Rozprawy doktorskie na temat „Muscle regeneration”

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.

Thesis (Ph. D.)--West Virginia University, 2007.
Title from document title page. Document formatted into pages; contains xii, 270 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Satellite cells are a distinct lineage of myogenic precursors that are responsible for the growth of muscle during post-natal life and for its repair after damage. During muscle growth and regeneration satellite cells are activated in response to growth signals from the environment, which induces the expression of one or both of the two MRFs, Myf-5 or MyoD. Activated satellite cells migrate to the site of injury and proliferate before these transcription factors go on to activate transcription of myogenic genes. The myoblasts can then adopt one of two fates. Some myoblasts initiate terminal differentiation and are able to either fuse into existing myofibres to repair them, or fuse with other myoblasts to form new fibres. Other myoblasts do not differentiate but instead return to quiescence and adopt a satellite cell position on repaired or newly formed fibres. Mighty, a downstream target of myostatin that was discovered by the Functional Muscle Genomics Laboratory has recently been shown to induce cell hypertrophy in cell culture through enhanced differentiation and fusion of myoblasts. Myostatin-null mice have hypertrophic muscles and an improved muscle regeneration phenotype. These mice have also been shown to have higher basal levels of Mighty in skeletal muscle than wild-type mice. In this thesis the expression profile of Mighty during skeletal muscle regeneration was characterised in relation to MyoD. During regeneration Mighty gene expression was induced at day five post-injury in both wild-type and myostatin-null mice. In the myostatin-null mice Mighty gene expression remained elevated at day seven post injury in contrast to the levels in the wild-type, which had decreased at this time point. By day-14 and day-28 post-injury Mighty levels were decreased. The up-regulation of Mighty occurs at the time of peak myotube formation in regenerating skeletal muscle, consistent with a role for Mighty in enhancing differentiation and fusion of myoblasts. The extended up-regulation of Mighty in the myostatin-null muscle may be responsible for the enhanced regeneration phenotype of these mice. Analysis of the myotube and reserve cell populations, which are an in vitro model of satellite cells, from both C2C12 cells and Mighty over-expressing clones (Clone 7 and Clone 11) showed that Mighty expression down-regulates two satellite cell markers, CD34 and Sca-1. Both these molecules have been recently shown to be involved in myoblast fusion and reserve cell specification, although their exact role in these processes is not yet known. Expression of Sca-1 is associated with a slowly proliferating non-dividing state while CD34 is associated with the population of reserve cells that do not fuse when notch signalling is inhibited. The results of this thesis indicate that Mighty over-expression may cause the enhanced fusion phenotype by regulating these two molecules. In conclusion the data in this thesis supports a role for Mighty in the myotube formation phase of regeneration and may be able to enhance regeneration by recruiting more myoblasts to terminal differentiation by altering CD34 and Sca-1 expression.

Thesis (Ph. D.)--Ohio State University, 2004.
Document formatted into pages. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2005 Aug. 2.

Skeletal muscle plays fundamental roles for locomotion, posture maintenance and breathing and to preserve its function, skeletal muscle has developed a remarkable capacity to regenerate also after severe damage. Several studies aimed at understanding the cellular and molecular mechanisms involved in muscle repair that are deregulated in muscular dystrophy-associated fibrosis and in aging-related muscle dysfunction. However, the cellular and molecular effectors of muscle repair remain largely unknown. This doctoral thesis aims at understanding molecular mechanisms and the interplay between different muscle populations, by perturbing muscle regeneration and differentiation with small molecules. Recent studies have suggested that muscle regenerative process is improved when AMPK is activated. In the muscle of young and old mice a low calorie diet, which activates AMPK, markedly enhances muscle regeneration. Remarkably, intraperitoneal injection of AICAR, an AMPK agonist, improves the structural integrity of muscles of dystrophin-deficient mdx mice. Building on these observations we asked whether metformin, a powerful anti-hyperglycemic drug, which indirectly activates AMPK, affects the response of skeletal muscle to damage. In our conditions, metformin treatment did not significantly influence muscle regeneration. On the other hand we observed that the muscles of metformin treated mice are more resilient to cardiotoxin injury displaying lesser muscle damage. Accordingly myotubes, originated in vitro from differentiated C2C12 myoblast cell line, become more resistant to cardiotoxin damage after pre-incubation with metformin. Our results indicate that metformin limits cardiotoxin damage by protecting myotubes from necrosis. Although the details of the molecular mechanisms underlying the protective effect remain to be elucidated, we report a correlation between the ability of metformin to promote resistance to damage and its capacity to counteract the increment of intracellular calcium levels induced by cardiotoxin treatment. Since increased cytoplasmic calcium concentrations characterize additional muscle pathological conditions, including dystrophies, metformin treatment could prove a valuable strategy to ameliorate the conditions of patients affected by dystrophies. Moreover, in order to understand and control the differentiation decisions of muscle cell populations, we used automated fluorescence microscopy to screen the Prestwick library of small molecules 100% approved by the U.S. Food and Drug Administration (FDA). We have developed fluorescence microscopy readouts to monitor cell proliferation and differentiation into skeletal muscle, adipocyte or osteoblasts both in mesoangioblasts (MABS), a muscle multipotent cell line, and in a heterogeneous mixture of diverse muscle cell populations. We performed the high-throughput and highcontent screening, in order to identify compounds that either promote or inhibit the differentiation process. To date we have screened 240 molecules and we produced a list of drugs that affect the differentiation of muscle cells in skeletal muscle, adipocytes or osteoblasts. We have performed experiments to validate the list of putative interfering small molecules and in parallel we have built a similarity tree from the collection of transcriptional expression profile data from cultured human cells treated with bioactive small molecules, developed by the Connectivity Map team in The Broad Institute of MIT. By highlighting the obtained putative drugs in the similarity tree, we can discriminate if drugs affecting a specific differentiation phenotype may do so via similar or different molecular pathway and we can identify molecular mechanisms involved in differentiation decision.

Duchenne muscular dystrophy (DMD) is a fatal degenerative disorder of locomotor and respiratory muscles, in which myofibers are progressively replaced by non-muscular fibrotic tissue. Here, we show that fibrin/ogen accumulates in dystrophic muscles of DMD patients and of the mdx mouse model of DMD. Genetic loss or pharmacological depletion of fibrin/ogen in mdx mice attenuated muscular dystrophy progression and improved locomotor capacity. More importantly, fibrin/ogen depletion reduced fibrosis in mdx mouse diaphragm. Our data indicate that fibrin/ogen, through induction of IL-1 Ò, drives the synthesis of TGF Ò by mdx macrophages, which in turn, induces collagen production in mdx fibroblasts. Fibrin/ogen-produced TGF Ò further amplifies collagen accumulation through recruitment and activation of pro-fibrotic alternatively activated macrophages. Fibrin/ogen also stimulated collagen synthesis directly in mdx fibroblasts, via Ñv Ò3 integrin engagement. In addition, when analyzing a group of 39 DMD patients, fibrin/ogen accumulation in locomotor muscles was found associated with fibrosis and disease severity. These data unveil a novel role of fibrin/ogen in muscular dystrophy and, importantly, in the replacement of muscle by fibrotic tissue.

Volumetric muscle loss (VML) typically results from traumatic incidents; such as those presented from combat missions, where soft-tissue extremity injuries account for approximately 63% of diagnoses. These injuries lead to a devastating loss of function due to the complete destruction of large amounts of tissue and its native basement membrane, removing important biochemical cues such as hepatocyte growth factor (HGF), which initiates endogenous muscle regeneration by recruiting progenitor cells. Clinical strategies to treat these injuries consist of autologous tissue transfer techniques, requiring large amounts of healthy donor tissue and extensive surgical procedures that can result in donor site morbidity and limited functional recovery. As such, there is a clinical need for an off-the-shelf, bioactive scaffold that directs patient’s cells to align and differentiate into muscle tissue in situ. In this thesis, we developed fibrin microthreads, scaffolds composed of aligned fibrin material that direct cell alignment along the longitudinal axis of the microthread structure, with specific structural and biochemical properties to recreate structural cues lost in VML injuries. We hypothesized that fibrin microthreads with an increased resistance to proteolytic degradation and loaded with HGF would enhance the functional, mechanical regeneration of skeletal muscle tissue in a VML injury. We developed a crosslinking strategy to increase fibrin microthread resistance to enzymatic degradation, and increased their tensile strength and stiffness two- to three-fold. This crosslinking strategy enhanced the adsorption of HGF, facilitated its rapid release from microthreads for 2 to 3 days, and increased the chemotactic response of myoblasts twofold in 2D and 3D assays. Finally, we implanted HGF-loaded, crosslinked (EDCn-HGF) microthreads into a mouse model of VML to evaluate tissue regeneration and functional recovery. Fourteen days post-injury, we observed more muscle ingrowth along EDCn-HGF microthreads than untreated controls, suggesting that released HGF recruited additional progenitor cells to the injury site. Sixty days post-injury, EDCn-HGF microthreads guided mature, organized muscle to replace the microthreads in the wound site. Further, EDCn-HGF microthreads restored the contractile mechanical strength of the tissue to pre-injured values. In summary, we designed fibrin microthreads that recapitulate regenerative cues lost in VML injuries and enhance the functional regeneration of skeletal muscle.

Skeletal muscle comprises a large percentage of the human body mass and plays an essential role in locomotion, postural support, and breathing. Unfortunately, severe muscle injuries can lead to extensive and irreversible fibrosis, scarring, and loss of function without therapeutic intervention. In these cases, the repair of damaged muscle may be improved by a material system capable of on-demand, spatiotemporally controlled biologic delivery. The hypothesis guiding this thesis is that the regeneration of injured skeletal muscle can be controlled by an active ferrogel scaffold that provides a microenvironment suitable for myogenic cell survival and is capable of delivering these cells to injured muscle tissue in a noninvasive and precisely timed manner. In this thesis, a new magnetically responsive biomaterial capable of triggered drug and cell delivery was developed to enhance the regeneration of severely injured skeletal muscle. By redistributing the iron oxide content of the conventional monophasic ferrogel, biphasic ferrogels were fabricated that were appropriate in size and mechanical properties for in vivo implantation and on-demand triggered release in small animal models. Strikingly, magnetic actuation of empty biphasic ferrogel scaffolds resulted in uniform cyclic compressions that enhanced muscle regeneration without the use of cells or growth factors. Reduced fibrous capsule formation around the implant, as well as reduced fibrosis and inflammation in the injured muscle, demonstrated a potential immunomodulatory role for ferrogel-driven cyclic compressions. Biphasic ferrogel scaffolds were also used to deliver cells and growth factors precisely timed with inflammation in vivo to enhance functional muscle regeneration. Cells and growth factors were delivered by ferrogel scaffold to severely injured muscle immediately following injury and at delayed time points. Significant reductions in fibrosis and increases in angiogenesis were observed following delayed delivery. More importantly, delayed scaffold treatment of injured muscle led to enhanced engraftment efficiency and functional muscle regeneration. Together, these results demonstrate the therapeutic potential of this new magnetically responsive biomaterial.
Engineering and Applied Sciences - Engineering Sciences

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.

