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.     

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.     

Nuclear localized Akt limits skeletal muscle derived fibrotic signaling

Skeletal muscle regeneration following injury is a complex multi-stage process involving the recruitment of inflammatory cells, the activation of muscle resident fibroblasts, and the differentiation of activated myoblasts into myocytes. Dysregulation of these cellular processes is associated with ineffective myofiber repair and excessive deposition of extracellular matrix proteins leading to fibrosis. PI3K/Akt signaling is a critical integrator of intra- and intercellular signals connecting nutrient availability to cell survival and growth. Activation of the PI3K/Akt pathway in skeletal muscle leads to hypertrophic growth and a reversal of the changes in body composition associated with obesity and advanced age. Though the molecular mechanisms mediating these effects are incompletely understood, changes in paracrine signaling are thought to play a key role. Here, we utilized modified RNA to study the biological role of the transient translocation of Akt to the myonuclei of maturing myotubes. Using a conditioned medium model system, we show that ectopic myonuclear Akt suppresses fibrogenic paracrine signaling in response to oxidative stress, and that interventions that increase or restore myonuclear Akt may impair fibrosis.     

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     

Nesprin provides elastic properties to muscle nuclei by cooperating with spectraplakin and EB1

Muscle nuclei are exposed to variable cytoplasmic strain produced by muscle contraction and relaxation, but their morphology remains stable. Still, the mechanism responsible for maintaining myonuclear architecture, and its importance, is currently elusive. Herein, we uncovered a unique myonuclear scaffold in Drosophila melanogaster larval muscles, exhibiting both elastic features contributed by the stretching capacity of MSP300 (nesprin) and rigidity provided by a perinuclear network of microtubules stabilized by Shot (spectraplakin) and EB1. Together, they form a flexible perinuclear shield that protects myonuclei from intrinsic or extrinsic forces. The loss of this scaffold resulted in significantly aberrant nuclear morphology and subsequently reduced levels of essential nuclear factors such as lamin A/C, lamin B, and HP1. Overall, we propose a novel mechanism for maintaining myonuclear morphology and reveal its critical link to correct levels of nuclear factors in differentiated muscle fibers. These findings may shed light on the underlying mechanism of various muscular dystrophies.     

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     

An autoradiographic study of the role of satellite cells and myonuclei during myogenesis in vitro

Satellite cells and myonuclei of neonatal rat muscles were differentially labeled with 3H-thymidine according to the procedure of Moss and Leblond (1971). Minced muscles fragments containing either labeled satellite cells or labeled myonuclei were cultured until multinucleated myotubes grew out from the explants. Reutilzation of isotope released from degenerating nuclei was competitively inhibited by using a culture medium containing excess (0.32-0.41 mM) cold thymidine. after an 8-10 day growth period, the explants were fixed and prepared for autoradiographic (ARG) examination to determine whether labeled satellite cells or myonuclei had contributed to the myonuclear population of the developing myotubes. Counts were made of the number of labeled myotubes in the explants and compared with the number of labeled satellite cells and myonuclei in samples of the original muscle tissues fixed at the time of explantation. The original muscles showed a mean satellite cell labeling index of 51.7% and gave rise to myotubes with a mean labeling incidence of 40%. In contrast, myonuclear labeling in the original muscle tissues showed no correlation with subsequent myotube labeling. Only 3.4% myotube labeling was found in explants in which over 30% of the original tissue myonuclei had been labeled. Under conditions controlled for isotope reutilization, these observations confirm results of in vivo ARG studies indicating that satellite cells are the only significant source of regenerating myoblasts in injured muscle tissue.     

Syd/JIP3 and JNK Signaling Are Required for Myonuclear Positioning and Muscle Function

