Altered secondary myogenesis in transgenic animals expressing the neural cell adhesion molecule under the control of a skeletal muscle alpha-actin promoter

The majority of skeletal muscle fibers are generated through the process of secondary myogenesis. Cell adhesion molecules such as NCAM are thought to be intricately involved in the cell-cell interactions between developing secondary and primary myotubes. During secondary myogenesis, the expression of NCAM in skeletal muscle is under strict spatial and temporal control. To investigate the role of NCAM in the regulation of primary-secondary myotube interactions and muscle fusion in vivo, we have examined muscle development in transgenic mice expressing the 125-kD muscle-specific, glycosylphosphatidylinositol-anchored isoform of human NCAM, under the control of a human skeletal muscle alpha-actin promoter that is active from about embryonic day 15 onward. Analysis of developing muscle from transgenic animals revealed a significantly lower number of myofibers encased by basal lamina at postnatal day 1 compared with nontransgenic littermates, although the total number of developing myofibers was similar. An increase in muscle fiber size and decreased numbers of VCAM-1-positive secondary myoblasts at postnatal day 1 was also found, indicating enhanced secondary myoblast fusion in the transgenic animals. There was also a significant decrease in myofiber number but no increase in overall muscle size in adult transgenic animals; other measurements such as the number of nuclei per fiber and the size of individual muscle fibers were significantly increased, again suggesting increased secondary myoblast fusion. Thus the level of NCAM in the sarcolemma is a key regulator of cell-cell interactions occurring during secondary myogenesis in vivo and fulfills the prediction derived from transfection studies in vitro that the 125-kD NCAM isoform can enhance myoblast fusion.     

Indirect fluorescence of primary and secondary myofibers in developing porcine muscle

Cytochemical differentiation of two populations of developing skeletal myofibers has been demonstrated in fetal muscle with metachromatic fluorescence of ribonucleic acid and deoxyribonucleic acid by staining fresh frozed cryostat sections of developing porcine skeletal muscle with acridine orange (CL. 46005). Evidence is presented that supports the hypothesis that first-formed myofibers (primary myofibers) serve as a structural framework upon which myoblasts proliferate, fuse in linear sequence and give rise to a second population (secondary myofibers) of myofibers.     

The superior gluteal artery perforator flap

The neuronal isoform of nitric oxide synthase (nNOS, termed also NOS-I) is expressed in normal adult skeletal muscle, suggesting important functions for NO in muscle biology. However, the expression and subcellular localization of NOS in muscle development and myoblast differentiation are largely unknown. In the present study, NOS was immunolocalized with isoform-specific antibodies in developing muscle and in differentiated myoblast cultures (mouse C2C12) together with histochemical NADPH-dependent diaphorase activity that is blocked by specific NOS inhibitors and therefore designated as NOS-associated diaphorase activity (NOSaD). Western blot analysis revealed immunoreactive bands for NOS-I-III in lysates from perinatal and adult muscle tissue and C2C12-myotubes that comigrated with prototypical proteins. In embryonic skeletal muscle, but not in adult myofibers, diffuse cytosolic staining and lack of sarcolemmal NOSaD activity and NOS-I immunoreaction were evident. In both myoblasts and fusioned myotubes, NOSaD and NOS isoforms I-III colocalize in the cytosol. Additionally, members of the sarcolemmal dystrophin-glycoprotein complex (i.e., dystrophin, adhalin, beta1-dystroglycan) immunolocalize in the cytosol of differentiating myoblasts, whereas anti-dystrophin and anti-beta1-dystroglycan clearly delineate the sarcolemma in myotubes. Thus, expression of NOS isoforms I-III and NOSaD is cytosolic in fusion-competent myoblasts during myotube formation in vitro. Interaction of NOSaD/NOS-I with the sarcolemmal dystrophin-complex known from mature myofibers is apparently lacking in prenatal muscle development and differentiating myoblasts. Localization of NOS isoforms thus characterized in myogenic cultures may help further to investigate regulated NO formation in muscle cells in vitro.     

Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular myocytes

Neuregulins (i.e. neuregulin-1 (NRG1), also called neu differentiation factor, heregulin, glial growth factor, and acetylcholine receptor-inducing activity) are known to induce growth and differentiation of epithelial, glial, neuronal, and skeletal muscle cells. Unexpectedly, mice with loss of function mutations of NRG1 or of either of two of their cognate receptors, ErbB2 and ErbB4, die during midembryogenesis due to the aborted development of myocardial trabeculae in ventricular muscle. To examine the role of NRG and their receptors in developing and postnatal myocardium, we studied the ability of a soluble NRG1 (recombinant human glial growth factor 2) to promote proliferation, survival, and growth of isolated neonatal and adult rat cardiac myocytes. Both ErbB2 and ErbB4 receptors were found to be expressed by neonatal and adult ventricular myocytes and activated by rhGGF2. rhGGF2 (30 ng/ml) provoked an approximate 2-fold increase in embryonic cardiac myocyte proliferation. rhGGF2 also promoted survival and inhibited apoptosis of subconfluent, serum-deprived myocyte primary cultures and also induced hypertrophic growth in both neonatal and adult ventricular myocytes, which was accompanied by enhanced expression of prepro-atrial natriuretic factor and skeletal alpha-actin. Moreover, NRG1 mRNA could be detected in coronary microvascular endothelial cell primary cultures prepared from adult rat ventricular muscle. NRG1 expression in these cells was increased by endothelin-1, another locally acting cardiotropic peptide within the heart. The persistent expression of both a neuregulin and its cognate receptors in the postnatal and adult heart suggests a continuing role for neuregulins in the myocardial adaption to physiologic stress or injury.     

The Marburg parent-child spelling training

Early skeletal muscle development is accompanied by changes in the composition of the cytoskeleton. In this report we analyze the distribution of the intermediate filament nestin in the developing mouse limb buds in vivo and in mesenchymal cells isolated from limb buds in vitro. The subcellular distribution of nestin mRNA and protein in muscle cells was also analyzed. We find a shift in nestin expression during early limb bud development. At embryonic day 11 (E11), low levels of nestin (protein) were expressed in the mesenchymal cells of the developing limb bud. Later, nestin mRNA and protein were down-regulated in the mesenchymal condensations undergoing chondrogenesis (E12 and E13), but remained expressed predominantly in the ectodermal cells and in the differentiating myoblasts. At E18, only muscle fibres, endothelial cells and nerves were nestin positive. This shift in expression was reproduced in vitro, in micro-mass cultures of mesenchymal cells. In E11 cultures, nestin protein was initially expressed in all cells. Upon formation of cartilage foci (after 2-3 days in culture), nestin immunoreactivity was not observed in cartilage, and low levels were detected in the cells located between the foci. A subpopulation of mono- and multinucleated cells, peripheral to the cartilage nodules, expressed the muscle-specific intermediate filament desmin protein together with high levels of nestin protein. The proportion of nestin-expressing cells could be changed by addition of specific signalling molecules. Insulin-like growth factors I and II (IGF I and II) increased the number of nestin-positive cells, while basic fibroblast growth factor (FGF) reduced the number of nestin-expressing cells. Finally, we present evidence for a different subcellular localization of nestin protein and mRNA: the mRNA is predominantly located in the ends of the muscle cell, whereas the protein is found in the central region. Intracellular localization of nestin mRNA may constitute an additional level of regulation of the cytoskeleton during muscle development.     

Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro

Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiation in vitro. We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis.     

Caveolin-3 associates with developing T-tubules during muscle differentiation

Caveolae, flask-shaped invaginations of the plasma membrane, are particularly abundant in muscle cells. We have recently cloned a muscle-specific caveolin, termed caveolin-3, which is expressed in differentiated muscle cells. Specific antibodies to caveolin-3 were generated and used to characterize the distribution of caveolin-3 in adult and differentiating muscle. In fully differentiated skeletal muscle, caveolin-3 was shown to be associated exclusively with sarcolemmal caveolae. Localization of caveolin-3 during differentiation of primary cultured muscle cells and development of mouse skeletal muscle in vivo suggested that caveolin-3 is transiently associated with an internal membrane system. These elements were identified as developing transverse-(T)-tubules by double-labeling with antibodies to the alpha 1 subunit of the dihydropyridine receptor in C2C12 cells. Ultrastructural analysis of the caveolin-3-labeled elements showed an association of caveolin-3 with elaborate networks of interconnected caveolae, which penetrated the depths of the muscle fibers. These elements, which formed regular reticular structures, were shown to be surface-connected by labeling with cholera toxin conjugates. The results suggest that caveolin-3 transiently associates with T-tubules during development and may be involved in the early development of the T-tubule system in muscle.     

