Change of voltage-gated sodium channel repertoire in skeletal muscle of a MuSK myasthenia gravis mouse model.

Muscle-specific kinase myasthenia gravis (MuSK MG) is caused by autoantibodies against MuSK in the neuromuscular junction (NMJ). MuSK MG patients have fluctuating, fatigable skeletal muscle weakness, in particular of bulbar muscles. Severity differs greatly between patients, in spite of comparable autoantibody levels. One explanation for inter-patient and inter-muscle variability in sensitivity might be variations in compensatory muscle responses. Previously, we developed a passive transfer mouse model for MuSK MG. In preliminary ex vivo experiments, we observed that muscle contraction of some mice, in particular those with milder myasthenia, had become partially insensitive to inhibition by µ-Conotoxin-GIIIB, a blocker of skeletal muscle NaV1.4 voltage-gated sodium channels. We hypothesised that changes in NaV channel expression profile, possibly co-expression of (µ-Conotoxin-GIIIB insensitive) NaV1.5 type channels, might lower the muscle fibre's firing threshold and facilitate neuromuscular synaptic transmission. To test this hypothesis, we here performed passive transfer in immuno-compromised mice, using 'high', 'intermediate' and 'low' dosing regimens of purified MuSK MG patient IgG4. We compared myasthenia levels, µ-Conotoxin-GIIIB resistance and muscle fibre action potential characteristics and firing thresholds. High- and intermediate-dosed mice showed clear, progressive myasthenia, not seen in low-dosed animals. However, diaphragm NMJ electrophysiology demonstrated almost equal myasthenic severities amongst all regimens. Nonetheless, low-dosed mouse diaphragms showed a much higher degree of µ-Conotoxin-GIIIB resistance. This was not explained by upregulation of Scn5a (the NaV1.5 gene), lowered muscle fibre firing thresholds or histologically detectable upregulated NaV1.5 channels. It remains to be established which factors are responsible for the observed µ-Conotoxin-GIIIB insensitivity and whether the NaV repertoire change is compensatory beneficial or a bystander effect.

Development and characterization of agonistic antibodies targeting the Ig-like 1 domain of MuSK.

Muscle-specific kinase (MuSK) is crucial for acetylcholine receptor (AChR) clustering and thereby neuromuscular junction (NMJ) function. NMJ dysfunction is a hallmark of several neuromuscular diseases, including MuSK myasthenia gravis. Aiming to restore NMJ function, we generated several agonist monoclonal antibodies targeting the MuSK Ig-like 1 domain. These activated MuSK and induced AChR clustering in cultured myotubes. The most potent agonists partially rescued myasthenic effects of MuSK myasthenia gravis patient IgG autoantibodies in vitro. In an IgG4 passive transfer MuSK myasthenia model in NOD/SCID mice, MuSK agonists caused accelerated weight loss and no rescue of myasthenic features. The MuSK Ig-like 1 domain agonists unexpectedly caused sudden death in a large proportion of male C57BL/6 mice (but not female or NOD/SCID mice), likely caused by a urologic syndrome. In conclusion, these agonists rescued pathogenic effects in myasthenia models in vitro, but not in vivo. The sudden death in male mice of one of the tested mouse strains revealed an unexpected and unexplained role for MuSK outside skeletal muscle, thereby hampering further (pre-) clinical development of these clones. Future research should investigate whether other Ig-like 1 domain MuSK antibodies, binding different epitopes, do hold a safe therapeutic promise.

A bioassay for neuromuscular junction-restricted complement activation by myasthenia gravis acetylcholine receptor antibodies.

