Immunoelectron microscopic localization of carbonic anhydrase III in rat skeletal muscle

Summary The subcellular distribution of carbonic anhydrase III in rat soleus and vastus lateralis muscles was studied using an immunogold technique. The enzyme protein was found to be distributed diffusely in the cytoplasm of skeletal muscle cells. Red skeletal muscle (mainly type I fibers) revealed very strong immunogold staining whereas in white muscle (mainly type II fibers) gold particles were almost completely absent. No immunoreaction was observed in mitochondria or in other intracellular organelles.     

Lipid accumulation and mitochondrial abnormalities are associated with fiber atrophy in the skeletal muscle of rats with collagen-induced arthritis

Rheumatoid arthritis (RA) has a negative impact on muscle mass, and reduces patient's mobility and autonomy. Furthermore, RA is associated with metabolic comorbidities, notably in lipid homeostasis by unknown mechanisms. To understand the links between the loss in muscle mass and the metabolic abnormalities, arthritis was induced in male Sprague Dawley rats (n = 11) using the collagen-induced arthritis model. Rats immunized with bovine type II collagen were compared to a control group of animals (n = 11) injected with acetic acid and complete Freund's adjuvant. The clinical severity of the ensuing arthritis was evaluated weekly by a semi-quantitative score. Skeletal muscles from the hind limb were used for the histological analysis and exploration of mitochondrial activity, lipid accumulation, metabolism and regenerative capacities. A significant atrophy in tibialis anterior muscle fibers was observed in the arthritic rats despite a non-significant decrease in the weight of the muscles. Despite moderate inflammation, accumulation of triglycerides (P < 0.05), reduced mitochondrial DNA copy number (P < 0.05) and non-significant dysfunction in mitochondrial cytochrome c oxidase activity were found in the gastrocnemius muscle. Concomitantly, our results suggested an activation of the muscle specific E3 ubiquitin ligases MuRF-1 and MAFbx. Finally, the adipose tissue from the arthritic rats exhibited decreased PPARγ mRNA suggesting reduced adipogenic capacities. In conclusion, the reduced adipose tissue adipogenic capacity and skeletal muscle mitochondrial capacity are probably involved in the activation of protein catabolism, inhibition of myogenesis, accumulation of lipids and fiber atrophy in the skeletal muscle during RA.     

Role of glutathione peroxidase in rheumatoid arthritis: analysis of enzyme activity and DNA polymorphism.

Aberrant expression of the antioxidant enzyme glutathione peroxidase (GPx) could contribute to the etiology of rheumatoid arthritis (RA). However, previous enzyme activity studies examining this relationship were inconclusive. Indirect evidence for this relationship derives from the known efficacy of gold therapy in RA, since gold compounds specifically inhibit GPx. The hypothesis that variants of GPx are associated with RA was examined by two approaches: enzyme activity analysis and restriction fragment length polymorphism (RFLP) association analysis. No significant difference was found in whole blood GPx activity between 28 RA patients and 36 controls. GPx activity appeared to be independent of sex, race, or type of drug treatment. However, a statistically significant difference was found with respect to treatment responsiveness. RA patients classified as good responders to gold therapy, but who were no longer taking gold, had a significantly higher GPx activity compared to both the controls and good responders currently on gold therapy. Aberrantly high GPx activity could contribute to RA by generating excess oxidized glutathione, a potent collagenase activator. Gold therapy would reduce GPx activity to normal levels. The restriction enzyme Pvu II in conjunction with a GPx gene probe identified a useful RFLP (Al, 22 kbp; A2, 15 kbp) with allelic frequencies of A1 and A2 equal to 0.11 and 0.89, respectively, in the control population. No statistically significant association, however, could be demonstrated between this allelic variant of the GPx gene and RA.     

Changes in expiratory muscle function following spinal cord section.

