Tomaž Marš

Functional innervation of cultured human skeletal muscle proceeds by two modes with regard to agrin effects

Nerve-derived agrin is a specific isoform of agrin that promotes clustering of nicotinic acetylcholine receptors (AChR) and other components of the neuromuscular junction (NMJ). We investigated the effects of agrin on functional maturation of NMJs at the early stages of synaptogenesis in human muscle. Specifically, we assessed the importance of agrin for the differentiation of developing NMJs to the stage where they are able to transmit signals that result in contractions of myotubes. We utilized an in vitro model in which human myotubes are innervated by neurons extending from spinal cord explants of fetal rat. This model is suitable for functional studies because all muscle contractions are the result of neuromuscular transmission and can be quantitated. An anti-agrin antibody, Agr 33, was applied to co-cultures during de novo NMJ formation. Quantitative analyses demonstrated that Agr 33 reduced the number of AChR clusters to 20% and their long axes to 50% of control, yet still permitted early, NMJ-mediated muscle contractions that are normally observed in 7-10-day-old co-cultures. However, at later times of development, the same treatment completely prevented the increase in the number of contracting units as compared with untreated co-cultures. It is concluded that there are two modes of functional maturation of NMJs with regard to agrin effects: one that is insensitive and the other that is sensitive to agrin blockade. Agrin-insensitive mode is limited to the small population of NMJs that become functional at the earlier stages of functional innervation. However, most of the NMJs become contraction-competent at the later stages of the innervation process. These NMJs become functional only if agrin action is uncompromised. This is the first characterization of the contribution of agrin to NMJ development on human muscle.

Total plasma sulfide as a marker of shock severity in nonsurgical adult patients.

Previous animal and human studies have suggested that total plasma sulfide plays a role in the pathophysiology of shock. This studys aim was to determine the value of total plasma sulfide as a marker of shock severity in nonsurgical adult patients admitted to the ICU. Forty-one patients, with various types of shock (septic, cardiogenic, obstructive, and hypovolemic), were included in the study, with an average total plasma sulfide concentration of 23.2 ± 26.3 µM. Survivors (of shock) had lower total plasma sulfide concentrations than nonsurvivors (13.0 ± 26.3 vs. 31.9 ± 31.5 µM; P = 0.02). Total plasma sulfide correlated with dose of administered norepinephrine (R2 linear = 0.829; P = 0.001) and with Acute Physiology and Chronic Health Evaluation II (APACHE II) score (R2 cubic = 0.767; P = 0.001). Area under the receiver operating characteristic for total plasma sulfide as a predictor of ICU mortality was 0.739 (confidence interval, 0.587-0.892; P = 0.009). Even after correcting for APACHE II score and lactate values, total plasma sulfide correlated with mortality (odds ratio, 1.058; 95% confidence interval, 1.001-1.118; P = 0.045). The study provides evidence that, in nonsurgical adult ICU patients admitted because of any type of shock, total plasma sulfide correlates with administered norepinephrine dose at admission, severity of disease (APACHE II score ≥30 points), and survival outcome.

Aging impairs collateral sprouting of nociceptive axons in the rat

Sprouting of uninjured nociceptive axons was examined in young adult, middle aged and aged rats. Axon sprouting from the spared sural nerve, both into adjacent denervated skin and into end-to-side coapted nerve graft, was significantly higher in young rats than in aged rats. Cross-transplantations of the end-to-side coapted nerve grafts between young and aged rats demonstrated that axon sprouting from young recipient nerves into aged donor nerve grafts was significantly deteriorated, whereas the axon sprouting from aged recipient nerves into young donor nerve grafts was not statistically significantly affected. The levels of laminin polypeptides in peripheral nerves were 50-100% higher in young adult than in aged rats. However, the levels of peripherin, NGF isoforms and TrkA in skin, peripheral nerves and DRG, respectively, were not significantly reduced in aged rats. Therefore, impaired sprouting of nociceptive axons in aged rats is due rather to the alterations in peripheral neural pathways, than to the limited sprouting capacity of aged sensory neurons. Decreased levels of extracellular matrix components might be important in this respect.

The cholinergic and non-cholinergic effects of organophosphates and oximes in cultured human myoblasts