BACKGROUND: Myasthenia gravis (MG) is an autoimmune neuromuscular disorder hallmarked by fluctuating fatigable muscle weakness. Most patients have autoantibodies against acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). These are thought to have three possible pathogenic mode-of-actions: 1) cross-linking and endocytosis of AChRs, 2) direct block of AChRs and 3) complement activation. The relative contributions of these mechanisms to synaptic block and muscle weakness of individual patients cannot be determined. It likely varies between patients and perhaps also with disease course, depending on the nature of the circulating AChR antibodies. NEW METHOD: We developed a new bioassay which specifically enables functional characterization and quantification of complement-mediated synaptic damage at NMJs, without interference of the other pathogenic mechanisms. To this end, we pre-incubated mouse hemi-diaphragm muscle-nerve preparations with mAb35-hG1, a humanized rat AChR monoclonal and subsequently exposed the preparation to normal human serum as a complement source. NMJ-restricted effects were studied. RESULTS: Clearly NMJ-restricted damage occurred. With immunohistology we showed complement deposition at NMJs, and synaptic electrophysiological measurements demonstrated transmission block. In whole-muscle contraction experiments we quantified the effect and characterized its onset and progression during the incubation with normal human serum. COMPARISON WITH EXISTING METHODS: With this new assay the complement-mediated component of myasthenic NMJ pathology can be studied separately. CONCLUSIONS: Our assay will be of importance in detailed mechanistic studies of local complement activation at NMJs, investigations of new complement inhibitors, and laboratory pre-screening of therapeutic efficacy for individual MG patients to optimize care with clinically approved complement inhibitors.

Advances in the understanding of disease mechanisms of autoimmune neuromuscular junction disorders.

Muscle weakness and fatigue are the hallmarks of autoimmune neuromuscular junction disorders. Although a plethora of immunosuppressive treatments exist, no cure is available to date and many patients are left with debilitating muscle weakness. Recent advances in the understanding of the structure and function of the neuromuscular junction, and the development of novel in vitro and in vivo models, have been instrumental in unravelling the pathophysiology of these autoimmune diseases. These advances are providing the rationale for the development of new therapeutic strategies. Restoration of the immune imbalance in these diseases, in parallel with symptomatic therapeutic approaches at the neuromuscular junction, will be crucial to obtain long-term remission or even cure.

Functional monovalency amplifies the pathogenicity of anti-MuSK IgG4 in myasthenia gravis.

Human immunoglobulin (Ig) G4 usually displays antiinflammatory activity, and observations of IgG4 autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, IgG4 naturally engages in a stochastic process termed "Fab-arm exchange" in which unrelated IgG4s exchange half-molecules continuously. The resulting IgG4 antibodies are composed of two different binding sites, thereby acquiring monovalent binding and inability to cross-link for each antigen recognized. Here, we demonstrate that this process amplifies autoantibody pathogenicity in a classic IgG4-mediated autoimmune disease: muscle-specific kinase (MuSK) myasthenia gravis. In mice, monovalent anti-MuSK IgG4s caused rapid and severe myasthenic muscle weakness, whereas the same antibodies in their parental bivalent form were less potent or did not induce a phenotype. Mechanistically this could be explained by opposing effects on MuSK signaling. Isotype switching to IgG4 in an autoimmune response thereby may be a critical step in the development of disease. Our study establishes functional monovalency as a pathogenic mechanism in IgG4-mediated autoimmune disease and potentially other disorders.

Simvastatin Treatment Does Not Ameliorate Muscle Pathophysiology in a Mouse Model for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy is an X-linked, recessive muscular dystrophy in which the absence of the dystrophin protein leads to fibrosis, inflammation and oxidative stress, resulting in loss of muscle tissue. Drug repurposing, i.e. using drugs already approved for other disorders, is attractive as it decreases development time. Recent studies suggested that simvastatin, a cholesterol lowering drug used for cardiovascular diseases, has beneficial effects on several parameters in mdx mice. To validate properly the effectiveness of simvastatin, two independent labs tested the effects of 12-week simvastatin treatment in either young (starting at 4 weeks of age) or adult (starting at 12 weeks of age) mdx mice. In neither study were benefits of simvastatin treatment observed on muscle function, histology or expression of genes involved in fibrosis, regeneration, oxidative stress and autophagy. Unexpectedly, although the treatment protocol was similar, simvastatin plasma levels were found to be much lower than observed in a previous study. In conclusion, in two laboratories, simvastatin did not ameliorate disease pathology in mdx mice, which could either be due to the ineffectiveness of simvastatin itself or due to the low simvastatin plasma levels following oral administration via the food.

Cross-sectional study into age-related pathology of mouse models for limb girdle muscular dystrophy types 2D and 2F.