Following spinal cord injury, muscles below the level of injury develop variable degrees of disuse atrophy. The present study assessed the physiological changes of the expiratory muscles in a cat model of spinal cord injury. Muscle fiber typing, cross-sectional area, muscle weight, and changes in pressure-generating capacity were assessed in five cats spinalized at the T(6) level. Airway pressure (P)-generating capacity was monitored during lower thoracic spinal cord stimulation before and 6 mo after spinalization. These parameters were also assessed in five acute animals, which served as controls. In spinalized animals, P fell from 41 +/- l to 28 +/- 3 cm H2O (means +/- SE; P < 0.001). Muscle weight of the external oblique, internal oblique, transversus abdominis, and internal intercostal muscles decreased significantly (P < 0.05 for each). Muscle weight of the external oblique, internal oblique, transversus abdominis, and internal intercostal, but not rectus abdominis (RA), correlated linearly with P (r > 0.7 for each; P < 0.05 for each). Mean muscle fiber cross-sectional area of these muscles was significantly smaller (P < 0.05 for each; except RA) and also correlated linearly with P (r > 0.55 for each; P < 0.05 for each, except RA). In spinalized animals, the expiratory muscles demonstrated a significant increase in the population of fast muscle fibers. These results indicate that, following spinalization, 1) the expiratory muscles undergo significant atrophy and fiber-type transformation and 2) the P-generating capacity of the expiratory muscles falls significantly secondary to reductions in muscle mass.     

The effect of glucocorticoids on the myosin heavy chain isoforms' turnover in skeletal muscle

The purpose of this study was to find the effect of dexamethasone on the myosin heavy chain (MyHC) isoforms' composition in different skeletal muscles and glycolytic (G) fibres in relation with their synthesis rate and degradation of MyHC isoforms by alkaline proteinases. Eighteen-week-old male rats of the Wistar strain were treated with dexamethasone (100 microg/100 g bwt) during 10 days. The forelimb strength decreased from 9.52 to 6.19 N (P<0.001) and hindlimb strength from 15.54 to 8.55 N (P<0.001). Daily motor activity decreased (total activity from 933 to 559 and ambulatory activity from 482 to 226 movements/h, P<0.001). The degradation rate of muscle contractile proteins increased from 2.0 to 5.9% per day (P<0.001), as well as the myosin heavy chain IIB isoform degradation with alkaline proteinase in fast-twitch (F-T) muscles (12 +/- 0.9%; P<0.05) and glycolytic muscle fibres (15 +/- 1.1%; P<0.001). The synthesis rate of MyHC type II isoforms decreased in Pla muscles (P<0.05) and MyHC IIA (P<0.05) and IIB in EDL muscle and G fibres (P<0.001). The relative content of MyHC IIB isoform decreased in F-T muscles (P<0.001) and in G fibres (P<0.01), and the relative content of IIA and IID isoforms increased simultaneously. Dexamethasone decreased the MyHC IIB isoform synthesis rate and increased the sensibility of MyHC IIB isoform to alkaline proteinase, which in its turn led to the decrease of MyHC IIB isoform relative content in F-T muscles with low oxidative potential and G muscle fibres.     

Fiber-type-specific alphaB-crystallin distribution and its shifts with T(3) and PTU treatments in rat hindlimb muscles.

Changes in alphaB-crystallin content in adult rat soleus and extensor digitorum longus (EDL) were examined after 8 wk of 3,5, 3'-triiodothyronine (T(3)) and propylthiouracil (PTU) treatments. Cellular distributions of alphaB-crystallin expression related to fiber type, and distribution shifts with these treatments were also examined in detail from the gray level of reactivity to specific anti-alphaB-crystallin antibody. alphaB-crystallin content in both soleus and EDL muscles was significantly decreased after T(3), and that in EDL was significantly increased over twofold after PTU treatment. In both control soleus and EDL muscles, the gray level of type I fibers was higher than that of type II fibers. alphaB-crystallin expression among type II subtypes was muscle specific; the order was type I > IIa > IIx > IIb in control EDL muscle and type IIx > or = IIa in soleus muscle. The relation was basically unchanged in both muscles after T(3) treatment and was, in particular, well maintained in EDL muscle. Under hypothyroidism conditions with PTU, the mean alphaB-crystallin levels of type IIa and IIx fibers were significantly lower than levels under control conditions. Thus the relation between fiber type and the expression manner of stress protein alphaB-crystallin is muscle specific and also is well regulated under thyroid hormone, especially in fast EDL muscle.     

