Effect of nutrient restriction on calpain and calpastatin content of skeletal muscle from cows and fetuses

Calpains are crucial for the degradation of myofibrillar proteins in muscle. Calpastatin is a specific inhibitor of calpains. The objective of this study was to elucidate the effect of nutrient restriction on the activity of calpains and calpastatin in the skeletal muscle of both cows and fetuses. Beginning 30 d after conception, 20 cows were fed either a control diet consisting of native grass hay fortified with vitamins and minerals at recommendations for a mature cow to gain 0.72 kg/d or half the vitamins and minerals and millet straw at 68.1% of NEm requirements. Cows were slaughtered on d 125 of gestation, and the LM was sampled at the 12th rib for calpain and calpastatin measurement. When comparing the muscle samples from nutrient-restricted and control cows, no difference in the activity of calpain I and II was observed; however, there was a significant difference (P < 0.05) in calpastatin activity. Muscle samples from control cows had greater calpastatin content than those of nutrient-restricted cows (P < 0.05); in contrast, the calpastatin content of fetal muscle was greater in fetuses gestated by nutrient-restricted cows than those of control cows (P < 0.05). Further, there were three calpastatin isoforms of 125, 110, and 70 kD detected in fetal muscle, whereas only the110-kD isoform was detected for cow muscle. These results indicate that the activity of the calpain system in skeletal muscle is mainly controlled through the expression of calpastatin. Alternating the calpastatin content in muscle and thereby modulating calpain activity may provide a mechanism for the maintenance of fetal muscle growth during nutrient restriction, whereas skeletal muscle loss in cows is upregulated.     

Effects of cimaterol on rabbit growth and myofibrillar protein degradation and on calcium-dependent proteinase and calpastatin activities in skeletal muscle

The objectives of this study were to examine effects of a beta-adrenergic agonist (cimaterol) on growth and muscle development in rabbits and to examine cimaterol's effects on myofibrillar protein degradation (MPD) and on activities of several proteolytic enzymes including the calcium-dependent proteinases (CDP). Twelve New Zealand White rabbits were assigned to either control diets or to diets containing cimaterol for 35 d, after which they were killed and effects on performance and tissue weight gains were determined. Urine was collected from d 21 through 28 from each rabbit for assessment of N tau-methylhistidine (NMH) excretion. Cimaterol increased rates of gain, efficiency of gain and skeletal muscle weights. Enhancement in muscle weight was associated with an increase in total DNA and with a reduction in NMH. Cimaterol did not affect activities of cathepsin B, cathepsin D or neutral serine proteinase, but it reduced activities of the millimolar and micromolar forms of the CDP by 58 and 57%, respectively, and it reduced activity of the inhibitor of the CDP (calpastatin) by 52%. Cimaterol-dependent myofibrillar protein accretion was likely mediated, at least in part, by a reduction in MPD. The change in MPD was associated with a reduction in muscle CDP activities. Cimaterol-dependent muscle hypertrophy therefore may involve changes in calcium-dependent proteolysis of myofibrillar proteins. The significance of the effects of cimaterol on calpastatin activity is not known.     

Effects of age and castration on activities of calpains and calpastatin in sheep skeletal muscle.

The objectives of this experiment were to assess effects of animal age and castration on activities of calpain I, calpain II, and calpastatin in sheep skeletal muscle. Ten newborn male lambs (2.9 kg), six weaned wethers (23.2 kg), six weaned rams (22.2 kg), six market wethers (55.4 kg), and six market rams (60.2 kg) were slaughtered and samples of biceps femoris were taken for assay of calpain I (micromolar calcium-dependent proteinase), calpain II (millimolar calcium-dependent proteinase), and calpastatin. Preweaning weight gain was similar for rams and wethers; however, postweaning ram growth exceeded (P less than .05) that of wethers. Ram biceps femoris weights at market were greater than those of wethers (P less than .05). Irrespective of age or gender, activity of calpain II was two- to threefold greater than that of calpain I. Muscle calpastatin activity was severa fold higher than calpain I and II activities. Activities of calpains and calpastatin declined (P less than .05) between birth and weaning. A portion of these losses were due to a dilution effect caused by accumulation of muscle proteins. Neonatal attenuation of calpain activities may underlie age-related attenuation of fractional rates of muscle protein degradation. Although ram muscle growth exceeded that of wethers, no differences (P greater than .05) in activities of muscle calpains or calpastatin were detected between these two groups at weaning or at market weight. Hence, castration did not influence lamb muscle growth by altering muscle calpain or calpastatin activities.     