Limb girdle muscular dystrophy (LGMD) types 2D and 2F are caused by mutations in the genes encoding for α- and δ-sarcoglycan, respectively, leading to progressive muscle weakness. Mouse models exist for LGMD2D (Sgca-/-) and 2F (Sgcd-/-). In a previous natural history study, we described the pathology in these mice at 34 weeks of age. However, the development of muscle pathology at younger ages has not been fully characterised yet. We therefore performed a study into age-related changes in muscle function and pathology by examining mice at different ages. From 4 weeks of age onwards, male mice were subjected to functional tests and sacrificed at respectively 8, 16 or 24 weeks of age. Muscle histopathology and expression of genes involved in muscle pathology were analysed for several skeletal muscles, while miRNA levels were assessed in serum. In addition, for Sgcd-/- mice heart pathology was assessed. Muscle function showed a gradual decline in both Sgca-/- and Sgcd-/- mice. Respiratory function was also impaired at all examined timepoints. Already at 8 weeks of age, muscle pathology was prominent, and fibrotic, inflammatory and regenerative markers were elevated, which remained relatively constant with age. In addition, Sgcd-/- mice showed signs of cardiomyopathy from 16 weeks of age onwards. These results indicate that Sgca-/- and Sgcd-/- are relevant disease models for LGMD2D and 2F.

Natural disease history of the D2-mdx mouse model for Duchenne muscular dystrophy.

The C57BL/10ScSn-Dmdmdx/J (BL10-mdx) mouse has been the most commonly used model for Duchenne muscular dystrophy (DMD) for decades. Their muscle dysfunction and pathology is, however, less severe than in patients with DMD, which complicates preclinical studies. Recent discoveries indicate that disease severity is exacerbated when muscular dystrophy mouse models are generated on a DBA2/J genetic background. Knowledge on the natural history of animal models is pivotal for high-quality preclinical testing. However, for BL10-mdx mice on a DBA2/J background (D2-mdx), limited data are available. We addressed this gap in the natural history knowledge. First, we compared histopathological aspects in skeletal muscles of young D2-mdx, BL10-mdx, and wild-type mice. Pathology was more pronounced in D2-mdx mice and differed in severity between muscles within individuals. Secondly, we subjected D2-mdx mice to a functional test regime for 34 weeks and identified that female D2-mdx mice outperform severely impaired males, making females less useful for functional preclinical studies. Direct comparisons between 10- and 34-wk-old D2-mdx mice revealed that disease pathology ameliorates with age. Heart pathology was progressive, with some features already evident at a young age. This natural history study of the D2-mdx mouse will be instrumental for experimental design of future preclinical studies.-Van Putten, M., Putker, K., Overzier, M., Adamzek, W. A., Pasteuning-Vuhman, S., Plomp, J. J., Aartsma-Rus, A. Natural disease history of the D2-mdx mouse model for Duchenne muscular dystrophy.

An automated hybrid bioelectronic system for autogenous restoration of sinus rhythm in atrial fibrillation.

Because of suboptimal therapeutic strategies, restoration of sinus rhythm in symptomatic atrial fibrillation (AF) often requires in-hospital delivery of high-voltage shocks, thereby precluding ambulatory AF termination. Continuous, rapid restoration of sinus rhythm is desired given the recurring and progressive nature of AF. Here, we present an automated hybrid bioelectronic system for shock-free termination of AF that enables the heart to act as an electric current generator for autogenous restoration of sinus rhythm. We show that local, right atrial delivery of adenoassociated virus vectors encoding a light-gated depolarizing ion channel results in efficient and spatially confined transgene expression. Activation of an implanted intrathoracic light-emitting diode device allows for termination of AF by illuminating part of the atria. Combining this newly obtained antiarrhythmic effector function of the heart with the arrhythmia detector function of a machine-based cardiac rhythm monitor in the closed chest of adult rats allowed automated and rapid arrhythmia detection and termination in a safe, effective, repetitive, yet shock-free manner. These findings hold translational potential for the development of shock-free antiarrhythmic device therapy for ambulatory treatment of AF.

