Katarina Mis

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.

Origin of acetylcholinesterase in the neuromuscular junction formed in the in vitro innervated human muscle.

Synaptic basal lamina is interposed between the pre‐ and postsynaptic membrane of the neuromuscular junction (NMJ). This position permits deposition of basal lamina‐bound NMJ components of both neuronal and muscle fibre origin. One such molecule is acetylcholinesterase (AChE). The origin of NMJ AChE has been investigated previously as the answer would elucidate the relative contributions of muscle fibers and motor neurons to NMJ formation. However, in the experimental models used in prior investigations either the neuronal or muscular components of the NMJs were removed, or the NMJs were poorly differentiated. Therefore, the question of AChE origin in the intact and functional NMJ remains open. Here, we have approached this question using an in vitro model in which motor neurons, growing from embryonic rat spinal cord explants, form well differentiated NMJs with cultured human myotubes. By immunocytochemical staining with species‐specific anti‐AChE antibodies, we are able to differentiate between human (muscular) and rat (neuronal) AChE at the NMJ. We observed strong signal at the NMJ after staining with human AChE antibodies, which suggests a significant muscular AChE contribution. However, a weaker, but still clearly recognizable signal is observed after staining with rat AChE antibodies, suggesting a smaller fraction of AChE was derived from motor neurons. This is the first report demonstrating that both motor neuron and myotube contribute synaptic AChE under conditions where they interact with each other in the formation of an intact and functional NMJ.

Acetylcholinesterase is involved in apoptosis in the precursors of human muscle regeneration

The best established role of acetylcholinesterase (EC 3.1.1.7, AChE) is termination of neurotransmission at cholinergic synapses. However, AChE is also located at sites, where no other cholinergic components are present and there is accumulating evidence for non-cholinergic functions of this protein. In the process of skeletal muscle formation, AChE is expressed already at the stage of mononuclear myoblast, which is long before other cholinergic components can be demonstrated in this tissue. Myoblast proliferation is an essential step in muscle regeneration and is always accompanied by apoptosis. Since there are several reports demonstrating AChE participation in apoptosis one can hypothesize that early AChE expression in myoblasts reflects the development of the apoptotic apparatus in these cells. Here we tested this hypothesis by following the effect of siRNA AChE silencing on apoptotic markers and by determination of AChE level after staurosporine-induced apoptosis in cultured human myoblasts. Decreased apoptosis in siRNA AChE silenced myoblasts and increased AChE expression in staurosporine-treated myoblasts confirmed AChE involvement in apoptosis. The three AChE splice variants were differently affected by staurosporine-induced apoptosis. The hydrophobic (H) variant appeared unaffected, a tendency towards increase of tailed (T) variant was detected, however the highest, 8-fold increase was observed for readthrough (R) variant. In the light of these findings AChE appears to be a potential therapeutic target at muscle injuries including organophosphate myopathy.

Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo.

Localization of mRNAs encoding acetylcholinesterase and butyrylcholinesterase in the rat spinal cord by nonradioactive in situ hybridization.

In spite of intensive investigations, the roles of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) in the central nervous system (CNS) remain unclear. A role recently proposed for BuChE as an explanation for survival of AChE knockout mice is compensation for AChE activity if it becomes insufficient. Neuronal contribution of both enzymes to the cholinesterase pool in the neuromuscular junction has also been suggested. These proposals imply that BuChE expression follows that of AChE and that, in addition to AChE, BuChE is also expressed in α-motor neurons. However, these assumptions have not yet been properly tested. Histochemical approaches to these problems have been hampered by a number of problems that prevent unambiguous interpretation of results. In situ hybridization (ISH) of mRNAs encoding AChE and BuChE, which is the state-of-the-art approach, has not yet been done. Here we describe rapid nonradioactive ISH for the localization of mRNAs encoding AChE and BuChE. Various probes and experimental conditions had been tested to obtain reliable localization. In combination with RT-PCR, ISH revealed that, in rat spinal cord, cells expressing AChE mRNA also express BuChE mRNA but in smaller quantities. α-Motor neurons had the highest levels of both mRNAs. Virtual absence of transcripts encoding AChE and BuChE in glia might reflect a discrepancy between mRNA and enzyme levels previously reported for cholinesterases.

HIF-1α Response to Hypoxia is Functionally Separated from the Glucocorticoid Stress Response in the in vitro Regenerating Human Skeletal Muscle

