Metabolic aspects of glycogenolysis with special attention to McArdle disease.

The physiological function of muscle glycogen is to meet the energy demands of muscle contraction. The breakdown of glycogen occurs through two distinct pathways, primarily cytosolic and partially lysosomal. To obtain the necessary energy for their function, skeletal muscles utilise also fatty acids in the ß-oxidation. Ketogenesis is an alternative metabolic pathway for fatty acids, which provides an energy source during fasting and starvation. Diseases arising from impaired glycogenolysis lead to muscle weakness and dysfunction. Here, we focused on the lack of muscle glycogen phosphorylase (PYGM), a rate-limiting enzyme for glycogenolysis in skeletal muscles, which leads to McArdle disease. Metabolic myopathies represent a group of genetic disorders characterised by the limited ability of skeletal muscles to generate energy. Here, we discuss the metabolic aspects of glycogenosis with a focus on McArdle disease, offering insights into its pathophysiology. Glycogen accumulation may influence the muscle metabolic dynamics in different ways. We emphasize that a proper treatment approach for such diseases requires addressing three important and interrelated aspects, which include: symptom relief therapy, elimination of the cause of the disease (lack of a functional enzyme) and effective and early diagnosis.

The natural origins of cytostatic compounds used in rhabdomyosarcoma therapy.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and represents a high-grade neoplasm of skeletal myoblast-like cells. About 40% of all registered soft tissue tumors are RMSs. This paper describes our current understanding of the RMS subtypes (alveolar (ARMS), embryonic (ERMS), pleomorphic (PRMS), and spindle cell/sclerosing (s/scRMS)), diagnostic methods, molecular bases, and characteristics. We also present the currently used treatment methods and the potential use of natural substances in the treatment of this type of cancer. Natural cytotoxic substances are compounds that have been the subject of numerous studies and discussions in recent years. Since anti-cancer therapies are often limited by a low therapeutic index and cancer resistance to pharmacotherapy, it is very important to search for new, effective compounds. Additionally, compounds of a natural origin are usually readily available and have a reduced cytotoxicity. Thus, the undiscovered potential of natural anti-cancer compounds makes this field of research a very important area. The introduction of model species into research examining the use of natural cytostatic therapies for RMS will allow for further assessment of the effects of these compounds on cancerous and healthy tissues.

Unique Features of River Lamprey (Lampetra fluviatilis) Myogenesis.

The river lamprey (L. fluviatilis) is a representative of the ancestral jawless vertebrate group. We performed a histological analysis of trunk muscle fiber differentiation during embryonal, larval, and adult musculature development in this previously unstudied species. Investigation using light, transmission electron (TEM), and confocal microscopy revealed that embryonal and larval musculature differs from adult muscle mass. Here, we present the morphological analysis of L. fluviatilis myogenesis, from unsegmented mesoderm through somite formation, and their differentiation into multinucleated muscle lamellae. Our analysis also revealed the presence of myogenic factors LfPax3/7 and Myf5 in the dermomyotome. In the next stages of development, two types of muscle lamellae can be distinguished: central surrounded by parietal. This pattern is maintained until adulthood, when parietal muscle fibers surround the central muscles on both sides. The two types show different morphological characteristics. Although lampreys are phylogenetically distant from jawed vertebrates, somite morphology, especially dermomyotome function, shows similarity. Here we demonstrate that somitogenesis is a conservative process among all vertebrates. We conclude that river lamprey myogenesis shares features with both ancestral and higher vertebrates.

Assessment of the Preventive Effect of L-carnitine on Post-statin Muscle Damage in a Zebrafish Model.

Statins, such as lovastatin, are lipid-lowering drugs (LLDs) that have been used to treat hypercholesterolaemia, defined as abnormally elevated cholesterol levels in the patient's blood. Although statins are considered relatively safe and well tolerated, recipients may suffer from adverse effects, including post-statin myopathies. Many studies have shown that supplementation with various compounds may be beneficial for the prevention or treatment of side effects in patients undergoing statin therapy. In our study, we investigated whether L-carnitine administered to zebrafish larvae treated with lovastatin alleviates post-statin muscle damage. We found that exposure of zebrafish larvae to lovastatin caused skeletal muscle disruption observed as a reduction of birefringence, changes in muscle ultrastructure, and an increase in atrogin-1. Lovastatin also affected heart performance and swimming behaviour of larvae. Our data indicated that the muscle-protective effect of L-carnitine is partial. Some observed myotoxic effects, such as disruption of skeletal muscle and increase in atrogin-1 expression, heart contraction could be rescued by the addition of L-carnitine. Others, such as slowed heart rate and reduced locomotion, could not be mitigated by L-carnitine supplementation.

