Kindlin-2 loss in condylar chondrocytes causes spontaneous osteoarthritic lesions in the temporomandibular joint in mice / 国际口腔科学杂志·英文版

The progressive destruction of condylar cartilage is a hallmark of the temporomandibular joint (TMJ) osteoarthritis (OA); however, its mechanism is incompletely understood. Here, we show that Kindlin-2, a key focal adhesion protein, is strongly detected in cells of mandibular condylar cartilage in mice. We find that genetic ablation of Kindlin-2 in aggrecan-expressing condylar chondrocytes induces multiple spontaneous osteoarthritic lesions, including progressive cartilage loss and deformation, surface fissures, and ectopic cartilage and bone formation in TMJ. Kindlin-2 loss significantly downregulates the expression of aggrecan, Col2a1 and Proteoglycan 4 (Prg4), all anabolic extracellular matrix proteins, and promotes catabolic metabolism in TMJ cartilage by inducing expression of Runx2 and Mmp13 in condylar chondrocytes. Kindlin-2 loss decreases TMJ chondrocyte proliferation in condylar cartilages. Furthermore, Kindlin-2 loss promotes the release of cytochrome c as well as caspase 3 activation, and accelerates chondrocyte apoptosis in vitro and TMJ. Collectively, these findings reveal a crucial role of Kindlin-2 in condylar chondrocytes to maintain TMJ homeostasis.

The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts

Fibroblasts are a highly heterogeneous population of cells, being found in a large number of different tissues. These cells produce the extracellular matrix, which is essential to preserve structural integrity of connective tissues. Fibroblasts are frequently engaged in migration and remodeling, exerting traction forces in the extracellular matrix, which is crucial for matrix deposition and wound healing. In addition, previous studies performed on primary myoblasts suggest that the E3 ligase MuRF2 might function as a cytoskeleton adaptor. Here, we hypothesized that MuRF2 also plays a functional role in skeletal muscle fibroblasts. We found that skeletal muscle fibroblasts express MuRF2 and its siRNA knock-down promoted decreased fibroblast migration, cell border accumulation of polymerized actin, and down-regulation of the phospho-Akt expression. Our results indicated that MuRF2 was necessary to maintain the actin cytoskeleton functionality in skeletal muscle fibroblasts via Akt activity and exerted an important role in extracellular matrix remodeling in the skeletal muscle tissue.

Myostatin promotes distinct responses on protein metabolism of skeletal and cardiac muscle fibers of rodents

Myostatin is a novel negative regulator of skeletal muscle mass. Myostatin expression is also found in heart in a much less extent, but it can be upregulated in pathological conditions, such as heart failure. Myostatin may be involved in inhibiting protein synthesis and/or increasing protein degradation in skeletal and cardiac muscles. Herein, we used cell cultures and isolated muscles from rats to determine protein degradation and synthesis. Muscles incubated with myostatin exhibited an increase in proteolysis with an increase of Atrogin-1, MuRF1 and LC3 genes. Extensor digitorum longus muscles and C2C12 myotubes exhibited a reduction in protein turnover. Cardiomyocytes showed an increase in proteolysis by activating autophagy and the ubiquitin proteasome system, and a decrease in protein synthesis by decreasing P70S6K. The effect of myostatin on protein metabolism is related to fiber type composition, which may be associated to the extent of atrophy mediated effect of myostatin on muscle.

Loss of Ovarian Function Results in Increased Loss of Skeletal Muscle in Arthritic Rats / Perda da função ovariana resulta em perda de músculo esquelético aumentada em ratos artríticos

