Peter Steinbacher

Impact of Oxidative Stress on Exercising Skeletal Muscle

It is well established that muscle contractions during exercise lead to elevated levels of reactive oxygen species (ROS) in skeletal muscle. These highly reactive molecules have many deleterious effects, such as a reduction of force generation and increased muscle atrophy. Since the discovery of exercise-induced oxidative stress several decades ago, evidence has accumulated that ROS produced during exercise also have positive effects by influencing cellular processes that lead to increased expression of antioxidants. These molecules are particularly elevated in regularly exercising muscle to prevent the negative effects of ROS by neutralizing the free radicals. In addition, ROS also seem to be involved in the exercise-induced adaptation of the muscle phenotype. This review provides an overview of the evidences to date on the effects of ROS in exercising muscle. These aspects include the sources of ROS, their positive and negative cellular effects, the role of antioxidants, and the present evidence on ROS-dependent adaptations of muscle cells in response to physical exercise.

Neutrophil extracellular trap (NET) formation characterises stable and exacerbated COPD and correlates with airflow limitation

BackgroundCOPD is a progressive disease of the airways that is characterized by neutrophilic inflammation, a condition known to promote the excessive formation of neutrophil extracellular traps (NETs). The presence of large amounts of NETs has recently been demonstrated for a variety of inflammatory lung diseases including cystic fibrosis, asthma and exacerbated COPD.ObjectiveWe test whether excessive NET generation is restricted to exacerbation of COPD or whether it also occurs during stable periods of the disease, and whether NET presence and amount correlates with the severity of airflow limitation.Patients, materials and methodsSputum samples from four study groups were examined: COPD patients during acute exacerbation, patients with stable disease, and smoking and non-smoking controls without airflow limitation. Sputum induction followed the ECLIPSE protocol. Confocal laser microscopy (CLSM) and electron microscopy were used to analyse samples. Immunolabelling and fluorescent DNA staining were applied to trace NETs and related marker proteins. CLSM specimens served for quantitative evaluation.ResultsSputum of COPD patients is clearly characterised by NETs and NET-forming neutrophils. The presence of large amounts of NET is associated with disease severity (p < 0.001): over 90 % in exacerbated COPD, 45 % in stable COPD, and 25 % in smoking controls, but less than 5 % in non-smokers. Quantification of NET-covered areas in sputum preparations confirms these results.ConclusionsNET formation is not confined to exacerbation but also present in stable COPD and correlates with the severity of airflow limitation. We infer that NETs are a major contributor to chronic inflammatory and lung tissue damage in COPD.

MyoD and Myogenin expression during myogenic phases in brown trout: A precocious onset of mosaic hyperplasia is a prerequisite for fast somatic growth

Muscle cell recruitment (hyperplasia) during myogenesis in the vertebrate embryo is known to occur in three consecutive phases. In teleost fish (including zebrafish), however, information on myogenic precursor cell activation is largely fragmentary, and comprehensive characterization of the myogenic phases has only been fully undertaken in a single slow‐growing cyprinid species by examination of MEF2D expression. Here, we use molecular techniques to provide a comprehensive characterization of MyoD and Myogenin expression during myogenic cell activation in embryos and larvae of brown trout, a fast‐growing salmonid with exceptionally large embryos. Results confirm the three‐phase pattern, but also demonstrate that the second and third phases begin simultaneously and progress vigorously, which is different from the previously described consecutive activation of these phases. Furthermore, we suggest that Pax7 is expressed in myogenic progenitor cells that account for second‐ and third‐phase myogenesis. These findings are discussed in relation to teleost myotome development and to teleost growth strategies. Developmental Dynamics 236:1106–1114, 2007.