Zebrafish as a Model for the Study of Lipid-Lowering Drug-Induced Myopathies.

Drug-induced myopathies are classified as acquired myopathies caused by exogenous factors. These pathological conditions develop in patients without muscle disease and are triggered by a variety of medicaments, including lipid-lowering drugs (LLDs) such as statins, fibrates, and ezetimibe. Here we summarise the current knowledge gained via studies conducted using various models, such as cell lines and mammalian models, and compare them with the results obtained in zebrafish (Danio rerio) studies. Zebrafish have proven to be an excellent research tool for studying dyslipidaemias as a model of these pathological conditions. This system enables in-vivo characterization of drug and gene candidates to further the understanding of disease aetiology and develop new therapeutic strategies. Our review also considers important environmental issues arising from the indiscriminate use of LLDs worldwide. The widespread use and importance of drugs such as statins and fibrates justify the need for the meticulous study of their mechanism of action and the side effects they cause.

Muscle Glycogen Phosphorylase and Its Functional Partners in Health and Disease.

Glycogen phosphorylase (PG) is a key enzyme taking part in the first step of glycogenolysis. Muscle glycogen phosphorylase (PYGM) differs from other PG isoforms in expression pattern and biochemical properties. The main role of PYGM is providing sufficient energy for muscle contraction. However, it is expressed in tissues other than muscle, such as the brain, lymphoid tissues, and blood. PYGM is important not only in glycogen metabolism, but also in such diverse processes as the insulin and glucagon signaling pathway, insulin resistance, necroptosis, immune response, and phototransduction. PYGM is implicated in several pathological states, such as muscle glycogen phosphorylase deficiency (McArdle disease), schizophrenia, and cancer. Here we attempt to analyze the available data regarding the protein partners of PYGM to shed light on its possible interactions and functions. We also underline the potential for zebrafish to become a convenient and applicable model to study PYGM functions, especially because of its unique features that can complement data obtained from other approaches.

What is unknown in skeletal muscles? / Czego jeszcze nie wiemy o miesniach szkieletowych?

Skeletal muscles are a highly specialized animal tissue whose basic function is the contraction, which leads into animal movement. One of the types of skeletal muscles are trunk (myotomal) muscles, which in vertebrates belong to the oldest phylogenetically group of muscles. The comparative studies of myotomal myogenesis have shown that these muscles, despite a similar structure plan and under the control of the same genetic factors, may differentiate differently in individual species of vertebrates (both in model and non-model species). The understanding of the skeletal muscle development mechanisms seem to be a precondition for understanding the muscle tissue diseases observed in humans. This paper summarizes the current knowledge on the skeletal muscles differentiation in animals, pathological states of muscles caused by mutations in the genes of structural and metabolic proteins.

Characterization of Hspb8 in Zebrafish.

Hspb8 is a member of the small heat shock protein (sHSP) family. Its expression is known to be upregulated under heat shock. This protein interacts with different partners and can, therefore, be involved in various processes relevant to tissue integrity and functioning. In humans, mutations in the gene encoding Hspb8 can lead to the development of various diseases such as myopathies and neuropathies. In our study, we aimed to perform an in-depth characterization of zebrafish Hspb8 during zebrafish development. We applied techniques such as RT-qPCR, Western blot, immunofluorescence, co-immunoprecipitation, LC-MS, and morpholino-mediated knockdown. We broadened the knowledge regarding zebrafish hspb8 expression during development under normal and heat shock conditions as well as its tissue- and subcellular-specific localization. A co-IP analysis allowed us to conclude that zebrafish Hspb8 can interact with proteins such as Bag3 and Hsc70, which are crucial for formation of an autophagy-inducing complex. We also demonstrated that hspb8 morpholino-mediated knockdown has an impact on zebrafish embryos' morphology, muscle ultrastructure, and motility behavior. Our research provides a valuable resource for the potential use of the zebrafish as a model for studying pathological conditions associated with hspb8 disorders.

Everybody wants to move-Evolutionary implications of trunk muscle differentiation in vertebrate species.

