Temporal response pattern of biochemical analytes in experimental diabetes

The activities of the enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LD), creatine kinase (CK), amylase (AMS) and angiotensin converting enzyme (ACE) have been used to assess the toxic effects of xenobiotics that have hypoglycaemic action in hepatic, pancreatic, renal and muscle tissue. Using a validated experimental model of diabetes mellitus in rats, we ascertained whether this syndrome itself affected the serum activities of these enzymes over a 53-day period. Levels of hepatic enzymes AST, ALT and ALP were higher in the streptozotocin (STZ)diabetic rats (group D), but were controlled by insulin therapy (group DI). AMS was reduced in group D and unchanged in group DI rats. Proteinuria was detected 1 day after STZ administation and partially controlled by insulin (group DI); its early presence in group D rats, and the lack of any change in serum ACE in this group, indicates that proteinuria is the better marker for microangiopathy. Microscopic examination of liver, kidney, heart and skeletal muscles (soleus and extensor digitorum longus) revealed various alterations in group D rat tissues, which were less pronounced in group DI. The liver, pancreas and kidney tissue-damage was consistent with the altered serum levels of AST, ALT, ALP and AMS and proteinuria. We conclude that: (i) rigorous control is required when these serum-enzyme levels are used as indicators of tissue toxicity in experimental diabetes, and (ii) LD, CK and bilirubin serum levels, which are unaffected by diabetes, can be used when testing effects of xenobiotics on tissues.

Differential morphofunctional characteristics and gene expression in fast and slow muscle of rats with monocrotaline-induced heart failure

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Paralyzing and myotoxic effects of a recombinant bothropstoxin-1 (BthTX-I) on mouse neuromuscular preparations

As a first step to investigate the structure-function relationship of bothropstoxin-1 (BthTX-1), a myotoxin from Bothrops jararacussu snake venom, Our group previously cloned a recombinant toxin (rBthTX-1) in Escherichia coli. The aim or this work was to characterize the biological activities of this rBthTX-1 (1.0 mu M) in both phrenic-diaphragm and extensor digitorum longus preparations in vitro, by means of myographic and morphologic techniques. Native BthTX-1 (1.0 mu M) was used as a standard. The influence of heparin (27.5 mu g/ml) upon the biological activities of both toxins was also investigated. rBthTX-1 had similar effects to the native toxin inducing blockage of both directly and indirectly evoked contractions in phrenic-diaphragm preparations, and muscle damage characterized by edema, round fibers, and cell areas devoid of myofibrils. Interestingly the paralyzing activity of rBthTX-1 was slightly more potent than the native toxin. Heparin prevented paralyzing and myotoxic effects of both the native and recombinant toxins. This work shows that rBthTX-1 was expressed in a fully active form, and presents a biological profile similar to the native toxin. (c) 2005 Elsevier GmbH All rights reserved.

Reduction of crotoxin-induced neuromuscular blockade by gamma radiation

A comparative study between crotoxin and gamma irradiated crotoxin was performed on the indirectly evoked twitches and tetani of sciatic nerve-extensor digitorum longus muscle of rats. Crotoxin (3 to 14 mu g/ml) decreased the amplitude of twitches and induced a slight tetanic fade, and irradiated crotoxin did not significantly affect either twitch amplitude or tetanic tension. Since gamma radiation reduced the neurotoxicity of crotoxin it may be useful for the production of anticrotalic serum. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Antagonism of myotoxic and paralyzing activities of bothropstoxin-I by surarnin

