No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
The Transition from Proliferation to Differentiation Is Delayed in Satellite Cells from Mice Lacking MyoD
Abstract
Satellite cells from adult rat muscle coexpress proliferating cell nuclear antigen and MyoD upon entry into the cell cycle, suggesting that MyoD plays a role during the recruitment of satellite cells. Moreover, the finding that muscle regeneration is compromised in MyoD-/- mice, has provided evidence for the role of MyoD during myogenesis in adult muscle. In order to gain further insight into the role of MyoD during myogenesis in the adult, we compared satellite cells from MyoD-/- and wildtype mice as they progress through myogenesis in single-myofiber cultures and in tissue-dissociated cell cultures (primary cultures). Satellite cells undergoing proliferation and differentiation were traced immunohistochemically using antibodies against various regulatory proteins. In addition, an antibody against the mitogen-activated protein kinases ERK1 and ERK2 was used to localize the cytoplasm of the fiber-associated satellite cells regardless of their ability to express specific myogenic regulatory factor proteins. We show that during the initial days in culture the myofibers isolated from both the MyoD-/- and the wildtype mice contain the same number of proliferating, ERK+ satellite cells. However, the MyoD-/- satellite cells continue to proliferate and only a very small number of cells transit into the myogenin+ state, whereas the wildtype cells exit the proliferative compartment and enter the myogenin+ stage. Analyzing tissue-dissociated cultures of MyoD-/- satellite cells, we identified numerous cells whose nuclei were positive for the Myf5 protein. In contrast, quantification of Myf5+ cells in the wildtype cultures was difficult due to the low level of Myf5 protein present. The Myf5+ cells in the MyoD-/- cultures were often positive for desmin, similar to the MyoD+ cells in the wildtype cultures. Myogenin+ cells were identified in the MyoD-/- primary cultures, but their appearance was delayed compared to the wildtype cells. These "delayed" myogenin+ cells can express other differentiation markers such as MEF2A and cyclin D3 and fuse into myotubes. Taken together, our studies suggest that the presence of MyoD is critical for the normal progression of satellite cells into the myogenin+, differentiative state. It is further proposed that the Myf5+/MyoD- phenotype may represent the myogenic stem cell compartment which is capable of maintaining the myogenic precursor pool in the adult muscle.
... In tissue samples from ST, the mRNA expression of Pax7 and Myf5 was similar over all groups, whereas the expression level of MyoD was markedly reduced in samples from both LC and PCS piglets compared with HCs. A large number of differentiation-related genes, e.g., creatine kinase, acetylcholine receptor, MyoG, the cyclindependent kinase inhibitor p21, and myosin light chain, are MyoD targets and can be negatively affected by MyoD repression (31,(64)(65)(66)(67)(68). Thus, lower MyoD expression in LC and PCS groups means that all piglets from affected litters have an increased risk of developing a visible splay leg phenotype due to regulatory disorders at various levels of the differentiation process. ...
... Indeed, we found low levels of MyoD and MyoG, a higher proportion of proliferative Pax7 + /Ki67 + progenitor cells, and increased apoptotic activity in muscle samples from PCS piglets. These results are in accord with those found in studies with MyoD − or p21-deficient mice, indicating delayed myoblast-to-myocyte transition (66,75,78). In fact, our in vitro assays with isolated cells confirmed delayed differentiation kinetics in the PCS group. ...
... This is in accordance with data from others showing that lowering of MyoD is associated with Myf5 upregulation (79) and that reduction of Pax7 has no effect on Myf5. Most importantly, Myf5 can activate MyoG directly, whereas activation of myogenesis by Pax7 is MyoD-dependent (25,66). Therefore, Myf5 can partly compensate for MyoD in myogenesis regulation. ...
Introduction
Porcine congenital splay leg syndrome (PCS) is a major birth defect in piglets, resulting in lameness and high mortality rates. The multifactorial pathogenesis of PSC is not well understood but includes a polygenic inheritance.
Methods
Here, in addition to morphological investigations, we characterized the expression of myogenic genes and functional (proliferation and differentiation) properties of myogenic precursor/satellite cells (SATCs) in 1 day-old PCS piglets, non-affected littermates (LCs), and piglets from PCS-free healthy litters (HCs). In addition, PCS phenotypes were related to the SNP Homer1_rs325197091 within the Homer1 locus, which has been identified as a potential hereditary cause of PCS.
Results and discussion
Samples from musculus semitendinosus (ST) of PCS piglets had a higher proportion of type II fibers, reflecting myofiber immaturity. In addition, myofiber atrophy, a lower number of myonuclei per fiber (ST), and a higher apoptotic activity (in ST and longissimus dorsi muscle; LD) were found in the PCS group. A higher proportion of cycling committed myoblasts (Pax7⁺/Ki67⁺ cells) occurred in samples from PCS-affected piglets, and on the other hand, the mRNA expression of genes involved in differentiation (muscle differentiation 1; MyoD, myogenin; MyoG) was repressed compared with HCs. Cultured SATCs from PCS-affected animals showed a temporal shift in peak expression of Pax7, MyoD, and MyoG toward days 3 and 4 of their 7 days differentiation regime. In vitro experiments with isolated SATCs confirmed the lower differentiation potential and the delayed progression of the myogenic processes in cells from piglets with PCS phenotype. In addition, Pax7 and desmin were differently expressed in Homer1_rs325197091 genotype variants (GG, GA, and AA). Both genes showed the lowest expression in the homozygous AA-variant, which was most frequently found in PCS-affected animals. The homozygous AA-variant was also associated with lower expression of the truncated Homer1-subtype 205. Thus, we hypothesize that in PCS, the balance between Homer1 proteins and its signaling functions is changed in a way detrimental to the myogenic differentiation program. Our results demonstrated direct negative effects of the Homer1 AA genotype on Pax7 expression, but the exact mode of action still needs to be elucidated.
... Cells in which no further MRFs are produced-and in which MyoD1 and Myf5 levels are decreased-revert to the pool of quiescent cells [32]. It is believed that MyoD1 expression determines MPC differentiation [33]. ...
... In another study, another team of researchers showed that MyoD1 −/− muscles have branched muscle fibers, indicating an abnormal regeneration process within these myofibers [36]. These abnormalities may result from an imbalance between the satellite cells that stop proliferating, returning to the G0 phase of the cell cycle, and those that rapidly divide [33][34][35]. ...
... As an example of the dominance of MyoD1 factor expression, Myf5 −/− myoblasts showed early cell differentiation [21]. In contrast, the behavior of MyoD1 −/− myoblasts-in which increased proliferation and differentiation occurred with a significant delay-may serve as an example of a program path that is presumably followed by cells with Myf5 overexpression [32,33]. ...
The population of satellite cells (mSCs) is highly diversified. The cells comprising it differ
in their ability to regenerate their own population and differentiate, as well as in the properties they exhibit. The heterogeneity of this group of cells is evidenced by multiple differentiating markers that enable their recognition, classification, labeling, and characterization. One of the main tasks of satellite cells is skeletal muscle regeneration. Myofibers are often damaged during vigorous exercise in people who participate in sports activities. The number of satellite cells and the speed of the regeneration processes that depend on them affect the time structure of an athlete’s training. Thisprocess depends on inflammatory cells. The multitude of reactions and pathways that occur during the regeneration process results in the participation and control of many factors that are activated and secreted during muscle fiber damage and at different stages of its regeneration. However, not all of them are well understood yet. This paper presents the current state of knowledge on satellite cells dependent skeletal muscle regeneration. Studies describing the effects of various forms of exercise and age on this process were reviewed.
... Although it is now well established that satellite cells are essential for muscle regeneration (McCarthy et al., 2011;Murphy et al., 2011;Sambasivan et al., 2011), the mechanisms by which myogenic programming is established and maintained, and the functions of Myod1 (MyoD) and Myf5 in satellite cell determination and differentiation, remain unknown. Satellite cells lacking either MyoD or Myf5 exhibit relatively mild differentiation and growth defects and remain stably committed to the myogenic fate (Gayraud-Morel et al., 2007;Megeney et al., 1996;Starkey et al., 2011;Ustanina et al., 2007;Yablonka-Reuveni et al., 1999). Determining whether this reflects genetic redundancy between these MRF family members, as in the embryo (Kassar-Duchossoy et al., 2004;Rudnicki et al., 1993), or the engagement of alternative or compensatory regulatory pathways in muscle regeneration, requires analyses of mice lacking both MRFs. ...
... Using conditional mutagenesis, we demonstrated an absolute requirement for either MyoD or Myf5 in skeletal muscle regeneration. Whereas mutations in either MyoD or Myf5 cause comparatively modest regeneration and stem cell deficits (Asakura et al., 2007;Cornelison et al., 2000;Gayraud-Morel et al., 2007;Megeney et al., 1996;Sabourin et al., 1999;Schuierer et al., 2005;Ustanina et al., 2007;Yablonka-Reuveni et al., 1999), satellite cells homozygousnull for both MRFs exhibited profound stem cell dysfunction, resulting in the complete failure of injured skeletal muscle to regenerate. As the great majority of activated satellite cells express both MyoD and Myf5 (Cornelison and Wold, 1997;Zammit et al., 2002), genetic redundancy between these factors likely reflects functional compensatory activities within individual satellite cells rather than regulative behavior of distinct MyoD-or Myf5-dependent populations. ...
... Although either MyoD or Myf5 is sufficient to support regeneration, satellite cell defects are much more pronounced in MyoD mutant mice (Asakura et al., 2007;Cornelison et al., 2000;Gayraud-Morel et al., 2007;Megeney et al., 1996;Sabourin et al., 1999;Schuierer et al., 2005;Ustanina et al., 2007;Yablonka-Reuveni et al., 1999). The differential capacity of these MRFs to support regeneration was also shown here under conditions of reduced gene dosage in which satellite cells carried only a single functional allele of either MyoD or Myf5. ...
MyoD and Myf5 are fundamental regulators of skeletal muscle lineage determination in the embryo, and their expression is induced in satellite cells following muscle injury. MyoD and Myf5 are also expressed by satellite cell precursors developmentally, although the relative contribution of historical and injury-induced expression to satellite cell function is unknown. We show that satellite cells lacking both MyoD and Myf5 (double knockout [dKO]) are maintained with aging in uninjured muscle. However, injured muscle fails to regenerate and dKO satellite cell progeny accumulate in damaged muscle but do not undergo muscle differentiation. dKO satellite cell progeny continue to express markers of myoblast identity, although their myogenic programming is labile, as demonstrated by dramatic morphological changes and increased propensity for non-myogenic differentiation. These data demonstrate an absolute requirement for either MyoD or Myf5 in muscle regeneration and indicate that their expression after injury stabilizes myogenic identity and confers the capacity for muscle differentiation.
... [16][17][18][19][20][21] In addition, MyoD plays a unique role in adult muscle regeneration, as reported by the delayed onset of myogenic differentiation and increased propensity for self-renewal in MyoD knockout (KO) mice, suggesting that MYOD regulates the balance between self-renewal and differentiation during regeneration. [22][23][24][25] Furthermore, MyoD KO mice on a mdx background, a model of Duchenne muscular dystrophy, shows poor survival and die around the age of 12 months 22 MYOD has also been used as an activation marker for MuSCs during myogenic development and regeneration. The combination of MYOD and PAX7 immunostaining is frequently used to define the divergent fates of MuSCs during myogenesis and culture. ...
... 22,25 Therefore, we interpret these data to suggest that the MyoD-tdTomato low population expressing high levels of Pax7 and Myf5 contains a rare intermediate state between quiescent and activated MuSCs. 23,25,54 Our whole transcriptome data will be a great database to decipher the new molecular mechanism for a small unique subset of self-renewing MuSCs and to determine their fate in future studies. We found that the MyoD-tdTomato low population expresses genes associated with endothelial cell development. ...
Myoblast determination protein 1 (MyoD) dynamics define the activation status of muscle stem cells (MuSCs), aiding in muscle tissue regeneration after injury. However, the lack of experimental platforms to monitor MyoD dynamics in vitro and in vivo has hampered the investigation of fate determination and heterogeneity of MuSCs. Herein, we report a MyoD knock-in (MyoD-KI) reporter mouse expressing tdTomato at the endogenous MyoD locus. Expression of tdTomato in MyoD-KI mice recapitulated the endogenous MyoD expression dynamics in vitro and during the early phase of regeneration in vivo. Additionally, we showed that tdTomato fluorescence intensity defines MuSC activation status without immunostaining. Based on these features, we developed a high-throughput screening system to assess the effects of drugs on the behavior of MuSCs in vitro. Thus, MyoD-KI mice are an invaluable resource for studying the dynamics of MuSCs, including their fate decisions and heterogeneity, and for drug screening in stem cell therapy.
... MyoD, a key transcription factor and the most investigated MRF, is present in proliferating SCs. In the absence of MyoD, SCs showed an increased propensity for self-renewal rather than differentiation, which results in a deficit in muscle regeneration [31][32][33]. Myostatin, a growth and differentiation factor, is a negative regulator of myogenesis, and it was shown as an inhibitor of myoblast differentiation by down-regulating the expression of MyoD [34], mediated through Smad3 (mothers against decapentaplegic homolog 3). A subset of activated SCs downregulates MyoD and does not differentiate, maintaining an inactive state almost identical with the quiescence, process depending on Sprouty1 [21]. ...
... A subset of activated SCs downregulates MyoD and does not differentiate, maintaining an inactive state almost identical with the quiescence, process depending on Sprouty1 [21]. Myostatin is increased in aged muscle [33], so it is explicable that muscle mass decreases with the inhibition of cellular differentiation through myostatin/Smad3/MyoD signaling pathway. ...
In aged muscle, satellite cells’ symmetric and asymmetric divisions are impaired, and intrinsic and extrinsic complex mechanisms govern these processes. This review presents many updated aspects regarding muscle stem cells’ fate in normal and aging conditions. The balance between self-renewal and commitment divisions contributes to muscle regeneration, muscle homeostasis, aging, and disease. Stimulating muscle regeneration in aging could be a therapeutic target, but there is still a need to understand the many mechanisms that influence each other in satellite cells and their niche. We highlight here the general outlines regarding satellite cell divisions, the primary markers present in muscle stem cells, the aging aspects concerning signaling pathways involved in symmetric/asymmetric divisions, the regenerative capacity of satellite cells and their niche alteration in senescent muscle, genetics and epigenetics mechanisms implied in satellite cells aging and exercise effect on muscle regeneration in the elderly.
... 15 Satellite cell numbers increase when quiescent, nonproliferative satellite cells that express the Pax7 transcription factor become activated to a proliferative population that expresses Pax7 and the transcription factor MyoD. MyoD plays a central role in regulating the early stages of muscle differentiation. [16][17][18] Those Pax7+/MyoD+ cells can continue to proliferate or they can return to a Pax7+/MyoDquiescent state, or they can withdraw from the cell cycle and express myogenin. 19 Myogenin, also a transcription factor, regulates the terminal differentiation of myogenic cells and their fusion into mature muscle fibers. ...
... RNA quality was determined by the clear separation of 28S and 18S ribosomal RNA by electrophoresis. Two micrograms of total RNA were reverse transcribed with Super Script Reverse Transcriptase II (Invitrogen, Waltham, MA, USA) using Oligo(dT) [12][13][14][15][16][17][18] Primers (Invitrogen, Waltham, MA, USA) for product extension. cDNA was used to measure the expression for the genes of interest using SYBR Green qPCR Master Mix (Bio-Rad, Hercules, CA, USA) or iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). ...
Modulating the number of muscle stems cells, called satellite cells, during early postnatal development produces long‐term effects on muscle growth. We tested the hypothesis that high expression levels of the anti‐aging protein Klotho in early postnatal myogenesis increase satellite cell numbers by influencing the epigenetic regulation of genes that regulate myogenesis. Our findings show that elevated klotho expression caused a transient increase in satellite cell numbers and slowed muscle fiber growth, followed by a period of accelerated muscle growth that leads to larger fibers. Klotho also transcriptionally downregulated the H3K27 demethylase Jmjd3, leading to increased H3K27 methylation and decreased expression of genes in the canonical Wnt pathway, which was associated with a delay in muscle differentiation. In addition, Klotho stimulation and Jmjd3 downregulation produced similar but not additive reductions in the expression of Wnt4, Wnt9a, and Wnt10a in myogenic cells, indicating that inhibition occurred through a common pathway. Together, our results identify a novel pathway through which Klotho influences myogenesis by reducing the expression of Jmjd3, leading to reductions in the expression of Wnt genes and inhibition of canonical Wnt signaling.
... However, in this study TRF did not increase MyoD expression in senescent myoblasts as has been observed on its mRNA expression [15], suggesting that MyoD protein expression did not correlate completely with its gene expression. MyoD has an important regulatory role in initiating myogenic differentiation [38,40]. Defects in differentiation and at the proliferation-differentiation transition were observed in satellite cells derived from adult MyoD -/mice [40]. ...
... MyoD has an important regulatory role in initiating myogenic differentiation [38,40]. Defects in differentiation and at the proliferation-differentiation transition were observed in satellite cells derived from adult MyoD -/mice [40]. Thus, increased MyoD expression in young myoblasts with TRF treatment at day 1 and day 3 of differentiation induction observed in this study revealed the effectiveness of TRF in promoting the differentiation process in myoblasts. ...
Introduction
Replicative senescence results in dysregulation of cell proliferation and differentiation, which plays a role in the regenerative defects observed during age-related muscle atrophy. Vitamin E is a well-known antioxidant, which potentially ameliorates a wide range of age-related manifestations. The aim of this study was to determine the effects of tocotrienol-rich fraction (TRF) in modulating the expression of proliferation- and differentiation-associated proteins in senescent human myoblasts during the differentiation phase.
