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The Effects of Organophosphates in the Early Stages of Human Muscle Regeneration
Abstract
Myopathy is a frequent condition with organophosphate (OP) poisoning. Skeletal muscle responds to it by activating a regeneration process that starts from dormant mononuclear satellite cells located under the basal lamina of adult muscle fibers. The step that decisively determines the mass of regenerated muscle is proliferation of myoblasts, which develop from activated satellite cells. Our investigations were focused on the effects of OPs on these early myogenic precursors. The OP effects-diisopropylphosphorofluoridate (DFP) was used in most of our experiments-were studied in the in vitro model that reproduces all stages of human muscle regeneration. We found decreased IL-6 release from myoblasts exposed to DFP, which might directly impair muscle regeneration because IL-6 promotes myoblast proliferation. A cellular response to stress indicated by increased levels of heat shock proteins was observed in DFP-treated myoblasts. Hypoxia, which can be severe in OP poisoning, led to a marked increase of hypoxia-inducible factor-1α expression in myoblasts. Recently, we identified a neuropathy target esterase-related enzyme in our muscle cultures; its inhibition by DFP opens new explanations of the OP effects in skeletal muscle. However, de novo AChE synthesis in human myoblasts was not altered by DFP. The described interactions of OPs with various systems in human myoblasts might have important consequences for muscle regeneration.
... B. Micrographic illustrations of coculture stages: mononucleated myoblasts, multinucleated myotubes, myotubes co-cultured with embryonic rat spinal cord explant with innervation area; mature innervated and contracting myotube with cross striations seen at higher magnification (insert). Adopted from [42,46,132]. For definition of the contracting unit see Table 1. ...
... B. Micrographic illustrations of co-culture stages: mononucleated myoblasts, multinucleated myotubes, myotubes co-cultured with embryonic rat spinal cord explant with innervation area; mature innervated and contracting myotube with cross striations seen at higher magnification (insert). Adopted from [42,46,132]. For definition of the contracting unit see Table 1. ...
... These data suggest that AChE-R variant might play a key role in modulation of myoblast apoptosis. However, on the other hand, in cultured myoblasts AChE-T is the most abundant AChE isoform and represents a major fraction of the total AChE activity [132]. A small increase of its expression may therefore have a greater impact on myoblast apoptosis than a marked increase in expression of AChE-R. ...
Acetylcholinesterase (AChE) and agrin, a heparan-sulfate proteoglycan, reside in the basal lamina of the neuromuscular junction (NMJ) and play key roles in cholinergic transmission and synaptogenesis. Unlike most NMJ components, AChE and agrin are expressed in skeletal muscle and α-motor neurons. AChE and agrin are also expressed in various other types of cells, where they have important alternative functions that are not related to their classical roles in NMJ. In this review, we first focus on co-cultures of embryonic rat spinal cord explants with human skeletal muscle cells as an experimental model to study functional innervation in vitro. We describe how this heterologous rat-human model, which enables experimentation on highly developed contracting human myotubes, offers unique opportunities for AChE and agrin research. We then highlight innovative approaches that were used to address salient questions regarding expression and alternative functions of AChE and agrin in developing human skeletal muscle. Results obtained in co-cultures are compared with those obtained in other models in the context of general advances in the field of AChE and agrin neurobiology.
... PNPLA research is yet to gather information about the basic kinetic properties, substrate preferences, and the mechanisms of regulation at the gene and protein level to help design prospective anti-PNPLA acting drugs (1,12). One direction in kinetic research, for example, could be to use tissue or cell lysates with overexpressed enzyme and a substrate specific enough to determine and distinguish PNPLA catalytic activity (16,142,143). However, a better approach would be to purify the active form of these enzymes for kinetic characterisation. ...
This paper brings a brief review of the human patatin-like phospholipase domain-containing protein (PNPLA) family. Even though it consists of only nine members, their physiological roles and mechanisms of their catalytic activity are not fully understood. However, the results of a number of knock-out and gain- or loss-of-function research models suggest that these enzymes have an important role in maintaining the homeostasis and integrity of organelle membranes, in cell growth, signalling, cell death, and the metabolism of lipids such as triacylglycerol, phospholipids, ceramides, and retinyl esters. Research has also revealed a connection between PNPLA family member mutations or irregular catalytic activity and the development of various diseases. Here we summarise important findings published so far and discuss their structure, localisation in the cell, distribution in the tissues, specificity for substrates, and their potential physiological role, especially in view of their potential as drug targets.