Polyanionic substances are known to inhibit the myotoxic effects of some crotalide snake venoms. Bothropstoxin-I (BthTX-I), a basic Lys49 phospholipase (PLA(2)) homologue from Bothrops jararacussu venom, besides inducing muscle damage, also promotes the blockade of both directly and indirectly evoked contractions in mouse neuromuscular preparation. In this work, we evaluated the ability of suramin, a polysulfonated naphtylurea derivative, to antagonize the myotoxic and the paralyzing activities of BthTX-I on mice neuromuscular junction in vitro. Myotoxicity was assessed by light and electronic microscopic analysis of extensor digitorum longus (EDL) muscles; paralyzing activity was evaluated through the recording of both directly and indirectly evoked contractions of phrenic-diaphragm (PD) preparations. BthTX-I (1 muM) alone, or pre-incubated with suramin (10 muM) at 37degreesC for 15 min was added to the preparations for 120 min. BthTX-I induced histological alterations typical of myonecrosis in 14.6 +/- 1.0% of EDL muscle fibers. In addition, BthTX-I blocked 50% of both directly and indirectly evoked contractions in PD preparations in 72.1 +/- 9.1 and 21.1 +/- 2.0 min, respectively. Pre-incubation with suramin abolished both the muscle-damaging and muscle-paralyzing activities of BthTX-I. Since suramin is a polyanionic substance, we suggested that its effects result from the formation of inactive acid-base complexes with BthTX-I. (C) 2003 Elsevier Ltd. All rights reserved.

Nandrolone Stimulates Myod Expression During Muscle Regeneration in the Condition of Myonecrosis Induced by Bothrops jararacussu Venom Poisoning

Myonecrosis with permanent loss of muscle mass is a relevant local toxic effect following envenomation with Bothrops jararacussu snake venom. Regeneration of adult skeletal muscle involves the activation of satellite cells, a process regulated by myogenic regulatory factors (MRF). MyoD is an MRF involved in both proliferation and differentiation of satellite cells. Androgens are modulators of skeletal muscle, known to increase muscle mass and strength. This study examined the hypothesis that anabolic androgens improve the muscle regeneration process in mice following envenomation by Bothrops jararacussu snake venom. Myonecrosis was induced by venom injection (30 g/50 l in physiological solution) over the extensor digitorum longus (EDL) muscles of mice. Nandrolone (ND) (6 mg/kg, sc) was administered after 12 h, 7 d, and 14 d following venom injection. The histological changes in EDL muscle at 1, 3, 7, and 21 d after muscle injury were analyzed by light microscopy. Cross-sectional areas of fibers were measured. MyoD was evaluated by immunofluorescence technique. Histological examination revealed the presence of a regeneration process in ND-treated animals, characterized by the appearance of some myotubes at 3 d, and numerous myotubes at 7 d from venom injection. Nandrolone treatment reduced the frequency of small fibers at 7 and 21 d after venom administration, and increased the frequency of large fibers at 7 d postinjury. Nandrolone also significantly augmented the expression of MyoD-positive cells at 7 and 21 d after envenomation. These results suggest that ND accelerates muscle regeneration and indicate the involvement of MyoD in this process.

Heart failure increases atrogin-1 and MuRF1 gene expression in skeletal muscle with fiber type-specific atrophy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Histochemical differences in the responses of predominantly fast-twitch glycolytic muscle and slow-twitch oxidative muscle to veratrine

The aim of this study was to investigate if the Na+-channel activating alkaloid veratrine is able to change the oxidative and m-ATPase activities of a fast-twitch glycolytic muscle (EDL, extensor digitorum longus) and slow-twitch oxidative muscle (SOL, soleus) in mice. Oxidative fibers and glycolytic fibers were more sensitive to veratrine than oxidative-glycolytic fibers 15, 30 and 60 min after the i.m. injection of veratrine (10 ng/kg) with both showing an increase in their metabolic activity in both muscles. In EDL, the m-ATPase reaction revealed a significant (p < 0.001) decrease (50%) in the number of type IIB fibers after 30 min while the number of type I fibers increased by 550%. Type I fibers decreased from 34% in control SOL to 17% (50% decrease) in veratrinized muscles, with a 10% decrease in type IIA fibers within 15 min. A third type of fiber appeared in SOL veratrinized muscle, which accounted for 28% of the fibers. Our work gives evidence that the changes in the percentage of the fiber types induced by veratrine may be the result, at least partially, from a direct effect of veratrine on muscle fibers and else from an interaction with the muscle type influencing distinctively the response of a same fiber type. Based on the results obtained in the present study the alterations in EDL may be related to the higher number of Na+ channels present in this muscle whereas those in SOL may involve an action of veratrine on mitochondria. Although it is unlikely that the shift of enzymes activities induced by veratrine involves genotypic expression changes an alternative explanation for the findings cannot be substantiated by the present experimental approach. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Co-60 gamma irradiation prevents Bothrops jararacussu venom neurotoxicity and myotoxicity in isolated mouse neuromuscular junction

