Disintegration of the NuRD Complex in Primary Human Muscle Stem Cells in Critical Illness Myopathy.

Critical illness myopathy (CIM) is an acquired, devastating, multifactorial muscle-wasting disease with incomplete recovery. The impact on hospital costs and permanent loss of quality of life is enormous. Incomplete recovery might imply that the function of muscle stem cells (MuSC) is impaired. We tested whether epigenetic alterations could be in part responsible. We characterized human muscle stem cells (MuSC) isolated from early CIM and analyzed epigenetic alterations (CIM n = 15, controls n = 21) by RNA-Seq, immunofluorescence, analysis of DNA repair, and ATAC-Seq. CIM-MuSC were transplanted into immunodeficient NOG mice to assess their regenerative potential. CIM-MuSC exhibited significant growth deficits, reduced ability to differentiate into myotubes, and impaired DNA repair. The chromatin structure was damaged, as characterized by alterations in mRNA of histone 1, depletion or dislocation of core proteins of nucleosome remodeling and deacetylase complex, and loosening of multiple nucleosome-spanning sites. Functionally, CIM-MuSC had a defect in building new muscle fibers. Further, MuSC obtained from the electrically stimulated muscle of CIM patients was very similar to control MuSC, indicating the impact of muscle contraction in the onset of CIM. CIM not only affects working skeletal muscle but has a lasting and severe epigenetic impact on MuSC.

Statins Aggravate the Risk of Insulin Resistance in Human Muscle.

Beside their beneficial effects on cardiovascular events, statins are thought to contribute to insulin resistance and type-2 diabetes. It is not known whether these effects are long-term events from statin-treatment or already triggered with the first statin-intake. Skeletal muscle is considered the main site for insulin-stimulated glucose uptake and therefore, a primary target for insulin resistance in the human body. We analyzed localization and expression of proteins related to GLUT4 mediated glucose uptake via AMPKα or AKT in human skeletal muscle tissue from patients with statin-intake >6 months and in primary human myotubes after 96 h statin treatment. The ratio for AMPKα activity significantly increased in human skeletal muscle cells treated with statins for long- and short-term. Furthermore, the insulin-stimulated counterpart, AKT, significantly decreased in activity and protein level, while GSK3ß and mTOR protein expression reduced in statin-treated primary human myotubes, only. However, GLUT4 was normally distributed whereas CAV3 was internalized from plasma membrane around the nucleus in statin-treated primary human myotubes. Statin-treatment activates AMPKα-dependent glucose uptake and remains active after long-term statin treatment. Permanent blocking of its insulin-dependent counterpart AKT activation may lead to metabolic inflexibility and insulin resistance in the long run and may be a direct consequence of statin-treatment.

[Diagnostics and treatment of statin-associated muscle symptoms]. / Diagnostik und Therapie von statinassoziierten Muskelsymptomen.

Statins are among the most frequently prescribed drugs in Germany. Their benefits in lowering cardiovascular risk are beyond dispute. Nevertheless, many patients complain of side effects from statin therapy, including statin-associated muscle symptoms (SAMS) in particular. Despite their relative frequency, it is difficult to objectively diagnose them, as the time until appearance of first symptoms, the nature of the complaints and the severity of muscle problems vary widely. This narrative review summarizes the causes of SAMS as well as new possibilities regarding their diagnosis and therapy.

Statin-induced myopathic changes in primary human muscle cells and reversal by a prostaglandin F2 alpha analogue.

Statin-related muscle side effects are a constant healthcare problem since patient compliance is dependent on side effects. Statins reduce plasma cholesterol levels and can prevent secondary cardiovascular diseases. Although statin-induced muscle damage has been studied, preventive or curative therapies are yet to be reported. We exposed primary human muscle cell populations (n = 22) to a lipophilic (simvastatin) and a hydrophilic (rosuvastatin) statin and analyzed their expressome. Data and pathway analyses included GOrilla, Reactome and DAVID. We measured mevalonate intracellularly and analyzed eicosanoid profiles secreted by human muscle cells. Functional assays included proliferation and differentiation quantification. More than 1800 transcripts and 900 proteins were differentially expressed after exposure to statins. Simvastatin had a stronger effect on the expressome than rosuvastatin, but both statins influenced cholesterol biosynthesis, fatty acid metabolism, eicosanoid synthesis, proliferation, and differentiation of human muscle cells. Cultured human muscle cells secreted ω-3 and ω-6 derived eicosanoids and prostaglandins. The ω-6 derived metabolites were found at higher levels secreted from simvastatin-treated primary human muscle cells. Eicosanoids rescued muscle cell differentiation. Our data suggest a new aspect on the role of skeletal muscle in cholesterol metabolism. For clinical practice, the addition of omega-n fatty acids might be suitable to prevent or treat statin-myopathy.

