Satellite cell proliferation in low frequency-stimulated fast muscle of hypothyroid rat

Satellite cell proliferation was assessed inlow-frequency-stimulated hypothyroid rat fast-twitch muscle by5-bromo-2'-deoxyuridine (BrdU) labeling and subsequent staining oflabeled muscle nuclei, and by staining for proliferating cell nuclearantigen (PCNA). BrdU labeling and PCNA staining were highly correlatedand increased approximately fourfold at 5 days of stimulation, decayedthereafter, but remained elevated over control in 10- and 20-daystimulated muscles. Myogenin mRNA was ~4-fold elevated at 5 days and1.5-fold at 10 days. Staining for myogenin protein yielded resultssimilar to that for PCNA and BrdU. Furthermore, a detailed examination of the pattern of myogenin staining revealed that the number of myogenin-positive nuclei was elevated in the fast pure IIB fiber population at 5 and 10 days of chronic low-frequencystimulation. By 20 days, myogenin staining was observed intransforming fast fibers that coexpressed embryonic and adult myosinheavy chain isoforms. In the slower fiber populations (i.e., IIA andI), myogenin-positive transforming fibers that coexpressed embryonicmyosin heavy chain, appeared already at 5 days. Thus the satellite cellprogeny on slower fibers seemed to proliferate less and to fuse earlierto their associated fibers than the satellite cell progeny on fast fibers. We suggest that the increase in muscle nuclei of the fast fibers might be a prerequisite for fast-to-slow fiber type transitions.

     

Mechanisms leading to restoration of muscle size with exercise and transplantation after spinal cord injury

We have shown thatcycling exercise combined with fetal spinal cord transplantationrestored muscle mass reduced as a result of complete transection of thespinal cord. In this study, mechanisms whereby this combinedintervention increased the size of atrophied soleus and plantarismuscles were investigated. Rats were divided into five groups(n = 4, per group): control, nontransected; spinal cordtransected at T10 for 8 wk (Tx); spinal cord transected for 8 wk andexercised for the last 4 wk (TxEx); spinal cord transected for 8 wkwith transplantation of fetal spinal cord tissue into the lesion site 4 wk prior to death (TxTp); and spinal cord transected for 8 wk,exercised for the last 4 wk combined with transplantation 4 wk prior todeath (TxExTp). Tx soleus and plantaris muscles were decreased in sizecompared with control. Exercise and transplantation alone did notrestore muscle size in soleus, but exercise alone minimized atrophy inplantaris. However, the combination of exercise and transplantationresulted in a significant increase in muscle size in soleus andplantaris compared with transection alone. Furthermore, myofibernuclear number of soleus was decreased by 40% in Tx and was notaffected in TxEx or TxTp but was restored in TxExTp. A strongcorrelation (r = 0.85) between myofiber cross-sectional area and myofiber nuclear number was observed in soleus, but not inplantaris muscle, in which myonuclear number did not change with any ofthe experimental manipulations. 5'-Bromo-2'-deoxyuridine-positive nuclei inside the myofiber membrane were observed in TxExTp soleus muscles, indicating that satellite cells had divided and subsequently fused into myofibers, contributing to the increase in myonuclear number. The increase in satellite cell activity did not appear to becontrolled by the insulin-like growth factors (IGF), as IGF-I andIGF-II mRNA abundance was decreased in Tx soleus and plantaris, and wasnot restored with the interventions. These results indicate that,following a relatively long postinjury interval, exercise andtransplantation combined restore muscle size. Satellite cell fusion andrestoration of myofiber nuclear number contributed to increased musclesize in the soleus, but not in plantaris, suggesting that cellularmechanisms regulating muscle size differ between muscles with differentfiber type composition.

