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A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle.

An exceptional muscle development commonly referred to as 'double-muscled' (Fig. 1) has been seen in several cattle breeds and has attracted considerable attention from beef producers. Double-muscled animals are characterized by an increase in muscle mass of about 20%, due to general skeletal-muscle hyperplasia-that is, an increase in the number of muscle fibers rather than in their individual diameter. Although the hereditary nature of the double-muscled condition was recognized early on, the precise mode of inheritance has remained controversial; monogenic (domainant and recessive), oligogenic and polygenic models have been proposed. In the Belgian Blue cattle breed (BBCB), segregation analysis performed both in experimental crosses and in the outbred population suggested an autosomal recessive inheritance. This was confirmed when the muscular hypertrophy (mh) locus was mapped 3.1 cM from microsatellite TGLA44 on the centromeric end of bovine chromosome 2 (ref. 5). We used a positional candidate approach to demonstrate that a mutation in bovine MSTN, which encodes myostatin, a member of the TGF beta superfamily, is responsible for the double-muscled phenotype. We report an 11-bp deletion in the coding sequence for the bioactive carboxy-terminal domain of the protein causing the muscular hypertrophy observed in Belgian Blue cattle.

Amino Acid Sequence↗

The effects of in ovo administration of testosterone or an antiandrogen on growth of chick embryos and embryonic muscle characteristics.

Based on earlier studies from our laboratory, we hypothesized that higher levels of plasma androgens in male embryos stimulate greater muscle development and are responsible for the greater muscle mass of male chickens after hatching. The results of these studies show that androgen supplementation by in ovo injection of testosterone prior to incubation had no effect on weight of 12-, 16-, or 20-d-old male chicken embryos or on characteristics of their Pectoralis superficialis muscle. In contrast, weight of 12 d-old female embryos was depressed and the protein concentration and protein content of the P. superficialis was reduced in 16-d-old female embryos. Interference with the actions of endogenous androgens by preincubation in ovo injection of Flutamide, an antiandrogen, resulted in significant linear and quadratic relationships between the dose of Flutamide injected and the weight, protein content, and DNA content of the P. superficialis of 16-d-old female embryos. Increases over the lower part of the dose range (0 to 1.74 micromol per egg) were followed by a decrease at the highest dose (2.9 micromol per egg). The DNA content of the P. superficialis of 16-d-old female embryos was similarly affected when Flutamide was injected on Day 8 of incubation, whereas the protein content and protein concentration of the muscle was increased in 20-d-old female embryos. There were no significant changes in the weight, protein content, or DNA content of the P. superficialis of male embryos when Flutamide was injected before or on Day 8 of incubation.

Androgen Antagonists↗

Medial gastrocnemius muscle flap in limb-sparing surgery of malignant bone tumors of the proximal tibia: mid-term results in 25 patients.

Twenty-five patients in whom the medial head of the gastrocnemius muscle was transposed to cover defects in limb-sparing surgery of malignant bone tumors in the proximal tibia were evaluated prospectively in a tumor register. Surgical technique and mid-term clinical-functional results after a mean follow-up of 4 years 6 months are presented in this study. Twenty-four of the 25 gastrocnemius muscle flaps survived without complications. In 1 patient, infection and necrosis of the transposed muscle developed 10 days after surgery, requiring revisional muscle transposition. The total revision rate after primary tumor surgery was high (48%), but this had no impact on the viability of the transposed muscle flap. The mean Musculoskeletal Tumor Society score at follow-up was 75.1% (range, 20-97%) and the mean postoperative range of motion in the operated knee was 60 deg (range, 30-100 deg). Because of its easy, safe, and quick preparation, the medial gastrocnemius muscle flap is exceptionally valuable as a means of covering defects arising during the course of this extensive tumor surgery. It guarantees a high survival rate of the transposed muscle and supports vascularity of the skin flaps, helps to reestablish joint function and mobility by stabilization and reinforcement of the extensor mechanism, and provides protection against infection.

Bone Neoplasms↗

Functional effects of myoblast implantation into histoincompatible mice with or without immunosuppression.

