Necrosis Of Skeletal Muscle Fibers Research Articles

Snake venom Lys49 myotoxins: From phospholipases A2 to non-enzymatic membrane disruptors

Snake venoms often contain toxins that cause a rapid necrosis of skeletal muscle fibers, referred to as myotoxins. The most common among them are phospholipases A2 (PLA2s), enzymes that have two independent evolutionary origins in snake venoms. Within the group II PLA2s found in viperid venoms, a particular subgroup emerged, in which the otherwise conserved Asp49 of their catalytic center is replaced by Lys49. These intriguing proteins, referred to as Lys49 myotoxins, lost the ability to catalyze phospholipid hydrolysis, but still induce myonecrosis by a non-enzymatic mechanism based on membrane permeabilization as the critical event. Such mechanism is only partially understood. This review briefly describes the general structural and functional characteristics of the Lys49 myotoxins, and summarizes four proposed models of their functional “toxic site”. Finally, it discusses some novel insights into their mode of action, in particular examining arguments and experimental observations that could shed light on the possible nature of their membrane target on skeletal muscle cells, which remains elusive.

Aspectos clinicopatológicos e laboratoriais do envenenamento experimental por Bothrops moojeni e Bothropoides neuwiedi em ovinos

O envenenamento ofídico espontâneo, ou acidente ofídico, é descrito como causa de morte em animais domésticos. No entanto, dados concretos relativos ao gênero e espécie de serpente envolvida, à evolução do quadro clínico, e às alterações clinicopatológicas desenvolvidas, são escassos. Assim sendo, este trabalho teve como objetivo determinar as alterações clinicopatológicas e laboratoriais provocadas pelo veneno de Bothrops moojeni e Bothropoides neuwiedi em ovinos no intuito de fornecer informações adicionais referentes a acidentes ofídicos em animais de produção, auxiliando o estabelecimento do diagnóstico dessa condição. Os venenos liofilizados foram diluídos em 1 ml de solução fisiológica e administrados a quatro ovinos por via subcutânea na face direita, nas doses de 0,41mg/kg e 0,82mg/kg do veneno de B. moojeni em dois ovinos, e de 1,0mg/kg do veneno de B. neuwiedi em dois ovinos. Apenas o ovino que recebeu a menor dose (0,41mg/kg) sobreviveu, apesar de ter desenvolvido quadro clínico muito severo e semelhante aos demais. Os sinais clínicos iniciaram nos primeiros 10 minutos após a inoculação em todos os ovinos. O período de evolução variou de dois a quatro dias. O quadro clínico dos quatro ovinos caracterizou-se por apatia, acentuado aumento de volume da face, da porção ventral do pescoço e do peito, leve aumento de volume da porção proximal dos membros anteriores, tempo de sangramento aumentado, taquicardia, mucosas pálidas e grande quantidade de sangue não digerido nas fezes. Ao exame laboratorial observou-se principalmente redução das proteínas plasmáticas e aumento de creatinaquinase em todos os ovinos. À necropsia, foram observados extensos hematomas nas áreas correspondentes ao aumento de volume subcutâneo. Observaram-se petéquias, equimoses e sufusões leves a moderadas na serosa de diversos órgãos e acúmulo de sangue em meio às fezes na porção final do reto. Além de hemorragias, a principal alteração histopatológica observada foi necrose das fibras musculares esqueléticas e da parede de vasos, nas áreas próximas à inoculação do veneno. Nos ovinos deste estudo o aumento de volume, observado na face, pescoço, peito e membros, era constituído por sangue.

Anti-TNFalpha (Remicade) therapy protects dystrophic skeletal muscle from necrosis.