Objective We studied the effects of loss of ovarian function (ovariectomy) onmuscle mass of gastrocnemius and themRNA levels of IGF-1, atrogin-1, MuRF-1, andmyostatin in an experimental model of rheumatoid arthritis in rats. Methods We randomly allocated 24 female Wistar rats (9 weeks, 195.3±17.4 grams) into four groups: control (CT-Sham; n = 6); rheumatoid arthritis (RA; n = 6); ovariectomy without rheumatoid arthritis (OV; n = 6); ovariectomy with rheumatoid arthritis (RAOV; n = 6). We performed the ovariectomy (OV and RAOV) or Sham (CTSham or RA) procedures at the same time, fifteen days before the rheumatoid arthritis induction. The RA and RAOV groups were immunized and then were injected with Met- BSA in the tibiotarsal joint. After 15 days of intra-articular injections the animals were euthanized. We evaluated the external manifestations of rheumatoid arthritis (perimeter joint) as well as animal weight, and food intake throughout the study. We also analyzed the cross-sectional areas (CSA) of gastrocnemius muscle fibers in 200 fibers (H&E method). In the gastrocnemius muscle, we analyzed mRNA expression by quantitative real time PCR followed by the Livak method (ΔΔCT). Results The rheumatoid arthritis induced reduction in CSA of gastrocnemius muscle fibers. The RAOV group showed a lower CSA of gastrocnemius muscle fibers compared to RA and CT-Sham groups. Skeletal muscle IGF-1 mRNA increased in arthritics and ovariectomized rats. The increased IGF-1 mRNA was higher in OV groups than in the RA and RAOV groups. Antrogin-1 mRNA also increased in the gastrocnemius muscle of arthritic and ovariectomized rats. However, the increased atrogin-1 mRNA was higher in RAOV groups than in the RA and OV groups. Gastrocnemius muscle MuRF-1 mRNA increased in the OVand RAOVgroups, but not in the RA and Shamgroups. However, the RAOV group showed higher MuRF-1 mRNA than the OV group. The myostatin gene expression was similar in all groups. Conclusion Loss of ovarian function results in increased loss of skeletal musclerelated ubiquitin ligases atrogin-1 and MuRF-1 in arthritic rats.

Objetivo Foram estudados os efeitos da perda da função ovariana (ovariectomia) sobre músculo esquelético e os níveis de RNAm de IGF-1, atrogina-1, MuRF-1, e de miostatina em modelo experimental de artrite reumatóide em ratos. Métodos 24 ratos Wistar (9 semanas, 195,3±17,4 gramas) foram distribuídos aleatoriamente em quatro grupos: controle (CT-Sham, n = 6); artrite reumatóide (RA, n = 6); ovariectomia sem artrite reumatóide (OV; n = 6); ovariectomia com artrite reumatóide (RAOV; n = 6). Os procedimentos da ovariectomia (OV e RAOV) ou simulação da ovariectomia (CT-Shamou RA) foramrealizados aomesmo tempo, quinze dias antes da indução da artrite reumatóide. Os grupos RA e RAOV foramimunizados e, em seguida, foram injetados com Met-BSA na articulação tibiotársica. Após 15 dias das injeções intra-articulares, os animais foram eutanasiados. Foram avaliadas as manifestações externas da artrite reumatóide (perimetria articular), bem como o peso dos animais e a ingestão de alimentos ao longo do estudo. Além disso, as áreas de secção transversa (CSA) do músculo gastrocnêmio foram analisadas em 200 fibras (método H & E). No músculo gastrocnêmio, a expressão de RNAm foi analisada por PCR quantitativo em tempo real, seguido pelo método Livak (ΔΔCT). Resultados A artrite reumatoide reduziu a CSA das fibras do músculo gastrocnêmio. O grupo RAOV mostrou uma CSA menor nas fibras do músculo gastrocnêmio em comparação com os grupos RA e CT-Sham. O RNAm do IGF-1 do músculo esquelético aumentou nos ratos artríticos e ovariectomizados. O RNAm do IGF-1 foi maior nos grupos OV do que nos grupos RA e RAOV. A expressão de antrogina-1 também aumentou no músculo gastrocnêmio dos ratos artríticos e ovariectomizados. No entanto, o aumento do RNAm da atrogina-1 foi maior no grupo RAOV do que nos grupos RA e OV. O RNAm da MuRF-1 aumentou nos grupos OV e RAOV, mas não nos grupos RA e CT-Sham. Porém, o grupo RAOV apresentou maior expressão gênica de MuRF-1 do que o grupo OV. A expressão do gene da miostatina foi semelhante em todos os grupos. Conclusão A perda de função ovariana resulta em perda de músculo esquelético associado às ubiquitina-ligases atrogina-1 e MuRF-1 em ratos artríticos.

Exercise improved lipid metabolism and insulin sensitivity in rats fed a high-fat diet by regulating glucose transporter 4 (GLUT4) and musclin expression

This study aimed to evaluate the effects of exercise training on triglyceride deposition and the expression of musclin and glucose transporter 4 (GLUT4) in a rat model of insulin resistance. Thirty male Sprague-Dawley rats (8 weeks old, weight 160±10 g) were fed a high-fat diet (40% calories from fat) and randomly divided into high-fat control group and swimming intervention group. Rats fed with standard food served as normal control. We found that 8-week swimming intervention significantly decreased body weight (from 516.23±46.27 to 455.43±32.55 g) and visceral fat content (from 39.36±2.50 to 33.02±2.24 g) but increased insulin sensitivity index of the rats fed with a high-fat diet. Moreover, swimming intervention improved serum levels of TG (from 1.40±0.83 to 0.58±0.26 mmol/L) and free fatty acids (from 837.80±164.25 to 556.38±144.77 μEq/L) as well as muscle triglycerides deposition (from 0.55±0.06 to 0.45±0.02 mmol/g) in rats fed a high-fat diet. Compared with rats fed a standard food, musclin expression was significantly elevated, while GLUT4 expression was decreased in the muscles of rats fed a high-fat diet. In sharp contrast, swimming intervention significantly reduced the expression of musclin and increased the expression of GLUT4 in the muscles of rats fed a high-fat diet. In conclusion, increased musclin expression may be associated with insulin resistance in skeletal muscle, and exercise training improves lipid metabolism and insulin sensitivity probably by upregulating GLUT4 and downregulating musclin.