La régénération musculaire s’appuie sur une réserve de cellules souches résidant dans le muscle appelées cellules satellites (MuSCs). Les MuSCs sont quiescentes et peuvent s’activer à la suite d’une blessure du muscle afin de former des progéniteurs amplificateurs (myoblastes) qui se différencieront et fusionneront pour former de nouvelles myofibres. Durant ce processus, un réseau complexe de voies de signalisation est impliqué, parmi lequel la signalisation du facteur de croissance transformant bêta (TGFβ) joue un rôle fondamental. Précédents rapports ont proposé de nombreuses fonctions pour la signalisation TGFβ dans les cellules musculaires, comme leur quiescence, activation et différenciation, mais l’impact de TGFβ sur la fusion de myoblastes n’a jamais été étudié. Dans cette étude, nous avons montré que cette signalisation réduit la fusion des cellules musculaires indépendamment de leur différenciation. Au contraire, l’inhibition de la signalisation TGFβ accroît la fusion cellulaire et favorise les ramifications entre myotubes. Une pharmaco-modulation de la voie in vivo perturbe la régénération musculaire après blessure. Une addition exogène de la protéine TGFβ conduit à une perte de fonction du muscle, tandis que l’inhibition de la voie induit la formation de myotubes géants. Les analyses transcriptomiques et fonctionnelles ont montré que TGFβ agit sur la dynamique de l’actine afin de réduire la diffusion cellulaire à travers une modulation des protrusions à base d’actine. Nos résultats ont donc révélé une voie de signalisation qui limite la fusion de myoblastes et ajoutent un nouveau niveau de compréhension sur la régulation moléculaire de la myogenèse
Muscle regeneration relies on a pool of muscle-resident stem cells called satellite cells (MuSCs). MuSCs are quiescent and can activate following muscle injury to give rise to transient amplifying progenitors (myoblasts) that will differentiate and finally fuse together to form new myofibers. During this process, a complex network of signalling pathways is involved, among which, Transforming Growth Factor beta (TGFβ) signalling cascade plays a fundamental role. Previous reports proposed several functions for TGFβ signalling in muscle cells including quiescence, activation and differentiation. However, the impact of TGFβ on myoblast fusion has never been investigated. In this study, we show that TGFβ signalling reduces muscle cell fusion independently of the differentiation step. In contrast, inhibition of TGFβ signalling enhances cell fusion and promotes branching between myotubes. Pharmacological modulation of the pathway in vivo perturbs muscle regeneration after injury. Exogenous addition of TGFβ protein results in a loss of muscle function while inhibition of the TGFβ pathway induces the formation of giant myofibres. Transcriptome analyses and functional assays revealed that TGFβ acts on actin dynamics to reduce cell spreading through modulation of actin-based protrusions. Together our results reveal a signalling pathway that limits mammalian myoblast fusion and add a new level of understanding to the molecular regulation of myogenesis

La atrofia muscular por desuso en el contexto de las enfermedades crónicas, como enfermedades respiratorias, cáncer o un reposo prolongado, se caracteriza por la pérdida de masa y función muscular. Además, el desgaste muscular asociado a la caquexia cancerosa, puede verse agravado tras un período de desuso muscular. Un tratamiento con compuestos polifenólicos como la curcumina o el resveratrol puede atenuar la pérdida de masa muscular. En esta tesis, un modelo de inmovilización de una extremidad trasera en ratones, dio lugar a una disminución del peso y función en el músculo gastrocnemio, junto con una activación de la regeneración muscular. Mientras que un período de recuperación, sin la inmovilización de la extremidad trasera, conllevó a un aumento en el peso y la función del músculo gastrocnemio además de la regulación del proceso de regeneración muscular tardía. Asimismo, la inmovilización trasera en ratones con caquexia cancerosa, contribuyó aún más al desgaste muscular de estos ratones. El tratamiento con curcumina o resveratrol en animales expuestos a inmovilización seguida de períodos de recuperación, atenuó la pérdida de masa del músculo gastrocnemio a través de la vía de la sirtuina-1 y la regulación de los mecanismos proteolíticos, junto con una activación de las células progenitoras musculares.
Disuse muscle atrophy in the context of chronic diseases, such as respiratory diseases, cancer or prolonged bed rest, is characterized by the loss of muscle mass and function. Furthermore, muscle wasting due to cancer associated cachexia may be further aggravated by periods of muscle disuse. Treatment with polyphenolic compounds such as curcumin or resveratrol may attenuate muscle mass loss. In the present thesis, a model of hindlimb immobilization in mice induced a decline in gastrocnemius muscle weights and function along with an activation in muscle regeneration. While a period of muscle recovery, without hindlimb immobilization, elicited an increase in gastrocnemius muscle weight and function with an upregulation of muscle late regeneration process. Moreover, hindlimb immobilization in cancercachectic animals, further contributed to the muscle wasting in those mice. Treatment with either curcumin or resveratrol in animals exposed to immobilization followed by periods of recovery, ameliorate gastrocnemius muscle mass loss through activation of sirtuin-1 and regulation of proteolytic mechanisms, together with an activation of muscle progenitor cells.

Skeletal muscles have a remarkable capacity to repair and regenerate in response to injury by virtue of their unique population of resident muscle stem cells (MuSCs). Recently, several studies have reported that mitochondria are important regulators of fate and function in various types of stem cells including MuSCs. Furthermore, emerging evidence has shown that accumulation of dysfunctional mitochondria leads to stem cell aging, premature commitment and impaired self-renewal. Preliminary evidence from publicly available transcriptomics datasets processed by our lab showed that Phosphatase and tensin homolog (PTEN)-induced putative kinase 1(PINK1) and Parkin/PARK2 genes, two key regulators of mitophagy are expressed in quiescent MuSCs and are transiently down-regulated as MuSCs activate. This led us to hypothesize that maintenance of an optimally functioning population of mitochondria through mitophagy would be important for self-renewal and muscle repair. In vitro single myofiber cultures isolated from mitophagy reporter mice (mito-QC mice), show that mitophagy is active in quiescent MuSCs and is transiently decreased upon MuSCs activation. We also show that mitophagy is re-activated in differentiating and self-renewing MuSCs. To further study muscle regeneration, we used a cardiotoxin (CTX) injury model of the Tibialis anterior (TA) muscle in mouse models harboring a knockout (KO) of PINK1 and Parkin. We show that loss of PINK1 in vivo promotes commitment of MuSCs in response to acute injury and ultimately leads to depletion of the MuSC pool and impaired muscle regeneration compared to wild type (WT) mice following repetitive injuries. Similarly, loss of Parkin in MuSCs in vivo impaired their self-renewal capacity. Consistent with these results, in vitro single myofiber cultures isolated from PINK1-deficient mice showed increased MuSCs commitment and impaired self-renewal. In vitro preliminary results from MuSCs-specific KO of Parkin revealed altered lineage progression, differentiation and self-renewal of MuSCs. Together, these findings suggest that PINK1/Parkin-dependent mitophagy acts as an important mitochondrial quality control mechanism which could be required for regulating MuSCs fate and function during muscle regeneration.

This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019
Cataloged from student-submitted PDF version of thesis. Vita.
Includes bibliographical references.
Regeneration requires both new cell production and patterning information to correctly place new tissue. Planarians are flatworms with remarkable capacity to regenerate after nearly any injury and to indefinitely maintain tissue homeostasis. Dividing cells, neoblasts, are the source of all new tissue, whereas positional information is hypothesized to be harbored by post-mitotic muscle, including the subepidermal body wall musculature. Single-muscle-cell mRNA sequencing along the anterior-posterior axis revealed regional gene expression within muscle cells. The resulting axial gene expression map included FGF receptor-like (FGFRL) homologs and genes encoding components of Wnt signaling. Two distinct FGFRL-Wnt circuits, involving juxtaposed anterior FGFRL and posterior Wnt expression domains, controlled head and trunk patterning.
Inhibition of FGFRL-Wnt circuit components led to the formation of ectopic posterior eyes or secondary pharynges, indicating their importance in maintaining the anterior-posterior axis. Inhibition of different myogenic transcription factors specifically ablated orthogonal subsets of the body wall musculature. Longitudinal fibers, oriented along the anterior-posterior axis, are required for regeneration initiation. Circular fibers maintained medial-lateral patterning during head regeneration. During early regeneration, transcriptional changes in muscle cells comprised part of a generic wound response displayed by all injuries, from incisions to decapitations. The sole exception to this generic response was the expression in body-wall muscle of the Wnt inhibitor notum, which occurs preferentially at anterior-facing wounds in longitudinal muscle fibers. Therefore, anterior-posterior polarity, the choice of head or tail regeneration, involves longitudinal body wall muscle fibers.
Planarian muscle were found to be highly secretory. Combining an in silico definition of the planarian matrisome and recent whole animal single-cell transcriptome data revealed that muscle is a major source of extracellular matrix (ECM). Inhibition of hemicentin-1 (hmcn-1), which encodes a highly conserved ECM glycoprotein expressed in body wall muscle, resulted in ectopic localization of internal cells, including neoblasts, outside of the muscle fiber layer. ECM secretion and maintenance of tissue separation indicated that muscle functions as planarian connective tissue. Whereas muscle is often viewed as a strictly contractile tissue, these findings reveal that planarian muscle has specific regulatory roles in axial patterning, wound signaling, and tissue architecture to enable correct regeneration.
by Lauren E. Cote.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology

Extracellular matrices (ECMs) play important structural and mechanical roles in muscle tissue. They are also critical for normal muscle homeostasis, but it is poorly understood how individual matrix proteins, or the mix of ECM proteins found in vivo affect myoblast behaviour. Aspects of this question have been examined in this thesis using both matrices from skeletal muscle and artificial scaffolds prepared from silk proteins. The ability of these surfaces to support myoblast proliferation and differentiation was examined using a murine myoblast cell line and primary human skeletal muscle myoblasts.