Efgartigimod improves muscle weakness in a mouse model for muscle-specific kinase myasthenia gravis.

Myasthenia gravis is hallmarked by fatigable muscle weakness resulting from neuromuscular synapse dysfunction caused by IgG autoantibodies. The variant with muscle-specific kinase (MuSK) autoantibodies is characterized by prominent cranial and bulbar weakness and a high frequency of respiratory crises. The majority of MuSK MG patients requires long-term immunosuppressive treatment, but the result of these treatments is considered less satisfactory than in MG with acetylcholine receptor antibodies. Emergency treatments are more frequently needed, and many patients develop permanent facial weakness and nasal speech. Therefore, new treatment options would be welcome. The neonatal Fc receptor protects IgG from lysosomal breakdown, thus prolonging IgG serum half-life. Neonatal Fc receptor antagonism lowers serum IgG levels and thus may act therapeutically in autoantibody-mediated disorders. In MuSK MG, IgG4 anti-MuSK titres closely correlate with disease severity. We therefore tested efgartigimod (ARGX-113), a new neonatal Fc receptor blocker, in a mouse model for MuSK myasthenia gravis. This model involves 11 daily injections of purified IgG4 from MuSK myasthenia gravis patients, resulting in overt myasthenic muscle weakness and, consequently, body weight loss. Daily treatment with 0.5 mg efgartigimod, starting at the fifth passive transfer day, reduced the human IgG4 titres about 8-fold, despite continued daily injection. In muscle strength and fatigability tests, efgartigimod-treated myasthenic mice outperformed control myasthenic mice. Electromyography in calf muscles at endpoint demonstrated less myasthenic decrement of compound muscle action potentials in efgartigimod-treated mice. These substantial in vivo improvements of efgartigimod-treated MuSK MG mice following a limited drug exposure period were paralleled by a tendency of recovery at neuromuscular synaptic level (in various muscles), as demonstrated by ex vivo functional studies. These synaptic improvements may well become more explicit upon longer drug exposure. In conclusion, our study shows that efgartigimod has clear therapeutic potential in MuSK myasthenia gravis and forms an exciting candidate drug for many autoantibody-mediated neurological and other disorders.

MuSK myasthenia gravis monoclonal antibodies: Valency dictates pathogenicity.

Objective: To isolate and characterize muscle-specific kinase (MuSK) monoclonal antibodies from patients with MuSK myasthenia gravis (MG) on a genetic and functional level. Methods: We generated recombinant MuSK antibodies from patient-derived clonal MuSK-specific B cells and produced monovalent Fab fragments from them. Both the antibodies and Fab fragments were tested for their effects on neural agrin-induced MuSK phosphorylation and acetylcholine receptor (AChR) clustering in myotube cultures. Results: The isolated MuSK monoclonal antibody sequences included IgG1, IgG3, and IgG4 that had undergone high levels of affinity maturation, consistent with antigenic selection. We confirmed their specificity for the MuSK Ig-like 1 domain and binding to neuromuscular junctions. Monovalent MuSK Fab, mimicking functionally monovalent MuSK MG patient Fab-arm exchanged serum IgG4, abolished agrin-induced MuSK phosphorylation and AChR clustering. Surprisingly, bivalent monospecific MuSK antibodies instead activated MuSK phosphorylation and partially induced AChR clustering, independent of agrin. Conclusions: Patient-derived MuSK antibodies can act either as MuSK agonist or MuSK antagonist, depending on the number of MuSK binding sites. Functional monovalency, induced by Fab-arm exchange in patient serum, makes MuSK IgG4 antibodies pathogenic.

Low dystrophin levels are insufficient to normalize the neuromuscular synaptic abnormalities of mdx mice.