Adaptations of diaphragm and medial gastrocnemius muscles to inactivity.

The effects of 2 wk of inactivity on in vitro contractile properties of diaphragm and medial gastrocnemius (MG) muscles were examined in adult hamsters. In addition, inactivity effects on fiber-type proportions and cross-sectional areas were studied. Inactivity of the right hemidiaphragm or MG muscle was induced by either tetrodotoxin (TTX) blockade of nerve impulses or denervation (DNV). Inactivity effects on diaphragm or MG were compared with corresponding sham (saline-treated or untreated control) muscles. After both TTX- and DNV-induced inactivity, isometric twitch contraction and half-relaxation times were prolonged, maximum tetanic force decreased, and fatigue resistance improved. Proportions of type I and II fibers in both diaphragm and MG were unaffected by TTX- and DNV-induced inactivity. However, in both muscles, type I fibers hypertrophied, whereas type II fibers atrophied. In diaphragm, contractile and morphometric adaptations after DNV were generally more pronounced than those induced by TTX. In addition, compared with corresponding untreated or saline-treated control groups, inactivity effects (both TTX and DNV) on MG were generally greater than those induced in diaphragm, with the exception of hypertrophy of type I fibers. We conclude that inactivity exerts differential effects on type I and II fibers in both diaphragm and MG. Yet, these morphometric adaptations cannot completely account for the adaptations in muscle contractile and fatigue properties after inactivity.     

Cytochrome c mRNA and alpha-actin mRNA in muscles of rats fed beta-GPA

A diet of 1% beta-guanidinopropionic acid (beta-GPA) fed to rats for weeks results in decreased muscle adenosine triphosphate and creatine phosphate concentrations (J. Biol. Chem. 249: 1060-1063, 1974), increased activities of selected mitochondrial enzymes (Biochem. J. 232: 125-131, 1985), and atrophied type IIb fibers (Lab. Invest. 33: 151-158, 1975). The hypothesis of the present study was that chronic beta-GPA feeding would increase cytochrome c mRNA in muscle and would decrease alpha-skeletal actin mRNA in type IIb muscle. Data collected supported, in part, the hypothesis. After 22 days of a 1% beta-GPA diet, cytochrome c mRNA was increased 60-67% in muscles with inherently low cytochrome c mRNA but was not altered in muscles with higher cytochrome c mRNA levels. alpha-Skeletal actin mRNA was unchanged in muscles with low and high cytochrome c mRNA after 22 days of 1% beta-GPA. After 66 days of beta-GPA feeding, both cytochrome c mRNA and alpha-skeletal actin mRNA were decreased 18 and 26%, respectively, per unit of total RNA, in white quadriceps muscle. At the same time muscles composed of predominantly type II fibers atrophied 22%, whereas type I muscle size was unaltered. These data suggest that high-energy phosphate levels could play some role in adaptive changes in muscle composition.     

The rib cage in normal and emphysematous subjects: a roentgenographic approach

The configuration and motion of the bony rib cage were studied from lateral chest roentgenograms in 10 young normal subjects (YN), 12 elderly normal subjects, and 12 hyperinflated emphysematous patients [chronic obstructive pulmonary disease subjects (COPD), mean total lung capacity (TLC) 133% of predicted]. The acute angles formed by the fourth through seventh ribs with an axial reference plane were measured at residual volume, functional residual capacity, and TLC in both supine and standing positions and correlated with corresponding lung volumes. both rib angles (RA) and changes in RA with lung volume were greatest with the fourth rib and decreased progressively going down (caudad) the chest. At TLC the RA of upper ribs was significantly less in EN and significantly greater in COPD than in YN. RA's were greater supine than standing. When RA information was used together with autopsy data on the angles formed by intercostal muscles with adjacent ribs, intercostal muscle lengths in hyperinflation could be calculated. Computed intercostal muscle length data suggested that hyperinflation should not be associated with degrees of intercostal muscle shortening or overstretching, that would interfere seriously with tension generation.     