La régénération tissulaire est un processus de remplacement des cellules perdues ou endommagées lors d'une blessure. La sénescence cellulaire est une réponse physiologique au stress caractérisée par un arrêt du cycle cellulaire stable. Mon doctorat vise à étudier la sénescence in vivo lors de la régénération musculaire, y compris son identité, ses fonctions et sa dynamique. Nous avons précédemment démontré que la sénescence induite par blessure favorise la reprogrammation dans le muscle squelettique. Mon projet vise à étudier la biologie de la sénescence lors de la régénération musculaire de manière systémique. Premièrement, j'ai montré que la sénescence atteignait son maximum au 3ème jour après la blessure et se terminait au 15ème jour après la blessure. Ensuite, j'ai trouvé que les FAPs (Fibro-Adipogenic Progenitors) sont les principales cellules qui deviennent sénescentes. Par ailleurs, les FAPs sénescents améliorent peut-être la myogenèse in vitro de manière paracrine. P57 est le principal inhibiteur de CDK qui permet l’induction de la sénescence associée aux FAPs. En effet, il y a une réduction significative de la sénescence induite par blessure chez les souris P57KO. Enfin, nous avons trouvé que Mcl-1 est surexprimé dans les FAPs sénescents, ce qui suggère l'utilisation potentielle d’inhibiteurs de MCL-1 comme drogues sénolytiques. Dans l’ensemble, mon doctorat montre que les FAPs sont sénescents lors d'une lésion musculaire, via P57 et pourraient être important pour la régénération musculaire en favorisant la myogenèse. Ces découvertes soutiennent fortement le rôle bénéfique de la sénescence lors de la régénération musculaire
Tissue regeneration is a process of replacing lost or damaged cells upon injury. Cellular senescence is a physiological response to stress characterized by a stable cell cycle arrest and is associated with various biological and pathological processes. My Ph.D. project aims to investigate in vivo senescence during muscle regeneration, including its identities, functions, and dynamics. It has been demonstrated that cellular senescence could facilitate optimal wound healing. We previously demonstrated that injury-induced senescence promotes reprogramming in the skeletal muscle, notably through IL-6 (Chiche et al., 2017). Firstly, I showed that senescence was peaking at day 3 post-injury and terminated by day 15 post injury. Then, I found that FAPs is the primary cell type that becomes senescent by RNA sequencing (both bulk population and single-cell). Besides, senescent FAPs enhance myogenesis in vitro in a paracrine manner. Of note, P57 is the major CKI which mediates the FAPs-associated senescence. Importantly, there is a significant reduction of the injury-induced senescence in the P57 null mice. Lastly, we found Mcl-1 is overexpressed in the senescent FAPs, suggesting the potential usage of MCL-1 inhibitors as senolytic drugs.Taken together, my ph.D show that FAPs are senescent upon muscle injury, which is P57-dependent and might be important to muscle regeneration by enhancing myogenesis. These findings strongly support a beneficial role of senescence during muscle regeneration, which has direct implications in muscular degenerative diseases and muscle aging

Le muscle strié squelettique régénère ad integrum après une lésion aigüe stérile grâce aux cellules satellites qui sont les cellules souches du muscle strié squelettique. L'inflammation, et notamment les macrophages, joue un rôle important durant ce processus. En effet, après une lésion, les monocytes sanguins infiltrent le tissu et deviennent des macrophages avec un phénotype pro-inflammatoire associé à la lésion. Ces macrophages phagocytent les débris cellulaires et promeuvent la prolifération des cellules souches musculaires. Ensuite, les macrophages changent leur phénotype vers un phénotype anti-inflammatoire associé à la restauration du tissu. Ils promeuvent la différenciation, puis la fusion des cellules souches musculaires et la croissance des myofibres. Cette séquence de phénotypes inflammatoires est essentielle pour une régénération musculaire efficace. Le laboratoire a montré que ce changement de phénotype est dépendant d'un senseur énergétique majeur de la cellule qui contrôle le métabolisme cellulaire, l'AMP kinase (AMPK)al. Par ailleurs, les glucocorticoïdes sont utilisés depuis des décennies pour leurs effets anti-inflammatoires sur l'inflammation. Leur action est médiée par le Récepteur aux Glucocorticoïdes qui induit ou réprime l'expression de gènes par interaction directe ou indirecte à l'ADN. Comme l'AMPKal et les glucocorticoïdes induisent des effets anti-inflammatoires similaires sur les macrophages, nous avons posé l'hypothèse que ces 2 voies de signalisation pourraient être interconnectées dans les macrophages afin de permettre leur changement de phénotype et la régénération musculaire. Les données issues d'un modèle in vitro de lésion musculaire utilisant des macrophages dérivés de la moelle osseuse de souris ont montré que : i) les glucocorticoïdes induisaient la phosphorylation de l'AMPKal ; ii) l'AMPKal était requise pour l'acquisition fonctionnelle du statut anti-inflammatoire des macrophages induit par les glucocorticoïdes puisque des macrophages déficients pour l'AMPKal ne modifiaient pas leur phénotype et ne stimulaient pas la myogenèse. Les expériences in vivo utilisant des souris LysMCre/+;AMPKalfl/fl dans lesquelles l'AMPKal est invalidée uniquement dans les cellules myéloïdes ont montré que l'AMPKal dans les macrophages régulait les effets bénéfiques des glucocorticoïdes au cours de la régénération du muscle strié squelettique. En effet, en absence d'AMPKal dans les macrophages, les glucocorticoïdes induisaient un retard de régénération et une modification de la maturation des fibres attestée par une modification de l'expression des isoformes des chaînes lourdes de myosines. En conclusion, ces données montrent que l'AMPKal est requise pour le changement de phénotype des macrophages induit par les glucocorticoïdes et une régénération musculaire efficace
Skeletal muscle regenerates ad integrum after a sterile acute injury thanks to satellite cells (muscle stem cells). Inflammation, and notably macrophages, plays important roles during this process. Just after injury, monocytes infiltrate the tissue from the blood and convert into pro-inflammatory damaged associated macrophages. These macrophages phagocyte muscle debris and promote the proliferation of muscle stem cells. Then, macrophages switch their phenotype toward an anti-inflammatory restorative profile and promote muscle stem differentiation, fusion and myofiber growth. This sequence of macrophage profile is essential for an efficient skeletal muscle regeneration. The lab has shown that this phenotype switch is dependent of AMP kinase (AMPK)a1, a major energetic sensor in the cell controlling cellular metabolism. Besides, glucocorticoids have been used for decades for their anti-inflammatory effects on inflammation. Their actions are mediated by the Glucocorticoid Receptor which induces or represses gene expression by direct or indirect DNA-binding. As AMPKa1 and glucocorticoids induce similar anti-inflammatory effects on macrophages, we hypothesized that these 2 pathways could be interconnected in macrophages to allow the resolution of inflammation and muscle repair. Data from an in vitro model of skeletal muscle injury using bone marrow derived macrophages showed that: i) glucocorticoids induce AMPK phosphorylation; ii) AMPKa1 is required for the functional acquisition of the anti-inflammatory phenotype induced by glucocorticoids. Indeed, AMPKa1-deficient macrophages did not switch their phenotype and did not sustain myogenesis. In vivo experiments using LysMCre/+;AMPKa1fl/fl mice in which AMPKa1 is depleted only in myeloid cells, showed that macrophagic AMPK drove the beneficial effects of glucocorticoids during skeletal muscle regeneration. Inversely, in absence of AMPK in macrophages, glucocorticoids induced a delayed muscle regeneration and a modification in myofiber maturation, assessed by the alteration of myosin heavy chain expression. Altogether, these data show that glucocorticoids need AMPKa1 in macrophages for the resolution of inflammation and an efficient skeletal muscle regeneration

