Дисертації з теми "Skeletal muscle satellite cells"

The primary cilium has recently been recognised as an essential regulator of the Sonic hedgehog (Shh) signalling pathway. Mutations that disrupt cilia function in humans can cause conditions known as ciliopathies. A wide range of phenotypes is observed in chick and mouse ciliopathy models,including polydactyly, craniofacial defects and polycystic kidneys. The Shh pathway and therefore primary cilia are vital for many developmental processes, including embryonic muscle development, with recent evidence suggesting they may also play a role in adult muscle regeneration. Our studies focus on the Talpid3 gene, which encodes a centrosomal protein required for primary cilia formation and Shh signalling. The Talpid3 loss-of-function mutant has perturbed ciliogenesis and displays many of the phenotypes that are typically associated with developmental Shh mutants and with ciliopathies. Talpid3 mutants have defects in Shh signalling, and processing of Gli transcription factors is affected in structures such as the developing limb buds and the neural tube. However, the role of Talpid3 in muscle development and regeneration remains unknown. The role of Talpid3 in adult muscle regeneration was investigated using a tamoxifen inducible, satellite cell specific knock-out of Talpid3 in mice. This mouse model was generated by crossing Talpid3 floxed mice to a mouse carrying an inducible Pax7-CreERT2 allele. To determine whether loss of Talpid3 affects muscle regeneration a cardiotoxin injury model was used. This showed that loss of Talpid3 in satellite cells results in a regeneration defect as fibres were smaller after 5, 10, 15 and 25 days of regeneration compared to control mice. This defect may be due to a reduced ability of Talpid3 mutant satellite cells to differentiate. We also show that Talpid3 plays a role in satellite cell self-renewal as we observe a complete loss of regeneration in some areas of the muscle following repeat injuries. We provide the first evidence that Talpid3 is critical for the regeneration of skeletal muscle following injury.

Young female rats treated with trenbolone demonstrated an increase in weight gain per day and overall weight increase during the treatment period. Trenbolone treated rats also experienced improved feed efficiency. Muscles removed from the lower hind limb of trenbolone treated rats had a greater DNA to protein ratio than muscles from control animals. However, there was no significant difference in wet muscle weight between trenbolone treated and control muscles. Satellite cells from untreated female rats were not responsive to trenbolone added in vitro. In studies utilizing serum free medium, trenbolone alone, and in the presence of growth factors, could not stimulate proliferation above controls. In similar serum free medium studies, satellite cells from trenbolone treated rats were more responsive to growth factors than cells from control rats.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2006" Includes bibliographical references.

Satellite cells are defined by their position beneath the basal lamina of myofibres, and are a source of new myonuclei in adult skeletal muscles. However, other phenotypes also contribute to muscle regeneration, and the relative importance of satellite cells is not known. This work aimed to analyse the stem cell potential of satellite cells by formally investigating their contribution to muscle regeneration. Myofibres isolated from extensor digitorum longus, soleus, and tibialis anterior muscles were found to have respective means of 7,22 and 10 associated satellite cells. When a single myofibre was grafted into an irradiated dystrophic mouse muscle, the associated satellite cells underwent extensive, stem cell-like proliferation, generating progeny which sometimes gave rise to a cluster of more than 100 new myofibres. Cluster size varied according to the muscle group from which the graft was derived, but was not proportional to satellite cell number. Primary myoblasts derived from equivalent muscle groups did not undergo such extensive proliferation, or show inter-muscle variability, suggesting that stem cell activity is critically dependent on a component of the satellite cell niche. Single myofibres isolated from irradiated muscles were non-myogenic after grafting. Satellite cells associated with single myofibres were found to generate new satellite cells in engrafted muscles, demonstrating that satellite cell compartment is maintained by self-renewal. When single myofibre-engrafted muscles were damaged with myotoxin, graft-derived cells underwent rapid clonal expansion to regenerate compact clusters of donor-derived myofibres. The percentage of engrafted muscles containing identifiable donor-derived nuclei was increased after damage, showing that previously inactive cells had been recruited into an active myogenic program. Without experimentally-induced damage, frequency of muscle formation and cluster size were spontaneously augmented over time. These findings demonstrate that satellite cells have several stem cell-like qualities, and thus constitute a self-sufficient and sustainable source of regeneration in adult muscles.

The project is divided in two sections: the formeris focused on the study of the relationship between the NO system, mitochondrial structure/activity and skeletal muscle wasting, paying attention on the role of autophagy and using a mouse model in which NOS1 (nNOSμ) is absent. My data demonstrate that an altered NO signaling leads to mitochondrial dysfunction resulting in enhanced autophagy and reduced muscle growth, however not associated with atrophy induction. Furthermore autophagy is essential to maintain muscle mass, but its role on regenerating population and its impact on muscle growth and development has not been evaluated yet. Starting form conclusion in the second part of my PhD project i focalized my attention on the role of autophagy specifically on satellite cells population, generating a transgenic mice in which autophagy is selectively inhibited in satellite cells and studying their impact on muscle growth. My data suggest that autophagy is involved in the controlling of satellite cells functions. Autophagy loss of function in satellite cells impairs their proliferation rate as well as their capability to fuse and differentiate. The study of these two different transgenic mice reveals as in muscle, unbalanced autophagy from the early phase of growth affects satellite cells population causing muscle wasting and suggests how during skeletal muscle development is important to have appropriate levels of autophagy.

In spite of its post mitotic nature, skeletal muscle maintains remarkable plasticity. Muscle fibres (myofibres) are capable of large alterations in their size as well as an enormous ability to regenerate following injury – thanks to a potent population of resident stem cells (satellite cells). Deciphering the molecular signalling networks responsible for skeletal muscle growth and regeneration is of key scientific interest – not least because of the therapeutic potential these pathways may hold for the treatment of diseases such as muscular dystrophy. In this thesis, the transcriptional co-factor Yes-Associated protein (Yap), the downstream effector of the Hippo Pathway, was investigated in skeletal muscle. Using gain and loss of function approaches within in vitro, ex vivo and in vivo models, the contribution of Yap in regulating both satellite cell behaviour and myofibre growth was investigated. Yap expression and activity are dynamically regulated during satellite cell activation, proliferation and differentiation ex vivo. Overexpression of Yap increased satellite cell proliferation and maintained cells in a ‘naive’, ‘activated’ state by inhibiting myogenic commitment. Knock-down of Yap impaired satellite cell expansion, but did not influence myogenic differentiation. Yap interacts with Tead transcription factors in myoblasts to upregulate genes such as CyclinD1 and Myf5. Forced expression of Yap eventually led to the oncogenic transformation of myoblasts in vitro. Contrary to predictions, constitutive expression of Yap under an inducible muscle-specific promoter in adult mice failed to induce growth and instead led to muscle wasting, atrophy and degeneration – providing evidence against the notion that Yap represents a universal regulator of tissue growth. These data provide the first insight into the function of Yap in skeletal muscle. Results highlight a novel role for Yap in regulating myogenic progression in satellite cells, as well as its propensity to induce oncogenic transformation. The precise function of Yap in adult myofibres remains unclear however, data presented here demonstrates clear cell-type specific roles for Yap compared to observations made in other tissues.

Skeletal muscle satellite cells located between the plasma membrane and the basal lamina of muscle fibres, could for many years, only be studied in situ by electron microscopy. The introduction of immunohistochemistry and the discovery of molecular markers of satellite cells then made them accessible for light microscopic studies and a wealth of information is today available. Satellite cells are myogenic stem cells that can be activated from a quiescent state to proliferate for self-renewal or differentiate into myogenic cells. The satellite cells are involved in muscle growth during fetal and postnatal development and play a key role in repair and regeneration of damaged muscle fibres. The satellite cells are also essential for muscle fibre hypertrophy and maintenance of muscle mass in the adult. When the present thesis was initiated, studies on satellite cells in human skeletal muscle relied on the neuronal cell adhesion molecule (NCAM) as a marker for satellite cell identification. The results from different studies varied markedly. Therefore the aims of the present thesis were i) to develop a highly reliable method using light microscopy for satellite cell identification and quantification in biopsies of human skeletal muscle in normal and pathological conditions. A molecular marker for the myofibre basal lamina or plasma membrane to enhance the reliability of myonuclei and satellite cell identification were to be included. Furthermore unbiased morphometric methods should be used in the quantification process. ii) to evaluate which molecular markers which had been described for satellite cell and stem cell identification in different cell states (quiescence, activated or differentiated) are the most useful for studies on human skeletal muscle. iii) to further explore the function and heterogeneity of satellite cells with respect to different markers in human skeletal muscle by studying the effects of strength-training, intake of anabolic substances and pathological conditions. A new immunofluorescence method was developed where in the same tissue section, two satellite cell markers, the basal lamina and nuclei were monitored. From the evaluation of different markers it was found that both NCAM and Pax7 identified the majority of satellite cells but that both markers were needed for reliable identification. The members of the myogenic regulatory family were evaluated and by using the new method MyoD and myogenin were found to be useful markers to identify activated and differentiated satellite cells. Upon re-examination of biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects it was observed that the new results on satellite cell frequency were significantly different from those obtained when using staining for NCAM and nuclei alone. In addition three subtypes of satellite cells (94.4% NCAM+/Pax7+, 4.2% NCAM+/Pax7– and 1.4% NCAM–/Pax7+) were observed. Thus the multiple marker method gave more information about satellite cells heterogeneity in human muscle and we propose that this is more reliable than previous methods. Low numbers of MyoD or myogenin stained satellite cells were observed in both untrained and strength trained subjects. Other markers such as DLK1/FA1, a member of the EGF-like family and c-Met, the receptor for hepatocyte growth factor showed that satellite cell heterogeneity in human muscle is far greater than previously shown. Furthermore, new evidence is presented for so called fibre splitting observed in hypertrophic muscle fibres to be due to defect regeneration of partially damaged fibres.

The vast majority of horses engage in some form of exercise, whether it be for leisure or competition. Despite almost half of the approximately 7.2 million horses engaging in structured athletic work, very little is known about one of the most critical facets of recovery: satellite cells (SCs). Satellite cells lie adjacent to the myofiber of skeletal muscle, poised to enter the myogenic program and fuse to the nearby muscle after a damaging event. Hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) transcript abundance increased after an exhaustive bout of endurance exercise in concert with myogenic regulator factors and preceding increased SC abundance in a previous study. This suggests that SCs may participate in repair of exercise-induced muscle damage. To assess the role of HGF in this process, equine SCs (eqSCs) were isolated from the gluteus medius of mature thoroughbred geldings for activation, proliferation and differentiation assays. Activation was not accelerated by 1, 5, 10, or 25 ng/mL HGF. Instead, 25 ng/mL HGF increased the proliferation rate of eqSC via protein kinase C δ and decreased differentiation. The influence of dietary L-citrulline, an amino acid that has the potential to influence SC activity and nutrient availability by its metabolism to L-arginine, was assessed during recovery from exercise in unfit adult horses. To model submaximal exercise, horses were exercised for 1 h at an average heart rate of 116 bpm, suggested to be typical of a heavy exercise session by the National Research Council. L-citrulline decreased myogenin mRNA abundance compared to controls while exercise increased peroxisome proliferator-activated receptor gamma coactivator 1- α (PGC1α) mRNA abundance, a master regulator of energy metabolism, at 1 d post-exercise. Although SCs were not activated in response to a single bout of submaximal exercise, metabolic regulators increased in the early period of recovery. Through these studies eqSC dynamics during exercise are better defined.
Doctor of Philosophy
The horse is well-known as an athletic creature and is often used in amateur and professional athletic events. Despite its popularity as a pastime in low and high-stakes competition, certain facets directly related to performance during exercise remain relatively unstudied. One crucial component of recovery from exercise is the intrinsic ability of skeletal muscle to repair exercise-induced muscle damage. This is accomplished largely through the incorporation of new nuclei, which originate from a position orbiting the muscle, hence the name satellite cells. This cell is essential to muscle regeneration from injury as often demonstrated in rodent models, but the role of satellite cells in recovery from exercise remains elusive in all species, but particularly so in horses. For instance, whether satellite cells only contribute nuclei after exercise to stimulate gains in muscle mass or whether they may also play a role in the process of adaptation to exercise is not clearly understood. The purpose of my work was to define the response of satellite cells to hepatocyte growth factor, a factor present in skeletal muscle during exercise that is already well-studied in rodent models. Additionally, to determine whether the addition of the non-essential amino acid, citrulline, would influence satellite cells and nutrient reserves after a session of submaximal exercise. I found that hepatocyte growth factor does not influence satellite cells isolated from horses in the same way it influences those from rodents, nor through the same mechanisms. Additionally, I found that satellite cells were not stimulated after a session of submaximal exercise, but a factor involved in regulation of genetic expression that is associated with satellite cells and skeletal muscle was downregulated with the addition of citrulline. Together, these results suggest that satellite cells may behave like other species in some ways, such as some responses to hepatocyte growth factor and the lack of response to a submaximal bout of exercise, but that there is still much to be learned in order to begin to influence management and training decisions as regards skeletal muscle recovery.

Skeletal satellite cells are adult stem cells located among muscle fibers. Proliferation, migration and subsequent differentiation of these cells are critical steps in the repair of muscle injury. We document in this study the roles and mechanisms through which the NAPDH oxidase complex regulates skeletal satellite cell proliferation. The NADPH oxidase subunits Nox2, Nox4, p22phox, p47phox and p67 phox were detected in primary human and murine skeletal muscle satellite cells. In human satellite cells, NADPH oxidase-fusion proteins were localized in the cytosolic and membrane compartments of the cell, except for p47 phox, which was detected in the nucleus. In proliferating subconfluent satellite cells, both Nox2 and Nox4 contributed to O2- production. However, Nox4 expression was significantly attenuated in confluent cells and in differentiated myotubes. Proliferation of satellite cells was significantly reduced by antioxidants (N-acetylcysteine and apocynin), inhibition of p22phox expression using siRNA oligonucleotides, and reduction of Nox4 and p47phox activities with dominant-negative vectors resulted in attenuation of activities of the Erk1/2, PI-3 kinase/AKT and NFkappaB pathways and significant reduction in cyclin D1 levels. We conclude that NADPH oxidase is expressed in skeletal satellite cells and that its activity plays an important role in promoting proliferation of these cells.