1. The goals of this study were to evaluate the immunogenicity of myogenic cells (MCs) (1) immediately after implantation into regenerating muscles, and (2) following their maturation under initial immunosuppression. Implanted mouse soleus muscles were evaluated by isometric tension recordings in vitro followed by histological investigations on frozen sections. 2. Implantation of non-histocompatible myoblasts into cryodamaged soleus muscles of CBA/J mice induced immune rejection which caused large and permanent deficits in muscle force: 4-42 weeks postimplantation maximal tetanic tension was 50-60% that of intact or regenerated cryodamaged control muscles without tendency for recovery or histological signs of muscle regeneration. Specific tension (force per unit muscle weight) was also significantly reduced. 3. On frozen sections, only 62 +/- 12% of the total area was desmin-positive, that is, occupied by muscle fibres, versus 90 +/- 4% in regenerated and 92 +/- 3% in intact muscles. Also, the total number of muscle fibre profiles was significantly reduced. 4. Under immune suppression with cyclosporin A (CsA), large muscles developed within 4 weeks. Following CsA withdrawal, muscle weight and force, in addition to desmin-positive areas on cross-sections, gradually declined over several months despite continual regeneration, indicating retarded immune rejection. 5. Initial application of CsA for 8 weeks after implantation, instead of 4 weeks, did not result in better survival of the implants, nor did a higher initial dose of CsA (100 instead of 50 mg kg-1 day-1). Prolonged continuous application of a reduced dose (25 mg kg-1 day-1) did not prevent muscle wasting but caused an additional delay. 6. It is concluded that histoincompatible myoblasts are highly immunogenic and that immune rejection causes large and permanent muscle deficits indicating elimination of host muscle tissue. Initial transient immunosuppression protects the incompatible cells, but after withdrawal, prolonged immune rejection and retarded muscle wasting occur.

Animals↗

Calcium source diversity in feline lower esophageal sphincter circular and sling muscle.

Within muscular equivalents of cat lower esophageal sphincter (LES), the circular muscle develops greater spontaneous tone, whereas the sling muscle is more responsive to cholinergic stimulation. Smooth muscle contraction involves a combination of calcium release from stores and of calcium entry via several pathways. We hypothesized that there are differences in the sources of Ca(2+) used for contraction in sling and circular muscles and that these differences could contribute to functional asymmetry observed within LES. Contraction of muscle strips from circular and sling regions of LES was assessed in the presence of TTX. In Ca(2+)-free Krebs, tone was inhibited to a greater degree in circular than sling muscle. L-type Ca(2+) channel blockade with nifedipine or verapamil inhibited tone in LES circular but not sling muscle. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA) caused greater increase in tone in sling than in circular muscle. The phospholipase C inhibitor U-73122 and the SR inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptor blocker 2-aminoethoxydiphenyl borate (2-APB) inhibited tone in circular and sling muscles, demonstrating that continuous release of Ca(2+) from Ins(1,4,5)P(3)-sensitive stores is important in tone generation in both muscles. In Ca(2+)-free Krebs, ACh-induced contractions (AChC) were inhibited to a greater degree in sling than circular muscles. However, nifedipine and verapamil greatly inhibited AChC in the circular but not sling muscle. Depletion of SR Ca(2+) stores with CPA or inhibition of Ins(1,4,5)P(3)-mediated store release with either U-73122 or 2-APB inhibited AChC in both muscles. We demonstrate that LES circular and sling muscles 1) use intracellular and extracellular Ca(2+) sources to different degrees in the generation of spontaneous tone and AChC and 2) use different Ca(2+) entry pathways. These differences hold the potential for selective modulation of LES tone in health and disease.

Acetylcholine↗

Neuropeptide responses and mechanics of the proximal and distal feline colon in vitro.