Calcium ions play key roles in different intracellular mechanisms, ranging from biological processes such as proliferation, gene transcription, protein post-translational modifications and aerobic metabolism to physiological mechanisms such as muscle contraction, exocytosis, energy metabolism, chemotaxis and synaptic plasticity during learning and memory [1,2]. Key players in the spatio-temporal regulation of cytosolic calcium concentrations ([Ca2+]cyt) are mitochondria, intracellular organelles that can accumulate Ca2+ in the very rapid time-scale of hundreds of milliseconds [3] and reach values up to 100 µM in some cell types [4]. Mitochondrial Ca2+ overload, caused by either abnormal release from internal stores, physical damage of mitochondria or malfunction of receptors and channels present in their membrane, has been long known to be a critical event in the bioenergetic crisis associated with cell death by necrosis [2]. Furthermore, one of the most important roles that highlights the importance of mitochondrial Ca2+ in cell metabolism and survival is the mitochondrial Ca2+-dependent control of mitochondrial adenosine triphosphate (ATP) production, the main fuel for sustaining diverse cell functions [5,6]. Thus, the understanding of the role of mitochondria in regulating cellular Ca2+ homeostasis has become crucial for the understanding of a series of cell functions. Nevertheless, this analysis was severely limited due to the lack of molecular information on the identity of the protein responsible of mitochondrial Ca2+ uptake. The situation completely reversed in 2011 when Mootha’s and our laboratory identified the molecular identity of the mitochondrial Ca2+ uniporter (MCU) [7,8]. From that moment, we have witnessed an explosion of studies aimed to characterize the composition and regulation of the complex. It is now clear that MCU exists in a large protein complex that includes pore-forming and regulatory subunits [9]. The pore forming subunit includes MCU, the MCU dominant-negative subunit, MCUb, and the essential MCU regulator, EMRE, whereas the regulatory subunit includes the mitochondrial calcium uptake protein 1 and 2, MICU1, MICU2 [7,10–13]. One of the most peculiar subunits of the MCU complex is MCUb that was identified in our laboratory in 2013 as one of the components of the pore region of the MCU complex [10]. We have demonstrated that MCUb inhibits mitochondrial Ca2+ uptake by acting as a dominant-negative subunit of MCU [10]. Intriguingly, the ratio of expression between MCU and MCUb varies greatly between tissues (e.g.: 3:1 in heart or lung and 40:1 in skeletal muscle) [10], and this might contribute to the spatiotemporal regulation of mitochondrial Ca2+ uptake. Indeed, the MCU/MCUb ratio correlates with patch clump recording data of mitochondrial Ca2+ uptake of isolated mitochondria from different tissues [14]. Our Real Time-PCR experiments demonstrated that MCUb expression levels dramatically increase during skeletal muscle regeneration 3 days after cardiotoxin (CTX)-induced injury. This induction is specific, since the expression of the other components of the MCU complex is unchanged in this condition. Therefore, we hypothesized that MCUb might play a role in the progression of skeletal muscle regeneration after damage. In addition, high MCUb expression levels have been detected in anti-inflammatory macrophages (M2), one of the most important effectors of the later stages of tissue repair [15–17]. Importantly, our in vitro results demonstrated that MCUb silencing affects macrophages polarization towards an M2 pro-regenerative phenotype and that, during the progression of skeletal muscle regeneration in vivo, MCUb is induced selectively in macrophages. We therefore asked whether MCUb overexpression in M2 macrophages, occurring during skeletal muscle regeneration, could be crucial for their differentiation and thus for tissue repair. To answer to this question, we performed skeletal muscle regeneration experiments on a total MCUb knockout (KO) mouse model. We observed that MCUb ablation in vivo affects macrophage skewing from a pro-inflammatory (M1) to an M2 phenotype. It is widely accepted that M1 and M2 macrophages actively participate to skeletal muscle regeneration process by releasing cytokines and growth factors that promote skeletal muscle regeneration [15]. Specifically, M1 macrophages promote the activation and proliferation of satellite cells, while M2 macrophages are involved in the last stages of skeletal muscle repair by promoting the differentiation and fusion of myogenic precursor cells (MPCs) [15]. We hypothesized that an impairment in M1 to M2 transition might also influence skeletal muscle repair capacity by affecting the expression levels of myogenic regulatory factors. Strikingly, our results demonstrated that the lack of MCUb, by influencing macrophage plasticity and function, might negatively influence the expression of early myogenic regulatory genes Pax7 and Myod, crucial regulators of proliferation and differentiation of satellite cells [18,19], causing an impairment in skeletal muscle regeneration process. Our results strongly support the hypothesis that this altered muscular phenotype is caused by an impairment in macrophages skewing from the M1 to the M2 phenotype, since M2 macrophages are endowed with pro-regenerative capacity [15–17]. This is supported by our latest data showing that macrophages from MCUb KO animals present lower phagocytic capacity compared to wild type animals. These results are in line with published data, demonstrating that the phagocytic activity is fundamental for M2 polarization [20]. Intriguingly, we observed a significant reduction in the number of regenerating myofibers in regenerating muscles of MCUb KO mice compared with WT animals, parameter related to the efficiency of skeletal muscle regenerative capacity. Furthermore, it is well established that, during skeletal muscle regeneration, satellite cells that are not initiated to differentiation, return to quiescence to replenish the reserve population of satellite cells that will become activated during further rounds of muscle injures [21]. In order to evaluate whether MCUb KO mice show an alteration in the reconstitution of the satellite cells pool, we performed a triple skeletal muscle regeneration experiment, as already performed [22]. Intriguingly, we found a dramatic decrease in the cross-sectional area of regenerating muscle fibers in MCUb KO mice compared with WT animals. This result suggests that a possible exhaustion of the pool of satellite cells might occur in the MCUb KO animals by affecting the myogenesis process after damage. We also observed that MCUb KO mice show a decrease in collagen content suggesting that MCUb ablation, by affecting M2 polarization, and thus function, alters the reconstitution of muscle structure. These results are in line with data in literature that demonstrate the role of M2 macrophages in collagen production [16]. Finally, we analysed in detail the mechanism responsible for MCUb induction in M2 macrophages. We found that MCUb promoter, and thus its transcription in macrophages, is activated by IL-4. We thus hypothesized that the IL-4-STAT6 pathway, that is known to be involved in M2 macrophages polarization [23], might also regulate MCUb transcription. In the future, we will analyse also the 5' AMP-activated protein kinase (AMPK) phosphorylation state, the cellular metabolic sensor, that it is known to mediate macrophage skewing from an M1 to M2 phenotype [20]. We believe that MCUb, by regulating the entry of Ca2+ into mitochondria, and thus the rate of ATP production, might induce the activation of this pathway. It is known that perturbations of macrophage function and/or activation may result in impaired regeneration and fibrosis deposition, as described in several pathological disease [15], and that skeletal muscle repair system is compromised during ageing and in patients affected by muscular dystrophies [24]. Therefore, our research might be fundamental for the understanding of the molecular basis of chronic muscular diseases, such as the Duchenne Muscular Dystrophy (DMD).
Il Ca2+ riveste un ruolo fondamentale nella regolazione di un vasto spettro di processi biologici e fisiologici come la proliferazione cellulare, la trascrizione genica, la stimolazione del metabolismo aerobico, la regolazione della contrazione muscolare, l’esocitosi e la plasticità sinaptica [1,2]. I principali organelli intracellulari predisposti alla regolazione della concentrazione di Ca2+ citosolica ([Ca2+]cit) sono i mitocondri. Quest’ultimi hanno la capacità di accumulare molto velocemente elevate [Ca2+][3] che in alcuni tessuti possono raggiungere livelli superiori ai 100 µM [4]. L’alterato rilascio di Ca2+ dalle riserve intracellulari, il danno mitocondriale o il malfunzionamento dei recettori e canali presenti nella loro membrana possono portare ad un accumulo eccessivo di Ca2+ all’interno del mitocondrio, fenomeno che è stato dimostrato condurre ad una crisi energetica con conseguente induzione di morte cellulare [2]. In condizioni fisiologiche, il Ca2+ mitocondriale riveste un ruolo fondamentale nella sopravvivenza e metabolismo cellulare sostenendo la produzione dell’adenosina trifosfato (ATP), la principale molecola energetica della cellula, necessaria per lo svolgimento di diverse funzioni cellulari [5,6]. Di conseguenza, lo studio del ruolo dei mitocondri nella regolazione dell’omeostasi del Ca2+ è diventato cruciale per la comprensione di una elevata serie di funzioni cellulari. Sfortunatamente, l’analisi di questo aspetto è stata fortemente limitata dalla mancata caratterizzazione molecolare della proteina responsabile dell’accumulo di Ca2+ mitocondriale. Nel 2011, nel nostro laboratorio e in quello del Prof. Mootha è stata scoperta l’identità molecolare dell’uniporto del Ca2+ mitocondriale (MCU), proteina necessaria e sufficiente a permettere l’ingresso di Ca2+ nel mitocondrio [7,8]. Questa scoperta ha permesso l’identificazione di vari componenti dell’uniporto e dei meccanismi che regolano la sua funzione [9]. E’ ormai chiaro che MCU esiste sotto forma di un complesso, costituito da diverse subunità che compongono la regione del poro (MCU, MCUb, EMRE) e da subunità regolatorie (MICU1, MICU2) [7,10–13]. Tra le varie subunità che compongono il complesso, un ruolo rilevante è svolto da MCUb. Questa subunità è stata identificata nel 2013 nel nostro laboratorio come parte integrante della regione formante il poro del canale [10]. Abbiamo dimostrato che MCUb agisce come dominante negativo di MCU in quanto inibisce l’ingresso di Ca2+ nel mitocondrio [10]. Nonostante l’elevata similarità in sequenza e struttura, MCU e MCUb mostrano un diverso grado di espressione nei diversi tessuti (es: rapporto di 3:1 nel cuore e polmone e di 40:1 nel muscolo scheletrico). E’ stato ipotizzato che queste differenze possano contribuire alla regolazione spazio-temporale dell’ingresso di Ca2+ mitocondriale nei diversi tessuti [10]. Coerentemente, il diverso grado di espressione di MCU e MCUb nei vari tessuti correla con dati ottenuti da esperimenti di patch clamp in cui è stato misurato l’accumulo di Ca2+ in mitocondri isolati da diversi tessuti [14]. Esperimenti di Real Time-PCR da noi condotti hanno dimostrato una elevata induzione dell’espressione di MCUb durante il corso della rigenerazione del muscolo scheletrico 3 giorni dopo il danno indotto da cardiotossina (CTX). Questa induzione è specifica in quanto l’espressione delle altre componenti del canale è inalterata in questa condizione. Dal momento che, in condizioni basali, MCUb è poco espresso nel muscolo scheletrico e che la sua espressione aumenta significativamente e specificamente durante il corso della rigenerazione muscolare, abbiamo ipotizzato che MCUb potesse avere un ruolo fondamentale in questo processo. In aggiunta, MCUb è specificatamente espresso in macrofagi con fenotipo anti-infiammatorio (M2), facendo ipotizzare che l’aumento significativo di MCUb, osservato durante il corso della rigenerazione, potesse essere attribuito alla componente macrofagica. Infatti, i macrofagi di tipo M2, che infiltrano massivamente un muscolo rigenerante, partecipano attivamente durante le ultime fasi che caratterizzano questo processo [15–17]. Risultati ottenuti da esperimenti effettuati in vitro hanno dimostrato che il silenziamento di MCUb nei macrofagi impedisce a quest’ultimi di acquisire un fenotipo di tipo M2. Questo dato fa emergere l’ipotesi che MCUb, in vivo, possa rivestire un ruolo fondamentale nella polarizzazione dei macrofagi dal tipo M1 al tipo M2 e in questo modo influire sulla rigenerazione del muscolo scheletrico in seguito a danno. Per confermare le nostre ipotesi, sono stati condotti degli esperimenti di rigenerazione del muscolo scheletrico utilizzando un modello murino in cui MCUb è stato deleto in tutti i tessuti (MCUb knockout (KO)). I nostri risultati dimostrano che la mancanza di MCUb, durante il corso della rigenerazione muscolare, impedisce la polarizzazione dei macrofagi da un fenotipo pro-infiammatorio (M1) a quello anti infiammatorio (M2). E’ ormai ampiamente accettato il concetto che i macrofagi di tipo M1 e M2 influenzino il corso della rigenerazione muscolare attraverso il rilascio di citochine e fattori di crescita che facilitano la risoluzione del danno [15]. In particolare, i macrofagi di tipo M1 promuovono l’attivazione e proliferazione delle cellule satellite mentre gli M2 sono coinvolti negli ultimi stadi della rigenerazione promuovendo il differenziamento e la fusione dei precursori delle cellule muscolari (MPCs ) [15]. Abbiamo quindi ipotizzato che la mancata transizione dei macrofagi dal profilo pro-infiammatorio (M1) a quello anti-infiammatorio (M2) potesse influenzare negativamente l’espressione di geni codificanti fattori di trascrizione coinvolti nelle varie fasi di riparazione del muscolo scheletrico in seguito a trauma. Questa ipotesi sembra confermata. Infatti, l’espressione di Pax7 e di Myod, regolatori cruciali del processo di proliferazione e differenziamento delle cellule satelliti [18,19], appare essere drasticamente ridotta nei topi MCUb KO rispetto ai topi di controllo durante il corso della rigenerazione muscolare, suggerendo un’alterazione del processo di rigenerazione muscolare in mancanza di MCUb. Questi risultati supportano l’ipotesi che l’alterato fenotipo muscolare osservato nei topi KO possa essere ricondotto alla mancata transizione dei macrofagi dal fenotipo di tipo M1 a quello di tipo M2, considerato che quest’ultimi sono dotati di capacità pro-regenerative [15–17]. Ulteriore conferma del fatto che l’assenza di MCUb possa influenzare negativamente la transizione fenotipica dei macrofagi da M1 a M2, deriva dalla dimostrazione che i macrofagi derivanti dal topo MCUb KO, mostrano una riduzione dell’attività fagocitica, funzione essenziale per la polarizzazione dei macrofagi verso un fenotipo di tipo M2 [20]. Un altro parametro attraverso il quale è stato possibile valutare l’efficienza della rigenerazione del muscolo scheletrico, si basa sull’osservazione che il numero di fibre rigeneranti nei topi KO risulta significativamente ridotto rispetto alle fibre dei topi di controllo 14 giorni dopo l’induzione del danno. E’ noto che, durante il corso della rigenerazione muscolare, le cellule satelliti che non vanno incontro al processo di differenziamento, ritornino ad uno stato di quiescenza andando a ricostituire la nicchia di cellule satelliti originarie che verranno attivate in seguito ad ulteriore trauma [21]. Con lo scopo di valutare se i topi MCUb KO mostrino un’alterazione nella ricostituzione della riserva di cellule satelliti in seguito a danno muscolare, abbiamo condotto degli esperimenti di tripla rigenerazione, come precedentemente effettuato [22], e abbiamo osservato una sostanziale riduzione dell’area delle fibre rigeneranti dei topi KO rispetto a quelle dei topi controllo. Da questi risultati è emersa l’ipotesi che l’assenza di MCUb possa impedire la ricostituzione del pool di cellule satellite, alterando, in questo modo, il normale decorso della rigenerazione. In linea con questi dati abbiamo osservato come l’ablazione di MCUb, impedendo la polarizzazione dei macrofagi di tipo M2, influenzi negativamente la quantità di collagene, componente essenziale della matrice extracellulare [16], depositata durante la fase di riparazione muscolare. Questi risultati sono in linea con dati presenti in letteratura che dimostrano il ruolo dei macrofagi di tipo M2 nella produzione di collagene [16]. La dimostrazione che l’espressione di MCUb sia indotta nei macrofagi di tipo M2 ci ha spinti allo studio dei meccanismi che regolano la trascrizione di MCUb in questa sottopopolazione di cellule. In particolare, abbiamo dimostrato come l’IL-4, citochina di tipo anti-infiammatorio, attivi la trascrizione di MCUb. Dal momento che l’ablazione di MCUb nei macrofagi impedisce la loro polarizzazione verso un fenotipo di tipo M2, abbiamo ipotizzato che la via di segnale mediata dall’asse IL-4-STAT6, nota per essere coinvolta nella polarizzazione dei macrofagi M2 [23], possa essere coinvolta nell’attivazione della trascrizione di MCUb. In futuro, analizzeremo lo stato di fosforilazione di AMPK, uno dei principali sensori metabolici cellulari, che è stato dimostrato essere un giocatore essenziale nella transizione dei macrofagi dal fenotipo M1 a quello M2 [20]. La nostra ipotesi è che, MCUb, regolando negativamente l’ingresso di Ca2+ nel mitocondrio e quindi la produzione di ATP, possa indurre l’attivazione di vie di segnale mediate da AMPK influenzando, in questo modo, la polarizzazione dei macrofagi. E’ ormai noto che perturbazioni nella funzionalità e attivazione dei macrofagi possano portare ad alterazioni nel processo rigenerativo e alla deposizione di tessuto fibrotico, come descritto in diverse condizioni patologiche [15]. Questa condizione è una caratteristica comune a diverse patologie come le distrofie muscolari e durante l’invecchiamento [25]. Per questo motivo, la nostra ricerca potrebbe rivelarsi fondamentale per la comprensione delle basi molecolari di malattie croniche del muscolo scheletrico, come la distrofia di Duchenne (DMD) e potrebbe portare all’identificazione di nuovi bersagli ter