Three days of intermittent stretching after muscle disuse alters the proteins involved in force transmission in muscle fibers in weanling rats

The aim of this study was to determine the effects of intermittent passive manual stretching on various proteins involved in force transmission in skeletal muscle. Female Wistar weanling rats were randomly assigned to 5 groups: 2 control groups containing 21- and 30-day-old rats that received neither immobilization nor stretching, and 3 test groups that received 1) passive stretching over 3 days, 2) immobilization for 7 days and then passive stretching over 3 days, or 3) immobilization for 7 days. Maximal plantar flexion in the right hind limb was imposed, and the stretching protocol of 10 repetitions of 30 s stretches was applied. The soleus muscles were harvested and processed for HE and picrosirius staining; immunohistochemical analysis of collagen types I, III, IV, desmin, and vimentin; and immunofluorescence labeling of dystrophin and CD68. The numbers of desmin- and vimentin-positive cells were significantly decreased compared with those in the control following immobilization, regardless of whether stretching was applied (P<0.05). In addition, the semi-quantitative analysis showed that collagen type I was increased and type IV was decreased in the immobilized animals, regardless of whether the stretching protocol was applied. In conclusion, the largest changes in response to stretching were observed in muscles that had been previously immobilized, and the stretching protocol applied here did not mitigate the immobilization-induced muscle changes. Muscle disuse adversely affected several proteins involved in the transmission of forces between the intracellular and extracellular compartments. Thus, the 3-day rehabilitation period tested here did not provide sufficient time for the muscles to recover from the disuse maladaptations in animals undergoing postnatal development.

Expression of MuRF1 and MAFbx in donor and recipient muscles after musculocutaneous nerve transection and partial pectoralis major muscle transfer for reconstruction of elbow flexion in rats / Expresión de MuRF1 y MAFbx en músculos donantes y receptores después de transección del nervio musculocutáneo y transferencia parcial del músculo pectoral mayor para la reconstrucción de flexión de codo en ratas

The expression of MuRF1 and MAFbx in a denervated muscle has previously been studied. However, the expression of MuRF1 and MAFbx in the recipient and donor muscles after muscle transfer for reconstruction of joint function has not been sufficiently investigated. Forty-two adult Sprague-Dawley rats were divided into 7 groups: normal, 1 w post-, 2 w post-, and 4 w post-musculocutaneous nerve transection; and 1 w post-, 2 w post-, and 4 w post-reconstruction of elbow flexion. Muscle wet weights were assessed, and MuRF1 and MAFbx mRNA expressions were detected by polymerase chain reaction. The length of the oblique part of the pectoralis major of an SD rat is sufficient for suture to the insertion of the biceps brachii tendon. The muscle wet weight and the wet weight retention rate of the biceps brachii continued to decline after musculocutaneous nerve transection and a gradual increase was noted after the oblique part of the pectoralis major was transferred for reconstruction of elbow flexion. The oblique part of the pectoralis major showed a decrease of only 2­6%. The upregulated expression of MuRF1 and MAFbx in the biceps brachii reached a peak 2 w after denervation and 1 w after elbow flexion reconstruction, with an increase of 15% and 4%, respectively. This was followed by downregulation; however, the expression had not normalized at postoperative 4 w. The increased expression of MuRF1 (17%) and MAFbx (1%) in the oblique part of the pectoralis major at postoperative 1 w had decreased to below normal levels at postoperative 4 w. The transfer of the oblique part of the pectoralis major for elbow flexion reconstruction after musculocutaneous nerve transection can downregulate the expression of MuRF1 and MAFbx in the recipient muscle and causes only transient damage to the donor muscle in rats.