     

Changes in proliferation,differentiation, fibroblast growth factor 2 responsiveness and expression of syndecan‐4 and glypican‐1 with turkey satellite cell age

Myogenic satellite cells are heterogeneous multipotential stem cells that are required for muscle repair, maintenance, and growth. The membrane‐associated heparan sulfate proteoglycans syndecan‐4 and glypican‐1 differentially regulate satellite cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) signal transduction, and expression of the myogenic regulatory factors MyoD and myogenin. The objective of the current study was to determine the effect of age on syndecan‐4 and glypican‐1 satellite cell populations, proliferation, differentiation, FGF2 responsiveness, and expression of syndecan‐4, glypican‐1, MyoD, and myogenin using satellite cells isolated from the pectoralis major muscle of 1‐day‐old, 7‐week‐old and 16‐week‐old turkeys. Proliferation was significantly reduced in the 16‐week‐old satellite cells, while differentiation was decreased in the 7‐week‐old and the 16‐week‐old cells beginning at 48 h of differentiation. Fibroblast growth factor 2 responsiveness was highest in the 1‐day‐old and 7‐week‐old cells during proliferation; during differentiation there was an age‐dependent response to FGF2. Syndecan‐4 and glypican‐1 satellite cell populations decreased with age, but syndecan‐4 and glypican‐1 were differentially expressed with age during proliferation and differentiation. MyoD and myogenin mRNA expression was significantly decreased in 16‐week‐old cells compared to the 1‐day‐old and 7‐week‐old cells. MyoD and myogenin protein expression was higher during proliferation in the 16‐week‐old cells and decreased with differentiation. These data demonstrate an age‐dependent effect on syndecan‐4 and glypican‐1 satellite cell subpopulations, which may be associated with age‐related changes in proliferation, differentiation, FGF2 responsiveness, and the expression of the myogenic regulatory factors MyoD and myogenin.     

New fiber formation in the interstitial spaces of rat skeletal muscle during postnatal growth.

The purpose of this study was to determine whether fiber hyperplasia occurs in the rat plantaris muscle during postnatal weeks 3-20. Total muscle fiber number, obtained via the nitric acid digestion method, increased by 28% during the early postnatal rapid growth phase (3-10 weeks), whereas the number of branched fibers was consistently low. Whole-muscle mitotic activity and amino acid uptake levels showed an inverse relationship to the increase in total fiber number. The expression of MyoD mRNA (RT-PCR) levels decreased from 3 to 20 weeks of age, as did the detection of anti-BrdU- and MyoD-positive cells in histological sections. Immunohistochemical staining patterns for MyoD, myogenin, or developmental myosin heavy chain on sections stained for laminin (identification of the basal lamina) and electron micrographs clearly indicate that de novo fiber formation occurred in the interstitial spaces. Myogenic cells in the interstitial spaces were negative for the reliable specific satellite cell marker M-cadherin. In contrast, CD34 (an established marker for hematopoietic stem cells)-positive cells were located only in the interstitial spaces, and their frequency and location were similar to those of MyoD- and/or myogenin-positive cells. These findings are consistent with fiber hyperplasia occurring in the interstitial spaces of the rat plantaris muscle during the rapid postnatal growth phase. Furthermore, these data suggest that the new fibers may be formed from myogenic cells in the interstitial spaces of skeletal muscle and may express CD34 that is distinct from satellite cells.     

In vitro characterization of proliferation and differentiation of trout satellite cells

Fish satellite cells have been extracted from various species, but the myogenic characteristics of these cells in culture remain largely unknown. We show here that 60%-70% of the adherent cells are myogenic based on their immunoreactivity for the myogenic regulatory factor MyoD. In DMEM containing 10% fetal calf serum (FCS), trout myoblasts display rapid expression of myogenin (18% of myogenin-positive cells at day 2) combined with rapid fusion into myotubes (50% of myogenin-positive nuclei and 30% nuclei in myosin heavy chain [MyHC]-positive cells at day 7). These kinetics of differentiation are reminiscent of the behavior of fetal myoblasts in mammals. However, not all the myogenic cells differentiate; this subpopulation of cells might correspond to the previously named “reserve” cells. More than 90% of the BrdU-positive cells are also positive for MyoD, indicating that myogenic cells proliferate in vitro. By contrast, less than 1% of myogenin-positive cells are positive for BrdU suggesting that myogenin expression occurs only in post-mitotic cells. In order to maximize either the proliferation or the differentiation of cells, we have defined new culture conditions based on the use of a proliferation medium (F10+10%FCS) and a differentiation medium (DMEM+2%FCS). Three days after switching the medium, the differentiation index (% MyHC-positive nuclei) is 40-fold higher than that in proliferation medium, whereas the proliferation index (% BrdU-positive nuclei) is three-fold lower. Stimulation of cell proliferation by insulin-like growth factor 1 (IGF1), IGF2, and FGF2 is greater in F10 medium. The characterization of these extracted muscle cells thus validates the use of this in vitro system of myogenesis in further studies of the myogenic activity of growth factors in trout.     