... Besides, OP poisoning is associated with cardiac toxicity and irregular ECGs, which along with respiratory distress can lead to hypotension, hypoperfusion, and electrolyte disorders. These factors may be the main causes of respiratory and metabolic acidosis in acute OP poisoning Mars et al. 2015). According to the findings of a study assessing acid-base status in predicting mortality in patients with acute pesticide poisoning, all parameters related to acid-base balance, except partial pressure of carbon dioxide, might be a reliable predictor for mortality in patients with OP poisoning (Gil et al. 2021). ...
Poisoning with organophosphate compounds is a significant public health risk, especially in developing countries. Considering the importance of early and accurate prediction of organophosphate poisoning prognosis, the aim of this study was to develop a machine learning-based prediction model to predict the severity of organophosphate poisoning. The data of patients with organophosphate poisoning were retrospectively extracted and split into training and test sets in a ratio of 70:30. The feature selection was done by least absolute shrinkage and selection operator method. Selected features were fed into five machine learning techniques, including Histogram Boosting Gradient, eXtreme Gradient Boosting, K-Nearest Neighborhood, Support Vector Machine (SVM) (kernel = linear), and Random Forest. The Scikit-learn library in Python programming language was used to implement the models. Finally, the performance of developed models was measured using ten-fold cross-validation methods and some evaluation criteria with 95% confidence intervals. A total of 1237 patients were used to train and test the machine learning models. According to the criteria determining severe organophosphate poisoning, 732 patients were assigned to group 1 (patients with mild to moderate poisoning) and 505 patients were assigned to group 2 (patients with severe poisoning). With an AUC value of 0.907 (95% CI 0.89 to 0.92), the model developed using XGBoost outperformed other models. Feature importance evaluation found that venous blood gas-pH, white blood cells, and plasma cholinesterase activity were the top three variables that contribute the most to the prediction performance of the prognosis in patients with organophosphate poisoning. XGBoost model yield an accuracy of 90.1% (95% CI 0.891 to 0.918), specificity of 91.4% (95% CI 0.90 to 0.92), a sensitivity of 89.5% (95% CI 0.87 to 0.91), F-measure of 91.2% (95% CI 0.90 to 0.921), and Kappa statistic of 91.2% (95% CI 0.90 to 0.92). The machine learning-based prediction models can accurately predict the severity of organophosphate poisoning. Based on feature selection techniques, the most important predictors of organophosphate poisoning were VBG-pH, white blood cell count, plasma cholinesterase activity, VBG-BE, and age. The best algorithm with the highest predictive performance was the XGBoost classifier.
Diverse stress stimuli induce long-lasting cognitive deficits, but the underlying molecular mechanisms are still incompletely understood. Here, we report three different stress models demonstrating that stress-inducible increases in microRNA-132 (miR-132) and consequent decreases in its acetylcholinesterase (AChE) target are causally involved. In a mild model of predator scent-induced anxiety, we demonstrate long-lasting hippocampal elevation of miR-132, accompanied by and associated with reduced AChE activity. Using lentiviral-mediated suppression of “synaptic” AChE-S mRNA, we quantified footshock stress-inducible changes in miR-132 and AChE and its corresponding cognitive damages. Stressed mice showed long-lasting impairments in the Morris water maze. In contrast, pre-stress injected AChE-suppressing lentivirus, but not a control virus, reduced hippocampal levels of both miR-132 and AChE and maintained similar cognitive performance to that of naïve, non-stressed mice. To dissociate between miR-132 and synaptic AChE-S as potential causes for stress-inducible cognitive deficits, we further used engineered TgR mice with enforced over-expression of the soluble “readthrough” AChE-R variant without the 3′-untranslated region binding site for miR-132. TgR mice displayed excess AChE-R in hippocampal neurons, enhanced c-fos labeling and correspondingly intensified reaction to the cholinergic agonist pilocarpine. They further showed excessive hippocampal expression of miR-132, accompanied by reduced host AChE-S mRNA and the GTPase activator p250GAP target of miR-132. At the behavioral level, TgR mice showed abnormal nocturnal locomotion patterns and serial maze mal-performance in spite of their reduced AChE-S levels. Our findings attribute stress-inducible cognitive impairments to cholinergic-mediated induction of miR-132 and consequently suppressed ACHE-S, opening venues for intercepting these miR-132-mediated damages.
Electronic supplementary material
The online version of this article (doi:10.1007/s00429-011-0376-z) contains supplementary material, which is available to authorized users.