La expresión de MuRF1 y MAFbx en un músculo denervado ha sido estudiada previamente. Sin embargo, la expresión de MuRF1 y MAFbx en los músculos receptores y donantes después de la transferencia del músculo para la reconstrucción de la función articular no se ha investigado lo suficiente. Cuarenta y dos ratas adultas Sprague-Dawley fueron divididas en 7 grupos: normales, 1 semana post-, 2 semanas post- y 4 semanas post-transección del nervio musculocutáneo; y 1 semana post-, 2 semanas post-, y 4 semanas post-reconstrucción de la flexión del codo. Se evaluó el peso de los músculos húmedos, y las expresiones de MuRF1 y MAFbx mRNA fueron detectadas a través de reacción en cadena de la polimerasa. La longitud de la parte oblicua del músculo pectoral mayor de una rata Sprague-Dawley es suficiente para realizar la sutura en la inserción del tendón de músculo bíceps braquial. El peso húmedo del músculo bíceps braquial y su tasa de retención siguieron disminuyendo después de la sección del nervio musculocutáneo y un aumento gradual se observó después de la transferencia de la parte oblicua del músculo pectoral mayor para la reconstrucción de la flexión del codo. La parte oblicua del músculo pectoral mayor mostró una disminución de sólo 2-6%. La expresión regulada por incremento de MuRF1 y MAFbx en el bíceps braquial alcanzó un peak 2 semanas después de la denervación y 1 semana después de la reconstrucción de la flexión del codo, con un incremento del 15% y el 4%, respectivamente. Esto fue seguido por un regulación en baja. Sin embargo, la expresión no se normalizó en el postoperatorio de las 4 semanas. El aumento de la expresión de MuRF1 (17%) y MAFbx (1%) en la parte oblicua del músculo pectoral fue mayor en el postoperatorio de 1 semana, mientras que se encontró por debajo de los niveles normales en el postoperatorio de 4 semanas. La transferencia de la parte oblicua del músculo pectoral mayor para la reconstrucción de la flexión del codo después de la sección del nervio musculocutáneo puede regular a la baja la expresión de MuRF1 y MAFbx en el músculo receptor y provocar solo un daño transitorio en el músculo donado en ratas.

Expression of Glycolysis-Related Proteins in Solid Papillary Carcinoma of the Breast According to Basement Membrane Status

PURPOSE: The aim of this study was to investigate the differences of expression in glycolysis-related proteins such as Glut-1, carbonic anhydrase (CA) IX, and monocarboxylate transporter (MCT) 4 according to the myoepithelial cell (MEC) and basement membrane (BM) status in solid papillary carcinoma (SPC) of the breast. MATERIALS AND METHODS: Immunohistochemical evaluation of Glut-1, CAIX, and MCT4, as well as p63 and type IV collagen, were performed on 23 SPC cases. RESULTS: Six and nine cases of SPC showed the presence and absence of myoepithelial cells, respectively, and eight cases belonged to the borderline status (p63-positive MEC on some areas of the outer tumor surface but not in others). BM was partially or completely absent in 14 cases and present in nine cases. SPC lacking BM more frequently showed high expression of CAIX than SPC with BM (p=0.037). CONCLUSION: In SPC of the breast, a strong expression of CAIX seems to be associated with an increasing degree of loss of BM, which can be interpreted as BM degradation due to the induction of extracellular acidity with increasing expression of CAIX.

Effect of Anorexia and Neuropathic Pain Induced by Cisplatin on Hindlimb Muscles of Rat

PURPOSE: The purpose of this study was to examine the effect of anorexia and neuropathic pain induced by cisplatin on hindlimb muscles of rats. METHODS: Adult male Sprague-Dawley rats were divided into two groups, a cisplatin-treated group (n=10) and a control group (n=10). In the cisplatin-treated group, cisplatin at a dose of 2 mg/kg was injected intraperitoneally two times a week up to a cumulative dose of 20 mg/kg over 5 weeks, and in the control group saline (0.9% NaCl) was injected intraperitoneally at the same dose and duration as the cisplatin-treated group. At 34 days all rats were anesthetized, after which the soleus and plantaris muscles were dissected. Withdrawal threshold, body weight, food intake, activity, muscle weight, Type I and II fiber cross-sectional areas and myofibrillar protein content of the dissected muscles were determined. RESULTS: Compared with the control group, the cisplatin-treated group showed significant decreases (p<.05) in withdrawal threshold, activity, food intake, body weight, Type I and II fiber cross-sectional areas, myofibrillar protein content and weight of the soleus and plantaris muscles. CONCLUSION: Muscular atrophy in hindlimb occurs due to anorexia and neuropathic pain induced by the cisplatin treatment.