Pw1/Peg3 est un gène d’empreinte parental exprimé par l’allèle paternel. Il est exprimé dans l’ensemble des populations de cellules souches, y compris les cellules satellites du tissu musculaire. Nous avons découvert que la perte constitutive de Pw1/Peg3 entraîne une perte de la masse musculaire, résultat d’une diminution du nombre de fibres musculaires. Le nombre de fibres réduit est présent dès la naissance. De plus, les souris double KO ont un nombre de fibres encore inférieur, suggérant que l’allèle maternel est fonctionnel pendant le développement pré-natal, et des analyses de souris hybrides C57BL6J/CAST/Ei révèlent une expression bi-allélique de Pw1/Peg3 d’environ 10%. Pw1/Peg3 est également fortement exprimé après blessure du muscle squelettique. Chez les souris Pw1/Peg3 KO, nous avons observé que les cellules satellites montrent une réduction de leur capacité d’auto-renouvèlement à la suite d’une blessure. Pw1/Peg3 est également exprimé dans une sous-population de cellules souches interstitielles, les PICS. Afin de déterminer le rôle spécifique de Pw1/Peg3 dans les cellules satellites nous avons croisé notre allèle conditionnel Pw1/Peg3 avec la lignée Pax7-Cre-ER. Ces souris ont un phénotype présentant un défaut de régénération prononcé, montrant ainsi un rôle clair et direct de Pw1/Peg3 dans la fonction régénératrice des cellules satellites. En résumé, l’ensemble de ces données montre un rôle de Pw1/Peg3 dans le développement fœtal et la détermination du nombre de fibres musculaires par son action dans l’auto-renouvellement des cellules satellites du tissu musculaire
Pw1/Peg3 is a parentally imprinted gene expressed from the paternal allele. It is expressed in all adult progenitor/stem cell populations examined to date including muscle satellite cells. We examined the impact of loss-of-function of Pw1/Peg3 in skeletal muscle, a tissue that greatly contributes to body mass. We found that constitutive loss of Pw1/Peg3 results in reduced muscle mass resulting from a decrease in muscle fiber number. The reduced fiber number is present at birth. Mice lacking both the paternal and maternal alleles display a lower fiber number as compared to mice carrying the paternal deletion, suggesting that the maternal allele is functional during prenatal development. Hybrid analyses (C57BL6J and Cast/Ei) of muscle tissue reveal a bi-allelic expression of Pw1/Peg3 around 10%. Pw1/Peg3 is strongly up-regulated in response to muscle injury. Using the constitutive Pw1/Peg3 knock out mouse, we observed that satellite cells display a reduced self-renewal capacity following muscle injury. Pw1/Peg3 is expressed in satellite cells as well as a subset of muscle interstitial cells (PICs). To determine the specific role of Pw1/Peg3 in satellite cells, we crossed our conditional Pw1/Peg3 allele with the Pax7-CreER line. Interestingly, these mice displayed a more pronounced phenotype of impaired regeneration revealing a clear and direct role for Pw1/Peg3 in satellite cells. Taken together, our data show that Pw1/Peg3 plays a role during fetal development in the determination of muscle fiber number that is gene-dosage dependent and plays a specific role in muscle satellite cell self-renewal

About half of the human body mass is comprised of skeletal muscles, a component of the musculoskeletal system involved in maintaining body posture, gait and locomotion. Additionally, skeletal muscles have essential function in glucose metabolism and thermoregulation. Thus, maintenance of skeletal muscle homeostasis is critical for the health of organisms. Satellite cells are muscle-specific stem cells responsible for postnatal growth, regeneration upon injury, and maintenance of skeletal muscle homeostasis. Satellite cell's activity is regulated by a sophisticated network of signalling pathways, which act in a combinatorial manner to regulate satellite cell expansion and differentiation, and to preserve a pool of stem cells during the life course of skeletal muscles. Many of these signalling pathways are known to operate during embryonic myogenesis and are re-activated in adult myogenesis. One such signalling pathway, Sonic hedgehog (Shh) signalling, controls several aspects of myogenesis in the embryo and previous studies have indicated that it plays a role in adult myogenesis. However, it remains unclear whether Shh signalling acts upon satellite cells or non-myogenic resident cells. This study builds on previous work in the lab showing that Shh signalling is cell-autonomously required in satellite cells for efficient muscle regeneration. Through a combination of ex vivo and in vivo genetic approaches, I demonstrated that up-regulation of Shh signalling increased the proliferation of satellite cells by accelerating their entry into cell cycle and progression through the cycle program. Up-regulation of Shh signalling in satellite cells altered also the balance between self-renewal and differentiation, by promoting asymmetric cell division at the expense of symmetric cell division. Given the involvement of Shh signalling in tumour development in other systems and in skeletal muscle tumours i.e. Rhabdomyosarcoma (RMS), the present study may provide novel insights into the role of Shh signalling in the pathogenesis of RMS through the deregulation of satellite cell homeostasis.

Skeletal muscle possesses the remarkable capacity to complete a rapid and extensive regeneration, even following severe damage. The regenerative ability of skeletal muscle relies on Satellite Cells (SCs), a population of muscle specific adult stem cells. However, during aging or under several pathological conditions, the ability of skeletal muscle to fully regenerated is compromised. Here, a morphological and molecular study on SCs from patients affected by ALS is described. Moreover, the role of the cell cycle regulator P16Ink4a during skeletal muscle regeneration and aging has been investigated.

Skeletal muscle possesses the remarkable capacity to complete a rapid and extensive regeneration, even following severe damage. The regenerative ability of skeletal muscle relies on Satellite Cells (SCs), a population of muscle specific adult stem cells. However, during aging or under several pathological conditions, the ability of skeletal muscle to fully regenerated is compromised. Here, a morphological and molecular study on SCs from patients affected by ALS is described. Moreover, the role of the cell cycle regulator P16Ink4a during skeletal muscle regeneration and aging has been investigated.

C/EBPβ is a bZIP transcription factor known to be involved in various physiological processes, including adipogenesis, osteogenesis and liver development. Previous studies in this laboratory revealed an inhibition of myogenesis and reduced myogenic protein expression in 5-azacytidine treated mesenchymal stem cells retrovirally transduced to overexpress C/EBPβ. The goal of this thesis was to evaluate the role of C/EBPβ in myogenic differentiation by overexpression in C2C12 myoblasts and primary myoblasts. We demonstrate reduced MyoD protein expression and subsequent downregulation of myogenic proteins during differentiation following C/EBPβ overexpression. We localized C/EBPβ to the quiescent Pax7+ satellite cells associated with the muscle fiber. Upon satellite cell activation, we observed the downregulation of C/EBPβ protein expression prior to MyoD protein expression. Furthermore, the re-expression of C/EBPβ correlated with the loss of MyoD expression later in differentiation. Histological analysis of C/EBPβ-/- mice revealed smaller fibers and a reduced Pax7+ satellite cell population as compared to control animals. In this thesis, we propose that C/EBPβ is a negative regulator of skeletal muscle differentiation by inhibiting the expression of MyoD, thus impairing proper progression through the myogenic program. In addition, we propose a role for C/EBPβ in the maintenance of undifferentiatied satellite cells.

Skeletal muscle regeneration is a multifaceted process requiring the spatial and temporal coordination of myogenesis as well as angiogenesis. While these processes are often studied independently, recent evidence from our lab has shown that the resident adult stem cell population within skeletal muscle, called satellite cells, begins secreting soluble growth factors likely to contribute to the proangiogenic response. The overall aim of this study is to investigate the role of pro-angiogenic factors secreted by satellite cells during skeletal muscle regeneration. Results from the study indicate that Hepatocyte Growth Factor (HGF) is a critical protein for the proangiogenic effect of satellite cells. It was also shown that in hypoxic environments, such as those seen in an injury state, it appears that satellite cells decrease their proangiogenic effect if oxygen levels fall below a threshold level. This decrease in pro-angiogenic effect in the hypoxic environment appears to be due to the decrease in HGF expression and protein secretion and is not compensated for by the increase in Vascular Endothelial Growth Factor secretion also seen in the hypoxic response. Furthermore, the regulation of HGF in these hypoxic conditions appears to be in part due to increased levels of hypoxia inducible factor, which are acting on the hypoxia response element site found on the HGF promoter. In the last set of experiments, this injury response was further investigated as the effect of satellite cell mediated angiogenesis was examined in the disease state of muscular dystrophy. Here, we also observed a reduction in angiogenesis from media conditioned by satellite cells from dystrophic muscle compared to healthy muscle. Overall, this study further strengthens the case for satellite cells as important mediators of the angiogenic response in regenerating muscle and may serve as a potential site for therapeutic intervention in the future.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

Previous work from our laboratory in the mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that a fraction of muscle stem cells (i.e., satellite cells) (MuSCs) progressively lose the expression of myogenic markers during the progression of the disease. In the process of characterizing this aberrant behaviour, we serendipitously discovered that MuSCs might be separated into two distinct subpopulations based on the expression of the GPI-anchored surface protein CD90. Crucially, this separation does not correlate with a divergence from the myogenic lineage; instead, it separates the pool of MuSCs into two subpopulations, both maintaining myogenic properties in healthy muscles. These two newly identified subpopulations do not overlap with any previously reported subpopulation and may be prospectively isolated; present a different response in terms of kinetics of activation and differentiation during the regenerative process induced by acute muscle damage; show a different propensity to enter in GAlert state upon distal injury; display dissimilar pAMPK activity and contain a different amount of mitochondria; are present in different proportions in distinct muscle groups; differentially express ECM encoding genes during quiescence. Moreover, one of the two subpopulations can give rise to the other and therefore appears to be upstream in the lineage hierarchy. Notably, loss of function experiments, in which CD90 was downregulated in MuSCs, diminish the difference in activation displayed by the two subpopulations. This demonstrates that CD90 is a molecular determinant of MuSCs functional diversification. Importantly, although the two subpopulations of MuSCs are numerically similar in healthy limb muscles, one of the two subpopulations is lost with time in dystrophic mdx mice. Based on these data, we are hypothesizing that an imbalance between the two newly identified subpopulations may impair regeneration in the dystrophic muscles. These observations not only increase our knowledge of the molecular and cellular dynamics that are controlling normal and pathological muscle homeostasis but also open the possibility that restoring the proper functional equilibrium between subpopulations of MuSCs may counteract the progression of the dystrophic disease.

Skeletal muscle has a remarkable capacity for regeneration, mediated by muscle stem cells that can self-renew or differentiate to form the mature myofibers of the tissue. Several human diseases are characterized by a loss of function and strength in skeletal muscle, with impairments in the ability to regenerate and consequent decreases in quality of life and increases in mortality. The work in this dissertation has focused on developing methods for combating muscle disease. This goal has been approached through attempts at cell replacement therapy - by generating muscle cells that can be engrafted in vivo. I also investigated the influence on regeneration of the skeletal muscle microenvironment (skeletal muscle-resident fibroblasts), and systemic environment (inflammation in myogenic and non-myogenic tissues), both of which were found to affect skeletal muscle stem cell behavior and the efficiency of myogenic regeneration. Ultimately, these studies identified novel factors that impair or improve skeletal muscle differentiation, and that offer the potential to modulate the process of muscle regeneration. In the process of investigating if induced pluripotent stem cells from skeletal muscle retain an epigenetic memory conducive to myogenic differentiation, I discovered that precursor cells in skeletal muscle reprogram to a pluripotent state more efficiently. However, these induced pluripotent stem cells, like embryonic stem cells, retain strong barriers to skeletal muscle differentiation. Together, these findings offer insights into the process of muscle regeneration, and suggest new potential pathways towards treatment of muscle disease.

Les facteurs de croissance de la superfamille TGF-β jouent un rôle dans toutes les étapes de la myogenèse prénatale et régissent l'entretien des muscles adultes. Les protéines morphogénétiques osseuses (BMPs) sont membres de la sous-famille des TGF-β et sont à l’origine de signaux clés régulant le développement musculaire embryonnaire. Cette thèse étudie le rôle de la signalisation BMP dans les cellules souches musculaires, dénommées cellules satellites. J'ai montré qu’après la naissance les BMPs régulent la croissance des fibres musculaires dépendante des cellules satellites. Suite à l’inhibition de la voie BMP, j'ai observé que les précurseurs myogéniques deviennent quiescents et cessent de progresser vers la différenciation, tandis que le traitement avec BMP4 suffit pour réactiver leur programme myogénique. La signalisation BMP affecte aussi la taille du muscle indépendante des cellules satellites. J'ai observé que les BMPs fournissent un signal hypertrophique et protègent de l’atrophie musculaire suite à une dénervation. Dans les conditions précédentes, la voie BMP inhibe l'expression de l’ubiquitine ligase E3, Fbxo30. J'ai analysé l'interaction entre la myostatine et la signalisation BMP. La myostatine est un autre membre de la famille des TGF-β, mais elle se lie à des récepteurs différents de ceux des BMPs. En l'absence de myostatine, l’hypertrophie musculaire dépend entièrement de la signalisation BMP. La dénervation musculaire chez les souris déficientes en myostatine provoque une atrophie, aggravée par l’inhibition des BMPs. Par conséquent, la voie BMP est un signal hypertrophique essentiel dans le muscle adulte qui prédomine sur la signalisation de la myostatine
Growth factors of the TGF-β superfamily play a role in all stages of prenatal myogenesis and govern adult muscle maintenance. Bone morphogenetic proteins (BMPs) are members of the TGF-β subfamily and are key signals that regulate embryonic and fetal muscle development. This work investigates the role of BMP signaling in muscle stem cells of the postnatal muscle, the satellite cells. I showed that BMPs regulate satellite cell-dependent growth of postnatal fibers and the generation of the satellite cell pool. After inhibition of BMP signaling, I observed that myogenic precursor cells become quiescent and fail to progress towards differentiation, whereas treatment with BMP4 on its own is sufficient to reactivate the myogenic program. BMP signaling also affects the size of the muscle in a satellite cell-independent manner. I found that BMPs provide a hypertrophic signal and protect from denervation-induced muscle atrophy. Under such condition, BMP signaling inhibits the expression of the E3 ubiquitin ligase Fbxo30. I further analyzed the interaction between myostatin and BMP signaling. Myostatin is another member of TGF-β superfamily, but myostatin and BMPs bind to different receptors for signaling. Large muscles in absence of myostatin entirely depend on the presence of BMP signaling. Denervation of muscle in myostatin mutant mice causes a strong muscle atrophy, which is aggravated by the inhibition of BMP signaling. Therefore, the BMP pathway is a fundamental hypertrophic signal in adult muscle and is dominant over myostatin signaling

The age-related loss of skeletal muscle mass and function is accompanied by a decline in regenerative capacity. The processes that facilitate healthy muscle repair are complex, involving several phases of degradation and rebuilding of muscle tissue and the surrounding microenvironment. Specifically, myogenic progenitor cells known as satellite cells are the most influential in repairing damaged muscle tissue. Following injury, satellite cells become activated and migrate, proliferate and fuse with mature skeletal muscle fibers to restore homeostasis to the tissue. However, satellite cells do not act in isolation, a robust inflammatory response is necessary to facilitate successful and rapid healing. Macrophages are one of the first and most abundant immune cells to infiltrate damaged skeletal muscle tissue. Primarily, macrophages adapt to a proinflammatory state to clear the area of cellular debris, promote degradation of the extracellular matrix and stimulate satellite cell activation and proliferation. Afterwards, a timely transition to an anti-inflammatory state directs rebuilding of the extracellular matrix and terminal differentiation of satellite cells. Indeed, the inhibition of macrophage activity leads to impaired healing and loss of skeletal muscle function. Little is known regarding the behavior of macrophages in aged skeletal muscle following injury in humans. Thus, the objective of this dissertation is to investigate the age-related response of macrophages in human skeletal muscle, and their role in muscle repair.

Thoroughbred racehorses undergo strenuous exercise which often leads to the occurrence of exercise-induced pulmonary hemorrhage (EIPH), in which capillaries rupture within the alveoli in the lungs causing bleeding. Severe cases of EIPH lead to epistaxis and may result in fatality. Presently, the loop diuretic furosemide is the only medication approved to mitigate the effects of EIPH. Often regarded in the racing industry as "performance enhancing" due to 4% weight loss ensued by its diuretic effect, it is unknown what effects furosemide may have on muscle recovery. Therefore, the objective of this study was to determine the effects various doses of furosemide may have on equine satellite cell (eqSC) myogenesis and metabolism. Mitotic index was increased (P<0.05) as a result of treatment with 100 µg/mL furosemide, a 10-fold pharmacological dose, in comparison to vehicle, but was not different (P>0.05) compared to the physiological dose of 10 µg/mL furosemide. Average cell number decreased (P<0.05) in the excess furosemide group compared to all other groups. Pax7 expression did not differ (P>0.05) between groups. Expression of the differentiation transcription factor myogenin, and embryonic sarcomeric myosin heavy chain decreased (P<0.05) when cells were treated with 100 µg/mL furosemide. Fusion index and myotube area decreased (P<0.05) as a result of treatment with excess furosemide. Glycogen concentration in myotubes was lower (P<0.05) following treatment with 100 µg/mL furosemide, while IGF-1 was unsuccessful in rescuing the effects of furosemide. Excess furosemide decreased expression of muscle creatine kinase while increasing expression of phosphoglucomutase 1, glycogen synthase 1, and glycogen branching enzyme 1 (P<0.05). Excess furosemide decreased basal oxygen consumption rate (OCR) and increased OCR after addition of oligomycin (P<0.05). Excess furosemide did not affect myotube glycolysis rates in vitro. In conclusion, furosemide inhibits muscle differentiation and oxidative metabolism in eqSCs.
Master of Science
Thoroughbred racehorses often bleed from the lungs as a result of high-intensity exercise. This condition can oftentimes be fatal depending on severity. Furosemide, is used in the industry to reduce blood pressure within the lungs during racing to prevent bleeding. Furosemide, a diuretic given four hours prior to a race, causes a horse to excrete up to 4% of its body weight. This effect of furosemide decreases the weight a horse must carry during a race, thus allowing the horse to run faster. Therefore, deemed as a performance enhancing drug due to its effects on the kidney, to our knowledge, no research has been conducted on what effects furosemide might have on muscle generation. High-intensity exercise causes massive muscle damage and therefore must be repaired to prepare for the next bout of exercise. Muscle generation is called myogenesis. Stem cells, or satellite cells, that lie within the muscle become activated, recognizing the need for muscle repair. Satellite cells divide, increasing in cell number and then fuse together, forming new muscle fibers. Satellite cells undergo different types of metabolism depending on their state of development. For example, proliferating cells require glucose for energy, while cells fusing together forming myotubes, require oxidative metabolism for long-lasting energy. Therefore, the objective of this study was to determine the effects furosemide might have on muscle formation and metabolism. The excess furosemide dose (100 µg/mL) decreased cell proliferation. The expression of regulatory factors responsible for forming myotubes at different stages of muscle development are decreased when cells were treated with the defined excess furosemide dose. Furosemide decreased the ability of satellite cells to generate myotubes. Glycogen concentration was also decreased as a result of excess furosemide treatment. Gene expression of enzymes involved in glycogen synthesis were increased from treatment with our excess furosemide dose. No effect of furosemide was seen on glycolysis, whereas oxidative metabolism suffered as a result of treatment with excess furosemide. In conclusion, furosemide does indeed affect muscle generation and oxidative metabolism.