The Role of Reactive Oxygen Species (ROS) in the Formation of Extracellular Traps (ETs) in Humans

Extracellular traps (ETs) are reticulate structures of extracellular DNA associated with antimicrobial molecules. Their formation by phagocytes (mainly by neutrophils: NETs) has been identified as an essential element of vertebrate innate immune defense. However, as ETs are also toxic to host cells and potent triggers of autoimmunity, their role between pathogen defense and human pathogenesis is ambiguous, and they contribute to a variety of acute and chronic inflammatory diseases. Since the discovery of ET formation (ETosis) a decade ago, evidence has accumulated that most reaction cascades leading to ET release involve ROS. An important new facet was added when it became apparent that ETosis might be directly linked to, or be a variant of, the autophagy cell death pathway. The present review analyzes the evidence to date on the interplay between ROS, autophagy and ETosis, and highlights and discusses several further aspects of the ROS-ET relationship that are incompletely understood. These aspects include the role of NADPH oxidase-derived ROS, the molecular requirements of NADPH oxidase-dependent ETosis, the roles of NADPH oxidase subtypes, extracellular ROS and of ROS from sources other than NADPH oxidase, and the present evidence for ROS-independent ETosis. We conclude that ROS interact with ETosis in a multidimensional manner, with influence on whether ETosis shows beneficial or detrimental effects.

Phases of myogenic cell activation and possible role of dermomyotome cells in teleost muscle formation

Present knowledge indicates that fibre recruitment (hyperplasia) in developing teleost fish occurs in three distinct phases. However, the origin and relationship of the myogenic precursors activated during the different phases remains unclear. Here, we address this issue using molecular techniques on embryos and larvae of pearlfish, a large cyprinid species. Results provide comprehensive molecular characterisation of cell recruitment over the three phases of myogenesis, identifying muscle types as they arise. Specifically, we show that the myogenic cells arising during 2nd phase myogenesis are clearly different from the myogenic cells arising during the 3rd phase and that the dermomyotome is a major source of myogenic cells driving 2nd phase hyperplasia. These findings are discussed in relation to their implications for the generality of vertebrate developmental patterns. Developmental Dynamics 235:3132–3143, 2006.

New Aspects on the Structure of Neutrophil Extracellular Traps from Chronic Obstructive Pulmonary Disease and In Vitro Generation

Polymorphonuclear neutrophils have in recent years attracted new attention due to their ability to release neutrophil extracellular traps (NETs). These web-like extracellular structures deriving from nuclear chromatin have been depicted in ambiguous roles between antimicrobial defence and host tissue damage. NETs consist of DNA strands of varying thickness and are decorated with microbicidal and cytotoxic proteins. Their principal structure has in recent years been characterised at molecular and ultrastructural levels but many features that are of direct relevance to cytotoxicity are still incompletely understood. These include the extent of chromatin decondensation during NET formation and the relative amounts and spatial distribution of the microbicidal components within the NET. In the present work, we analyse the structure of NETs found in induced sputum of patients with acutely exacerbated chronic obstructive pulmonary disease (COPD) using confocal laser microscopy and electron microscopy. In vitro induced NETs from human neutrophils serve for purposes of comparison and extended analysis of NET structure. Results demonstrate that COPD sputa are characterised by the pronounced presence of NETs and NETotic neutrophils. We provide new evidence that chromatin decondensation during NETosis is most extensive and generates substantial amounts of double-helix DNA in ‘beads-on-a-string’ conformation. New information is also presented on the abundance and location of neutrophil elastase (NE) and citrullinated histone H3 (citH3). NE occurs in high densities in nearly all non-fibrous constituents of the NETs while citH3 is much less abundant. We conclude from the results that (i) NETosis is an integral part of COPD pathology; this is relevant to all future research on the etiology and therapy of the disease; and that (ii) release of ‘beads-on-a-string’ DNA studded with non-citrullinated histones is a common feature of in vivo NETosis; this is of relevance to both the antimicrobial and the cytotoxic effects of NETs.

Age‐related changes in the composition of the cornified envelope in human skin

The main function of the epidermis is to protect us against a multitude of hostile attacks from the environment. Its main cell type, the keratinocytes have a sophisticated system of different proteins and lipids available to form the cornified envelope, which is responsible for the barrier function of the skin. During ageing, dramatic changes are taking place. Some proteins of the SPRR‐, S100‐ and LCE3‐family are massively up‐regulated, whereas others like loricrin, filaggrin and the LCE1&2 protein families are significantly down‐regulated. The latter ones are known to be under control of calcium and/or ‘calcium response elements’. We were able to show that the calcium peak specific for the stratum granulosum, which is the site where loricrin and the LCE1&2 families are synthesized, is reduced during ageing. The resulting cornified envelope in old skin has an extensively changed composition on the molecular level compared to young skin. This knowledge is of critical importance to understand chronic wound formation and ulcers in old age.