In our review we have completed current knowledge on myotomal myogenesis in model and non-model vertebrate species (fishes, amphibians, reptiles, birds and mammals) at morphological and molecular levels. Data obtained from these studies reveal distinct similarities and differences between amniote and anamniote species. Based on the available data, we decided to present evolutionary implications in vertebrate trunk muscle development. Despite the fact that in all vertebrates muscle fibres are multinucleated, the pathways leading to them vary between vertebrate taxa. In fishes during early myogenesis myoblasts differentiate into multinucleated lamellae or multinucleate myotubes. In amphibians, myoblasts fuse to form multinucleated myotubes or, bypassing fusion, directly differentiate into mononucleated myotubes. Furthermore, mononucleated myotubes were also observed during primary myogenesis in amniotes. The mononucleated state of myogenic cells could be considered as an old phylogenetic, plesiomorphic feature, whereas direct multinuclearity of myotubes has a synapomorphic character. On the other hand, the explanation of this phenomenon could also be linked to the environmental conditions in which animals develop. The similarities observed in vertebrate myogenesis might result from a conservative myogenic programme governed by the Pax3/Pax7 and myogenic regulatory factor (MRF) network, whereas differences in anamniotes and amniotes are established by spatiotemporal pattern expression of MRFs during muscle differentiation and/or environmental conditions.

The effect of muscle glycogen phosphorylase (Pygm) knockdown on zebrafish morphology.

Muscle glycogen phosphorylase (PYGM) is a key enzyme in the first step of glycogenolysis. Mutation in the PYGM gene leads to autosomal recessive McArdle disease. Patients suffer from exercise intolerance with premature fatigue, muscle cramps and myalgia due to lack of available glucose in muscles. So far, no efficient treatment has been found. The zebrafish has many experimental advantages, and was successfully implemented as an animal model of human myopathies. Since zebrafish skeletal muscles share high similarity with human skeletal muscles, it is our animal of choice to investigate the impact of Pygm knockdown on skeletal muscle tissue. The two forms of the zebrafish enzyme, Pygma and Pygmb, share more than 80% amino acid sequence identity with human PYGM. We show that the Pygm level varies at both the mRNA and protein level in distinct stages of zebrafish development, which is correlated with glycogen level. The Pygm distribution in muscles varies from dispersed to highly organized at 72 hpf. The pygma and pygmb morpholino knockdown resulted in a reduced Pygm level in zebrafish morphants, which exhibited altered, disintegrated muscle structure and accumulation of glycogen granules in the subsarcolemmal region. Thus, lowering the Pygm level in zebrafish larvae leads to an elevated glycogen level and to morphological muscle changes mimicking the symptoms of human McArdle disease. The zebrafish model of this human disease might contribute to further understanding of its molecular mechanisms and to the development of appropriate treatment.

Models of amphibian myogenesis - the case of Bombina variegata.

Several different models of myogenesis describing early stages of amphibian paraxial myotomal myogenesis are known. Myoblasts of Xenopus laevis and Hymenochirus boettgeri change their position from perpendicular to parallel, in relation to axial organs, and differentiate into mononucleate myotubes. In Bombina variegate the myotomal myoblasts change their shape from round to elongate and then differentiate into mononuclear, morphologically mature myotubes. In Pelobates fuscus and Triturus vulgaris, myoblasts fuse into multinuclear myotubes. Mono- and multinucleate myotubes achieve morphological maturity during the differentiation process. During myogenesis of B. variegata, the nuclei of mononucleate, differentiating myotubes contain a tetraploid quantity of DNA (4C DNA). The stable quantity of DNA is confirmed by lack of 3H-thimidine incorporation into myotube nuclei. This outcome is a proof that myoblasts withdraw from the cell cycle in the G2 phase. The further development of myotomal myotubes involves myoblasts of mesenchymal origin. These myoblasts fuse with myotubes in X. laevis and B. variegate in the G1 phase. Secondary muscle fibres in amphibian myotomes have only mesenchymal origin. Mesenchymal myoblasts fuse into multinucleated myotubes. Myofibril development in the differentiating myotube and lack of DNA replication confirm the classical paradigm of myogenesis. Mesenchymal myoblasts are taking part in the myogenesis of musculus rectus abdominis and limb muscles. The mesenchymal cells in the myogenesis process show one model of myogenesis, which is a classical model of myogenesis. The mesenchymal cells probably come from dermatome.

Zebrafish: A Model for the Study of Toxicants Affecting Muscle Development and Function.

