Empagliflozin treatment rescues abnormally reduced Na+ currents in ventricular cardiomyocytes from dystrophin-deficient mdx mice.

Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a severe muscle illness caused by mutations in the gene encoding for the intracellular protein dystrophin. A major source for arrhythmia vulnerability in patients with DMD is impaired ventricular impulse conduction, which predisposes for ventricular asynchrony, decreased cardiac output, and the development of reentrant circuits. Using the dystrophin-deficient mdx mouse model for human DMD, we previously reported that the lack of dystrophin causes a significant loss of peak Na+ current (INa) in ventricular cardiomyocytes. This finding provided a mechanistic explanation for ventricular conduction defects and concomitant arrhythmias in the dystrophic heart. In the present study, we explored the hypothesis that empagliflozin (EMPA), an inhibitor of sodium/glucose cotransporter 2 in clinical use to treat type II diabetes and nondiabetic heart failure, rescues peak INa loss in dystrophin-deficient ventricular cardiomyocytes. We found that INa of cardiomyocytes derived from mdx mice, which had received clinically relevant doses of EMPA for 4 wk, was restored to wild-type level. Moreover, incubation of isolated mdx ventricular cardiomyocytes with 1 µM EMPA for 24 h significantly increased their peak INa. This effect was independent of Na+-H+ exchanger 1 inhibition by the drug. Our findings imply that EMPA treatment can rescue abnormally reduced peak INa of dystrophin-deficient ventricular cardiomyocytes. Long-term EMPA administration may diminish arrhythmia vulnerability in patients with DMD.NEW & NOTEWORTHY Dystrophin deficiency in cardiomyocytes leads to abnormally reduced Na+ currents. These can be rescued by long-term empagliflozin treatment.

Lesional Antibody Synthesis and Complement Deposition Associate With De Novo Antineuronal Antibody Synthesis After Spinal Cord Injury.

BACKGROUND AND OBJECTIVES: Spinal cord injury (SCI) disrupts the fine-balanced interaction between the CNS and immune system and can cause maladaptive aberrant immune responses. The study examines emerging autoantibody synthesis after SCI with binding to conformational spinal cord epitopes and surface peptides located on the intact neuronal membrane. METHODS: This is a prospective longitudinal cohort study conducted in acute care and inpatient rehabilitation centers in conjunction with a neuropathologic case-control study in archival tissue samples ranging from acute injury (baseline) to several months thereafter (follow-up). In the cohort study, serum autoantibody binding was examined in a blinded manner using tissue-based assays (TBAs) and dorsal root ganglia (DRG) neuronal cultures. Groups with traumatic motor complete SCI vs motor incomplete SCI vs isolated vertebral fracture without SCI (controls) were compared. In the neuropathologic study, B cell infiltration and antibody synthesis at the spinal lesion site were examined by comparing SCI with neuropathologically unaltered cord tissue. In addition, the CSF in an individual patient was explored. RESULTS: Emerging autoantibody binding in both TBA and DRG assessments was restricted to an SCI patient subpopulation only (16%, 9/55 sera) while being absent in vertebral fracture controls (0%, 0/19 sera). Autoantibody binding to the spinal cord characteristically detected the substantia gelatinosa, a less-myelinated region of high synaptic density involved in sensory-motor integration and pain processing. Autoantibody binding was most frequent after motor complete SCI (grade American Spinal Injury Association impairment scale A/B, 22%, 8/37 sera) and was associated with neuropathic pain medication. In conjunction, the neuropathologic study demonstrated lesional spinal infiltration of B cells (CD20, CD79a) in 27% (6/22) of patients with SCI, the presence of plasma cells (CD138) in 9% (2/22). IgG and IgM antibody syntheses colocalized to areas of activated complement (C9neo) deposition. Longitudinal CSF analysis of an additional single patient demonstrated de novo (IgM) intrathecal antibody synthesis emerging with late reopening of the blood-spinal cord barrier. DISCUSSION: This study provides immunologic, neurobiological, and neuropathologic proof-of-principle for an antibody-mediated autoimmunity response emerging approximately 3 weeks after SCI in a patient subpopulation with a high demand of neuropathic pain medication. Emerging autoimmunity directed against specific spinal cord and neuronal epitopes suggests the existence of paratraumatic CNS autoimmune syndromes.