Material and methods
Human skeletal muscle myoblasts were cultured until senescence. Young and senescent cells were treated with TRF for 24 h before and after differentiation induction, followed by evaluation of cellular morphology and efficiency of differentiation. Expression of cell proliferation marker Ki67 protein and myogenic regulatory factors MyoD and myogenin were determined.
Results
Our findings showed that treatment with TRF significantly improved the morphology of senescent myoblasts. Promotion of differentiation was observed in young and senescent myoblasts with TRF treatment as shown by the increased fusion index and larger size of myotubes. Increased Ki67 and myogenin expression with TRF treatment was also observed in senescent myoblasts, suggesting amelioration of the myogenic program by TRF during replicative senescence.
Conclusions
TRF modulates the expression of regulatory factors related to proliferation and differentiation in senescent human myoblasts and could be beneficial for ameliorating the regenerative defects during aging.
keywords:
tocotrienols, replicative senescence, myoblasts, myotubes, differentiation
... Having established that Staufen1 can interact with MyoD transcript in quiescent MuSCs and limit its translation, we asked whether this process has any impact on MuSC function in vivo. Analysis of MyoD knockout mice showed that MuSCs without MyoD are slower to divide compared with wild-type cells (31,32). We therefore asked whether the Staufen1-MyoD axis impacts the propensity of MuSCs to break quiescence and begin proliferating. ...
... Altogether, these observations suggest that MyoD can drive MuSCs into the cell cycle and trigger their differentiation program. Consistently, MyoD null cells are slower to enter the cell cycle and have a differentiation defect in vitro (31,32). ...
Significance
This work addresses a fundamental mechanism for the translational control of a master regulator of myogenic differentiation, MyoD, by the RNA binding protein Staufen1. We show that muscle stem cells express the MyoD transcript in the quiescent state in vivo but block its translation through direct repression by Staufen1. Loss of this translational repression leads to MyoD translation and cell cycle entry, highlighting a novel role for MyoD in regulating the exit from quiescence. This mechanism of direct translational repression enables the cells to exist poised for activation and cell cycle entry. These data provide insight in the translational control of muscle stem cell quiescence.
... 56 Research has shown that RA can block AKT activity, stimulating mRNA translation by phosphorylating the translation initiation repressor eIF4EBP1. 31,44 In this study, we found that RA + RARγ treatment of cells decreased AKT phosphorylated protein levels and eIF4EBP1 phosphorylated protein levels and blocked MYOD protein synthesis. The AKT activator SC79 treatment of RA + RARγ group cells promoted MYOD protein synthesis, rescued the cell proliferation inhibitory effect caused by RA + RARγ, it also significantly improved cell differentiation and formed more multinucleated myotubes, thereby enhancing myogenesis. ...
Vitamin A is an essential nutrient in animals, playing important roles in animal health. In the pig industry, proper supplementation of vitamin A in the feed can improve pork production performance, while deficiency or excessive intake can lead to growth retardation or disease. However, the specific molecular mechanisms through which vitamin A operates on pig skeletal muscle growth as well as muscle stem cell function remain unexplored. Therefore, in this study, we isolated the pig primary skeletal muscle stem cells (pMuSCs) and treated with retinoic acid (RA), the natural metabolite of vitamin A, and then examined the myogenic capacity of pMuSCs via immunostaining, real-time PCR, CCK8 and western-blot analysis. Unexpectedly, the RA caused a significant decrease in the proliferation and differentiation of pMuSCs. Mechanistically, the RA addition induced the activation of retinoic acid receptor gamma (RARγ), which inhibited the myogenesis through the blockage of protein translation of the master myogenic regulator myogenic differentiation 1 gene (MYOD). Specifically, RARγ inactivate AKT kinase (AKT) signalling and lead to dephosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (eIF4EBP1), which in turn repress the eukaryotic translation initiation factor 4E (eIF4E) complex and block mRNA translation of MYOD. Inhibition of AKT could rescue the myogenic defects of RA-treated pMuSCs. Our findings revealed that retinoid acid signalling inhibits the skeletal muscle stem cell proliferation and differentiation in pigs. Therefore, the vitamin A supplement in the feedstuff should be cautiously optimized to avoid the potential adverse consequences on muscle development associated with the excessive levels of retinoic acid.
... Myogenic progenitor cells, often called myoblasts, are highly proliferative cells characterized by the expression of myogenic regulatory factors (MRFs), especially MYOD, which is crucial for adult muscle regeneration [32,33]. Indeed, MYOD knockout (KO) mice show a delay in the onset of myogenic differentiation and an increased propensity for selfrenewal, suggesting its role in the balance between self-renewal and differentiation during regeneration [34][35][36][37]. In this context, Fujita, R. and colleagues have recently published that SCs with a low expression level of MYOD are undifferentiated. ...
Skeletal muscle regeneration is a complex process involving the generation of new myofibers after trauma, competitive physical activity, or disease. In this context, adult skeletal muscle stem cells, also known as satellite cells (SCs), play a crucial role in regulating muscle tissue homeostasis and activating regeneration. Alterations in their number or function have been associated with various pathological conditions. The main factors involved in the dysregulation of SCs’ activity are inflammation, oxidative stress, and fibrosis. This review critically summarizes the current knowledge on the role of SCs in skeletal muscle regeneration. It examines the changes in the activity of SCs in three of the most common and severe muscle disorders: sarcopenia, muscular dystrophy, and cancer cachexia. Understanding the molecular mechanisms involved in their dysregulations is essential for improving current treatments, such as exercise, and developing personalized approaches to reactivate SCs.
... A previous study also reported that the overexpression of Pgc-1α, a gene related to mitochondrial biosynthesis, had no effect on MyoD expression and MB proliferation [29], which is consistent with our observation. In addition, MyoD expression is upregulated in proliferating SCs whereas MyoD-deficient cells exhibit a high proliferative potential and a strong ability to form SCs [30,31]. Hence, the change in MyoD expression caused by Ndufs8 expression may be independent of the characteristics of proliferating and self-renewing cells. ...
Skeletal muscle comprises different muscle fibers, including slow- and fast-type muscles, and satellite cells (SCs), which exist in individual muscle fibers and possess different myogenic properties. Previously, we reported that myoblasts (MBs) from slow-type enriched soleus (SOL) had a high potential to self-renew compared with cells derived from fast-type enriched tibialis anterior (TA). However, whether the functionality of myogenic cells in adult muscles is attributed to the muscle fiber in which they reside and whether the characteristics of myogenic cells derived from slow- and fast-type fibers can be distinguished at the genetic level remain unknown. Global gene expression analysis revealed that the myogenic potential of MBs was independent of the muscle fiber type they reside in but dependent on the region of muscles they are derived from. Thus, in this study, proteomic analysis was conducted to clarify the molecular differences between MBs derived from TA and SOL. NADH dehydrogenase (ubiquinone) iron-sulfur protein 8 (Ndufs8), a subunit of NADH dehydrogenase in mitochondrial complex I, significantly increased in SOL-derived MBs compared with that in TA-derived cells. Moreover, the expression level of Ndufs8 in MBs significantly decreased with age. Gain- and loss-of-function experiments revealed that Ndufs8 expression in MBs promoted differentiation, self-renewal, and apoptosis resistance. In particular, Ndufs8 suppression in MBs increased p53 acetylation, followed by a decline in NAD/NADH ratio. Nicotinamide mononucleotide treatment, which restores the intracellular NAD ⁺ level, could decrease p53 acetylation and increase myogenic cell self-renewal ability in vivo. These results suggested that the functional differences in MBs derived from SOL and TA governed by the mitochondrial complex I-encoding gene reflect the magnitude of the decline in SC number observed with aging, indicating that the replenishment of NAD ⁺ is a possible approach for improving impaired cellular functions caused by aging or diseases.
... Mice that are null for either MyoD or Myf5 are viable, and therefore, it is widely believed that these two proteins can functionally substitute for one another during embryonic development (Le Grand and Rudnicki, 2007). However, this compensation may not be as efficient in the regeneration of muscle because cultures of MyoD / myoblasts, which apparently have 5-10 times more Myf5 protein, are dramatically delayed in their transition to a differentiated state (Sabourin et al., 1999;Yablonka-Reuveni et al., 1999). Moreover, these myoblasts also appear to be limited in their ability to expand in population when compared with wild type (Montarras et al., 2000), and this may reflect the fact that Myf5 is less effective than MyoD in regulating the same set of growth-phase genes (Montarras et al., 2000;Ishibashi et al., 2005). ...
MyoD is a transcriptional factor that is required for the differentiation of muscle stem cells (satellite cells). In this study, we describe a previously unknown function for MyoD in regulating a gene (Cdc6) that is vital to endowing chromatin with the capability of replicating DNA. In C2C12 and primary mouse myoblasts, we show that MyoD can occupy an E-box within the promoter of Cdc6 and that this association, along with E2F3a, is required for its activity. MyoD and Cdc6 are both expressed after quiescent C2C12 myoblasts or satellite cells in association with myofibers are stimulated for growth, but MyoD appears at least 2–3 h earlier than Cdc6. Finally, knockdown of MyoD impairs the ability of C2C12 cells to express Cdc6 after leaving quiescence, and as a result, they cannot fully progress into S phase. Our results define a mechanism by which MyoD helps myogenic satellite cells to enter into the first round of DNA replication after transitioning out of quiescence.
... Since their initial discoveries, these cells are often referred to as "satellite" cells, the term coined by Mauro, owing to their peripheral "satellite" location between the sarcolemma and basal lamina of muscle fibers. It was later demonstrated that upon activation, these single cells can undergo cell division, particularly following a stimulus or insult such as injury or muscle damage from heavy resistance exercise, where they then migrate to the site of damage and fuse to the existing muscle fiber and contribute new myonuclei (15)(16)(17)(18)(19)(20)(21)(22)(23). Indeed, satellite cells are the main source of new myonuclei in muscle after exercise (24)(25)(26). ...
Skeletal muscle memory is an exciting phenomenon gaining significant traction across several scientific communities, amongst exercise practitioners and the public. Research has demonstrated that skeletal muscle tissue can be 'primed' by earlier positive encounters with exercise training that can enhance adaptation to later training, even following significant periods of exercise cessation or detraining. This review will describe and discuss the most recent research investigating the underlying mechanisms of skeletal muscle memory: 1) 'cellular' muscle memory and, 2) 'epigenetic' muscle memory as well as the emerging evidence of how these theories may work in synergy. We will discuss both 'positive' and 'negative' muscle memory and highlight the importance of investigating muscle memory for optimising exercise interventions and training programmes as well as the development of therapeutic strategies for counteracting muscle wasting conditions and age-related muscle loss. Finally, important directions emerging in the field will be highlighted to advance the next generation of studies in skeletal muscle memory research into the future.
... miR-16 overexpression prevents myogenic cell differentiation and myotube formation in vitro (34), whereas knockdown enhances these processes (34,40). MyoD contributes to myogenic cell differentiation (41)(42)(43)(44)(45)(46)(47)(48). miR-16 knockdown in murine myofiberassociated myogenic cell culture may increase the proportion of MyoD þ satellite cells by 3 days but reduces it by 5 days, pointing to an effect on myogenic cell behavior and fate (6). ...
microRNAs (miRs) control stem cell biology and fate. Ubiquitously expressed and conserved miR-16 was the first miR implicated in tumorigenesis. miR-16 is low in muscle during developmental hypertrophy and regeneration. It is enriched in proliferating myogenic progenitor cells but is repressed during differentiation. The induction of miR-16 blocks myoblast differentiation and myotube formation while knockdown enhances it. Despite a central role for miR-16 in myogenic cell biology, how it mediates its potent effects is incompletely defined. In this investigation, global transcriptomic and proteomic analyses after miR-16 knockdown in proliferating C2C12 myoblasts revealed how miR-16 influences myogenic cell fate. Eighteen hours after miR-16 inhibition, ribosomal protein gene expression levels were higher relative to control myoblasts and p53 pathway-related gene abundance was lower. At the protein level at this same timepoint, miR-16 knockdown globally upregulated TCA cycle proteins while downregulating RNA metabolism-related proteins. miR-16 inhibition induced specific proteins associated with myogenic differentiation such as ACTA2, EEF1A2, and OPA1. We extend prior work in hypertrophic muscle tissue and show that miR-16 is lower in mechanically overloaded muscle in vivo. Our data collectively point to how miR-16 is implicated in aspects of myogenic cell differentiation. A deeper understanding of the role of miR-16 in myogenic cells has consequences for muscle developmental growth, exercise-induced hypertrophy, and regenerative repair after injury, all of which involve myogenic progenitors.
... A low Pax7/MyoD ratio was suggested to induce the commitment of proliferating cells to differentiation (Olguin et al., 2007). Studies have shown that MPCs deficient for MyoD do not differentiate properly showing the essential role of this transcription factor in myogenic cell differentiation (Wood et al., 2013;Yablonka-Reuveni et al., 1999). While MyoD appears to be necessary for the early phase of differentiation commitment, the MRF myogenin is expressed at the end of the differentiation process to specifically trigger the expression of myogenic genes . ...
Idiopathic inflammatory myopathies (IIM) constitute a heterogeneous group of diseases defined by non-infectious nor toxic acquired myopathies. Amongst them, dermatomyositis (DM) is the most frequent IIM, occurring during child and adulthood, and is potentially life-threatening. Its diagnosis is based on the association of muscle weakness, inflammatory infiltrates on histological muscle sections and auto-antibodies in the serum. Etiopathogenesis, that has mainly focused on adaptative immunity, remains largely unknown. In DM, the necrosis/regeneration balance is detrimental to muscle integrity, with a chronic course and poor clinical outcome for some patients. Nevertheless, the capacity of muscle regeneration has been barely studied. In normal injured muscle, regeneration is achieved by myogenic precursor cells (MPCs) that undergo myogenesis process to build new myofibers. The aim of our project was to evaluate DM-derived MPC capacity to undertake myogenesis. Methods. After establishing specific selection criteria, I selected DM (severe [n=5] and mild [n=3]), and healthy controls (HC [n=5], sex and age paired) from the Hospices Civils de Lyon database and obtained corresponding samples from the hospital biobank. Supplemental investigation was performed on other IIM derived-MPCs: immune-mediated necrotizing myopathy (IMNM) (n=3), inclusion body myositis (IBM) (n=3), and anti-synthetase syndrome (ASS) (n=3). Myogenesis was explored in vitro using specific MPC culture for the evaluation of proliferation, differentiation and fusion. Proliferation was evaluated as the incorporation of a thymidine analog into DNA during proliferation. Terminal myogenic differentiation was evaluated by myogenin expression, an essential transcription factor of myogenic differentiation. Cell fusion was quantified as the number of nuclei incorporated into myotubes. Quantitative analysis was performed using Image J software, and statistical analysis was performed by mean comparison (t test or ANOVA). Similar methods were used to evaluate the impact of interferon blockers (JAK / STAT inhibitor) on myogenesis. Senescence was analyzed by flow cytometry with fluorogenic substrate of SA-βGalactosidase, a senescence-specific marker. Results. Analysis of MPC proliferation indicates that DM-derived MPCs exhibited a significantly lower proliferation than HC-derived MPCs (p=0.0020). Terminal myogenic differentiation was also strongly impaired in severe DM-derived MPCs while less altered in mild-DM-derived cells (p=0.001). MPC fusion was significantly altered in DM-derived MPCs with a dramatic decreased observed in severe DM and to a lesser degree in mild DM (p<0.0001 for each). These results were recapitulated for other IIM, with altered myogenesis identified for IMNM (n=3), IBM (n=3), but not for ASS (n=3). Through cellular and molecular investigations, we found increased senescence in DM derived-MPCs, and potential involvement of interferon type I in myogenesis impairment. Indeed, sDM and mDM derived-MPCs myogenesis was improved even restored when culture was performed with JAK/STAT inhibitor, a downstream interferon pathway blocker or IFN receptor blocking antibody. Conclusion. These results show that skeletal muscle regeneration is impaired in DM due to altered capacities of MPCs to proliferate, to differentiate and to fuse. This may explain in part the muscle alterations and muscle weakness that are observed in patients. These results support the hypothesis of intrinsic alterations of MPC properties in DM, potentially linked with interferon pathway, and participating to the pathogenesis of the disease
... When tissue is damaged or injured, SCs change to a proliferative state, which enables the production of a large cell pool suited for myogenic differentiation [35]. Our research shows that, similar to SCs from other species, SCs from horses exclusively express Pax7, MyoD1, and Desmin [36][37][38]. In the population of adult SCs, Pax7 is expressed in both quiescent and active conditions [39]. ...
Myostatin (MSTN), a member of the transforming growth factor-β superfamily, inhibits the activation of muscle satellite cells. However, the role and regulatory network of MSTN in equine muscle cells are not well understood yet. We discovered that MSTN knockdown significantly reduces the proliferation rate of equine muscle satellite cells. In addition, after the RNA sequencing of equine satellite cells transfected with MSTN-interference plasmid and control plasmid, an analysis of the differentially expressed genes was carried out. It was revealed that MSTN regulatory networks mainly involve genes related to muscle function and cell-cycle regulation, and signaling pathways, such as Notch, MAPK, and WNT. Subsequent real-time PCR in equine satellite cells and immunohistochemistry on newborn and adult muscle also verified the MSTN regulatory network found in RNA sequencing analysis. The results of this study provide new insight into the regulatory mechanism of equine MSTN.
