Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy

Myostatin regulates skeletal muscle size via the activin receptor IIB (ActRIIB). However, its effect on muscle energy metabolism and energy dependent muscle function remains largely unexplored. This question needs to be solved urgently since various therapies for neuromuscular diseases based on blockade of ActRIIB signaling are being developed. Here we show in mice that four months of pharmacological abrogation of ActRIIB signaling by treatment with soluble ActRIIB-Fc triggers extreme muscle fatigability. This is associated with elevated serum lactate levels and a severe metabolic myopathy in the mdx mouse, an animal model of Duchenne muscular dystrophy. Blockade of ActRIIB signaling down-regulates Porin, a crucial ADP/ATP shuttle between cytosol and mitochondrial matrix leading to a consecutive deficiency of oxidative phosphorylation as measured by in vivo Phophorus Magnetic Resonance Spectroscopy (31P-MRS). Further, ActRIIB blockade reduces muscle capillarization, which further compounds the metabolic stress. We show that ActRIIB regulates key determinants of muscle metabolism, such as Pparβ, Pgc1α, and Pdk4 thereby optimizing different components of muscle energy metabolism. In conclusion, ActRIIB signaling endows skeletal muscle with high oxidative capacity and low fatigability. The severe metabolic side effects following ActRIIB blockade caution against deploying this strategy, at least in isolation, for treatment of neuromuscular disorders.

Muscle hypertrophy driven by myostatin blockade does not require stem/precursor-cell activity

Myostatin, a member of the TGF-beta family, has been identified as a powerful inhibitor of muscle growth. Absence or blockade of myostatin induces massive skeletal muscle hypertrophy that is widely attributed to proliferation of the population of muscle fiber-associated satellite cells that have been identified as the principle source of new muscle tissue during growth and regeneration. Postnatal blockade of myostatin has been proposed as a basis for therapeutic strategies to combat muscle loss in genetic and acquired myopathies. But this approach, according to the accepted mechanism, would raise the threat of premature exhaustion of the pool of satellite cells and eventual failure of muscle regeneration. Here, we show that hypertrophy in the absence of myostatin involves little or no input from satellite cells. Hypertrophic fibers contain no more myonuclei or satellite cells and myostatin had no significant effect on satellite cell proliferation in vitro, while expression of myostatin receptors dropped to the limits of detectability in postnatal satellite cells. Moreover, hypertrophy of dystrophic muscle arising from myostatin blockade was achieved without any apparent enhancement of contribution of myonuclei from satellite cells. These findings contradict the accepted model of myostatin-based control of size of postnatal muscle and reorient fundamental investigations away from the mechanisms that control satellite cell proliferation and toward those that increase myonuclear domain, by modulating synthesis and turnover of structural muscle fiber proteins. It predicts too that any benefits of myostatin blockade in chronic myopathies are unlikely to impose any extra stress on the satellite cells.

Neuromuscular junction morphology, fiber-type proportions, and satellite-cell proliferation rates are altered in MyoD(-/-) mice

Gene compensation by members of the myogenic regulatory factor (MRF) family has been proposed to explain the apparent normal adult phenotype of MyoD(-/-) mice. Nerve and field stimulation were used to investigate contraction properties of muscle from MyoD(-/-) mice, and molecular approaches were used to investigate satellite-cell behavior. We demonstrate that MyoD deletion results in major alterations in the organization of the neuromuscular junction, which have a dramatic influence on the physiological contractile properties of skeletal muscle. Second, we show that the lineage progression of satellite cells (especially initial proliferation) in the absence of MyoD is abnormal and linked to perturbations in the nuclear localization of beta-catenin, a key readout of canonical Wnt signaling. These results show that MyoD has unique functions in both developing and adult skeletal muscle that are not carried out by other members of the MRF family.

