Concomitant increases in myonuclear and satellite cell content in female trapezius muscle following strength training

A skeletal muscle fibre maintains its cytoplasmic volume by means of hundreds of myonuclei distributed along its entire length. Therefore it is hypothesised that changes in fibre size would involve modifications in myonuclear number. In this study, we have examined whether 10 weeks of strength training can induce changes in the number of myonuclei and satellite cells in female trapezius muscles. Biopsies were taken pre- and posttraining from the upper part of the descending trapezius muscle of nine subjects. Muscle samples were analysed for fibre area and myonuclear and satellite cell number using immunohistochemistry. There was a 36% increase in the cross-sectional area of muscle fibres. The hypertrophy of muscle fibres was accompanied by an approximately 70% increase in myonuclear number and a 46% increase in the number of satellite cells. Myonuclei number was positively correlated to satellite cell number indicating that a muscle with an increased concentration of myonuclei will contain a correspondingly higher number of satellite cells. The acquisition of additional myonuclei appears to be required to support the enlargement of multinucleated muscle cells following 10 weeks of strength training. Increased satellite cell content suggests that mitotic divisions of satellite cells produced daughter cells that became satellite cells. Accepted: 30 November 1999     

Switching of actin isoforms in skeletal muscle differentiation using mouse ES cells

Among six actin isoforms, α-skeletal and α-cardiac actins have similar amino acid components and are highly conserved. Although skeletal muscles essentially express α-skeletal actins in the adult tissue, α-cardiac isoform actin is prominent in the embryonic muscle tissue. Switching of actin isoforms from α-cardiac to α-skeletal actin occurs during skeletal muscle differentiation. The cardiac type α-actin is expressed in the regeneration and patho-physiological states of the skeletal muscles as well. In the present study, we demonstrate the morphological switching of α-type actin isoforms from α-cardiac to α-skeletal actin in vitro using mouse ES cells for the first time. Immunofluorescent double staining with two specific antibodies revealed that α-cardiac actin appeared first in myoblasts. After cell fusion to form myotubes, the cardiac type actin decreased and α-skeletal actin conversely increased. Finally, the α-skeletal isoform remained as a main actin component in the fully mature skeletal muscle fibers. The exchange of isoforms is not directly linked to the sarcomere formation. As a result, ES cells provide a useful in vitro system for exploring skeletal muscle differentiation.     

Cellular adaptation of the trapezius muscle in strength-trained athletes

 The aim of this study was to elucidate the cellular events that occur in the trapezius muscle following several years of strength training. In muscle biopsies from ten elite power lifters (PL) and six control subjects (C), several parameters were studied: cross-sectional area of muscle fibres, myosin heavy chain composition (MHC) and capillary supply [capillaries around fibres (CAF) and CAF/fibre area]. A method was also developed for counting the number of myonuclei and satellite cell nuclei. The proportion of fibres expressing MHC IIA, the cross-sectional area of each fibre type and the number of myonuclei, satellite cells and fibres expressing markers for early myogenesis were significantly higher in PL than in C (P<0.05). A significant correlation between the myonuclear number and the cross-sectional area was observed. Since myonuclei in mature muscle fibres are not able to divide, we suggest that the incorporation of satellite cell nuclei into muscle fibres resulted in the maintenance of a constant nuclear to cytoplasmic ratio. The presence of small diameter fibres expressing markers for early myogenesis indicates the formation of new muscle fibres. Accepted: 17 November 1998     

Effects of one bout of endurance exercise on the expression of myogenin in human quadriceps muscle

The objective of this study was to investigate the cellular localisation of MyoD and myogenin in human skeletal muscle fibres as well as the possible alterations in the expression of MyoD and myogenin in response to a single bout of endurance exercise at 40% and 75% of maximum oxygen uptake (VO₂ max). Twenty-five biopsies (5 per subject) from the vastus lateralis muscle were obtained before exercise, from the exercising leg at 40% and 75% of VO₂ max and from the resting leg following these exercise bouts. The tyramide signal amplification-direct and the Vectastain ABC methods using specific monoclonal antibodies were used to determine the exact location of myogenin and MyoD, to identify muscle satellite cells and to determine myosin heavy chain (MyHC) composition. At rest, myonuclei did not express MyoD or myogenin. Following a single bout of exercise at 40% and 75% of VO₂ max, an accumulation of myogenin in myonuclei and not in satellite cells was observed in biopsies from the exercised leg but not in biopsies before exercise and from the resting leg. The number of myogenin-positive myonuclei varied among individuals indicating differences in the response to a single exercise bout. In conclusion, this immunohistochemical study showed that a rapid rearrangement of myogenin expression occurs in exercised human skeletal muscles in response to a single bout of exercise.     

Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene.