... Satellite cells, the skeletal muscle stem cells, remain in a state of quiescence until their activation is triggered ( Figure 16). This will lead to an overexpression of MYOD and Myf5, that will set cells for differentiation into myoblasts [327][328][329] . This indicates that to maintain a good regeneration potential through the life of an individual, the preservation of the stem cell pool is critical 332,333 . ...
Almost half of the human genome derives from transposable elements (TE). Among them, the Long INterspersed Element-1 (LINE-1 or L1) forms the only currently active and autonomous transposable element family in humans. Although hundreds of thousands L1 copies are dispersed in the human genome, only 80-100 of them are still retrotransposition competent, i.e. able to replicate by a “copy-and-paste” mechanism via an RNA intermediate and a reverse transcription step. On the one hand, L1 activity can have deleterious consequences, such as insertional mutagenesis, and is tightly regulated at the transcriptional or post-transcriptional levels. However, specific host factors are necessary for completion of L1 replication cycle. When occurring in the germline or in the early embryo, L1 insertions can be transmitted to the next generation. Somatic retrotransposition has been also described in epithelial tumors and in the brain, both in neural progenitor cells and differentiated neurons. Nevertheless, the extent of L1 expression and mobilization in other somatic tissues remains unclear.Here, we investigated the activity of L1 retrotransposons in human and mouse skeletal muscle cells. We show that the most abundant L1 protein, ORF1p, which is essential to retrotransposition, is undetectable under our experimental conditions, in mouse or human muscle samples, while it is readily detected in cancer cells or in testis. Similarly, it was undetected in immortalized mouse or human myoblasts. However, we found that L1 is capable of retrotransposition in human and mouse myoblasts when expressed from a plasmid or from an integrated copy with a constitutive or inducible promoter, respectively. In conclusion, while L1 expression is under the limit of detection in muscle, myoblasts are permissive to retrotransposition, indicating that these cells express all the cellular factors necessary to achieve this process, and do not express significant restriction factors that would prevent retrotransposition.Altogether, our findings suggest that somatic L1 activity could not be confined to the brain or cancer cells, but could also occur in muscles under environmental or pathological conditions that would unleash L1 expression.
... Their differentiation produces myoblasts that can rebuild damaged fibers and their self-renewal to supplement the muscle stem cell bank for subsequent injury and repair [6]. Satellite cell activation is partly mediated by the induced expression of MyoD and Myf5 [42,43], and in our regenerating CNF we found down-regulated expression levels of MyoD, Myf5 and MyHC in 1,25(OH) 2 D deficient mice Therefore, our results indicate that 1,25(OH) 2 D deficiency inhibits the regeneration of skeletal muscle cells by inhibiting the proliferation and differentiation of skeletal muscle cells. ...
To determine if 1,25(OH)2D deficiency can induce age-related sarcopenia, the skeletal muscular phenotype of male wild-type (WT) and Cyp27b1 knockout (KO) mice were compared at 3 and 6 months of age. We found that muscle mass, grip strength and muscle fiber size were significantly decreased in aging Cyp27b1 KO male mice. The expression levels of genes related to mitochondrial metabolic activity, and antioxidant enzymes including SOD1, catalase, Nqo1 and Gcs were significantly down-regulated in skeletal muscle tissue of Cyp27b1 KO male mice; in contrast, the percentage of p16+ and p21+ myofibers, and the expression of p16, p19, p21, p53, TNFα, IL6 and MMP3 at mRNA and/or protein levels were significantly increased. We then injected tibialis anterior muscle of WT and Cyp27b1+/- male mice with BaCl2, and analyzed the regenerative ability of skeletal muscle cells 7 days later. The results revealed that the numbers of newly formed regenerating central nucleated fibers (CNF), the percentage of BrdU+ cells and the expression of MyoD, MyHC and Myf5 at mRNA levels were significantly down-regulated in the injured skeletal muscle tissue of Cyp27b1+/- mice. In summary, our studies indicate that 1,25(OH)2D deficiency can result in the development of age-related sarcopenia by inducing oxidative stress, skeletal muscular cell senescence and SASP, and by inhibiting skeletal muscle regeneration. Cyp27b1 KO mice can therefore be used as an animal model of age-related sarcopenia in order to investigate the pathogenesis of age-related sarcopenia and potentially to test intervention measures for treatment of sarcopenia.
... It is known that the proliferation of skeletal muscle stem cells was promoted through keeping MYOD expression at low levels (Conboy and Rando, 2002). MYOD deficiency in satellite cells caused them remaining in proliferative state (Yablonka-Reuveni et al., 1999). MYOD-null myoblasts were more resistant to apoptosis during proliferation (Asakura et al., 2007). ...
Identifying the genes relevant for muscle development is pivotal to improve meat production and quality in pigs. Insulin-degrading enzyme (IDE), a thiol zinc-metalloendopeptidase, has been known to regulate the myogenic process of mouse and rat myoblast cell lines, while its myogenic role in pigs remained elusive. Therefore, the current study aimed to identify the effects of IDE on the proliferation and apoptosis of porcine skeletal muscle stem cells (PSMSCs) and underlying molecular mechanism. We found that IDE was widely expressed in porcine tissues, including kidney, lung, spleen, liver, heart, and skeletal muscle. Then, to explore the effects of IDE on the proliferation and apoptosis of PSMSCs, we subjected the cells to siRNA-mediated knockdown of IDE expression, which resulted in promoted cell proliferation and reduced apoptosis. As one of key transcription factors in myogenesis, MYOD, its expression was also decreased with IDE knockdown. To further elucidate the underlying molecular mechanism, RNA sequencing was performed. Among transcripts perturbed by the IDE knockdown after, a downregulated gene myostatin (MSTN) which is known as a negative regulator for muscle growth attracted our interest. Indeed, MSTN knockdown led to similar results as those of the IDE knockdown, with upregulation of cell cycle-related genes, downregulation of MYOD as well as apoptosis-related genes, and enhanced cell proliferation. Taken together, our findings suggest that IDE regulates the proliferation and apoptosis of PSMSCs via MSTN/MYOD pathway. Thus, we recruit IDE to the gene family of regulators for porcine skeletal muscle development and propose IDE as an example of gene to prioritize in order to improve pork production.
... Early investigations of such cells concentrated mainly on the in vitro behaviour of the distinguishable cell types present in these populations, principally on their myogenic proclivities versus those favouring formation of fibrous scar tissue or fatty tissue. This approach had become progressively sophisticated by development of cellsorting techniques made available by the advent of a range of antigenic markers [42][43][44][45][46][47][48], supplemented, more recently, by in situ RNA hybridization to locate sites of expression of specific genes and by editing of specific genes to provide visualizable signals of their active expression [39,[49][50][51]. These advances have greatly enhanced the precision of cell identification and accelerated the overall aim of relating molecular and structural data to the clinical and pathological status of the muscle. ...
Careful quantitative analysis of histological preparations of muscle samples is crucial to accurate investigation of myopathies in man and of interpretation of data from animals subjected to experimental or potentially therapeutic treatments. Protocols for measuring cell numbers are subject to problems arising from biases associated with preparative and analytical techniques. Prominent among these is the effect of polarized structure of skeletal muscle on sampling bias. It is also common in this tissue to collect data as ratios to convenient reference dominators, the fundamental bases of which are ill-defined, or unrecognized or not accurately assessable. Use of such ‘floating’ denominators raises a barrier to estimation of the absolute values that assume practical importance in medical research, where accurate comparison between different scenarios in different species is essential to the aim of translating preclinical research findings in animal models to clinical utility in Homo sapiens. This review identifies some of the underappreciated problems with current morphometric practice, some of which are exacerbated in skeletal muscle, and evaluates the extent of their intrusiveness into the of building an objective, accurate, picture of the structure of the muscle sample. It also contains recommendations for eliminating or at least minimizing these problems. Principal among these, would be the use of stereological procedures to avoid the substantial counting biases arising from inter-procedure differences in object size and section thickness. Attention is also drawn to the distortions of interpretation arising from use of undefined or inappropriate denominators.
... 27 Experiments performed in a MyoD-null adult mouse showed that satellite cells have a lesser proliferative capacity and a delayed transition to myogenic differentiation even when they were cultured under myogenic conditions. 28 MyoD expression is low in undifferentiated myoblasts and increases during the early stages of the differentiation process. 26 It acts as a transcriptional factor that enhances the transcription of MyoG and MEF2C genes, among others. ...
Background
Hyperphosphatemia has been related to the development of sarcopenia in aging mice. We describe the intracellular mechanisms involved in the impairment of the myogenic differentiation promoted by hyperphosphatemia and analyse these mechanisms in the muscle from older mice.
Methods
C2C12 cells were grown in 2% horse serum in order to promote myogenic differentiation, in the presence or absence of 10 mM beta-glycerophosphate (BGP) for 7 days. Troponin T, paired box 7 (Pax-7), myogenic factor 5 (Myf5), myogenic differentiation 1 (MyoD), myogenin (MyoG), myocyte enhancer factor 2 (MEF2C), P300/CBP-associated factor (PCAF), histone deacetylase 1 (HDAC1), fibronectin, vimentin, and collagen I were analysed at 48, 72, and 168 h, by western blotting or by immunofluorescence staining visualized by confocal microscopy. Studies in mice were performed in 5- and 24-month-old C57BL6 mice. Three months before sacrifice, 21-month-old mice were fed with a standard diet or a low phosphate diet, containing 0.6% or 0.2% phosphate, respectively. Serum phosphate concentration was assessed by a colorimetric method and forelimb strength by a grip test. Fibrosis was observed in the tibialis anterior muscle by Sirius Red staining. In gastrocnemius muscle, MyoG, MEF2C, and fibronectin expressions were analysed by western blotting.
Results
Cells differentiated in the presence of BGP showed near five times less expression of troponin T and kept higher levels of Pax-7 than control cells indicating a reduced myogenic differentiation. BGP reduced Myf5 about 50% and diminished MyoD transcriptional activity by increasing the expression of HDAC1 and reducing the expression of PCAF. Consequently, BGP reduced to 50% the expression of MyoG and MEF2C. A significant increase in the expression of fibrosis markers as collagen I, vimentin, and fibronectin was found in cells treated with BGP. In mice, serum phosphate (17.24 ± 0.77 mg/dL young; 23.23 ± 0.81 mg/dL old; 19.09 ± 0.75 mg/dL old with low phosphate diet) correlates negatively (r = −0.515, P = 0.001) with the muscular strength (3.13 ± 0.07 gf/g young; 1.70 ± 0.12 gf/g old; 2.10 ± 0.09 gf/g old with low phosphate diet) and with the expression of MyoG (r = −0.535, P = 0.007) and positively with the expression of fibronectin (r = 0.503, P = 0.001) in gastrocnemius muscle. The tibialis anterior muscle from old mice showed muscular fibrosis. Older mice fed with a low phosphate diet showed improved muscular parameters relative to control mice of similar age.
Conclusions
Hyperphosphatemia impairs myogenic differentiation, by inhibiting the transcriptional activity of MyoD, and enhances the expression of fibrotic genes in cultured myoblasts. Experiments carried out in older mice demonstrate a close relationship between age-related hyperphosphatemia and the decrease in the expression of myogenic factors and the increase in factors related to muscle fibrosis.
... During the immediate post-hatch period, muscle growth is dependent on the adult myoblast satellite cells fusing with existing myofibers, which leads to muscle growth through hypertrophy (Stockdale and Holtzer 1961;Moss and Leblond 1971). The satellite cells require the expression of myogenic transcriptional regulatory factors, such as myogenic determination factor 1 (MYOD1) and myogenin (MYOG), for their proliferation and differentiation (Brunetti and Goldfine 1990;Yablonka-Reuveni et al. 1999). One of the transmembrane extracellular matrix heparan sulphate proteoglycans, syndecan-4 (SDC4), plays an essential role in muscle growth and development through regulating satellite cell proliferation, migration and differentiation (Velleman et al. 2007;Shin et al. 2013). ...
1. The objective of this study was to identify the effects of the antioxidant alpha-tocopherol acetate (ATA) and alpha lipoic acid (ALA) which have anti-inflammatory effects on developmental onset, severity and the progression of wooden breast (WB) based on Pectoralis major (P. major) muscle morphology and expression of genes associated with WB during the first three weeks post-hatch.
2. A total of 160 newly hatched Ross 708 broiler chicks were randomly assigned in a replicated trial to either a control group or three dietary treatments (ATA 160 mg/kg feed, ALA 500 mg/kg feed or in combination).
3. Microscopic changes associated with WB began at one week of age in all groups. The ATA acetate and ALA fed in combination decreased WB severity at two weeks age (P=0.05) and ATA alone or in combination reduced severity at three weeks of age compared to the control group (P=0.05). Expression of myogenic determination factor 1 and peroxisome proliferator-activated receptor gamma was reduced in all dietary treatments compared to the control at three weeks of age (P≤ 0.05), which suggested reduced muscle degeneration and lipid deposition.
4. ATA and ALA fed both independently and in combination had a positive effect on mitigating WB severity microscopically as early as two weeks age.
... It is known that the proliferation of skeletal muscle stem cells was promoted through keeping MyoD expression at low levels [26]. MyoD de ciency in satellite cells caused them remaining in proliferative state [27]. MyoD-null myoblasts were more resistant to apoptosis during proliferation [28]. ...
Background: Identifying the genes relevant for muscle development is pivotal to improve meat production and quality in pigs. Insulin-degrading enzyme (IDE), a thiol zinc-metalloendopeptidase, has been known to regulate the myogenic process of mouse and rat myoblast cell lines, while its myogenic role in pigs remained elusive. Therefore, the current study aimed to identify the effects of IDE on the proliferation and apoptosis of porcine skeletal muscle stem cells and underlying molecular mechanism.
Results: We found in the present study that IDE was widely expressed in porcine tissues, including kidney, lung, spleen, liver, heart, and skeletal muscle. Then, to explore the effects of IDE on the proliferation and apoptosis of porcine skeletal muscle stem cells, we subjected the cells to siRNA-mediated knockdown of IDE expression, which resulted in promoted cell proliferation and reduced apoptosis. As one of key transcription factors in myogenesis, MYOD, its expression was also decreased with IDE knockdown. To further elucidate the underlying molecular mechanism, RNA sequencing was performed. Among transcripts perturbed by the IDE knockdown after, a down-regulated gene myostatin (MSTN) which is known as a negative regulator for muscle growth attracted our interest. Indeed, MSTN knockdown led to similar results as those of the IDE knockdown, with upregulation of cell cycle-related genes, downregulation of MYOD as well as apoptosis-related genes, and enhanced cell proliferation.
Conclusion: Our findings suggest that IDE regulates the proliferation and apoptosis of porcine skeletal muscle stem cells through MSTN/MYOD pathway. Thus, we recruit IDE to the gene family of regulators for porcine skeletal muscle development, and propose IDE as an example of gene to prioritize in order to improve pork production.
... Activated MuSCs are now poised to rapidly expand their population through continued cell cycle progression in response to environmental signaling cues in the damaged muscle. As the MuSCs start to accumulate, PAX7 and MYF5 expression becomes repressed while MYOG becomes expressed to drive cell cycle exit and form myocytes (PAX7 --FOXO -MYF5 -MYOD1 + MYOG + ) ( Fig. 1) [13,18]. Finally, the formation of multinucleated myofibers results in the decreased expression of MYOD1 while MYF6/MRF4 becomes highly expressed in the functional muscle fiber [19][20][21]. ...
In response to muscle injury, muscle stem cells integrate environmental cues in the damaged tissue to mediate regeneration. These environmental cues are tightly regulated to ensure expansion of muscle stem cell population to repair the damaged myofibers while allowing repopulation of the stem cell niche. These changes in muscle stem cell fate result from changes in gene expression that occur in response to cell signaling from the muscle environment.
Integration of signals from the muscle environment leads to changes in gene expression through epigenetic mechanisms. Such mechanisms, including post-translational modification of chromatin and nucleosome repositioning, act to make specific gene loci more, or less, accessible to the transcriptional machinery. In youth, the muscle environment is ideally structured to allow for coordinated signaling that mediates efficient regeneration. Both age and disease alter the muscle environment such that the signaling pathways that shape the healthy muscle stem cell epigenome are altered. Altered epigenome reduces the efficiency of cell fate transitions required for muscle repair and contributes to muscle pathology. However, the reversible nature of epigenetic changes holds out potential for restoring cell fate potential to improve muscle repair in myopathies.
In this review, we will describe the current knowledge of the mechanisms allowing muscle stem cell fate transitions during regeneration and how it is altered in muscle disease. In addition, we provide some examples of how epigenetics could be harnessed therapeutically to improve regeneration in various muscle pathologies.
... For instance, the loss of MyoD in vivo can trigger an up-regulation of Myf5 expression that can efficiently compensate and support myogenesis (25,27,38). In comparison, myogenic defects in primary cultures of mouse MyoD KO myoblasts were pronounced, although low-level fusions were still observed (27,(54)(55)(56). Our results showed that deletion of MyoD in human myoblasts did not affect the expression level of MYF5, yet it also failed to safeguard a myogenic program for MyoD KO myoblasts. ...