Molecular requirements for L-type Ca2+ channel blockade by testosterone

Despite being generally perceived as detrimental to the cardiovascular system, testosterone has marked beneficial vascular effects; most notably it acutely and directly causes vasodilatation. Indeed, men with hypotestosteronaemia can present with myocardial ischemia and angina which can be rapidly alleviated by infusion of testosterone. To date, however, in vitro studies have failed to provide a convincing mechanism to account for this clinically important effect. Here, using whole-cell patch-clamp recordings to measure current flow through recombinant human L-type Ca2+ channel alpha(1C) subunits (Ca(v)1.2), we demonstrate that testosterone inhibits such currents in a concentration-dependent manner. Importantly, this occurs over the physiological range of testosterone concentrations (IC50 34 nM), and is not mimicked by the metabolite 5alpha-androstan-17beta-ol-3-one (DHT), nor by progesterone or estradiol, even at high (10 microM) concentration. L-type Ca2+ channels in the vasculature are also important clinical targets for vasodilatory dihydropyridines. A single point mutation (T1007Y) almost completely abolishes nifedipine sensitivity in our recombinant expression system. Crucially, the same mutation renders the channels insensitive to testosterone. Our data strongly suggest, for the first time, the molecular requirements for testosterone binding to L-type Ca2+ channels, thereby supporting its beneficial role as an endogenous Ca2+ channel antagonist in the treatment of cardiovascular disease.

Voltage-gated sodium (NaV) channel blockade by plant cannabinoids does not confer anticonvulsant effects per se

Cannabidiol (CBD) is a non-psychoactive, well-tolerated, anticonvulsant plant cannabinoid, although its mechanism(s) of seizure suppression remains unknown. Here, we investigate the effect of CBD and the structurally similar cannabinoid, cannabigerol (CBG), on voltage-gated Na+ (NaV) channels, a common anti-epileptic drug target. CBG’s anticonvulsant potential was also assessed in vivo. CBD effects on NaV channels were investigated using patch-clamp recordings from rat CA1 hippocampal neurons in brain slices, human SH-SY5Y (neuroblastoma) cells and mouse cortical neurons in culture. CBG effects were also assessed in SH-SY5Y cells and mouse cortical neurons. CBD and CBG effects on veratridine-stimulated human recombinant NaV1.1, 1.2 or 1.5 channels were assessed using a membrane potential-sensitive fluorescent dye high-throughput assay. The effect of CBG on pentyleneterazole-induced (PTZ) seizures was assessed in rat. CBD (10M) blocked NaV currents in SH-SY5Y cells, mouse cortical neurons and recombinant cell lines, and affected spike parameters in rat CA1 neurons; CBD also significantly decreased membrane resistance. CBG blocked NaV to a similar degree to CBD in both SH-SY5Y and mouse recordings, but had no effect (50-200mg/kg) on PTZ-induced seizures in rat. CBD and CBG are NaV channel blockers at micromolar concentrations in human and murine neurons and recombinant cells. In contrast to previous reports investigating CBD, CBG had no effect upon PTZ-induced seizures in rat, indicating that NaV blockade per se does not correlate with anticonvulsant effects.

Molecular, cellular and physiological investigation of myostatin propeptide-mediated muscle growth in adult mice

Inhibition of myostatin signalling or its biological activity has recently emerged as a potential remedial approach against muscle wasting and degenerative diseases such as muscular dystrophies. In the present study we systemically administered a recombinant AAV8 vector expressing a mutated myostatin propeptide (AAV8ProMyo) to healthy mice in order to assess its impact on the histological, cellular and physiological properties of the skeletal muscle, exploiting the fact that myostatin is naturally inhibited by its own propeptide. We report that a single intravenous administration of AAV8ProMyo leads to increases in muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection. Moreover, treatment resulted in muscle fibre hypertrophy but not hyperplasia, with IIB myofibres responding to the greatest extent following propeptide-induced myostatin inhibition. Additionally, myofibre nuclear: cytoplasmic ratio was decreased in the AAV8ProMyo treated animals. Importantly, the hypertrophic EDL muscle 8 weeks after AAV8ProMyo treatment did not show the dramatic decrease in specific force displayed by the germline myostatin null mice. (C) 2009 Elsevier B.V. All rights reserved.