Protein turnover, amino acid requirements and recommendations for athletes and active populations

Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

Comparative proteomics analysis of chronic atrophic gastritis: changes of protein expression in chronic atrophic gastritis without Helicobacter pylori infection

Chronic atrophic gastritis (CAG) is a very common gastritis and one of the major precursor lesions of gastric cancer, one of the most common cancers worldwide. The molecular mechanism underlying CAG is unclear, but its elucidation is essential for the prevention and early detection of gastric cancer and appropriate intervention. A combination of two-dimensional gel electrophoresis and mass spectrometry was used in the present study to analyze the differentially expressed proteins. Samples from 21 patients (9 females and 12 males; mean age: 61.8 years) were used. We identified 18 differentially expressed proteins in CAG compared with matched normal mucosa. Eight proteins were up-regulated and 10 down-regulated in CAG when compared with the same amounts of proteins in individually matched normal gastric mucosa. Two novel proteins, proteasome activator subunit 1 (PSME1), which was down-regulated in CAG, and ribosomal protein S12 (RPS12), which was up-regulated in CAG, were further investigated. Their expression was validated by Western blot and RT-PCR in 15 CAG samples matched with normal mucosa. The expression level of RPS12 was significantly higher in CAG than in matched normal gastric mucosa (P < 0.05). In contrast, the expression level of PSME1 in CAG was significantly lower than in matched normal gastric mucosa (P < 0.05). This study clearly demonstrated that there are some changes in protein expression between CAG and normal mucosa. In these changes, down-regulation of PSME1 and up-regulation of RPS12 could be involved in the development of CAG. Thus, the differentially expressed proteins might play important roles in CAG as functional molecules.

Proteomic Investigation of Changes in Rat Skeletal Muscle after Exercise-Induced Fatigue

The mechanisms of exercise-induced fatigue have not been investigated using proteomic techniques, an approach that could improve our understanding and generate novel information regarding the effects of exercise. In this study, the proteom alterations of rat skeletal muscle were investigated during exercise-induced fatigue. The proteins were extracted from the skeletal muscle of SD rat thigh, and then analyzed by two-dimensional electrophoresis and PDQuest software. Compared to control samples, 10 significantly altered proteins were found in exercise samples, two of them were upregulated and eight of them were downregulated. These proteins were identified by MALDI TOF-MS. The two upregulated proteins were identified as MLC1 and myosin L2 (DTNB) regulatory light-chain precursors. The eight decreased proteins are Glyceraldehyde-3-phosphate Dehydrogenas (GAPDH); Beta enolase; Creatine kinase M chain (M-CK); ATP-AMP Transphosphorylase (AK1); myosin heavy chain (MHC); actin; Troponin I, fast-skeletal muscle (Troponin I fast-twitch isoform), fsTnI; Troponin T, fast-skeletal muscle isoforms (TnTF). In these proteins, four of the eight decreased proteins are related directly or indirectly to exercise induced fatigue. The other proteins represent diverse sets of proteins including enzymyes related to energy metabolism, skeletal muscle fabric protein and protein with unknown functions. They did not exhibit evident relationship with exercise-induced fatigue. Whereas the two identified increased proteins exhibit evident relationship with fatigue. These findings will help in understanding the mechanisms involved in exercise-induced fatigue.

Treinamento resistido reduz inflamação em músculo esquelético e melhora a sensibilidade à insulina periférica em ratos obesos induzidos por dieta hiperlipídica / Resistive training reduces inflammation in skeletal muscle and improves the peripheral insulin sensitivity in obese rats induced by hyperlipidic diet

OBJETIVO: Investigar em ratos obesos o efeito da prática de exercício resistido sobre a sensibilidade à insulina e sobre a expressão de citocinas pró-inflamatórias e de transportador de glicose em músculo solear. MATERIAIS E MÉTODOS: Ratos Wistar alimentados com dieta hiperlipídica (grupos obesos) foram submetidos ao protocolo de exercício tipo jump squat. A sensibilidade à insulina e a expressão gênica de Tnf-α, SOCS3 e GLUT4 foram comparadas entre os grupos obesos sedentários (OS) e exercitados (OE) e controles sedentários (CS) e exercitados (CE). RESULTADOS: A sensibilidade à insulina estava reduzida no grupo OS e elevada no OE. Os conteúdos de RNAm de Tnf-α e de SOCS3 estavam aumentados no músculo esquelético do grupo OS e reduzidos no OE. O conteúdo proteico e de RNAm de GLUT4 não diferiu entre os grupos. CONCLUSÃO: O exercício resistido reverte o quadro de resistência à insulina periférica e de inflamação no músculo esquelético de obesos induzidos por dieta.