Ivabradine acutely improves cardiac Ca handling and function in a rat model of Duchenne muscular dystrophy.

The muscular dystrophies caused by dystrophin deficiency, the so-called dystrophinopathies, are associated with impaired cardiac contractility and arrhythmias, which considerably contribute to disease morbidity and mortality. Impaired Ca handling in ventricular cardiomyocytes has been identified as a causative factor for complications in the dystrophic heart, and restoration of normal Ca handling in myocytes has emerged as a promising new therapeutic strategy. In the present study, we explored the hypothesis that ivabradine, a drug clinically approved for the treatment of heart failure and stable angina pectoris, improves Ca handling in dystrophic cardiomyocytes and thereby enhances contractile performance in the dystrophic heart. Therefore, ventricular cardiomyocytes were isolated from the hearts of adult dystrophin-deficient DMDmdx rats, and the effects of acutely applied ivabradine on intracellular Ca transients were tested. In addition, the drug's acute impact on cardiac function in DMDmdx rats was assessed by transthoracic echocardiography. We found that administration of ivabradine to DMDmdx rats significantly improved cardiac function. Moreover, the amplitude of electrically induced intracellular Ca transients in ventricular cardiomyocytes isolated from DMDmdx rats was increased by the drug. We conclude that ivabradine enhances Ca release from the sarcoplasmic reticulum in dystrophic cardiomyocytes and thereby improves contractile performance in the dystrophic heart.

Glycerol-3-phosphate shuttle is a backup system securing metabolic flexibility in neurons.

Electrical activity in neurons is highly energy demanding and accompanied by rises in cytosolic Ca2+ Cytosolic Ca2+, in turn, secures energy supply by pushing mitochondrial metabolism either through augmented NADH transfer into mitochondria via the malate aspartate shuttle (MAS) or via direct activation of dehydrogenases of the TCA cycle after passing into the matrix through the mitochondrial Ca2+ uniporter (MCU). Another Ca2+-sensitive booster of mitochondrial ATP synthesis is the glycerol-3-phosphate shuttle (G3PS) whose role in neuronal energy supply has remained elusive. Essential components of G3PS are expressed in hippocampal neurons. Single neuron metabolic measurements in primary hippocampal cultures derived from rat pups of either sex reveal only moderate, if any, constitutive activity of G3PS. However, during electrical activity neurons fully rely on G3PS when MAS and MCU are unavailable. Under these conditions, G3PS is required for appropriate action potential firing. Accordingly, G3PS safeguards metabolic flexibility of neurons to cope with energy demands of electrical signaling.SIGNIFICANCE STATEMENT:Ca2+ ions are known to provide a link between the energy-demanding electrical activity and an adequate ATP supply in neurons. To do so, Ca2+ acts both, from outside and inside of the mitochondrial inner membrane. Neuronal function critically depend on this regulation and its defects are often found in various neurological disorders. Although interest in neuronal metabolism increases, many aspects thereof have remained unresolved. In particular, a Ca2+-sensitive NADH shuttling system, the glycerol-3-phosphate shuttle, has been largely ignored with respect to its function in neurons. Our results demonstrate that this shuttle is functional in hippocampal neurons and safeguards ATP supply and appropriate action potential firing when malate aspartate shuttle and mitochondrial Ca2+ uniporter are unavailable, thereby ensuring neuronal metabolic flexibility.

Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice.