Endotoxin stimulation of the immune system produces marked alterations in memory functioning. However, molecular links between this cognitive response and infection-responding neurotransmission pathways are still unknown. The cytokine and memory responses of volunteers injected with 0.8 ng/kg Salmonella endotoxin were compared with changes in plasma levels and integrity of the stress-induced acetylcholinesterase variant, AChE-R. Vascular endothelial cells were found to express AChE-R messenger RNA and protein both in healthy and inflamed human tissues. Plasma AChE activity was reduced after endotoxin treatment, but not placebo treatment, parallel to the decline in cortisol after the endotoxin-induced peak and inversely to the accumulation of a C-terminal immunopositive AChE-R peptide of 36 amino acid residues. AChE-R cleavage coincided with significant endotoxin-induced improvement in working memory and impairment in declarative memory. By 3 h posttreatment, working memory improvement was negatively correlated with AChE-R cleavage, which showed association to proinflammatory cytokine levels. By 9 h posttreatment, declarative memory impairment was negatively correlated with AChE-R cleavage and positively correlated with the suppressed AChE activity. Endotoxin-induced peripheral cholinergic stress responses are hence associated with greater impairment in declarative memory and lower improvement in working memory, pointing at AChE-R as a surrogate marker of psychoneuroimmunological stress.
Cholinergic Status, the total soluble circulation capacity for acetylcholine hydrolysis, was tested for putative involvement in individual variabilities of the recruitment of immune cells in response to endotoxin challenge. Young (average age 26) and elderly (average age 70) volunteers injected with either Escherichia coli endotoxin or saline on two different occasions were first designated Enhancers and Suppressors if they showed increase or decrease, respectively, in plasma acetylcholinesterase (AChE) activity 1.5 h after endotoxin administration compared to saline. Enhancers showed significant co-increases in plasma butyrylcholinesterase (BChE) and paraoxonase (PON1) activities, accompanied by rapid recovery of lymphocyte counts. Young Enhancers alone showed pronounced post-exposure increases in the pro-inflammatory cytokine interleukin-6 (IL-6), and upregulation of the normally rare, stress-induced AChE-R variant, suggesting age-associated exhaustion of the cholinergic effects on recruiting innate immune reactions to endotoxin challenge. Importantly, IL-6 injected to young volunteers or administered in vitro to primary mononuclear blood cells caused upregulation of AChE, but not BChE or PON1, excluding it from being the sole cause for this extended response. Interestingly, Suppressors but not Enhancers showed improved post-exposure working memory performance, indicating that limited cholinergic reactions may be beneficial for cognition. Our findings establish Cholinergic Status modulations as early facilitators and predictors of individual variabilities in the peripheral response to infection.
In the terminal axon of the motor end-plate in the diaphragm of albino rat we observed synaptic vesicles, mitochondria, and in some cases neurofilaments. Possibly the fibrillar components of the axoplasm are connected with the synaptic vesicles, which are located near the praesynaptic membrane. Probably there exists a functional relation between vesicles and neurofilaments.
In the diaphragm there appear degenerative changes when Soman (methyl-pinacolyloxy-phosphonylfluoride) has been applicated intraperitoneally. These are locally limited and extend to neuromuscular junctions as well as to muscle fibres. The extent of injury is very different in the various regions of necrosis, but in most cases there appears a dissolution of sarcolemma in the subneural infoldings probably preventing the production of acetylcholinesterase. In a more progressive state of poisoning the ultrastructural components of the muscle fiber are also injured. The destruction of muscle tissue caused by Soman is characterized by swelling of mitochondria, dissolving of its cristae, formation of myelin figures in mitochondria, pyknosis of nuclei, disappearing of the striation of muscle, and finally the decay of myofilaments.
Recovery of AChE activity in the motor end plate region and end plate free region of the rat diaphragm was studied after irreversible inhibition by soman. Recovery was slow during the first 2 days and only 4 S and 10 S molecular forms of AChE were present in the end plate region. However, cytochemical evidence indicates that synaptic AChE has already started to accumulate and that the synthesis of AChE in muscle and Schwann cell might even be enhanced. Tubular structures, observed underneath the motor end plate, may serve to transport the enzyme from its sites of synthesis in the sarcoplasmic reticulum. Asymmetric molecular forms of AChE in he end plate region appeared later during recovery and, one week after poisoning, their activity was only about 50% of normal value. The limited ability of newly synthesized AChE to attach to the subcellular structures and, therefore, be retained in the muscle, may explain the phase of slow recovery. In accordance with this view, AChE activity in brain recovered in a similar way as in muscle, whereas soluble plasma cholinesterases recovered faster, apparently without a slow initial phase.