Comparison of calpain and calpastatin activities in skeletal muscle of broiler and layer chickens

1. The objective of this study was to estimate the difference between broiler and layer chicks in the activities of calpain and calpastatin (inhibitor of calpain) in breast muscle. Differences between broilers and layers in body weight, daily gain at 3 weeks of age and fractional growth rate (FGR) during 2 and 3 weeks of age were statistically significant (P < 0.01).

2. Calpain and calpastatin activities were measured at three weeks of age with alkali‐denatured casein as a substrate. The m‐calpain (calpain activated by millimolar calcium concentration) activities in units/g muscle and units/ mg extractable muscle protein were 0.779 and 0.353 for broilers, and 1.042 and 0.440 for layers, respectively. The calpastatin activities in units/g muscle and units/mg extractable muscle protein were 0.332 and 0.153 for broilers, and 0.262 and 0.112 for layers, respectively.

3. Broilers with high FGR showed low m‐calpain and high calpastatin activities. In contrast, layers with low FGR showed high m‐calpain and low calpastatin activities.

4. These results suggest that m‐calpain and calpastatin activities in skeletal muscle vary between breeds which have different rates of muscle production.     

Effects of dietary corticosterone and trilostane on growth and skeletal muscle protein turnover in broiler cockerels

1. The effects of dietary corticosterone and trilostane, an inhibitor of glucocorticoid synthesis, on: growth, rates of synthesis and breakdown of skeletal muscle protein, and content of abdominal fat were studied in broiler chickens.

2. Dietary corticosterone (5, 10 or 20 mg/kg) depressed body weight gain and increased abdominal fat content in a dose‐dependent manner while dietary trilostane (1.4 or 7.0 mg/kg) had no effect.

3. The rate of protein breakdown in skeletal muscle estimated from N‐methylhistidine excretion was increased in a dose‐dependent manner by dietary corticosterone but it was decreased by trilostane.

4. The rate of skeletal muscle protein synthesis was not affected by corticosterone although it was decreased by trilostane.

5. Plasma corticosterone concentration was increased in a dose‐dependent manner by dietary corticosterone and decreased by treatment with 7 mg trilostane/kg diet.

6. The results indicate that higher concentrations of plasma corticosterone increase protein breakdown in skeletal muscle but do not affect muscle protein synthesis while both the rates of synthesis and breakdown are decreased when plasma corticosterone concentration is reduced.     

不同蛋白水平饲粮对肉兔骨骼肌发育及相关因子表达的影响

体况相近的断奶伊拉肉兔30只,随机分为3组:高蛋白饲粮组(HPD)、中蛋白饲粮组(MPD)和低蛋白饲粮组(LPD),每组10个重复,每个重复1只兔。结果显示:随着饲粮中蛋白含量的增加,家兔的体质量、胴体质量、前腿质量、后腿质量、前腿质量/体质量和后腿质量/体质量显著增加(P0.05);与MPD比,HPD显著增加血浆中总蛋白、尿素和胰岛素的含量(P0.05);与MPD和LPD相比,HPD组家兔显著增加了骨骼肌中胰岛素样生长因子1(IGF1)、IGF1R、肌原性决定因子(MYOD)、肌原因子5(MYF5)、肌生成抑制蛋白(MSTN)、MYF5和包WW包含体E3泛素蛋白酶体连接酶1(WWP)的mRNA水平(P0.05)。从整体上看,饲粮中蛋白水平能显著影响家兔骨骼肌发育。高蛋白饲粮促进骨骼肌发育可能与IGF1、MYOD和MYF5基因有关;低蛋白饲粮抑制骨骼肌发育可能与IGF1的低表达有关。     

Technique for physiological examination of canine skeletal muscle in vivo

A technique is described for studying the physiological function of canine skeletal muscle in vivo. The contractile properties of the tarsal flexor muscles were examined in three beagle dogs under general anaesthesia. The force responses to electrical stimulation of the common peroneal nerve were measured at various frequencies to determine the frequency:force relationship for this muscle group. Fatigue characteristics were also examined during intermittent stimulated activity delivered in a set pattern of frequencies. The results provide quantitative characterisation of muscle function which is repeatable. The technique described could be applied to other animals and is a potentially powerful tool for evaluating the effects of drugs on muscle performance.     