Highlighting the importance of proper intracellular organization, many muscle diseases are characterized by mispositioned myonuclei. Proper positioning of myonuclei is dependent upon the microtubule motor proteins, Kinesin-1 and cytoplasmic Dynein, and there are at least two distinct mechanisms by which Kinesin and Dynein move myonuclei. The motors exert forces both directly on the nuclear surface and from the cell cortex via microtubules. How these activities are spatially segregated yet coordinated to position myonuclei is unknown. Using Drosophila melanogaster, we identified that Sunday Driver (Syd), a homolog of mammalian JNK-interacting protein 3 (JIP3), specifically regulates Kinesin- and Dynein-dependent cortical pulling of myonuclei without affecting motor activity near the nucleus. Specifically, Syd mediates Kinesin-dependent localization of Dynein to the muscle ends, where cortically anchored Dynein then pulls microtubules and the attached myonuclei into place. Proper localization of Dynein also requires activation of the JNK signaling cascade. Furthermore, Syd functions downstream of JNK signaling because without Syd, JNK signaling is insufficient to promote Kinesin-dependent localization of Dynein to the muscle ends. The significance of Syd-dependent myonuclear positioning is illustrated by muscle-specific depletion of Syd, which impairs muscle function. Moreover, both myonuclear spacing and locomotive defects in syd mutants can be rescued by expression of mammalian JIP3 in Drosophila muscle tissue, indicating an evolutionarily conserved role for JIP3 in myonuclear movement and highlighting the utility of Drosophila as a model for studying mammalian development. Collectively, we implicate Syd/JIP3 as a novel regulator of myogenesis that is required for proper intracellular organization and tissue function.     

Regenerative potential of human skeletal muscle during aging

In this study, we have investigated the consequences of aging on the regenerative capacity of human skeletal muscle by evaluating two parameters: (i) variation in telomere length which was used to evaluate the in vivo turn-over and (ii) the proportion of satellite cells calculated as compared to the total number of nuclei in a muscle fibre. Two skeletal muscles which have different types of innervation were analysed: the biceps brachii, a limb muscle, and the masseter, a masticatory muscle. The biopsies were obtained from two groups: young adults (23 +/- 1.15 years old) and aged adults (74 +/- 4.25 years old). Our results showed that during adult life, minimum telomere lengths and mean telomere lengths remained stable in the two muscles. The mean number of myonuclei per fibre was lower in the biceps brachii than in the masseter but no significant change was observed in either muscle with increasing age. However, the number of satellite cells, expressed as a proportion of myonuclei, decreased with age in both muscles. Therefore, normal aging of skeletal muscle in vivo is reflected by the number of satellite cells available for regeneration, but not by the mean number of myonuclei per fibre or by telomere lengths. We conclude that a decrease in regenerative capacity with age may be partially explained by a reduced availability of satellite cells.     

Analysis of the Arabidopsis nuclear proteome and its response to cold stress

The nucleus is the subcellular organelle that contains nearly all the genetic information required for the regulated expression of cellular proteins. In this study, we comprehensively characterized the Arabidopsis nuclear proteome. Nuclear proteins were isolated and analyzed using two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Approximately 500-700 spots were detected in reference 2D gels of nuclear proteins. Proteomic analyses led to the identification of 184 spots corresponding to 158 different proteins implicated in a variety of cellular functions. We additionally analyzed the changes in the nuclear proteome in response to cold stress. Of the 184 identified proteins, 54 were up- or downregulated with a greater than twofold change in response to cold treatment. Among these, six proteins were selected for further characterization. Northern analysis data revealed that gene expression of these proteins was also altered by cold stress. Following transient expression in BY-2 protoplasts, two proteins were detected in both the cytoplasm and the nucleus and four others were detected exclusively in the nucleus, which correlates well with the nuclear localization patterns of the proteomic data. Our study provides an initial insight into the Arabidopsis nuclear proteome and its response to cold stress.     

Em evidence of myoblast origin in regenerating human skeletal muscle explants

EM study of cultured human skeletal muscle explants on 10 consecutive days after incubation made possible a record for the first time, the early events occurring during regeneration. After incubation, normal myonuclei underwent activation and dense granulation. Some myonuclei showed early transformation to presumptive myoblasts. The conclusion was that myonuclei transformed into myoblasts which developed into satellite cells (SC). These SC of myonuclear origin, proliferated, and fused forming myotubes that matured into myofibres, replacing damaged muscle. The findings have new implications for the current myoblast / cell transplant and gene transfer therapy research which may provide possible answers for muscular dystrophy in the future.     

Distribution of myonuclei and microtubules in live muscle fibers of young, middle-aged, and old mice.