Dystrophin is a sub-sarcolemmal component of skeletal muscle fibres and is enriched at the postsynaptic membrane of the neuromuscular junction (NMJ). In the mdx mouse, dystrophin absence not only causes muscle damage but also mild synaptic dysfunctions and clear morphological aberrations at NMJs. In particular, reduction of postsynaptic sensitivity for the neurotransmitter acetylcholine and extra exhaustion of presynaptic acetylcholine release during intense synaptic activity exists. Current experimental therapeutic approaches in Duchenne muscular dystrophy aim to restore dystrophin expression. An important question is what dystrophin levels are needed to improve muscle function. Recent experimental and clinical studies suggested that levels as low as a few percent of normal can be beneficial. Similarly, it is of interest to know how dystrophin levels relate to NMJ function and morphology. We investigated NMJs of a series of mdx-XistΔhs mice, which expressed dystrophin between ~2% and 19% of normal. Most functional and morphological NMJ parameters of these mice remained comparable to mdx. On the other hand, mdx+/- mice (expressing ~50% dystrophin) showed normal NMJ features. Thus, the minimal dystrophin level required for normal NMJ function and morphology lies between 19% and 50% of normal when expression of dystrophin is not uniform.

IgG4-mediated autoimmune diseases: a niche of antibody-mediated disorders.

Immunoglobulin 4 (IgG4) is one of four human IgG subclasses and has several unique functional characteristics. It exhibits low affinity for complement and for most Fc receptors. It furthermore has generally high affinity for its antigen, with binding occurring in a monovalent fashion, as IgG4 can exchange Fab-arms with other IgG4 molecules. Because of these characteristics, IgG4 is believed to block its targets and prevent inflammation, which, depending on the setting, can have a protective or pathogenic effect. One example of IgG4 pathogenicity is muscle-specific kinase (MuSK) myasthenia gravis (MG), in which patients develop IgG4 MuSK autoantibodies, resulting in muscle weakness. As a consequence of the distinct IgG4 characteristics, the pathomechanism of MuSK MG is very different from IgG1-and IgG3-mediated autoimmune diseases, such as acetylcholine receptor MG. In recent years, new autoantibodies in a spectrum of autoimmune diseases have been discovered. Interestingly, some were found to be predominantly IgG4. These IgG4-mediated autoimmune diseases share many pathomechanistic aspects with MuSK MG, suggesting that IgG4-mediated autoimmunity forms a separate niche among the antibody-mediated disorders. In this review, we summarize the group of IgG4-mediated autoimmune diseases, discuss the role of IgG4 in MuSK MG, and highlight interesting future research questions for IgG4-mediated autoimmunity.

Passive transfer models of myasthenia gravis with muscle-specific kinase antibodies.

Myasthenia gravis (MG) with antibodies to muscle-specific kinase (MuSK) is characterized by fluctuating fatigable weakness. In MuSK MG, involvement of bulbar muscles, neck, and shoulder and respiratory weakness are more prominent than in acetylcholine receptor (AChR) MG. MuSK autoantibodies are mainly of the IgG4 subclass, and as such are unable to activate complement, have low affinity for Fc receptors, and are functionally monovalent. Therefore, the pathogenicity of IgG4 MuSK autoantibodies was initially questioned. A broad collection of in vitro active immunization and passive transfer models has been developed that have shed light on the pathogenicity of MuSK autoantibodies. Passive transfer studies with purified IgG4 from MuSK MG patients confirmed that IgG4 is sufficient to reproduce clear clinical, electrophysiological, and histological signs of myasthenia. In vitro experiments revealed that MuSK IgG4 autoantibodies preferably bind the first Ig-like domain of MuSK, correlate with disease severity, and interfere with the association between MuSK and low-density lipoprotein receptor-related protein 4 and collagen Q. Some patients have additional IgG1 MuSK autoantibodies, but their role in the disease is unclear. Altogether, this provides a rationale for epitope-specific or IgG4-specific treatment strategies for MuSK MG and emphasizes the importance of the development of different experimental models.

Neuromuscular synapse electrophysiology in myasthenia gravis animal models.