Skeletal muscle regeneration is a critical process that replaces damaged muscle fibers with new fibers. The regenerative process can be segmented into four main phases: necrosis, inflammation, regeneration, and maturation. While many of the key signaling molecules are known and characterized, there are still gaps in our understanding of how this process is regulated. While it is reported that growth arrest specific 6 (Gas6) and its receptor Axl are expressed in mature muscle tissue, nothing is known about the effect that Gas6 and Axl have on regulating skeletal muscle regeneration. In this study we investigated the regenerative process in a Gas6/Axl double knockout (dKO) mouse model. The tibialis anterior (TA) muscle was chemically injured with BaCl2 and allowed to recover for 3, 7, or 14 days. We investigated satellite cell (SC) activation and muscle growth. We found that the dKO injured muscle has fewer SCs at 3-days post-injury, but the percentage of mitotically active SCs were no different between WT and dKO injured muscle. Interestingly, basal and injured dKO muscle has an increased cross-sectional area compared to wild type in male mice. Together this may suggest that in the absence of Gas6/Axl signaling may lead to impaired regeneration and compensatory fiber hypertrophy. The mechanism behind the hypertrophy remains unknown, but ultimately our findings suggest that Gas6/Axl signaling has an effect on skeletal muscle regeneration.

One of the most exciting challenges in tissue engineering is the complete reconstruction of an organ, with all its components. This depends on finding the most effective combination of stem cells and a biocompatible scaffold, able to support cell proliferation and differentiation, other than a physical structure. During my PhD study, I investigated about the possibility to reconstruct a muscle after severe ablation, and I designed and developed an experimental protocol for the delivery of either muscle progenitor cells (MPCs) or SCs via an injectable and in situ photo-crosslinkable hyaluronan-based hydrogel (HA-PI). Engrafting partially ablated tibialis anterioris (TA) muscles of C57BL/6J mice with freshly isolated GFP+ SCs embedded in hydrogel, I found a huge recovery of muscle mass, when compared to TAs receiving hydrogel+MPCs or hydrogel alone. Moreover, freshly isolated SCs embedded in HA-PI were also able to promote functional recovery, as monitored by contractile force measurements. The latter was associated with generation of both neural and vascular networks and the reconstitution of a functional satellite cell niche. This work constitutes the first realization of an in vivo tissue engineering approach, that hopefully would overcome previous limitations in skeletal muscle tissue engineering. Following these results, I also focused on finding a subpopulation of SCs, endowed with great capacity of proliferation and migration in the host muscle, and able to promote the best regeneration after damage. SCs were initially considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs has been accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. In my study, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle, I was able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (~75%) and the high proliferative clones (HPC), present instead in minor amount (~25%). LPC spontaneously generated myotubes whilst HPC differentiated into adipocytes even though might skip the adipogenic program if co-cultured with LPC. LPC and HPC differed also for mitochondrial membrane potential (ΔΨm), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that preceded differentiation. Notably, SCs heterogeneity was retained in vivo. I could conclude that SCs may therefore comprise of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation.
Una delle sfide più intriganti nell’ambito dell’ingegneria tissutale è la ricostruzione completa di un organo. Questa dipende dal trovare la combinazione più efficace tra cellule staminali e uno scaffold biocompatibile, in grado di fornire supporto per la proliferazione e la migrazione cellulare, oltre ad una struttura fisica. Durante i miei studi per il dottorato di ricerca mi sono concentrato specificamente nella ricostruzione del muscolo in seguito ad una consistente rimozione di tessuto, e ho disegnato e sviluppato un protocollo sperimentale sia per l’impianto di cellule precursori muscolari (MPCs) che di cellule satelliti (SCs) attraverso un idrogel a base di acido ialuronico iniettabile e fotopolimerizzabile in situ. Impiantando muscoli tibialis anterioris (TA), parzialmente ablati, di topi C57BL/6J con SCs isolate a fresco e GFP-positive, inserite in idrogel, ho potuto documentare un consistente recupero di massa muscolare, se paragonato a TAs che avevano ricevuto idrogel+MPCs o il solo idrogel. Inoltre le SCs isolate a fresco e inserite in idrogel hanno portato ad un recupero funzionale, monitorato attraverso misurazione della forza contrattile. Questo recupero è stato associato alla generazione sia di un network neurale che vascolare e alla ricostituzione di una nicchia di SCs funzionale. Questo lavoro costituisce la prima realizzazione di un approccio di ingegneria tissutale in vivo, con le potenzialità per superare le limitazioni incontrate in precedenza nell’ingegnerizzazione del tessuto muscolare. In seguito a questi risultati mi sono focalizzato nella caratterizzazione di una sottopopolazione di SCs, con elevata capacità proliferativa e di migrazione nel muscolo ricevente, nonché in grado di promuovere una efficace rigenerazione in seguito a danno. Le SCs sono state inizialmente considerate cellule staminali unipotenti, in grado cioé di dare origine ad un unico fenotipo specializzato. In seguito è stato dimostrato in topo come anche il differenziamento in senso alternativo verso le pathways osteogenica e adipogenica fosse possibile. Anche se si conviene che la popolazione delle SCs sia la maggiore, e probabilmente l’unica fonte di mionuclei per il muscolo, è verosimile che le cellule satelliti non siano tutte cellule staminali multipotenti, in quanto evidenze di diversità all’interno del compartimento miogenico sono state descritte sia in vitro che in vivo. Nel mio studio, attraverso l’isolamento di singole fibre da muscolo flexor digitorum brevis (FDB) di ratto, ho potuto identificare e caratterizzare clonalmente due principali sottopopolazioni di SCs: i cloni a bassa proliferazione (LPC), presenti in proporzione maggiore (~75%), e i cloni ad alta proliferazione (HPC), presenti invece in minor quantità (~25%). I LPC generano spontaneamente miotubi mentre i HPC differenziano in adipociti, con la proprietà di skippare il programma adipogenico se messi in co-coltura con LPC. LPC e HPC differiscono anche per il potenziale di membrana (ΔΨm), il bilancio dell’ATP e la generazione di specie reattive dell’ossigeno (ROS), mettendo in evidenza diversità nel metabolismo che precedono il differenziamento. Inoltre l’eterogeneità delle SCs è mantenuta anche in vivo. Posso così concludere che il pool delle SCs sembra comprendere due popolazioni cellulari distinte, anche se non irreversibilmente determinate, distinguibili in base a notevoli differenze riguardo a parametri biologici basali, come proliferazione, metabolismo e differenziamento.

Satellite cells are muscle-specific progenitor cells involved in the routine maintenance of skeletal muscle homeostasis, growth and regeneration. They are activated by various stimuli (myotrauma, growth factors etc), undergo rounds of proliferation as skeletal muscle myoblasts, to differentiate and fuse with each other to generate new myotubes or onto existing myofibres to augment growth or repair damaged fibres. Satellite cells contribute to hypertrophy by facilitating nuclear addition, which maintains contractile protein synthetic capacity. Conversely, during atrophy the dysregulation of satellite cells (e.g., via myogenic suppression), causes an opposing deficit in nuclear supplementation/contractile protein synthesis. The ‘activity status’ of satellite cells, an important determinant of muscle regenerative capacity is not routinely addressed in studies characterising mechanisms of muscle hypertrophy and atrophy. Therefore, the investigations described within this thesis examined the satellite cell specific signalling events that contribute to muscle loss or gain, in rodent models experiencing non-mechanically-induced muscle hypertrophy or atrophy. Chronic administration of an anabolic agent (BRL-47672, the pro-drug of clenbuterol) increased the expression of early components of satellite cell myogenesis (pax7, ki-67, myoD) but caused no alteration in myogenin expression, relative to control in rat soleus muscle. Pro-drug administration increased myostatin expression, with no concomitant change in follistation mRNA; this is likely a compensatory mechanism to check excessive muscle growth. These results provided evidence of increase satellite cell activity in hypertrophying muscle. In a lipopolysaccharide (LPS)-infusion model of muscle atrophy, satellite cells were inhibited in an inflammatory-dependent manner. LPS infusion caused early (<2hr) elevations inflammatory cytokines TNF-, IL-6 and NF-B. LPS-induced elevation in cytokine transcript levels paralleled increased myostatin and decreased pax7 and myoD mRNA and protein expression. The differential increase in cytokines also paralleled the reduction in the number of pax7+ and myoD+ satellite cells. These results suggest that alterations in satellite cell activity may contribute to the progression of muscle atrophy, due to the suppression of muscle compensatory mechanisms, which include satellite cell activation, differentiation and fusion for nuclear supplementation. Co-infusion with an anti-inflammatory agent, dexamethasone (Dex), blunted LPS-induced increase in inflammatory cytokines but had an additive effect on myogenic suppression. Dex+LPS infusion prevented LPS-induced increase in myogenin and resulted in an additional suppression of pax7 and myoD, greater than that elicited by either substance alone. Negative regulation of satellite cells by glucocorticoids could impede their efficacy in the treatment of inflammatory muscle disorders. The research within this thesis emphasise satellites are important for maintenance of muscle homeostasis and their activation/inhibition, may determine the magnitude of muscle loss or gain. This was demonstrated by the pattern of pax7 and myoD expression in hypertrophying muscle, where both markers were up-regulated and in atrophying muscle, where they were down-regulated. Down-regulation of these markers in atrophy could have implications for muscle regenerative capacity, especially myoD, whose expression was continuously inhibited across all time-points sampled in septic muscles. Satellite cells are a major source of compensatory action in skeletal muscle, their activation and subsequent myogenesis represents an auxiliary mechanism by which muscle responds to damaging stimuli; therefore their dysregulation (through the alteration of key myogenic markers) results in an alteration of normal function. Such dysregulation, as frequently reported in cases or progressive muscle degeneration and sarcopenia, limits the efficacy of muscle compensatory processes (i.e. satellite cell activation/proliferative or differentiation potential), thereby contributing to the progression of muscle atrophy and myopathy.

Skeletal muscle satellite cells are important for muscle repairing and muscle mass growth. For a successful muscle regenerative process, satellite cells have to sequentially undergoing different stages of myogenic process, i.e. proliferative state and differentiation state. To support this process, the presence of different circulating factors, such as immune cells, cytokines, and hormones, at the appropriate time course is critical. Among these factors, hormones, such as testosterone, cortisol, and IGF-1, have shown to play an important role in satellite cell proliferation and differentiation. Studies investigated the effect of testosterone on satellite cell using a supraphysiological dose in human or in cell culture demonstrated that testosterone is critical in satellite cell myogenic process. Due to the anabolic effect of testosterone on muscle, studies had been focused on the physiological means to increase the circulating testosterone concentration in the body to maximize the muscle mass growth from resistance exercise. The acute and transient increase in testosterone has shown to be beneficial to muscle mass growth and strength gain; however, this change in physiological testosterone concentration on satellite cell myogenesis is not known. Therefore the purpose of this dissertation is to first determine the effect of acute change in exercise-induced hormones on satellite cell myogenic state, then to determine if testosterone promotes satellite cell proliferation.