T-type Ca channels are strongly expressed and important in the developing heart. In the adult heart, these channels play a significant role in pacemaker tissues, but there is uncertainty about their presence and physiological relevance in the working myocardium. Here, we show that the T-type Ca channel isoforms Cav3.1 and Cav3.2 are expressed at a protein level in ventricular cardiomyocytes from healthy adult C57/BL6 mice. Myocytes isolated from adult wild-type and Cav3.2 KO mice showed considerable whole cell T-type Ca currents under beta-adrenergic stimulation with isoprenaline. We further show that the detectability of basal T-type Ca currents in murine wild-type cardiomyocytes depends on the applied experimental conditions. Together, these findings reveal the presence of functional T-type Ca channels in the membrane of ventricular myocytes. In addition, electrically evoked Ca release from the sarcoplasmic reticulum was significantly impaired in Cav3.2 KO compared to wild-type cardiomyocytes. Our work implies a physiological role of T-type Ca channels in the healthy adult murine ventricular working myocardium.

The Bradycardic Agent Ivabradine Acts as an Atypical Inhibitor of Voltage-Gated Sodium Channels.

Background and purpose: Ivabradine is clinically administered to lower the heart rate, proposedly by inhibiting hyperpolarization-activated cyclic nucleotide-gated cation channels in the sinoatrial node. Recent evidence suggests that voltage-gated sodium channels (VGSC) are inhibited within the same concentration range. VGSCs are expressed within the sinoatrial node and throughout the conduction system of the heart. A block of these channels thus likely contributes to the established and newly raised clinical indications of ivabradine. We, therefore, investigated the pharmacological action of ivabradine on VGSCs in sufficient detail in order to gain a better understanding of the pro- and anti-arrhythmic effects associated with the administration of this drug. Experimental Approach: Ivabradine was tested on VGSCs in native cardiomyocytes isolated from mouse ventricles and the His-Purkinje system and on human Nav1.5 in a heterologous expression system. We investigated the mechanism of channel inhibition by determining its voltage-, frequency-, state-, and temperature-dependence, complemented by a molecular drug docking to the recent Nav1.5 cryoEM structure. Automated patch-clamp experiments were used to investigate ivabradine-mediated changes in Nav1.5 inactivation parameters and inhibition of different VGSC isoforms. Key results: Ivabradine inhibited VGSCs in a voltage- and frequency-dependent manner, but did not alter voltage-dependence of activation and fast inactivation, nor recovery from fast inactivation. Cardiac (Nav1.5), neuronal (Nav1.2), and skeletal muscle (Nav1.4) VGSC isoforms were inhibited by ivabradine within the same concentration range, as were sodium currents in native cardiomyocytes isolated from the ventricles and the His-Purkinje system. Molecular drug docking suggested an interaction of ivabradine with the classical local anesthetic binding site. Conclusion and Implications: Ivabradine acts as an atypical inhibitor of VGSCs. Inhibition of VGSCs likely contributes to the heart rate lowering effect of ivabradine, in particular at higher stimulation frequencies and depolarized membrane potentials, and to the observed slowing of intra-cardiac conduction. Inhibition of VGSCs in native cardiomyocytes and across channel isoforms may provide a potential basis for the anti-arrhythmic potential as observed upon administration of ivabradine.

Psilocybin Therapy of Psychiatric Disorders Is Not Hampered by hERG Potassium Channel-Mediated Cardiotoxicity.

Psilocybin, a hallucinogen contained in "magic" mushrooms, holds great promise for the treatment of various psychiatric disorders, and early clinical trials are encouraging. Adverse cardiac events after intake of high doses of psilocybin and a trial reporting QT interval prolongation in the electrocardiogram attributed to the drug's main metabolite, psilocin, gave rise to safety concerns. Here we show that clinical concentrations of psilocin do not cause significant human ether-a-go-go-related gene (hERG) potassium channel inhibition, a major risk factor for adverse cardiac events. We conclude that hERG channel blockage by psilocin is not liable for psilocybin- associated cardiotoxic effects.