Histochemical differentiation of fiber types in neonatal canine skeletal muscle

Differentiation of fiber types in developing canine skeletal muscle was studied, using morphologic, morphometric, and histochemical techniques. Sample collections were made from 6 muscles from the pectoral and pelvic limbs of 16 healthy pups between 1 day and 12 weeks of age. In newborn pups, 90% to 95% of the fibers in the 6 muscles were classified as undifferentiated or type IIC; the remaining fibers were classified either normal or large-size type I. Large-size type I fibers usually accounted for 2% to 4% of the total population and were considered analogous with the B fiber of Wohlfart. These fibers were larger than all other fiber types and disappeared after pups reached 4 to 5 weeks of age. After 2 to 4 weeks, the number of undifferentiated fibers decreased with the appearance of, and the concomitant numerical increases of, normal size type I and type IIA fibers. The percentages of type I and IIA fibers approached proportions of the adult dog by 12 weeks, at which time a type IIA fiber predominance was present in biceps femoris, lateral head of the gastrocnemius, cranial tibial, and long head of the triceps. Type I fibers predominated in medial head of the triceps and superficial digital flexor after 4 to 5 weeks. The mean fiber diameters of type I and IIA fibers were similar to any given muscle throughout the postnatal development. All fiber types stained uniformly with the oxidative stain nicotinamide adeninedinucleotide-tetrazolium reductase during the first 12 weeks of life, whereas a distinction between type I and II fibers was evident after 3 to 4 weeks with the periodic acid-Schiff stain reaction.     

Histochemical identification of fiber types in canine skeletal muscle.

Proximal and distal skeletal muscles from pectoral and pelvic limbs were histochemically examined in 18 neuromuscular disease-free dogs. On the basis of the human system of classification and nomenclature and results of standard adenosine triphosphatase (ATPase) and glycine-formaldehyde preincubation procedures, the fiber types identified in immature and mature canine skeletal muscles were I, IIA, and IIC.     

Biochemical evaluation of mitochondrial respiratory chain enzymes in canine skeletal muscle

OBJECTIVE: To perform respiratory chain enzymatic activity assays on canine skeletal muscle biopsy specimens and establish reference range values of skeletal muscle enzyme activities for dogs. SAMPLE POPULATION: Biopsy specimens from the vastus lateralis muscle were obtained from 24 dogs (8 sexually intact males and 14 sexually intact females) ranging from 15 months to 6 years of age. PROCEDURE: Mean values of citrate synthase, cytochrome-c oxidase, succinate dehydrogenase, succinate dehydrogenase-cytochrome-c reductase, nicotinamide adenine dinucleotide (NADH) dehydrogenase, and NADH dehydrogenase-cytochrome-c reductase activities were established by use of 6 standard spectrophotometric assays for respiratory chain enzyme analysis. RESULTS: Compared with published data for skeletal muscle enzyme activities in humans, skeletal muscle enzyme activities in dogs were 2- to 4-fold higher. Additionally, citrate synthase activity, a marker for mitochondrial volume, was positively correlated with age in dogs, suggesting that mitochondrial volume increases with age, although no apparent change in respiratory chain enzymatic activity with an increase in age was found. CONCLUSIONS AND CLINICAL RELEVANCE: Reference range values for skeletal muscle enzyme activities of dogs are needed to accurately interpret results of respiratory chain enzymatic activity assays. During investigation of metabolic myopathies, if skeletal muscle biopsy specimens are evaluated for respiratory chain enzyme kinetics, they should be performed and evaluated in concert with skeletal muscle biopsy specimens from clinically normal animals of the same species.     

The effect of severe dietary protein restriction on skeletal muscle fiber number, area and composition in weanling rats

The purpose of the present investigation was to determine the effects of severe dietary protein restriction on soleus (S) and tibialis anterior (TA) fiber number and S muscle fiber area, composition and length. The S and TA muscles were removed from one leg of 12 male Sprague-Dawley rats at 21 d of age. Six of these animals were placed on a 1% protein diet until 42 d of age, while six served as age-matched controls. Muscle fiber number was determined by the nitric acid digestion method for S muscles and the mean fiber dry-weight estimation method for the TA muscles. Mean fiber numbers for the S muscles were 2,655 +/- 42 and 2,669 +/- 71 for the treatment group at 21 and 42 d of age, respectively, and 15,989 +/- 899 and 16,067 +/- 695 at 21 and 42 d of age, respectively, for the TA muscle. For the age-matched control group, fiber numbers for the S muscle were 2,928 +/- 78 and 2,949 +/- 76 at 21 and 42 d, respectively, and 17,964 +/- 281 and 18,445 +/- 296 at 21 and 42 d, respectively, for the TA muscle. The S muscle fiber area, composition and length were studied using 12 male Sprague-Dawley rats. Six animals were placed on the 1% protein diet from 21 to 42 d of age, while six animals served as age-matched controls. The S muscle fiber area was 33.1 and 51.5% smaller for type I and type II fibers, respectively, for animals fed the 1% protein diet. The S fiber length was 27.9% less in animals fed the 1% protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of protein turnover in skeletal muscle strips