Human muscle-derived CLEC14A-positive cells regenerate muscle independent of PAX7.

Skeletal muscle stem cells, called satellite cells and defined by the transcription factor PAX7, are responsible for postnatal muscle growth, homeostasis and regeneration. Attempts to utilize the regenerative potential of muscle stem cells for therapeutic purposes so far failed. We previously established the existence of human PAX7-positive cell colonies with high regenerative potential. We now identified PAX7-negative human muscle-derived cell colonies also positive for the myogenic markers desmin and MYF5. These include cells from a patient with a homozygous PAX7 c.86-1G > A mutation (PAX7null). Single cell and bulk transcriptome analysis show high intra- and inter-donor heterogeneity and reveal the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7-negative cell populations, including PAX7null, form myofibers after transplantation into mice, and regenerate muscle after reinjury. Transplanted PAX7neg cells repopulate the satellite cell niche where they re-express PAX7, or, strikingly, CLEC14A. In conclusion, transplanted human cells do not depend on PAX7 for muscle regeneration.

A Molecular Signature of Myalgia in Myotonic Dystrophy 2.

BACKGROUND: Chronic muscle pain affects close to 20% of the population and is a major health burden. The underlying mechanisms of muscle pain are difficult to investigate as pain presents in patients with very diverse histories. Treatment options are therefore limited and not tailored to underlying mechanisms. To gain insight into the pathophysiology of myalgia we investigated a homogeneous group of patients suffering from myotonic dystrophy type 2 (DM2), a monogenic disorder presenting with myalgia in at least 50% of affected patients. METHODS: After IRB approval we performed an observational cross-sectional cohort study and recruited 42 patients with genetically confirmed DM2 plus 20 healthy age and gender matched control subjects. All participants were subjected to an extensive sensory-testing protocol. In addition, RNA sequencing was performed from 12 muscle biopsy specimens obtained from DM2 patients. FINDINGS: Clinical sensory testing as well as RNA sequencing clearly separated DM2 myalgic from non-myalgia patients and also from healthy controls. In particular pressure pain thresholds were significantly lowered for all muscles tested in myalgic DM2 patients but were not significantly different between non-myalgic patients and healthy controls. The expression of fourteen muscle expressed genes in myalgic patients was significantly up or down-regulated in myalgic compared to non-myalgic DM2 patients. INTERPRETATION: Our data support the idea that molecular changes in the muscles of DM2 patients are associated with muscle pain. Further studies should address whether muscle-specific molecular pathways play a significant role in myalgia in order to facilitate the development of mechanism-based therapeutic strategies to treat musculoskeletal pain. FUNDING: This study was funded by the German Research Society (DFG, GK1631), KAP programme of Charité Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine.

Hormesis is induced in the red flour beetle Tribolium castaneum through ingestion of charred toast.

PURPOSE: Charred foods are generally suspected to exert health threats by providing toxicants, such as acrylamide or polycyclic aromatic hydrocarbons. Using the red flour beetle Tribolium castaneum as a model organism, we tested its survival under heat stress in response to feeding charred toast. METHODS: Survival of beetles was measured at 42 °C after a pre-feeding phase with flour enriched with increasing concentrations of charred toast. In order to assess the influence of key transcription factors for phase-I and phase-II xenobiotic metabolism, gene homologs for ahr and nrf-2, respectively, were knocked down by the use of RNA interference (RNAi). RESULTS: Beetles fed only charred toast died off much earlier than control beetles fed on flour, whereas beetles fed flour enriched with 5% charred toast survived significantly longer than the control. Both, ahr and nrf-2 proved essential in order to enable the increase in survival by the feeding of 5% charred toast. Moreover, functional loss of ahr and nrf-2 made the beetles hypersensitive versus the feeding of 100% charred toast. Finally, at the transcriptional level, it was shown that RNAi for ahr blocked the inducing activities of charred toast on nrf-2. CONCLUSIONS: Our studies suggest a hormetic response of the red flour beetle to feeding of charred toast that causes an increased stress resistance through the activation of ahr and nrf-2. Those adaptations, however, are saturable and accordingly the hormetic effects at increasing concentrations of the toxicants become expended.