We have recently published a new technique for visualizing nuclei in living muscle fibers of intact animals, based on microinjection of labeled DNA into single myofibers, excluding satellite cells (Bruusgaard JC, Liestol K, Ekmark M, Kollstad K, and Gundersen K. J Physiol 551: 467-478, 2003). In the present study, we use this technique to study fiber segments of soleus and extensor digitorum longus (EDL) muscles from mice aged 2, 14, and 23 mo. As the animals maturing from 2 to 14 mo, they displayed an increase in size and number of nuclei. Soleus showed little change in nuclear domain size, whereas this increased by 88% in the EDL. For 14-mo-old animals, no significant correlation between fiber size and nuclear number was observed (R2=0.18, P=0.51) despite a fourfold variation in cytoplasmic volume. This suggests that size and nuclear number is uncoupled in middle-aged mice. When animals aged from 14 to 23 mo, EDL IIb, but not soleus, fibers atrophied by 41%. Both EDL and soleus displayed a reduction in number of nuclei: 20 and 16%, respectively. A positive correlation between number of nuclei and size was observed at 2 mo, and this reappeared in old mice. The atrophy in IIb fibers at old age was accompanied by a disturbance in the orderly positioning of nuclei that is so prominent in glycolytic fibers at younger age. In old animals, changes in nuclear shape and in the peri- and internuclear microtubule network were also observed. Thus changes in myonuclear number and distribution, perhaps related to alterations in the microtubular network, may underlie some of the adverse consequences of aging on skeletal muscle size and function.     

Effective fiber hypertrophy in satellite cell-depleted skeletal muscle

An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca(2+) sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.     

Myonuclear domain size varies along the lengths of maturing skeletal muscle fibers

In a skeletal muscle fiber, each myonucleus is responsible for gene expression in its surrounding cytoplasm. The region of cytoplasm associated with an individual myonucleus is termed myonuclear domain. However, little is known about domain size variation within individual muscle fibers. This study tests the hypothesis that myonuclear domains expressing neonatal myosin within end regions of maturing fibers will be smaller than domains elsewhere in the fibers. The model used is chicken pectoralis, where we have previously shown that during development repression of neonatal myosin radiates from the central region towards the fiber ends. Samples excised from birds aged nine through to 115 days after hatching were sectioned transversely. Using computer image analysis and immunocytochemistry, fiber profiles were classified as neonatal, transforming or adult. Each profile was also located in an adjacent dystrophin-labelled section, where myonuclei were visualized using haematoxylin and bisbenzamide. Variation in myonuclear length with age was not found to be significant (p = 0.925). Myonuclei were counted, and formulae used to calculate mean myonuclear domain size for each profile type. Myonuclear number/mm fiber was calculated to be adult (mean = 108.57 myonuclei/mm), transforming (65.82) and neonatal (25.23). Transforming profiles had significantly (p=0.027) more myonuclei/mm than neonatal, as did adult (p=0.005). Volume of cytoplasm/myonucleus was adult (mean = 16,132 microm3/myonucleus), transforming (12,899) and neonatal (8,130). Transforming and adult profiles had significantly (p<0.001) larger myonuclear domains than did neonatal profiles. Transforming and adult profiles did not differ in either myonuclei/mm (p=0.302) or volume of cytoplasm/myonucleus (p=0.413). This study demonstrates smaller domains at the terminal tips of maturing muscle fibers.     

Satellite cell regulation of muscle mass is altered at old age.

Muscle mass is decreased with advancing age, likely due to altered regulation of muscle fiber size. This study was designed to investigate cellular mechanisms contributing to this process. Analysis of male Fischer 344 X Brown Norway rats at 6, 20, and 32 mo of age demonstrated that, even though significant atrophy had occurred in soleus muscle by old age, myofiber nuclear number did not change, resulting in a decreased myonuclear domain. Also, the number of centrally located nuclei was significantly elevated in soleus muscle of 32-mo-old rats, correlating with an increase in gene expression of MyoD and myogenin. Whereas total 5'-bromo-2'deoxyuridine (BrdU)-positive nuclei were decreased at older ages, BrdU-positive myofiber nuclei were increased. These results suggest that, with age, loss of muscle mass is accompanied by increased myofiber nuclear density that involves fusion of proliferative satellite cells, resembling ongoing regeneration. Interestingly, centrally located myofiber nuclei were not BrdU labeled. Rats were subjected to hindlimb suspension (HS) for 7 or 14 days and intermittent reloading during HS for 1 h each day (IR) to investigate how aging affects the response of soleus muscle to disuse and an atrophy-reducing intervention. After 14 days of HS, soleus muscle size was decreased to a similar extent at all three ages. However, myofiber nuclear number and the total number of BrdU-positive nuclei decreased with HS only in the young rats. IR was associated with an attenuation of atrophy in soleus muscles of 6- and 20- but not 32-mo-old rats. Furthermore, IR was associated with an increase in BrdU-positive myofiber nuclei only in young rats. These data indicate that altered satellite cell function with age contributes to the impaired response of soleus muscle to an intervention that attenuates muscle atrophy in young animals during imposed disuse.     

