The expression patterns of Pax7 in satellite cells during overload-induced rat adult skeletal muscle hypertrophy

Aim: Activated satellite cells (SCs) have the ability to reacquire a quiescent, undifferentiated state. Pax7 plays a crucial role in allowing activated SCs to undergo self‐renewal. Because the increase in the SC population is induced during overload‐induced skeletal muscle hypertrophy, it is possible that Pax7‐regulated SC self‐renewal is involved in the modulation of the SC population during the functional overload of skeletal muscles. However, the characteristics of the expression patterns of Pax7 in SCs during the functional overload of adult skeletal muscles are poorly understood. Methods: Using immunohistochemical approaches, we examined the temporal and spatial expression patterns of Pax7 expressed in SCs during the functional overloading of rat skeletal muscles. Results: The time course of Pax7 expression in SCs was similar to that of the expression of the differentiation regulatory factor myogenin during the early stage of functional overload. However, the percentage of SCs that expressed Pax7 was markedly higher than that of the SCs that expressed myogenin. Coexpression of Pax7 and myogenin was not detected in SCs. In addition, the expression of cyclin‐dependent kinase inhibitor p21, which regulates cell cycle arrest and differentiation, was not detected in Pax7‐positive SCs. Conclusion: These results suggest that Pax7‐regulated self‐renewal of SCs may be induced during the early stage of functional overload and may contribute to modulating the SC population in hypertrophied muscles. Furthermore, it was suggested that the numbers of SCs which underwent self‐renewal may be higher than that of SCs which were provided as the additional myonuclei for hypertrophying myofibres.     

Voluntary resistance wheel exercise during post-natal growth in rats enhances skeletal muscle satellite cell and myonuclear content at adulthood

Aim: To determine whether voluntary free wheel (FW) or resistance wheel (RW) exercise or reduced muscle activity would influence maturational increases in muscle mass and the number of satellite cells (SCs) and myonuclei (MN) accrued by adulthood. Methods: Hind limb muscles of male rats housed with, or without, FWs from 4 to 5, 7 or 10 weeks of age, and rats housed with RWs from 4 to 10 week of age, were evaluated. To assess the effect of reduced muscle activity, gastrocnemius muscles of 4‐week‐old rats were injected with botulinum toxin (Btx) and collected at 7 weeks of age. Muscle fibre size and the frequency of Pax7‐positive SCs and MN were determined in 7‐ and 10‐week‐old muscles via immunohistochemical methods. Results: Free wheel exercise enhanced muscle growth and the frequency of SCs in the medial gastrocnemius (MG) (threefold) and vastus lateralis (VL) (twofold) of rats at 10 week of age. Resistance wheel exercise increased the number of SCs and MN (22–30%), with more muscle fibre nuclei being associated with larger fibre size, in the soleus, MG and VL muscles. Btx impaired the normal increases in muscle fibre size and the accrual of MN but not SCs. Conclusion: A greater volume of exercise during maturational growth was important for enhancing SC numbers, whereas their conversion to MN required higher‐intensity exercise. The enhanced muscle fibre nuclear populations may influence the capacity of the muscle to adapt to exercise, injury or disuse in later adulthood.     

Alterations of M-cadherin, neural cell adhesion molecule and beta-catenin expression in satellite cells during overload-induced skeletal muscle hypertrophy

Aim: Neural cell adhesion molecule (NCAM) and M‐cadherin are cell adhesion molecules expressed on the surface of skeletal muscle satellite cell (SC). During myogenic morphogenesis, M‐cadherin participates in mediating terminal differentiation and fusion of myoblasts by forming a complex with β ‐catenin and that NCAM contributes to myotube formation by fusion of myoblasts. Hypertrophy and hyperplasia of functionally overloaded skeletal muscle results from the fusion with SCs into the existing myofibres or new myofibre formation by SC–SC fusion. However, the alterations of NCAM, M‐cadherin and β ‐catenin expressions in SCs in response to functional overload have not been investigated. Methods: Using immunohistochemical approaches, we examined the temporal and spatial expression patterns of these factors expressed in SCs during the functional overload of skeletal muscles. Results: Myofibres with SCs showing NCAM+/M‐cadherin?, NCAM+/M‐cadherin+ or NCAM?/M‐cadherin+ were detected in overloaded muscles. The percentage changes of myofibres with SCs showing NCAM+/M‐cadherin?, NCAM+/M‐cadherin+ or NCAM?/M‐cadherin+ were elevated in day‐3 post‐overloaded muscles, and then only the percentage changes of myofibres with SCs showing NCAM?/M‐cadherin+ were significantly increased in day‐7 post‐overload muscles (P < 0.05). Both β ‐catenin and M‐cadherin were co‐localized throughout quiescent, proliferation and differentiation stages of SCs. Conclusion: These results suggested that the expressions of NCAM, M‐cadherin and β ‐catenin in SCs may be controlled by distinct regulatory mechanisms during functional overload, and that interactions among NCAM, M‐cadherin and β ‐catenin in SCs may play important roles to contribute to overload‐induced muscle hypertrophy via fusion with each other or into the existing myofibres of SCs.     