Cardiac and skeletal myopathy in beta myosin heavy-chain simian virus 40 tsA58 transgenic mice

The mechanisms regulating cardiac muscle differentiation and development are incompletely understood. To examine the relationships between cardiocyte proliferation and differentiation, we tested the ability of a fragment from the rat beta myosin heavy-chain (MHC beta) gene to correctly target expression of a thermolabile simian virus 40 large tumor antigen allele (tsA58) in the developing mouse. Transgene expression in the heart was observed as early as 10 days postconception and was developmentally regulated in parallel with the endogenous MHC beta gene. Expression was also detected in developing skeletal muscle, although at low levels. Despite the temperature sensitivity of the mutant large tumor antigen protein, a subset of transgenic mice in several lineages developed marked cardiac and skeletal myopathies.     

Dihydropyridine receptor gene expression in skeletal muscle from mdx and control mice

The expression of isoform-specific dihydropyrine receptor-calcium channel (DHPR) alpha 1-subunit genes was investigated in mdx and control mouse diaphragm (DIA) and tibialis anterior (TA). RNase protection assays were carried out with a rat DHPR cDNA probe specific for skeletal muscle and a mouse DHPR cDNA probe specific for cardiac muscle. The level of expression of the gene encoding the cardiac DHPR was very weak in TA muscle from both control and mdx mice. Compared to TA, DIA expressed mRNA for the cardiac isoform at significantly higher levels, but mdx and control mouse DIA levels were similar to one another. In contrast, mRNA expression levels for the DHPR skeletal muscle isoform were lower in control DIA than TA. However, there was a dramatic increase in the expression for the DHPR skeletal muscle isoform in mdx DIA compared with control DIA, reaching the TA expression level, whereas dystrophy did not affect TA expression. [3H]-PN200-110 binding was used to further assess DIA DHPR expression at the protein level. The density of binding sites for the probe was not significantly affected in DIA muscles of mdx vs. control mice, but it was reduced in older mdx and control mice. The increase in DHPR mRNA levels without a consequent increase in DHPR protein expression could be secondary to possible enhanced protein degradation which occurs in mdx DIA. The altered DHPR expression levels found here do not appear to be responsible for the severe deficits in contractile function of the mdx DIA.     

Permeability and stability in buffer and in human serum of fluorinated phospholipid-based liposomes

Calsequestrin is the major Ca(2+)-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its alpha-helical content and the intrinsic fluorescence upon binding of Ca2+. Calsequestrin binds Ca2+ with high-capacity and with moderate affinity and it functions as a Ca2+ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca2+ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsequestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart.     

Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy

The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity--suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.     

Force production in mechanically isolated cardiac myocytes from human ventricular muscle tissue

OBJECTIVE: The expression of contractile isoforms changes during various pathological conditions but little is known about the consequences of these changes for the mechanical properties in human ventricular muscle. We investigated the feasibility of simultaneous determination of protein composition and isometric force development in single cardiac myocytes from human ventricular muscle tissue obtained from small biopsies taken during open heart surgery. METHODS: Small biopsies of about 3 mg wet weight were taken during open heart surgery from patients with aortic valve stenosis. These biopsies were divided in two parts. One part (approximately 2 mg) was used for mechanical isolation of single myocytes and subsequent force measurement while the remaining part was used, in aliquots of 1 microgram dry weight, for protein analysis by polyacrylamide gel electrophoresis. The myocytes were attached with silicon glue to a sensitive force transducer and a piezoelectric motor, mounted on an inverted microscope and permeabilized by means of Triton X-100. Force development was studied at various free calcium concentrations. RESULTS: From all biopsies, myocytes could be obtained and the composition of contractile proteins could be determined. The average isometric force (+/- s.e.m.) at saturating calcium concentration obtained on 20 myocytes from 5 patients amounted to 51 +/- 8 kN/m2. Force was half maximal at a calcium concentration of 2.47 +/- 0.10 microM. CONCLUSION: These measurements indicate that it is possible to study the correlation between mechanical properties and protein composition in small biopsies from human ventricular muscle.     

Memory consolidation and the hippocampal system

The dihydropyridines (DHP) receptor forms a high threshold L-type calcium channel in various excitable cells. In skeletal and cardiac muscle cells, a DHP receptor antagonist blocks not only the voltage-gated calcium current but also immobilizes the charge movement linked to the receptor. The DHP receptor is also present in cerebellar Purkinje neurons. Previously, we showed that nifedipine immobilizes a part of the charge movement but has no effect on the calcium channel current recorded in freshly dissociated mice Purkinje neurons. We report here the effect of other families of DHP receptor antagonists, benzothiazepines and phenylalkylamines, on the physiological properties of this receptor in mouse Purkinje neurons.     