We have recently characterized two distinct populations of Satellite Cells (SCs), defined as Low Proliferative Clones (LPC) and High Proliferative Clones (HPC), that differ for proliferation, egenerative potential and mitochondrial coupling efficiency. In here, we have deep investigated their cell biology and characterized features that remark their intrinsic differences retrievable also at the initial phases of their cloning. LPC and HPC can indeed be istinguished for characteristic mitochondrial membrane potential (ΔΨm) just after isolation from their parental fibre. This is merged by mitochondrial redox state measured via NAD+/NADH analysis- and alternative respiratory CO2 production in cloned cells, which are accountable for metabolic differences reflected by alternative expression of the glycolytic enzyme Pfkfb3. In addition also mitochondrial Ca2+ handling and the sensitivity to apoptosis triggered via the intrinsic pathway are modified as well as the size of the mitochondrial network. In conclusion, we were able to determine which clone represents the suitable stem cell within the SCs population. These further experimental observations report novel physiological features in the cell biology of SCs populations before and after cloning, highlighting an intrinsic heterogeneity on which the stemness of the satellite cell is likely to depend. In the second part of my work we have also investigated their potential to trans-differentiate into smooth muscle cells. Enteric Nervous System normally interacts with muscle cells to control the peristaltic and secretory activity of the gut wall. Incomplete gut colonization by neural crest cells causes Hirschsprung’s disease, characterized by aganglionosis of the distal bowel. Multipotent, self-renewing enteric precursor neurosphere-like bodies (NLBs) -capable of generating neurons and glia derived from the neural crest- can be isolated from the gut of mice, rats, and human and they are able to colonize the gut after transplantation. Our aim is to understand the relationship between satellite cells-derived muscle precursor cells (MPCs) and NLBs using an in vitro co-culture model: this will be useful in perspective of a tissue engineering approach for bowel regeneration and skeletal muscle. Our records highlighted that NLBs were able to form new myotubes in presence of MPCs. Co-cultures in myogenic medium showed a remarkable improvement of MPCs ifferentiation by NLBs, promoting the formation of sarcomeric striatures onto myotubes and increasing the desmin expression of MPCs. On the other side, using neurogenic medium MPCs-NLBs showed a neural-like phenotype. As future perspectives, we need to understand the relationship between MPCs and NLBs and if the synapses are involved in this process; to verify if the seeding on a biocompatible polymer influences the behaviour of neural cells; and we must confirm these data with an in vivo skeletal and smooth muscle differentiation. We have finally explored the possibility of deriving smooth muscle cells from a different source, taking in consideration the difficulties related to the expansion of both skeletal and smooth muscle progenitors. Therefore, we aim to derive functional smooth muscle cells (SMCs) from non-muscle cells, such as human Amniotic Fluid Stem (hAFSC) cells. hAFSC were transduced using vector encoding ZsGreen under the αSMA promoter. SMhAFSC expressed significantly higher level of smooth muscle genes (such as αSMA, desmin, calponin and smoothelin expression) after selective culture condition. These features were confirmed by immunofluorescence, demonstrating a single lineage commitment; TEM established increased intermediate filaments, dense bodies and glycogen deposits in SMhAFSC, similar pattern compared to SMCs; and sequential imaging analyses demonstrated that SMhAFSC have a higher contractile potential than hAFSC. Consecutive single cell sampling showed the presence of voltagedependent calcium activated potassium channels on differentiated SMhAFSC and showed a higher production of carbon dioxide. In conclusion, we were able to generate to functional SMCs starting from a non-muscle precursor; secondly the transduction process may represent a valuable tool to select SM committed population. This step may eventually overcome the well-known problem of expanding SM progenitors, making these cells amenable to tissue engineering.
Il nostro gruppo ha recentemente caratterizzato due distinte popolazioni di cellule satelliti, classificate come cloni a bassa proliferazione (LPC) e ad alta proliferazione (HPC), che si differenziano in termini di proliferazione, potenziale rigenerativo e metabolismo mitocondriale. Nel mio lavoro di dottorato, abbiamo valutato e caratterizzato la loro biologia cellulare con particolare attenzione a quelle differenze intrinseche presenti anche prima della loro clonazione. Infatti, ambo le tipologie clonali possono essere distinte mediante il potenziale di membrana mitocondriale (ΔΨm) subito dopo l’isolamento dalla fibra. Questo dato è in accordo con lo stato ossido riduttivo mitocondriale misurato tramite NAD+/NADH e la quantificazione della produzione di CO2. Questi risultati sono responsabili delle differenze metaboliche e possono essere spiegati dalla diversa espressione dell’enzima glicolitico Pfkfb3. Inoltre la concentrazione mitocondriale del Ca2+ e la sensibilità all’apoptosi sono modificate così come la dimensione della rete mitocondriale. In conclusione, siamo stati in grado di determinare quale clone rappresenta la cellula staminale all’interno della popolazione di cellule satelliti. Queste nuove osservazioni sperimentali rivelano caratteristiche fisiologiche della biologia delle popolazioni delle cellule satelliti prima e dopo la clonazione, mettendo in luce un’eterogeneità intrinseca della cellula satellite. Nella seconda parte della mia tesi abbiamo esplorato la possibilità che le cellule satelliti possano, se opportunamente stimolate, trans-differenziarsi in cellule muscolari lisce. Il sistema nervoso enterico normalmente interagisce con le cellule muscolari per controllare l’attività peristaltica e secretoria della parete intestinale. L’incompleta colonizzazione dell’intestino da parte delle cellule della cresta neurale provoca la malattia di Hirschsprung, caratterizzata da aganglionosi del colon distale. Le neurosfere (NLBs), precursori enterici in grado di auto-rinnovarsi, possono generare neuroni e glia; essere isolate dall’intestino di topi, ratti e umani e sono in grado di colonizzare l'intestino dopo il trapianto. Il nostro obiettivo è di capire la relazione tra i precursori di cellule satelliti (MPCs) e NLBs utilizzando un modello in vitro di co-coltura: questo sarà utile in prospettiva di un approccio di ingegneria tissutale per la rigenerazione intestinale e muscolo scheletrico. I nostri dati hanno evidenziato che NLBs, in presenza di MPCs, sono in grado di formare nuovi miotubi. L’uso di terreni di coltura miogenici ha evidenziato un notevole aumento della differenziazione in senso muscolare, promuovendo la formazione di striature ed aumentando l’espressione di desmina. Dall’altra parte, l’utilizzo di terreni di coltura neurogenici ha mostrato un fenotipo simil neurale. Come prospettive future, dobbiamo comprendere ulteriormente la relazione tra MPCs e NLBs e se le sinapsi sono coinvolte in questo processo; si deve verificare se un loro utilizzo su polimeri biocompatibili ne possa influenzare il comportamento, ed infine è necessaria una conferma dei suddetti dati tramite un’analisi di differenziazione in vivo in muscolo scheletrico e liscio. Nella terza ed ultima fase del mio lavoro, ci siamo focalizzati ad esplorare la possibilità che cellule non-muscolari possano, se opportunamente stimolate, differenziare in senso muscolare liscio. Il nostro obiettivo è stato quello di ottenere cellule muscolari lisce (SMCs) partendo da cellule staminali del fluido amniotico umano (hAFSC). hAFSC sono state trasdotte utilizzando un virus codificante per ZsGreen sotto il promotore αSMA. SMhAFSC così ottenute hanno evidenziate un alto livello d’espressione dei geni del muscolo liscio (come αSMA, desmina, calponina e smoothelin). Queste caratteristiche sono state confermate da molteplici analisi: di immunofluorescenza, dimostrando la positività a marcatori specifici per il muscolo liscio; microscopia a trasmissione elettronica (TEM), dove si verificava l’aumento della presenza di filamenti intermedi, di corpi densi e depositi di glicogeno, modello simile rispetto alle SMCs. Analisi in timelapse di SMhAFSC hanno dimostrato che queste possiedono un potenziale contrattile superiore rispetto hAFSC e studi su singola cellula hanno evidenziato la presenza di canali calcio voltaggio-dipendenti attivati da potassio solamente su SMhAFSC. In conclusione, siamo stati in grado di generare di cellule muscolari lisce funzionali da un precursore nonmuscolare ed in secondo luogo il processo di trasduzione può rappresentare un valido strumento per distinguere e selezionare differenti popolazioni. Questa fase può eventualmente superare il ben noto problema dell’espansione di progenitori di cellule muscolari lisce, rendendo queste cellule suscettibili per approcci d’ingegneria tessutale.

The neonatal period in mammals is characterized by high rates of growth, attributed to rapid myonuclear accretion and protein deposition in muscle. Low-birth-weight (LBWT) neonates experience restricted muscle development, which leads to impaired postnatal growth and metabolic disorders later in life. The overall hypothesis of this dissertation was that dysfunction of myogenic satellite cells and aberrant regulation of protein synthesis and degradation signaling predispose LBWT neonatal pigs to slower postnatal growth. We sought to determine the proliferation and differentiation of satellite cells (SCs) derived from skeletal muscle of LBWT neonatal pigs and to elucidate the cellular mechanisms that regulate protein synthesis and degradation in LBWT pig muscles. Newborn pigs were considered as normal-birth-weight (NBWT) or LBWT when weight at birth was within 0.5 SD and below 2 SD of litter average respectively. SCs isolated from longissimus dorsi (LD) muscle of NBWT and LBWT neonatal pigs displayed similar proliferation rates. Fusion was modestly diminished in SCs from muscle of LBWT pigs compared with their NBWT siblings, suggesting SCs were not intrinsically different between the two groups and were unlikely a major contributor to the impaired muscle growth of LBWT pigs. Plasma and muscle insulin-like growth factor (IGF)-I was diminished in LBWT compared with NBWT pigs. In addition, reduced activation of key components of IGF-I downstream signaling pathway in LBWT pigs muscle may lead to diminished translation initiation signaling and thus decreased protein synthesis in these animals. However, IGF-I receptor expression and myostatin signaling inversely correlated to LBWT, indicating they may participate in compensatory responses for the reduction in protein synthesis signaling. Expression of eukaryotic initiation factor (eIF) 4F complex subunits, eIF4E, eIF4G, and eIF4A was reduced in LBWT compared with NBWT pigs. This would suggest that diminished translation initiation signaling in skeletal muscle of LBWT pigs is the main factor that predisposes LBWT pigs to slower growth rates in the neonatal period. In contrast, changes in protein degradation signaling do not appear to affect protein turnover in LBWT neonatal pigs.
PHD

[Résumé traduit automatiquement par le site web Reverso : Un problème majeur dans la biologie de cellule souche pour la recherche de base et clinique est l'accessibilité aux cellules souches de sain ou des individus de malade et le maintien(la maintenance) de leur puissance pour l'expérimentation, des écrans de médicament(drogue) thérapeutiques, ou des transplantations. Ici nous rapportons des conditions pour l'isolement de cellules myogenic squelettiques viables et fonctionnelles de l'homme jusqu'à 17 jours et la souris jusqu'à 14 jours post-mortem, significativement au-delà des rapports précédents. Les cellules souches de muscle ont été enrichies dans le mouchoir en papier(le tissu) post-mortem suggérant un avantage de survie sélectif comparé à d'autres types cellulaires. Les transplantations de 4 jour le muscle de souris post-mortem et des cellules souches haematopoietic ont régénéré des mouchoirs en papier(des tissus) solidement. La quiétude cellulaire contribue à cette viabilité de cellule souche de muscle où les cellules adoptent un état inerte réversible caractérisé par l'activité métabolique réduite, une phase de décalage prolongée avant la première division cellulaire et un statut transcriptional moins prêt pour l'engagement. Plus loin(De plus), nous montrons que la réponse de stress(d'accent) de cellules souches à l'environnement post-mortem est NF-? B-independent et que des cellules souches de muscle post-mortem sont caractérisées par les niveaux élevés de ROS, plus haut mitochondrial le contenu d'ADN et l'activité inférieure d'oxyde super dismutases, pourtant ils ne montrent(n'affichent) pas de changements(monnaies) de niveaux de redox. Finalement, l'hypoxie sévère(grave), ou l'anoxie sont critiques pour maintenir(entretenir) la viabilité de cellule souche et la capacité régénératrice robuste. Ces découvertes ont des implications majeures pour des études fondamentales et cliniques sur des cellules souches et ils peuvent aussi être prolongés(étendus) à d'autres systèmes de cellule souche de normal et des patients de malade (comme un exemple que nous avons aussi montré que des cellules souches hematopoietic survivent pendant une période prolongée(étendue) après la mort dans des mouchoirs en papier(des tissus) et restent fonctionnelles in vivo).]
A major issue in stem cell biology for basic and clinical research is the accessibility to stem cells from healthy or diseased individuals, and the maintenance of their potency for experimentation, therapeutic drug screens, or transplantations. Here we report conditions for the isolation of viable and functional skeletal myogenic cells from human up to 17 days, and mouse up to 14 days post mortem, significantly beyond previous reports. Muscle stem cells were enriched in post mortem tissue suggesting a selective survival advantage compared to other cell types. Transplantations of 4 day post mortem mouse muscle and haematopoietic stem cells regenerated tissues robustly. Cellular quiescence contributes to this muscle stem cell viability where cells adopt a reversible dormant state characterised by reduced metabolic activity, a prolonged lag phase before the first cell division, and a transcriptional status less primed for commitment. Further, we show that the stress response of stem cells to the post mortem environment is NF-κB-independent, and that post mortem muscle stem cells are characterised by elevated levels of ROS, higher mitochondrial DNA content, and lower activity of super oxide dismutases, yet they do not display changes in redox levels. Finally, severe hypoxia, or anoxia is critical for maintaining stem cell viability and robust regenerative capacity. Ces découvertes ont des implications majeures pour des études fondamentales et cliniques sur des cellules souches et ils peuvent aussi être prolongés(étendus) à d'autres systèmes de cellule souche de normal et des patients de malade (comme un exemple que nous avons aussi montré que des cellules souches hematopoietic survivent pendant une période prolongée(étendue) après la mort dans des mouchoirs en papier(des tissus) et restent fonctionnelles in vivo)

Thesis (Ph. D.)--West Virginia University, 2001.
Title from document title page. Document formatted into pages; contains ix, 109 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 88-104).

Le muscle squelettique adulte est un tissu avec une grande plasticité étant donné qu’il adapte sa taille suite à la surcharge fonctionnelle et il régénère suite à une lésion. La base de cette plasticité est la myofibre et les cellules souches associées, les cellules satellites (CS). Suite aux stimuli, les CS sortent de la quiescence, elles s’activent, proliférent, s’engagent dans la voie myogénique et fusionnent entre elles ou bien avec la fibre pre-éxistante. Une partie des CS retourne à la quiescence afin de maintenir le « pool » de progéniteurs. Ce projet a pour objectif de mieux caractériser des voies de signalisation responsables des adaptations des CS au cours de la régénération et le l’hypertrophie compensatoire. Srf est un facteur de transcription, particulièrement exprimé dans les muscles. Les gènes cibles de Srf sont des gènes qui participent à la régulation de la prolifération cellulaire et des gènes codant des protéines sarcomériques du muscle ou bien des gènes ayant un rôle dans l’adhésion cellulaire, la migration et l’organisation du cytosquelette. Il a été montré que la perte de fonction de Srf dans la lignée de cellules musculaire C2C12 inhibe leur prolifération et leur différenciation et que Srf contrôle l’expression de MyoD qui est un gène de détermination myogénique. Aucune donnée n’est disponible à ce jour concernant la fonction de Srf dans les CS in vivo. Nous avons généré des souris dépourvues de Srf spécifiquement dans les CS adultes. Les CS ont été recruitées par l’hypertrophie et la régénération musculaire. En parallèle des études ex vivo ont été menées afin de préciser si les phénotypes observés sont cellule-autonomes et afin de disséquer les mécanismes sous-jacents. Nous montrons que la perte de Srf dans les CS affecte fortement les processus de régénération et d’hypertrophie suggérant un rôle de Srf dans le contrôle du destin cellulaire de CS. Nos études montrent que la perte le Srf dans les SC n’affecte pas leur prolifération et leur engagement dans la différenciation myogénique. Par contre, leur motilité et leur capacité de fusion sont fortement réduites. Afin d’identifier les effecteurs de Srf impliqués dans la motilité et le défaut de fusion des CS mutantes, nous avons réalisé des études transcriptomiques et identifié le set de gènes dont l’expression est altérée par la perte de Srf dans des conditions de prolifération et de différenciation. L’analyse des fonctions altérées nous a indiqué que la voie de signalisation du cytosquelette d’actine était perturbée. En effet les CS dépourvues de Srf expriment moins d’actine et présentent une organisation du cytosquelette d’actine perturbée. Des expériences de sauvetage utilisant un modèle de souris permettant la surexpression inductible d’actine alpha dans les CS dépourvues de Srf ont montré que la surexpression d’actine chez les mutants Srf était suffisante pour rétablir partiellement l’organisation du cytosquelette et améliorer les capacités de fusion des CS. De manière intéressante, seule la fusion hétérotypique (entre une cellule contrôle et une cellule mutante), et pas la fusion homotypique (entre deux cellules mutantes), est rétablie par l’expression de l’actine. In vivo, le rétablissement de la fusion hétérotypique restaure la croissance hypertrophique des muscles alors que l’altération de la régénération chez les mutants Srf n’est que faiblement améliorée par la surexpression de l’actine. Cette étude nous a permis d’avoir une vision d’ensemble et mécanistique de la contribution du facteur de transcription Srf dans la biologie des CS et de mettre en évidence l’importance structurale du maintien du cytosquelette d’actine pour la fusion des cellules musculaires
The adult skeletal muscle is a high plastic tissue as it adapts its size upon overload and it is capable of regeneration upon muscle lesion. The skeletal muscle is composed of a specialized syncytium, the myofiber, which is the functional unit of the muscle and a small population of myogenic progenitors, residing adjacent to the myofibers, termed as satellite cells (SCs). SCs are the muscle-specific stem cells which endow the skeletal muscle with its remarkable capacity to repair and to maintain homeostasis during muscle turnover. In resting adult muscles, SCs are quiescent but they activate upon exposure to stimuli. The activated SCs (myoblasts) proliferate extensively and subsequently differentiate and fuse between them or pre-existing myofibers, a series of cellular events called myogenesis. In parallel to the myogenesis, a reserve population of SCs escapes the myogenic program and self-renews to replenish the SC pool. The current project aims to further characterize the signalling pathways involved in SC functions during muscle regeneration and compensatory hypertrophy (CH). Srf is a muscle-enriched transcription factor with Srf-target genes implicated in cell proliferation, differentiation (sarcomeric proteins), adhesion, migration and cellular cytoskeleton. Studies in C2C12 mouse myogenic cell line showed that Srf loss prevent the myoblast proliferation and differentiation by down-regulating the expression of the myogenic determinant MyoD gene. We used a genetic murine model for adult SC-specific Srf-loss in order to conduct in vivo and ex vivo studies for the Srf role in SCs. Compensatory hypertrophy and regeneration are the two means by which SCs were recruited. We show that loss of Srf in SCs affects the regeneration process and the CH suggesting the Srf role in the SC fate. Srf-depleted SCs display probably no defect in their proliferation and differentiation but reduced capacity in motility and fusion. Transcriptomic analysis revealed altered actin cytoskeleton and signalling. Srf-depleted SCs show reduced actin expression and altered actin cytoskeleton. Rescue of actin expression in Srf-depleted SCs partially restored the cytoskeleton organization and the fusion process. Interestingly by actin overexpression only the heterotypic/asymmetric fusion was established but not the homotypic/symmetric fusion. Therefore actin overexpression restored the hypertrophic growth in the CH (in vivo model of heterotypic fusion) but failed to do so in the regeneration (in vivo model of homotypic fusion). This study contributed to the in vivo investigation of the Srf mechanistic role in adult SCs and underlined the importance of actin cytoskeleton maintenance in the fusion of myogenic cells