The satellite cell in male and female, developing and adult mouse muscle: distinct stem cells for growth and regeneration

Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration.     

Changes in calpain and caspase gene expression at the mRNA level during bovine muscle satellite cell myogenesis and the correlation between the cell model and the muscle tissue

The calpain proteolytic system plays a central role in cell death and cell signaling. Caspases are a family of proteases implicated in apoptosis. The objective of this study was to explore the regulation and change trend of calpains (CAPN1 and CAPN3) and caspases (caspase-3, caspase-7, and caspase-9) expression at the mRNA level in Luxi cattle skeletal muscle satellite cells during proliferation and differentiation into myotubes. We also sought to assess whether there is a relationship between the muscle satellite cell model and skeletal muscle tissue. Satellite cells were isolated from longissimus dorsi muscle from Luxi cattle and cultured in vitro. Immunofluorescence was used to characterize satellite cells. Our study was divided into three groups: stage one, satellite cells proliferated at 50- and 80-% confluence; stage two, satellite cells differentiated at days 1, 3, 5, 7, and 15; stage three, not the satellite cells but the skeletal muscle tissue. Real-time PCR was used to quantify expression of calpains and the caspases at the mRNA level. These data demonstrated that CAPN1, CAPN3, CASP7, Myf5, and MyoG gene expression significantly increased from satellite cell proliferation to differentiation phases (P < 0.05). In contrast, CASP3 and CASP9 gene expression was significantly down-regulated during myogenesis (P < 0.05). Moreover, we put the CAPN1, CAPN3, CASP3, CASP7, CASP9, Myf5, and MyoG together to say that these genes expression had no significant correlation between the satellite cell model and the skeletal muscle tissue (P > 0.05). Here, we conclude that calpains (CAPN1 and CAPN3), caspases (caspase-3, caspase-7, and caspase-9), and Myf5 and MyoG all have important roles in satellite cell myogenesis. However, there is no relationship between the cell model and muscle tissue.     

Unloading of juvenile muscle results in a reduced muscle size 9 wk after reloading.

The role of satellite cells and DNA unit size in determining muscle size was examined by inhibiting postnatal skeletal muscle development by using hindlimb suspension. Satellite cell mitotic activity and DNA unit size were determined in the soleus muscles from hindlimb-suspended and age-matched weight-bearing rats before the initiation of hindlimb suspension, at the conclusion of a 28-day hindlimb-suspension period, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-suspended rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb suspension, but they were the same (P > 0. 05) as those of weight-bearing rats 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, nuclei per millimeter, and DNA unit size for the hindlimb-suspended rats were significantly (P < 0.05) smaller than for the weight-bearing rats at all recovery times. Satellite cell mitotic activity was significantly (P < 0.05) higher in the soleus muscles from hindlimb-suspended rats 2 wk after reloading, but it was the same (P > 0.05) as in weight-bearing rats 9 wk after reloading. Juvenile soleus muscles failed to achieve normal muscle size 9 wk after reloading because there was incomplete compensation for the hindlimb-suspension-induced interruptions in myonuclear accretion and DNA unit size expansion.     

The COX-2 pathway regulates growth of atrophied muscle via multiple mechanisms

Loss of muscle mass occurs with disease, injury, aging, and inactivity. Restoration of normal muscle mass depends on myofiber growth, the regulation of which is incompletely understood. Cyclooxygenase (COX)-2 is one of two isoforms of COX that catalyzes the synthesis of prostaglandins, paracrine hormones that regulate diverse physiological and pathophysiological processes. Previously, we demonstrated that the COX-2 pathway regulates early stages of myofiber growth during muscle regeneration. However, whether the COX-2 pathway plays a common role in adult myofiber growth or functions specifically during muscle regeneration is unknown. Therefore, we examined the role of COX-2 during myofiber growth following atrophy in mice. Muscle atrophy was induced by hindlimb suspension (HS) for 2 wk, followed by a reloading period, during which mice were treated with either the COX-2-selective inhibitor SC-236 (6 mg·kg^–1·day^–1) or vehicle. COX-2 protein was expressed and SC-236 attenuated myofiber growth during reloading in both soleus and plantaris muscles. Attenuated myofiber growth in the soleus was associated with both decreased myonuclear addition and decreased inflammation, whereas neither of these processes mediated the effects of SC-236 on plantaris growth. In addition, COX-2^–/– satellite cells exhibited impaired activation/proliferation in vitro, suggesting direct regulation of muscle cell activity by COX-2. Together, these data suggest that the COX-2 pathway plays a common regulatory role during various types of muscle growth via multiple mechanisms. cyclooxygenase-2; prostaglandins; myonuclear number; satellite cells; inflammation     