The concept of skeletal muscle homeostasis - often viewed as the net balance between two separate processes, namely protein degradation and protein synthesis - are not occurring independently of each other, but are finely co-ordinated by a web of intricate signalling networks (Nader, 2005). Such signalling networks are in charge of executing environmental and cellular cues that ultimately determine whether muscle proteins are synthesised or degraded. Prolonged elevations of proinflammatory cytokines are closely associated with muscle wasting that occurs during the sarcopenia of ageing and in cachectic AIDS and cancer patients (Strle et a/. 2007). These clinical disorders occur along with a decline in IGF-I anabolic activity, which is consistent with in vitro findings in muscle progenitor cells (Strle et a/. 2007). Very low concentrations ofTNF-a (0.01-1 ng.ml") inhibit IGF-I-induced protein synthesis (Broussard et a/. 2003; Strle et al. 2004) and expression of the critical muscle differentiation factors, MyoD (Strle et a/., 2004) and myogenin (Broussard et al. 2003; Strle et a/. 2004). Potential treatments that might overcome TNF-a-induced hormone resistance in myoblasts are unknown. Increased activation of the IGF/insulin pathway is an attractive target for combating many of the cachectic conditions associated with muscle wasting. Using rodent skeletal muscle cell lines we have investigated TNF-a/IGF-I interactions, in an attempt to mimic and understand mechanisms underlying the wasting process. We hypothesised that treatment of mouse myoblasts with TNF-a at specific doses ranging from high (20 ng.ml') to low (1.25 ng.ml") would result in dose-dependent block of differentiation and induction of apoptosis and that subsequent IGF-I co-incubations would stimulate myoblast survival and myotube formation. Objectives were to ascertain signalling pathways underpinning these outcomes. In contrast to our hypothesis, a novel role of IGF-I has been identified whereby eo-incubation of skeletal muscle C2 cells with IGF-I (1.5 ng.ml') and a non- apoptotic dose of TNF-a (1.25 ng.ml"; sufficient to block differentiation) unexpectedly were shown to facilitate a significant four-fold increase in myoblast death (P < 0.05). Specificity of the apoptotic potential of this growth factor was confirmed when neither bFGF-2 nor PDGF-BB (10 or 30 ng.ml' and 1.25 or 5 ng.rnl", respectively) were able to reveal the apoptotic potential of low dose TNF-a. By contrast, but in line with our II hypothesis, dosing with 10 ng.ml" TNF-a resulted in a block of differentiation and initiation of apoptosis, which was rescued by IGF-1. Preliminary signalling studies suggest that MAPK activation rather than the caspases are involved in the induction of death associated with low dose TNF-a (1.25 ng.mrl)/IGF-I incubation and therefore blocking the caspases would be without effect in this circumstance. The PI(3)K pathway is involved in the survival effects of high TNF-a (10 ng.mrl)/IGF co-incubations. Importantly, the rescue of death (regardless of the means required) did not facilitate differentiation and did not rescue the block of expression of IGF-ll or IGFBP-5 (produced by skeletal myoblasts as early events in their terminal differentiation and associated with preventing cell death) in our models. Using array technology we further established potential insulin survival and apoptotic genes that were upregulated in the above conditions and confirmed their expression with qRT-PCR. Of these genes three were selected to conduct gene silencing experiments. The gene silencing studies were effective in reducing expression of Adrald, Birc2 and Sirtl. Our findings suggest that inhibition of Adrald leads to an increase in myoblast death in conditions that are associated with myoblast survival and include basal conditions. This novel finding indicates Adrald expression to be essential for the general maintenance of myoblasts. This may be due to the multiple signalling pathways which the al-ARs regulate which include the PI(3)K-Akt pathway that is associated with growth and anti-apoptosis. Birc2 expression, which is upregulated in our cell model under conditions of myotoxic stress showed no significant effect on myoblast survival when suppressed. Associated with inhibition of apoptosis, it was hypothesised that inhibition of Birc2 would result in an increase in myoblast death however levels of damage were comparable to control myoblasts. Recent articles have stated that Birc, only when overexpressed above physiological levels, is associated with anti-apoptosis and consequently have proposed an alternative nomenclature that names the family after its distinctive structural feature, the BIR, rather than by inhibitor of apoptosis proteins lAPs (Silke & Vaux, 200 l; for review Srinivasula & Ashwell, 2008). Finally Sirtl, similar to Birc2 was highly expressed in conditions that induced the greatest incidence of myoblast death. Subsequent inhibition resulted in further increase in death which was not observed under basal conditions where myoblasts received DM alone. Unlike Adrald, this implicates Sirtl expression as a III survival mechanism which is specific for conditions associated with myotoxic stress. The mammalian Sirtl deacetylase was originally shown to modulate life-span in various species. However, the molecular mechanisms by which Sirtl increases longevity and with regard to the present study, survival, are largely unknown. In mammalian cells, Sirtl appears to control the cellular response to stress by regulating the FOXO family of Forkhead transcription factors. The FOXO family members are negatively regulated by the PI(3)K-Akt signalling pathway. Mammalian FOXOs control various biological functions, including cell cycle arrest, differentiation, repair of damaged DNA and apoptosis. Because the ability to regulate apoptosis and repair damage is correlated with increased organismal longevity and survival in many species these particular functions of FOXO transcription factors may be relevant to Sirtl ability to control longevity These experiments in myoblasts show that IGF-I (Lcng.ml') can facilitate apoptosis in the presence of non-a pop to tic doses ofTNF-a (1.25ng.mr\ which appears to depend not only on the upregulation of specific apoptosis genes (potentially downstream of MAPK) but also on the suppression of survival factors IGF-ll and IGFBP-5 which may also lie downstream of MAPK. These studies highlight the complex regulation of cell survival and cell death at the signalling level, as a consequence of interactions of one cytokine, TNF-a, and one growth factor, IGF-I. More information regarding the pathways involved in regulating their expression and activity will be necessary to fully understand the action of these molecules.