Low glucose-1, 6-bisphosphate and high fructose-2, 6-bisphosphate concentrations in muscles of patients with glycogenosis types VII and V

The level of glucose-1, 6-bisphosphate, a potent allosteric activator of phosphofructokinase, was markedly decreased in muscles of patients with glycogenosis type VII (muscle phosphofructokinase deficiency) and type V (muscle phosphorylase deficiency). Glucose-1-phosphate kinase activity in muscle was virtually absent in a patient with glycogenosis type VII, whereas it was normal in a patient with type V glycogenosis. Glucose-1-phosphate level was increased in type VII glycogenosis, whereas it was decreased in type V glycogenosis. Another activator of phosphofructokinase, fructose-2, 6-bisphosphate was increased in muscles of patients with both types of glycogenosis although it was much higher in type VII than in type V. This finding may be partly related to the difference of fructose-6-phosphate concentrations. The results suggest that phosphofructokinase would contribute to the major glucose-1-phosphate kinase activity in normal human muscle and would also form a kind of self-activating system.     

Trypanosoma cruzi: histochemical characterization of parasitized skeletal muscle fibers

C57B1/6 mice were infected with Brasil strain Trypanosoma cruzi trypomastigotes. The leg muscles of the mice were serial-sectioned with a cryostat, and individual fibers were classified histochemically as type I or type II on the basis of succinic dehydrogenase or adenosine triphosphatase activity. Although markedly more type II fibers were present in the leg muscles, the percentage of infected type I fibers was nearly five-fold higher than type II. This is the first demonstration of a preferential in vivo distribution of T. cruzi in muscle fibers based upon muscle type.     

Analysis of the composition of skeletal muscle fibers in skaters' muscles

Muscle fibers composition was investigated in vastus lateralis muscles of 103 skaters. In the total population of skaters muscle fibers composition was as follows: muscle fibers, type I--in 54 +/- 2%, muscle fibers, type II A--in 35 +/- 1% and muscle fibers, type II B--in 11 +/- 1%. 13 +/- 8% muscle fibers, type I; 1.56 +/- 6% muscle fibers, type II A and 31 +/- 7% muscle fibers, type II B were observed in the muscles of sprinters, while the muscles of long-distance skaters contained 60 +/- 4%, 33 +/- 4% and 7 +/- 2% muscle fibers, respectively.     

Effect of extracellular osmolality on cell volume and resting metabolism in mammalian skeletal muscle

The purpose of the present investigation was to establish an in vitro mammalian skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated, whole muscles [soleus and extensor digitorum longus (EDL)] were dissected from Long-Evans rats and incubated for 60 min in Sigma medium 199 (1 g of resting tension, bubbled with 95% O(2)-5% O(2), 30 +/- 2 degrees C, and pH 7.4). Medium osmolality was altered to simulate hyposmotic (190 +/- 10 mmol/kg) or hyperosmotic conditions (400 +/- 10 mmol/kg), whereas an isosmotic condition (290 +/- 10 mmol/kg) served as a control. After incubation, relative water content of the muscle decreased with hyperosmotic and increased with hyposmotic condition in both muscle types (P < 0.05). The cross-sectional area of soleus type I and type II fibers increased (P < 0.05) in hyposmotic, whereas hyperosmotic exposure led to no detectable changes. The EDL type II fiber area decreased in the hyperosmotic condition and increased after hyposmotic exposure, whereas no change was observed in EDL type I fibers. Furthermore, exposure to the hyperosmotic condition in both muscle types resulted in decreased muscle ATP and phosphocreatine (P < 0.05) contents and increased creatine and lactate contents (P < 0.05) compared with control and hyposmotic conditions. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acute alterations in muscle water content and resting muscle metabolism.     