Myoblast fusion is essential for formations of myofibers, the basic cellular and functional units of skeletal muscles. Recent genetic studies in mice identified two long-sought membrane proteins, Myomaker and Myomixer, which cooperatively drive myoblast fusion. It is unknown whether and how human muscles, with myofibers of tremendously larger size, use this mechanism to achieve multinucleations. Here, we report an interesting fusion model of human myoblasts where Myomaker is sufficient to induce low-grade fusion, while Myomixer boosts its efficiency to generate giant myotubes. By CRISPR mutagenesis and biochemical assays, we identified MyoD as the key molecular switch of fusion that is required and sufficient to initiate Myomixer and Myomaker expression. Mechanistically, we defined the E-box motifs on promoters of Myomixer and Myomaker by which MyoD induces their expression for multinucleations of human muscle cells. Together, our study uncovered the key molecular apparatus and the transcriptional control mechanism underlying human myoblast fusion.
... In myogenic differentiation, most satellite cells downregulate PAX7 and maintain MYOD expression to initiate myogenesis through upregulation of myogenin (Halevy et al., 2004;Zammit et al., 2004). MyoD-null mice exhibit reduced regeneration ability after muscle injury due to defects in population expansion and differentiation, indicating that MYOD plays an important role in the activation of satellite cells at the initial stages of regeneration (Cornelison et al., 2000;Megeney et al., 1996;Yablonka-Reuveni et al., 1999;Yamamoto et al., 2018). ...
Muscle satellite cells are normally quiescent but are rapidly activated following muscle damage. Here, we investigated whether damaged myofibers influence the activation of satellite cells. Our findings revealed that satellite cells are directly activated by damaged-myofiber-derived factors (DMDFs). DMDFs induced satellite cells to enter the cell cycle; however, the cells stayed at the G1 phase and did not undergo S phase, and these cells were reversible to the quiescent-like state. Proteome analysis identified metabolic enzymes, including GAPDH, as DMDFs, whose recombinant proteins stimulated the activation of satellite cells. Satellite cells pre-exposed to the DMDFs demonstrated accelerated proliferation ex vivo. Treatment with recombinant GAPDH prior to muscle injury promoted expansion of the satellite cell population in vivo. Thus, our results indicate that DMDFs are not only a set of biomarkers for muscle damage, but also act as moonlighting proteins involved in satellite cell activation at the initial step of muscle regeneration.
... The importance of MyoD in muscle regeneration is dem onstrated by the fact that it is required for efficient muscle regeneration (Megeney et al., 1996). Muscle regeneration is im paired in MyoD deficient mice and the activated satellite cells fail to exit the cell cycle and enter the differentiation program efficiently (Sabourin et al., 1999;Yablonka-Reuveni et al., 1999). A requirem ent for MyoD could occur during developm ent to establish the satellite cell or could be necessary only subsequent to its activation for entry into the myogenic pathw ay. ...
Urodele amphibians regenerate various structures including the limb, tail, lens, and heart. Underlying this regenerative ability may be the plasticity of cells at the site of injury. Muscle is a key tissue to study these issues because myogenesis proceeds by fusion of mononucleate precursor cells into a multinucleate syncytium which is in a state of stable post-mitotic arrest. Following implantation into a regenerating limb, newt myotubes re-enter the cell cycle and give rise to proliferating mononucleate progeny. In culture, generation of mononucleate cells has never been observed, but the myotube nuclei re-enter the cell cycle and arrest in G2. This is in contrast to their mammalian counterparts, which are refractory to growth factor stimulation, and suggests that cell cycle re-entry may be one aspect of myotube plasticity. In initial experiments mouse and newt myoblasts were fused to create hybrid myotubes. In these hybrids, DNA synthesis was observed in both mouse and newt nuclei demonstrating that the post-mitotic arrest of mammalian nuclei can be destabilised. Other experiments addressed the hypothesis that mononucleate cells are generated from multinucleate myotubes by fragmentation of the syncytium, as the nuclei progress through mitosis and cytokinesis. I pursued several strategies to overcome the G2 arrest of cultured myotubes. Transfection with SV-40 large T antigen induced endoreplication of DNA in myotubes, but did not lead to mitosis. Treatment with caffeine resulted in the appearance of fragmented nuclei, which are indicative of aberrant mitosis. This response was dependent on traversal of S-phase by the myotube nuclei. These observations suggested that the block to mitosis is stable in cultured myotubes. A critical question is whether cell cycle re-entry is required for generation of mononucleate cells following implantation of myotubes into regenerating limbs. Cell-cycle re-entry was blocked in myotubes by X-irradiation or expression of the cdk4/6 inhibitor pl6. These myotubes were fluorescently labelled and implanted. Both arrested and control myotubes gave rise to mononucleate progeny, demonstrating that cell cycle re-entry is not required for generation of mononucleate cells.
... In agreement with this idea, myoblasts isolated from MyoD -/mice have four-fold higher levels of Myf5, but show reduced differentiation potential (Sabourin et al. 1999). Furthermore, MyoD-deficient satellite cells resist differentiation and retain their proliferative status (Yablonka-Reuveni et al. 1999). This indicates that efficient progression through the myogenic program requires the coordinated action of Myf5 and MyoD to balancing commitment, proliferation, and differentiation. ...
... A decline in Pax7 expression along with the concomitant induction of MyoG is indicative of myoblasts that have entered terminal differentiation and initiated withdrawal from the cell cycle. Concurrent with the increase in MyoG expression, differentiating myoblasts also initiate transcription of various structural proteins, including actin and myosin, which facilitates the fusion of myoblasts into de novo myotubes or into existing myofibers (Smith et al. 1994, Yablonka-Reuveni et al. 1999, Andres and Walsh 1996. ...
... In addition, high level of helix-loop-helix muscle-specific transcription factor, MyoD, is involved in myogenic differentiation and SCs fusion with adult muscle fibers. Study proved that the murine MyoD −/− isolated satellite cells continue to proliferate and only a very small amount of cells transit into the myogenin-positive state, whereas the wild type cells exit the proliferative compartment and enter the myogenin-positive stage (20). Therefore, SCs expressing more MyoD than Myf5 confers for differentiation. ...
Space traveling is imperative for mankind in the future. Expectedly, hibernation will become an option for space traveler to overcome the endless voyage. With regard to some of the studies pointed out that during hibernation, muscle will undergo atrophy and meantime neurogenesis will reduce, these obstacles were frequently related with stem cell regeneration. Thus, investigation on whether hibernation will lead to dysfunction of stem cell becomes an important issue. By going through four main systems in this article, such as, hematopoietic system, skeletal muscle system, central nervous system and orthopedic system, we are expecting that stem cells regeneration capacity will be affected by hibernation. To date, these researches are majorly the read-out from short term or seasonal hibernating mammals. Proposing and creating a simulated long-term hibernation animal model is turning essential for the further investigation on the effect of longer period of hibernation to human stem cells.
... 7,75 It has been shown that the lack of MyoD delay proliferation to differentiation transition time. 112 If SCs maintain PAX7 expression and suppress MyoD level, then the progression toward differentiation is impeded. However, once SCs express Myogenin, it can directly suppress PAX7 expression and differentiation ensues, which suggesting a reciprocal inhibitory mechanism between MRFs and PAX7. ...
Prenatal and postnatal myogenesis share many cellular and molecular aspects. Myogenic regulatory factors are basic Helix-Loop-Helix transcription factors that indispensably regulate both processes. These factors (Myf5, MyoD, Myogenin, and MRF4) function as an orchestrating cascade, with some overlapped actions. Prenatally, myogenic regulatory factors are restrictedly expressed in somite-derived myogenic progenitor cells and their derived myoblasts. Postnatally, myogenic regulatory factors are important in regulating the myogenesis process via satellite cells. Many positive and negative regulatory mechanisms exist either between myogenic regulatory factors themselves or between myogenic regulatory factors and other proteins. Upstream factors and signals are also involved in the control of myogenic regulatory factors expression within different prenatal and postnatal myogenic cells. Here, the authors have conducted a thorough and an up-to-date review of the myogenic regulatory factors since their discovery 30 years ago. This review discusses the myogenic regulatory factors structure, mechanism of action, and roles and regulations during prenatal and postnatal myogenesis.
Impact statement
Myogenic regulatory factors (MRFs) are key players in the process of myogenesis. Despite a considerable amount of literature regarding these factors, their exact mechanisms of actions are still incompletely understood with several overlapped functions. Herein, we revised what has hitherto been reported in the literature regarding MRF structures, molecular pathways that regulate their activities, and their roles during pre- and post-natal myogenesis. The work submitted in this review article is considered of great importance for researchers in the field of skeletal muscle formation and regeneration, as it provides a comprehensive summary of all the biological aspects of MRFs and advances a better understanding of the cellular and molecular mechanisms regulating myogenesis. Indeed, attaining a better understanding of MRFs could be utilized in developing novel therapeutic protocols for multiple myopathies.
... This same redundancy of Myf5 and MyoD in development and muscle injury has now been reported in zebrafish (Hinits et al., 2009;Siegel et al., 2013), Drosophila (Abmayr and Keller, 1998) and Xenopus (Chanoine and Hardy, 2003), showing a high conservation of the MYOD/MYF5 transcriptional machinery in regulating muscle specification and development throughout evolution. These same MRFs are also required for self-renewal of adult muscle satellite cells (Cooper et al., 1999;Ustanina et al., 2007;Yablonka-Reuveni et al., 1999), yet roles for these factors in individually regulating muscle fate and self-renewal are now just emerging in the literature. For example, a subset of muscle progenitors are specified by MYOD without the contribution of MYF5 (Haldar et al., 2008(Haldar et al., , 2014. ...
Rhabdomyosarcoma (RMS) is a pediatric malignacy of muscle with myogenic regulatory transcription factors MYOD and MYF5 being expressed in this disease. Consensus in the field has been that expression of these factors likely reflects the target cell of transformation rather than being required for continued tumor growth. Here, we used a transgenic zebrafish model to show that Myf5 is sufficient to confer tumor-propagating potential to RMS cells and caused tumors to initiate earlier and have higher penetrance. Analysis of human RMS revealed that MYF5 and MYOD are mutually-exclusively expressed and each is required for sustained tumor growth. ChIP-seq and mechanistic studies in human RMS uncovered that MYF5 and MYOD bind common DNA regulatory elements to alter transcription of genes that regulate muscle development and cell cycle progression. Our data support unappreciated and dominant oncogenic roles for MYF5 and MYOD convergence on common transcriptional targets to regulate human RMS growth.
... Satellite cell activation plays an important role in muscular hypertrophy. We investigated the satellite cell activator HGF [30], Pax7 expression in resting satellite cells, which is required for satellite cell pool formation in skeletal muscles [31], and the proliferation/differentiation markers of satellite cells MyoD and Myogenin [32][33][34]. Unexpectedly, satellite cell activator and proliferation/differentiation-related factors were significantly increased in the T/T genotype than in the C/C genotype. It has recently been reported that satellite cells secrete soluble capillary blood vessel growth factor, which is associated with capillary angiogenesis [35,36]. ...
Variants of the Myostatin gene have been shown to have an influence on muscle hypertrophy phenotypes in a wide range of mammalian species. Recently, a Thoroughbred horse with a C-Allele at the g.66493737C/T single-nucleotide polymorphism (SNP) has been reported to be suited to short-distance racing. In this study, we examined the effect of the Myostatin SNP on muscle fiber properties in young Thoroughbred horses during a training period. To investigate the effect of the Myostatin SNP on muscle fiber before training, several mRNA expressions were relatively quantified in biopsy samples from the middle gluteal muscle of 27 untrained male Thoroughbred horses (1.5 years old) using real-time RT-PCR analysis. The remaining muscle samples were used for immunohistochemical analysis to determine the population and area of each fiber type. All measurements were revaluated in biopsy samples of the same horses after a 5-month period of conventional training. Although the expressions of Myostatin mRNA decreased in all SNP genotypes, a significant decrease was found in only the C/C genotype after training. While, expression of VEGFa, PGC1α, and SDHa mRNAs, which relate to the biogenesis of mitochondria and capillaries, was significantly higher (54-82%) in the T/T than the C/C genotypes after training. It is suggested that hypertrophy of muscle fiber is directly associated with a decrease in Myostatin mRNA expression in the C/C genotype, and that increased expressions of VEGFa, PGC1α, and SDHa in the T/T genotype might be indirectly caused by the Myostatin SNP.
... This emerging theme suggests that a function of the duplicated vertebrate MRF genes within fibres is robust regulation of muscle size. Although Myod is well known to promote MPC terminal differentiation (Kablar et al., 1997;Yablonka-Reuveni et al., 1999), our data are inconsistent with a simple delay in myogenesis in myod mutants. Fibre number is reduced, but does not recover when excess MPCs subsequently differentiate, suggesting that a critical window for fibre formation has been missed. ...
Balancing the number of stem cells and their progeny is crucial for tissue development and repair. Here we examine how cell numbers and overall muscle size are tightly regulated during zebrafish somitic muscle development. Muscle stem/precursor cell (MPCs) expressing Pax7 are initially located in the dermomyotome (DM) external cell layer, adopt a highly stereotypical distribution and thereafter a proportion of MPCs migrate into the myotome. Regional variations in the proliferation and terminal differentiation of MPCs contribute to growth of the myotome. To probe the robustness of muscle size control and spatiotemporal regulation of MPCs, we compared the behaviour of wild type (wt) MPCs with those in mutant zebrafish that lack the muscle regulatory factor Myod. Myod(fh261) mutants form one third fewer multinucleate fast muscle fibres than wt and show a significant expansion of the Pax7(+) MPC population in the DM. Subsequently, myod(fh261) mutant fibres generate more cytoplasm per nucleus, leading to recovery of muscle bulk. In addition, relative to wt siblings, there is an increased number of MPCs in myod(fh261) mutants and these migrate prematurely into the myotome, differentiate and contribute to the hypertrophy of existing fibres. Thus, homeostatic reduction of the excess MPCs returns their number to normal levels, but fibre numbers remain low. The GSK3 antagonist BIO prevents MPC migration into the deep myotome, suggesting that canonical Wnt pathway activation maintains the DM in zebrafish, as in amniotes. BIO does not, however, block recovery of the myod(fh261) mutant myotome, indicating that homeostasis acts on fibre intrinsic growth to maintain muscle bulk. The findings suggest the existence of a critical window for early fast fibre formation followed by a period in which homeostatic mechanisms regulate myotome growth by controlling fibre size. The feedback controls we reveal in muscle help explain the extremely precise grading of myotome size along the body axis irrespective fish size, nutrition and genetic variation and may form a paradigm for wider matching of organ size.
... Chronic resistance training promotes muscle anabolism via a complex interaction of multiple competing pathways (Marcotte et al., 2015). A number of myogenic regulatory factors including MyoD and MyoG are upregulated in individuals after chronic resistance exercise and are involved in myogenic remodeling and programming following exercise (Yablonka-Reuveni et al., 1999). Conversely, resistance exercise reduces expression of some genes which promote muscle degradation such as myostatin and atrogin1, while increasing the expression others such as MuRF1 (Zanchi et al., 2009;Fernandez-Gonzalo et al., 2013). ...
Powerlifters are the epitome of muscular adaptation and are able to generate extreme forces. The molecular mechanisms underpinning the significant capacity for force generation and hypertrophy are not fully elucidated. MicroRNAs (miRs) are short non-coding RNA sequences that control gene expression via promotion of transcript breakdown and/or translational inhibition. Differences in basal miR expression may partially account for phenotypic differences in muscle mass and function between powerlifters and untrained age-matched controls. Muscle biopsies were obtained from m. vastus lateralis of 15 national level powerlifters (25.1 ± 5.8 years) and 13 untrained controls (24.1 ± 2.0 years). The powerlifters were stronger than the controls (isokinetic knee extension at 60°/s: 307.8 ± 51.6 Nm vs. 211.9 ± 41.9 Nm, respectively P < 0.001), and also had larger muscle fibers (type I CSA 9,122 ± 1,238 vs. 4,511 ± 798 μm2p < 0.001 and type II CSA 11,100 ± 1,656 vs. 5,468 ± 1,477 μm2p < 0.001). Of the 17 miRs species analyzed, 12 were differently expressed (p < 0.05) between groups with 7 being more abundant in powerlifters and five having lower expression. Established transcriptionally regulated miR downstream gene targets involved in muscle mass regulation, including myostatin and MyoD, were also differentially expressed between groups. Correlation analysis demonstrates the abundance of eight miRs was correlated to phenotype including peak strength, fiber size, satellite cell abundance, and fiber type regardless of grouping. The unique miR expression profiles between groups allow for categorization of individuals as either powerlifter or healthy controls based on a five miR signature (miR-126, -23b, -16, -23a, -15a) with considerable accuracy (100%). Thus, this unique miR expression may be important to the characterization of the powerlifter phenotype.