Lack of myostatin results in excessive muscle growth but impaired force generation

The lack of myostatin promotes growth of skeletal muscle, and blockade of its activity has been proposed as a treatment for various muscle-wasting disorders. Here, we have examined two independent mouse lines that harbor mutations in the myostatin gene, constitutive null (Mstn(-/-)) and compact (Berlin High Line, BEH(c/c)). We report that, despite a larger muscle mass relative to age-matched wild types, there was no increase in maximum tetanic force generation, but that when expressed as a function of muscle size (specific force), muscles of myostatin-deficient mice were weaker than wild-type muscles. In addition, Mstn(-/-) muscle contracted and relaxed faster during a single twitch and had a marked increase in the number of type IIb fibers relative to wild-type controls. This change was also accompanied by a significant increase in type IIB fibers containing tubular aggregates. Moreover, the ratio of mitochondrial DNA to nuclear DNA and mitochondria number were decreased in myostatin-deficient muscle, suggesting a mitochondrial depletion. Overall, our results suggest that lack of myostatin compromises force production in association with loss of oxidative characteristics of skeletal muscle.

Lymph heart in chick - somitic origin, development and embryonic oedema

The lymph heart is a sac-like structure on either side of avian tail. In some adult birds, it empties the lymph from the copulatory organ; however, during embryonic development, it is thought to circulate extra-embryonic lymph. Very little is known about the origin, innervation and the cellular changes it undergoes during development. Using immunohistochemistry and gene expression profiling we show that the musculature of the lymph heart is initially composed solely of striated skeletal muscle but later develops an additional layer composed of smooth myofibroblasts. Chick-quail fate-mapping demonstrates that the lymph heart originates from the hypaxial compartments of somites 34-41. The embryonic lymph heart is transiently innervated by somatic motoneurons with no autonomic input. In comparison to body muscles, the lymph heart has different sensitivity to neuromuscular junction blockers (sensitive only to decamethonium). Furthermore, its abundant bungarotoxin-positive acetylcholinesterase receptors are unique as they completely lack specific acetylcholinesterase activity. Several lines of evidence suggest that the lymph heart may possess an intrinsic pacing mechanism. Finally, we assessed the function of the lymph heart during embryogenesis and demonstrate that it is responsible for preventing embryonic oedema in birds, a role previously thought to be played by body skeletal muscle contractions.

Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). In vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.

The effects of paeonol on the electrophysiological properties of cardiac ventricular myocytes

Previous studies have shown that "Mudanpi", a Chinese herbal medicine, has a significant cardioprotective effect against myocardial ischaemia. Based on these findings we hypothesised that paeonol, the main component of Mudanpi, might have an effect on the cellular electrophysiology of cardiac ventricular myocytes. The effects of paeonol on the action potential and ion channels of cardiac ventricular myocytes were studied using the standard whole-cell configuration of the patch-clamp technique. Ventricular myocytes were isolated from the hearts of adult guinea-pig by enzymic dispersion. The myocytes were continuously perfused with various experimental solutions at room temperature and paeonol applied in the perfusate. Action potentials and membrane currents were recorded using both current and voltage clamp modes of the patch-clamp technique. Paeonol, at concentrations 160 mu M and 640 mu M, decreased the action potential upstroke phase, an action associated with the blockade of the voltage-gated, fast sodium channel. The effects of paeonol on both action potential and Na+ current were concentration dependent. Paeonol had a high affinity for inactivated sodium channels. Paeonol also shortened the action potential duration, in a manner not associated with the blockade of the calcium current, or the enhancement of potassium currents. These findings suggest that paeonol, and therefore Mudanpi, may possess antiarrhythmic activity, which may confer its cardioprotective effects. (c) 2006 Elsevier B.V All rights reserved.