Altered expression of skeletal muscle myosin isoforms in cancer cachexia

Cachexia is commonly seen in cancer and ischaracterized by severe muscle wasting, but little is known about theeffect of cancer cachexia on expression of contractile protein isoforms such as myosin. Other causes of muscle atrophy shift expression ofmyosin isoforms toward increased fast (type II) isoform expression. Weinjected mice with murine C-26 adenocarcinoma cells, a tumor cell linethat has been shown to cause muscle wasting. Mice were killed 21 daysafter tumor injection, and hindlimb muscles were removed. Myosin heavychain (MHC) and myosin light chain (MLC) content was determined inmuscle homogenates by SDS-PAGE. Body weight was significantly lower intumor-bearing (T) mice. There was a significant decrease in muscle massin all three muscles tested compared with control, with the largestdecrease occurring in the soleus. Although no type IIb MHC was detectedin the soleus samples from control mice, type IIb comprised 19% of thetotal MHC in T soleus. Type I MHC was significantly decreased in T vs. control soleus muscle. MHC isoform content was not significantly different from control in plantaris and gastrocnemius muscles. Thesedata are the first to show a change in myosin isoform expression accompanying muscle atrophy during cancer cachexia.

     

Mechanical restitution at different temperatures in papillary muscles from rabbit, rat, and hedgehog

Mechanical restitution curves, i.e., peak isometric force as a function of the duration of the preceding test interval, were investigated in papillary muscles from rabbit, rat, and hedgehog. Peak force of rabbit papillary muscle increased with prolongation of the test interval from about 0.3 s to about 1.0 s and for longer intervals peak force declined (called type I mechanical restitution). On the other hand, in rat and hedgehog, papillary muscles' force reached a maximum value at intervals of 30-120 s (called type II mechanical restitution). When temperature was decreased from 35 to 15 degrees C, maximum force of type I mechanical restitution shifted from 1.0 to 10 s, whereas maximum force of type II restitution did not change significantly. Type II mechanical restitution consisted of two different phases, designated phase A and phase B, respectively. As temperature was decreased from 35 to 0 degree C in the hedgehog preparation, the two phases became even more separated. At 35 degrees C, the rising part of mechanical restitution in the rabbit muscle could not be distinguished from phase A of the hedgehog preparation and was also very similar to phase A of the rat muscle. Phase A is thus present in both type I and type II mechanical restitution, but phase B is a special feature of type II mechanical restitution. Phase A and phase B might be a manifestation of activator calcium originating from two different sources, e.g., the sarcoplasmic reticulum and the sarcolemma.     

Sonic hedgehog (SHH) specifies muscle pattern at tissue and cellular chick level, in the chick limb bud.

Development of the musculature in chick limbs involves tissue and cellular patterning. Patterning at the tissue level leads to the precise arrangement of specific muscles; at the cellular level patterning gives rise to the fibre type diversity in muscles. Although the data suggests that the information controlling muscle patterning is localised within the limb mesenchyme and not in the somitic myogenic precursor cells themselves, the mechanisms underlying muscle organisation have still to be elucidated. The anterior-posterior axis of the limb is specified by a group of cells in the posterior region of the limb mesenchyme, called the zone of polarizing activity (ZPA). When polarizing-region cells are grafted to the anterior margin of the bud, they cause mirror-image digit duplications to be produced. The effect of ZPA grafts can be reproduced by application of retinoic acid (RA) beads and by grafting sonic hedgehog (SHH)-expressing cells to the anterior margin of the limb. Although most previous studies have looked at changes of the skeletal patterning, ZPA and RA also affect muscle patterning. In this report, we investigated the role of SHH in tissue and cellular patterning of forearm wing muscles. Ectopic application of a localised source of SHH to the anterior margin of the wing, leading to complete digit duplication, is able to transform anterior forearm muscles into muscles with a posterior identity. Moreover, the ectopic source of SHH induces a mirror image duplication of the normal posterior muscles fibre types in the new posterior muscles. The reorganisation of the slow fibres can be detected before muscle mass cleavage has started; suggesting that the appropriate fibre type arrangement is in place before the splitting process can be observed.     

Trypanosoma cruzi: Histochemical Characterization of Parasitized Skeletal Muscle Fibers1

C57B1/6 mice were infected with Brasil strain Trypanosoma cruzi trypomastigotes. The leg muscles of the mice were serial-sectioned with a cryostat, and individual fibers were classified histochemically as type I or type II on the basis of succinic dehydrogenase or adenosine triphosphatase activity. Although markedly more type II fibers were present in the leg muscles, the percentage of infected type I fibers was nearly five-fold higher than type II. This is the first demonstration of a preferential in vivo distribution of T. cruzi in muscle fibers based upon muscle type.     