The impact of sarcopenia and exercise training on skeletal muscle satellite cells

It has been well-established that the age-related loss of muscle mass and strength, or sarcopenia, impairs skeletal muscle function and reduces functional performance at a more advanced age. Skeletal muscle satellite cells (SC), as precursors of new myonuclei, have been suggested to be involved in the development of sarcopenia. In accordance with the type II muscle fiber atrophy observed in the elderly, recent studies report a concomitant fiber type specific reduction in SC content. Resistance type exercise interventions have proven effective to augment skeletal muscle mass and improve muscle function in the elderly. In accordance, recent work shows that resistance type exercise training can augment type II muscle fiber size and reverse the age-related decline in SC content. The latter is supported by an increase in SC activation and proliferation factors that generally appear following exercise training. Present findings strongly suggest that the skeletal muscle SC control myogenesis and have an important, but yet unresolved, function in the loss of muscle mass with aging. This review discusses the contribution of skeletal muscle SC in the age-related loss of muscle mass and the efficacy of exercise training as a means to attenuate and/or reverse this process.     

Nrf2 augments skeletal muscle regeneration after ischaemia–reperfusion injury

Skeletal muscles harbour a resident population of stem cells, termed satellite cells (SCs). After trauma, SCs leave their quiescent state to enter the cell cycle and undergo multiple rounds of proliferation, a process regulated by MyoD. To initiate differentiation, fusion and maturation to new skeletal muscle fibres, SCs up‐regulate myogenin. However, the regulation of these myogenic factors is not fully understood. In this study we demonstrate that Nrf2, a major regulator of oxidative stress defence, plays a role in the expression of these myogenic factors. In both promoter studies with myoblasts and a mouse model of muscle injury in Nrf2‐deficient mice, we show that Nrf2 prolongs SC proliferation by up‐regulating MyoD and suppresses SC differentiation by down‐regulating myogenin. Moreover, we show that IL‐6 and HGF, both factors that facilitate SC activation, induce Nrf2 activity in myoblasts. Thus, Nrf2 activity promotes muscle regeneration by modulating SC proliferation and differentiation and thereby provides implications for tissue regeneration.Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Steep Increase in Myonuclear Domain Size During Infancy

We investigated whether myonuclear number increases in proportion to the increase in fiber size during maturational growth of skeletal muscle. Thoraco‐abdominal muscle tissue was obtained from twenty 6‐day to 15‐year‐old boys and girls during cardiothoracic surgery. Cross‐sections were stained by anti‐laminin for the basal lamina and by DAPI to identify nuclei. Basal lamina was traced on digital images to measure the fiber cross‐sectional area (FCSA). Nuclei located within the basal lamina were considered myonuclei if pax7‐negative and satellite cell nuclei if pax7‐positive. Samples of two children were excluded from analysis because of clear signs of hypoxia as shown by positive carbonic anhydrase IX staining. Linear regression showed that FCSA increased with age by 187 μm²·per annum (R² = 0.90; P < 0.001). Satellite cell density showed a dramatic decrease in the first months of life, but this was not accompanied by an increase in myonuclei per muscle fiber cross‐section. Till four years of age the number of myonuclei per muscle fiber cross‐section remained relatively constant but increased thereafter. Myonuclear domain size showed a steep increase during infancy and reached adult values in the young adolescent phase. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.     

A single bout of exercise activates skeletal muscle satellite cells during subsequent overnight recovery