Mechanical properties and responses to neuropeptides were compared for proximal and distal feline colonic muscle. Proximal longitudinal (PL), proximal circular (PC), distal longitudinal (DL), and distal circular (DC) muscles were studied in vitro under isometric conditions. Total tension in DL [1.636 +/- 0.009 (SE) kg/cm2] was greater than in DC (0.699 +/- 0.004 kg/cm2) or PC (0.710 +/- 0.005 kg/cm2, P less than 0.05). Longitudinal muscle developed proportionately more active tension than circular muscle at each region (80.9% in DL vs. 54.1% in DC and 77.1% in PL vs. 52.3% in PC, P less than 0.01). Neuropeptides varied in potency and efficacy. Cholecystokinin octapeptide (CCK-8) was the most potent and efficacious in PL and substance P was the most efficacious in PC muscle (P less than 0.05). Substance P was more efficacious whereas CCK-8 and neurotensin were less efficacious in PC than PL muscle (P less than 0.01). DL muscle did not respond to CCK-8. DC muscle did not respond to CCK-8 or neurotensin. Isometric contractions to each neuropeptide were insensitive to tetrodotoxin. We conclude that 1) mechanical properties of circular and longitudinal colonic muscle differ and 2) responses to neuropeptides depend on anatomic region and intrinsic properties.

Acetylcholine↗

[Objective evaluation of selective peripheral denervation for spasmodic torticollis].

The authors evaluated the results of selective peripheral denervation (SPD) of posterior rami of the cervical spinal nerves and/or the accessory nerve for spasmodic torticollis. Five patients underwent seven operations in total and the results were evaluated with the modified Tsui's score which was used in the clinical trial of botulinum toxin (BTX) for torticollis in Japan. The preoperative score was 10.8 +/- 2.2 (mean +/- S.D.) and the postoperative score was 1.4 +/- 1.7. The score changes indicated the effects of the operation as "excellent" in four cases and "good" in one case. These results indicate that SPD is superior to BTX in terms of control of symptoms in spasmodic torticollis. After the initial operation, however, two patients showed the so-called "mole-hitting game phenomenon" in which normal muscles develop abnormal contraction after denervation of abnormal muscles. This forced us to perform the second operations. Although this phenomenon was first described in botulinum toxin treatment, this is probably the first report in surgically denervated cases.

Adult↗

Expression of adenylyl cyclase mRNAs in the denervated and in the developing mouse skeletal muscle.

Changes in the activity and in the expression of adenylyl cyclase (AC) were examined in mouse skeletal muscle after denervation and during development. Four isoforms of AC (AC2, AC6, AC7, and AC9) were detected by Northern blot analysis in gastrocnemius muscle, AC9 being the most abundant. After denervation, the levels of AC2 and AC9 mRNA decreased, whereas those of AC6 and AC7 increased. AC activity in response to several neurotransmitters was increased after denervation. During development, AC activity was high in fetus and neonate and declined in the adult; the sensitivity of AC activity to various neurotransmitters was the highest on the third postnatal day. The levels of AC6 and AC7 mRNAs were high on the third postnatal day and then decreased in adult, paralleling the decline in AC activity. All the characteristics of AC expression and activity in fetus and neonate resembled those observed in denervated adult muscle. These results indicate that changes in AC activity and AC mRNAs play an important role in the various physiopathological states of skeletal muscle, especially during muscle atrophy.

Adenylyl Cyclases↗

Spatiotemporal distribution of heparan sulfate epitopes during myogenesis and synaptogenesis: a study in developing mouse intercostal muscle.

Formation of a basal lamina (BL) ensheathing developing skeletal muscle cells is one of the earliest events in mammalian skeletal muscle myogenesis. BL-resident heparan sulfate proteoglycans have been implicated in various processes during myogenesis, including synaptic differentiation. However, attention has focused on the proteoglycan protein core, ignoring the glycosaminoglycan moiety mainly because of a lack of appropriate tools. Recently, we selected a panel of anti-heparan sulfate antibodies applied here to study the spatiotemporal distribution of specific heparan sulfate (HS) epitopes during myogenesis. In mouse intercostal muscle at embryonic day (E14), formation of acetylcholine receptor clusters at synaptic sites coincides with HS deposition. Although some HS epitopes show a general appearance throughout the BL, one epitope preferably clusters at synaptic sites but does so only from E16 onward. During elongation and maturation of primary myotubes, a process preceding secondary myotube development, significant changes in the HS epitope constitution of both synaptic and extrasynaptic BL were observed. As a whole, the data presented here strengthen previous observations on developmental regulation by BL components, and add to the putative roles of specific HS epitopes in myogenesis and synaptogenesis.

Animals↗

Embryonic and fetal myogenic programs act through separate enhancers at the MLC1F/3F locus.