The red flour beetle Tribolium castaneum allows for the convenient determination of fitness and survival as a measure of toxic effects of the food contaminant acrylamide.

Acrylamide  is a toxic ingredient generated as a by-product of the Maillard reaction in starchy foods processed at temperatures above 120°C. Here we used the red flour beetle Tribolium castaneum as a model organism to test the effects of acrylamide on fitness and survival. Beetles were fed on flour spiked with acrylamide between 0.5% and 10% at 32°C over 2 weeks. Fitness of the beetles was tested by measuring the running distance and survival was recorded after 2 weeks of feeding at increased temperatures at 42°C. Both parameters were dose-dependent reduced by acrylamide. Knockdown of gene homologues of ahr, the arylhydrocarbon receptor, and of nrf-2, the nuclear factor erythroid 2-related factor 2, both reduced fitness and survival. Application of 0.5% acrylamide under knockdown of each factor further reduced fitness and survival, suggesting that ahr and nrf-2 are important for an adequate response to the toxicant. RNA-interference for ahr blocked completely the increase in nrf-2 mRNA levels, suggesting that the actions of ahr on acrylamide detoxification are mediated via Nrf-2.

The Red Flour Beetle Tribolium castaneum as a Model to Monitor Food Safety and Functionality.

: Food quality is a fundamental issue all over the world. There are two major requirements to provide the highest quality of food: having the lowest reachable concentrations of health-threatening ingredients or contaminants and having the optimal concentrations of health-improving functional ingredients. Often, the boundaries of both requirements are blurred, as might be best exemplified by nutraceuticals (enriched food products invented to prevent or even treat diseases), for which undesirable side effects have been reported sometimes. Accordingly, there is an increasing need for reliable methods to screen for health effects of wanted or unwanted ingredients in a complex food matrix before more complex model organisms or human probands become involved. In this chapter, we present the red flour beetle Tribolium castaneum as a model organism to screen for effects of complex foods on healthspan or lifespan by assessing the survival of the beetles under heat stress at 42 °C after feeding different diets. There is a higher genetic homology between T. castaneum and humans when compared to other invertebrate models, such as Drosophila melanogaster or Caenorhabditis elegans. Therefore, the red flour beetle appears as an interesting model to study interactions between genes and food ingredients, with relevance for stress resistance and lifespan. In that context, we provide data showing reduced lifespans of the beetles when the food-relevant contaminant benz(a)pyrene is added to the flour they were fed on, whereas a lifespan extension was observed in beetles fed on flour enriched with an extract of red wine.

Longevity in the red flour beetle Tribolium castaneum is enhanced by broccoli and depends on nrf-2, jnk-1 and foxo-1 homologous genes.

Diet is generally believed to affect the aging process. The effects of complex foods on life span can be investigated using simple models that produce rapid results and allow the identification of food-gene interactions. Here, we show that 1 % lyophilized broccoli, added to flour as a dietary source, significantly increases the life span of the red flour beetle (Tribolium castaneum) under physiological conditions (32 °C) and under heat stress (42 °C). The beneficial effects of broccoli could also be reproduced by supplementing flour with the isothiocyanate sulforaphane at concentrations found in the broccoli-supplemented diet. We identified stress-resistant genes responsible for these effects on longevity by microinjecting pupae with double-stranded RNA to induce RNA interference (RNAi). The knockdown of transcripts encoding homologs of Nrf-2, Jnk-1 and Foxo-1 reduced the life span of beetles and abrogated the beneficial effects of broccoli, whereas the knockdown of Sirt-1 and Sirt-3 had no impact in either scenario. In conclusion, T. castaneum is a suitable model organism to investigate food-gene interactions that affect stress resistance and longevity, and RNAi can be used to identify functionally relevant genes. As a proof of principle, we have shown here that broccoli increases the longevity of beetles and mediates its effect through signaling pathways that include key stress-resistant factors such as Nrf-2, Jnk-1 and Foxo-1.