Autoimmune Global Amnesia as Manifestation of AMPAR Encephalitis and Neuropathologic Findings.

OBJECTIVE: To report an unusual clinical phenotype of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) encephalitis and describe associated neuropathologic findings. METHODS: We retrospectively investigated 3 AMPAR encephalitis patients with autoimmune global hippocampal amnesia using comprehensive cognitive and neuropsychologic assessment, antibody testing by in-house tissue-based and cell-based assays, and neuropathologic analysis of brain autopsy tissue including histology and immunohistochemistry. RESULTS: Three patients presented with acute-to-subacute global amnesia without affection of cognitive performance, attention, concentration, or verbal function. None of the patients had epileptic seizures, change of behavior, personality changes, or psychiatric symptoms. The MRI was normal in 1 patient and showed increased fluid-attenuated inversion recovery/T2 signal in the hippocampus in the other 2 patients. Two patients showed complete remission after immunotherapy. The one patient who did not improve had an underlying adenocarcinoma of the lung and died 3.5 months after disease onset because of tumor progression. Neuropathologic analysis of the brain autopsy revealed unilateral hippocampal sclerosis accompanied by mild inflammatory infiltrates, predominantly composed of T lymphocytes, and decrease of AMPAR immunoreactivity. CONCLUSION: AMPAR antibodies usually associate with limbic encephalitis but may also present with immune responsive, acute-to-subacute, isolated hippocampal dysfunction without overt inflammatory CSF or MRI changes.

On the Origin of Paroxysmal Depolarization Shifts: The Contribution of Cav1.x Channels as the Common Denominator of a Polymorphous Neuronal Discharge Pattern.

Since their discovery in the 1960s, the term paroxysmal depolarization shift (PDS) has been applied to a wide variety of reinforced neuronal discharge patterns. Occurrence of PDS as cellular correlates of electrographic spikes during latent phases of insult-induced rodent epilepsy models and their resemblance to giant depolarizing potentials (GDPs) nourished the idea that PDS may be involved in epileptogenesis. Both GDPs and - in analogy - PDS may lead to progressive changes of neuronal properties by generation of pulsatile intracellular Ca2+ elevations. Herein, a key element is the gating of L-type voltage gated Ca2+ channels (LTCCs, Cav1.x family), which may convey Ca2+ signals to the nucleus. Accordingly, the present study investigates various insult-associated neuronal challenges for their propensities to trigger PDS in a LTCC-dependent manner. Our data demonstrate that diverse disturbances of neuronal function are variably suited to induce PDS-like events, and the contribution of LTCCs is essential to evoke PDS in rat hippocampal neurons that closely resemble GDPs. These PDS appear to be initiated in the dendritic sub-compartment. Their morphology critically depends on the position of recording electrodes and on their rate of occurrence. These results provide novel insight into induction mechanisms, origin, variability, and co-existence of PDS with other discharge patterns and thereby pave the way for future investigations regarding the role of PDS in epileptogenesis.

Cardiovascular phenotype of the Dmdmdx rat - a suitable animal model for Duchenne muscular dystrophy.

Besides skeletal muscle abnormalities, Duchenne muscular dystrophy (DMD) patients present with dilated cardiomyopathy development, which considerably contributes to morbidity and mortality. Because the mechanisms responsible for the cardiac complications in the context of DMD are largely unknown, evidence-based therapy approaches are still lacking. This has increased the need for basic research efforts into animal models for DMD. Here, we characterized in detail the cardiovascular abnormalities of Dmdmdx rats, with the aim of determining the suitability of this recently established dystrophin-deficient small animal as a model for DMD.Various methods were applied to compare cardiovascular properties between wild-type and Dmdmdx rats, and to characterize the Dmdmdx cardiomyopathy. These methods comprised echocardiography, invasive assessment of left ventricular hemodynamics, examination of adverse remodeling and endothelial cell inflammation, and evaluation of vascular function, employing wire myography. Finally, intracellular Ca2+ transient measurements, and recordings of currents through L-type Ca2+ channels were performed in isolated single ventricular cardiomyocytes. We found that, similar to respective observations in DMD patients, the hearts of Dmdmdx rats show significantly impaired cardiac function, fibrosis and inflammation, consistent with the development of a dilated cardiomyopathy. Moreover, in Dmdmdx rats, vascular endothelial function is impaired, which may relate to inflammation and oxidative stress, and Ca2+ handling in Dmdmdx cardiomyocytes is abnormal.These findings indicate that Dmdmdx rats represent a promising small-animal model to elucidate mechanisms of cardiomyopathy development in the dystrophic heart, and to test mechanism-based therapies aiming to combat cardiovascular complications in DMD.