Embryonic and fetal stages of skeletal muscle development are characterized by the differential expression of a number of muscle-specific genes. These include the products of independent promoters at the fast myosin light chain 1F/3F locus. In the mouse embryo MLC1F transcripts accumulate in embryonic skeletal muscle from E9, 4-5 days before high-level accumulation of MLC3F transcripts. A 3' enhancer can activate MLC1F and MLC3F promoters in differentiated muscle cells in vitro and in transgenic mice; both promoters, however, are activated at the time of MLC1F transcript accumulation. We now demonstrate the presence of a second muscle-specific enhancer at this locus, located in the intron separating the MLC1F and MLC3F promoters. Transgenic mice containing the intronic, but lacking the 3' enhancer, express high levels of an nlacZ reporter gene from the MLC3F promoter in adult fast skeletal muscle fibers. In contrast to the 3' enhancer, the intronic element is inactive both in embryonic muscle cells in vivo and in embryonic myocyte cultures. The intronic enhancer is activated at the onset of fetal development in both primary and secondary muscle fibers, at the time of endogenous MLC3F transcript accumulation. Late-activated MLC3F transgenes thus provide a novel in toto marker of fetal myogenesis. These results suggest that temporal regulation of transcription at the MLC1F/3F locus is controlled by separate enhancers which are differentially activated during embryonic and fetal development.

Animals↗

Alterations in sarcomere structure, collagen organization, mitochondrial activity, and protein metabolism in the avian low score normal muscle weakness.

Skeletal muscle fibers are surrounded by an extracellular matrix. The extracellular matrix is composed of glycoproteins, collagen, and proteoglycans. Proteoglycans have been suggested to play an important functional role in tissue differentiation. However, an understanding of how the extracellular matrix affects skeletal muscle development and function is largely unknown. In the avian genetic muscle weakness, low score normal (LSN), a late embryonic increase in the expression of decorin is followed by a subsequent increase in collagen crosslinking. The sarcomere organization, collagen fibril diameter and organization were investigated using transmission electron microscopy. Measurements were made at 20 days of embryonic development and 6 weeks posthatch. These studies showed changes in sarcomere organization and deterioration of muscle fibril structure in the LSN pectoral muscle. In vitro satellite cell cultures were developed and assayed for mitochondrial activity, and protein synthesis and degradation. In these analyses, mitochondrial activity from LSN satellite cells was significantly higher than those from normal pectoral muscle satellite cells. Protein synthesis rates between the normal and LSN satellite cell-derived myotubes were similar, but protein degradation rates were higher in the LSN cultures. Based on the reported functions of decorin as a regulator of cell proliferation and collagen fibril organization, it is possible that the late embryonic increase in decorin may be influencing the alterations in LSN sarcomere and collagen organization.

Animals↗

Dysplastic cardiac development presenting as cardiomyopathy.

Multiple manifestations of disordered or arrested cardiac muscle development are reported in a 14-year-old boy with clinical evidence of heart disease. These include persistent noncommunicating intramyocardial sinusoids, anomalous right ventricular muscle bands, muscular deficiency of ventricular septum, and papillary muscle underdevelopment. To our knowledge, this complex of findings has not been previously described.

Adolescent↗

Determination and development of the larval muscle pattern in Drosophila melanogaster.

This review describes briefly what is known about the early steps of mesoderm differentiation in the fruitfly Drosophila melanogaster. After a summary of general aspects including mesoderm differentiation, mesoderm cell migration and subdivision of the mesoderm, more detail is given about the specification of muscle progenitor cells, due to their role as the earliest obvious landmarks in muscle fiber development in Drosophila. Particular focus is given to recent results on the role of asymmetric cell division in muscle differentiation. Furthermore a short summary of myoblast fusion is provided.

Animals↗

Switching of subunit composition of muscle spectrin during myogenesis in vitro.