The rapid progress in medicine, agriculture, and allied sciences has enabled the development of a large amount of potentially useful bioactive compounds, such as drugs and pesticides. However, there is another side of this phenomenon, which includes side effects and environmental pollution. To avoid or minimize the uncontrollable consequences of using the newly developed compounds, researchers seek a quick and effective means of their evaluation. In achieving this goal, the zebrafish (Danio rerio) has proven to be a highly useful tool, mostly because of its fast growth and development, as well as the ability to absorb the molecules diluted in water through its skin and gills. In this review, we focus on the reports concerning the application of zebrafish as a model for assessing the impact of toxicants on skeletal muscles, which share many structural and functional similarities among vertebrates, including zebrafish and humans.

ß- and γ-Actins in the nucleus of human melanoma A375 cells.

Actin is a highly conserved protein that is expressed in all eukaryotic cells and has essential functions in the cytoplasm and the nucleus. Nuclear actin is involved in transcription by all three RNA polymerases, chromatin remodelling, RNA processing, intranuclear transport, nuclear export and in maintenance of the nuclear architecture. The nuclear actin level and polymerization state are important factors regulating nuclear processes such as transcription. Our study shows that, in contrast to the cytoplasm, the majority of endogenous nuclear actin is unpolymerized in human melanoma A375 cells. Most mammalian cells express the two non-muscle ß- and γ-actin isoforms that differ in only four amino acids. Despite their sequence similarity, studies analysing the cytoplasmic functions of these isoforms demonstrated that ß- and γ-actins show differences in localization and function. However, little is known about the involvement of the individual actin isoforms in nuclear processes. Here, we used the human melanoma A375 cell line to analyse actin isoforms in regard to their nuclear localization. We show that both ß- and γ-non-muscle actin isoforms are present in nuclei of these cells. Immunolocalization studies demonstrate that both isoforms co-localize with RNA polymerase II and hnRNP U. However, we observe differences in the ratio of cytoplasmic to nuclear actin distribution between the isoforms. We show that ß-actin has a significantly higher nucleus-to-cytoplasm ratio than γ-actin.

Model organisms in the fight against muscular dystrophy: lessons from drosophila and Zebrafish.

Muscular dystrophies (MD) are a heterogeneous group of genetic disorders that cause muscle weakness, abnormal contractions and muscle wasting, often leading to premature death. More than 30 types of MD have been described so far; those most thoroughly studied are Duchenne muscular dystrophy (DMD), myotonic dystrophy type 1 (DM1) and congenital MDs. Structurally, physiologically and biochemically, MDs affect different types of muscles and cause individual symptoms such that genetic and molecular pathways underlying their pathogenesis thus remain poorly understood. To improve our knowledge of how MD-caused muscle defects arise and to find efficacious therapeutic treatments, different animal models have been generated and applied. Among these, simple non-mammalian Drosophila and zebrafish models have proved most useful. This review discusses how zebrafish and Drosophila MD have helped to identify genetic determinants of MDs and design innovative therapeutic strategies with a special focus on DMD, DM1 and congenital MDs.

Identification and characterization of a novel myosin Ic isoform that localizes to the nucleus.

In vertebrates, two myosin Ic isoforms that localize to the cytoplasm and to the nucleus have been characterized. The isoform that predominantly localizes to the nucleus is called nuclear myosin I (NMI). NMI has been identified as a key factor involved in nuclear processes such as transcription by RNA polymerases I and II and intranuclear transport processes. We report here the identification of a previously uncharacterized third MYOIC gene product that is called isoform A. Similar to NMI, this isoform contains a unique N-terminal peptide sequence, localizes to the nucleus and colocalizes with RNA polymerase II. However, unlike NMI, upon exposure to inhibitors of RNA polymerase II transcription the newly identified isoform translocates to nuclear speckles. Furthermore, in contrast to NMI, this new isoform is absent from nucleoli and does not colocalize with RNA polymerase I. Our results suggest an unexpected diversity among nuclear myosin Ic isoforms in respect to their intranuclear localization and interaction with nuclear binding partners that could provide new insights into the regulation of myosin-dependent nuclear processes.

[Nuclear actin]. / Aktyna w jadrze komórkowym.

Actin is multifunctional protein occurring in all eukaryotic cells. It appears in cytoplasm and in nucleus. Organization and functions of nuclear actin are not well recognized to distinguish from cytoplasmic actin. Actin monomers are shuttling between cytoplasm and nucleus, integrating both of those compartments. Monomers, oligomers and short polimers are observed as the result of the limited space within the nucleus. Actin participates in nuclear structure organization, chromatin remodeling, transcription and signal transduction. Nuclear actin level, organization and functions are under control of ABPs (similarly as it takes place in cytoplasm), including some proteins appearing only in the nucleus.