The thesis is divided in two parts. Part I describes effects of unacyated ghrelin, a small peptide mainly produced by stomach with multiple biological effects, on muscle regeneration in dystrophic condition using the murine mdx model. This study shows that unacylated ghrelin stimulates the polarity complex-mediated assymetric division of satellite cells, stem cells of muscle, leading to increase regenerative potential of satellite cells that in turn leads to the reduction of muscle degeneration and overall improvement of muscle function. These data were published in "Unacylated ghrelin enhances satellite cell function and relieves the dystrophic phenotype in Duchenne muscular dystrophy mdx model" (Reano et al., 2017, Stem cells) and a detailed protocol describing satellite cell handling in "Mouse satellite cell isolation and transplantaion" (Angelino et al., 2018, Bio-protocol). Moreover, Part I contains a study of the effect of unacylated ghrelin on cardiac muscle in mdx mice willing to establish if unacylated ghrelin treatment improved cardiac function in dystrophic mice. To complete information about the endogenous role of ghrelin peptides in muscle regeneration, Ghrl-/- mice, which lack all ghrelin gene products, have been investigated resulting in "Ghrelin knockout mice display defective skeletal muscle regeneration and impaired satellite cell self-renewal" (Angelino et al., Endocrine, under revision). Part II describes the in vitro study of the effect of vitamin D metabolites 25(OH)D and 1,25(OH)2D on skeletal muscle, both in normal condition and upon cytokine-induced cachexia, showing that these two metabolites have opposite effects: in basal condition, 25(OH)D induces hypertrophy while 1,25(OH)2D causes atrophy, and upon cytokine-induced atrophy, only 25(OH)D has a protective effect.

Occurrence of obesity has steadily increased in the human population and, along with it, associated health complications such as systemic insulin resistance, which can lead to the development of type 2 diabetes mellitus. Obesity is a complex metabolic disorder that often leads to chronic inflammation and an overall decline in human and animal health. In mouse skeletal muscle, obesity has been shown to impair muscle regeneration after injury, however, the mechanism underlying these changes in satellite cell (SC) biology have yet to be explored. To test the negative impacts of obesity on SC behaviors, we fed C57BL/6 mice normal chow (NC, control) or high-fat diet (HFD) for 10 wks and performed SC proliferation and differentiation assays in vitro. SCs from HFD mice formed colonies with smaller numbers (P < 0.001) compared to those isolated from NC mice, and this observation was confirmed (P < 0.05) by BrdU incorporation. Moreover, in vitro differentiation assays consisting of equally seeded SCs derived from NC and HFD muscles showed that HFD SCs exhibited compromised (P < 0.001) differentiation capacity compared to NC SCs. Immunocytochemical staining of cultured SCs demonstrated that the percentage of Pax7+/MyoD- (self-renewed) SC subpopulation decreased (P < 0.001) with HFD treatment group compared to the control. In single fiber explants, a higher ratio of SCs experienced apoptotic events as revealed by the expression of cleaved caspase 3 (P < 0.001). To investigate further the impact of obesity on SC quiescence and cycling properties in vivo, we used an inducible H2B-GFP mouse model to trace the turnover rate of GFP and thus cell division under normal and obese conditions. Flow cytometric analysis revealed that SCs from HFD treatment cycled faster (P < 0.001) than their NC counterparts, as reflected by the quicker loss of the GFP intensity. To test for SC muscle regenerative capacity in vivo, we used cardiotoxin (CTX) to induce wide-spread muscle damage in the tibialis anterior muscle. After analysis we found that HFD leads to a compromised, though mild, impairment in muscle regeneration. Taken together, these findings suggest that obesity negatively affects SC quiescence, proliferation, differentiation, and self-renewal in vitro, ex vivo and in vivo.
MS

Muscle regeneration is a complex process that involves many different types of cells, both myogenic and non-myogenic. In particular, macrophages play a fundamental role, since they exert many different functions: they phagocyte fiber debris, release cytokines to regulate the inflammatory response and stimulate satellite cells through the secretion of myogenic factors. Importantly, inflammatory processes mediated by macrophages are known to play a major role in the pathophysiology of many inherited muscular dystrophies, such as Duchenne Muscular Dystrophy (DMD). There are two main classes of activated macrophages: M1 macrophages, which have a pro-inflammatory action, and M2 macrophages that are anti-inflammatory. The exact effect of these two types of macrophages on myogenic cells is still debated, although all Authors state that if given type has a pro-proliferation effect it does not exert a pro-differentiation effect, and vice versa. In order to study the effects of macrophage-released factors onto myogenic cells we use the murine macrophage cell line J774 to obtain a serum-free, macrophage- conditioned medium (mMCM). We previously found that mMCM could enhance the proliferation rate and prevent the trans-differentiation of rat satellite cells, as well as human myoblasts from both normal and dystrophic muscles. Besides, it can greatly enhance the repair processes in muscles that underwent large surgical ablations. As these features could all be very useful in clinical terms to treat inherited and traumatic muscle diseases, we are now trying to clarify the mechanism(s) of action of mMCM. To do so, we have moved to murine in vitro and in vivo models, both wild type (wt) and dystrophic (mdx). In this work, we confirmed the pro-proliferative effect of mMCM on murine satellite cells, both wt and mdx: mMCM significantly shortened their average duplication time from 90 to 48 hours and from 100 to 20 hours, respectively. The results were also confirmed with FACS-sorted wt satellite cells (duplication time reduced from 45 to 31 hours), suggesting that the effects were not limited to a specific subpopulation of satellite cells. At the same time, though, not only mMCM did not inhibit the differentiation process, but in some instances it seemed to enhance them. We also tested the effect of mMCM on the proliferation of primary wt and mdx fibroblasts, finding that mMCM consistently had a clear anti-proliferative effect on mdx fibroblast, while it did not affect wt fibroblasts. Experiments on the effect of mMCM pro-proliferative action on satellite cell transplantation in dystrophic muscle were carried out using GFP+ satellite cells, showing that when recipient muscles were treated with mMCM the number of GFP-positive fibers was higher that in controls. In vivo experiments on wt mice muscles showed that in intact tissue delivery of mMCM does not elicit an inflammatory response; on the other hand, in chemically pre-injured muscles mMCM injections lowered the expression levels of various macrophagic markers, both for M1 and M2, thus indicating that mMCM can interfere with the inflammatory process, apparently reducing the total number of macrophages. In order to further elucidate the possible interactions between mMCM and activated macrophages we also investigated the effects of mMCM on macrophages polarization. To this aim, we used a model in which human monocytes obtained from blood that were differentiated into macrophages and then stimulated with cytokines to acquire either M1 or M2 phenotype. We then tested the effects on mMCM on non-stimulated, M1 and M2 macrophages. These analyses showed that mMCM alone cannot influence macrophages polarization but, when combined with polarization stimuli, it seems to enhance the M1 phenotype. Finally, we began an analysis of mMCM composition, finding that it contains various cytokines, some pro- and other anti-inflammatory. We are also starting the mass spectrometry analysis, and the preliminary results identified some interesting candidates. mMCM also contain exosomes, whose content is however still under investigation. Our data confirm the potential of mMCM as a therapeutic tool for muscular pathologies, but also underline the need of improving our understanding of the complex interplay between macrophages and muscle environment
La rigenerazione muscolare è un processo complesso che coinvolge diversi tipi cellulari, sia miogenici sia non miogenici. In particolare, i macrofagi ricoprono un ruolo fondamentale, svolgendo diverse funzioni: fagocitano i detriti cellulari, rilasciano citochine che regolano la risposta infiammatoria e stimolano le cellule satelliti attraverso la secrezione di fattori miogenici. I processi infiammatori mediati dai macrofagi hanno un ruolo fondamentale nella patofisiologia di molte distrofie muscolari ereditarie, come la Distrofia Muscolare di Duchenne (DMD). Esistono due classi di macrofagi attivati: i macrofagi M1, che hanno un effetto pro-infiammatorio, e i macrofagi M2, che hanno un’azione anti-infiammatoria. L’esatto effetto di questi due tipi di macrofagi sulle cellule miogeniche è ancora oggetto di dibattito, sebbene tutti gli Autori concordino nell’affermare che se un certo tipo ha un’azione pro-proliferativa sulle cellule, non avrà anche un effetto pro-differenziativo sulle stesse e viceversa. Per studiare l’effetto dei fattori rilasciati dai macrofagi sulle cellule miogeniche, abbiamo utilizzato la linea cellulare di macrofagi murini J774, per ottenere un medium condizionato da macrofagi privo di siero, chiamato murine Macrophage Conditioned Medium (mMCM). In lavori precedenti abbiamo dimostrato che mMCM può aumentare la proliferazione e impedire il trans-differenziamento di cellule satelliti di ratto, nonché di mioblasti umani, sia di individui sani che di pazienti distrofici. Può inoltre migliorare grandemente la rigenerazione in muscoli che hanno subito grosse rimozioni chirurgiche di massa. Tutte queste caratteristiche potrebbero essere molto utili nel trattamento clinico di patologie muscolari sia traumatiche sia ereditarie e stiamo quindi cercando di chiarire quale sia il meccanismo (o i meccanismi) di azione del mMCM. Per far ciò, abbiamo deciso di utilizzare il modello murino in vitro e in vivo, sia selvatico (wild type, wt), sia distrofico (mdx). In questo lavoro, abbiamo confermato l’effetto pro-proliferativo del mMCM sulle cellule satelliti di topo, sia wt che mdx: mMCM ha ridotto significativamente il loro tempo di duplicazione medio rispettivamente da 90 a 48 ore e da 100 a 20 ore. Tali risultati sono stati anche confermati con cellule satelliti wt isolate tramite FACS (tempo di duplicazione ridotto da 45 a 31 ore), indicando che gli effetti visti sulle cellule non sono limitati ad una specifica subpopolazione di cellule satelliti. Allo stesso tempo, mMCM non solo non ha inibito il processo di differenziamento delle cellule, ma in alcuni casi è anche sembrato che lo migliorasse. Abbiamo anche studiato l’effetto del mMCM sulla proliferazione di fibroblasti primari, sia wt che mdx, trovando che mMCM ha un significativo effetto anti- proliferativo sui fibroblasti distrofici, mentre non ha effetto sui wt. Abbiamo poi condotto alcuni esperimenti in vivo per capire l’effetto del mMCM sul trapianto di cellule satelliti in muscolo distrofico, usando satelliti GFP+, e abbiamo dimostrato che se il muscolo ricevente viene trattato con mMCM, il numero di fibre GFP-positive è più alto dei controlli. Gli esperimenti condotti in vivo su muscoli wt intatti hanno mostrato che mMCM da solo non stimola una risposta infiammatoria, mentre la somministrazione di mMCM in muscoli danneggiati chimicamente ha diminuito l’espressione di vari marcatori macrofagici, sia M1 che M2, indicando che mMCM può interferire con il processo infiammatorio, apparentemente riducendo il numero totale di macrofagi. Per indagare più approfonditamente sulle possibili interazioni fra mMCM e i macrofagi attivati, abbiamo poi investigato gli effetti del mMCM sulla polarizzazione di macrofagi. Abbiamo dunque usato un modello in cui monociti umani ottenuti dal sangue sono differenziati in macrofagi e poi stimolati con citochime per acquisire un fenotipo M1 o M2. Abbiamo poi studiato gli effetti del mMCM sui macrofagi già polarizzati o ancora vergini. Queste analisi hanno mostrato che mMCM da solo non può influenzare la polarizzazione dei macrofagi, ma che quando è in combinazione con stimoli polarizzanti, sembra spingere verso il fenotipo M1. Infine, abbiamo iniziato l’analisi della composizione del mMCM. mMCM contiene diverse citochine, alcune pro- e altre anti-infiammatorie. Stiamo inoltre iniziando l’analisi con la spettrometria di massa sul mMCM e i risultati preliminari hanno identificato alcune proteine interessanti. mMCM contiene inoltre anche exosomi, il cui contenuto è ancora oggetto di analisi. I nostri dati confermano il potenziale del mMCM come strumento terapeutico per le patologie muscolari, ma evidenziano anche il bisogno di migliorare le nostre conoscenze sulle complesse interazioni fra macrofagi e ambiente muscolare