Necrosis of skeletal muscle fibers in the lethal childhood myopathy Duchenne muscular dystrophy (DMD) results from defects in the cell membrane-associated protein, dystrophin. This study tests the novel hypothesis that the initial sarcolemmal breakdown resulting from dystrophin deficiency is exacerbated by inflammatory cells and that cytokines, specifically tumor necrosis factor-alpha (TNFalpha), contribute to muscle necrosis. To block in vivo TNFalpha bioactivity, young dystrophic mdx mice (a model for DMD) were injected weekly from 7 days of age with the anti-TNFalpha antibody Remicade before the onset of muscle necrosis and dystropathology that normally occurs at 21 days postnatally. The extent of inflammation, muscle necrosis, and myotube formation was measured by histological analysis from 18 to 28 days and muscle damage was also visualized by penetration of Evans blue dye into myofibers. Data from Remicade-treated and control mdx mice were compared with mdx/TNFalpha-/- mice that lack TNFalpha. Pharmacological blockade of TNFalpha activity with Remicade clearly delayed and greatly reduced the breakdown of dystrophic muscle, in marked contrast to the situation in mdx and mdx/TNFalpha-/- mice. Remicade had no adverse effect on new muscle formation. Remicade is a highly specific anti-inflammatory intervention, and clinical application to muscular dystrophies is suggested by this marked protective effect against skeletal muscle breakdown.

Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3.

We produced and analyzed mice deficient for Na/Ca exchanger 3 (NCX3), a protein that mediates cellular Ca(2+) efflux (forward mode) or Ca(2+) influx (reverse mode) and thus controls intracellular Ca(2+) concentration. NCX3-deficient mice (Ncx3(-/-)) present a skeletal muscle fiber necrosis and a defective neuromuscular transmission, reflecting the absence of NCX3 in the sarcolemma of the muscle fibers and at the neuromuscular junction. The defective neuromuscular transmission is characterized by the presence of electromyographic abnormalities, including low compound muscle action potential amplitude, a decremental response at low-frequency nerve stimulation, an incremental response, and a prominent postexercise facilitation at high-frequency nerve stimulation, as well as neuromuscular blocks. The analysis of quantal transmitter release in Ncx3(-/-) neuromuscular junctions revealed an important facilitation superimposed on the depression of synaptic responses and an elevated delayed release during high-frequency nerve stimulation. It is suggested that Ca(2+) entering nerve terminals is cleared relatively slowly in the absence of NCX3, thereby enhancing residual Ca(2+) and evoked and delayed quantal transmitter release during repetitive nerve stimulation. Our findings indicate that NCX3 plays an important role in vivo in the control of Ca(2+) concentrations in the skeletal muscle fibers and at the neuromuscular junction.

Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3

Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3

Inflammatory effects of snake venom myotoxic phospholipases A 2

Snake venom phospholipases A 2 (PLA 2) show a remarkable functional diversity. Among their toxic activities, some display the ability to cause rapid necrosis of skeletal muscle fibers, thus being myotoxic PLA 2s. Besides myotoxicity, these enzymes evoke conspicuous inflammatory and nociceptive events in experimental models. Local inflammation and pain are important characteristics of snakebite envenomations inflicted by viperid and crotalid species, whose venoms are rich sources of myotoxic PLA 2s. Since the discovery that mammalian PLA 2 is a key enzyme in the release of arachidonic acid, the substrate for the synthesis of several lipid inflammatory mediators, much interest has been focused on this enzyme in the context of inflammation. The mechanisms involved in the proinflammatory action of secretory PLA 2s are being actively investigated, and part of the knowledge on secretory PLA 2 effects has been gained by using snake venom PLA 2s as tools, due to their high structural homology with human secretory PLA 2s. The inflammatory events evoked by PLA 2s are primarily associated with enzymatic activity and to the release of arachidonic acid metabolites. However, catalytically inactive Lys49 PLA 2s trigger inflammatory and nociceptive responses comparable to those of their catalytically active counterparts, thereby evidencing that these proteins promote inflammation and pain by mechanisms not related to phospholipid hydrolysis nor to mobilization of arachidonic acid. These studies have provided a boost to the research in this field and various approaches have been used to identify the amino acid residues and the specific sites of interaction of myotoxic PLA 2s with cell membranes potentially involved in the PLA 2-induced inflammatory and nociceptive effects. This work reviews the proinflammatory and nociceptive effects evoked by myotoxic PLA 2s and their mechanisms of action.