Isolated porcine and bovine muscle strips were incubated in Krebs Ringer bicarbonate buffer to determine in vitro protein synthesis (PS) and protein degradation (PD) rates to validate the in vitro system for use with livestock species. The addition of 5X plasma concentrations of amino acids to the medium stimulated PS 30%. Addition of 3.5 mM leucine to a leucine-deficient buffer supplemented with amino acids decreased PD 37% and stimulated PS 24%. The addition of .1 U/ml insulin reduced PD 28% and increased PS 30%. Protein degradation was elevated in longitudinally split rat soleus and extensor digitorum longus muscles compared to their contralateral intact muscles. Muscle strips must be removed within 15 min of exsanguination because PD rates become greatly elevated thereafter. ATP concentrations declined during incubation, but the addition of ATP or creatine had no effect on either PD or PS. Neither PD nor PS was affected by the addition of transferrin, fetuin, ascorbate, dexamethasone or indomethacin to the incubation medium. However, muscle strips were sensitive to the addition of triiodothyronine (T3), PD was increased up to 75% as T3 concentration was increased, and PS rates doubled compared to controls. Serum from mature barrows or gilts had no effect on protein turnover, but the addition of 10% and 15% serum from boars increased both PD and PS. With fasted pigs a continual decline in PS occurred over 5 d, whereas PD was elevated at 3 d and then declined to rates comparable to the fed state after 5 d. These data suggest that the in vitro system has application for assessing relative changes that occur in vivo following nutritional, physiological and endocrinological manipulation.     

A comparative histochemical and morphometric study of canine skeletal muscle.

The purpose of this study was to determine whether there were differences in skeletal muscle properties in the hindlimb muscles of different types of dogs. Muscle samples were obtained from the gracilis, sartorius cranial head, sartorius caudal head and tibialis anterior muscles of mixed-breed and hound-type dogs and Beagles. Fiber type, fiber size and capillary morphometry determinations of each muscle from each dog were made from sections stained for myofibrillar ATPase activity. Individual animals were bilaterally symmetric for all measured variables. Fiber type, fiber size and capillary geometry varied between dogs of a given type and muscles within a given dog. There were no differences between dog types for fiber type or fiber size; significant variation in log(muscle)/log(body) mass ratios between dog types was observed for all muscles. The results indicate that for a given muscle, significant variation can occur in skeletal muscle characteristics between different types of dogs and that these differences can be independent of differences in exercise history.     

Reduced dietary lysine enhances proportion of oxidative fibers in porcine skeletal muscle

The present study was conducted to elucidate the effects of dietary lysine levels on the proportion of oxidative muscle fibers in porcine muscle. Two 6-week-old barrows from each of five litters were used. Each littermate was assigned to one of two diets, control (lysine content: 1.16%) or low lysine (LL) diet (lysine content: 0.73%). The diets were iso-energetic and iso-protein, and contained all essential amino acids (apart from lysine) in the recommended amounts. The pigs were fed these diets for 3 weeks. Citrate synthase (EC 4.1.3.4) activity in longissimus dorsi and rhomboideus muscles was higher in the LL group ( P  < 0.05). In both muscles, pigs fed the LL diet had a higher proportion of muscle fiber with activities of reduced nicotinic amide adenine dinucleotide dehydrogenase (EC 1.6.5.3, P  < 0.01). The abundance of mRNA of peroxisome proliferator-activated receptor γ coactivator 1α in rhomboideus muscle was higher in the LL group ( P  < 0.05), and those of peroxisome proliferator-activated receptor γ2 were higher in both longissimus dorsi and rhomboideus muscles in the LL group ( P  < 0.05). We conclude that reduced intake of dietary lysine enhances proportion of oxidative muscle fiber, and hence oxidative capacity of porcine muscle.     

