validated using wild-type CNGA3 homomeric and heteromeric channels and ACHM2-associated homomeric mutant CNG channels, CNGA3-R427C, CNGA3-E590K, and CNGA3-L633P. Additionally, we examined two naturally occurring canine mutations causing day-blindness previously studied by patch clamp. We compared the CPT-cGMP K0.5 values of the channels with patch clamp values from previous studies. The assay provides a screen for modulation of gating and/or rescue of trafficking and/or misfolding defects in ACHM2-associated CNG channels. Importantly, the calcium flux assay is advantageous compared to patch clamp as it allows the ability to monitor CNG channel activity in the presence of calcium.