Organophosphorus compounds (OPs) and oximes may interfere with other molecules than AChE in the living systems, affecting in this way various cellular processes and underlying mechanisms. These non-cholinergic effects may contribute to the clinical status in OP poisoning and therefore deserve equal scientific attention. Here, we investigated the effects of tabun and oxime K048 on the processes known to be involved in muscle response to the environmental factors, like IL-6 release and the regulation of the heat shock proteins (HSPs). While IL-6 stimulates muscle regeneration, which follows well known OP-induced myopathy, HSPs have cytoprotective effect against various stress factors including xenobiotics. All our experiments were carried out on cultured human myoblasts, as the precursors of muscle regeneration. We found unchanged AChE mRNA level after tabun/K048 treatment meaning that tabun and K048 did not interfere with the transcription or stability of this mRNA in the time period tested, even if AChE catalytic activity was significantly affected. On the other hand, after myoblast exposure to tabun, we observed significant changes in the protein levels of HSP 27 and in the secretion of IL-6. Namely, secretion of IL-6 decreased to 53% and the level of HSP 27 increased by 34% compared to the control level. Both effects were attenuated if myoblasts were pretreated with oxime K048, but not if they were treated with K048 after exposure to tabun. The molecular mechanism underlying these effects remains to be elucidated. However, it seems that these effects could be associated with OPs and oximes as a specific group of compounds rather than as a specific compound itself. Overall, the effects of OPs and oximes demonstrated here might play an important role in muscle regeneration which importantly determines the final outcome of OP myotoxicity.

Apoptosis in co-cultures of human muscle and rat spinal cord during the formation of the neuromuscular junction

Abstract The motor neurons and skeletal muscle fibers they innervate are strongly dependent on each other. Important communication between both tissues is mediated through the neuromuscular junction. However, release and reception of various factors at other parts of both tissues must also be considered as means of mutual influences. In vitro innervated human muscle is the only experimental model to investigate nerve - muscle interactions during synaptogenesis of human neuromuscular junction. Here we studied the occurrence of programmed cell death in this model. In order to find out if mutual interactions between neurons and myotubes control the extent of apoptosis in both tissues, we compared the number of apoptotic cells in spinal cord explants and myotubes in co-cultures and mono-cultures of these tissues. Apoptotic cells were detected by TUNEL at selected time intervals (10, 21 and 30 days after co-culture) coresponding to various stages of synaptogenesis of neuromuscular junction. The number of apoptotic cells in spinal cord explants was higher at earlier (day 10) in comparison to later stages (days 21 and 30). Co-cultures and mono-cultures did not differ in this respect. TUNEL positive cells were not found in mono-cultured human myotubes under our experimental conditions. These results suggest that the process of apoptosis seems to be rather independent on neuron - muscle interactions at least at the time points examine.

Morphometric characteristics of myonuclear distribution in the normal and denervated fast rat muscle fiber

Unlike the majority of mammalian tissues, which have mononuclear cells as a basic unit of the tissue composition, skeletal muscle fiber is a polynuclear syncytium. Polynuclear organization offers an additional possibility for the regulation of protein expression: it can also be controlled at the level of myonuclear distribution and specialization. Distribution of myonuclei can be considered as the distribution of genes. Variability in gene concentration may have important impact on the regional differentiation of the muscle fiber, since it leads to different regional concentrations of various gene products including factors controlling their expression. The aim of the present study was: 1) to provide morphometrical data on the myonuclear distribution in the junctional and extrajunctional regions of the normal fast rat muscle fiber, and 2) to analyze, whether morphometrical parameters of nuclear distribution change after mechanical denervation of this muscle. Single muscle fibers were isolated from the normal and denervated fast rat m. sternomastoideus. Their neuromuscular junctions were stained by thiocholine histochemical procedure and myonuclei were fluorescently labeled by Hoechst 33342. Myonuclear distribution in each individual muscle fiber was morphometrically analyzed on the image analyzer. Synaptic concentrations of myonuclei were found to exceed extrasynaptic concentrations by a factor of 17. The number of myonuclei accumulated at the endplates did not change after one or two weeks of denervation, neither did the morphometric parameters of these nuclei. A higher concentration of myonuclei due to muscle atrophy was observed in the extrasynaptic region and the longitudinal axis of these nuclei also became significantly shorter. Unchanged morphometric parameters of the junctional myonuclei after denervation are indicative of either irreversibility of the nerve-induced formation of nuclear clusters in this region or persistence of the factors responsible for their formation and maintenance.

Heparin blocks functional innervation of cultured human muscle by rat motor nerve

In vitro innervated human muscle is the only experimental model to study synaptogenesis of the neuromuscular junction in humans. Cultured human muscle never contracts spontaneously but will if innervated and therefore is a suitable model to study the effects of specific neural factors on the formation of functional neuromuscular contacts. Here, we tested the hypothesis that nerve derived factor agrin is essential for the formation of functional synapses between human myotubes and motoneurons growing from the expiant of embryonic rat spinal cord. Agrin actions were blocked by heparin and the formation of functional neuromuscular contacts was quantitated. At a heparin concentration of 25 µg/ml, the number of functional contacts was significantly reduced. At higher concentrations, formation of such contacts was blocked completely. Except at the highest heparin concentrations (150 µg/ml) neuronal outgrowth was normal indicating that blockade of neuromuscular junction formation was not due to neuronal dysfunction. Our results are in accord with the concept that binding of neural agrin to the synaptic basal lamina is essential for the formation of functional neuromuscular junctions in the human muscle.