The ability of gamma radiation from Co-60 (2000 Gy) to attenuate the toxic effects of Bothrops jararacussu venom was investigated on mouse neuromuscular preparations in vitro. A comparative study between the effects of native and irradiated venoms was performed on both phrenic-diaphragm (PD) and extensor digitorum longus (EDL) preparations by means of myographic, biochemical and morphological techniques. Native venom (10 and 20 mug/ml) induced a concentration-dependent paralysis of both directly and indirectly evoked contractions on PD preparations. At 20 mug/ml, it also caused a pronounced myotoxic effect on the EDL muscle preparation that was characterized by an increase of creatine kinase release and by several morphological changes of this preparation. By contrast, irradiated venom, even at concentrations as high as 40 mug/ml, induced neither paralyzing nor myotoxic effects. It was concluded that Co-60 gamma radiation is able to abolish both the paralyzing and the myotoxic effects of B. jararacussu venom on the mouse neuromuscular junction. These findings support the hypothesis that gamma radiation could be an important toot to improve antisera production by reducing toxicity while preserving immunogenicity. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Skeletal muscles with antagonistic muscular actions: morphological, contractile and metabolic characteristics

The muscles can perform the same function in a specific segment (muscles of fast and slow contraction), and at the same time be antagonistic in relation to muscular action (flexors or extensors). The present research aimed to study the morphology, frequency and metabolism of fiber types and the contractile characteristics of extensor and flexors muscles of rabbit. We studied muscles anterior tibialis (AT), flexor digitorum supeficialis (FDS), extensor digitorum longus (EDL) and posterior tibialis (PT). The muscles were submitted to the techniques HE, NADH-TR and myofibrillar ATPase. In EDL and PT extensor muscles, the frequencies of red (SO + FOG) and white fibers (FG) were 68.77% and 31.23% versus 58.87% and 41.13%, respectively. In the AT and FDS flexor muscles, these frequencies were 75.14% and 24.86% versus 73.89% and 26.11%, respectively. In extensor muscles, the percentage of slow contraction fibers was 8.05% in EDL and 9.74% in PT, and in fast contraction, 91.95% in EDL and 90.26% in PT. In flexors, the slow contraction frequencies were 12.35% in AT and 8.17% in FDS, and in fast contraction, 87.65% and 91.83%, respectively. Skeletal muscles with antagonistic muscular actions (flexors and extensors) the morphological, contractile and metabolic characteristics are identical.

Maternal protein restriction induce skeletal muscle changes without altering the MRFs MyoD and myogenin expression in offspring

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tempo de início da atividade elétrica dos estabilizadores patelares na marcha em sujeitos com e sem síndrome de dor femoropatelar

INTRODUÇÃO: A síndrome de dor femoropatelar (SDFP) é um problema comum que afeta uma em cada quatro pessoas. A alteração no tempo de ativação e a intensidade de contração dos músculos vasto medial oblíquo (VMO) e vasto lateral (VL) são consideradas fatores importantes na etiologia da SDFP. No entanto, existem poucos estudos sobre a função da porção oblíqua do vasto lateral (VLO) e nenhum sobre o tempo de ativação (onset) do VLO em atividades funcionais em sujeitos normais e com SDFP. OBJETIVO: Assim, o objetivo do estudo foi investigar o tempo de início de atividade eletromiográfica nos músculos VMO, VLO e VL longo (VLL) durante a marcha. MATERIAIS E MÉTODOS: A amostra foi formada por 15 sujeitos sem e 12 com SDFP. Dados eletromiográficos foram obtidos dos músculos VMO, VLL e VLO durante caminhada na esteira sem inclinação. A diferença relativa no onset (DRO) entre VMO-VLL e VMO-VLO foi determinada a partir da média de três passadas. RESULTADOS: Houve diferença entre os sujeitos com e sem SDFP em relação à DRO entre VMO-VLL. Nos sujeitos com SDFP, a ordem de início da atividade elétrica foi VLL seguida por VLO e após VMO. Nos indivíduos sem a patologia, a ordem foi diferente: primeiro VMO após VLO e, por último, VLL. CONCLUSÃO: Os achados sugerem que a ativação do VMO após o VLL poderia auxiliar no desenvolvimento e na manutenção da SDFP, enquanto o tempo de ativação do VLO possui menor participação.