OBJECTIVE: To determine if resistive exercise protocol can modulate Tnf-α, SOCS3 and glucose transporter GLUT4 genes expression in skeletal muscle, and peripheral insulin sensitivity in obese rats induced by hyperlipidic diet. MATERIALS AND METHODS: Wistar obese rats induced by hyperlipidic diet were subjected a resistive exercise protocol as jump squat. Insulin sensitivity and mRNA content of Tnf-α, SOCS3 and GLUT4 were assayed and compared among the groups: obese sedentary (OS) and exercised (OE), control sedentary (CS) and exercised (CE). RESULTS: The mRNA content of Tnf-α and SOCS3 has increased in skeletal muscle from OS and has decreased in OE group. The protein and GLUT4 mRNA contents were correlated but they did not change among the groups. Peripheral insulin sensitivity has increased in the OE compared to OS group. CONCLUSION: The resistive exercise reverses the peripheral insulin resistance and the inflammatory state in skeletal muscle from diet-induced obese rats.

Effects of Nitric Oxide Synthase Inhibitor on Hindlimb Muscles in Rats with Neuropathic Pain Induced by Unilateral Peripheral Nerve Injury

PURPOSE: The purpose of this study was to examine effects of nitric oxide synthase (NOS) inhibitor on muscle weight and myofibrillar protein content of affected and unaffected hindlimb muscles in rats with neuropathic pain induced by unilateral peripheral nerve injury. METHODS: Neuropathic pain was induced by ligation and cutting of the left L5 spinal nerve. Adult male Sprague-Dawley rats were randomly assigned to one of two groups: The NOSI group (n=19) had NOS inhibitor (L-NAME) injections daily for 14 days, and the Vehicle group (n=20) had vehicle injections daily for 14 days. Withdrawal threshold, body weight, food intake and activity were measured every day. At 15 days all rats were anesthetized and soleus, plantaris and gastrocnemius muscles were dissected from hindlimbs. Muscle weight and myofibrillar protein content of the dissected muscles were determined. RESULTS: The NOSI group showed significant increases as compared to the Vehicle group for body weight at 15 days, muscle weight and myofibrillar protein content of the unaffected soleus and gastrocnemius. The NOSI group demonstrated a higher pain threshold than the vehicle group. CONCLUSION: NOSI for 14 days attenuates unaffected soleus and gastrocnemius muscle atrophy in neuropathic pain model.

Spinal Cord Injury Markedly Altered Protein Expression Patterns in the Affected Rat Urinary Bladder during Healing Stages

The influence of spinal cord injury (SCI) on protein expression in the rat urinary bladder was assessed by proteomic analysis at different time intervals post-injury. After contusion SCI between T9 and T10, bladder tissues were processed by 2-DE and MALDI-TOF/MS at 6 hr to 28 days after SCI to identify proteins involved in the healing process of SCI-induced neurogenic bladder. Approximately 1,000 spots from the bladder of SCI and sham groups were visualized and identified. At one day after SCI, the expression levels of three protein were increased, and seven spots were down-regulated, including heat shock protein 27 (Hsp27) and heat shock protein 20 (Hsp20). Fifteen spots such as S100-A11 were differentially expressed seven days post-injury, and seven proteins including transgelin had altered expression patterns 28 days after injury. Of the proteins with altered expression levels, transgelin, S100-A11, Hsp27 and Hsp20 were continuously and variably expressed throughout the entire post-SCI recovery of the bladder. The identified proteins at each time point belong to eight functional categories. The altered expression patterns identified by 2-DE of transgelin and S100-A11 were verified by Western blot. Transgelin and protein S100-A11 may be candidates for protein biomarkers in the bladder healing process after SCI.

Perfil eletrocardiográfico e conteúdo glicogênico muscular de ratos tratados com nandrolona / Electrocardiographic profile and muscle glycogen content of rats treated with nandrolone / Perfil electrocardiográfico y contenido glicogénico muscular de ratones tratados con nandrolona