L-type Ca2+ channel-mediated Ca2+ influx adjusts neuronal mitochondrial function to physiological and pathophysiological conditions.

L-type voltage-gated Ca2+ channels (LTCCs) are implicated in neurodegenerative processes and cell death. Accordingly, LTCC antagonists have been proposed to be neuroprotective, although this view is disputed, because intentional LTCC activation can also have beneficial effects. LTCC-mediated Ca2+ influx influences mitochondrial function, which plays a crucial role in the regulation of cell viability. Hence, we investigated the effect of modulating LTCC-mediated Ca2+ influx on mitochondrial function in cultured hippocampal neurons. To activate LTCCs, neuronal activity was stimulated by increasing extracellular K+ or by application of the GABAA receptor antagonist bicuculline. The activity of LTCCs was altered by application of an agonistic (Bay K8644) or an antagonistic (isradipine) dihydropyridine. Our results demonstrated that activation of LTCC-mediated Ca2+ influx affected mitochondrial function in a bimodal manner. At moderate stimulation strength, ATP synthase activity was enhanced, an effect that involved Ca2+-induced Ca2+ release from intracellular stores. In contrast, high LTCC-mediated Ca2+ loads led to a switch in ATP synthase activity to reverse-mode operation. This effect, which required nitric oxide, helped to prevent mitochondrial depolarization and sustained increases in mitochondrial Ca2+ Our findings indicate a complex role of LTCC-mediated Ca2+ influx in the tuning and maintenance of mitochondrial function. Therefore, the use of LTCC inhibitors to protect neurons from neurodegeneration should be reconsidered carefully.

Neuronal nitric oxide synthase regulation of calcium cycling in ventricular cardiomyocytes is independent of Cav1.2 channel modulation under basal conditions.

Neuronal nitric oxide synthase (nNOS) is considered a regulator of Cav1.2 L-type Ca2+ channels and downstream Ca2+ cycling in the heart. The commonest view is that nitric oxide (NO), generated by nNOS activity in cardiomyocytes, reduces the currents through Cav1.2 channels. This gives rise to a diminished Ca2+ release from the sarcoplasmic reticulum, and finally reduced contractility. Here, we report that nNOS inhibitor substances significantly increase intracellular Ca2+ transients in ventricular cardiomyocytes derived from adult mouse and rat hearts. This is consistent with an inhibitory effect of nNOS/NO activity on Ca2+ cycling and contractility. Whole cell currents through L-type Ca2+ channels in rodent myocytes, on the other hand, were not substantially affected by the application of various NOS inhibitors, or application of a NO donor substance. Moreover, the presence of NO donors had no effect on the single-channel open probability of purified human Cav1.2 channel protein reconstituted in artificial liposomes. These results indicate that nNOS/NO activity does not directly modify Cav1.2 channel function. We conclude that-against the currently prevailing view-basal Cav1.2 channel activity in ventricular cardiomyocytes is not substantially regulated by nNOS activity and NO. Hence, nNOS/NO inhibition of Ca2+ cycling and contractility occurs independently of direct regulation of Cav1.2 channels by NO.

Lipid-independent control of endothelial and neuronal TRPC3 channels by light.