Toxic Peripheral Neuropathy of Chicks FedSenna occidentalisSeeds

Plants of the genusSenna(formerlyCassia) are poisonous to livestock and other laboratory animals, leading to a syndrome of a widespread muscle degeneration, incoordination, recumbence, and death. The main histologic lesion is necrosis of skeletal muscle fibers. Recently, a mitochondrial myopathy with ragged-red and cytochrome oxidase (COX)-negative muscle fibers was recognized in hens chronically intoxicated with parts of seeds ofS. occidentalis. The purpose of the present work was to investigate if there was peripheral nerve involvement in the acute intoxication of chicks withS. occidentalisseeds. Teasing of individual fibers revealed signs of extensive axonal damage with myelin ovoids. Ultrathin sections confirmed the axonal damage. Axons were filled with membranes, some residual disorganized filaments, and enlarged mitochondria. In some instances the axon disappeared and there was secondary degeneration of the myelin sheath. The present work is the first description of the neurotoxic effect ofS. occidentalisintoxication. Future work should attempt to determine the mechanisms involved in this neuropathy.

Small-caliber skeletal muscle fibers do not suffer deleterious consequences of dystrophic gene expression.

In Duchenne dystrophy and in the genetic dystrophies of CHF-147 hamsters and MDX mice, the fundamental deleterious consequence of dystrophic gene expression is segmental necrosis of skeletal muscle fibers. The nature of the gene defects and the pathogenesis of muscle fiber damage are not known. However, clinical and experimental evidence indicates that muscle fibers, whose girth is below a certain level (estimated at approximately 20-25 microns in diameter in dystrophic hamsters and MDX mice) are not susceptible to necrosis. This apparent "immunity" has been observed in muscle fibers that are naturally of small girth (such as those in extraocular muscles), and in fibers that were prevented from growing normally by experimental procedures (hamsters and mice) or by pathological processes (Duchenne patients). The cellular or molecular basis by which small-caliber muscle fibers are resistant to the necrotizing effect of the dystrophic gene expression remains unknown. In small-caliber muscle fibers, the normal contraction-related mechanical strains per unit surface area are relatively less than in larger fibers; this could explain their relative resistance to necrosis in some dystrophies.

Autophagic response to strenuous exercise in mouse skeletal muscle fibers.

Strenuous physical exercise induces necrosis of skeletal muscle fibers and increases lysosomal enzyme activities in surviving muscle fibers. This study examines the ultrastructural basis of the stimulation of the lysosomal system in mouse vastus medialis muscle during the appearance and repair of exercise-induced (9 h of running) injuries. Necrotic fibers appeared the day after exercise and an inflammatory response with the replacement of necrotic fibers by phagocytes was highest 2-3 days after exertion. Ultrastructural study of surviving muscle fibers revealed numerous autophagic vacuoles, residual bodies, and spheromembranous structures at the periphery of myofibers, especially in fibers adjacent to necrotic fibers. The autophagic response was most prominent between 2 and 7 days after exertion. Autophagic vacuoles with double or single limiting membranes contained mitochondria at various stages of degradation. Vacuolar and multilamellar structures were also observed in regenerating muscle fibers. The structure of injured skeletal muscle fibers returned to normal within 2 weeks. It is proposed that increased autophagic activity could be related to the breakdown of cellular constituents of surviving muscle fibers to provide structural elements for regenerating muscle fibers.

Reinnervation is followed by necrosis in previously denervated skeletal muscles of dystrophic hamsters

Hind leg muscles of dystrophic hamsters were continually denervated by multiple crushes of the sciatic nerve to as long as 93 days of age. In these muscles, the prevalence of centronucleated fibers which is a cumulative index of prior necrosis, remained very low. In control dystrophic muscles the prevalence of centronucleated fibers increased steadily to approximately 80% where it leveled off. By omitting further crushes in other groups of animals, previously denervated muscles became adequately reinnervated. In the reinnervated muscles the prevalence of centronucleated fibers steadily increased throughout the necrotic phase of dystrophy at a rate that was comparable to corresponding stages of the natural history of the disease. These experiments indicated that continued denervation was effective in negating skeletal muscle fiber necrosis throughout the necrotic phase and that the electromechanical activity of muscle fibers which allows muscle fiber necrosis was not a time-locked factor.