Skeletal muscle satellite cell (SC) content has been reported to increase following a single bout of exercise. Data on muscle fibre type-specific SC content and/or SC activation status are presently lacking. The objective of the study was to determine the impact of a single bout of exercise on muscle fibre type-specific SC content and activation status following subsequent overnight recovery. Eight healthy men (age, 20 ± 1 years) performed a single bout of combined endurance- and resistance-type exercise. Muscle biopsies were collected before and immediately after exercise, and following 9 h of postexercise, overnight recovery. Muscle fibre type-specific SC and myonuclear content and SC activation status were determined by immunohistochemical analyses. Satellite cell activation status was assessed by immunohistochemical staining for both Delta-like homologue 1 (DLK1) and Ki-67. Muscle fibre size and fibre area per nucleus were greater in type II compared with type I muscle fibres (P < 0.05). At baseline, no differences were observed in the percentage of SCs staining positive for DLK1 and/or Ki67 between fibre types. No significant changes were observed in SC content following 9 h of postexercise, overnight recovery; however, the percentage of DLK1-positive SCs increased significantly during overnight recovery, from 22 ± 5 to 41 ± 5% and from 24 ± 6 to 51 ± 9% in the type I and II muscle fibres, respectively. No changes were observed in the percentage of Ki-67-positive SCs. A single bout of exercise activates both type I and II skeletal muscle fibre SCs within a single night of postexercise recovery, preceding the subsequent increase in SC content.     

Satellite cells express distinct patterns of myogenic proteins in immature skeletal muscle.

Satellite cells are the myogenic cells lying between the myofiber sarcolemma and basal lamina. The objective of this study was to determine the expression patterns of MyoD, myogenin, and Pax7 within the satellite cell population in the growing rat soleus and extensor digitorum longus (EDL) muscles. Secondly, the expression of the myogenic markers was also studied within the interstitial cell compartment and myonuclei. It was discovered that the soleus contained a higher number of Pax7, MyoD, or myogenin-positive nuclei compared with the EDL. Similarly, myogenin was expressed at a lower level in the myonuclei of the soleus compared with the EDL, and myogenin was expressed at a higher level in the interstitial compartment of the soleus compared with the EDL. When interstitial nuclei, myonuclei, and double-labeled nuclei were used in the estimate of the satellite cell population, it was discovered that approximately of 13% of the myofibers in a transverse section of the soleus muscle and 4.1% of EDL myofibers exhibit a labeled satellite cell nucleus. Overall, results from this study suggest that expression patterns of these markers vary predictably among muscles with different growth dynamics and phenotypic characteristics.     

Notch3 null mutation in mice causes muscle hyperplasia by repetitive muscle regeneration

Satellite cells are skeletal muscle stem cells responsible for growth, maintenance, and repair of postnatal skeletal muscle. Although several studies have demonstrated that Notch signaling plays a critical role in muscle regeneration through promoting proliferation and self-renewal of satellite cells, the function of Notch3 is yet to be elucidated. We analyzed muscle regeneration in Notch3-deficient mutant mice. We found a remarkable overgrowth of muscle mass in the Notch3-deficient mice but only when they suffered repetitive muscle injuries. Immunochemical analysis found that Notch3 was expressed in Pax7(+)/MyoD(-) quiescent satellite cells and also in Pax7(+)/MyoD(+)-activated satellite cells, but the expression was restricted to around half the population of each cell type. In Notch3-deficient mice, the number of sublaminar quiescent satellite cells was significantly increased compared with those in control mice. We also found that primary cultured myoblasts isolated from the Notch3-deficient mice proliferated faster than those from control mice. Analysis of cultured myofibers revealed that the number of self-renewing Pax7-positive satellite cells attached to the myofiber was increased in the Notch3-deficient mice when compared with control mice. The data obtained in this study suggested that Notch3 pathway might be distinct from Notch1 in muscle regeneration. Because overexpression of Notch3 activated the expression of Nrarp, a negative feedback regulator of Notch signaling, Notch3 might act as a Notch1 repressor by activating Nrarp.     

Adverse effect of the anabolic-androgenic steroid mesterolone on cardiac remodelling and lipoprotein profile is attenuated by aerobicz exercise training

Abuse of anabolic-androgenic steroids (AAS) for improving physical performance is associated with serious, sometimes fatal, adverse effects. The aim of the present work was to investigate the effects of AAS on the cardiac structure and the plasma lipoprotein profile isolated and in combination with exercise. Transgenic mice with a human lipaemic phenotype (expressing cholesteryl ester transfer protein on the LDL receptor knockout background) were used in this study. Sedentary and exercised mice (treadmill running, five times per week for 6 weeks) were treated with mesterolone (2 microg/g body weight) or vehicle (control-C) in the last 3 weeks. Four groups were compared: (i) exercise + mesterolone (Ex-M), (ii) exercise + vehicle (Ex-C), (iii) sedentary + mesterolone (Sed-M) and (iv) sedentary + vehicle (Sed-C). Arterial blood pressure and body mass increased in all groups along time, but Sed-M reached the highest values and Ex-C the lowest. Treatment with mesterolone increased total cholesterol, triglyceride, low-density lipoprotein cholesterol (LDL-c) and very LDL-c (VLDL-c) plasma levels. However, exercise blunted some of these deleterious effects by increasing high-density lipoprotein cholesterol and decreasing LDL-c, VLDL-c and triglycerides. Exercise training induced beneficial effects, such as physiological cardiomyocyte hypertrophy, increase in myocardial circulation and decrease in cardiac interstitium. However, mesterolone impaired such physiological gains and in addition increased troponin T plasma levels both in sedentary and exercised mice. Thus, while mesterolone induced pro-atherogenic lipoprotein profile and pathogenic cardiac hypertrophy, exercise counteracted these effects and modified favourably both the lipoprotein profile and the cardiac remodelling induced by mesterolone.     