A variety of differentiated cell types can be converted to skeletal muscle cells following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones, carrying a single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 gene but not the endogenous MyoD1, MRF4, Myf5, the skeletal muscle actin, or the myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers were observed only when the transfected cells were allowed to differentiate in vitro, via embryoid bodies, in low-mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and Myf5 genes and resulted in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle cells, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of the introduced gene was not required for myogenesis. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function, that MyoD1 functions in ES cells even under environmental conditions that favor differentiation is not dominant (incomplete penetrance), that MyoD1 expression is required for the establishment of the myogenic program but not for its maintenance, and that the exogenous MyoD1 gene can trans-activate the endogenous myogenin and MLC2 genes in undifferentiated ES cells.     

Effects of aging and gender on the spatial organization of nuclei in single human skeletal muscle cells

The skeletal muscle fibre is a syncitium where each myonucleus regulates the gene products in a finite volume of the cytoplasm, i.e., the myonuclear domain (MND). We analysed aging‐ and gender‐related effects on myonuclei organization and the MND size in single muscle fibres from six young (21–31 years) and nine old men (72–96 years), and from six young (24–32 years) and nine old women (65–96 years), using a novel image analysis algorithm applied to confocal images. Muscle fibres were classified according to myosin heavy chain (MyHC) isoform expression. Our image analysis algorithm was effective in determining the spatial organization of myonuclei and the distribution of individual MNDs along the single fibre segments. Significant linear relations were observed between MND size and fibre size, irrespective age, gender and MyHC isoform expression. The spatial organization of individual myonuclei, calculated as the distribution of nearest neighbour distances in 3D, and MND size were affected in old age, but changes were dependent on MyHC isoform expression. In type I muscle fibres, average NN‐values were lower and showed an increased variability in old age, reflecting an aggregation of myonuclei in old age. Average MND size did not change in old age, but there was an increased MND size variability. In type IIa fibres, average NN‐values and MND sizes were lower in old age, reflecting the smaller size of these muscle fibres in old age. It is suggested that these changes have a significant impact on protein synthesis and degradation during the aging process.     

Is the myonuclear domain size fixed?

It has been suggested that the number of myonuclei in a muscle fibre changes in proportion to the change in fibre size, resulting in a constant myonuclear domain size, defined as the cytoplasmic volume per myonucleus. The myonuclear domain size varies, however, between fibre types and is inversely related with the oxidative capacity of a fibre. Overall, the observations of an increase in myonuclear domain size during both maturational growth and overload-induced hypertrophy, and the decrease in myonuclear domain size during disuse- and ageing-associated muscle atrophy suggest that the concept of a constant myonuclear domain size needs to be treated cautiously. It also suggests that only when the myonuclear domain size exceeds a certain threshold during growth or overload-induced hypertrophy acquisition of new myonuclei is required for further fibre hypertrophy.     

Differential regulation of MyoD and myogenin mRNA levels by nerve induced muscle activity.

The levels of mRNAs coding for the myogenic factors MyoD and myogenin were measured during synapse formation in developing muscle and in adult muscle, after denervation and reinnervation and after muscle paralysis induced by blocking of neuromuscular transmission by neurotoxins known to alter the density and localization of synaptic proteins such as the acetylcholine receptor (AChR). The mRNA levels of both factors depend on usage of the neuromuscular synapses, but they change to different extents. Myogenin mRNA levels decrease drastically with innervation and increase strongly following blocking of transmission whereas the level of MyoD mRNA showed only a small decrease in response to innervation, denervation or muscle paralysis by neurotoxins. Neither mRNA showed a synapse-related cellular distribution. The results suggest that nerve-induced electrical muscle activity determines the cellular ratio of MyoD and myogenin mRNAs in adult muscle.     

Myogenic skeletal muscle satellite cells communicate by tunnelling nanotubes

Quiescent satellite cells sit on the surface of the muscle fibres under the basal lamina and are activated by a variety of stimuli to disengage, divide and differentiate into myoblasts that can regenerate or repair muscle fibres. Satellite cells adopt their parent's fibre type and must have some means of communication with the parent fibre. The mechanisms behind this communication are not known. We show here that satellite cells form dynamic connections with muscle fibres and other satellite cells by F‐actin based tunnelling nanotubes (TNTs). Our results show that TNTs readily develop between satellite cells and muscle fibres. Once developed, TNTs permit transport of intracellular material, and even cellular organelles such as mitochondria between the muscle fibre and satellite cells. The onset of satellite cell differentiation markers Pax‐7 and MyoD expression was slower in satellite cells cultured in the absence than in the presence of muscle cells. Furthermore physical contact between myofibre and satellite cell progeny is required to maintain subtype identity. Our data establish that TNTs constitute an integral part of myogenic cell communication and that physical cellular interaction control myogenic cell fate determination. J. Cell. Physiol. 223: 376–383, 2010. © 2010 Wiley‐Liss, Inc.     