Injury of skeletal muscle is followed by muscle regeneration in which new muscle tissue is formed from the proliferating mononuclear myoblasts, and by systemic response to stress that exposes proliferating myoblasts to increased glucocorticoid (GC) concentration. Because of its various causes, hypoxia is a frequent condition affecting skeletal muscle, and therefore both processes, which importantly determine the outcome of the injury, often proceed under hypoxic conditions. It is therefore important to identify and characterize in proliferating human myoblasts: 1) response to hypoxia which is generally organized by hypoxia-inducible factor-1α (HIF-1α); 2) response to GCs which is mediated through the isoforms of glucocorticoid receptors (GRs) and 11β-hydroxysteroid dehydrogenases (11β-HSDs), and 3) the response to GCs under the hypoxic conditions and the influence of this combination on the factors controlling myoblast proliferation. Using real-time PCR, Western blotting, and HIF-1α small-interfering RNA silencing, we demonstrated that cultured human myoblasts possess both, the HIF-1α-based response to hypoxia, and the GC response system composed of GRα and types 1 and 2 11β-HSDs. However, using combined dexamethasone and hypoxia treatments, we demonstrated that these two systems operate practically without mutual interactions. A seemingly surprising separation of the two systems that both organize response to hypoxic stress can be explained on the evolutionary basis: the phylogenetically older HIF-1α response is a protection at the cellular level, whereas the GC stress response protects the organism as a whole. This necessitates actions, like downregulation of IL-6 secretion and vascular endothelial growth factor, that might not be of direct benefit for the affected myoblasts.

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.

Acetylcholinesterase and agrin: different functions, similar expression patterns, multiple roles.

Acetylcholinesterase (AChE) and agrin play unique functional roles in the neuromuscular junction (NMJ). AChE is a cholinergic and agrin a synaptogenetic component. In spite of their different functions, they share several common features: their targeting is determined by alternative splicing; unlike most other NMJ components they are expressed in both, muscle and motor neuron and both reside on the synaptic basal lamina of the NMJ. Also, both were reported to play various nonjunctional roles. However, while the origin of basal lamina bound agrin is undoubtedly neural, the neural origin of AChE, which is anchored to the basal lamina with collagenic tail ColQ, is elusive. Hypothesizing that motor neuron proteins targeted to the NMJ basal lamina share common temporal pattern of expression, which is coordinated with the formation of basal lamina, we compared expression of agrin isoforms with the expression of AChE-T and ColQ in the developing rat spinal cord at the stages before and after the formation of NMJ basal lamina. Cellular origin of AChE-T and agrin was determined by in situ hybridization and their quantitative levels by RT PCR. We found parallel increase in expression of the synaptogenetic (agrin 8) isoform of agrin and ColQ after the formation of basal lamina supporting the view that ColQ bound AChE and agrin 8 isoform are destined to the basal lamina. Catalytic AChE-T subunit and agrin isoforms 19 and 0 followed different expression patterns. In accordance with the reports of other authors, our investigations also revealed various alternative functions for AChE and agrin. We have already demonstrated participation of AChE in myoblast apoptosis; here we present the evidence that agrin promotes the maturation of heavy myosin chains and the excitation-contraction coupling. These results show that common features of AChE and agrin extend to their capacity to play multiple roles in muscle development.

siRNA delivery into cultured primary human myoblasts--optimization of electroporation parameters and theoretical analysis.

Introduction of genetic material into muscle tissue has been extensively researched, including isolation and in vitro expansion of primary myoblasts as a potential source of cells for skeletal and heart muscle tissue engineering applications. In this study, we optimized the electroporation protocol for introduction of short interfering ribonucleic acid (siRNA) against messenger RNA for Hypoxia Inducible Factor 1α (HIF-1α) into cultured primary human myoblasts. We established optimal pulsing protocol for siRNA electro transfection, and theoretically analyzed the effect of electric field and pulse duration on silencing efficiency and electrophoretic displacement of siRNA. Silencing of HIF-1α was determined with quantitative polymerase chain reaction and Western Blot. The most efficient silencing (71% knockdown) was achieved with 8 × 2 ms pulses, E = 0.6 kV/cm. Viability was determined immediately, 1 h and 48 h after electroporation. In general, there was a trade-off between efficient silencing and preserved viability. Electric field and pulse duration are crucial parameters for silencing, since both increase membrane permeabilization and electrophoretic transfer of siRNA. Short-term viability showed immediate toxicity of pulses due to membrane damage, while indirect effects on cell proliferation were observed after 48 h. Presented results are important for faster optimization of electroporation parameters for ex vivo electrotransfer of short RNA molecules into primary human myoblasts.

Cell stress response to two different types of polymer coated cobalt ferrite nanoparticles

Potential nanoparticle (NP) toxicity is one of crucial problems that limit the applicability of NPs. When designing NPs for biomedical and biotechnological applications it is thus important to understand the mechanisms of their toxicity. In this study, we analysed the stress responses of previously designed polyacrylic acid (PAA) and polyethylenimine (PEI) coated NPs on primary human myoblasts (MYO) and B16 mouse melanoma cell line. Negatively charged PAA did not induce cell toxicity, reactive oxygen species (ROS) or activate the transcription factor NF-κB in either cell line even at high concentrations (100μg/ml). On the other hand, positively charged PEI NPs induced a concentration dependent necrotic cell death and an increase in ROS following 24h incubation already at low concentrations (>4μg/ml). Moreover, PEI NPs induced NF-κB activation 15-30min after incubation in MYO cells, most probably through activation of TLR4 receptor. Interestingly, there was no NF-κB response to PEI NPs in B16 cells.