Age-related changes in satellite cell proliferation by compensatory activation in rat diaphragm muscles

To investigate the age-related changes in satellite cell (SC) proliferation in vivo, we used a compensatory activation (CAC) model of the hemi-diaphragm muscle. Young (2-month), adult (14-month) and old (24-month) rats were randomly divided into control and CAC groups. In the CAC group, denervation surgery in the left hemi-diaphragm was performed to induce CAC of the right hemi-diaphragm. Six days after the surgery, the CAC diaphragm muscle was removed and separated into two blocks for immunohistochemical staining and real time RT-PCR procedures. The number of SCs in type I and IIa fibers were not affected significantly by the CAC in any age groups, but that in type IIx/b fibers was significantly increased in the young and adult groups. As compared to the age-matched control group, the Pax7 mRNA expression level was significantly higher in the young and adult CAC groups, but not in the old CAC group. These results may suggest that the mechanism of SC proliferation in type IIx/b fibers is impaired in aged diaphragm muscles.     

Ageing influences myonuclear domain size differently in fast and slow skeletal muscle of rats

Aim: In multinucleated skeletal muscle, a myonuclear domain is the region of cytoplasm governed by one nucleus, and myofibres are mosaics of overlapping myonuclear domains. Association of ageing and myonuclear domain is important in the understanding of sarcopenia and with prevention or combating age‐related muscle declines. This study examined the effects of age, fibre type and muscle on nucleo‐cytoplasmic (N/C) relationships as reflecting myonuclear domain size. Methods: The N/C was compared in fibre types of soleus and plantaris muscles from young (n = 6) and ageing (n = 8) male Fisher 344 rats. Results: There were no significant differences in fibre type composition or cross‐sectional area of the soleus across ages. The old soleus had significantly more myonuclei, resulting in a significantly smaller myonuclear domain size. The plantaris muscle showed a higher percentage of slow fibres in old compared with young fibres. There were no differences in the number of myonuclei or in myonuclear domain size between young and older animals. Conclusion: We found muscle‐specific differences in the effects of ageing on myonuclear domain, possibly as a result of reduced efficiency of the myonuclei in the slow muscles.     

Cellular mechanisms and local progenitor activation to regulate skeletal muscle mass

Skeletal muscle hypertrophy is a result of increased load, such as functional and stretch-overload. Activation of satellite cells and proliferation, differentiation and fusion are required for hypertrophy of overloaded skeletal muscles. On the contrary, a dramatic loss of skeletal muscle mass determines atrophy settings. The epigenetic changes involved in gene regulation at DNA and chromatin level are critical for the opposing phenomena, muscle growth and atrophy. Physiological properties of skeletal muscle tissue play a fundamental role in health and disease since it is the most abundant tissue in mammals. In fact, protein synthesis and degradation are finely modulated to maintain an appropriate muscle mass. When the molecular signaling is altered muscle wasting and weakness occurred, and this happened in most common inherited and acquired disorders such as muscular dystrophies, cachexia, and age-related wasting. To date, there is no accepted treatment to improve muscle size and strength, and these conditions pose a considerable anxiety to patients as well as to public health. Several molecules, including Magic-F1, myostatin inhibitor, IGF, glucocorticoids and microRNAs are currently investigated to interfere positively in the blueprint of skeletal muscle growth and regeneration.     