Control levels of acetylcholinesterase expression in the mammalian skeletal muscle

Protein expression can be controled at different levels. Understanding acetylcholinesterase (EC. 3.1.1.7, AChE) expression in the living organisms therefore necessitates: (1) determination and mapping of control levels of AChE metabolism; (2) identification of the regulatory factors acting at these levels; and (3) detailed insight into the mechanisms of action of these factors. Here we summarize the results of our studies on the regulation of AChE expression in the mammalian skeletal muscle. Three experimental models were employed: in vitro innervated human muscle, mechanically denervated adult fast rat muscle, and the glucocorticoid treated fast rat muscle. In situ hybridization of AChE mRNA, combined with AChE histochemistry, revealed that different distribution patterns of AChE, observed during in vitro ontogenesis and synaptogenesis of human skeletal muscle, reflect alterations in the distribution of AChE mRNA (Z. Grubic, R. Komel, W.F. Walker, A.F. Miranda, Myoblast fusion and innervation with rat motor nerve alter the distribution of acetylcholinesterase and its mRNA in human muscle cultures, Neuron 14 (1995) 317-327). To study the mechanisms of AChE mRNA loss in denervated adult rat skeletal muscle, we exposed deproteinated AChE mRNA to various subcellular fractions in vitro. Fractions were isolated from the normal and denervated rat sternomastoideus muscle. We found significantly increased, but non-specific AChE mRNA degradation capacities in the three fractions studied, suggesting that increased susceptibility of muscle mRNA to degradation might be at least partly responsible for the decreased AChE mRNA observed under such conditions (K. Zajc-Kreft, S. Kreft, Z. Grubic, Degradation of AChE mRNA in the normal and denervated rat skeletal muscle, Book of Abstracts, The Sixth International Meeting on Cholinesterases, La Jolla, CA, March 20-24, 1998, p. A3.). In adult fast rat muscle, treated chronically with glucocorticoids, we found the fraction of early synthesized AChE molecular forms to be reduced and AChE mRNA unchanged. This observation is consistent with the explanation that translation and/or early post-translational processes are impaired under such conditions (M. Brank, K. Zajc-Kreft, S. Kreft, R. Komel, Z. Grubic, Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle, Eur. J. Biochem. 251 (1998) 374-381). The AChE mRNA level is therefore important but not the only control level of AChE expression in the mammalian skeletal muscle.

The prognostic value of micrometastases found intraoperatively in the first drainig lymph node in gastric cancer patients.

Background: The concept of sentinel lymph node screening has been recently introduced in gastric cancer treatment. Even through micrometastases can be shown reliably by imunohystochemistry, such staining methods are lengthy and laborious, which precludes its intraoperative use. In this study, the clinical and prognostic implications of a new single sentinel lymph node screening for micrometastases concept were evaluated on a small study group. Methods: Twenty-three patients were included in our study. Nine were selected as a control group. The first stained lymph node was defined as the true sentinel lymph node. This lymph node was sent separately for RT-qPCR analysis to determine CEA and CK-20 expression as markers of micrometastases. Patient and tumor characteristics were analysed and possible correlations with micrometastatic involvement were determined. Results: Fourteen patients were found to be N0. Four patients (28.6 %) had micrometastases. Micrometastases were more prominent in patients with diffuse gastric cancer, with higher CA 19–9 values. Patients with micrometastases were also found to be older than those without them. Conclusions: Even through these results indicate the potential use of a single SNL in intraoperative decision making, the sensitivity and specificity of our method has to be evaluated on a larger series, supported by long-term recurrence and survival results.

Insulin-induced exocytosis in single, in vitro innervated human muscle fibres: a new approach.

We describe a new approach for studying insulin-induced exocytosis in individual, well-differentiated, innervated human muscle fibres. We used an in vitro system in which motor axons extending from embryonic rat spinal cord explants functionally innervate co-cultured human muscle fibres. Under such conditions, the human muscle fibres reach a high degree of differentiation that is never observed in non-innervated, cultured human muscle fibres. To monitor insulin-induced membrane dynamics, we used confocal microscopy to measure the fluorescence intensity changes of the styryl dye FM1-43, a marker for membrane area. The fluorescence intensity increased after insulin stimulation. This increase, as well as the intensity of staining for the glucose transporter 4 (GLUT4), was significantly higher in the innervated and contracting fibres than in myoblasts and myotubes. This shows that in vitro innervation of human muscle cells not only enhances the differentiation stage but also improves the insulin response. Our approach allows continuous monitoring and quantitative assessment of insulin-induced increase in cumulative exocytosis in individual human muscle fibres at a differentiation level practically corresponding to that of adult muscle. It is therefore a suitable system for studying various parameters affecting the mechanisms underlying insulin-induced GLUT4 translocation in human skeletal muscle.