The neuromuscular junction (NMJ) forms the synaptic connection between a motor neuron and a skeletal muscle fiber. In order to achieve a sustained muscle contraction, this synapse has to reliably transmit motor neuronal action potentials onto the muscle fiber. To guarantee successful transmission even during intense activation of the NMJ, a safety factor of neuromuscular transmission exists. In the neuromuscular disorder myasthenia gravis (MG), autoantibodies are directed against acetylcholine receptors or, in the rarer variants, against other postsynaptic NMJ proteins. This causes loss of functional acetylcholine receptors, which compromises the safety factor of neuromuscular transmission, leading to the typical fatigable muscle weakness of MG. With intracellular microelectrode measurement of (miniature) endplate potentials at NMJs in ex vivo nerve-muscle preparations from MG animal models, these functional synaptic defects have been determined in much detail. Here, we describe the electrophysiological events at the normal NMJ and the pathoelectrophysiology at NMJs of animal models for MG.

Acute and chronic effects of treatment with mesenchymal stromal cells on LPS-induced pulmonary inflammation, emphysema and atherosclerosis development.

BACKGROUND: COPD is a pulmonary disorder often accompanied by cardiovascular disease (CVD), and current treatment of this comorbidity is suboptimal. Systemic inflammation in COPD triggered by smoke and microbial exposure is suggested to link COPD and CVD. Mesenchymal stromal cells (MSC) possess anti-inflammatory capacities and MSC treatment is considered an attractive treatment option for various chronic inflammatory diseases. Therefore, we investigated the immunomodulatory properties of MSC in an acute and chronic model of lipopolysaccharide (LPS)-induced inflammation, emphysema and atherosclerosis development in APOE*3-Leiden (E3L) mice. METHODS: Hyperlipidemic E3L mice were intranasally instilled with 10 µg LPS or vehicle twice in an acute 4-day study, or twice weekly during 20 weeks Western-type diet feeding in a chronic study. Mice received 0.5x106 MSC or vehicle intravenously twice after the first LPS instillation (acute study) or in week 14, 16, 18 and 20 (chronic study). Inflammatory parameters were measured in bronchoalveolar lavage (BAL) and lung tissue. Emphysema, pulmonary inflammation and atherosclerosis were assessed in the chronic study. RESULTS: In the acute study, intranasal LPS administration induced a marked systemic IL-6 response on day 3, which was inhibited after MSC treatment. Furthermore, MSC treatment reduced LPS-induced total cell count in BAL due to reduced neutrophil numbers. In the chronic study, LPS increased emphysema but did not aggravate atherosclerosis. Emphysema and atherosclerosis development were unaffected after MSC treatment. CONCLUSION: These data show that MSC inhibit LPS-induced pulmonary and systemic inflammation in the acute study, whereas MSC treatment had no effect on inflammation, emphysema and atherosclerosis development in the chronic study.

Trans-synaptic homeostasis at the myasthenic neuromuscular junction.

Properly sustained impulse transmission at the neuromuscular junction (NMJ) is crucial for successful muscle contraction. To guarantee this, NMJs not only possess a considerable safety factor in transmission but also have the ability to adjust the presynaptic acetylcholine release level to cope with any changes in the postsynaptic neurotransmitter sensitivity. This review will provide overview on the discovery and characterization of this synaptic homeostatic mechanism, especially in the condition of the neuromuscular disorder myasthenia gravis (MG) where the postsynaptic transmitter sensitivity at the NMJ becomes severely reduced due to autoimmune attack of acetylcholine receptors. Because homeostatic signalling and adaptation is presumably maximally active in this condition, NMJs from MG animal models are important study objects. Although candidate post- and presynaptic factors as well as the retrograde signals have been proposed, the homeostatic mechanism at the MG NMJ is still incompletely understood. Further identification and functional characterization of key factors is important because these may form new therapeutic targets in MG.

Optogenetic termination of ventricular arrhythmias in the whole heart: towards biological cardiac rhythm management.