Thesis (MSc (Physiological Sciences)--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Although muscle tissue demonstrates a remarkable capacity for regeneration following injury, this process is slow and often accompanied by the formation of scar tissue and a subsequent decrease in contractile capacity following regeneration. Treatment options are few and mostly supportive in nature. This regeneration process involves muscle stem cells (satellite cells) which ultimately give rise to the regenerated muscle. The contentious field of low level laser therapy (LLLT) has made remarkable claims in facilitating wound healing in soft tissue injuries of various types. Yet, the mechanism(s) invoked in these beneficial effects are poorly understood. We have investigated the effects of LLLT using a 638 nm laser on satellite cells in culture and in-vivo. Using an array of techniques we have measured, amongst other things, metabolic responses to laser irradiation, signaling pathways activated/altered and antioxidant status. In response to laser irradiation satellite cells in culture showed an increase in MTT values (a measure of metabolic activity) and a decrease in antioxidant status (measured using the ORAC assay). In addition laser irradiation also altered the expression and phosphorylation state of several signaling pathways, including Akt and STAT-3. Following on from this the effects of laser irradiation on satellite cells in-vivo was assessed in a rat model of contusion injury. No significant differences in satellite cell number was found following laser irradiation, changes were seen in tissue antioxidant status and blood antioxidant status (measured using the ORAC assay). In the course of this study several standard techniques were used to investigate the effects of laser irradiation on satellite cells both in-vitro and invivo. It has become apparent that several of these techniques have problems associated with them that possibly make them inappropriate for vi further use in studies involving laser irradiation. However the results indicate that laser therapy is induces satellite cell behavior and further study is warranted in this field.
AFRIKAANSE OPSOMMING: Alhoewel spierweefsel merkwaardige regenerasie kapasiteit vertoon ten opsigte van besering, is hierdie proses stadig en word soms vergesel met die vorming van letselweefsel asook ‘n gevolglike afname in kontaktiele kapasiteit na afloop van regenerasie. Behandelingsmoontlikhede is skaars en meesal ondersteunend van aard. Hierdie proses sluit spierstamselle (satelietselle), wat uiteindelik die ontstaan van die regenerasie van spier tot gevolg het, in. Die kontroversiële veld van lae vlak laserterapie (Engels: Low level laser therapy (LLLT)) het merkwaardige aansprake in die fasilitering met verskeie sagteweefsel wondgenesing. Nietemin, die meganisme(s) wat voordelige effekte induseer, word nog nie goed begryp nie. Ons het die effek van LLLT, deur gebruik te maak van ‘n 638 nm laser op kultuur in vitro satelietselle sowel in-vivo, ondersoek. Deur gebruik te maak van verskeie tegnieke is onder meer die metaboliese, sowel die seinstransduksie weë en antioksidantstatus na laserbestraling, gemeet. In reaksie op die laserbestraling het satelietselle (in kultuur) ‘n toename in MTT waardes getoon (‘n maatstaf van die metaboliese aktiwiteit) en ‘n afname in die antioksidantstatus (gemeet deur van die ORAC toets). Addisioneel het laserbestraling ook uitdrukking en fosforilering van verskeie proteïene betrokke in seintransduksieweë beïnvloed, insluitend Akt, STAT-3). Na afloop van hierdie effekte op satelietselle na laserbestraling, is daar gebruik gemaak van ‘n kneusbeseringsrotmodel om hierdie effekte in vivo te ondersoek. Geen betekenisvolle verskille in die aantal satelietselle na laserbestraling is opgemerk nie, maar veranderings is wel opgemerk in weefsel- en bloed-antioksidantstatus (gemeet deur van die ORAC toets gebruik te maak). Gedurende die verloop van die studie is van verskeie standaardtegnieke gebruik gemaak om die effekte van laserbestraling op beide satelietselle in vitro en in vivo te ondersoek. iv Dit het duidelik na vore gekom dat daar wel gepaardgaande probleme met van hierdie tegnieke voorgekom het, en dat van hierdie tegnieke nie gepas is vir ondersoek in laserbestralingsstudies nie. Nietemin, die resultate toon wel dat laserbehandeling. satelietselgedrag induseer wat verdere studie in hierdie veld noodsaak

Skeletal muscle regeneration is dependent upon the influences of intrinsic and extrinsic factors that stimulate satellite cells. Regenerating proteins are upregulated at the onset of trauma or inflammation in the pancreas, gastrointestinal tract, liver, neural cells and other tissues. Studies have shown that Reg proteins have a mitogenic, anti-apoptotic and anti-inflammatory function in damaged tissues and is necessary for normal progression of regeneration. As skeletal muscle is also able to regenerate itself at a rapid rate, it seems highly likely that Reg proteins function to promote myogenesis in skeletal muscle regeneration. Therefore, the goal of our research was to characterize the expression of the Reg proteins and receptor in regenerating skeletal muscle and satellite cells, investigate the effect of exogenous Reg protein on myogenesis, and to examine direct Reg protein effect on satellite cell activity. To determine whether Reg proteins participate in skeletal muscle regeneration, mice were injected with marcaine in their tibialis anterior muscles to induce skeletal muscle damage. The gene expression analysis of undamaged and marcaine-damaged tibialis anterior muscles and mice satellite cells showed that Reg I, II, IIIα, IIIγ, IV and EXTL3 genes are present during skeletal muscle regeneration and satellite cells significantly express Reg I, IIIα, IIIγ and EXTL3. As Reg I and IIIα are most prevalent in vivo and in vitro respectively, we advocate these isoforms as the predominant candidates in skeletal muscle regeneration. To determine the effect of exogenous Reg protein on myogenesis, we performed gene expression and muscle morphometry analysis of Reg IIIα or PBS injected tibialis anterior muscles. Interestingly, our results indicate that the addition of Reg IIIα to damaged muscles inhibited myogenesis. To determine the direct effect of Reg protein on myogenic stem cell activity, Reg proteins were added to mice satellite cells and C2C12 cells. Results from these studies were inconclusive due to the failure of known positive and negative controls. Overall, our studies suggest that Reg proteins contribute to skeletal muscle regeneration; however, as an overabundance of Reg IIIα in regenerating tissues may have inhibited myogenesis, it is imperative that other isoforms or lower concentrations be investigated.

The STAC3 gene is a functionally undefined gene predicted to encode a protein containing two SH3 domains and one cysteine-rich domain. In this study, we determined the potential role of the STAC3 gene in proliferation and differentiation of bovine satellite cells. We isolated satellite cells from skeletal muscle of adult cattle and transfected them with STAC3 small interfering RNA (siRNA) or scrambled siRNA. Cell proliferation assays revealed that STAC3 knockdown had no effect on the proliferation rate of bovine satellite cells. We assessed the differentiation status of bovine satellite cells by quantifying the expression levels of myogenin and myosin heavy chain genes, and by quantifying fusion index. STAC3 knockdown stimulated mRNA and protein expression of myogenin, and myosin heavy chain 3 and 7, and increased fusion index of bovine satellite cells. These data together suggest that STAC3 inhibits differentiation of bovine satellite cells into myotubes. To determine the underlying mechanism, we identified and validated AP1?1 as a STAC3-interacting protein by yeast two-hybrid screening and co-immunoprecipitation. In C2C12 cells, STAC3 knockdown decreased the expression level of AP1?1 protein. In bovine satellite cells, STAC3 knockdown increased the membrane localization of glucose transporter 4 (GLUT4) and glucose uptake. These data together suggest the following mechanism by which STAC3 inhibits differentiation of bovine satellite cells: STAC3 increases AP1?1 stability in cells; a high level of AP1?1 keeps GLUT4 from translocating to the plasma membrane; reduced membrane localization of GLUT4 impedes glucose uptake; and restricted glucose uptake inhibits differentiation of satellite cells into myotubes.
Master of Science

Muscle aging, in particular, is characterized by the reduction of tissue mass and function, which are particularly prominent in geriatric individuals undergoing sarcopenia. The age-associated muscle wasting is also associated with a decline in regenerative ability and a reduction in resident muscle stem cell (satellite cell) number and function. Although sarcopenia is one of the major contributors to the general loss of physiological function, the mechanisms involved in age-related loss of muscle homeostasis and satellite cell activity are yet poorly understood. Using a microarray-based transcriptome analysis of muscle stem cells isolated from young and physiologically aged/geriatric mice, we uncovered specific changes in the gene expression profile that highlighted key biological processes and potential molecular markers associated with satellite cell aging, which included p16INK4a. We used Bmi1-deficient mice to further explore the implications of p16INK4a up-regulation in satellite cell function. We found premature p16INK4a up-regulation in young/adult Bmi1-deficient satellite cells correlating with defects in satellite cell number, proliferation and self-renewal capacity. In addition we have identified a number of overlapping biological processes dysregulated in physiologically aged and Bmi1-deficient satellite cells, suggesting that Bmi1-dependent epigenetic regulation may underlie many of the intrinsic changes taking place in chronologically aged satellite cells. In addition, we show that Bmi1 loss causes defects of late postnatal/adult muscle growth characterized by reduced muscle mass with smaller muscle fibers, typical of atrophying senescent/sarcopenic muscle. Since p16INK4a expression is specifically up-regulated in muscle satellite cells of geriatric, sarcopenic mice and in a mouse model of accelerated senescence/sarcopenia (SAMP8), we propose that the Bmi1/p16INK4a axis might be particularly operative in muscle stem cells from the elderly. Muscle wasting is one of the physiological consequences of sarcopenia and the identification of novel factors regulating muscle growth and atrophy is of potential relevance for therapeutical applications. We have uncovered a new role for Sestrins as skeletal muscle growth promoting factors in the adult. We found Sestrins expression regulated in mouse models of skeletal muscle atrophy and hypertrophy and in human myopathies. Through a gain of function approach we show that Sestrins induce skeletal muscle growth, by activating the IGF1/PI3K/AKT pathway.
El envejecimiento del tejido muscular está caracterizado concretamente por una reducción global de la masa muscular y un empeoramiento de la función de tejido, particularmente prominentes en individuos muy viejos (geriátricos) que padecen sarcopenia. La pérdida muscular asociado a la edad, se acompaña de una reducción en la capacidad de regeneración del músculo y en una reducción del número y la función de las células madre residentes en el músculo (células satélite). Aunque la sarcopenia sea una de las causas principales de la pérdida general de función fisiológica del músculo, los mecanismos implicados en la reducción de la homeostasis muscular y de actividad de las células satélite no han sido completamente caracterizados. Mediante el análisis comparativo del transcriptoma de células madre musculares aisladas de ratones jóvenes y de ratones viejos (geriátricos), hemos encontrado cambios específicos en su perfil de expresión génica que apuntan a los procesos biológicos dominantes y a los marcadores moleculares potencialmente asociados con el envejecimiento de las células satélite, entre los que destaca p16INK4a. Por ello, hemos utilizado ratones deficientes en Bmi1 para explorar más profundamente las implicaciones de la sobreexpresión de p16INK4a en la función de las células satélite. Hemos encontrado que células satélite jóvenes del ratón Bmi1-/- presentan sobrexpresión de p16INK4a, que correlacionan con una reducción en el número de la células, y en su capacidad de proliferación y autorenovación. Además hemos identificado un grupo de procesos biológicos comunes entre las células satélite viejas y las deficientes en Bmi1, sugiriendo que la regulación epigenética mediada por Bmi1 puede ser la base de muchos de los cambios intrínsecos que ocurren en células envejecidas fisiológicamente. Además, demostramos que la pérdida Bmi1 causa defectos en el crecimiento postnatal/adulto del músculo, caracterizado por pérdida de masa muscular con fibras más pequeñas, típico del músculo atrofiado senescente o sarcopénico. Puesto que la expresión de p16 está aumentada específicamente en el músculo de ratones viejos, sarcopénicos y en un modelo del ratón con envejecimiento (senescencia) acelerado (SAMP8), proponemos que el eje Bmi1/p16 puede actuar particularmente en las células madre musculares de los ancianos. La pérdida de masa muscular es una de las consecuencias fisiológicas de la sarcopenia y la identificación de nuevos factores que regulen el crecimiento y atrofia del músculo es de gran importancia para aplicaciones terapéuticas. Hemos descubierto un nuevo papel de las Sestrinas como factores promotores del crecimiento del músculo esquelético en el adulto. Hemos encontrado que la expresión de las Sestrinas se regula en modelos del ratón de atrofia y de hipertrofia muscular y en miopatías humanas. Mediante experimentos de ganacia de función hemos demostrado que las Sestrinas inducen el crecimiento del músculo esquelético, activando el ruta de señalización de IGF1/PI3K/AKT

Efficient muscle regeneration is essential in mammals in order to overcome daily stress such as wounds, exercise and pathologic processes. This regeneration relies on muscle stem cells, the satellite cells. After a lesion, satellite cells are activated, proliferate and differentiate in fonctionnal muscle fibers. Our laboratory has previously shown that the transcription factor GATA-6 is expressed in the satellite cells. The present thesis confirms the expression of this factor in this cell type. Also, it seems that GATA-6 could be implicated in the maintaining of quiescence of these cells. The GATA-6 heterozygous mouse muscle is characterized by an increase level of Myf5 and Pax7+ cells. Moreover, suppression of one copy of the GATA-6 gene in a muscular dystrophy model mouse, the mdx mice, alleviates its phenotype. Further experiments on a muscle-specific GATA-6 null mouse will allow a better understanding of the role of GATA-6 in muscle regeneration.
Keywords. GATA-6, muscle regeneration, mdx, satellite cells