Petri net modelling of gene regulation of the Duchenne muscular dystrophy.

UNLABELLED: Searching for therapeutic strategies for Duchenne muscular dystrophy, it is of great interest to understand the responsible molecular pathways down-stream of dystrophin completely. For this reason we have performed real-time PCR experiments to compare mRNA expression levels of relevant genes in tissues of affected patients and controls. To bring experimental data in context with the underlying pathway theoretical models are needed. Modelling of biological processes in the cell at higher description levels is still an open problem in the field of systems biology. In this paper, a new application of Petri net theory is presented to model gene regulatory processes of Duchenne muscular dystrophy. We have developed a Petri net model, which is based mainly on own experimental and literature data. We distinguish between up- and down-regulated states of gene expression. The analysis of the model comprises the computation of structural and dynamic properties with focus on a thorough T-invariant analysis, including clustering techniques and the decomposition of the network into maximal common transition sets (MCT-sets), which can be interpreted as functionally related building blocks. All possible pathways, which reflect the complex net behaviour in dependence of different gene expression patterns, are discussed. We introduce Mauritius maps of T-invariants, which enable, for example, theoretical knockout analysis. The resulted model serves as basis for a better understanding of pathological processes, and thereby for planning next experimental steps in searching for new therapeutic possibilities. AVAILABILITY: Free availability of the Petri net editor and animator Snoopy and the clustering tool PInA via http://www-dssz.informatik.tu-cottbus.de/~ wwwdssz/. The Petri net models used can be accessed via http://www.tfh-berlin.de/bi/duchenne/.

Modularization of biochemical networks based on classification of Petri net t-invariants.

BACKGROUND: Structural analysis of biochemical networks is a growing field in bioinformatics and systems biology. The availability of an increasing amount of biological data from molecular biological networks promises a deeper understanding but confronts researchers with the problem of combinatorial explosion. The amount of qualitative network data is growing much faster than the amount of quantitative data, such as enzyme kinetics. In many cases it is even impossible to measure quantitative data because of limitations of experimental methods, or for ethical reasons. Thus, a huge amount of qualitative data, such as interaction data, is available, but it was not sufficiently used for modeling purposes, until now. New approaches have been developed, but the complexity of data often limits the application of many of the methods. Biochemical Petri nets make it possible to explore static and dynamic qualitative system properties. One Petri net approach is model validation based on the computation of the system's invariant properties, focusing on t-invariants. T-invariants correspond to subnetworks, which describe the basic system behavior.With increasing system complexity, the basic behavior can only be expressed by a huge number of t-invariants. According to our validation criteria for biochemical Petri nets, the necessary verification of the biological meaning, by interpreting each subnetwork (t-invariant) manually, is not possible anymore. Thus, an automated, biologically meaningful classification would be helpful in analyzing t-invariants, and supporting the understanding of the basic behavior of the considered biological system. METHODS: Here, we introduce a new approach to automatically classify t-invariants to cope with network complexity. We apply clustering techniques such as UPGMA, Complete Linkage, Single Linkage, and Neighbor Joining in combination with different distance measures to get biologically meaningful clusters (t-clusters), which can be interpreted as modules. To find the optimal number of t-clusters to consider for interpretation, the cluster validity measure, Silhouette Width, is applied. RESULTS: We considered two different case studies as examples: a small signal transduction pathway (pheromone response pathway in Saccharomyces cerevisiae) and a medium-sized gene regulatory network (gene regulation of Duchenne muscular dystrophy). We automatically classified the t-invariants into functionally distinct t-clusters, which could be interpreted biologically as functional modules in the network. We found differences in the suitability of the various distance measures as well as the clustering methods. In terms of a biologically meaningful classification of t-invariants, the best results are obtained using the Tanimoto distance measure. Considering clustering methods, the obtained results suggest that UPGMA and Complete Linkage are suitable for clustering t-invariants with respect to the biological interpretability. CONCLUSION: We propose a new approach for the biological classification of Petri net t-invariants based on cluster analysis. Due to the biologically meaningful data reduction and structuring of network processes, large sets of t-invariants can be evaluated, allowing for model validation of qualitative biochemical Petri nets. This approach can also be applied to elementary mode analysis.