FUNDAMENTO: Considerou-se o uso indiscriminado de esteroides tanto por atletas de elite quanto por praticantes de atividades físicas. OBJETIVO: Avaliar os efeitos do decanoato de nandrolona sobre o perfil eletrocardiográfico, conteúdo glicogênico e de proteínas totais dos músculos cardíacos e esqueléticos, bem como as concentrações plasmática de albumina. MÉTODOS: Os animais do grupo tratado receberam a droga na concentração 5 mg/kg pela via subcutânea, duas vezes por semana, durante três semanas. Uma vez por semana, os ratos foram anestesiados com Pentobarbital sódico (50 mg/kg, ip) e submetidos à avaliação por meio do eletrocardiograma (ECG). Após o período experimental, amostras dos músculos cardíaco (ventrículo esquerdo - VE), sóleo (S), gastrocnêmio branco (GB), gastrocnêmio vermelho (GV), peitoral (P), intercostal (IC) e diafragma (D) foram prontamente coletadas e analisadas. Os dados (média ± epm) foram avaliados de acordo com ANOVA, segundo teste de Tukey (p>0,05). RESULTADOS: Os ratos do grupo tratado apresentaram alterações nos seguintes parâmetros cardíacos: intervalo QRS, intervalo QTc e frequência cardíaca, caracterizados por um aumento desses, tendo o ápice no intervalo da semana de pré-tratamento para a primeira semana. As reservas de glicogênio no VE apresentaram aumento de 127 por cento. Em relação à quantidade de proteínas totais, a diferença significativa foi constatada no S, GV e D. Quanto ao perfil bioquímico e ao hematócrito, foi observado um aumento na porcentagem de eritrócitos. CONCLUSÃO: O estudo mostra que importantes alterações cardíacas são deflagradas precocemente, sugerindo uma hierarquia na sequência de modificações que comprometem a homeostasia do organismo.

BACKGROUND: We considered both the indiscriminate use of steroids by top athletes and by physically active individuals. OBJECTIVE: To evaluate the effects of nandrolone decanoate on the electrocardiographic profile, glycogen content and total-protein profile of skeletal and cardiac muscles, as well as the plasma albumin concentrations. METHODS: The drug was administered subcutaneously, at a concentration of 5 mg/kg, twice a week for three weeks, to animals in the treated group. Once a week, the rats were anesthetized with sodium pentobarbital (50 mg/kg, ip) and they underwent an electrocardiogram (ECG). After the trial period, samples of the cardiac muscle (left ventricle - LV), soleus muscle (S), white gastrocnemius muscle (WG), red gastrocnemius muscle (RG), pectoral muscle (P), intercostal muscle (IC) and diaphragm muscle (D) were promptly collected and analyzed. An analysis of variance (ANOVA) and then a Tukey test (p>0.05) were carried out to assess the data (mean ± sem). RESULTS: There were changes in the following parameters of rats in the treated group: QRS interval, QTc interval and heart rate, characterized by an increase in these parameters, with the peak being reached in the period between the pre-treatment week and the first week. There was an increase of 127 percent in glycogen reserves in the LV. In relation to the total-protein amount, the significant difference was found in S, RG and D. As for the hematocrit and biochemical profile, it was possible to notice an increase in the percentage of erythrocytes. CONCLUSION: The study shows that major cardiac changes are triggered at an early stage, which indicates a hierarchy in the sequence of changes that compromise the homeostasis of the body.

FUNDAMENTO: Se consideró el uso indiscriminado de esteroides tanto por atletas de elite como por practicantes de actividades físicas. OBJETIVO: Evaluar los efectos del decanoato de nandrolona sobre el perfil electrocardiográfico, contenido glicogénico y de proteínas totales de los músculos cardíacos y esqueléticos, así como las concentraciones plasmática de albúmina. MÉTODOS: Los animales del grupo tratado recibieron la droga en la concentración 5mg/kg por vía subcutánea, dos veces por semana, durante tres semanas. Una vez por semana, los ratones fueron anestesiados con Pentobarbital sódico (50mg/Kg, ip) y sometidos a evaluación por medio de electrocardiograma (ECG). Después del período experimental, muestras de los músculos cardíaco (ventrículo izquierdo - VI), sóleo (S), gastrocnemio blanco (GB), gastrocnemio rojo (GV), pectoral (P), intercostal (IC) y diafragma (D) fueron colectadas y analizadas. Los datos (media±epm) fueron evaluados de acuerdo con ANOVA, segundo test de Tukey (p>0,05). RESULTADOS: Los ratones del grupo tratado presentaron alteraciones en los siguientes parámetros cardíacos: intervalo QRS, intervalo QTc y frecuencia cardíaca, caracterizados por un aumento de estos, teniendo el ápice en el intervalo de la semana de pretratamiento a la primera semana. Las reservas de glicógeno en el VI presentaron aumento de 127 por ciento. En relación a la cantidad de proteínas totales, una diferencia significativa fue constatada en el S, GV y D. En cuanto al perfil bioquímico y al hematocrito, fue observado un aumento en el porcentaje de eritrocitos. CONCLUSIÓN: El estudio muestra que importantes alteraciones cardíacas son provocadas precozmente, sugiriendo una jerarquía en la secuencia de modificaciones que comprometen la homeostasia del organismo.