Postembryonic fast muscle growth of teleost fish depends upon a nonuniformly distributed population of mitotically active Pax7+ precursor cells.

Muscle development in teleost embryos has been shown to depend on myogenic cell recruitment from the dermomyotome (DM). However, little is known as to the cellular mechanisms that account for myotome growth after the dissociation of the DM. Here we combine immunolabeling for cell‐specific markers with quantitative analysis to determine the sources and patterns of activation of myogenic cells in pearlfish larvae. Results demonstrate that appearance of mitotically active myogenic precursors inside the myotome coincides with the dissociation of the DM. Such cells are preferentially aggregated within the posterior lateral fast muscle. We therefore propose a growth model in which a pool of proliferative DM‐derived precursors transferred to the posterior lateral fast muscle functions as an important source of myogenic cell spread to carry forward stratified fast muscle hyperplasia. This indicates that postembryonic teleost muscle growth includes a cellular mechanism that has no direct equivalent in the amniotes. Developmental Dynamics 238:2442–2448, 2009.

Temperature-dependent modification of muscle precursor cell behaviour is an underlying reason for lasting effects on muscle cellularity and body growth of teleost fish.

SUMMARY Temperature is an important factor influencing teleost muscle growth, including a lasting (‘imprinted’) influence of embryonic thermal experience throughout all further life. However, little is known about the cellular processes behind this phenomenon. The study reported here used digital morphometry and immunolabelling for Pax7, myogenin and H3P to quantitatively examine the effects of thermal history on muscle precursor cell (MPC) behaviour and muscle growth in pearlfish (Rutilus meidingeri) until the adult stage. Fish were reared at three different temperatures (8.5, 13 and 16°C) until hatching and subsequently kept under the same (ambient) thermal conditions. Cellularity data were combined with a quantitative analysis of Pax7+ MPCs including those that were mitotically active (Pax7+/H3P+) or had entered differentiation (Pax7+/myogenin+). The results demonstrate that at hatching, body lengths, fast and slow muscle cross-sectional areas and fast fibre numbers are lower in fish reared at 8.5 and 13°C than at 16°C. During the larval period, this situation changes in the 13°C-fish, so that these fish are finally the largest. The observed effects can be related to divergent cellular mechanisms at the MPC level that are initiated in the embryo during the imprinting period. Embryos of 16°C-fish have reduced MPC proliferation but increased differentiation, and thus give rise to larger hatchlings. However, their limited MPC reserves finally lead to smaller adults. By contrast, embryos of 13°C-fish and, to a lesser extent, 8.5°-fish, show enhanced MPC proliferation but reduced differentiation, thus leading to smaller hatchlings but allowing for a larger MPC pool that can be used for enhanced post-hatching growth, finally resulting in larger adults.

Lateral Fast Muscle Fibers Originate From the Posterior Lip of the Teleost Dermomyotome

The predominant source of myogenic cells in vertebrates is the dermomyotome (DM). In teleost fish, recent research has provided a useful but limited picture of how myogenic precursors originate from the DM and how they develop into muscle fibers. Here, we combine detailed morphological analysis with examination of molecular markers in trout to describe the cellular mechanisms by which the lateral fast muscle growth zone is created during second phase myogenesis. Results suggest that this occurs by lateral‐to‐medial immigration of myogenic cells de‐epithelializing from the posterior DM lip. These cells then appear to stop proliferation and migrate anteriorly to finally differentiate into muscle fibres. This seems to be a continuation of the rotational cell movement that creates the teleost DM during early somite development. These findings suggest an evolutionary conserved role of the posterior DM lip in amniotes and fish. Developmental Dynamics 237:3233–3239, 2008.