Motor axons extending from embryonic rat spinal cord explants form fully mature neuromuscular junctions with cocultured human muscle. This degree of maturation is not observed in muscle innervated by dissociated motor neurons. Glial cells present in the spinal cord explants seem to be, besides remaining interneurons, the major difference between the two culture systems. In light of this observation and the well documented role of glia in neuronal development, it can be hypothesized that differentiated and long-lived neuromuscular junctions form in vitro only if their formation is accompanied by codifferentiation of neuronal and glial cells and if this codifferentiation follows the spatial and temporal pattern observed in vivo. Investigation of this hypothesis necessitates the characterization of neuronal and glial cell development in spinal cord explant-muscle cocultures. No such study has been reported, although these cocultures have been used in numerous studies of neuromuscular junction formation. The aim of this work was therefore to investigate the temporal relationship between neuromuscular junction formation and the differentiation of neuronal and glial cells during the first 3 weeks of coculture, when formation and development of the neuromuscular junction occurs in vitro. The expression of stage-specific markers of neuronal and glial differentiation in these cocultures was characterized by immunocytochemical and biochemical analyses. Differentiation of astrocytes, Schwann cells, and oligodendrocytes proceeded in concert with the differentiation of motor neurons and neuromuscular junction formation. The temporal coincidence between maturation of the neuromuscular junction and lineage progression of neurons and glial cells was similar to that observed in vivo. These findings support the hypothesis that glial cells are a major contributor to maturity of the neuromuscular junction formed in vitro in spinal cord explant-muscle cocultures.
Oxidative stress, as determined by increased lipid peroxidation, has been implicated in the pathology of myotoxicity. As a model system to study the response of muscle to oxidative insults, we have studied the effects of diisopropylphosphorofluoridate (DFP)-induced muscle hyperactivity on levels of nitric oxide (NO) and energy metabolites in rat skeletal muscles. In in vivo experiments, citrulline levels as indicators of NO and NO synthase (NOS), and ATP and phosphocreatine (PCr) as indicators of mitochondrial dysfunction, were determined using HPLC methods 15 min, 30 min, 60 min, 2 h, and 24 h after intoxication. Within 15 min of DFP exposure, with onset of fasciculations, citrulline levels were significantly elevated in all three muscles [soleus, extensor digitorum longus (EDL), and diaphragm]. Maximum increases in citrulline (272-288%) were noted 60 min after DFP injection. At this time point, acetylcholinesterase activity was reduced by 90-96% (soleus < diaphragm < EDL). The levels of ATP and PCr were maximally reduced (30-43%), and total adenine nucleotides, and total creatine compounds showed declines. The findings revealed that the increase in NOS activity and NO was greater than the decrease of ATP and PCr. Since memantine (MEM) has been shown to reduce nerve and muscle hyperactivity, we have studied the possible protective effect of MEM on the DFP-induced biochemical changes. Pretreatment with MEM (18 mg/kg s.c.) and atropine sulfate (16 mg/kg s.c.), 60 min and 15 min, respectively, before DFP injection prevented the increase in citrulline and muscle hyperactivity and the decrease in ATP and PCr. These data suggest that free radical reactions by depleting high-energy phosphates may be initiating the cascade of events leading to myotoxicity during DFP-induced muscle hyperactivity.
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.
Critical illness, more precisely defined as the systemic inflammatory response syndrome (SIRS), occurs in 20%-50% of patients who have been on mechanical ventilation for more than 1 week in an intensive care unit. Critical illness polyneuropathy (CIP) and myopathy (CIM), singly or in combination, occur commonly in these patients and present as limb weakness and difficulty in weaning from the ventilator. Critical illness myopathy can be subdivided into thick-filament (myosin) loss, cachectic myopathy, acute rhabdomyolysis, and acute necrotizing myopathy of intensive care. SIRS is the predominant underlying factor in CIP and is likely a factor in CIM even though the effects of neuromuscular blocking agents and steroids predominate in CIM. Identification and characterization of the polyneuropathy and myopathy depend upon neurological examination, electrophysiological studies, measurement of serum creatine kinase, and, if features suggest a myopathy, muscle biopsy. The information is valuable in deciding treatment and prognosis.
Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that functions as a master regulator of oxygen homeostasis in all metazoan species. O(2)-dependent hydroxylation of two proline residues in the HIF-1alpha subunit is necessary for the binding of the von Hippel-Lindau (VHL) protein, which is a component of a ubiquitin protein ligase that ubiquitinates HIF-1alpha, leading to its degradation by the proteasome. In the majority of cases of the clear cell type of renal carcinoma, both VHL genes are inactivated by mutation or epigenetic silencing, leading to dysregulated HIF-1 transcriptional activity. VHL loss-of-function leads, under aerobic conditions, to a HIF-1-dependent reprogramming of glucose and energy metabolism that includes increased glucose uptake, glycolysis, and lactate production accompanied by a reciprocal decrease in respiration. These findings delineate for the first time the molecular mechanisms underlying the Warburg effect in a human cancer.