Loss of ovarian function in mice results in abrogated skeletal muscle PPARδ and FoxO1-mediated gene expression

Menopause, the age-related loss of ovarian hormone production, promotes increased adiposity and associated metabolic pathology, but molecular mechanisms remain unclear. We previously reported that estrogen increases skeletal muscle PPARδ expression in vivo, and transgenic mice overexpressing muscle-specific PPARδ are reportedly protected from diet-induced obesity. We thus hypothesized that obesity observed in ovariectomized mice, a model of menopause, may result in part from abrogated expression of muscle PPARδ and/or downstream mediators such as FoxO1. To test this hypothesis, we ovariectomized (OVX) or sham-ovariectomized (SHM) 10-week old female C57Bl/6J mice, and subsequently harvested quadriceps muscles 12 weeks later for gene expression studies. Compared to SHM, muscle from OVX mice displayed significantly decreased expression of PPARδ (3.4-fold), FoxO1 (4.5-fold), PDK-4 (2.3-fold), and UCP-2 (1.8-fold). Consistent with studies indicating PPARδ and FoxO1 regulate muscle fiber type, we observed dramatic OVX-specific decreases in slow isoforms of the contractile proteins myosin light chain (11.1-fold) and troponin C (11.8-fold). In addition, muscles from OVX mice expressed 57% less myogenin (drives type I fiber formation), 2-fold more MyoD (drives type II fiber formation), and 1.6-fold less musclin (produced exclusively by type II fibers) than SHM, collectively suggesting a shift towards less type I oxidative fibers. Finally, and consistent with changes in PPARδ and FoxO1 activity, we observed decreased expression of atrogin-1 (2.3-fold) and MuRF-1 (1.9-fold) in OVX mice. In conclusion, muscles from ovariectomized mice display decreased PPARδ and FoxO1 expression, abrogated expression of downstream targets involved in lipid and protein metabolism, and gene expression profiles indicating less type I oxidative fibers.     

Isoform diversity of giant proteins in relation to passive and active contractile properties of rabbit skeletal muscles

The active and passive contractile performance of skeletal muscle fibers largely depends on the myosin heavy chain (MHC) isoform and the stiffness of the titin spring, respectively. Open questions concern the relationship between titin-based stiffness and active contractile parameters, and titin's importance for total passive muscle stiffness. Here, a large set of adult rabbit muscles (n = 37) was studied for titin size diversity, passive mechanical properties, and possible correlations with the fiber/MHC composition. Titin isoform analyses showed sizes between approximately 3300 and 3700 kD; 31 muscles contained a single isoform, six muscles coexpressed two isoforms, including the psoas, where individual fibers expressed similar isoform ratios of 30:70 (3.4:3.3 MD). Gel electrophoresis and Western blotting of two other giant muscle proteins, nebulin and obscurin, demonstrated muscle type-dependent size differences of < or =70 kD. Single fiber and single myofibril mechanics performed on a subset of muscles showed inverse relationships between titin size and titin-borne tension. Force measurements on muscle strips suggested that titin-based stiffness is not correlated with total passive stiffness, which is largely determined also by extramyofibrillar structures, particularly collagen. Some muscles have low titin-based stiffness but high total passive stiffness, whereas the opposite is true for other muscles. Plots of titin size versus percentage of fiber type or MHC isoform (I-IIB-IIA-IID) determined by myofibrillar ATPase staining and gel electrophoresis revealed modest correlations with the type I fiber and MHC-I proportions. No relationships were found with the proportions of the different type II fiber/MHC-II subtypes. Titin-based stiffness decreased with the slow fiber/MHC percentage, whereas neither extramyofibrillar nor total passive stiffness depended on the fiber/MHC composition. In conclusion, a low correlation exists between the active and passive mechanical properties of skeletal muscle fibers. Slow muscles usually express long titin(s), predominantly fast muscles can express either short or long titin(s), giving rise to low titin-based stiffness in slow muscles and highly variable stiffness in fast muscles. Titin contributes substantially to total passive stiffness, but this contribution varies greatly among muscles.