Copper (Cu) is an essential trace element required for respiration, neurotransmitter synthesis, oxidative stress response, and transcriptional regulation. Imbalance in Cu homeostasis can lead to several pathological conditions, affecting neuronal, cognitive, and muscular development. Mechanistically, Cu and Cu-binding proteins (Cu-BPs) have an important but underappreciated role in transcription regulation in mammalian cells. In this context, our lab investigates the contributions of novel Cu-BPs in skeletal muscle differentiation using murine primary myoblasts. Through an unbiased synchrotron X-ray fluorescence-mass spectrometry (XRF/MS) metalloproteomic approach, we identified the murine cysteine rich intestinal protein 2 (mCrip2) in a sample that showed enriched Cu signal, which was isolated from differentiating primary myoblasts derived from mouse satellite cells. Immunolocalization analyses showed that mCrip2 is abundant in both nuclear and cytosolic fractions. Thus, we hypothesized that mCrip2 might have differential roles depending on its cellular localization in the skeletal muscle lineage. mCrip2 is a LIM-family protein with 4 conserved Zn2+-binding sites. Homology and phylogenetic analyses showed that mammalian Crip2 possesses histidine residues near two of the Zn2+-binding sites (CX2C-HX2C) which are potentially implicated in Cu+-binding and competition with Zn2+. Biochemical characterization of recombinant human hsCRIP2 revealed a high Cu+-binding affinity for two and four Cu+ ions and limited redox potential. Functional characterization using CRISPR/Cas9-mediated deletion of mCrip2 in primary myoblasts did not impact proliferation, but impaired myogenesis by decreasing the expression of differentiation markers, possibly attributed to Cu accumulation. Transcriptome analyses of proliferating and differentiating mCrip2 KO myoblasts showed alterations in mRNA processing, protein translation, ribosome synthesis, and chromatin organization. CUT&RUN analyses showed that mCrip2 associates with a select set of gene promoters, including MyoD1 and metallothioneins, acting as a novel Cu-responsive or Cu-regulating protein. Our work demonstrates novel regulatory functions of mCrip2 that mediate skeletal muscle differentiation, presenting new features of the Cu-network in myoblasts.
Organismal homeostasis and regeneration are predicated on committed stem cells that can reside for long periods in a mitotically dormant but reversible cell-cycle arrest state defined as quiescence. Premature escape from quiescence is detrimental, as it results in stem cell depletion, with consequent defective tissue homeostasis and regeneration. Here, we report that Polycomb Ezh1 confers quiescence to murine muscle stem cells (MuSCs) through a non-canonical function. In the absence of Ezh1, MuSCs spontaneously exit quiescence. Following repeated injuries, the MuSC pool is progressively depleted, resulting in failure to sustain proper muscle regeneration. Rather than regulating repressive histone H3K27 methylation, Ezh1 maintains gene expression of the Notch signaling pathway in MuSCs. Selective genetic reconstitution of the Notch signaling corrects stem cell number and re-establishes quiescence of Ezh1-/- MuSCs.
Somatic stem cells are tissue-specific reserve cells tasked to sustain tissue homeostasis in adulthood and/or effect tissue regeneration after traumatic injury. The stem cells of skeletal muscle tissue are the satellite cells, which were originally described and named after their localization beneath the muscle fiber lamina and attached to the multi-nucleated muscle fibers. During adult homeostasis, satellite cells are maintained in quiescence, a state of reversible cell cycle arrest. Yet, upon injury, satellite cells are rapidly activated, becoming highly mitotically active to generate large numbers of myoblasts that differentiate and fuse to regenerate the injured muscle fibers. A subset self-renews to replenish the pool of muscle stem cells.Complex intrinsic gene regulatory networks maintain the quiescent state of satellite cells, or upon injury, direct their activation, proliferation, differentiation and self-renewal. Molecular cues from the satellite cells' environment provide the essential information as to when and where satellite cells are to stay quiescent or break quiescence and effect regenerative myogenesis. Predominantly, these cues are secreted, diffusible or membrane-bound ligands that bind to and activate their specific cognate receptors on the satellite cell to activate downstream signaling cascades and elicit context-specific cell behavior. This review aims to offer a concise overview of major intercellular signaling pathways regulating satellite cells during quiescence and in injury-induced skeletal muscle regeneration.
In general, the concept of a mechanism in biology has three distinct meanings. It may refer to a philosophical thesis about the nature of life and biology, to the internal workings of a machine-like structure, or to the causal explanation of a particular phenomenon [1].
Understanding the biological mechanisms that justify acute and chronic physiological responses to exercise interventions determines the development of training principles and training methods. A strong understanding of the effects of exercise in humans may help
researchers to identify what causes specific biological changes and to properly identify the
most adequate processes for implementing a training stimulus [1].
Despite the significant body of knowledge regarding the physiological and physical effects of different training methods (based on load dimensions), some biological causes of those changes are still unknown. Additionally, few studies have focused on natural biological variability in humans and how specific human properties may underlie different responses to the same training intervention. Thus, more original research is needed to
provide plausible biological mechanisms that may explain the physiological and physical effects of exercise and training in humans.
In this Special Issue, we discuss/demonstrate the biological mechanisms that underlie the beneficial effects of physical fitness and sports performance, as well as their importance and their role in/influences on physical health.
A total of 28 manuscripts are published here, of which 25 are original articles, two are reviews, and one is a systematic review.
Two papers are on neuromuscular training programs (NMTs), training monotony (TM), and training strain (TS) in soccer players [2,3]; five articles provide innovative findings about testosterone and cortisol [4,5], gastrointestinal hormones [6], spirulina [7], and concentrations of erythroferrone (ERFE) [8]; another five papers analyze fitness and
its association with other variables [7,9–12]; three papers examine body composition in elite female soccer players [2], adolescents [6], and obese women [7]; five articles examines the effects of high-intensity interval training (HIIT) [7,10,13–15]; one paper examines the
acute effects of different levels of hypoxia on maximal strength, muscular endurance, and cognitive function [16]; another article evaluates the efficiency of using vibrating exercise equipment (VEE) compared with using sham-VEE in women with CLBP (chronic lowback pain) [17]; one article compares the effects of different exercise modes on autonomic modulation in patients with T2D (type 2 diabetes mellitus) [14]; and another paper analyzes the changes in ABB (acid–base balance) in the capillaries of kickboxers [18]. Other studies evaluate: the effects of resistance training on oxidative stress and muscle damage in spinal cord-injured rats [19]; the effects of muscle training on core muscle performance in rhythmic
gymnasts [20]; the physiological profiles of road cyclist in different age categories [21]; changes in body composition during the COVID-19 [22]; a mathematical model capable of predicting 2000 m rowing performance using a maximum-effort 100 m indoor rowing
ergometer [23]; the effects of ibuprofen on performance and oxidative stress [24]; the associations of vitamin D levels with various motor performance tests [12]; the level of knowledge on FM (Fibromyalgia) [25]; and the ability of a specific BIVA (bioelectrical
impedance vector analysis) to identify changes in fat mass after a 16-week lifestyle program in former athletes [26]. Finally, one review evaluates evidence from published systematic reviews and meta-analyses about the efficacy of exercise on depressive symptoms in cancer patients [27]; another review presents the current state of knowledge on satellite cell dependent skeletal muscle regeneration [28]; and a systematic review evaluates the effects of exercise on depressive symptoms among women during the postpartum period [29]
Crisp grass carp (CGC), the most representative improved varieties of grass carp (GC), features higher muscle hardness after feeding faba bean ( Vicia faba L.) for 90–120 days. DNA methylation, a most widely studied epigenetic modification, plays an essential role in muscle development. Previous studies have identified numerous differentially expressed genes (DEGs) between CGC and GC. However, it remains unknown if the expression levels of these DEGs are influenced by DNA methylation. In the present study, we performed a comprehensive analysis of DNA methylation profiles between CGC and GC, and identified important candidate genes related to muscle development coupled with the transcriptome sequencing data. A total of 9,318 differentially methylated genes (DMGs) corresponding to 155,760 differentially methylated regions (DMRs) were identified between the two groups under the CG context in promoter regions. Combined with the transcriptome sequencing data, 14 key genes related to muscle development were identified, eight of which (gsk3b, wnt8a, wnt11, axin2, stat1, stat2, jak2, hsp90) were involved in muscle fiber hyperplasia, six of which (tgf-β1, col1a1, col1a2, col1a3, col4a1, col18a1) were associated with collagen synthesis in crisp grass carp. The difference of methylation levels in the key genes might lead to the expression difference, further resulting in the increase of muscle hardness in crisp grass carp. Overall, this study can help further understand how faba bean modulates muscle development by the epigenetic modifications, providing novel insights into the texture quality improvement in other aquaculture fish species by nutritional programming.
Background
The 4-octyl itaconate (OI) is a type of cell-permeable itaconate derivative. Studies have shown that with an anti-fibrotic effect in systemic sclerosis, the OI also affects osteoclast differentiation. The aim of this study was to explore the molecular mechanisms underlying the effects of OI on myoblast differentiation by RNA-seq analysis.
Methods
Myoblast proliferation, differentiation, and muscle regulatory factors were examined in C2C12 myoblasts treated with OI of various concentrations (2.5, 10, 25, 50, and 100 μmol/L). Cells were treated with the PI3K-Akt activator IGF-1 to explore the role of the PI3K-Akt pathway in OI inhibition of myogenic differentiation. The regulatory mechanisms of OI in myogenesis were further investigated by RNA-seq and subsequent gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG) and, gene set enrichment analysis (GSEA).
Results
OI of various concentrations did not show any effect during cell proliferation. During differentiation, OI inhibited the expressions of the marker of mature myotubes myosin heavy chain (MHC) and myogenin in a dose-dependent manner. OI inhibited muscle differentiation by affecting MyoD-regulated activity through inhibition of AKT1 phosphorylation. The results of the KEGG enrichment analysis and GSEA showed that OI affected multiple metabolic pathways during myogenic differentiation, including PI3K-Akt signaling, calcium signaling, and PPAR signaling.
Conclusions
Our study broadens the understanding of the OI inhibition of myogenic differentiation. OI plays its functions by targeting multiple molecules and pathways, providing novel insights into the understanding of the overall effect of OI.
The loading of copper (Cu) into cytochrome c oxidase (COX) in mitochondria is essential for energy production in cells. Extensive studies have been performed with mitochondrial cuproenzymes, such as Sco1, Sco2 and Cox17, which contributes to the metallation of the oxidase. However, limited information is available on the upstream mechanism of Cu transport and delivery to mitochondria, especially through Cu-impermeable membranes, in mammalian cells. The mitochondrial phosphate transporter SLC25A3, also known as PiC2, is also able to bind Cu+ and acts as an active copper transporter in eukaryotic cells through these membranes, and ultimately aid in the metallation of COX. We used a well-established differentiation model of primary myoblasts derived from mouse satellite cells, where Cu availability is necessary for growth and maturation, and showed PiC2 is a target of MTF1, its expression is induced during myogenesis and favored by Cu supplementation. PiC2 deletion using CRISPR/Cas9 showed that the transporter is required for proliferation and differentiation of primary myoblasts, as both processes are delayed upon PiC2 knock-out. The effects of PiC2 deletion were ameliorated by the addition of Cu to the growth medium, implying the deleterious effects of PiC2 knockout in myoblasts may be in part due to a failure to deliver sufficient Cu to the mitochondria, which can be compensated by other mitochondrial cuproproteins. Co-localization and co-immunoprecipitation of PiC2 and COX also strongly suggest that PiC2 may act to directly load Cu into COX, which was verified by in vitro Cu+-transfer experiments. The data indicate an important role for PiC2 in both the delivery of Cu to the mitochondria, COX and, subsequently, the differentiation of primary myoblasts.
The resident stem cell for skeletal muscle is the satellite cell. On the 50th anniversary of its discovery in 1961, we described the history of skeletal muscle research and the seminal findings made during the first 20 years in the life of the satellite cell (Scharner and Zammit 2011, doi: 10.1186/2044-5040-1-28). These studies established the satellite cell as the source of myoblasts for growth and regeneration of skeletal muscle. Now on the 60th anniversary, we highlight breakthroughs in the second phase of satellite cell research from 1980 to 2000. These include technical innovations such as isolation of primary satellite cells and viable muscle fibres complete with satellite cells in their niche, and generation of many useful reagents including genetically modified organisms and antibodies still in use today. New methodologies were combined with description of endogenous satellite cells markers, notably Pax7. Discovery of the muscle regulatory factors Myf5, MyoD, Myogenin, and MRF4 in the late 1980s revolutionized understanding of the control of both developmental and regerenative myogenesis. Emergence of genetic lineage markers facilitated identification of satellite cells in situ, but also empowered transplantation studies to examine satellite cell function. Finally, satellite cell heterogeneity and the non-satellite cell support of muscle regeneration were described. These major advances in methodology and in understanding satellite cell biology provided the foundations for the dramatic escalation of work on muscle stem cells in the 21st century.
The degree of intramuscular adipose tissue accumulation is one of the factors affecting meat quality. Accumulation of adipocytes is also observed under the pathological condition of skeletal muscle such as muscular dystrophy and sarcopenia. The origin of adipocytes seen in skeletal muscle is mesenchymal progenitor cells that can give rise to both adipocytes and fibroblasts. In the present study, we demonstrated that siRNA-mediated suppression of MyoD expression in rat skeletal muscle progenitor cell culture, which comprises both myogenic satellite cells and mesenchymal progenitor cells, resulted in diminished myotube formation and an unexpected spontaneous appearance of white adipocytes. Suppressing myomaker expression also resulted in complete absence of myotube formation without reducing MyoD expression, but no adipogenesis was seen in this scenario, indicating that decline in MyoD expression rather than decreased myotube formation is necessary to induce adipogenesis. In addition, spontaneous adipogenesis induced by suppressing MyoD expression in culture was inhibited by the conditioned medium from control culture, indicating that anti-adipogenic factor(s) are secreted from MyoD-positive myogenic cells. These results indicate the presence of regulatory mechanism on adipogenesis by myogenic cells.
Single trauma injuries or isolated fractures are often manageable and generally heal without complications. In contrast, high-energy trauma results in multi/poly-trauma injury patterns presenting imbalanced pro- and anti- inflammatory responses often leading to immune dysfunction. These injuries often exhibit delayed healing, leading to fibrosis of injury sites and delayed healing of fractures depending on the intensity of the compounding traumas. Immune dysfunction is accompanied by a temporal shift in the innate and adaptive immune cells distribution, triggered by the overwhelming release of an arsenal of inflammatory mediators such as complements, cytokines and damage associated molecular patterns (DAMPs) from necrotic cells. Recent studies have implicated this dysregulated inflammation in the poor prognosis of polytraumatic injuries, however, interventions focusing on immunomodulating inflammatory cellular composition and activation, if administered incorrectly, can result in immune suppression and unintended outcomes. Immunomodulation therapy is promising but should be conducted with consideration for the spatial and temporal distribution of the immune cells during impaired healing. This review describes the current state of knowledge in the spatiotemporal distribution patterns of immune cells at various stages during musculoskeletal wound healing, with a focus on recent advances in the field of Osteoimmunology, a study of the interface between the immune and skeletal systems, in long bone fractures. The goals of this review are to (1) discuss wound and fracture healing processes of normal and delayed healing in skeletal muscles and long bones; (2) provide a balanced perspective on temporal distributions of immune cells and skeletal cells during healing; and (3) highlight recent therapeutic interventions used to improve fracture healing. This review is intended to promote an understanding of the importance of inflammation during normal and delayed wound and fracture healing. Knowledge gained will be instrumental in developing novel immunomodulatory approaches for impaired healing.
Intrauterine growth restriction (IUGR) represents a rate of fetal growth that is less than average for the population and the growth potential of a specific infant. IUGR produces infants who are small for gestational age (SGA) but also appropriate for gestational age (AGA). It refers to growth less than expected for gestational age and is most often under 10th percentiles for age. It develops during the late second and third trimesters of gestation. The etiology of IUGR is multifactorial. One of the most important factors which leads to IUGR is a decrease of nutrients and oxygen delivered to the fetus by the placenta. The growth of adipose tissue and skeletal muscle is limited by the declined fetal nutrient supply later in gestation. IUGR affects about 24% of babies born in developing countries. Worldwide, IUGR is the second cause of perinatal morbidity and mortality behind the premature birth and a major predisposing factor to metabolic disorders throughout postnatal life, even at adult age. Skeletal muscle represents about 35–40% of the body mass and plays an essential role in metabolic homeostasis, being responsible for 65% of fetal glucose consumption. A reduction in skeletal muscle growth characterizes IUGR fetuses compared to normal weight neonates. The decrease in muscle mass is not compensated after birth and persists until adulthood. This is a review of the literature, a neonatological, clinical point of view on the effects of IUGR on striated muscles. The available studies on this subject are currently the results of experimental research on animals, and information about the human fetus and newborn are scarce.
Discovery of the myogenic regulatory factor family of transcription factors MYF5, MYOD, Myogenin and MRF4 was a seminal step in understanding specification of the skeletal myogenic lineage and control of muscle differentiation during development. These factors are also involved in specification of the muscle satellite cell lineage, which becomes the resident stem cell compartment in adult skeletal muscle. While MYF5, MYOD, Myogenin and MRF4 have subtle roles in mature muscle, they again play a crucial role in directing satellite cell function to regenerate skeletal muscle: linking the genetic control of developmental and regenerative myogenesis. Here, I review the role of the myogenic regulatory factors in developing and mature skeletal muscle, satellite cell specification and muscle regeneration.
Myf5 is the earliest-known muscle-specific factor to be expressed in vivo and its expression is associated with determination of the myoblast lineage. In C2 cells, we show by immunocytolocalization that Myf5 disappears rapidly from cells in which the differentiation program has been initiated. In proliferating myo-blasts, the levels of Myf5 and MyoD detected from cell to cell are very heterogeneous. We find that some of the heterogeneity of Myf5 expression arises from a posttranscriptional regulation of Myf5 by the cell cycle. Immunoblotting of extracts from synchronized cultures reveals that Myf5 undergoes periodic fluctuations during the cell cycle and is absent from cells blocked early in mitosis by use of nocodazole. The disappearance of Myf5 from mitotic cells involves proteolytic degradation of a phosphorylated form of Myf5 specific to this phase of the cell cycle. In contrast, MyoD levels are not depleted in mitotic C2 cells. The mitotic destruction of Myf5 is the first example of a transcription factor showing cell cycle–regulated degradation. These results may be significant in view of the possible role of Myf5 in maintaining the determination of proliferating cells and in timing the onset of differentiation.