During my PhD I focused on the role of collagen VI in skeletal muscle regeneration and in intestinal homeostasis. Collagen VI is a glycoprotein of the extracellular matrix (ECM) containing three genetically distinct chains and forming an extended microfilamentous network that interacts with the cells and with other ECM components. Mutations of collagen VI genes in humans cause several muscle diseases, including Bethlem myopathy and Ullrich congenital muscle dystrophy. The generation of a collagen VI knockout mouse model was fundamental for clarifying the pathomolecular defects caused by the absence of this protein and provided a valuable tool for developing novel therapeutic opportunities in patients. During the past decade, studies on collagen VI null mice revealed an increasingly important role for this ECM component in a plethora of different cell and tissue processes, including apoptosis and oxidative damage, autophagy, cell differentiation, maintenance of stemness, regulation of tissue regeneration and biomechanical properties. In the first part of my PhD work, I was involved in a large project aimed at characterizing the role of collagen VI in muscle regeneration and in the regulation of the activities of satellite cells (SCs), the main adult stem cell population of skeletal muscles. Our studies revealed that collagen VI is a key component of the SC niche, and its lack affects muscle regeneration and impairs SC self-renewal in collagen VI null mice. Interestingly, we found that the absence of collagen VI affects the in vivo mechanical properties of skeletal muscles. Furthermore, in vitro studies revealed that SC stemness and regenerative capabilities are strongly compromised when SCs are cultured on biomimetic substrates with the abnormal stiffness displayed by collagen VI deficient muscles, compared to SCs cultured on biomimetic substrates with the normal muscle stiffness. Both the biomechanical properties value and the regenerative capability of collagen VI null muscle are improved after in vivo grafting with wild-type muscle fibroblasts, the main cell type producing collagen VI, thus pointing out that a key mechanism by which collagen VI regulates SC activity is via modulation of muscle mechanical properties. In a subsequent work, we found that pharmacological treatment of collagen VI null mice with cyclosporin A is able to stimulate myogenesis in physiological conditions by increasing the percentage of regenerating myofibers, and to improve muscle regeneration and SC homeostasis after cardiotoxin-induced injury. In the second part of my PhD, I investigated the role of collagen VI in intestine. Despite its broad distribution within the mucosa and the smooth muscle layers of the digestive tract, lack of collagen VI does not trigger any gross abnormality in the intestinal architecture of knockout mice. However, studies of the gastrointestinal functionality revealed that the lack of this ECM component lead to increased motility and decreased paracellular permeability. Induction of experimental acute colitis by administration of dextran sodium sulphate showed that collagen VI deficient mice have a decreased responsiveness and severity to acute colitis, with a lower body weight loss and decreased colonic inflammation when compared to wild-type subjected to the same treatment. Moreover, wild-type mice displayed an increased recruitment of inflammatory cells, in association with increased macrophage number and neutrophil activity, which decreased during 10 days of recovery subsequent to acute colitis, thus allowing proper tissue repair. Conversely, collagen VI deficient mice were unable to efficiently turn off inflammation during post-colitis recovery, displaying a high number of pro-inflammatory M1 colonic macrophages, an increased neutrophil activity and a higher body weight loss when compared to wild-type. Moreover, lack of collagen VI affected both macrophage polarization and activity in physiological conditions and during mild inflammation. Further studies allowed to reveal that lack of collagen VI affects the behavior of intestinal macrophages, both in physiological conditions and during mild inflammation, whose activity is essential to ensure intestinal mucosa homeostasis. These findings point at a role for collagen VI as a chemoattractant during acute inflammation and in tissue regeneration in the subsequent recovery phase. Immunofluorescence studies revealed an increased deposition of collagen VI in the colonic mucosa during acute colitis, and the protein was found in contact with macrophages. Interestingly, ileal biopsies of Crohn’s disease patients displayed increased expression and deposition of collagen VI, in association with a high number of macrophages, suggesting that the dysregulation of this ECM component may play an active role in the onset and/or maintenance of inflammatory bowel diseases. In conclusion, my PhD work provided novel information on the in vivo roles of collagen VI in cell and tissue homeostasis. In more general terms, these findings highlight the importance of a specific defined ECM microenvironment to ensure tissue homeostasis, and demonstrate that lack of one of the major ECM component may have a severe impact on cell behavior.
Durante il mio percorso di dottorato mi sono occupata di studiare il ruolo del collagene VI nella rigenerazione del muscolo scheletrico e nell’omeostasi dell’intestino. Il collagene VI è una glicoproteina della matrice extracellulare (MEC) costituita da tre catene geneticamente distinte, le quali si organizzano in modo da formare un’estesa rete di microfilamenti in grado di connettere cellule e altri componenti della MEC. Mutazioni a carico dei geni codificanti le catene del collagene VI causano diverse patologie muscolari, principalmente la miopatia di Bethlem e la distrofia muscolare congenita di Ullrich. Gli studi condotti sul modello knockout murino privo di collagene VI hanno permesso di chiarire i difetti patomolecolari causati dall’assenza di questa proteina, dimostrandosi utile anche per l’identificazione di nuovi trattamenti farmacologici per le malattie umane. Nel coso degli anni, molteplici studi hanno messo in luce le diverse funzioni esercitate dal collagene VI nel regolare diversi eventi cellulari e tissutali, tra cui l’apoptosi e il danno ossidativo, l’autofagia, il differenziamento cellulare, il mantenimento della staminalità ai fini rigenerativi e le proprietà biomeccaniche. Nel corso del mio dottorato ho partecipato inizialmente alla caratterizzazione del ruolo del collagene VI durante la rigenerazione del muscolo scheletrico e la sua influenza sull’attività delle cellule satelliti, la popolazione principale di cellule staminali adulte nei muscoli scheletrici. Da tali studi è emerso che il collagene VI è un componente essenziale della nicchia delle cellule satelliti. La mancanza di tale proteina determina una ridotta rigenerazione tissutale e una diminuita capacità delle cellule satelliti di compiere self-renewal in seguito a danni muscolari multipli. I muscoli dei topi privi di collagene VI sono caratterizzati da una minore stiffness e approfondite analisi condotte in vitro hanno rivelato che le proprietà staminali e rigenerative delle cellule satelliti sono fortemente compromesse quando coltivate su biomateriali con un modulo elastico che mima la condizione patologica. Le capacità rigenerative e le proprietà meccaniche dei muscoli di topi privi di collagen VI vengono ripristinate in seguito alla deposizione di collagene VI, ristabilita tramite grafting di fibroblasti muscolari isolati da topi wild-type. Complessivamente, questi studi hanno dimostrato che modulando le proprietà meccaniche del muscolo, il collagene VI è in grado di regolare l’attività delle cellule satelliti. Abbiamo inoltre dimostrato che la somministrazione di ciclosporina A è in grado di stimolare la miogenesi in condizioni fisiologiche, inducendo la formazione di nuove fibre muscolari, e di migliorare la rigenerazione muscolare e l’omeostasi delle cellule satelliti in seguito a danni muscolari nei topi privi di collagene VI. Successivamente mi sono dedicata ad indagare il ruolo del collagene VI nell’omeostasi dell’intestino. Sebbene questa proteina sia ampiamente distribuita nella mucosa e nello strato muscolare, la sua assenza sembra non comportare alterazioni macroscopiche sull’architettura intestinale. L’analisi della funzionalità del sistema gastrointestinale ha evidenziato un’aumentata motilità e una ridotta permeabilità paracellulare in assenza di collagene VI. Esperimenti di induzione di colite acuta mediante sodio solfato destano hanno rivelato che i topi privi di collagene VI presentano una ridotta risposta e severità, associate ad una minore perdita di peso corporeo e minore infiammazione della mucosa del colon rispetto ai topi wild-type. Inoltre, durante la fase di colite acuta il reclutamento di cellule infiammatorie è risultato essere aumentato nei topi wild-type, comportando un aumento del numero di macrofagi e di attività dei neutrofili, mentre si riduce durante la fase di recupero seguente la colite acuta, favorendo la rigenerazione tissutale. Di contro, nei topi privi di collagene VI l’infiammazione è risultata essere ancora attiva durante la fase di recupero, con un elevato numero di macrofagi pro-infiammatori M1, un’alta attività dei neutrofili e un peggioramento della perdita di peso. Inoltre l’assenza di collagene VI ha dimostrato influenzare il comportamento dei macrofagi della mucosa del colon, sia in condizioni fisiologiche sia durante i primi giorni di infiammazione acuta, la cui attività è essenziale per assicurare l’omeostasi della mucosa intestinale. Nel complesso, da questi studi è emerso che il collagene VI esercita un ruolo da chemoattrattore per le cellule infiammatorie durante la fase di colite acuta, mentre nella successiva fase di risoluzione la sua presenza è necessaria nell’indurre una corretta rigenerazione tissutale. Studi di immunofluorescenza hanno inoltre rivelato nei topi wild-type un’elevata espressione di collagene VI in stretto contatto con i macrofagi della mucosa del colon durante la fase di colite acuta. L’evidenza di un’aumentata espressione di collagene VI su biopsia di ileo di paziente affetto da morbo di Crohn, associata ad un elevato numero di macrofagi rispetto al controllo sano, suggerisce un coinvolgimento di questo componente della MEC nel decorso delle malattie infiammatorie intestinali. In conclusione, le evidenze emerse in questo mio lavoro di tesi avvalorano l’importanza del ruolo della matrice extracellulare nell’omeostasi tissutale.

Satellite cells derived from normal donor mice contribute to muscle regeneration and restore dystrophin expression when transplanted into dystrophin-deficient mice (mdxnu/nu). However, unless the local host muscle environment has been modulated with high doses of gamma-radiation to incapacitate host satellite cells, but maintaining a functional niche, donor satellite cell engraftment is negligible. This work aimed to determine the cells and pathway(s) within host muscle which are responsible for mediating the radiation-induced effect. I first investigated whether this effect was mediated by apoptotic cells, by quantifying the percentage of TUNEL positive cells in muscles at basal levels and at different time points after irradiation. There was a correlation between the percentage of TUNEL positive cells and the time for optimal engraftment in mdxnu/nu host muscles. This suggests that apoptotic cells within host muscle might be mediators of the radiation-induced promotion of donor satellite cell engraftment. Then I performed a series of co-transplantation experiments to determine whether different cell preparations within the pre-irradiated mdxnu/nu muscle would enhance donor satellite cell transplantation. Three cell preparations (satellite cells, monocytic cell suspension, and single myofibres) were isolated from pre-irradiated mdxnu/nu donors and grafted with donor 3F-nLacZ-2E satellite cells into mdxnu/nu hosts. None of these preparations significantly enhanced donor satellite cell engraftment in non-irradiated hosts. Finally, I performed RNA sequencing on differentially treated muscles to investigate possible signalling pathways involved in enhancing satellite cell engraftment in pre-irradiated muscles. This revealed a phenotype consistent with type I and type II interferon responses after irradiation, leading to the secretion of the IL-6 family of cytokines. Further investigation confirmed an upregulation of LIF in pre-irradiated muscle. Overall, my findings suggest that irradiation of host muscle alters the inflammatory phenotype and elicits the secretion of the IL-6 family of cytokines, which are powerful regulators of satellite cell proliferation and differentiation.

La régénération du muscle fait appel à des cellules souches spécialisées mais elle nécessite également une action coordonnée d'éléments et de cellules du stroma et des tissus de soutien. L'étude de la régénération musculaire ne peut se borner à la seule étude de l'activation, la prolifération et la différenciation des cellules souches musculaires. L'objectif de mon travail de thèse a été d'approcher les mécanismes qui participent à la régénération harmonieuse du muscle à côté des cellules satellites à savoir les différents éléments cellulaires des tissus de soutien et aussi le stroma au travers de l'étude de la chimiokine CXCL12 et de son ancrage à la matrice extra-cellulaire musculaire. Dans un premier temps, nous avons fait le constat que les modèles de lésions musculaires étaient nombreux et étaient utilisés de façon indistincte avec une méconnaissance de leurs spécificités propres. Ainsi, la première partie de ce travail de thèse a consisté en la comparaison des modèles les plus utilisés dans la littérature afin de connaître leurs cibles potentielles et de choisir le mieux adapté aux questions scientifiques posées. Dans un deuxième temps, nous avons utilisé un modèle d'animaux génétiquement invalidés pour l'ancrage de l'isoforme gamma de CXCL12 à la matrice pour étudier le muscle strié squelettique, son développement, les cellules souches et l'organisation de leur niche et enfin, sa réparation. Bien que dans tous les modèles la lésion du muscle évolue à terme vers une restitution ad integrum, les processus mis en oeuvre varient en fonction du type et de l'ampleur de l'atteinte. En outre, nous avons montré que les paramètres histologiques seuls ne sont pas entièrement suffisants pour affirmer que la régénération musculaire est achevée et qu'il faut savoir considérer chaque type cellulaire en détail ainsi que des paramètres fonctionnels qu'il conviendra de mesurer dans les suites de ce travail. Nous avons ensuite étudié l'influence de l'adhésion de la chimiokine CXCL12 aux glycosaminoglycanes dans sa capacité à réguler la réparation musculaire. Pour ce faire nous avons utilisé comme modèle d'étude la souris knock in CXCL12Gagtm/Gagtm, récemment développée au laboratoire et dans laquelle le gène CXCL12 a été muté dans la région du site contrôlant l'ancrage de la molécule CXCL12 aux HS. Chez cette souris, CXCL12 est présent mais incapable de se fixer aux HS de la matrice extracellulaire tout en gardant son activité via CXCR4. Dans ce cas précis CXCL12 est donc incapable de générer un gradient responsable de l'attraction, la rétention et la migration de cellules cibles.Même si cette mutation n'altère pas le bon développement de la souris et que le muscle à l'état basal est normal, nous avons montré un défaut de régénérescence musculaire chez ces souris mutées ayant subit l'agression musculaire la plus sévère avec la présence d'un tissus fibreux cicatriciel et une infiltration d'adipocytes. Nous avons montré que l'absence de gradient de CXCL12 aboutit à une dérégulation de l'angiogenèse dont certains stigmates sont visibles à l'état basal, mais dont la pleine anomalie ne se mesure qu'en conditions d'agression. Cette dérégulation pourrait s'expliquer par la présence de vaisseaux non stabilisés par des cellules murales (cellules musculaires lisses et péricytes). Le développement de ce modèle de fibrose ouvre la voie à différentes questions sur le déroulement de la fibrose en général, de la réparation musculaire, et des relations qu'entretient l'arbre vasculaire les cellules de soutien
Muscle regeneration needs specialized stem cells but it also requires coordinated action of stromal cells and supporting tissue. The study of muscle regeneration can not be only limited to the study of the activation, proliferation and differentiation of muscle stem cells. The aim of this thesis was to approach the mechanisms involved in the harmonious regeneration of the muscle beside satellite cells to know the different cellular elements of the supporting tissues and also the stroma through the study of CXCL12 chemokine and its anchorage to the GAG of the muscle extracellular matrix.First, we made the observation that muscle damage models were numerous and were used indistinctly with ignorance of their own specificities. Thus, the first part of this thesis consisted of comparing different injury models commonly used in the literature to determine their potential targets and choose the most adapted to scientific questions asked. secondarily, we used an animal model genetically invalidated for anchoring of CXCL12 gamma isoform to the matrix to study the skeletal muscle development, stem cells and the organization of their niche and finally, the repair.We showed initially that the initial choice of the injury model is important during pathophysiological studies. Although all muscle injury models lead to an ad integrum restitution, regeneration processes vary considerably and the impact on different cell types also varies widely. In addition, we have shown that the only histological parameters, are not entirely sufficient to say that muscle regeneration is complete and each cell type should be considering in detail as well as functional parameters that should be measured in perspectives of this work.We used as a study model, mice knock in CXCL12Gagtm/Gagtm recently developed in the laboratory and in which CXCL12 gene has been mutated for the region coding the controlling anchoring of CXCL12 to HS. In this mouse, CXCL12 is present but unable to bind to the extracellular matrix HS while keeping its activity via CXCR4. In this case CXCL12 is unable to generate a gradient responsible for the attraction, retention and migration of target cells.Although this change does not affect the development of the mouse and the muscle at basal state is normal, we have shown a lack of muscle regeneration in these mice with fibrosis and fat infiltartion.The muscle stem cell compartment seems not to be altered in the mutant mice in the basal state and during the regeneration of the muscle. We have shown that the absence of CXCL12 gradient leads to deregulated angiogenesis through vascular hyperproliferation at the basal state. This deregulation seems to be responsible of an altered vascular regeneration after injury with the presence of non-stabilized mural cells (smooth muscle cells and pericytes). This lack of vascular regeneration appears to be responsible for a muscle regeneration failure

La sarcopénie est une maladie dégénérative liée à l’âge qui se caractérise par une perte progressive et involontaire de la masse et de la force musculaire. Elle s’accompagne d’une atteinte de la régénération musculaire et d’une accumulation des espèces réactives de l’oxygène. Les cavéoles sont des invaginations de la membrane plasmique. Dans le muscle, elles jouent un rôle dans la différenciation des cellules satellites et dans le maintien de l’unité contractile dans le muscle différencié. Certaines formes de myopathies sont consécutives à l’absence de cavéoles dans le muscle. Elles sont également impliquées dans la médiation de signaux liés à la régulation du stress oxydant. Afin de mieux comprendre les mécanismes régulant la mise en place de la sarcopénie, nous avons étudié ici les relations existant entre le stress oxydant et les cavéoles. Des cellules musculaires de souris ont été traitées par l’H2O2 et une diminution du taux des cavéolines-1et -3 a été mise en évidence dans des myoblastes et les myotubes, respectivement. Il apparaît donc que les protéines constitutives des cavéoles sont effectivement sensibles au stress oxydant dans les cellules musculaires. En présence d’H2O2, la fonction des cavéoles (endocytose et résistance au stress mécanique) était également significativement altérée dans des myoblastes. L’ensemble des résultats obtenus suggère que le stress oxydant aurait un effet sur les cavéoles, ce qui pourrait entraîner des conséquences sur la régénération et le maintien de l’intégrité musculaire au cours du vieillissement
Sarcopenia is an age-related degenerative disease which is characterized by a progressive and involuntary loss of muscle mass and strength. It is accompanied by an impairment of muscle regeneration and accumulation of reactive oxygen species. Caveolae are invaginations of the plasma membrane. In muscle, they play a role in the differentiation of satellite cells and in maintaining the contractile unit of the differentiated skeletal muscle. Some myopathies are resulting from the absence of caveolae in muscle. Caveolae are also involved in mediating signals related to the regulation of oxidative stress. To better understand the mechanisms involved in the development of sarcopenia, we investigated here the relationship between oxidative stress and caveolae. Mouse muscle cells were treated with H2O2 and decreased levels of caveolin-1 and -3 were demonstrated in myoblasts and myotubes, respectively. It therefore appears that caveolae constituent proteins are actually sensitive to oxidative stress in muscle cells. In the presence of H2O2, caveolae functions (endocytosis and resistance to mechanical stress) were also significantly degraded in myoblasts. Altogether, these data suggest that oxidative stress would affect caveolae, which could have consequences on regeneration and maintenance of muscle integrity during aging