Growth and sex effects on the expression of syndecan-4 and glypican-1 in turkey myogenic satellite cell populations

The adult skeletal muscle stem cells, satellite cells, are responsible for skeletal muscle growth and regeneration. Satellite cells represent a heterogeneous cell population that differentially express cell surface markers. The membrane-associated heparan sulfate proteoglycans, syndecan-4, and glypican-1, are differentially expressed by satellite cells during the proliferation and differentiation stages of satellite cells. However, how the population of syndecan-4- or glypican-1-positive satellite cells changes during proliferation and differentiation, and how sex and muscle growth potential affect the expression of these genes is unknown. Differences in the amount of satellite cells positive for syndecan-4 or glypican-1 would affect the process of proliferation and differentiation which would impact both muscle mass accretion and the regeneration of muscle. In the current study, the percentage of satellite cells positive for syndecan-4 or glypican-1 from male and female turkeys from a Randombred Control Line 2 and a line (F) selected for increased 16-week body weight were measured during proliferation and differentiation. Growth selection altered the population of syndecan-4- and glypican-1-positive satellite cells and there were sex differences in the percentage of syndecan-4- and glypican-1-positive satellite cells. This study provides new information on dynamic changes in syndecan-4- and glypican-1-positive satellite cells showing that they are differentially expressed during myogenesis and growth selection and sex affects their expression.     

Gene expression patterns of the fibroblast growth factors and their receptors during myogenesis of rat satellite cells.

Satellite cells are the myogenic precursors in postnatal muscle and are situated beneath the myofiber basement membrane. We previously showed that fibroblast growth factor 2 (FGF2, basic FGF) stimulates a greater number of satellite cells to enter the cell cycle but does not modify the overall schedule of a short proliferative phase and a rapid transition to the differentiated state as the satellite cells undergo myogenesis in isolated myofibers. In this study we investigated whether other members of the FGF family can maintain the proliferative state of the satellite cells in rat myofiber cultures. We show that FGF1, FGF4, and FGF6 (as well as hepatocyte growth factor, HGF) enhance satellite cell proliferation to a similar degree as that seen with FGF2, whereas FGF5 and FGF7 are ineffective. None of the growth factors prolongs the proliferative phase or delays the transition of the satellite cells to the differentiating, myogenin(+) state. However, FGF6 retards the rapid exit of the cells from the myogenin(+) state that routinely occurs in myofiber cultures. To determine which of the above growth factors might be involved in regulating satellite cells in vivo, we examined their mRNA expression patterns in cultured rat myofibers using RT-PCR. The expression of all growth factors, excluding FGF4, was confirmed. Only FGF6 was expressed at a higher level in the isolated myofibers and not in the connective tissue cells surrounding the myofibers or in satellite cells dissociated away from the muscle. By Western blot analysis, we also demonstrated the presence of FGF6 protein in the skeletal musle tissue. Our studies therefore suggest that the myofibers serve as the main source for the muscle FGF6 in vivo. We also used RT-PCR to analyze the expression patterns of the four tyrosine kinase FGF receptors (FGFR1-FGFR4) and of the HGF receptor (c-met) in the myofiber cultures. Depending on the time in culture, expression of all receptors was detected, with FGFR2 and FGFR3 expressed only at a low level. Only FGFR4 was expressed at a higher level in the myofibers but not the connective tissue cell cultures. FGFR4 was also expressed at a higher level in satellite cells compared to the nonmyogenic cells when the two cell populations were released from the muscle tissue and fractionated by Percoll density centrifugation. The unique localization patterns of FGF6 and FGFR4 may reflect specific roles for these members of the FGF signaling complex during myogenesis in adult skeletal muscle.     

Is the early postnatal rise of testosterone responsible for a later male pattern of growth hormone secretion in pigs?

