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.

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.

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.

St. Thomas Modified Cardioplegia Effects on Myoblasts' Viability and Morphology.

Background and Objectives: The cardioplegic arrest of the heart during cardiosurgical procedures is the crucial element of a cardioprotection strategy. Numerous clinical trials compare different cardioplegic solutions and cardioprotective protocols, but a relatively small number of papers apply to in vitro conditions using cultured cells. This work aimed to analyze whether it is possible to use the rat heart myocardium cells as an in vitro model to study the protective properties of St. Thomas cardioplegia (ST2C). Methods: The rat heart myocardium cells-H9C2 were incubated with cold cardioplegia for up to 24 h. After incubation, we determined: viability, confluency, and cell size, the thiol groups' level by modifying Ellman's method, Ki67, and Proliferating Cell Nuclear Antigen expression (PCNA). The impact on cells' morphology was visualized by the ultrastructural (TEM) study and holotomograpic 3D imaging. Results: The viability and confluency analysis demonstrated that the safest exposure to ST2C, should not exceed 4h. An increased expression of Ki67 antigen and PCNA was observed. TEM and 3D imaging studies revealed vacuolization after the longest period of exposure (24). Conclusions: According to obtained results, we conclude that STC can play a protective role in cardiac surgery during heart arrest.

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.

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.

Primary myogenesis in the sand lizard (Lacerta agilis) limb bud.

Our studies conducted on reptilian limb muscle development revealed, for the first time, early forelimb muscle differentiation at the morphological and molecular level. Sand lizard skeletal muscle differentiation in the early forelimb bud was investigated by light, confocal, and transmission electron microscopy as well as western blot. The early forelimb bud, filled with mesenchymal cells, is surrounded by monolayer epithelium cells. The immunocytochemical analysis revealed the presence of Pax3- and Lbx-positive cells in the vicinity of the ventro-lateral lip (VLL) of the dermomyotome, suggesting that VLL is the source of limb muscle progenitor cells. Furthermore, Pax3- and Lbx-positive cells were observed in the dorsal and ventral myogenic pools of the forelimb bud. Skeletal muscle development in the early limb bud is asynchronous, which is manifested by the presence of myogenic cells in different stages of differentiation: multinucleated myotubes with well-developed contractile apparatus, myoblasts, and mitotically active premyoblasts. The western blot analysis revealed the presence of MyoD and Myf5 proteins in all investigated developmental stages. The MyoD western blot analysis showed two bands corresponding to monomeric (mMyoD) and dimeric (dMyoD) fractions. Two separate bands were also detected in the case of Myf5. The observed bands were related to non-phosphorylated (Myf5) and phosphorylated (pMyf5) fractions of Myf5. Our investigations on sand lizard forelimb myogenesis showed that the pattern of muscle differentiation in the early forelimb bud shares many features with rodents and chicks.

Drosophila Hsp67Bc hot-spot variants alter muscle structure and function.

The Drosophila Hsp67Bc gene encodes a protein belonging to the small heat-shock protein (sHSP) family, identified as the nearest functional ortholog of human HSPB8. The most prominent activity of sHSPs is preventing the irreversible aggregation of various non-native polypeptides. Moreover, they are involved in processes such as development, aging, maintenance of the cytoskeletal architecture and autophagy. In larval muscles Hsp67Bc localizes to the Z- and A-bands, which suggests its role as part of the conserved chaperone complex required for Z-disk maintenance. In addition, Hsp67Bc is present at neuromuscular junctions (NMJs), which implies its involvement in the maintenance of NMJ structure. Here, we report the effects of muscle-target overexpression of Drosophila Hsp67Bc hot-spot variants Hsp67BcR126E and Hsp67BcR126N mimicking pathogenic variants of human HSPB8. Depending on the substitutions, we observed a different impact on muscle structure and performance. Expression of Hsp67BcR126E affects larval motility, which may be caused by impairment of mitochondrial respiratory function and/or by NMJ abnormalities manifested by a decrease in the number of synaptic boutons. In contrast, Hsp67BcR126N appears to be an aggregate-prone variant, as reflected in excessive accumulation of mutant proteins and the formation of large aggregates with a lesser impact on muscle structure and performance compared to the Hsp67BcR126E variant.