Identification of the Acetylation and Ubiquitin-Modified Proteome during the Progression of Skeletal Muscle Atrophy

Skeletal muscle atrophy is a consequence of several physiological and pathophysiological conditions including muscle disuse, aging and diseases such as cancer and heart failure. In each of these conditions, the predominant mechanism contributing to the loss of skeletal muscle mass is increased protein turnover. Two important mechanisms which regulate protein stability and degradation are lysine acetylation and ubiquitination, respectively. However our understanding of the skeletal muscle proteins regulated through acetylation and ubiquitination during muscle atrophy is limited. Therefore, the purpose of the current study was to conduct an unbiased assessment of the acetylation and ubiquitin-modified proteome in skeletal muscle during a physiological condition of muscle atrophy. To induce progressive, physiologically relevant, muscle atrophy, rats were cast immobilized for 0, 2, 4 or 6 days and muscles harvested. Acetylated and ubiquitinated peptides were identified via a peptide IP proteomic approach using an anti-acetyl lysine antibody or a ubiquitin remnant motif antibody followed by mass spectrometry. In control skeletal muscle we identified and mapped the acetylation of 1,326 lysine residues to 425 different proteins and the ubiquitination of 4,948 lysine residues to 1,131 different proteins. Of these proteins 43, 47 and 50 proteins were differentially acetylated and 183, 227 and 172 were differentially ubiquitinated following 2, 4 and 6 days of disuse, respectively. Bioinformatics analysis identified contractile proteins as being enriched among proteins decreased in acetylation and increased in ubiquitination, whereas histone proteins were enriched among proteins increased in acetylation and decreased in ubiquitination. These findings provide the first proteome-wide identification of skeletal muscle proteins exhibiting changes in lysine acetylation and ubiquitination during any atrophy condition, and provide a basis for future mechanistic studies into how the acetylation and ubiquitination status of these identified proteins regulates the muscle atrophy phenotype.     

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.     

Characterization of the human skeletal muscle proteome by one-dimensional gel electrophoresis and HPLC-ESI-MS/MS

Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I-V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most comprehensive characterization of the human skeletal muscle proteome to date. These data hold promise for future global assessment of quantitative changes in the muscle proteome of patients affected by disorders involving skeletal muscle.     

Muscle fiber structure in an aging long-lived seabird,the black-legged kittiwake (Rissa tridactyla)

Many long-lived animals do not appear to show classic signs of aging, perhaps because they show negligible senescence until dying from “catastrophic” mortality. Muscle senescence is seldom examined in wild animals, yet decline in muscle function is one of the first signs of aging in many lab animals and humans. Seabirds are an excellent study system for physiological implications of aging because they are long-lived animals that actively forage and reproduce in the wild. Here, we examined linkages between pectoralis muscle fiber structure and age in black-legged kittiwakes (Rissa tridactyla). Pectoralis muscle is the largest organ complex in birds, and responsible for flight and shivering. We obtained and fixed biopsies from wild black-legged kittiwakes of known age. We then measured muscle fiber diameter, myonuclear domain and capillaries per fiber area among birds of differing ages. All muscle parameters were independent of age. Number of nuclei per mm of fiber showed a positive correlation with muscle fiber cross-sectional area, and myonuclear domain increased with muscle fiber diameter. Thus, as muscle fibers increased in size, they may not have recruited satellite cells, increasing the protein turnover load per nuclei. We conclude that senescence in a long-lived bird with an active lifestyle, does not entail mammalian-like changes in muscle structure.