Quantitative morphometric analyses of the exercised extensor digitorum longus muscle of the mouse

The metric properties of several important components of muscle, the capillaries, the myonuclei and also the cross-sectional areas of type I and type II fibres in the extensor digitorum longus muscle of the mouse have been studied using techniques appropriate for anisotropic tissues. Myonuclear volume density (Nv) was found to be increased by 70% whilst capillary length density (Lv) was increased by 50%. The results were analysed using multivariate statistical analysis (MANOVA) which is necessary for the multiple comparisons which inevitably occur in any morphometric investigation. The mitochondrial distribution within type I fibres was analysed using two-way ANOVA to study the effect of exercise on the mitochondrial contents of the inner, intermediate and outer zones of the muscle fibre. Significant differences in the mitochondrial content in the various zones were found. Exercise was found to increase mitochondrial contents in all three zones.     

Tissue specific expression of an α-skeletal actin-lacZ fusion gene during development in transgenic mice

Transgenic mice carrying a chimaeric transgene containing 730 bp of the 5-flanking sequences and the entire first intron of the rat -skeletal actin gene fused to thelacZ reporter gene have been produced by microinjection. ThelacZ reporter gene was used to verify the suitability of using the rat -actin promoter elements to target expression of genes of agricultural and therapeutic value exclusively to skeletal and heart muscle cells and fibres of transgenic mice. Expression of the transgene indicates a tightly regulated developmental and muscle specific control of the rat -skeletal actin gene, making it a useful promoter for gene targeting to muscle tissues. The cells destined to form muscle tissues in these transgenic mice are readily visualized in intact embryos by staining for -galactosidase activity, making them a suitable animal model for studying the origin and development of skeletal and cardiac muscle tissues.     

Nuclear DNA fragmentation and morphological alterations in adult rabbit skeletal muscle after short-term immobilization

Nuclear DNA fragmentation and ultrastructural changes, indicative of myonuclear apoptosis, were examined in adult skeletal muscle in response to short-term immobilization. Adult rabbits were allocated to 2 days (n=5) or 6 days (n=5) of unilateral casting of the ankle in full plantar flexion or were used as untreated controls (n=2). Atrophy of the soleus muscle was apparent by significant reductions in wet mass of 15% and 26% after 2 days and 6 days of casting (P< or =0.05), respectively. Mean fibre cross-sectional area and myonuclear number per section were also lower (17% and 9.1%, respectively) after 6 days of casting, in comparison with contralateral control muscles (P< or =0.05). Electron-microscopic examination showed condensed chromatin and irregularly shaped myonuclei in muscles immobilized for either 2 days or 6 days. Myofibrillar disruption and abnormalities of the subsarcolemmal mitochondria were also apparent in the absence of inflammation or plasma membrane alterations in cast muscles. Longitudinal and transverse sections showed abundant in situ end-labelling of DNA strand breaks (TUNEL) after 2 days, with less after 6 days, of immobilization. Positive labelling corresponded to myonuclear locations within fibres, yet the number of TUNEL-positive nuclei indicated DNA fragmentation in additional cell types such as capillary endothelial cells or fibroblasts. The data indicate that the immobilization of slow-twitch skeletal muscle in a shortened position rapidly induces morphological alterations consistent with mitochondrial injury and apoptotic myonuclear elimination.     

Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products.

The development of muscle cells involves the action of myogenic determination factors. In this report, we show that human skeletal muscle tissue contains, besides the previously described Myf-5, two additional factors Myf-3 and Myf-4 which represent the human homologues of the rodent proteins MyoD1 and myogenin. The genes encoding Myf-3, Myf-4 and Myf-5 are located on human chromosomes 11, 1, and 12 respectively. Constitutive expression of a single factor is sufficient to convert mouse C3H 10T1/2 fibroblasts to phenotypically normal muscle cells. The myogenic conversion of 10T1/2 fibroblasts results in the activation of the endogenous MyoD1 and Myf-4 (myogenin) genes. This observation suggests that the expression of Myf proteins leads to positive autoregulation of the members of the Myf gene family. Individual myogenic colonies derived from MCA C115 cells (10T1/2 fibroblast transformed by methylcholanthrene) express various levels of endogenous MyoD1 mRNA ranging from nearly zero to high levels. The Myf-5 gene was generally not activated in 10T1/2 derived myogenic cell lines but was expressed in some MCA myoblasts. In primary human muscle cells Myf-3 and Myf-4 mRNA but very little Myf-5 mRNA is expressed. In mouse C2 and P2 muscle cell lines MyoD1 is abundantly synthesized together with myogenin. In contrast, the rat muscle lines L8 and L6 and the mouse BC3H1 cells express primarily myogenin and low levels of Myf-5 but no MyoD1. Myf-4 (myogenin) mRNA is present in all muscle cell lines at the onset of differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)