Regeneration of skeletal muscle tissue includes sequential processes of muscle cell proliferation and commitment, cell fusion, muscle fiber differentiation, and communication between cells of various tissues of origin. Central to the process is the myosatellite cell, a quiescent precursor cell located between the mature muscle fiber and its sheath of external lamina. To form new fibers in a muscle damaged by disease or direct injury, satellite cells must be activated, proliferate, and subsequently fuse into an elongated multinucleated cell. Current investigations in the field concern modulation of the effectiveness of skeletal muscle regeneration, the regeneration-specific role of myogenic regulatory gene expression distinct from expression during development, the impact of growth and scatter factors and their respective receptors in amplifying precursor numbers, and promoting fusion and maturation of new fibers and the ultimate clinical therapeutic applications of such information to alleviate disease. One approach to muscle regeneration integrates observations of muscle gene expression, proliferation, myoblast fusion, and fiber growth in vivo with parallel studies of cell cycling behaviour, endocrine perturbation, and potential biochemical markers of steps in the disease-repair process detected by magnetic resonance spectroscopy techniques. Experiments on muscles from limb, diaphragm, and heart of the mdx dystrophic mouse, made to parallel clinical trials on human Duchenne muscular dystrophy, help to elucidate mechanisms underlying the positive treatment effects of the glucocorticoid drug deflazacort. This review illustrates an effective combination of in vivo and in vitro experiments to integrate the distinctive complexities of post-natal myogenesis in regeneration of skeletal muscle tissue.Key words: satellite cell, cell cycling, HGF/SF, c-met receptor, MyoD, myogenin, magnetic resonance spectroscopy, mdx dystrophic mouse, deflazacort.
The activation of mononuclear muscle precursor cells after crush injury to mouse tibialis anterior muscles was monitored in vivo by in situ hybridization with MyoD1 and myogenin probes. These genes are early markers of skeletal muscle differentiation and have been extensively studied in vitro. The role in vivo of these regulatory proteins during myogenesis of mature muscle has not been studied previously. MyoD1 and myogenin mRNA were present in occasional mononuclear cells of uninjured muscle. Increased MyoD1 and myogenin mRNA sequences in mononuclear cells were detected as early as 6 h after injury, peaked between 24 and 48 h, and thereafter declined to pre-injury levels at about 8 days. The mRNAs were detected in mononuclear cells throughout the muscle, with the majority of cells located some distance from the site of crush injury. The presence of MyoD1 and myogenin mRNA at 6 to 48 h indicates that transcription of these genes is occurring at the same time as replication of muscle precursor cells in vivo. At no time were significant levels of mRNA for these genes detected in myotubes. MyoD1 and myogenin provide precise markers for the very early identification and study of mononuclear skeletal muscle precursor cells in muscle regenerating in vivo.
Monoclonal antibodies (MoAbs) were developed against recombinant wild-type murine MyoD1 protein. Each of 4 MoAbs was immunologically reactive with recombinant MyoD1 protein by enzyme-linked immunosorbent assay, and each specifically stained the nuclei of myogenic cells. Epitopes were mapped using fusion protein constructs with specific deletions of defined regions of the MyoD1 molecule. MoAb 5.2F recognized an epitope in the amino terminal region between amino acid residues (AAR) 3 and 56, whereas epitopes for MoAbs 1.1A, 5.4G, and 5.8A were in the carboxyl terminus (AAR 167-318) of the MyoD1 protein. The epitope for MoAb 5.8A was further delineated to AAR 170-209 by Western analysis and immunoprecipitation of in vivo transcribed and translated MyoD1 protein having specific deletions in the carboxyl terminus. The 5.8A epitope was ultimately localized to the region between AAR 180 and 189 of the protein by enzyme-linked immunosorbent assay using 10-amino acid residue synthetic peptides. This sequence is apparently unique to MyoD1 and has little homology to other myogenic regulatory proteins (myogenin, Myf5, Myf6, and MRF4). Transfection of cDNA for murine MyoD1 into a nonmuscle cell line conferred 5.8A reactivity, confirming the specificity of this reagent. MoAb 5.8A was then used to examine the expression of MyoD1 in normal and malignant human tissues. MyoD1 was not detected in any normal adult tissue but was detected in 25 of 25 histologically confirmed rhabdomyosarcomas. Staining was localized to the nucleus and showed marked heterogeneity between cells as well as differential staining within nuclei. Specific subcellular localization of 5.8A was further determined by immunoelectron microscopy, where antibody was found to localize to electron-dense areas, more frequently associated with the nuclear submembranous region. In addition to rhabdomyosarcomas, MoAb 5.8A stained 2 of 5 Wilms' tumors and one ectomesenchymoma, neoplasms known to contain myogenic elements. The 5.8A reagent was also of value in the accurate histopathological classification of 2 of 4 tumors previously diagnosed as extraosseous Ewing's sarcoma and 2 of 3 tumors diagnosed as undifferentiated sarcomas.
Satellite cells of adult muscle are quiescent myogenic stem cells that can be induced to enter the cell cycle by an extract of crushed muscle (Bischoff, R. 1986. Dev. Biol. 115:140-147). Here, evidence is presented that the extract acts transiently to commit cells to enter the cell cycle. Satellite cells associated with both live and killed rat myofibers in culture were briefly exposed to muscle extract and the increase in cell number was determined at 48 h in vitro, before the onset of fusion. An 8-12-h exposure to extract with killed, but not live, myofibers was sufficient to produce maximum proliferation of satellite cells. Continuous exposure for over 40 h was needed to sustain proliferation of satellite cells on live myofibers. The role of serum factors was also studied. Neither serum nor muscle extract alone was able to induce proliferation of satellite cells. In the presence of muscle extract, however, satellite cell proliferation was directly proportional to the concentration of serum in the medium. These results suggest that mitogens released from crushed muscle produce long-lasting effects that commit quiescent satellite cells to divide, whereas serum factors are needed to maintain progression through the cell cycle. Contact with a viable myofiber modulates the response of satellite cells to growth factors.
During the terminal stage of skeletal myogenesis, myoblasts stop replicating, fuse to form multinucleate fibers, and express the genes that encode the proteins that convey contractile capacity. Because of this dramatic shift in proliferative state, morphology, and gene expression, it has been possible to readily identify and quantitate terminally differentiating myoblasts. In contrast, it is not clear whether the proliferating cells that give rise to postmitotic myoblasts are equally distinct in their phenotype and in fact whether distinct stages in skeletal myogenesis precede the onset of terminal differentiation. To address these questions, monoclonal antibodies and immunofluorescence microscopy were used to determine that replicating myoblasts from newborn rats do express a muscle-specific phenotype. To identify replicating cells, incorporation of 5-bromo-2'-deoxyuridine (BrdUrd) into DNA was assayed by using anti-BrdUrd antibody. The developmentally regulated, muscle-specific, integral membrane protein H36 and the intermediate-filament protein desmin were scored as markers of the myogenic phenotype. The percentage of BrdUrd+ (i.e., proliferative) cells among H36+ and desmin+ myoblasts was equal to the percentage of BrdUrd+ cells in the entire population, indicating that the expression of H36 and desmin is uniformly characteristic of replicating myoblasts. Inhibition of protein synthesis before and during growth in BrdUrd did not alter the frequency of desmin and H36 immunofluorescence in BrdUrd+ cells. Thus, desmin and H36 were present in the replicating myoblasts prior to the onset of growth in BrdUrd. These results were confirmed using H36+ cells selected by flow cytometry: these purified H36+ myoblasts replicate, express desmin, and differentiate. Similar results were obtained with mouse myoblasts. Desmin expression in these mammalian cells differs from that in chicken embryo myoblasts: only a small proportion of replicating chicken embryo myoblasts express desmin. That replicating mammalian myoblasts have a muscle-specific phenotype serves to define a distinct stage in myogenic development and a specific cell in the myogenic lineage. Further, it implies that there is a regulatory event activated during myogenesis that precedes terminal differentiation and that is required for expression of those genes whose products distinguish the replicating myoblast.
Monoclonal antibodies ( McAbs ) have been generated against a preparation of intermediate filament proteins (IFP) from adult chicken gizzard. Two antibodies, D3 and D76 , have been characterized in detail. They bind specifically to desmin but recognize different epitopes. In the adult chicken, both McAbs produced equivalent immunofluorescent staining patterns, reacting in frozen sections with all forms of muscle tissue, including vascular smooth muscle, but with no other tissue types. In isolated skeletal myofibrils and in longitudinal frozen sections of cardiac and skeletal muscle, desmin was detected with both McAbs at the Z-band and in longitudinally-oriented filament bundles between myofibrils. In contrast to these results in the adult, the intermediate filaments (IF) of embryonic cardiac myocytes in primary cultures were decorated only with McAb D3, whereas McAb D76 was completely unreactive with these cells. Similarly, frozen sections through the heart at early stages of embryonic chick development (Hamburger-Hamilton stages 17-18) revealed regions of myocytes, identified by double immunofluorescence with myosin-specific McAbs , that were unstained with McAb D76 even though similar regions were stained by McAb D3. That McAb D76 reacted with desmin in all adult cardiac myocytes but not with all embryonic heart cells indicates that embryonic and adult cardiac IF are immunologically distinct and implies a conversion in IF immunoreactivity during cardiac development.
The expression pattern of myogenic regulatory factors and myotome-specific contractile proteins was studied during embryonic development of Myf-5 mutant mice by in situ hybridization and immunohistochemistry.
In contrast to somites in wild-type embryos, no expression of myogenin and Myf-6 (MRF4), or any other myotomal markers was detected in mutant animals at E9.0 and E10.0 indicating that Myf-5 plays a crucial role during this developmental period. Significantly, the onset of MyoD expression in rostral somites of E10.5 embryos was unaffected by the Myf-5 mutation suggesting that the activation of the MyoD gene occurs independently of Myf-5 at the correct developmental time. Immediately after the activation of MyoD myogenin transcripts and protein accumulated within the myotome. The first contractile proteins of the sarcomeric apparatus appeared slightly later. By E11.5 the expression of muscle markers were indistinguishable between wild-type and Myf-5 mutant mice.
The migration of muscle precursor cells that leave the somites to form limb musculature was monitored in Myf-5-mutant mice by Pax-3 expression. Pax-3-positive cells were equally found in somites and limbs of E10.0 wild-type and mutant mice indicating that myogenic factor expression at the level of somites is not a prerequisite for determination and subsequent migration of limb precursor cells.
Differentiation of skeletal myoblasts in culture is negatively regulated by certain growth factors, including basic fibroblast
growth factor (bFGF) and transforming growth factor β (TGFβ). We investigated the effects of bFGF and TGFβ on D-type cyclin
expression in skeletal myoblasts. When myoblasts were induced to differentiate in low mitogen medium, expression of cyclin
D1 rapidly fell below detectable levels. In contrast, expression of cyclin D3 increased to levels exceeding those present
in myoblasts. Expression of cyclin D1 was induced in myoblasts by bFGF and TGFβ (albeit with different kinetics for each factor),
while induction of cyclin D3 expression was inhibited by these growth factors. Although these results are consistent with
other reports showing induction of cyclin D1 by growth factors, induction of cyclin D3 expression during terminal differentiation
of myoblasts and inhibition of this induction by growth factors is surprising. These results suggest that cyclin D3, previously
thought to be only a positive regulator of cell cycle progression, may also function in the cellular context of terminal differentiated
muscle. Stable expression of cyclin D1 from an ectopic viral promoter inhibits C2C12 myoblast differentiation, but only in
those clones where the level of cyclin D1 expression does not significantly exceed that present in control myoblasts stimulated
by bFGF. Together, these result suggest that cyclin D1 expression functions in the inhibition of myoblast differentiation
by certain growth factors.
Mitogen-activated protein kinase kinase (MAPKK) is a dual specificity protein kinase that exhibits a high degree of specificity toward its downstream target, mitogen-activated protein kinase (MAPK). In this study, we used stable overexpression of MAPKK and its mutants in NIH 3T3 cells to study effects on downstream components of the MAPK signaling cascade and to correlate them to physiological responses. We have mutated the potential regulatory serine residue 222 to alanine (S222A) or to glutamate (S222E) and serines 212 and 218 together to alanine residues (S212A,S218A). Lysine 97 was mutated to alanine (K97A) to provide an inactive enzyme. Overexpression of the wild type MAPKK had no effect on any of the parameters examined. The K97A and S222A mutants served as dominant negatives by suppressing MAPKK, MAPK, and p90rsk activation in vivo. S222E enhanced all of these activities, and S212A,S218A had a small inhibitory effect. A similar trend was observed when cellular proliferation was examined and the different effects were accompanied by altered cellular shape. Taken together, our results demonstrate a direct linkage between the MAPK signaling pathway and the control of cellular proliferation and morphology and also establish that phosphorylation of serine 222 is essential for MAPKK activation together with the phosphorylation of an additional serine(s) (probably serine 218).
Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.
To investigate the function of MyoD in adult skeletal muscle, we interbred MyoD mutant mice with mdx mice, a model for Duchenne and Becker muscular dystrophy. Mice lacking both MyoD and dystrophin displayed a marked increase in severity of myopathy leading to premature death, suggesting a role for MyoD in muscle regeneration. Examination of MyoD mutant muscle revealed elevated numbers of myogenic cells; however, myoblasts derived from these cells displayed normal differentiation potential in vitro. Following injury, MyoD mutant muscle was severely deficient in regenerative ability, and we observed a striking reduction in the in vivo proliferation of myogenic cells during regeneration. Therefore, we propose that the failure of MyoD-deficient muscle to regenerate efficiently is not caused by a reduction in numbers of satellite cells, the stem cells of adult skeletal muscle, but results from an increased propensity for stem-cell self-renewal rather than progression through the myogenic program.
The myogenic regulatory factors (MRFs) form a family of basic helix-loop-helix transcription factors consisting of Myf-5, MyoD, myogenin, and MRF4. The MRFs play key regulatory roles in the development of skeletal muscle during embryogenesis. Sequence homology, expression patterns, and gene-targeting experiments have revealed a two-tiered subclassification within the MRF family. Myf-5 and MyoD are more homologous to one another than to the others, are expressed in myoblasts before differentiation, and are required for the determination or survival of muscle progenitor cells. By contrast, myogenin and MRF4 are more homologous to one another than to the others and are expressed upon differentiation, and myogenin is required in vivo as a differentiation factor while the role of MRF4 remains unclear. On this basis, MyoD and Myf-5 are classified as primary MRFs, as they are required for the determination of myoblasts, and myogenin and MRF4 are classified as secondary MRFs, as they likely function during terminal differentiation.
The myogenic progenitors of epaxial (paraspinal and intercostal) and hypaxial (limb and abdominal wall) musculature are believed to originate in dorsal-medial and ventral-lateral domains, respectively, of the developing somite. To investigate the hypothesis that Myf-5 and MyoD have different roles in the development of epaxial and hypaxial musculature, we further characterized myogenesis in Myf-5- and MyoD-deficient embryos by several approaches. We examined expression of a MyoD-lacZ transgene in Myf-5 and MyoD mutant embryos to characterize the temporal-spatial patterns of myogenesis in mutant embryos. In addition, we performed immunohistochemistry on sectioned Myf-5 and MyoD mutant embryos with antibodies reactive with desmin, nestin, myosin heavy chain, sarcomeric actin, Myf-5, MyoD and myogenin. While MyoD(−/−) embryos displayed normal development of paraspinal and intercostal muscles in the body proper, muscle development in limb buds and brachial arches was delayed by about 2.5 days. By contrast, Myf-5(−/−) embryos displayed normal muscle development in limb buds and brachial arches, and markedly delayed development of paraspinal and intercostal muscles. Although MyoD mutant embryos exhibited delayed development of limb musculature, normal migration of Pax-3-expressing cells into the limb buds and normal subsequent induction of Myf-5 in myogenic precursors was observed. These results suggest that Myf-5 expression in the limb is insufficient for the normal progression of myogenic development. Taken together, these observations strongly support the hypothesis that Myf-5 and MyoD play unique roles in the development of epaxial and hypaxial muscle, respectively.
Myogenic precursors in adult skeletal muscle (satellite cells) are mitotically quiescent but can proliferate in response to a variety of stresses including muscle injury. To gain further understanding of adult myoblasts, we are analyzing myogenesis of satellite cells on fibers isolated from adult rat muscle. In this culture model, satellite cells are maintained in their in situ position underneath the fiber basement membrane. Employing two different approaches to monitor proliferation of satellite cells on isolated fibers (autoradiography following (3)H-thymidine incorporation and immunofluorescence of cells positive for proliferating cell nuclear antigen (PCNA)), we show in the present study that satellite cells initiate cell proliferation at 12 to 24 hours following fiber culture establishment and that cell proliferation is reduced to minimal levels by 60 to 72 hours in culture. Maximal number of proliferating cells is seen at 36 to 48 hours in culture. These PCNA+ satellite cells transit into the differentiated, myogenin+ state following about 24 hours in the proliferative state. Continuous exposure of the fiber culture to FGF2 (basic FGF; added at the time of culture establishment) leads to a 2 fold increase in the number of PCNA+ cells by 48 hours in culture but the overall schedule of proliferation and transition into the myogenin+ state is not affected. Delaying the addition of FGF2 until 15 to 18 hours following the initiation of the fiber culture does not reduce its effect. However, the addition of FGF2 at 24 hours or later results in a progressive reduction in the number of proliferating satellite cells. Exposure of fiber cultures to transforming growth factor β (TGFβ1) leads to a reduction in the number of proliferating cells in both the absence or presence of FGF2. We propose that FGF2 enhances the number of proliferating cells by facilitating the recruitment of additional satellite cells from the quiescent state. However, satellite cells on isolated fibers conform to a highly coordinated program and rapidly transit from proliferation to differentiation regardless of the presence of FGF. The identification of agents that can prolong the proliferative state of satellite cells when the cells undergo myogenesis in their native position by the intact myofiber might be useful in improving myoblast transplantation into skeletal muscle for cell-mediated gene therapy.