"Engineered muscle tissue offers a promising solution for the treatment of large muscle defects. Three-dimensional tissue engineered matrices, such as microthreads, can be used to grow new myofibers that will reduce scar formation and integrate easily into native myofibers. We hypothesize that adsorbing growth factors to the surface of braided collagen scaffolds using crosslinking strategies will promote muscle derived fibroblastic cell (MDFC) attachment and growth, which will serve as a platform for delivering cells to large muscle defects for muscle regeneration. To test this hypothesis, self-assembled type I collagen threads were braided and crosslinked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) with and without heparin and 5 ng/mL, 10 ng/mL, or 50 ng/mL fibroblast growth factor (FGF-2) bound to the surface. Using immunhistochemistry, braided collagen scaffolds showed the presence of FGF-2 on the surface, and braiding the microthreads increased the mechanical properties compared to single threads. To determine the effect of FGF-2 on MDFC attachment, growth, and alignment, scaffolds were seeded with a MDFC cell suspension for 4 hours using a PDMS mold with a sealed 1 mm by 12 mm channel and cultured for 1, 5, or 7 days. After 1 day of culture, the results show a significant increase in cell attachment on braids crosslinked with EDC/NHS with heparin and no significant difference in attachment between the different concentrations of FGF-2 and EDC/NHS crosslinked scaffolds. After 7 days in culture, the MDFCs responded to FGF-2 with a positive linear correlation between growth rate and concentration of FGF-2 on the surface. Additionally, all control scaffolds showed cellular alignment after 7 days, while MDFCs on FGF-2 modified scaffolds showed limited alignment. These results show braided collagen scaffolds crosslinked with EDC/NHS with heparin delivering a controlled quantity of FGF-2 can support MDFC attachment and growth, which may serve as an exciting new approach to facilitate the growth and ultimately the delivery of cells to large defects in muscle regeneration."

La transplantation de cellules souches de muscle possède un grand potentiel pour la réparation à long terme du muscle dystrophique. Cependant, la croissance ex vivo des cellules souches musculaires réduit de manière significative l'efficacité de leur greffe puisque le potentiel myogénique est considérablement réduit lors de la mise en culture. La voie de signalisation Notch a émergé comme un régulateur majeur des cellules souches musculaires (MuSCs) et il a également été décrit que la sur-activation de Notch est crucial pour le maintien du caractère souche des MuSC. Cette découverte pourrait être traduite comme un bénéfice thérapeutique potentiel. Des MuSCs murines ont été fraîchement isolées et ensemencées sur des boîtes de culture recouverte de Dll1-Fc, le domaine extracellulaire de Delta-like-1 est fusionné au fragment Fc humain, afin d'activer la voie de signalisation Notch et avec un IgG hu-main comme contrôle. Nous avons utilisé le rAAV afin d’exprimer le Dll1 spécifique-ment dans les muscles de souris. Les souris P3 ont été traitées avec de l’AAV pendant 3 semaines et 6 semaines afin d’étudier l'effet de Dll1 au cours du développement postnatal. Afin d’étudier le processus de régénération, l'AAV a également été injecté dans les muscles de souris mdx alors que les souris de type sauvage ont été utilisées comme contrôle. Un potentiel caractère souche supérieur (marquée avec le Pax7) est observé dans les cultures des MuSCs qui sont recouverte de Dll1-Fc par rapport à leurs homologues contrôles, par contre le taux de proliférer est réduit. Au cours du développement postnatal, la sur-activation de la voie de signalisation Notch par Dll1 sur les fibres musculaires a été en mesure d'élargir le pool des cellules Pax7+, cependant elle entraîne une diminution de la masse musculaire avec réduction de la taille des fibres et ceci sans affecter l'accumulation des myonuclei. Dans les MuSCs quiescentes (de type sauvage), la sur-activation de la voie de signalisation Notch ne présente pas de réel effet. La surexpression de Dll1 dans le muscle mdx a diminué la masse musculaire et agrandit le pool de cellules souches musculaires, ce-pendant le taux de régénération n'a pas été affecté. L’augmentation des MuSCs est attribuée à une différenciation entravée des cellules souches musculaires. En étudiant la stimulation de la voie de signalisation Notch dans les MuSCs à la fois in vitro et in vivo, nous démontrons que sur-activation de Notch préserve le caractère souche des cellules via l’inhibition de la prolifération et de la différenciation myogénique des MuSCs
Muscle stem cell transplantation possesses great potential for long-term repair of dys-trophic muscle. However expansion of muscle stem cells ex vivo significantly reduces their engraftment efficiency since the myogenic potential is dramatically lost in culture. The Notch signaling pathway has emerged as a major regulator of muscle stem cells (MuSCs) and it has recently been discovered that high Notch activity is crucial for maintaining stemness in MuSCs. This feature might be exploited and developed into a novel therapeutic approach.Murine MuSCs were freshly isolated and seeded on culture vessels coated with Dll1-Fc, which fused Delta-like-1 extracellular domain with human Fc, to activate Notch sig-naling and with human IgG as a control. The rAAV gene delivery system was em-ployed to express Dll1 in murine muscles. P3 mice were treated with AAV for 3 weeks and 6 weeks to investigate the effect of Dll1 during postnatal development. To investi-gate the regeneration process, AAV were injected into mdx muscles whereas wild-type mice were used as control.Higher potential stemness (marked by Pax7 positivity) was observed in MuSCs grow-ing on a Dll1-Fc surface as compared to their counterparts on the control surface, while their proliferation rate was reduced. During postnatal development, overstimulation of Notch signaling by Dll1 on the mus-cle fibers was able to enlarge the Pax7+ cell pool, while also resulting in decreased muscle mass and smaller muscle fibers without affecting the accretion of myonuclei into the fiber. In quiescent (wild-type) MuSCs, overstimulation of Notch signaling did not have any discernible effect. Overexpression of Dll1 in mdx muscle decreased the muscle mass and enlarged the muscle stem cell pool, while muscle regeneration re-mained unaffected. By investigating Notch stimulation in MuSCs both in vitro and in vivo, we demonstrate that high Notch activity preserves stemness via inhibition of MuSCs proliferation and myogenic differentiation. Our findings point out that the Dll1 molecule, as a canonical Notch ligand, might have a therapeutic potential in cell-based therapies against muscu-lar dystrophies