Drosophila small heat shock protein CryAB ensures structural integrity of developing muscles, and proper muscle and heart performance.

Molecular chaperones, such as the small heat shock proteins (sHsps), maintain normal cellular function by controlling protein homeostasis in stress conditions. However, sHsps are not only activated in response to environmental insults, but also exert developmental and tissue-specific functions that are much less known. Here, we show that during normal development the Drosophila sHsp CryAB [L(2)efl] is specifically expressed in larval body wall muscles and accumulates at the level of Z-bands and around myonuclei. CryAB features a conserved actin-binding domain and, when attenuated, leads to clustering of myonuclei and an altered pattern of sarcomeric actin and the Z-band-associated actin crosslinker Cheerio (filamin). Our data suggest that CryAB and Cheerio form a complex essential for muscle integrity: CryAB colocalizes with Cheerio and, as revealed by mass spectrometry and co-immunoprecipitation experiments, binds to Cheerio, and the muscle-specific attenuation of cheerio leads to CryAB-like sarcomeric phenotypes. Furthermore, muscle-targeted expression of CryAB(R120G), which carries a mutation associated with desmin-related myopathy (DRM), results in an altered sarcomeric actin pattern, in affected myofibrillar integrity and in Z-band breaks, leading to reduced muscle performance and to marked cardiac arrhythmia. Taken together, we demonstrate that CryAB ensures myofibrillar integrity in Drosophila muscles during development and propose that it does so by interacting with the actin crosslinker Cheerio. The evidence that a DRM-causing mutation affects CryAB muscle function and leads to DRM-like phenotypes in the fly reveals a conserved stress-independent role of CryAB in maintaining muscle cell cytoarchitecture.

Developmental Expression and Functions of the Small Heat Shock Proteins in Drosophila.

Heat shock proteins (Hsps) form a large family of evolutionarily conserved molecular chaperones that help balance protein folding and protect cells from various stress conditions. However, there is growing evidence that Hsps may also play an active role in developmental processes. Here, we take the example of developmental expression and function of one class of Hsps characterized by low molecular weight, the small Hsps (sHsps). We discuss recent reports and genome-wide datasets that support vital sHsps functions in the developing nervous system, reproductive system, and muscles. This tissue- and time-specific sHsp expression is developmentally regulated, so that the enhancer sequence of an sHsp gene expressed in developing muscle, in addition to stress-inducible elements, also carries binding sites for myogenic regulatory factors. One possible reason for sHsp genes to switch on during development and in non-stress conditions is to protect vital developing organs from environmental insults.

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.

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.

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.

Effects of Nanosecond Pulsed Electric Field on Immune Checkpoint Receptors in Melanoma Cells.

Checkpoint molecules such as PD-1, LAG-3, and TIM-3 are currently under extensive investigation for their roles in the attenuation of the immune response in cancer. Various methods have been applied to overcome the challenges in this field. This study investigated the effects of nanosecond pulsed electric field (nsPEF) treatment on the expression of immune checkpoint molecules in A375 and C32 melanoma cells. The researchers found that the nsPEF treatment was able to enhance membrane permeabilization and morphological changes in the cell membrane without being cytotoxic. We found that the effects of nsPEFs on melanoma included (1) the transport of vesicles from the inside to the outside of the cells, (2) cell contraction, and (3) the migration of lipids from inside the cells to their peripheries. The treatment increased the expression of PD-1 checkpoint receptors. Furthermore, we also observed potential co-localization or clustering of MHC class II and PD-1 molecules on the cell surface and the secretion of cytokines such as TNF-α and IL-6. These findings suggest that nsPEF treatment could be a viable approach to enhance the delivery of therapeutic agents to cancer cells and to modulate the tumor microenvironment to promote an antitumor immune response. Further studies are needed to explore the mechanisms underlying these effects and their impacts on the antitumor immune response, and to investigate the potential of nsPEF treatment in combination with immune checkpoint inhibitors to improve clinical outcomes for cancer patients.

Betulin and Its Derivatives Reduce Inflammation and COX-2 Activity in Macrophages.