Physiological role of phosphorylation of the cyclic AMP response element binding protein in rat cardiac nuclei

We determine whether the cyclic AMP signal transduction pathway affects phosphorylation of cyclic AMP response element binding protein and increases muscle gene expression in the heart. Elevation of cyclic AMP results in phosphorylation of the binding protein which is detected using an antibody specific for the phosphorylated, but not the unphosphorylated, form. The protein is present, but not phosphorylated, within the nuclei of myocytes in intact neonatal rat hearts and in high-density cultures. It is not expressed in low-density cultures. Increasing the amount of phosphorylated cyclic AMP with either isoproterenol or forskolin also increases the frequency and force of the beating. The phosphorylated form of the response element binding protein is visible in the nuclei by 10 min and persists for 2 h of drug treatment. A 1.5-fold increase in skeletal alpha-actin and alpha-myosin gene expression is detected after 48 h of isoproterenol treatment. However, blockage of beating with a calcium channel blocker (verapamil) in the presence of cyclic AMP results in a similar increased gene expression. This suggests that muscle gene expression can be regulated directly by the cyclic AMP pathway, probably via phosphorylation of the cyclic AMP response element binding protein but independent of contractile activity.     

Stereotactic microsurgical craniotomy for the treatment of third ventricular colloid cysts

The process of myoblast fusion during skeletal myogenesis is calcium regulated. Both dihydropyridine receptor and ryanodine receptor are already present on muscle precursors, at the prefusional stage, before they are required for excitation-contraction coupling. Previous pharmacological studies have shown the need for a special pool of Ca2+ associated with the membrane for the fusion process to occur. We hypothesized that this pool of Ca2+ is mobilized via a machinery similar to that involved in excitation-contraction coupling. The process of fusion in rat L6 muscle precursors was either totally or partially abolished in the presence of the L-type calcium channel inhibitors SR33557 and nifedipine (half inhibition towards 2 microM), respectively. The inhibition was reversible and dose-dependent. Drugs able to deplete internal calcium stores (caffeine, ryanodine, and thapsigargin) were also tested on the fusion. Both caffeine and thapsigargin drastically inhibited fusion whereas ryanodine had no effect. This suggests that fusion may be controlled by internal pools of Ca2+ but that its regulation may be insensitive to ryanodine. We presumed that an early form of the ryanodine receptor may exist, with different pharmacological properties than the adult forms. Indeed, Western blot analysis of pre- and postfusional L6 cells demonstrated the presence, at the prefusional stage, of a transient form of the ryanodine receptor protein with an apparent molecular weight slightly different from those of the classical skeletal and cardiac forms. Taken together, these results support the hypothesis that the fusion process is driven by a mechanism involving both the dihydropyridine receptor (alpha1 subunit of the L-type Ca2+ channel) and the internal stores of Ca2+. The machinery underlying this mechanism might consist of slightly different forms of the classic molecules that in adult muscle ensure excitation-contraction coupling. It remains to be seen, however, whether the mobilization of the internal pool of Ca2+ is triggered by the type of mechanism already described in skeletal muscle.     

FK-binding protein is associated with the ryanodine receptor of skeletal muscle in vertebrate animals

The ryanodine receptor/calcium release channel (RyR1) of sarcoplasmic reticulum from rabbit skeletal muscle terminal cisternae (TC) contains four tightly associated FK506-binding proteins (FKBP12). Dissociation and reconstitution studies have shown that RyR1 can be modulated by FKBP12, which helps to maintain the channel in the quiescent state. In this study, we found that the association of FKBP with RyR1 of skeletal muscle is common to each of the five classes of vertebrates. TC from skeletal muscle representing animals from different vertebrates, i.e. mammals (rabbit), birds (chicken), reptiles (turtle), fish (salmon and rainbow trout), and amphibians (frog), were isolated. For each, we find the following: 1) FKBP12 is localized to the TC (there are four FKBP binding sites/ryanodine receptor); 2) soluble FKBP exchanges with the bound form on RyR1 of TC; 3) release of FKBP from terminal cisternae by drug (FK590) treatment leads to a significant reduction in the net calcium loading rate, consistent with channel activation (the calcium loading rate is restored to the control value by reconstitution with FKBP12); and 4) RyR1 of skeletal muscle TC can bind to and exchange with either FKBP12 or FKBP12.6 (FKBP12.6 is the novel FKBP isoform found selectively associated with RyR2 of dog cardiac sarcoplasmic reticulum). We conclude that FKBP is an integral part of the RyR1 of skeletal muscle in each of the classes of vertebrate animals. The studies are consistent with a role for FKBP in skeletal muscle excitation-contraction coupling.