The majority of multipotent epidermal stem cells do not protect their genome by asymmetrical chromosome segregation

The maintenance of genome integrity in stem cells (SCs) is critical for preventing cancer formation and cellular senescence. The immortal strand hypothesis postulates that SCs protect their genome by keeping the same DNA strand throughout life by asymmetrical cell divisions, thus avoiding accumulation of mutations that can arise during DNA replication. The in vivo relevance of this model remains to date a matter of intense debate. In this study, we revisited this long-standing hypothesis, by analyzing how multipotent hair follicle (HF) SCs segregate their DNA strands during morphogenesis, skin homeostasis, and SC activation. We used three different in vivo approaches to determine how HF SCs segregate their DNA strand during cell divisions. Double-labeling studies using pulse-chase experiments during morphogenesis and the first adult hair cycle showed that HF SCs incorporate two different nucleotide analogs, contradictory to the immortal strand hypothesis. The co-segregation of DNA and chromatin labeling during pulse-chase experiments demonstrated that label retention in HF SCs is rather a mark of relative quiescence. Moreover, DNA labeling of adult SCs, similar to labeling during morphogenesis, also resulted in label retention in HF SCs, indicating that chromosome segregation occurs randomly in most of these cells. Altogether, our results demonstrate that DNA strand segregation occurs randomly in the majority of HF SCs during development, tissue homeostasis, and following SC activation. Disclosure of potential conflicts of interest is found at the end of this article.     

Analysis of human muscle stem cells reveals a differentiation‐resistant progenitor cell population expressing Pax7 capable of self‐renewal

Studies using mouse models have established a critical role for resident satellite stem cells in skeletal muscle development and regeneration, but little is known about this paradigm in human muscle. Here, using human muscle stem cells, we address their lineage progression, differentiation, migration, and self‐renewal. Isolated human satellite cells expressed α7‐integrin and other definitive muscle markers, were highly motile on laminin substrates and could undergo efficient myotube differentiation and myofibrillogenesis. However, only a subpopulation of the myoblasts expressed Pax7 and displayed a variable lineage progression as measured by desmin and MyoD expression. Analysis identified a differentiation‐resistant progenitor cell population that was Pax7⁺/desmin^? and capable of self‐renewal. This study extends our understanding of the role of Pax7 in regulating human satellite stem cell differentiation and self‐renewal. Developmental Dynamics 238:138–149, 2009. © 2008 Wiley‐Liss, Inc.     

Prevention of muscle aging by myofiber-associated satellite cell transplantation

Skeletal muscle is dynamic, adapting to environmental needs, continuously maintained, and capable of extensive regeneration. These hallmarks diminish with age, resulting in a loss of muscle mass, reduced regenerative capacity, and decreased functionality. Although the mechanisms responsible for this decline are unclear, complex changes within the local and systemic environment that lead to a reduction in regenerative capacity of skeletal muscle stem cells, termed satellite cells, are believed to be responsible. We demonstrate that engraftment of myofiber-associated satellite cells, coupled with an induced muscle injury, markedly alters the environment of young adult host muscle, eliciting a near-lifelong enhancement in muscle mass, stem cell number, and force generation. The abrogation of age-related atrophy appears to arise from an increased regenerative capacity of the donor stem cells, which expand to occupy both myonuclei in myofibers and the satellite cell niche. Further, these cells have extensive self-renewal capabilities, as demonstrated by serial transplantation. These near-lifelong, physiological changes suggest an approach for the amelioration of muscle atrophy and diminished function that arise with aging through myofiber-associated satellite cell transplantation.     

Changes in skeletal muscle size, fibre-type composition and capillary supply after chronic venous occlusion in rats

Aim: We have previously shown that surgical occlusion of some veins from skeletal muscle results in muscle hypertrophy without mechanical overloading in the rat. The present study investigated the changes in muscle‐fibre composition and capillary supply in hypertrophied muscles after venous occlusion in the rat hindlimb. Methods: Sixteen male Wistar rats were randomly assigned into two groups: (i) sham operated (sham‐operated group; n = 7); (ii) venous occluded for 2 weeks (2‐week‐occluded group; n = 9). At the end of the experimental period, specimens of the plantaris muscle were dissected from the hindlimbs and subjected to biochemical and histochemical analyses. Results: Two weeks after the occlusion, both the wet weight of plantaris muscle relative to body weight and absolute muscle weight showed significant increases in the 2‐week‐occluded group (～15%) when compared with those in the sham‐operated group. The concentrations of muscle glycogen and lactate were higher in the 2‐week‐occluded group, whereas staining intensity of muscle lipid droplets was lower in the 2‐week‐occluded group than those in the sham‐operated group. The percentage of type I muscle fibre decreased, whereas that of type IIb fibre increased in the 2‐week‐occluded group when compared with the sham‐operated group. Although the expression of vascular endothelial growth factor‐188 mRNA increased, the number of capillaries around the muscle fibres tended to decrease (P = 0.07). Conclusion: Chronic venous occlusion causes skeletal muscle hypertrophy with fibre‐type transition towards faster types and changes in contents of muscle metabolites.