MyoD belongs to a family of helix-loop-helix proteins that control myogenic differentiation. Transfection of various non-myogenic cell lines with MyoD transforms them into myogenic cells. In normal embryonic development MyoD is upregulated at the time when the hypaxial musculature begins to form, but its role in the function of adult muscle remains to be elucidated. In this study we examined the cellular locations of MyoD protein in normal and abnormal muscles to see whether the presence of MyoD protein is correlated with a particular cellular behaviour and to assess the usefulness of MyoD as a marker for satellite cells. Adult rats were anaesthetised and their tibialis anterior or soleus muscles either denervated, tenotomised, freeze lesioned, lesioned and denervated, or lesioned and tenotomised. At various intervals after the operations the rats were killed and their muscles removed, snap frozen, and sectioned with a cryostat along with muscles from unoperated neonatal and adult rats. The sections were processed for immunohistochemistry using a rabbit affinity-purified antibody to recombinant MyoD. MyoD proved to be an excellent marker for active satellite cells; satellite cells in neonatal and regenerating muscles contained high levels of MyoD protein. MyoD positive cells were not observed in the muscles of old adults, in which the satellite cells are fully quiescent. MyoD immunoreactivity was rapidly lost from satellite cell nuclei after they fused into myotubes and was not detected in either sub-synaptic or non-synaptic nuclei of mature fibers. Denervation, and to a lesser extent tenotomy, of lesioned muscles induced expression of MyoD in myotubal nuclei. Denervation of normal muscles also upregulated MyoD in muscle fiber nuclei, an effect which was maximal after 3 days. We conclude that MyoD protein is neurally regulated in both myotubes and muscle fibers. © 1995 Wiley-Liss, Inc.
The muscle regulatory factors MRF4, myogenin, myf-5, and MyoD constitute a family of proteins that can function as muscle-specific transcriptional activators. Although this gene family has been extensively studied, a specific role for each factor during myogenesis remains to be determined. Understanding how these factors function requires a detailed analysis of their expression patterns during development. Toward this goal, we examined the temporal pattern of expression of MRF4 and the other factors in the rat myogenic cell line L6J1-C, in newborn rat primary muscle cell cultures and in fetal and postnatal rat limb muscle. Our results demonstrate that MyoD, myogenin, and myf-5 transcripts accumulate maximally at various stages of myoblast differentiation and decline to low expression levels in adult muscle tissue. In contrast, MRF4 transcript accumulation is restricted to cell cultures containing multinucleate myofibers, and its expression in vivo increases sharply during late fetal muscle development. This level of MRF4 expression is maintained in the adult which, together with decreased expression of the other three muscle regulatory factors, makes MRF4 the predominant factor in adult muscle. In situ hybridization of mouse embryo tissue sections indicates that MRF4 transcripts accumulate in the limb beginning 13.5 days post coitum, which is 2 days later than the initial appearance of myogenin and MyoD transcripts. Hybridization to earlier stages of development reveals, however, that MRF4 mRNA initially is present in the myotomal compartment of the somites, just after myogenin but 2 days prior to MyoD expression. Unlike myogenin and MyoD, MRF4 expression declines in the myotomes at the time that multinucleate axial muscles begin to form in this region, although during later development MRF4 is expressed in the myofibers of axial muscles at levels comparable to those in the limb. Differences in the expression patterns for MRF4, myogenin, myf-5 and MyoD between myotomal and other skeletal muscle development suggest that the relative timing of expression for each muscle regulatory factor may control the distinct phenotypes associated with myotomal myocytes and multinucleate myofibers.
Regulation of skeletal muscle determination and differentiation in vertebrates centers on a core regulatory network which is composed of two families of transcription factors, the MyoD group basic helix-loop-helix (bHLH) muscle regulatory factors (MRFs) and the myocyte enhancer factor 2 (MEF2) group of MADS-box regulators. Members of this network interact with each other genetically and physically, and together they cooperate to positively regulate transcription of downstream muscle-specific differentiation genes. During development, the myogenic network can be activated or repressed in response to patterning signals, some of which have recently been identified. Once activated, the powerful myogenic activity of the core network can be modulated and held in check by a remarkably large group of negative regulators that operate on network components by diverse mechanisms. Recent discoveries highlight extensive parallels between myogenesis and peripheral neurogenesis in the structures of their respective regulatory networks and in the interaction of their bHLH networks with other regulatory circuits. Comparisons with Drosophila indicate that these ensembles of interacting molecular circuits have been highly conserved during evolution.
Differentiation properties of a cell line, L84, which originated from a non-fusing clone isolated from the myogenic line L8, are described. In nutritional medium supplemented with 10% serum used routinely with L8 cells, L84 cells continue to proliferate to very high densities and fail to form multinucleated fibres. When grown in medium supplemented with 2% horse serum or 2% horse serum plus 0.1 μg/ml insulin, L84 cells behave very similarly to L8 cells grown in medium supplemented with 10% horse serum: when the cultures reach confluency, proliferation decreases and cells start to fuse and form a dense network of fibres.
Large increases in creatine kinase activity and synthesis of myosin are associated with cell fusion. Under conditions in which L84 cells do not fuse the increase in these synthetic activities is not observed, even after extremely high cell densities are reached. The data show that L84 cells retain the programme for their differentiation into muscle fibres. The difference between L84 and its progenitor line L8 lies in the sensitivity to the environmental conditions which trigger the expression of this programme.
THE muscular dystrophies are a group of hereditary disorders manifested by a progressive wasting of the skeletal muscles. In spite of extensive studies, the nature of the primary lesion is unknown (for review see ref. 1). Because of the complex interaction between tissues, it is difficult to study this question in vivo. Therefore attempts have been made to investigate this question in cultures of dystrophic muscles of human or animal origin. Tissue explants as well as monolayer primary cell cultures contain, in addition to the myogenic cells, a heterogeneous cell population, the composition of which might differ in normal and dystrophic muscle cultures. It is difficult in such experiments to distinguish between properties intrinsic to the myogenic cells and effects exerted by other cell types. Indeed, previous experiments have yielded conflicting conclusions2-6. We therefore tested the possibility of obtaining cell cultures consisting of pure populations of myogenic cells obtained from dystrophic muscles. The present report describes the isolation of a cloned population of such cells, derived from adult dystrophic mouse muscle, that can proliferate and differentiate in cell culture.
Normal adult mouse tibialis anterior muscles were perfused continuously with 3H-thymidine for nine days. Quantitation of the satellite cell population in these muscles reveals that not only is the frequency of satellite cell nuclei low, but that those present are mitotically quiescent.
A clonal cell line exhibiting many of the properties of skeletal muscle has been derived from embryonic BDIX rat heart tissue. Multinucleated myotubes, formed by fusion of mononucleated myoblasts, simultaneously contract and produce regenerative action potentials in response to electrical stimulation or iontophoretic application of acetylcholine. The acetylcholine response is inhibited by 1−3 × 10−7 M d-tubocurarine, 10−7 g/ml α-neurotoxin or 1 × 10−4 M atropine. The specific activities of the enzymes myokinase and creatine phosphokinase (CPK) increase 3-fold and 20-fold, respectively, after myotube formation, but only CPK activity parallels the extent of fusion. Exponentially dividing myoblasts synthesize a predominantly brain-type CPK isoenzyme while fused myotubes synthesize a muscle type CPK isoenzyme. Electron microscopic analysis reveals that the myotubes contain elaborately branched tubular systems and numerous bundles of thick filaments with distinct M-bands. Some of the thick filament bundles are associated with thin filaments and organized into sarcomere-like structures with faint Z-regions, but no distinct Z-bands are observed.
The differential expression of genes triggering myogenesis might cause or reflect differences among myoblasts. Little is known about the presence of MyoD1 and myogenin during the process of regeneration. We therefore examined the expression of MyoD1 and myogenin in muscle regeneration after grafting. Immunostaining of regenerating skeletal muscle of the mouse revealed the presence of both MyoD1 and myogenin. In mononucleated cells the proteins were not detected until shortly before fusion into myotubes. They persisted in the nuclei of regenerated muscle fibers for at least 2 weeks. MyoD1 and myogenin were not detected in nonregenerating control muscle.
In muscle cells, as in a variety of cell types, proliferation and differentiation are mutually exclusive events controlled by a balance of opposing cellular signals. Members of the MyoD family of muscle-specific helix-loop-helix proteins which, in collaboration with ubiquitous factors, activate muscle differentiation and inhibit cell proliferation function at the nexus of the cellular circuits that control proliferation and differentiation of muscle cells. The activities of these myogenic regulators are negatively regulated by peptide growth factors and activated oncogenes whose products transmit growth signals from the membrane to the nucleus. Recent studies have revealed multiple mechanisms through which intracellular growth factor signals may interfere with the functions of the myogenic regulators. When expressed at high levels, members of the MyoD family can override mitogenic signals and can cause growth arrest independent of their effects on differentiation. The ability of these myogenic regulators to inhibit proliferation of normal as well as transformed cells from multiple lineages suggests that they interact with conserved components of the cellular machinery involved in cell cycle progression and that similar types of regulatory factors participate in differentiation and cell cycle control in diverse cell types.
The Myf-5 gene, a member of the myogenic basic HLH factor family, has been inactivated in mice after homologous recombination in ES cells. Mice lacking Myf-5 were unable to breathe and died immediately after birth, owing to the absence of the major distal part of the ribs. Other skeletal abnormalities, except for complete ossification of the sternum, were not apparent. Histological examination of skeletal muscle from newborn mice revealed no morphological abnormalities. Northern blot analysis demonstrated normal levels of muscle-specific mRNAs including MyoD, myogenin, and Myf-6. However, the appearance of myotomal cells in early somites was delayed by several days. These results suggest that while Myf-5 plays a crucial role in the formation of lateral sclerotome derivatives, Myf-5 is dispensable for the development of skeletal muscle, perhaps because other members of the myogenic HLH family substitute for Myf-5 activity.
Satellite cell activity was examined in the stretch-enlarge anterior latissimus dorsi muscle (ALD) of the adult quail. Thirty-seven birds had a weight equal to 10% of their body mass attached to one wing while the contralateral wing served as an intra-animal control. At various time intervals after application of the wing weight (from 1 to 30 days), the birds were injected with tritiated thymidine and killed 1 h later. Stretched muscle length was greater by day 1 and mass by day 3 when compared with the contralateral muscle. Satellite cells actively synthesizing DNA were quantitated in fiber segments of the control and stretched ALD. A minimum of 1,500 muscle nuclei (satellite cell nuclei and myonuclei) were counted in each muscle. Labeling in stretched muscle was expressed by the percent labeled nuclei per total nuclei counted. Satellite cell labeling was initiated by day 1, peaked between days 3 and 7, and was not statistically different from control values at day 30. These results demonstrate that satellite cells are induced to enter the cell cycle in the stretch-enlarged ALD muscle from the adult quail, and the peak of proliferative activity is within the first week of stretch.
Single myofibers with attached satellite cells isolated from adult rats were used to study the influence of the mature myofiber on the proliferation of satellite cells. The satellite cells remain quiescent when cultured in serum containing medium but proliferate when exposed to mitogen from an extract of crushed adult muscle. The response of satellite cells to mitogen was measured under three situations with respect to cell contact: (1) in contact with a viable myofiber and its basal lamina, (2) detached from the myofiber by centrifugal force and deposited on the substratum and (3) beneath the basal lamina of a Marcaine killed myofiber. The results show that satellite cells in contact with the plasmalemma of a viable myofiber have reduced mitogenic response. Since inhibiting growth may induce differentiation, I tested whether satellite cells proliferating on the surface of a myofiber would fuse. Although the satellite cell progeny were fusion competent, they did not fuse with the myofiber. To determine whether fusion competence of the myofiber changes with time in culture, embryonic myoblasts were challenged to fuse with myofibers that had been stripped of satellite cells and cultured for several days. The myoblasts fused with pseudopodial sprouts growing from the ends of the myofiber, but did not fuse with the original myofiber surface. These results indicate that contact with the surface of a mature myofiber suppresses proliferation of myogenic cells but the cells do not fuse with the myofiber.
Desmin expression by myoblasts cultured from embryonic and adult chicken breast muscle was examined employing indirect immunofluorescence. The study was performed in conjunction with [3H]thymidine autoradiography and analysis of skeletal myosin expression in order to determine whether the desmin-expressing cells were terminally differentiated. Following 2 h of labeling with [3H]thymidine, 0.55%, 2.60%, and 15.10% of the cells in mass cultures from 10-day-old embryos, 18-day-old embryos and adults, respectively, incorporated [3H]thymidine and were desmin-positive but did not express skeletal-muscle-specific myosin. Using the same approach we determined that 0.07%, 1.25%, and 7.59% of the mononucleated cells in myogenic clones from 10-day-old embryos, 18-day-old embryos and adults, respectively, were desmin-positive, myosin-negative, [3H]thymidine-positive. We suggest that these desmin-positive, myosin-negative myoblasts are proliferating cells, and we conclude that the progeny of adult myoblasts exhibit more desmin-expressing cells of this type than embryonic myoblasts do.
Although the role of satellite cells has been confirmed during skeletal muscle growth and regeneration, their involvement during work-induced muscle growth remains uncertain. In this study, chronically overloaded rat soleus muscles were ultrastructurally monitored following surgical ablation of synergists to examine cytological adaptations of satellite cells and myofibers. The left soleus muscle of 20 female Sprague-Dawley rats (7 weeks of age) was induced to hypertrophy by excising the contralateral plantaris and gastrocnemius muscles under pentobarbital anesthesia. Right limbs were sham-operated and served as controls. On days 3, 7, 14, 21, and 30 after surgery, the soleus muscles were removed and processed for electron microscopy. Two morphologically distinct phases were noted in the surgically overloaded muscles. The first stage (week 1) was characterized by a significant increase in the number of satellite cells, and by more than half of the experimental muscle fibers displaying myofibrillar disruptions, mitochondrial alterations and glycogen pooling. The second phase (weeks 2-4) featured mostly normal, although larger appearing muscle fibers, with the satellite cell frequency remaining slightly elevated. These findings suggest that muscle fiber structural abnormalities, rather than an increase in muscle activity, may play a more significant role in the early activation of satellite cells during compensatory hypertrophy, whereas activation of satellite cells during the later stages may be in response to increased levels of muscle activity.
In this report, we describe the isolation, sequence, and initial characterization of the cDNA for the muscle-specific regulatory factor skeletal myogenin. Transfection of myogenin into the mesenchymal cell line C3H10T1/2 produces cells expressing muscle-specific markers. Myogenin is absent in undifferentiated cells, peaks, and then declines following a stimulus to differentiate, and is overexpressed in myoblasts selected with 5-bromodeoxyuridine for the overproduction of factors that regulate the decision to differentiate. High levels of myogenin transcripts are present in the myotomal region of somites at 8.5 days of gestation in the mouse. Although myogenin and MyoD are different genes, they share the myc homology domain. Myogenin and MyoD thus form part of a gene family regulating myogenesis, and together with myd may constitute a set of factors that interact to regulate the determination and differentiation of muscle cells.
Proliferation of satellite cells is responsible for formation of new muscle cells during development and regeneration. Signals governing satellite cell growth were studied using a tissue culture system consisting of single myofibers with attached satellite cells. The cells remain quiescent in basal medium but enter the cell cycle in response to growth factor obtained from injured muscle. The growth factor exhibits both source and target specificity and appears to be a polypeptide greater than 30 Kd. Satellite cells exposed to growth factor enter the S phase of the cell cycle by 18 h and proliferate with a generation time of 12 h. The growth factor is active in vivo and increases the proliferation and fusion of satellite cells to myofibers when injected intramuscularly in rat pups or adults. In addition to positive signals, satellite cell growth is modulated by negative feedback from the mature myofiber, as satellite cells removed from single myofibers by centrifugation show increased sensitivity to growth factor. The mitotic inhibition imposed on satellite cells results from contact with the myofiber plasmalemma and not the basal lamina, as shown by comparison of satellite cell mitogenesis in killed and living myofibers.
Muscle satellite cells are quiescent myogenic stem cells situated between the basal lamina and plasmalemma of mature skeletal muscle fibers. Injury to the fiber triggers the activation and proliferation of satellite cells whose progeny subsequently fuse to form new myotubes during regeneration. In this paper we report the proliferation of satellite cells on single muscle fibers isolated from adult rats and placed in culture. Viable fibers were liberated from muscle with collagenase and purified from non-muscle cells. The fibers were covered with a basal lamina and retained normal morphological characteristics. Each fiber contained two to three satellite cells per 100 myonuclei. Satellite cells showed little proliferative activity in medium with 10% serum but could be induced to enter the cell cycle by chick embryo extract or fibroblast growth factor. Other polypeptide mitogens such as epidermal growth factor, multiplication stimulating activity, and platelet-derived growth factor were ineffective. Mitogen-stimulated satellite cells fused to form new myotubes after 4-5 days in culture. These results imply that satellite cells are under positive growth control since they proliferate in contact with viable mature fibers when stimulated with mitogen. The mature fibers remained viable in culture but did not give rise to mononucleated cells. After several days, however, the fibers began to extend sarcoplasmic sprouts and underwent dedifferentiative changes that led to the formation of multinucleated cells resembling myotubes. These cells reexpressed embryonic isozymes of creatine kinase not made by the mature fibers.