Spectrin comprises a family of polypeptides thought to be involved in mediating linkage of actin filaments to the plasma membrane in a wide variety of cell types (for reviews see refs 1-3). Spectrin is present as a tetramer composed of two non-identical subunits. Most cells express a common subunit with a molecular weight (Mr) of 240,000 (240 K; termed alpha-spectrin) in association with a polymorphic cell type-specific subunit: Mr 260 K in the intestinal terminal web (termed TW260), Mr 235 K in nervous tissue., liver, lymphocytes and fibroblasts (termed gamma-spectrin; also referred to as fodrin), and Mr 225/220 K in erythrocytes and adult cardiac and skeletal muscle (termed beta'- and beta-spectrin, respectively). We show here that primary chicken myoblasts express predominantly alpha gamma-spectrin, but on terminal differentiation in vitro the cells gradually switch to alpha beta-spectrin as a result of the onset of beta- and beta'-spectrin synthesis and by the subsequent differential stabilization of beta- and gamma-spectrin. This switching correlates with known changes in the biophysical properties and function of the developing muscle sarcolemma and cytoskeleton.

Animals↗

Exertional fatigue due to skeletal muscle dysfunction in patients with heart failure.

BACKGROUND: Exertional fatigue in patients with chronic heart failure is usually attributed to skeletal muscle underperfusion. Recently, skeletal muscle atrophy, abnormal muscle metabolic responses, and reduced muscle enzyme levels have been noted in such patients, raising the possibility that some patients may develop muscle fatigue due to intrinsic muscle abnormalities. The present study was undertaken to determine if a subpopulation of patients with heart failure develops exertional fatigue due to skeletal muscle dysfunction rather than to reduced muscle flow. METHODS AND RESULTS: All exercise hemodynamic studies performed in our laboratory on patients with heart failure were reviewed to identify those who exhibited peak exercise VO2 levels < or = 18 ml.min-1 x kg-1 due to leg fatigue and who underwent insertion of a Swan-Ganz catheter and leg blood flow catheter. Thirty-four patients were identified. Six normal subjects were also studied to define normal leg flow and femoral venous lactate responses to exercise. Patients with peak exercise leg flow levels within the normal mean flow level +/- 2 SEM were considered to have normal skeletal muscle flow during exercise. Nine of the 34 patients with heart failure were found to have normal leg blood flow during exercise. All of these patients terminated exercise due to leg fatigue, and all exhibited abnormal increases in femoral venous lactate concentrations (slope of work load versus femoral venous lactate: normal, 0.33 +/- 0.07 mg/W; heart failure with normal flow, 0.81 +/- 0.08 mg/W; p < 0.002). There was no significant difference between patients with normal leg flows and those with reduced flow in age, ejection fraction, and resting hemodynamic measurements. However, patients with normal flows exhibited more normal cardiac output responses to exercise and tended to have higher peak exercise VO2 (14.1 +/- 0.9 versus 11.5 +/- 0.7 ml.min-1 x kg-1, p < 0.05). CONCLUSIONS: A substantial percentage of patients with chronic heart failure develop exertional fatigue due to skeletal muscle dysfunction rather than to reduced skeletal muscle blood flow. In such patients, therapeutic interventions probably should be directed at improving the skeletal muscle abnormalities rather than at improving skeletal muscle flow.

Adult↗

A method to establish co-cultures of myotubes and preadipocytes from collagenase digested neonatal pig semitendinosus muscles.

The relationships between adipocyte and muscle cell development within muscle are important in the study of factors or agents that may improve meat quality. Neonatal porcine muscle has the potential to yield both cell types for cell culture because it contains developing adipocytes and a high number of muscle satellite cells. Therefore, we modified a conventional collagenase-based procedure to digest neonatal porcine muscle and subsequently cultured the resultant muscle stromal-vascular (SV) cells on several substrata in basal and dexamethasone (DEX)-containing media. Developing myotubes and preadipocytes were present in muscle SV cell cultures on laminin substrata following seeding and plating with fetal bovine serum (FBS) with or without DEX. Myotube number was much higher (P < 0.05) on laminin substrata compared with all other substrata, whereas preadipocyte number in muscle SV cell cultures was independent of substrata, as we have shown previously. This approach can be used to establish co-cultures of differentiating adipocytes and myotubes from collagenase-digested neonatal pig muscle. Because the comparison is within the same culture dish, this method allows for a direct comparison of the responses of adipogenic and myogenic cells to growth and differentiation factors. For example, DEX did not alter myogenesis (i.e., 11 +/- 3 vs. 11 +/- 4 myotubes per unit area for control and DEX-treated cultures, respectively), but it has been shown to markedly increase preadipocyte number in muscle SV cell cultures.

Adipocytes↗