Myotonia, a new inherited muscle disease in mice

Electromyographic (EMG) and histological studies were carried out with the new mouse mutant myotonia (mto, autosomal recessive). Affected animals exhibited behavioral myotonia which was apparent at 2 weeks of age. EMG studies revealed myotonic discharges (prolonged repetitive discharges with recurrent variation in amplitude and frequency) in all skeletal muscles tested. These discharges were not affected by peripheral nerve section or by pancuronium bromide, a neuromuscular synapse inhibitor. Qualitative histological study revealed no evidence of skeletal muscle fiber necrosis, grouped atrophy, or inflammation compared toi sex-, age-, and weight-matched controls. Degeneration of myelinated axons in the ventrolateral funiculus of the spinal cord occurred only in older animals and does not explain the functional abnormality of muscle. Both the clinical and electrophysiological features of classical myotonia can be inherited as a single locus autosomal recessive disease in mice.

Prevention of skeletal muscle fiber necrosis in hamster dystrophy.

Necrosis and widespread central nucleation of skeletal muscle cells, which are the main features of skeletal muscle pathology in genetically dystrophic hamsters (UMX-7.1 strain), were not present in muscles of 45- to 65-day-old animals which had been surgically denervated at 15 to 18 days of age. Necrosis and widespread central nucleation were also absent in hind leg muscles at 30 to 60 days of age after transection of the high thoracic spinal cord at 15 to 18 days. Normal excitation or contraction of the skeletal muscle fibers in early age appears necessary for the microscopic pathological expression of the dystrophic gene in skeletal muscles in the UMX-7.1 strain hamsters. These experiments constitute a rare and clear example of consistent prevention of skeletal muscle cell destruction in vivo in an inherited myopathy.

Studies on Nutritional Myopathy Syndrome in Cultured Fishes-I

In 1974, 1975 and 1976, nutritional myopathy occurred among cultured young puffers (Fugu rubripes) fed frozen fish meat, in a farm in Kagawa Pref. They were very thin and slender in appearance. One hundred and fifty three diseased puffers were examined histopathologically.This case was characterized by degeneration and necrosis of skeletal muscle fibers, proliferation of connective tissue and appearance of ceroid-laden macrophages in the stroma. It was found that such changes advanced more severely and quickly in erector and depressor muscle tissues than in lateral muscle tissues. In advanced cases most of the skeletal muscle fibers disappeared, and the connective tissue proliferated and ceroid-laden macrophages appeared markedly in the stroma.The lipopigment was deposited in parenchymal cells in the liver. This lipopigment was similar to ceroid in histochemical nature except that the pigment had no acid-fastness. Ceroid-laden macrophages appeared in the spleen, kidney and intestine.The present authors consider the above-mentioned changes closely resemble yellow-fat disease in mammals and aves.

The crush injury: A high risk wound

A standardized experimental impact injury model was developed to produce soft tissue trauma that simulated impact injuries to soft tissue overlying the cranium. An aluminum impact instrument was constructed so as to deliver a measured amount of energy to a finite area of soft tissue over a fixed foundation. Impact injury resulted in readily demonstrable changes in the morphology of the tissue and its blood flow. As measured by the distribution of fluorescein dye, the blood flow to the impact site was considerably less than that to unwounded tissue. The magnitude of this reduction in blood flow to the site of injury was proportional to the level of energy absorbed by the tissue. The injury to skin resulting from the impact was restricted primarily to the subcutaneous tissue and panniculus carnosus. Loss of adipose tissue and necrosis of skeletal muscle fibers in these layers were associated with the development of a dense inflammatory infiltrate. The structural damage to the tissue and its reduced perfusion were correlated with the tissue's increased susceptibility to infection by bacteria delivered either by direct infection or as a result of a bacteremia. The magnitude of this damage to the host's defenses is, again, directly related to the amount of energy absorbed per unit area of soft tissue. Antibiotics did suppress the growth of bacteria in these experimental soft tissue impact wounds, even when treatment was delayed eight hours. While the therapeutic efficacy of antibiotics was readily apparent in soft tissue impact wounds, the degree to which they could suppress bacteria in these wounds was less than that encountered in the treated control wounds not subjected to impact.