TRPC3 cation channel plays an important role in proliferation and differentiation of skeletal muscle myoblasts

During membrane depolarization associated with skeletal excitation-contraction (EC) coupling, dihydropyridine receptor [DHPR, a L-type Ca2+ channel in the transverse (t)-tubule membrane] undergoes conformational changes that are transmitted to ryanodine receptor 1 [RyR1, an internal Ca2+-release channel in the sarcoplasmic reticulum (SR) membrane] causing Ca2+ release from the SR. Canonical-type transient receptor potential cation channel 3 (TRPC3), an extracellular Ca2+-entry channel in the t-tubule and plasma membrane, is required for full-gain of skeletal EC coupling. To examine additional role(s) for TRPC3 in skeletal muscle other than mediation of EC coupling, in the present study, we created a stable myoblast line with reduced TRPC3 expression and without alpha1SDHPR (MDG/TRPC3 KD myoblast) by knock-down of TRPC3 in alpha1SDHPR-null muscular dysgenic (MDG) myoblasts using retrovirus-delivered small interference RNAs in order to eliminate any DHPR-associated EC coupling-related events. Unlike wild-type or alpha1SDHPR-null MDG myoblasts, MDG/TRPC3 KD myoblasts exhibited dramatic changes in cellular morphology (e.g., unusual expansion of both cell volume and the plasma membrane, and multi-nuclei) and failed to differentiate into myotubes possibly due to increased Ca2+ content in the SR. These results suggest that TRPC3 plays an important role in the maintenance of skeletal muscle myoblasts and myotubes.

Nemaline myopathy: clinical, histochemical and immunohistochemical features / Miopatia nemalínica: achados clínicos, histoquímicos e imuno-histoquímicos

Nemaline myopathy (NM) is a congenital disease that leads to hypotonia and feeding difficulties in neonates. Some cases have a more benign course, with skeletal abnormalities later in life. We analyzed a series of eight patients with NM obtained from a retrospective analysis of 4300 muscle biopsies. Patients were classified as having the typical form in five cases, intermediate form in two cases and severe form in one case. Histochemical analysis showed mixed rods distribution in all cases and predominance of type I fibers in five cases. Immunohistochemical analysis showed abnormal nebulin expression in all patients (four heterogeneous and four absent), homogeneous desmin expression in four cases, strongly positive in three and absent in one, fast myosin expression in a mosaic pattern in six cases and absent in two cases. There was no specific relation between these protein expression patterns and the clinical forms of NM.

Miopatia nemalínica (NM) é uma doença congênita que leva a hipotonia e dificuldade de sugar em neonatos. Alguns casos possuem uma evolução benigna, com deformidades ósseas tardias. Nós analisamos uma série de oito pacientes com NM obtidos da análise retrospectiva de 4300 biópsias musculares. Os pacientes foram classificados como forma típica em cinco casos, forma intermediária em dois casos e forma severa em um caso. Análise histoquímica mostrou distribuição mista dos rods em todos os casos e predominância de fibras tipo I em cinco casos. Análise imuno-histoquímica mostrou expressão anormal da nebulina em todos os pacientes (quatro heterogênea e quatro ausente), expressão homogenea da desmina em quatro casos, fortemente positiva em tres e ausente em um, expressão da miosina (rápida) com padrão em mosaico em seis casos e ausente em dois casos. Não há relação específica entre a expressão destas proteínas e as formas clínicas da NM.

The inhibitory role of sympathetic nervous system in the Ca2+-dependent proteolysis of skeletal muscle

Mammalian cells contain several proteolytic systems to carry out the degradative processes and complex regulatory mechanisms to prevent excessive protein breakdown. Among these systems, the Ca2+-activated proteolytic system involves the cysteine proteases denoted calpains, and their inhibitor, calpastatin. Despite the rapid progress in molecular research on calpains and calpastatin, the physiological role and regulatory mechanisms of these proteins remain obscure. Interest in the adrenergic effect on Ca2+-dependent proteolysis has been stimulated by the finding that the administration of β2-agonists induces muscle hypertrophy and prevents the loss of muscle mass in a variety of pathologic conditions in which calpains are activated. This review summarizes evidence indicating that the sympathetic nervous system produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed, which indicate that epinephrine secreted by the adrenal medulla and norepinephrine released from adrenergic terminals have inhibitory effects on Ca2+-dependent protein degradation, mainly in oxidative muscles, by increasing calpastatin levels. Evidence is also presented that this antiproteolytic effect, which occurs under both basal conditions and in stress situations, seems to be mediated by β2- and β3-adrenoceptors and cAMP-dependent pathways. The understanding of the precise mechanisms by which catecholamines promote muscle anabolic effects may have therapeutic value for the treatment of muscle-wasting conditions and may enhance muscle growth in farm species for economic and nutritional purposes.