Lipid-gated TRPC channels are highly expressed in cardiovascular and neuronal tissues. Exerting precise pharmacological control over their activity in native cells is expected to serve as a basis for the development of novel therapies. Here we report on a new photopharmacological tool that enables manipulation of TRPC3 channels by light, in a manner independent of lipid metabolism and with higher temporal precision than lipid photopharmacology. Using the azobenzene photoswitch moiety, we modified GSK1702934A to generate light-controlled TRPC agonists. We obtained one light-sensitive molecule (OptoBI-1) that allows us to exert efficient, light-mediated control over TRPC3 activity and the associated cellular Ca2+ signaling. OptoBI-1 enabled high-precision, temporal control of TRPC3-linked cell functions such as neuronal firing and endothelial Ca2+ transients. With these findings, we introduce a novel photopharmacological strategy to control native TRPC conductances.

Rescue by 4-phenylbutyrate of several misfolded creatine transporter-1 variants linked to the creatine transporter deficiency syndrome.

Diseases arising from misfolding of SLC6 transporters have been reported over recent years, e.g. folding-deficient mutants of the dopamine transporter and of the glycine transporter-2 cause infantile/juvenile Parkinsonism dystonia and hyperekplexia, respectively. Mutations in the coding sequence of the human creatine transporter-1 (hCRT-1/SLC6A8) gene result in a creatine transporter deficiency syndrome, which varies in its clinical manifestation from epilepsy, mental retardation, autism, development delay and motor dysfunction to gastrointestinal symptoms. Some of the mutations in hCRT-1 occur at residues, which are highly conserved across the SLC6 family. Here, we examined 16 clinically relevant hCRT-1 variants to verify the conjecture that they were misfolded and that this folding defect was amenable to correction. Confocal microscopy imaging revealed that the heterologously expressed YFP-tagged mutant CRTs were trapped in the endoplasmic reticulum (ER), co-localised with the ER-resident chaperone calnexin. In contrast, the wild type hCRT-1 reached the plasma membrane. Preincubation of transiently transfected HEK293 cells with the chemical chaperone 4-phenylbutyrate (4-PBA) restored ER export and surface expression of as well as substrate uptake by several folding-deficient CRT-1 mutants. A representative mutant (hCRT-1-P544L) was expressed in rat primary hippocampal neurons to verify pharmacochaperoning in a target cell: 4-PBA promoted the delivery of hCRT-1-P544L to the neurite extensions. These observations show that several folding-deficient hCRT-1 mutants can be rescued. This proof-of-principle justifies the search for additional pharmacochaperones to restore folding of 4PBA-unresponsive hCRT-1 mutants. Finally, 4-PBA is an approved drug in paediatric use: this provides a rationale for translating the current insights into clinical trials. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

The Paroxysmal Depolarization Shift: Reconsidering Its Role in Epilepsy, Epileptogenesis and Beyond.

Paroxysmal depolarization shifts (PDS) have been described by epileptologists for the first time several decades ago, but controversy still exists to date regarding their role in epilepsy. In addition to the initial view of a lack of such a role, seemingly opposing hypotheses on epileptogenic and anti-ictogenic effects of PDS have emerged. Hence, PDS may provide novel targets for epilepsy therapy. Evidence for the roles of PDS has often been obtained from investigations of the multi-unit correlate of PDS, an electrographic spike termed "interictal" because of its occurrence during seizure-free periods of epilepsy patients. Meanwhile, interictal spikes have been found to be associated with neuronal diseases other than epilepsy, e.g., Alzheimer's disease, which may indicate a broader implication of PDS in neuropathologies. In this article, we give an introduction to PDS and review evidence that links PDS to pro- as well as anti-epileptic mechanisms, and to other types of neuronal dysfunction. The perturbation of neuronal membrane voltage and of intracellular Ca2+ that comes with PDS offers many conceivable pathomechanisms of neuronal dysfunction. Out of these, the operation of L-type voltage-gated calcium channels, which play a major role in coupling excitation to long-lasting neuronal changes, is addressed in detail.