Tipos de miosinas de linhagens de frangos de corte criados em sistemas de confinamento e semiconfinamento

O objetivo neste trabalho foi avaliar o peso vivo, o peso de pernas, os aspectos morfológicos das fibras musculares do músculo flexor longo do hálux e o perfil eletroforético das miosinas de cadeia pesada de quatro linhagens de frangos de corte criados nos sistemas de confinamento e semiconfinamento. Foram utilizados 1.440 pintos distribuídos em delineamento inteiramente casualisado em esquema fatorial 4 × 2, composto de quatro linhagens (Ross 305, Máster Gris, Label Rouge e Vermelhão Pesado) e dois sistemas de criação (confinamento e semiconfinamento), cada combinação avaliada com quatro repetições. Aos 28 e 84 dias de idade, foram abatidas quatro aves por tratamento, totalizando 64 aves. A eletroforese identificou a presença das três isoformas de miosinas, tipo MyHC-I, MyHC-IIa e MyHC-IIb, no músculo flexor longo do hálux dos frangos de corte. Com aumento da idade, a isoforma de miosina MyHC-II aumenta, enquanto a MyHC-I diminui. Somente aos 84 dias de idade, a expressão das isoformas de miosina do tipo MyHC-II foram influenciadas pela linhagem, confirmando o reflexo da seleção na linhagem Ross no músculo mais glicolítico.A linhagem Ross apresenta maior peso vivo, peso de perna, peso e área do músculo flexor longo do hálux em comparação às linhagens tipo caipira.

Effects of nandrolone decanoate on the neuromuscular junction of rats submitted to swimming

This study addressed the effects of nandrolone decanoate (ND) on contractile properties and muscle fiber characteristics of rats submitted to swimming. Male Wistar rats were grouped in sedentary (S), swimming (Sw), sedentary+ND (SND), and swimming+ND (SwND), six animals per group. ND (3 mg/kg) was injected (subcutaneously) 5 days/week, for 4 weeks. Swimming consisted of 60-min sessions (load 2%), 5 days/week, for 4 weeks. After this period, the sciatic nerve extensor digitorum longus (EDL) muscle was isolated for myographic recordings. Fatigue resistance was assessed by the percent (%) decline of 180 direct tetanic contractions (30 Hz). Safety margin of synaptic transmission was determined from the resistance to the blockade of indirectly evoked twitches (0.5 Hz) induced by pancuronium (5 to 9 x 10(-7) M). EDL muscles were also submitted to histological and histochemical analysis (haematoxylin-eosin (HE); nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR)). Significant differences were detected by two-way ANOVA (p<0.05). ND did not change body mass, fatigue resistance or kinetic properties of indirect twitches in either sedentary or swimming rats. In contrast, ND reduced the safety margin of synaptic transmission in sedentary animals (SND=53.3+/-4.7% vs. S=75.7+/-2.0%), but did not affect the safety margin in the swimming rats (SwND=75.81+/-3.1% vs. Sw=71.0+/-4.0%). No significant difference in fiber type proportions or diameters was observed in EDL muscle of any experimental group. These results indicate that ND does not act as an ergogenic reinforcement in rats submitted to 4 weeks of swimming. on the other hand, this study revealed an important toxic effect of ND, that it reduces the safety margin of synaptic transmission in sedentary animals. Such an effect is masked when associated with physical exercise. (C) 2004 Elsevier B.V. All rights reserved.

beta(2)-Agonists and cAMP inhibit protein degradation in isolated chick (Gallus domesticus) skeletal muscle

1. The role of beta(2)-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours.2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective beta(2)-agonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP.3. The decrease in the rate of total protein degradation induced by 10(-5) M clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10(-5) M ICI 118.551, a selective beta(2)-antagonist.4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10(-4) M dibutyryl-cAMP or isobutylmethylxanthine.5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.