AIMS: Current treatments of ventricular arrhythmias rely on modulation of cardiac electrical function through drugs, ablation or electroshocks, which are all non-biological and rather unspecific, irreversible or traumatizing interventions. Optogenetics, however, is a novel, biological technique allowing electrical modulation in a specific, reversible and trauma-free manner using light-gated ion channels. The aim of our study was to investigate optogenetic termination of ventricular arrhythmias in the whole heart. METHODS AND RESULTS: Systemic delivery of cardiotropic adeno-associated virus vectors, encoding the light-gated depolarizing ion channel red-activatable channelrhodopsin (ReaChR), resulted in global cardiomyocyte-restricted transgene expression in adult Wistar rat hearts allowing ReaChR-mediated depolarization and pacing. Next, ventricular tachyarrhythmias (VTs) were induced in the optogenetically modified hearts by burst pacing in a Langendorff setup, followed by programmed, local epicardial illumination. A single 470-nm light pulse (1000 ms, 2.97 mW/mm2) terminated 97% of monomorphic and 57% of polymorphic VTs vs. 0% without illumination, as assessed by electrocardiogram recordings. Optical mapping showed significant prolongation of voltage signals just before arrhythmia termination. Pharmacological action potential duration (APD) shortening almost fully inhibited light-induced arrhythmia termination indicating an important role for APD in this process. CONCLUSION: Brief local epicardial illumination of the optogenetically modified adult rat heart allows contact- and shock-free termination of ventricular arrhythmias in an effective and repetitive manner after optogenetic modification. These findings could lay the basis for the development of fundamentally new and biological options for cardiac arrhythmia management.

Continuous infusion of manganese improves contrast and reduces side effects in manganese-enhanced magnetic resonance imaging studies.

The ability to administer systemically high doses of manganese as contrast agent while circumventing its toxicity is of particular interest for exploratory MRI studies of the brain. Administering low doses either repeatedly or continuously over time has been shown to enable the acquisition of satisfactory MRI images of the mouse brain without apparent side effects. Here we have systematically compared the obtained MRI contrast and recorded potential systemic side effects such as stress response and muscle strength impairment in relation to the achieved contrast. We show in mice that administering MnCl2 via osmotic infusion pumps allows for a side-effect free delivery of a high cumulative dose of manganese chloride (480mg/kg bodyweight in 8 days). High contrast in MRI was achieved while we did not observe the weight loss or distress seen in other studies where mice received manganese via fractionated intraperitoneal injections of lower doses of manganese. As the normal daily conduct of the mice was not affected, this new manganese delivery method might be of particular use to study brain activity over several days. This may facilitate the phenotyping of new transgenic mouse models, the study of chronic disease models and the monitoring of changes in brain activity in long-term behavioral studies.

Characterization of neuromuscular synapse function abnormalities in multiple Duchenne muscular dystrophy mouse models.

Duchenne muscular dystrophy (DMD) is an X-linked myopathy caused by dystrophin deficiency. Dystrophin is present intracellularly at the sarcolemma, connecting actin to the dystrophin-associated glycoprotein complex. Interestingly, it is enriched postsynaptically at the neuromuscular junction (NMJ), but its synaptic function is largely unknown. Utrophin, a dystrophin homologue, is also concentrated at the NMJ, and upregulated in DMD. It is possible that the absence of dystrophin at NMJs in DMD causes neuromuscular transmission defects that aggravate muscle weakness. We studied NMJ function in mdx mice (lacking dystrophin) and wild type mice. In addition, mdx/utrn(+/-) and mdx/utrn(-/-) mice (lacking utrophin) were used to investigate influences of utrophin levels. The three Duchenne mouse models showed muscle weakness when comparatively tested in vivo, with mdx/utrn(-/-) mice being weakest. Ex vivo muscle contraction and electrophysiological studies showed a reduced safety factor of neuromuscular transmission in all models. NMJs had ~ 40% smaller miniature endplate potential amplitudes compared with wild type, indicating postsynaptic sensitivity loss for the neurotransmitter acetylcholine. However, nerve stimulation-evoked endplate potential amplitudes were unchanged. Consequently, quantal content (i.e. the number of acetylcholine quanta released per nerve impulse) was considerably increased. Such a homeostatic compensatory increase in neurotransmitter release is also found at NMJs in myasthenia gravis, where autoantibodies reduce acetylcholine receptors. However, high-rate nerve stimulation induced exaggerated endplate potential rundown. Study of NMJ morphology showed that fragmentation of acetylcholine receptor clusters occurred in all models, being most severe in mdx/utrn(-/-) mice. Overall, we showed mild 'myasthenia-like' neuromuscular synaptic dysfunction in several Duchenne mouse models, which possibly affects muscle weakness and degeneration.