2012/2013
The skeletal muscle is a terminally differentiated tissue. Its capacity to repair following injury or disease depends on a population of myogenic precursors, named satellite cells. These cells are localized beneath the skeletal muscle fiber, in a specialized microenvironment, the niche. The niche preserves the homeostatic conditions of satellite cell quiescence, but at the same time, it ensures their responsiveness to mechanical, physical and chemical triggers from the surrounding environment. Therefore, the composition of the external milieu is critical in determining satellite cell behavior. As a matter of fact, during aging or under pathological conditions, alterations of the extracellular environment entail a severe impairment of satellite cell ability to sustain regeneration and repair of the skeletal muscle tissue. The general goal of this thesis was to focus on some of the trophic factors potentially present in the satellite cell niche in vivo and to characterize their role on the modulation of satellite cell functions in vitro. The first part of the research activity dealt with the study of the trophic effect of ATP on mouse myoblast proliferation. From literature, it emerged that ATP is a potential regulator of the skeletal muscle regenerative program, however the signalling mechanism remained partially unknown. We observed that ATP increased myoblast growth rate, effect that was mimicked by low concentrations of H2O2. Reactive oxygen species (ROS) imaging revealed that ATP induced H2O2 production, at concentrations comparable to those effective in triggering myoblast proliferation. Interestingly, the exposure to equimolar concentrations of adenosine did not mimic the effect of ATP, excluding any role for the main hydrolysis product of ATP in the control of cell cycling. This result was in agreement with data reporting that the specific enzymes responsible for ATP degradation are poorly expressed in myoblasts and become upregulated after cell differentiation. In line with the latter observation, it appeared reasonable that the differentiating skeletal muscle cells were more exposed to ATP-derived adenosine than proliferating myoblasts, and this suggested a potential physiological role for the nucleoside adenosine in the later phases of myogenesis. Taking into account that adenosine receptors (ARs) are present in mouse myotubes, in a second study we hypothesized a crosstalk between nAChRs and ARs. Using the Ca2+-imaging technique, we observed that the pharmacological modulation of ARs triggered variations in the nAChR-driven ([Ca2+]i) spikes. Moreover, our preliminary results suggest not only an interplay between the two receptors but also that endogenous adenosine is tonically released by twitching myotubes and activates its receptors. The third research project was aimed at exploring the role of neural agrin, a heparan sulphate proteoglycan, so far known as the key organizer of post-synaptic elements during skeletal muscle differentiation/regeneration. Besides agrin’s canonical effect on the maturation of the NMJ, novel roles have been discovered in the recent years, suggesting that the neurotrophic factor has pleiotropic effects. In this new context, we pursued the identification of potential new roles for neural agrin in the determination of satellite cell behaviour. Firstly, the analysis of different cell models, including C2C12 cell line and primary mouse and human cells, and revealed an increase in IL-6 secretion following exposure to agrin. Secondly, we addressed the hypothesis of agrin as a potential modulator of human myoblasts proliferation. Our preliminary results demonstrate that agrin enhances the proliferative capacity of human satellite cells and suggest the potential mechanism involved in the signaling cascade.
Il muscolo scheletrico è un tessuto terminalmente differenziato. La sua capacità rigenerativa in seguito a danno o patologia dipende da una popolazione di precursori miogenici, le cellule satelliti. Esse sono localizzate sulla superficie della fibra muscolare, racchiuse in un ambiente altamente specializzato, la nicchia. La nicchia assicura il mantenimento della quiescenza cellulare, ma allo stesso tempo fa sì che la cellula satellite risponda a stimoli meccanici, fisici o chimici, provenienti dall’ambiente esterno. Per questo motivo, la composizione dell’ambiente circostante condiziona altamente il comportamento della cellula satellite. Infatti, le alterazioni che si verificano con l’invecchiamento o in seguito a patologia compromettono la capacità dei precursori miogenici di sostenere la rigenerazione del tessuto. Lo scopo di questo lavoro di tesi è stato quello di individuare e caratterizzare alcuni dei fattori trofici che compongono il microambiente della cellula satellite in vivo, per cercare di capire come essi modulino le funzioni dei precursori miogenici durante la rigenerazione in vitro. La prima parte dell’attività di ricerca ha riguardato lo studio dell’effetto trofico dell’ATP sulla proliferazione mioblastica. Studi in letteratura hanno fatto emergere il potenziale ruolo regolatore dell’ATP nella rigenerazione muscolare, anche se i meccanismi attraverso cui opera non sono ancora chiari. I risultati da noi ottenuti hanno dimostrato che l’ATP aumenta la proliferazione mioblastica e che un effetto simile si osserva in presenza di H2O2. L’imaging per le specie reattive dell’ossigeno (ROS) ha inoltre dimostrato che l’ATP induce la produzione di H2O2, a concentrazioni paragonabili a quelle capaci di aumentare la proliferazione. In presenza di concentrazioni equimolari di adenosina non è stato osservato alcun effetto sulla proliferazione cellulare, fatto che suggerisce che il ruolo dell’ATP non sia attribuibile all’adenosina, il suo principale prodotto di degradazione. Questo risultato è in accordo con quanto già riportato da altri Autori a proposito degli enzimi responsabili dell’idrolisi dell’ATP: essi sono poco espressi nei mioblasti in proliferazione, mentre la loro espressione aumenta notevolmente con il differenziamento cellulare. Alla luce di queste osservazioni, risultava verosimile che i miotubi fossero più esposti rispetto ai mioblasti all’adenosina derivante dall’ATP e che pertanto l’adenosina avesse un ruolo fisiologico preponderante nelle fasi avanzate della miogenesi. Dal momento che i recettori per l’adenosina (ARs) sono espressi nei miotubi murini, un secondo lavoro ha avuto come obiettivo quello di investigare un possibile “crosstalk” tra i ARs e i nAChRs. Esperimenti di Ca2+- imaging hanno dimostrato come la modulazione farmacologica dei ARs si traduca in una variazione nelle oscillazioni di [Ca2+]i indotte dall’attività del nAChR. Questi risultati, sebbene preliminari, suggeriscono non solo che i due recettori interagiscono tra loro, ma anche che l’adenosina è tonicamente secreta dai miotubi in contrazione e agisca attivando i suoi recettori. Il terzo progetto di ricerca è stato finalizzato allo studio del ruolo dell’agrina neuronale, un proteoglicano eparan solfato, già noto per la sua capacità di aggregare elementi sinaptici durante la fase di differenziamento e di rigenerazione del muscolo scheletrico. Accanto al ruolo canonico che la vede coinvolta nella maturazione della giunzione neuromuscolare, negli ultimi anni sono state descritte nuove funzioni per l’agrina neuronale, che la indicano come un fattore pleiotropico. In questo contesto, abbiamo esplorato nuove proprietà dell’agrina. In primo luogo, l’analisi di diversi modelli cellulari, incluse la linea cellulare C2C12 e cellule primarie murine e umane, ha dimostrato che il fattore neurotrofico potenzia il rilascio di IL-6. In un secondo studio, è stato ipotizzato un potenziale effetto di modulazione della proliferazione di mioblasti umani da parte dell’agrina neuronale. Risultati preliminari hanno dimostrato che l’agrina aumenta la capacità proliferativa delle cellule satelliti umane. Inoltre, sono stati individuati alcuni dei fattori molecolari che partecipano alla cascata di segnalazione.
XXVI Ciclo
1986

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2011.
Includes bibliography.
ENGLISH ABSTRACT: Background: IL-6 belongs to a cytokine super-family known to affect cell proliferation, although other family members are better characterized. Proliferation promoting factors (IL-6) compete with differentiation promoting factors (myogenic regulatory factors: MyoD and myogenin) to affect cell cycle. Cell cycle progression is assessed by determining the proportion of cells shifting from arrest to chromatin synthesis and mitosis phases (G0/G1 and S and G2/M respectively). Methods: This study assessed the effects of IL-6 on cell cycle progression and proliferation vs. differentiation of C2C12 skeletal myoblasts. Physiological doses (10 or 100 pg/ml) were compared to a high dose (10 ng/ml), with exposure lasting 48 hours (addition of IL-6 dose to proliferation medium at 0 and 24 hours). Acute signaling downstream of the IL-6 gp130 receptor was assessed after the first exposure. Results: Propidium iodide analysis of nuclear material using flow cytometry indicated shifts in forward scatter. Both Low and Medium doses shifted a greater proportion (p<0.05) of cells from G0/G1 to S and G2M phases at 24 hours and all doses resulted in the same shift (p<0.05) at the 48 hour time point. However, the High dose significantly (p<0.05) increased myogenin expression at the 48 hour time point. Microscopy indicated that confluence was prevented by low seeding density and did not influence the result. Cells harvested at 5 minutes post stimulation indicated that all doses significantly increased STAT3 phosphorylation. 10 minutes post stimulation the High dose group sustained elevated levels of STAT3 phosphorylation. Conclusions: Low and medium doses of IL-6 increase proliferation in a muscle satellite cell line by activating cell division and allowing myoblasts to remain in the active cell cycle. High doses of IL-6 increase differentiation by mediating upregulation of myogenic regulatory factors and this is thought to be due to prolonged STAT3 activation. Physiological control of myoblast behaviour by cytokines is evident and such control would be influenced by the severity of the endogenous cytokine response to various stimuli.
AFRIKAANSE OPSOMMING: Agtergrond: IL-6 behoort aan n sitokien super-familie bekend vir die affektering van sel verspreiding, alhoewel ander familie lede beter gekenmerk is. Bevordering van verspreiding faktore (IL-6) kompeteer met bevordering van differensiasie fatore (myogenic regulatory factors: MyoD en myogenin) om die sel siklus te affekteer. Sel siklus progressie word geassesseer deur die bepaling van die proporsie selle wat verskuif van arrestasie na chromatien sintese en mitose fases (G0/G1 en S en G2/M onderskeidelik). Metodes: Hierdie studie het die effekte van IL-6 op die progressie van die sel siklus geassesseer asook die proliferasie vs. differensiasie van C2C12 skelet spier satelliet selle. Fisiologiese dosisse (10 en 100 pg/ml) was vergelyk tot n hoog dose (10 ng/ml), met blywende blootstelling van 48 uur (byvoeging van IL-6 dose tot verspreidings medium op 0 and 24 uur). Akute sein stroomaf van die IL-6 gp130 reseptor was ook geassesseer na die eerste blootstelling. Resultate: Propidium iodide analise van kern materiaal deur vloei sitometrie het voorwaarts verskuiwing aangedui. Beide Laag and Medium doses het n groter proporsie (p<0.05) selle verskuif van die G0/G1 tot die S en G2M fases na 24 uur en alle dosisse het gelei in die selfde verskuiwing (p<0.05) by die 48 huur tyd punt. Alhoewel die Hoog dose myogenin uitdrukking aansienlik (p<0.05) verhoog het na 48 uur. Mikroskopie het aangedui dat samevloeiing voorkom was deur n lae loting digtheid en dit het nie resultate geaffekteer nie. Selle wat geoes was 5 minute na stimulasie het aangedui dat alle dosisse STAT3 fosforilasie laat toeneem het. 10 minute na stimulasie het die Hoog dose groep volgehoue vlakke van STAT3 fosforilasie besit. Gevolgtrekkings: Laag en Medium dosisse van IL-6 verhoog verspreiding in n spier satelliet sel lyn deur die aktivering van sel deling en deur selle toe te laat om in die aktiewe sel siklus te bly. Hoog dosisse van IL-6 verhoog differensiase deur bemiddelende opstoot van myogenic regulatory factors en die gedagte is dat dit bewerkstellig word deur aanhoudende aktivering van STAT3. Fisiologies beheer van satelliet selle deur sitokiene is duidelik en die beheer sal beinvloed word deur die erns van die endogene sitokien reaksie op verskillende stimuli.

Thesis (MSc)--University of Stellenbosch, 2005.
ENGLISH ABSTRACT: Cryotherapy is one of the popular treatments used to alleviate muscle soreness, especially in the competitive sports arena. However, the therapeutic use of cryotherapy is unsubstantiated because of a lack of proper investigations in the literature, especially a hypothesised effect on muscle recovery. Thus, our aims were to characterise satellite cell (SC) activity in human subjects with delayed onset muscle soreness (DOMS) and to shed light on the effect of cryotherapy on SC activity. DOMS was induced in six male subjects (24 ± 3 years) by completion of a downhill-run (DHR) protocol (5 x 8 min bouts, 2 min rest between bouts) at 70 or 80% of their individual peak treadmill speed. Ice application was applied to only one leg per subject for 3 days: 30 min every 2 hours, 5 times per day. In total 5 muscle biopsies were obtained from each subject: 1 baseline and 4 post-DHR. Post-DHR biopsies: 1 from each leg on day 1 and 7 (1st group, n=3) and 1 from each leg on day 2 and 9 (2nd group, n=3). DOMS was successfully induced as indicated by significant increases in muscle soreness at days 1 and 2 post-DHR (P < 0.01), and creatine kinase activity at day 1 post-DHR (P < 0.01). No difference in muscle soreness was found between treated and untreated legs. SC quiescence and activation were characterised by their expression of the cell surface markers CD34 and CD56 respectively. No significant change in quiescent SC was observed in the untreated or treated legs over time. However, at day 1 post-DHR the number of quiescent SC was significantly lower in the untreated compared with the treated leg (P < 0.05). There was a significant increase in activated SC numbers at day 2 post-DHR in the untreated leg, which was sustained up to day 9 post-DHR (P < 0.01). However, no such increase was found in biopsies taken on days 1 and 7. Also, no change was found in the treated leg, however a significant difference between the number of activated SC in untreated and treated legs on days 2 and 9 post-DHR (P < 0.01) was seen. No significant effect of DOMS or ice treatment was observed for the expression of the myogenic regulatory factors, MyoD and myogenin. C2C12 cell cultures induced to differentiate, however, did stain using these antibodies. This is the first study to report an effect of cryotherapy at the tissue level. In conclusion, this study highlights many unanswered questions on the SC response to DOMS at tissue level, and lays a good foundation for future studies.
AFRIKAANSE OPSOMMING: Kreoterapie is een van die gewilde behandelings wat gebruik word om spierseerheid te verlig, veral in die kompeterende sport arena, maar die gebruik van kreoterapie is onbevestig as gevolg van ‘n gebrek aan voldoende ondersoeke in die literatuur, veral ‘n hipotese oor die effek op spier-herstel. Ons doelstellings was dus om satellietsel (SC) aktiwiteit te ondersoek in mens proefpersone met vertraagde aanvang spierseerheid (DOMS) en ook om lig te werp op die effek van kreoterapie op SC aktiwiteit. DOMS was in ses mans proefpersone (24 ± 3 jare) geїnduseer deur voltooїng van ‘n afdraend-hardloop (DHR) protokol (5 x 8 min rondtes, 2 min rus tussen rondtes) teen 70 of 80% van elkeen se individuele maksimum trapmeul-spoed. Ys was vir 3 dae op net een been per proefpersoon aangewend: 30 min elke 2 ure, 5 keer per dag. 5 spierbiopsies in totaal was van elke proefpersoon verkry: 1 basislyn en 4 post-DHR. Post-DHR biopsies: 1 van elke been op dae 1 en 7 (1ste groep, n=3) en 1 van elke been op dae 2 en 9 (2de groep, n=3). DOMS was suksesvol geїnduseer soos aangedui deur die betekenisvolle verhogings in spierseerheid op dae 1 en 2 post-HR (P < 0.01) en kreatien kinase aktiwiteit op dag 1 post-DHR (P < 0.01). Geen verskil in spierseerheid is gevind tussen die onbehandelde en behandelde bene nie. SC dormansie en aktivering was gekarakteriseer deur die onderskeidelike uitdrukking van die sel oppervlak merkers CD34 en CD56. Geen betekenisvolle verandering is in SC dormansie in die onbehandelde en behandelde bene waargeneem nie, maar op dag 1 post-DHR was die getal dormante SC betekenisvol laer in die onbehandelde been as in die behandelde been (P < 0.05). Daar was ‘n betekenisvolle verhoging in die getalle geaktiveerde SC op dag 2 post-DHR in die onbehandelde been wat volgehou was tot op dag 9 post-DHR (P < 0.01), maar so ‘n verhoging was nie in biopsies wat op dae 1 en 7 geneem is gevind nie. Daar is ook geen verandering in die behandelde been gevind nie, maar ‘n betekenisvolle verskil in die getal geaktiveerde SC is tussen die onbehandelde en behandelde bene op dae 2 en 9 post-DHR gevind(P < 0.01). Geen betekenisvolle effek van DOMS en ys-aanwending vir die uitdrukking van die miogeniese (myogenic) regulatoriese faktore, MyoD en myogenin, is waargeneem nie. C2C12 sel kulture wat geїnduseer is om te differensieer het wel gekleur vir hierdie antiliggame. Dit is die eerste studie wat ‘n effek van kreoterapie op weefselvlak rapporteer. Ten slotte, hierdie studie beklemtoon baie onbeantwoorde vrae oor die SC respons op DOMS op weefselvlak en dit lê ‘n goeie grondslag neer vir toekomstige studies.