Betulin is a heavily studied natural compound for its use as an anticancer or pro-regenerative agent. The structural similarity between betulin to steroids gives rise to the idea that the substance may as well act as an anti-inflammatory drug. This study is the first to describe the anti-inflammatory properties of betulinic acid, betulin, and its derivatives with amino acids 1,4-diaminebutane (Dab), 1,3-diaminepropane (Dap), Ornithine (Orn), and lysine (Lys) on murine macrophages from lymphoma site. The compounds were compared to dexamethasone. To establish the response of the macrophages to the natural compounds, we tested the viability as well as sensitivity to the inflammatory signaling (IFNγR). IL-6 secretory properties and HSP-70 content in the cells were examined. Furthermore, we characterized the effects of compounds on the inhibition of cyclooxygenase-2 (COX-2) activity both in the enzymatic assays and molecular docking studies. Then, the changes in COX-2 expression after betulin treatment were assessed. Betulin and betulinic acid are the low-cytotoxicity compounds with the highest potential to decrease inflammation via reduced IL-6 secretion. To some extent, they induce the reorganization of IFNγR with nearly no effect on COX-2 activity. Conversely, Bet-Orn and Bet-Lys are highly cytotoxic and induce the aggregation of IFNγR. Besides, Bet-Lys reduces the activity of COX-2 to a higher degree than dexamethasone. Bet-Orn is the only one to increase the HSP-70 content in the macrophages. In case of IL-6 reduction, all compounds were more potent than dexamethasone.

Lipid droplets in skeletal muscle during grass snake (Natrix natrix L.) development.

Lipid droplets (LDs) are common organelles observed in Eucaryota. They are multifunctional organelles (involved in lipid storage, metabolism, and trafficking) that originate from endoplasmic reticulum (ER). LDs consist of a neutral lipid core, made up of diacyl- and triacylglycerols (DAGs and TAGs) and cholesterol esters (CEs), surrounded by a phospholipid monolayer and proteins, which are necessary for their structure and dynamics. Here, we report the protein and lipid composition as well as characterization and dynamics of grass snake (Natrix natrix) skeletal muscle LDs at different developmental stages. In the present study, we used detailed morphometric, LC-MS, quantitative lipidomic analyses of LDs isolated from the skeletal muscles of the snake embryos, immunofluorescence, and TEM. Our study also provides a valuable insight concerning the LDs' multifunctionality and ability to interact with a variety of organelles. These LD features are reflected in their proteome composition, which contains scaffold proteins, metabolic enzymes signalling polypeptides, proteins necessary for the formation of docking sites, and many others. We also provide insights into the biogenesis and growth of muscle LDs goes beyond the conventional mechanism based on the synthesis and incorporation of TAGs and LD fusion. We assume that the formation and functioning of grass snake muscle LDs are based on additional mechanisms that have not yet been identified, which could be related to the unique features of reptiles that are manifested in the after-hatching period of life, such as a reptile-specific strategy for energy saving during hibernation.

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.

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.

The Australian lungfish (Neoceratodus forsteri) - fish or amphibian pattern of muscle development?

The Australian lungfish Neoceratodus forsteri (Dipnoi-Sarcoterygians) is a likely candidate for the extant sister group of Tetrapoda. Transmission electron and light microscopy analysis revealed that the arrangement of somite cells of the lungfish resembles the structure of the urodelan somite. On the other hand, the pattern of early muscle formation in N. forsteri is similar to that found in the Siberian sturgeon (Acipenser baeri). During the early stages of myogenesis of N. forsteri, somite-derived cells fuse to form multinucleated muscle lamellae. During later stages, mononucleated undifferentiated cells are first observed in the intermyotomal fissures and subsequently in the myotomes, among white muscle lamellae. The cells from the intermyotomal fissure differentiate into fibroblasts. The cells which have migrated into the myotomes, differentiate into mesenchyme-derived myoblasts. After hatching, white muscle lamellae are successively converted into polygonal muscle fibres. Conversion of lamellae into fibres may occur through splitting of muscle lamellae, or cylindrical muscle fibres may arise de novo as a result of fusion of mesenchyme-derived myoblasts. No increase in the number of muscle fibre nuclei is observed either in embryonic or juvenile musculature of N. forsteri. We suggest that until the 53 stage of embryonic development, the increase in muscle mass is accomplished mainly through hyperplasy. Thus, lungfish muscle represents the organizational intermediate between fishes and amphibians. This makes it a useful model to study the evolutionary implications of the mechanisms of muscle development.