The paroxysmal depolarization shift in epilepsy research.

Several shortcomings with currently available pharmacotherapy of epilepsy necessitate the search for new drug targets. Paroxysmal depolarization shifts (PDS) represent the cellular correlates of electrographic (e.g. interictal) spikes. While the ionic basis of PDS is understood in great detail, controversy exists regarding their proposed implication in epilepsy. To address this issue and to consider potential targetability, this mini-review gives an overview of the ionic conductances contributing to PDS and reflects on the hypotheses of their potential pro-epileptic (epileptogenic) and anti-epileptic roles.

Detection Methods for Autoantibodies in Suspected Autoimmune Encephalitis.

This review provides an overview on different antibody test methods that can be applied in cases of suspected paraneoplastic neurological syndromes (PNS) and anti-neuronal autoimmune encephalitis (AIE) in order to explain their diagnostic value, describe potential pitfalls and limitations, and discuss novel approaches aimed at discovering further autoantibodies. Onconeuronal antibodies are well-established biomarkers for PNS and may serve as specific tumor markers. The recommended procedure to detect onconeuronal antibodies is a combination of indirect immunohistochemistry on fixed rodent cerebellum and confirmation of the specificity by line assays. Simplification of this approach by only using line assays with recombinant proteins bears the risk to miss antibody-positive samples. Anti-neuronal surface antibodies are sensitive and specific biomarkers for AIE. Their identification requires the use of test methods that allow the recognition of conformation dependent epitopes. These commonly include cell-based assays and tissue based assays with unfixed rodent brain tissue. Tissue based assays can detect most of the currently known neuronal surface antibodies and thus enable broad screening of biological samples. A complementary testing on live neuronal cell cultures may confirm that the antibody recognizes a surface epitope. In patients with peripheral neuropathy, the screening may be expanded to teased nerve fibers to identify antibodies against the node of Ranvier. This method helps to identify a novel subgroup of peripheral autoimmune neuropathies, resulting in improved immunotherapy of these patients. Tissue based assays are useful to discover additional autoantibody targets that play a role in diverse autoimmune neurological syndromes. Antibody screening assays represent promising avenues of research to improve the diagnostic yield of current assays for antibody-associated autoimmune encephalitis.

Calcium current properties in dystrophin-deficient ventricular cardiomyocytes from aged mdx mice.

Duchenne muscular dystrophy (DMD), caused by mutations in the gene encoding for the cytoskeletal protein dystrophin, is linked with severe cardiac complications including cardiomyopathy development and cardiac arrhythmias. We and others recently reported that currents through L-type calcium (Ca) channels were significantly increased, and channel inactivation was reduced in dystrophin-deficient ventricular cardiomyocytes derived from the mdx mouse, the most commonly used animal model for human DMD. These gain-of-function Ca channel abnormalities may enhance the risk of Ca-dependent arrhythmias and cellular Ca overload in the dystrophic heart. All studies, which have so far investigated L-type Ca channel properties in dystrophic cardiomyocytes, have used hearts from either neonatal or young adult mdx mice as cell source. In consequence, the dimension of the Ca channel abnormalities present in the severely-diseased aged dystrophic heart has remained unknown. Here, we have studied potential abnormalities in Ca currents and intracellular Ca transients in ventricular cardiomyocytes derived from aged dystrophic mdx mice. We found that both the L-type and T-type Ca current properties of mdx cardiomyocytes were similar to those of myocytes derived from aged wild-type mice. Accordingly, Ca release from the sarcoplasmic reticulum was normal in cardiomyocytes from aged mdx mice. This suggests that, irrespective of the presence of a pronounced cardiomyopathy in aged mdx mice, Ca currents and Ca release in dystrophic cardiomyocytes are normal. Finally, our data imply that dystrophin- regulation of L-type Ca channel function in the heart is lost during aging.