Sexual differentiation in Placentalia consists of several consecutive steps during fetal, postnatal, and premature development. It is known from male rats that an elevation in testosterone synthesis is observable within 2 d of birth, which leads to a male pattern of growth hormone (GH) secretion with low base levels and high amplitudes compared with that in females. In the male pig, a transient rise in testosterone concentration occurs about 4 wk after birth, but it is unknown whether it results in a later male pattern of GH secretion. In this study, male pigs (sus scrofa) were castrated either at 1 wk of age (Group 1, n = 8) or at 6 wk of age (Group 2, n = 8). Blood was sampled daily via cephalic vein catheters between 17 and 29 wk of age and analyzed for testosterone, GH, insulin-like growth factor-1 (IGF-1), and urea. High-frequency blood sampling (every 20 min over 24 h) for determination of GH pulsatility was performed at ages 19 and 24 wk. Total fat content and protein synthesis were determined at age 25 wk and at slaughter, respectively. Comparing Groups 1 and 2, there were no differences in daily GH concentrations or pulsatile secretion patterns, but in both groups, mean GH levels and pulsatility decreased from Week 19 to Week 24. Consequently, IGF-1, protein synthesis, urea, and body fat showed no differences when comparing both groups. It is concluded that the postnatal rise of testicular steroidogenesis in male pigs is not responsible for the later male pattern of GH secretion.     

Apoptosis in capillary endothelial cells in ageing skeletal muscle

The age‐related loss of skeletal muscle mass and function (sarcopenia) is a consistent hallmark of ageing. Apoptosis plays an important role in muscle atrophy, and the intent of this study was to specify whether apoptosis is restricted to myofibre nuclei (myonuclei) or occurs in satellite cells or stromal cells of extracellular matrix (ECM). Sarcopenia in mouse gastrocnemius muscle was characterized by myofibre atrophy, oxidative type grouping, delocalization of myonuclei and ECM fibrosis. Terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL) indicated a sharp rise in apoptosis during ageing. TUNEL coupled with immunostaining for dystrophin, paired box protein‐7 (Pax7) or laminin‐2α, respectively, was used to identify apoptosis in myonuclei, satellite cells and stromal cells. In adult muscle, apoptosis was not detected in myofibres, but was restricted to stromal cells. Moreover, the age‐related rise in apoptotic nuclei was essentially due to stromal cells. Myofibre‐associated apoptosis nevertheless occurred in old muscle, but represented < 20% of the total muscle apoptosis. Specifically, apoptosis in old muscle affected a small proportion (0.8%) of the myonuclei, but a large part (46%) of the Pax7⁺ satellite cells. TUNEL coupled with CD31 immunostaining further attributed stromal apoptosis to capillary endothelial cells. Age‐dependent rise in apoptotic capillary endothelial cells was concomitant with altered levels of key angiogenic regulators, perlecan and a perlecan domain V (endorepellin) proteolytic product. Collectively, our results indicate that sarcopenia is associated with apoptosis of satellite cells and impairment of capillary functions, which is likely to contribute to the decline in muscle mass and functionality during ageing.     

The depletion of skeletal muscle satellite cells with age is concomitant with reduced capacity of single progenitors to produce reserve progeny

Satellite cells are myogenic progenitors that reside on the myofiber surface and support skeletal muscle repair. We used mice in which satellite cells were detected by GFP expression driven by nestin gene regulatory elements to define age-related changes in both numbers of satellite cells that occupy hindlimb myofibers and their individual performance. We demonstrate a reduction in satellite cells per myofiber with age that is more prominent in females compared to males. Satellite cell loss also persists with age in myostatin-null mice regardless of increased muscle mass. Immunofluorescent analysis of isolated myofibers from nestin-GFP/Myf5^nLacZ/+ mice reveals a decline with age in the number of satellite cells that express detectable levels of βgal. Nestin-GFP expression typically diminishes in primary cultures of satellite cells as myogenic progeny proliferate and differentiate, but GFP subsequently reappears in the Pax7⁺ reserve population. Clonal analysis of sorted GFP⁺ satellite cells from hindlimb muscles shows heterogeneity in the extent of cell density and myotube formation among colonies. Reserve cells emerge primarily within high-density colonies, and the number of clones that produce reserve cells is reduced with age. Thus, satellite cell depletion with age could be attributed to a reduced capacity to generate a reserve population.