Determining feature relevance for the grouping of motor unit action potentials through generative topographic mapping

The study of motor unit action potential (MUAP) activity from electrornyographic signals is an important stage on neurological investigations that aim to understand the state of the neuromuscular system. In this context, the identification and clustering of MUAPs that exhibit common characteristics, and the assessment of which data features are most relevant for the definition of such cluster structure are central issues. In this paper, we propose the application of an unsupervised Feature Relevance Determination (FRD) method to the analysis of experimental MUAPs obtained from healthy human subjects. In contrast to approaches that require the knowledge of a priori information from the data, this FRD method is embedded on a constrained mixture model, known as Generative Topographic Mapping, which simultaneously performs clustering and visualization of MUAPs. The experimental results of the analysis of a data set consisting of MUAPs measured from the surface of the First Dorsal Interosseous, a hand muscle, indicate that the MUAP features corresponding to the hyperpolarization period in the physisiological process of generation of muscle fibre action potentials are consistently estimated as the most relevant and, therefore, as those that should be paid preferential attention for the interpretation of the MUAP groupings.

Upper limb tremor suppression in ADL via an orthosis incorporating a controllable double viscous beam actuator

Neuromuscular disorders affect millions of people world-wide. Upper limb tremor is a common symptom, and due to its complex aetiology it is difficult to compensate for except, in particular cases by surgical intervention or drug therapy. Wearable devices that mechanically compensate for limb tremor could benefit a considerable number of patients, but the technology to assist suffers in this way is under-developed. In this paper we propose an innovative orthosis that can dynamically suppress pathological tremor, by applying viscous damping to the affected limb in a controlled manner. The orthosis design utilises a new actuator design based on Magneto-Rheological Fluids that efficiently deliver damping action in response to the instantaneous tremor frequency and amplitude.

The application of the Hilbert spectrum to the analysis of electromyographic signals

This paper investigates the application of the Hilbert spectrum (HS), which is a recent tool for the analysis of nonlinear and nonstationary time-series, to the study of electromyographic (EMG) signals. The HS allows for the visualization of the energy of signals through a joint time-frequency representation. In this work we illustrate the use of the HS in two distinct applications. The first is for feature extraction from EMG signals. Our results showed that the instantaneous mean frequency (IMNF) estimated from the HS is a relevant feature to clinical practice. We found that the median of the IMNF reduces when the force level of the muscle contraction increases. In the second application we investigated the use of the HS for detection of motor unit action potentials (MUAPs). The detection of MUAPs is a basic step in EMG decomposition tools, which provide relevant information about the neuromuscular system through the morphology and firing time of MUAPs. We compared, visually, how MUAP activity is perceived on the HS with visualizations provided by some traditional (e.g. scalogram, spectrogram, Wigner-Ville) time-frequency distributions. Furthermore, an alternative visualization to the HS, for detection of MUAPs, is proposed and compared to a similar approach based on the continuous wavelet transform (CWT). Our results showed that both the proposed technique and the CWT allowed for a clear visualization of MUAP activity on the time-frequency distributions, whereas results obtained with the HS were the most difficult to interpret as they were extremely affected by spurious energy activity. (c) 2008 Elsevier Inc. All rights reserved.

Generative topographic mapping applied to clustering and visualization of motor unit action potentials

The identification and visualization of clusters formed by motor unit action potentials (MUAPs) is an essential step in investigations seeking to explain the control of the neuromuscular system. This work introduces the generative topographic mapping (GTM), a novel machine learning tool, for clustering of MUAPs, and also it extends the GTM technique to provide a way of visualizing MUAPs. The performance of GTM was compared to that of three other clustering methods: the self-organizing map (SOM), a Gaussian mixture model (GMM), and the neural-gas network (NGN). The results, based on the study of experimental MUAPs, showed that the rate of success of both GTM and SOM outperformed that of GMM and NGN, and also that GTM may in practice be used as a principled alternative to the SOM in the study of MUAPs. A visualization tool, which we called GTM grid, was devised for visualization of MUAPs lying in a high-dimensional space. The visualization provided by the GTM grid was compared to that obtained from principal component analysis (PCA). (c) 2005 Elsevier Ireland Ltd. All rights reserved.

The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.