Modulation of the heart's electrical properties by the anticonvulsant drug retigabine.

Retigabine, currently used as antiepileptic drug, has a wide range of potential medical uses. Administration of the drug in patients can lead to QT interval prolongation in the electrocardiogram and to cardiac arrhythmias in rare cases. This suggests that the drug may perturb the electrical properties of the heart, and the underlying mechanisms were investigated here. Effects of retigabine on currents through human cardiac ion channels, heterologously expressed in tsA-201 cells, were studied in whole-cell patch-clamp experiments. In addition, the drug's impact on the cardiac action potential was tested. This was done using ventricular cardiomyocytes isolated from Langendorff-perfused guinea pig hearts and cardiomyocytes derived from human induced pluripotent stem cells. Further, to unravel potential indirect effects of retigabine on the heart which might involve the autonomic nervous system, membrane potential and noradrenaline release from sympathetic ganglionic neurons were measured in the absence and presence of the drug. Retigabine significantly inhibited currents through hKv11.1 potassium, hNav1.5 sodium, as well as hCav1.2 calcium channels, but only in supra-therapeutic concentrations. In a similar concentration range, the drug shortened the action potential in both guinea pig and human cardiomyocytes. Therapeutic concentrations of retigabine, on the other hand, were sufficient to inhibit the activity of sympathetic ganglionic neurons. We conclude that retigabine- induced QT interval prolongation, and the reported cases of cardiac arrhythmias after application of the drug in a typical daily dose range, cannot be explained by a direct modulatory effect on cardiac ion channels. They are rather mediated by indirect actions at the level of the autonomic nervous system.

Cav 1.3 channels play a crucial role in the formation of paroxysmal depolarization shifts in cultured hippocampal neurons.

OBJECTIVE: An increase of neuronal Cav 1.3 L-type calcium channels (LTCCs) has been observed in various animal models of epilepsy. However, LTCC inhibitors failed in clinical trials of epileptic treatment. There is compelling evidence that paroxysmal depolarization shifts (PDSs) involve Ca2+ influx through LTCCs. PDSs represent a hallmark of epileptiform activity. In recent years, a probable epileptogenic role for PDSs has been proposed. However, the implication of the two neuronal LTCC isoforms, Cav 1.2 and Cav 1.3, in PDSs remained unknown. Moreover, Ca2+ -dependent nonspecific cation (CAN) channels have also been suspected to contribute to PDSs. Nevertheless, direct experimental support of an important role of CAN channel activation in PDS formation is still lacking. METHODS: Primary neuronal networks derived from dissociated hippocampal neurons were generated from mice expressing a dihydropyridine-insensitive Cav 1.2 mutant (Cav 1.2DHP-/- mice) or from Cav 1.3-/- knockout mice. To investigate the role of Cav 1.2 and Cav 1.3, perforated patch-clamp recordings were made of epileptiform activity, which was elicited using either bicuculline or caffeine. LTCC activity was modulated using the dihydropyridines Bay K 8644 (agonist) and isradipine (antagonist). RESULTS: Distinct PDS could be elicited upon LTCC potentiation in Cav 1.2DHP-/- neurons but not in Cav 1.3-/- neurons. In contrast, when bicuculline led to long-lasting, seizure-like discharge events rather than PDS, these were prolonged in Cav 1.3-/- neurons but not in Cav 1.2DHP-/- neurons. Because only the Cav 1.2 isoform is functionally coupled to CAN channels in primary hippocampal networks, PDS formation does not require CAN channel activity. SIGNIFICANCE: Our data suggest that the LTCC requirement of PDS relates primarily to Cav 1.3 channels rather than to Cav 1.2 channels and CAN channels in hippocampal neurons. Hence, Cav 1.3 may represent a new therapeutic target for suppression of PDS development. The proposed epileptogenic role of PDSs may allow for a prophylactic rather than the unsuccessful seizure suppressing application of LTCC inhibitors.