Masticatory and skeletal muscle myositis in canine leishmaniasis (Leishmania infantum)

Twenty-four dogs with a parasitologically and serologically established diagnosis of leishmaniasis were studied to investigate the atrophy of the masticatory muscles which commonly occurs in this disease, and to compare the lesions in the masticatory muscles with those in the cranial tibial muscles. The 24 animals were divided into three groups of eight, group A dogs with no muscular atrophy, group B dogs with different degrees of atrophy in the masticatory and skeletal muscles, and group C dogs with similar degrees of atrophy in the masticatory and skeletal muscles. Increased activities of creatine phosphokinase and lactate dehydrogenase were recorded in only some of the dogs in groups B and C, but there were no significant differences between the mean activities in the three groups. Electromyographic changes indicating myopathy and involving both the temporalis and cranial tibial muscles, were observed in two of the dogs in group A, seven of those in group B, and in all the dogs in group C. Muscle histopathology revealed a variable degree of muscle fibre necrosis and atrophy, mononuclear infiltrates and neutrophilic vasculitis in all the dogs except two in group A. Leishmanial amastigotes were found within macrophages and myofibres in 16 of the dogs, some in each group. IgG immune complexes were detected in muscle samples, and circulating antibodies against myofibres were detected in serum samples from all the 24 dogs.     

Effect of skeletal muscle decorin on collagen fibrillogenesis in vitro

The effect of skeletal muscle decorin on collagen fibrillogenesis was investigated, in order to provide background for understanding the functions of decorin in skeletal muscle. The self‐assembly of type I and III collagen with the addition of decorin or the core protein of decorin from bovine neonatal skeletal muscle was monitored using a spectrophotmeter. Time course changes in the absorbance of collagen solutions showed typical sigmoidal curves composed of three phases. The time of the initial phase was not different between the collagen solution with decorin and that without decorin. The increase rate of the absorbance in the second phase decreased with concentration of decorin added in collagen solutions. Similar effects on fibrillogenesis of type I and III collagens were observed when the core protein of decorin was added in collagen solutions. These results suggest that regulation of collagen fibrillogenesis by decorin depends on its core protein. The networks of reconstructed collagen fibrils with decorin were looser than those without decorin. Bovine skeletal muscle decorin could participate in the regulation of collagen fibrillogenesis and in the arrangement of collagen fibrils in the intramuscular connective tissue.     

Effects of dietary crude protein concentration during periods of feed restriction on performance, carcass characteristics, and skeletal muscle protein turnover in feedlot steers.

In Exp. 1, 36 individually penned steers (initial BW = 294 +/- 3.8 kg) were used to determine effects of dietary CP percentage and programming gain on performance and carcass characteristics. Steers were fed to achieve a predicted gain of 1.13 kg/d for the first 84 kg of gain and 1.36 kg/d for the next 124 kg of gain and were offered feed for ad libitum consumption for the final 58 kg of gain before slaughter. In these three phases of growth, steers were fed diets, sequentially, with the following CP percentages: HHH (16, 13.5, and 12.5%), LHH (9, 13.5, and 13%), or LLL (9, 9, and 9%). When predicted gain was 1.13 kg/d, ADG was greater (P < 0.01) for steers in the HHH (1.09 kg/d) vs LHH and LLL (0.83 kg/d) systems. When predicted gain was 1.36 kg/d, ADG and gain efficiency were greatest (P < 0.01) for steers in the LHH system. Overall ADG and gain efficiency were greater (P < 0.01) for steers in the HHH (1.46 kg/d, 0.194) and LHH systems (1.38 kg/d, 0.190), compared with steers in the LLL (1.21 kg/d and 0.166) system. Carcass fat thickness was lower for steers in the LHH (0.74 cm) system than for steers in the LLL system (1.09 cm). In Exp. 2, 18 individually penned steers (initial BW = 225 +/- 5.8 kg) were either offered a 13% CP diet for ad libitum intake (AL) throughout the 134-d experiment or fed a high- (16% CP; PI-HH) or low- (10% CP; PI-LH) CP diet and fed to achieve a predicted gain of 1.13 kg/d for the first 85 d of the experiment. Steers in the PI-HH and PI-LH feeding regimens were then offered a 13% CP diet for ad libitum consumption from d 86 to 134. Fractional protein accretion rate was greater (P < 0.01) for steers in the PI-HH and PI-LH feeding regimens than for steers in the AL regimen at d 92, 106, and 120. Fractional breakdown and synthesis rates were not affected (P = 0.63) by feeding regimen. Increased ADG and gain efficiency of steers during compensatory growth periods may in part be due to greater fractional accretion rates of skeletal muscle protein.