Satellite cells (SC) are muscle-specific stem cells involved in muscle growth and repair in adults. The niche of SC consists of basement membrane (basal lamina), muscle fibre and supporting cells. A major component of basement membranes (BM) is laminin, a heterotrimer protein composed of α, β and γ chains. During the transition from embryonic to foetal and to adult, the muscle basement membrane is extensively remodelled. Indeed, the embryonic basement membrane that is associated with the myotome contains Laminin-111 and -511. During foetal stages, Laminin-111 and -511 are progressively replaced by Laminin-211, which is the main Laminin constituent of the adult basement membrane surrounding muscle fibres. As similarities exist between the myogenic programme carried out by satellite cells and that executed by embryonic muscle progenitor cells, I hypothesized that the composition of the basal lamina in adult muscles may be dynamic at the site of satellite cells to support their activation and progression through myogenesis. In this study, immunofluorescence and quantitative PCR data on ex-vivo culture system of mouse extensor digitorum longus (EDL) muscle fibres revealed the expression of Laminin a1 at the site of activated satellite cells, in contrast to previous reports showing that the adult skeletal muscle basal lamina has a uniform Laminin composition (Laminin- 211). Laminin a1 association with activated satellite cells was also observed in vivo in two distinct models of muscle regeneration, the Dystrophin-deficient mdx mouse and in cardiotoxin-injured tibialis anterior (TA) muscles. Laminin a1, secreted by satellite cells, is also expressed at the surface of macrophages in vivo. Finally, I provide evidence that Integrin α6β1, the preferred receptor for Laminin a1, is expressed at the surface of activated satellite cells ex-vivo. Altogether these results reveal that a remodelling of the basal lamina occurs during skeletal muscle regeneration with the concomitant secretion and deposition of Laminin α1 in the basal lamina overlying activated satellite cells. Laminin a1 may signal through its specific receptor Integrin α6β1 to promote satellite cell progression through myogenesis. Finally, the presence of Laminin a1 may also act as a mediator between activated satellite cells and macrophages, promoting the recruitment of macrophages to the vicinity of satellite cells to support muscle regeneration. Thus, this study provides an insight into a mechanism allowing for the remodelling of the satellite cell niche during muscle regeneration.

Skeletal muscle is a primary contributor to body mass and whole-body energy metabolism. It is an adaptive tissue with the ability to fluctuate in size and mechanical properties in response to stimulus. Health conditions involving chronic elevated inflammation levels often feature metabolic inflexibility and losses in skeletal muscle mass. Mononuclear stem cells, termed satellite cells, are mitotic and serve to donate nuclei to muscle fibers to enable skeletal muscle adaptation. Despite the well-characterized nature of satellite cell activation, proliferation, and differentiation, the underlying mechanisms regulating this process is not fully understood. Recent characterization of cytokines secreted by skeletal muscle in an endocrine type fashion has led to discoveries of inflammatory cytokines influencing satellite cell function. However, how the autocrine production and secretion of these cytokines during proliferation and differentiation in humans and their correlation with myogenic transcription factors is not well understood. Our study used satellite cells cultured from the vastus lateralis of 12 male human research subjects, and ELISA analysis to measure levels of TNF-α and IL-6 across proliferation, early differentiation, and late differentiation. Additionally, mRNA levels of Pax7, MyoD, myogenin, IL-6, TNF-α, and TGF-β were assessed in satellite cells cultured from a subset of two endurance trained and two sedentary individuals from the larger group of 12 human subjects. The novelty of our study is the large number of human research subjects and simultaneous analysis of inflammatory cytokine secretion, mRNA inflammatory cytokine expression, and myogenic transcription factor mRNA expression. Results showed an 83% decrease in IL-6 protein secretion 24 hours after exposure to differentiation media (p-value <0.05) before increasing 50-fold after 7 day of exposure to differentiation media (p-value < 0.05). Myogenin and TGF-β mRNA expression levels were positively correlated (R2 = 0.5814, p-value < 0.0001). A negative correlation was found between IL-6 and MyoD (R2 = 0.2473, p – value = 0.0257). After 1 day of exposure to differentiation media, satellite cells from endurance trained subjects exhibited higher levels of TGF-β mRNA expression compared to sedentary satellite cells of sedentary subjects of the same age and levels of adiposity (p-value < 0.05). Results support a potential relationship in humans satellite cells between myogenic transcription factors and inflammatory cytokines, however, further study is necessary in order to investigate the underlying mechanisms behind the correlations.
Master of Science
Skeletal muscle is responsible for conscious, voluntary movement. In addition, the tissue is responsible for the majority of energy expenditure in the human body. Skeletal muscle is able to adapt to exercise programs through the fusion of undifferentiated stem cells – called satellite cells – in the skeletal muscle fiber. In long-term diseased conditions, the immune response involves chronic rises in inflammation and results in the loss of skeletal muscle and corresponding loss of ability to move. A shorter rise in inflammation is also linked with the positive exercise response. Our study features satellite cells harvested from muscle samples of 12 male human research participants. We were interested in evaluating the relationships between the expression and secretion of two proteins associated with inflammation and regulation of the satellite cell cycle. The two proteins of interest in our study are tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). We also measure the gene expression of another inflammatory protein, transforming growth factor beta (TGF-β). In order to know where the cells were in their life cycle, we measured expression of genes associated with the division (Pax7), early fusion (MyoD), and late fusion of satellite cells (myogenin). Our study found a decrease in IL-6 secretion and expression as the process of satellite cells turning into muscle fibers was initiated. Additionally, a 50-fold increase in IL-6 expression was found at day 7 compared to day 0 of the satellite cell cycle. Additionally, we found a positive correlation between TGF-β and myogenin and a negative correlation between IL-6 and MyoD. Although we found correlations between satellite cell cycle genes and inflammation genes, more research is necessary to see if there is a pathway causing this relationship.

Myotonic dystrophy (DM) is an autosomal dominant multisystemic disorder characterized by a variety of multisystemic features including myotonia, muscular dystrophy, cardiac dysfunctions, cataracts and insulin-resistance. DM1 is caused by an expanded (CTG)n in the 3’ UTR of the DMPK gene, while DM2 is caused by the expansion of a (CCTG)n repeat in the intron 1 of the CNBP gene. In both forms, the mutant transcripts accumulate in nuclear foci altering the function of some alternative splicing regulators which are necessary for the physiological processing of mRNAs. However, the downstream pathways by which these RNA binding proteins cause skeletal muscle alteration are not well understood. For these reasons the aim of my PhD project was to analyze the molecular mechanisms behind DM skeletal muscle atrophy. In the first part of my PhD we have performed different studies to better define the molecular pathogenesis of DM. In particular, we have analysed the histopathological and biomolecular features of skeletal muscle biopsies from a cohort of DM1 and DM2 patients presenting different phenotypes. The results indicated that the splicing and muscle pathological alterations observed are related to the clinical DM1 and DM2 phenotype and that CUGBP1 seems to play a role only in DM1, confirming that the molecular pathomechanism of DM is more complex than the one actually suggested. These data were confirmed by the analysis of two different biopsies obtained from 5 DM2 patients that showed that morphological alterations evolve more rapidly over time than the molecular changes suggesting that the molecular mechanisms that drive to skeletal muscle atrophy are still unclear and that these features cannot be explained only by spliceopathy. For all these reasons we decided to analyse DM satellite cells activity in vitro. Satellite cells are the muscle fibre precursor cells and our data indicated that both DM1 and DM2 skeletal muscle cells have lower proliferative capability than control myoblasts. Moreover, the premature proliferative growth arrest observed in DM cells appears to be caused by an overexpression of p16 in DM1 muscle cells, while DM2 muscle cells stop dividing with telomeres shorter than controls, suggesting that in these cells the signaling involved in premature senescence depend on a telomere-driven pathway. Finally, we decided to analyze the insulin pathway which is involved in the regulation of skeletal muscle atrophy. Our data have shown that DM1 and DM2 cells exhibit a lower glucose uptake and a lower proteins activation after 10 nM insulin stimulation when compared to controls suggesting that also this pathway could play a role in the molecular mechanisms that drive skeletal muscle atrophy in DM patients. In conclusion, we have shown that the molecular mechanisms behind skeletal muscle atrophy in DM1 and DM2 patients are more complicated than that previously suggested and further analysis are necessary to understand why skeletal muscle atrophy affect mainly type 1 fibres in DM1 patients, while on the contrary it affects selectively type 2 fibres in DM2 patients.

2006/2007
Aims: The mechanisms involved in sarcopenia, the decline in muscle mass with aging coupled with loss of force and function, has been actively investigated in animal and human models over the last years [reviewed in Di Iorio et al., Sarcopenia: age-related skeletal muscle changes from determinants to physical disability, Int. J. Immunopathol. Pharmacol. 19 (2006) 703-719]. An important age-associated deficit may be the alteration of the mechanisms controlling Ca2+ handling. Moreover, it has already been proposed that defective fibres in old humans could result from a reduced efficiency of aged satellite cells (a distinct muscle cell subtype, responsible for post-natal growth and repair of damaged fibres) in properly differentiating into myotubes with a mature E-C coupling mechanism [see: Lorenzon et al., Aging affects the differentiation potential of human myoblasts, Exp. Gerontol. 39 (2004) 1545-1554]. Proceeding from these results, the main goal of the present Ph.D. thesis was to investigate whether the inefficiency of aged satellite cells to generate functional skeletal muscle fibres could be partly due to defective voltage-dependent Ca2+ currents. Methods: The whole-cell patch clamp and the videoimaging techniques were employed to measure respectively T- and L-type Ca2+ currents and [Ca2+]i transients in myoblasts and/or myotubes derived from murine and human satellite cells, obtained respectively from young murine skeletal muscle and then aged in vitro under culture conditions, and from human skeletal muscle tissue of healthy donors aged 2, 12, 76 and 86 years. Results: First of all, I confirmed that both murine and human senescent satellite cells fuse more slowly and less efficiently, leading to smaller and thinner myotubes, as known from previous work. Moreover, I showed for the first time that both in myotubes derived from in vitro aged murine satellite cells and in human myotubes derived from satellite cells of old donors the functional expression and the biophysical properties of T- and L-type voltage-dependent Ca2+ channels are impaired. In fact, extensively, less Ca2+ can be available via T-type and L-type channels in old myotubes than in the young ones, and this can be put in relation to the age-related decrease in the quality of myoblast fusion. I also confirmed a specific responsibility of the decrease of the L-type channel number and/or activity for the age-related lowering of intracellular Ca2+ release (the so-called E-C uncoupling; see: Delbono et al., Excitation-calcium release uncoupling in aged single human skeletal muscle fibers, J. Membr. Biol. 148 (1995) 211-222]. Conclusions: From these results one can infer a clear parallelism between the results obtained with the in vitro aging of murine satellite cells model and that concerning the physiological process of human skeletal muscle aging in vivo. In the final analysis, aging effects on voltage-dependent L- and T-type currents could be one of the causes of the inability of old satellite cells to efficiently counteract age-related impairment in muscle force. So, a further strong evidence has been given that in humans, as in other mammals, the satellite cells and the regulation of Ca2+ homeostasis have a decisive role in the physiological process of skeletal muscle aging.
**************************************************************************************** Scopo della ricerca: Nel corso dell’invecchiamento il muscolo scheletrico subisce cambiamenti significativi, quali la perdita di forza e di massa muscolare (sarcopenia; per una rassegna recente vedere: Di Iorio et al., Sarcopenia: age-related skeletal muscle changes from determinants to physical disability, Int. J. Immunopathol. Pharmacol. 19 (2006) 703-719). Era già noto che le disfunzioni correlate all’età potrebbero essere almeno in parte dovute all’inabilità delle cellule satelliti, le cellule staminali per eccellenza del muscolo scheletrico, di rigenerare fibre muscolari funzionali nell’individuo anziano (vedere: Lorenzon et al., Aging affects the differentiation potential of human myoblasts, Exp. Gerontol. 39 (2004) 1545-1554). Il principale scopo di questa Tesi di Dottorato è stato quello di studiare le possibili modificazioni dei meccanismi che regolano l’omeostasi calcica in cellule satelliti murine ed umane, rispettivamente nel corso dell’invecchiamento in vitro (senescenza replicativa in coltura) ed in vivo. In particolare l’attenzione è stata focalizzata sull’effetto delle alterazioni, collegate all’età, delle correnti al Ca2+ voltaggio-dipendenti di tipo L e di tipo T in miotubi provenienti dalla proliferazione e dal differenziamento di cellule satelliti a vari stadi di invecchiamento. Metodologia: Le cellule satelliti murine utilizzate derivavano da una linea primaria espansa denominata i28; le cellule satelliti umane sono state ottenute da biopsie di individui di diversa età (2, 12, 76 e 86 anni). Esperimenti di elettrofisiologia e di videomicroscopia hanno permesso lo studio rispettivamente delle correnti al Ca2+ e dei transienti di Ca2+ intracellulare, nonché delle loro modifiche collegate all’invecchiamento in vitro e in vivo nei modelli murino ed umano. Risultati: Vengono confermati, sia nel modello murino di invecchiamento in vitro che nel modello umano di invecchiamento in vivo, i dati sulla relazione tra sarcopenia e difficoltà di cellule satelliti invecchiate nel formare un numero sufficiente di nuovi miotubi, che anche morfologicamente risultano diversi da quelli derivanti dalla fusione di cellule satelliti giovani. Inoltre, si dimostra per la prima volta che le correnti al Ca2+ in esame sono espresse in minor percentuale e densità, e più tardivamente nel corso del differenziamento, in miotubi derivati da cellule satelliti murine a stadi avanzati di senescenza replicativa, e in cellule umane da donatore anziano. Anche le proprietà biofisiche dei canali di tipo L e T, presenti in miotubi derivati da cellule satelliti invecchiate in vitro e in vivo, appaiono compromesse; complessivamente, meno Ca2+ può entrare attraverso i due tipi di canale e ciò può essere messo in relazione alla riduzione, correlata all’età, della capacità differenziativa e di fusione in miotubi. Viene ulteriormente messo in rilievo il ruolo determinante, nel corso dell’invecchiamento, del calo in numero e in attività dei canali di tipo L, come meccanismo alla base del minor rilascio di calcio intracellulare (fenomeno del disaccoppiamento eccitazione-contrazione; vedere: Delbono et al., Excitation-calcium release uncoupling in aged single human skeletal muscle fibers, J. Membr. Biol. 148 (1995) 211-222). Conclusioni: Dai risultati ottenuti si evince un netto parallelismo tra il modello dell’invecchiamento in vitro di cellule satelliti murine e l’invecchiamento in vivo di cellule satelliti umane. In ultima analisi, si avvalora l’ipotesi che alterazioni età-dipendenti delle correnti al Ca2+ voltaggio-attivate possano essere alla base dell’impossibilità di cellule satelliti invecchiate di contrastare efficacemente la riduzione di forza muscolare caratteristica dell’anziano.
XX Ciclo
1980