The source of the new nuclei appearing during the growth of muscle fibers was examined in the tibialis anterior muscle of young Sherman rats (14–17 days of age) using radioautography at various intervals after a single injection of a small, non‐toxic dose of ³ H‐thymidine (2 μCi/g body weight). Two techniques were employed: (1) labeled nuclei were detected in 1 μ thick radioautographs examined in the light microscope, and identified by simultaneous electron microscope examination of an adjacent section. The nuclei were then classified either as “true” muscle nuclei (within the plasmalemma of the fibers) or as belonging to “satellite cells” (which are mononucleated cells with scanty cytoplasm wedged between plasmalemma and basement membrane). (2) Muscle fibers freed by collagenase digestion were radioautographed one hour after ³ H‐thymidine injection in order to determine the total number of labeled nuclei (true muscle nuclei plus those of satellite cells) per unit length of fiber.
Certain nuclei within the basement membrane of muscle fibers are labeled one hour after ³ H‐thymidine and, therefore, synthesize DNA. The electron microscope demonstrates that these nuclei invariably belong to satellite cells, never to true muscle nuclei. Furthermore, the total number of labeled nuclei per unit length of fiber doubles between 1 and 24 hours; and, therefore, the labeled satellite cell nuclei undergo mitosis.
Following mitosis, half of the daughters of satellite cells are incorporated into the fibers to become true muscle nuclei. The remaining half divides again later; and half of their daughter cells are incorporated. Thus, satellite cells in young rats divide repeatedly and function as a source of true muscle nuclei.
The effect of hepatocyte growth factor (HGF) on the activation of quiescent rat skeletal muscle satellite cells was evaluated in vitro. Satellite cells from 9-month-old adult rats are quiescent in vivo and when cultured, display a protracted lag phase prior to division that is not present in satellite cells from neonatal or regenerating muscle. Under normal growth conditions, satellite cells divide for the first time between 42 and 60 hr. Hepatocyte growth factor increased proliferation in a dose-dependent fashion prior to 48 hr with half-maximal stimulation at approximately 3 ng/ml; in addition, heparin enhanced this activity. The time course of cyclin-D1 and proliferating cell nuclear antigen (PCNA) expression was accelerated in HGF-treated satellite cells, indicating that cells entered the cell cycle earlier. No significant effects on muscle-derived fibroblast proliferation was observed. The signalling receptor for HGF is the product of the c-met protooncogene, and rtPCR analysis of satellite cells 0-72 hr in culture demonstrated the presence of this message throughout this time period. The presence of c-met in quiescent satellite cells, the ability of HGF to stimulate precocious entry into the cell cycle, and the previously described localization of HGF message in regenerating muscle (Jennische et al., 1993) indicate that HGF could act as an activator of quiescent satellite cells in vivo.
It has been suggested that myogenin is an important factor for the differentiation of myoblasts and that its function in myogenesis is regulated by proto-oncogenes in in vitro experiments. We have characterized the spatial and temporal expression patterns of myogenin, c-fos, c-jun, and muscle creatine kinase mRNAs during the skeletal muscle regeneration process using in situ hybridization histochemistry. Myogenin transcripts are first detected in the myonuclei/nuclei of satellite cells at 6 h after induction of regeneration. Myogenin mRNA is expressed in desmin-positive myoblasts, yet no muscle creatine kinase mRNA is detected in this cell type. Both the muscle creatine kinase and myogenin mRNAs are expressed in the newly formed myotubes, but not at earlier stages. Transcripts for c-fos and c-jun mRNAs are expressed first in the myonuclei/nuclei of satellite cells at 3 h post-trauma. c-jun mRNA is expressed in both myoblasts and myotubes, while c-fos mRNA was not detected in these cells. These results suggest that myogenin plays important role in the regeneration of injured muscle and that c-jun and c-fos may have different roles in this process.
The myogenic precursor cells of postnatal and adult skeletal muscle are situated underneath the basement membrane of the myofibers. It is because of their unique positions that these precursor cells are often referred to as satellite cells. Such defined satellite cells can first be detected following the formation of a distinct basement membrane around the fiber, which takes place in late stages of embryogenesis. Like myoblasts found during development, satellite cells can proliferate, differentiate, and fuse into myofibers. However, in the normal, uninjured adult muscle, satellite cells are mitotically quiescent. In recent years several important questions concerning the biology of satellite cells have been asked. One aspect has been the relationship between satellite cells and myoblasts found in the developing muscle: are these myogenic populations identical or different? Another aspect has been the physiological cues that control the quiescent, proliferative, and differentiative states of these myogenic precursors: what are the growth regulators and how do they function? These issues are discussed, referring to previous work by others and further emphasizing our own studies on avian and rodent satellite cells. Collectively, the studies presented indicate that satellite cells represent a distinct myogenic population that becomes dominant in late stages of embryogenesis. Moreover, although satellite cells are already destined to be myogenic precursors, they do not express any of the four known myogenic regulatory genes unless their activation is induced in the animal or in culture. Furthermore, multiple growth factors are important regulators of satellite cell proliferation and differentiation. Our work on the role of one of these growth factors [platelet-derived growth factor (PDGF)] during proliferation of adult myoblasts is further discussed with greater detail and the possibility that PDGF is involved in the transition from fetal to adult myoblasts in late embryogenesis is brought forward.
L6 cells are committed skeletal muscle precursors which can be induced to differentiate into multinucleated, terminally differentiated myotubes. Upon differentiation, these immature skeletal myotubes enter a quiescent state and are unable to reenter the cell cycle. We have examined expression of a series of genes involved in regulation of progression through the G1/S boundary in undifferentiated L6 cells and during terminal differentiation of L6 myoblasts. While no change in the level of cyclin D1 transcript and a transient increase in cyclin D2 transcript were observed, a large increase in cyclin D3 expression was found. Immunohistochemistry demonstrated strong staining for cyclin D3 protein in the nuclei of the multinucleated myotubes from 4 independent myoblast cell lines; L6, L8, G8 and C2C12. Immunoprecipitation confirmed a greater than 20-fold increase in the levels of cyclin D3 protein in the differentiated L6 myotubes as well as its association with a number of proteins. Western assays demonstrated, further, that cyclin D3 was complexed with the cyclin dependent-kinases, cdk2 and cdk4, in differentiated L6 cells. However, while kinase activity specific for a GST-pRB fusion protein was seen for cyclin D3-containing complexes isolated from undifferentiated cells, the high levels of cyclin D3 in the differentiated myotubes had no associated kinase activity. These data demonstrate that cyclin D3 may also have a function in terminally differentiated, quiescent cells. The lack of cyclin D3-associated kinase activity and its association with a number of different proteins suggest that cyclin D3 may regulate the function of other proteins by direct interaction with these factors.
Skeletal muscle differentiation entails the coordination of muscle-specific gene expression and terminal withdrawal from the cell cycle. This cell cycle arrest in the G0 phase requires the retinoblastoma tumor suppressor protein (Rb). The function of Rb is negatively regulated by cyclin-dependent kinases (Cdks), which are controlled by Cdk inhibitors. Expression of MyoD, a skeletal muscle-specific transcriptional regulator, activated the expression of the Cdk inhibitor p21 during differentiation of murine myocytes and in nonmyogenic cells. MyoD-mediated induction of p21 did not require the tumor suppressor protein p53 and correlated with cell cycle withdrawal. Thus, MyoD may induce terminal cell cycle arrest during skeletal muscle differentiation by increasing the expression of p21.
Myogenic precursors in adult skeletal muscle (satellite cells) are mitotically quiescent but can proliferate in response to a variety of stresses including muscle injury. To gain further understanding of adult myoblasts, we analyzed myogenesis of satellite cells on intact fibers isolated from adult rat muscle. In this culture model, satellite cells are maintained in their in situ position underneath the fiber basement membrane. In the present study patterns of satellite cell proliferation, expression of myogenic regulatory factor proteins, and expression of differentiation-specific, cytoskeletal proteins were determined, via immunohistochemistry of cultured fibers. The temporal appearance and the numbers of cells positive for proliferating cell nuclear antigen (PCNA) or for MyoD were similar, suggesting that MyoD is present in detectable amounts in proliferating but not quiescent satellite cells. Satellite cells positive for myogenin, alpha-smooth muscle actin (alpha SMactin), or developmental sarcomeric myosin (DEVmyosin) appeared following the decline in PCNA and MyoD expression. However, expression of myogenin and alpha SMactin was transient, while DEV-myosin expression was continuously maintained. Moreover, the number of DEVmyosin + cells was only half of the number of myogenin + or alpha SMactin + cells--indicating, perhaps, that only 50% of the satellite cell descendants entered the phase of terminal differentiation. We further determined that the number of proliferating satellite cells can be modulated by basic FGF but the overall schedule of cell cycle entry, proliferation, differentiation, and temporal expression of regulatory and structural proteins was unaffected. We thus conclude that satellite cells conform to a highly coordinated program when undergoing myogenesis at their native position along the muscle fiber.
When skeletal muscle is damaged, it is repaired by the proliferation of mononuclear muscle precursor cells (mpc) which fuse either with one another to form young multinucleated muscle cells (myotubes) or with the ends of damaged myofibres (Robertson et al., 1990). The success of new muscle formation is related to the size of the injury, as after major trauma and extensive disruption of the external lamina of muscle fibres there is often significant replacement by fibrous and cellular connective tissue. Impaired muscle regeneration and progressive replacement by fat and connective tissue is a feature of myopathies such as Duchenne muscular dystrophy (DMD), although this results from many small discrete lesions constantly recurring over a long period of time rather than from a single large injury. Failed regeneration can be seen in simplistic terms as a failure of muscle precursor replication. In this review we shall concentrate on the biology of muscle precursor cells. For coverage of other aspects of regeneration such as resealing of damaged myofibres, revascularization and reinnervation, see Grounds (1991).
The time course of expression of the skeletal muscle-specific regulatory genes MyoD and myogenin was studied in primary cultures of skeletal muscle from adult SJL/J and BALB/c mice. In situ detection of expression with MyoD and myogenin riboprobes and myogenin antibody showed that the onset of expression of these genes occurred earlier in cells from SJL/J mice. Probe-positive cells and myotubes were also more frequent in cultures from SJL/J mice than in BALB/c. The onset of expression of MyoD and myogenin was delayed in cultured cells relative to the time course seen following injury in vivo. Myogenin protein was demonstrated in replicating cells and all myogenin-positive cells expressed desmin. The observed strain-specific difference infers a greater intrinsic myogenicity of cells in SJL/J muscle in vitro and reflects the superior capacity for new muscle formation previously reported in SJL/J mice in vivo.
The satellite cell is responsible for growth and repair of postnatal skeletal muscle. We investigated the expression of the myogenic regulatory gene (MRG) family in these cells in the stages from quiescence to fusion. Using polymerase chain reaction amplification of reverse-transcribed RNA (RT-PCR) isolated from adult rat satellite cells, we demonstrated a temporal sequence of gene activation, which is distinct from that previously observed in embryonic somatic cells. No MRG expression was detected in predominantly quiescent cells. MyoD is activated by 12 h in cell culture, prior to the first evidence of proliferation. MRF4 and myf-5 appear by 48 h and may be associated with the first division cycle. Myogenin is not detectable until 72 h after satellite cell recovery from the muscle fiber, coincidental with the first evidence of differentiation.
Evidence now suggests that satellite cells constitute a class of myogenic cells that differ distinctly from other embryonic myoblasts. Satellite cells arise from somites and first appear as a distinct myoblast type well before birth. Satellite cells from different muscles cannot be functionally distinguished from one another and are able to provide nuclei to all fibers without regard to phenotype. Thus, it is difficult to ascribe any significant function to establishing or stabilizing fiber type, even during regeneration. Within a muscle, satellite cells exhibit marked heterogeneity with respect to their proliferative behavior. The satellite cell population on a fiber can be partitioned into those that function as stem cells and those which are readily available for fusion. Recent studies have shown that the cells are not simply spindle shaped, but are very diverse in their morphology and have multiple branches emanating from the poles of the cells. This finding is consistent with other studies indicating that the cells have the capacity for extensive migration within, and perhaps between, muscles. Complexity of cell shape usually reflects increased cytoplasmic volume and organelles including a well developed Golgi, and is usually associated with growing postnatal muscle or muscles undergoing some form of induced adaptive change or repair. The appearance of activated satellite cells suggests some function of the cells in the adaptive process through elaboration and secretion of a product. Significant advances have been made in determining the potential secretion products that satellite cells make. The manner in which satellite cell proliferative and fusion behavior is controlled has also been studied. There seems to be little doubt that cellcell coupling is not how satellite cells and myofibers communicate. Rather satellite cell regulation is through a number of potential growth factors that arise from a number of sources. Critical to the understanding of this form of control is to determine which of the many growth factors that can alter satellite cell behavior in vitro are at work in vivo. Little work has been done to determine what controls are at work after a regeneration response has been initiated. It seems likely that, after injury, growth factors are liberated through proteolytic activity and initiate an activation process whereby cells enter into a proliferative phase. After myofibers are formed, it also seems likely that satellite cell behavior is regulated through diffusible factors arising from the fibers rather than continuous control by circulating factors.(ABSTRACT TRUNCATED AT 400 WORDS)
A unique pattern of expression of the four muscle regulatory factor (MRF) proteins was found to distinguish early somitic from embryonic, fetal and newborn limb myogenic cells in vitro. Expression of the myosin heavy chain (MHC), MyoD, myogenin, Myf-5, and MRF4 proteins was examined by immunocytochemistry in cultures of four distinct types of mouse myogenic cells: somitic (E8.5), embryonic (E11.5), fetal (E16.5) and newborn limb. In embryonic, fetal and newborn cultures, the MRF proteins were expressed in generally similar patterns: MyoD was the first MRF expressed; MyoD and myogenin were expressed by more cells than Myf-5 or MRF4; and each of the four MRFs was found both in cells that expressed MHC and in cells that did not express MHC. In cultures of somitic cells, in contrast, Myf-5 was expressed first and by more cells than MyoD or myogenin; MRF4 was not detected; and the MRFs were never found to be coexpressed with MHC in the same cell. Thus, some somitic cells had the unexpected ability to maintain MHC expression in the absence of detectable MRF protein expression. The different myogenic programs of embryonic, fetal and newborn myogenic cells are not, therefore, a simple result of qualitatively different MRF expression patterns, whereas myogenesis by somitic cells does include a unique pattern of MRF expression.
The formation of skeletal muscle during vertebrate embryogenesis requires commitment of mesodermal precursor cells to the skeletal muscle lineage, withdrawal of myoblasts from the cell cycle, and transcriptional activation of dozens of muscle structural genes. The myogenic basic helix-loop-helix (bHLH) factors - MyoD, myogenin, Myf5, and MRF4 - act at multiple points in the myogenic lineage to establish myoblast identity and to control terminal differentiation. Recent studies have begun to define the inductive mechanisms that regulate myogenic bHLH gene expression and muscle cell determination in the embryo. Myogenic bHLH factors interact with components of the cell cycle machinery to control withdrawal from the cell cycle and act combinatorially with other transcription factors to induce skeletal muscle transcription. Elucidation of these aspects of the myogenic program is leading to a detailed understanding of the regulatory circuits controlling muscle development.
The activity of the E2 F-family of transcription factors is tightly linked to control of the cell cycle. p107 and p130, two closely related members of the retinoblastoma protein-family of negative cell cycle regulators, modulate the activity of the E2f-family proteins by direct interaction with these factors. To understand the role of p107 and p130 in progression through or exit from the cell cycle, we have characterized the expression, phosphorylation state, cyclin-binding, and E2f-binding activity of p107 and p130 during terminal differentiation of rat myoblast cells into immature skeletal muscle (myotubes). In exponentially growing L6 myoblasts, p107 is phosphorylated in a cell cycle-dependent manner, and E2f-site binding complexes containing p107 is phosphorylated in a cell cycle-dependent manner, and E2f-site binding complexes containing p107 can be observed throughout the cell cycle. During differentiation of L6 cells, p107 levels are reduced, while p130 protein levels are increased 8-fold. Despite both p107 and p130 becoming hypophosphorylated during myogenesis, the E2F-site DNA-binding complexes containing p107 observed in exponentially growing myoblasts are quantitatively replaced in myotubes with complexes containing only p130. In myotubes, p107 is not associated with E2f-family proteins that are capable of binding DNA. The failure to observe p107-containing complexes in myotubes appears to be due to the differentiation-specific induction of both p130 and cyclin D3, p107 is found in complexes with cyclin D3 in myotubes, and the addition of exogenous cyclin D3 or p130 to lysates from undifferentiated L6 cells was able to disrupt p107-containing E2F-site binding complexes. In myotubes, p130 also forms complexes with cyclin D3 as well as cyclin E, cdk2, and cdk4. We are able to copurify cyclin D3 with cyclin E from myotubes, indicating the presence of a macromolecular complex containing both cyclin E and cyclin D3 simultaneously bound to p130. Thus, in myoblasts, p107 is normally involved in regulation of E2f-family proteins during cell cycle progression, while p130 is a differentiation-specific regulator of E2f activity. Our results also provide evidence that the apparent positive regulator of cell cycle progression, cyclin D3, has a function in terminally differentiated muscle cells.