La transplantation de cellules souches de muscle possède un grand potentiel pour la réparation à long terme du muscle dystrophique. Cependant, la croissance ex vivo des cellules souches musculaires réduit de manière significative l'efficacité de leur greffe puisque le potentiel myogénique est considérablement réduit lors de la mise en culture. La voie de signalisation Notch a émergé comme un régulateur majeur des cellules souches musculaires (MuSCs) et il a également été décrit que la sur-activation de Notch est crucial pour le maintien du caractère souche des MuSC. Cette découverte pourrait être traduite comme un bénéfice thérapeutique potentiel. Des MuSCs murines ont été fraîchement isolées et ensemencées sur des boîtes de culture recouverte de Dll1-Fc, le domaine extracellulaire de Delta-like-1 est fusionné au fragment Fc humain, afin d'activer la voie de signalisation Notch et avec un IgG hu-main comme contrôle. Nous avons utilisé le rAAV afin d’exprimer le Dll1 spécifique-ment dans les muscles de souris. Les souris P3 ont été traitées avec de l’AAV pendant 3 semaines et 6 semaines afin d’étudier l'effet de Dll1 au cours du développement postnatal. Afin d’étudier le processus de régénération, l'AAV a également été injecté dans les muscles de souris mdx alors que les souris de type sauvage ont été utilisées comme contrôle. Un potentiel caractère souche supérieur (marquée avec le Pax7) est observé dans les cultures des MuSCs qui sont recouverte de Dll1-Fc par rapport à leurs homologues contrôles, par contre le taux de proliférer est réduit. Au cours du développement postnatal, la sur-activation de la voie de signalisation Notch par Dll1 sur les fibres musculaires a été en mesure d'élargir le pool des cellules Pax7+, cependant elle entraîne une diminution de la masse musculaire avec réduction de la taille des fibres et ceci sans affecter l'accumulation des myonuclei. Dans les MuSCs quiescentes (de type sauvage), la sur-activation de la voie de signalisation Notch ne présente pas de réel effet. La surexpression de Dll1 dans le muscle mdx a diminué la masse musculaire et agrandit le pool de cellules souches musculaires, ce-pendant le taux de régénération n'a pas été affecté. L’augmentation des MuSCs est attribuée à une différenciation entravée des cellules souches musculaires. En étudiant la stimulation de la voie de signalisation Notch dans les MuSCs à la fois in vitro et in vivo, nous démontrons que sur-activation de Notch préserve le caractère souche des cellules via l’inhibition de la prolifération et de la différenciation myogénique des MuSCs
Muscle stem cell transplantation possesses great potential for long-term repair of dys-trophic muscle. However expansion of muscle stem cells ex vivo significantly reduces their engraftment efficiency since the myogenic potential is dramatically lost in culture. The Notch signaling pathway has emerged as a major regulator of muscle stem cells (MuSCs) and it has recently been discovered that high Notch activity is crucial for maintaining stemness in MuSCs. This feature might be exploited and developed into a novel therapeutic approach.Murine MuSCs were freshly isolated and seeded on culture vessels coated with Dll1-Fc, which fused Delta-like-1 extracellular domain with human Fc, to activate Notch sig-naling and with human IgG as a control. The rAAV gene delivery system was em-ployed to express Dll1 in murine muscles. P3 mice were treated with AAV for 3 weeks and 6 weeks to investigate the effect of Dll1 during postnatal development. To investi-gate the regeneration process, AAV were injected into mdx muscles whereas wild-type mice were used as control.Higher potential stemness (marked by Pax7 positivity) was observed in MuSCs grow-ing on a Dll1-Fc surface as compared to their counterparts on the control surface, while their proliferation rate was reduced. During postnatal development, overstimulation of Notch signaling by Dll1 on the mus-cle fibers was able to enlarge the Pax7+ cell pool, while also resulting in decreased muscle mass and smaller muscle fibers without affecting the accretion of myonuclei into the fiber. In quiescent (wild-type) MuSCs, overstimulation of Notch signaling did not have any discernible effect. Overexpression of Dll1 in mdx muscle decreased the muscle mass and enlarged the muscle stem cell pool, while muscle regeneration re-mained unaffected. By investigating Notch stimulation in MuSCs both in vitro and in vivo, we demonstrate that high Notch activity preserves stemness via inhibition of MuSCs proliferation and myogenic differentiation. Our findings point out that the Dll1 molecule, as a canonical Notch ligand, might have a therapeutic potential in cell-based therapies against muscu-lar dystrophies

Expression of the cell surface sialomucin CD34 is common to many adult stem cell types, including muscle satellite cells. However, no clear stem cell or regeneration-related phenotype has ever been reported in mice lacking CD34, and its function on these cells remains poorly understood. Here, we assess the functional role of CD34 on satellite cell-mediated muscle regeneration. Using an optimized flow cytometry-based method to analyze myogenic progenitors, we show that CD34’s expression is tightly regulated early during the muscle regeneration process. Following this, we show that Cd34⁻/⁻ mice, which have no obvious developmental phenotype, display a defect in muscle regeneration when challenged with either acute or chronic muscle injury, resulting in impaired myofibre hypertrophy. In vivo engraftment efficiency and BrdU proliferation assays comparing WT and Cd34⁻/⁻ myogenic progenitors attribute this defect to impaired myogenic progenitor cell function in Cd34⁻/⁻ animals. Lastly, the culture of isolated single myofibres demonstrate that this overall muscle regenerative defect is caused by a delay in the activation of satellite cells lacking CD34 as well as impaired proliferation following activation. Consistent with the reported anti-adhesive function of CD34, Cd34⁻/⁻ satellite cells also show decreased motility along their host myofibre. Altogether, our results identify a role for CD34 in the poorly understood early steps of satellite cell activation, and provide the first evidence that beyond being a stem cell marker, CD34 may play an important function in modulating satellite cell activity.

Galectin-1 is a p-galactoside binding lectin that has been implicated in a variety of cellular processes in different tissues, including skeletal muscle. Initial studies investigating the phenotype of a null mouse model for galectin-1 suggested that there were no obvious differences between the musculature of the null mouse and that of the wild-type mouse. However, in vitro studies suggested that the addition of galectin-1 Improved the fusion of myoblasts to myotubes and caused dermal fibroblasts to convert to the myogenic lineage.

The aim of this thesis was to develop a novel cross-linking strategy to prepare defined collagen-based hydrogel networks to investigate stiffness-induced cell differentiation for skeletal muscle tissue engineering. Volumetric muscle loss (VML) occurs with traumatic injury or aggressive tumour ablation and results in a diminished natural capacity for repair. Whilst autologous muscle transfer offers the gold standard treatment option, the level of success is limited by surgical expertise, availability of healthy tissue, donor site morbidity and a loss of muscle strength and function due to scar tissue formation. As a result, a clear need exists for therapeutic strategies that can enhance the innate ability of skeletal muscle to regenerate following VML. Thiol-ene photo-click collagen hybrid hydrogels were systematically developed and prepared via step-growth reaction using thiol-functionalised type-I collagen and 8-arm poly(ethylene glycol) norbornene terminated (PEG8NB). Collagen was thiol-functionalised by a ring opening reaction with 2-iminothiolane (2IT), whereby up to 80% functionalisation and 90% triple helical preservation were recorded in addition to improved solubility of the material. Type, i.e. Irgacure 2959 (I2959) or lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and concentration of photoinitiator were varied to ensure minimal photoinitiator- induced cytotoxicity in line with the in vitro study. Gelation kinetics proved to be largely affected by the specific photoinitiator and the concentration, with LAP- containing thiol-ene mixtures leading to 8 times faster gelation times compared to I2959-containing mixtures. Photo-click hydrogels with tunable storage moduli (G’: 0.54- 6.4 kPa), elastic modulus (Ec: 1.2- 12.5 kPa), gelation time (ԏ: 73- 331 s) and swelling ratio (SR: 1500- 3000 wt.%) were prepared. Three of these hydrogels (Ec: 7, 10, 13 kPa) were taken forward for in vitro tests with a myoblast cell line due to their similarity to the elasticity of natural muscle. These three hydrogels were shown to support cell attachment, spreading, proliferation and maturation/ differentiation of myoblasts into myotubes. A subcutaneous model was used to analyse the immune response of these new materials at 1, 4 and 7 days after implantation using Mucograft® as the control. Mucograft® was shown to present a lower immune response and reduced inflammation, whereas, the immune response from the hydrogel, promoted angiogenesis, which can be more beneficial for muscle regeneration.

Skeletal muscle is a direct target for the group of seco-steroids collectively termed Vitamin D. Skeletal muscle expresses both CYP27A1 and CYP27B1 encoding for the hydroxylases required to convert Vitamin D to 25[OH]D and subsequently the bioactive 1α-25-dihydroxyvitamin D3 (1α-25[OH]2D3) (Girgis et al., 2014b). Crucially, the Vitamin D receptor (VDR) is also present in skeletal muscle (Srikuea, Zhang, Park-Sarge, & Esser, 2012) and upon exposure, binds to its ligand 1α-25[OH]2D3 and initiates genomic and non-genomic rapid signalling responses. At present there is a global prevalence of low serum Vitamin D (25[OH]D) concentrations due to a lack of sun exposure (the primary route for Vitamin D synthesis) as a function of latitude and/or an indoor lifestyle coupled with few dietary sources of Vitamin D (Chen et al., 2007). Accumulating data are now suggestive that low 25[OH]D may be associated with perturbations in contractile activity and the regeneration of human skeletal muscle (Owens, Fraser, & Close, 2014), although a definitive causal relationship is yet to be established. Therefore, this thesis aimed to establish a more precise role for Vitamin D in human skeletal muscle function and regeneration. There were four overarching aims: 1. Explore the role of Vitamin D in human skeletal muscle contractile properties in humans in vivo. 2. Identify the role of Vitamin D in human skeletal muscle contractile properties ex vivo. 3. Investigate the role of Vitamin D in skeletal muscle regeneration following eccentric exercise induced muscle damage in vivo. 4. Elucidate cellular mechanisms of the muscle regeneration process that are responsive to Vitamin D in vitro. The main findings from this work imply that serum 25[OH]D concentrations across a broad range from 18 → 100 nmol.L-1 do not affect skeletal muscle contractile properties. Conversely elevating serum 25[OH]D from < 50 nmol.L-1 to > 75 nmol.L-1 resulted in significant improvements in the recovery of maximal voluntary contraction force following a bout of eccentric exercise. Implementing an in vitro model of muscle regeneration also identified potential cellular mechanisms for these observations: Muscle derived cells treated with a total amount of 10 nmol 1α-25[OH]2D3 following a mechanical scrape improved migration dynamics and fusion capability of skeletal muscle derived cells. Taken together, low Vitamin D concentrations are highly prevalent but can be easily corrected with supplementation of Vitamin D3. This may have the advantage of optimising the regenerative capacity of skeletal muscle amongst other health benefits previously characterised by others.

Le muscle strié squelettique possède de grandes capacités de régénération grâce à ses cellules souches, les cellules satellites. Après une lésion, le processus de régénération musculaire qui se met en place est finement régulé dans le temps et l’espace par le microenvironnement, constitué de cellules avoisinantes mais également par des éléments de la matrice extracellulaire (MEC). Cette dernière se compose de molécules structurales comme les collagènes et de composants possédant un rôle trophique comme les glycosaminoglycanes (GAGs). La MEC musculaire est peu étudiée à cause d’une organisation tridimensionnelle complexe rendant son exploration difficile. Lors d’une lésion avec perte de substance musculaire, la régénération est altérée, associée à une fibrose et une inflammation chronique. Ce type de lésion est fréquemment rencontré en traumatologie mais survient également chez le blessé de guerre. Malgré un traitement optimal, une invalidité fonctionnelle persiste chez ces patients. L’utilisation d’un biomatériau décellularisé, constitué de MEC pourrait fournir ce support physique et trophique faisant défaut dans ce type de lésion. Dans ce travail, nous avons entrepris l'établissement d'une MEC d’origine musculaire et nous avons établi un protocole de décellularisation permettant d’obtenir un biomatériau conservant l’architecture spécifique de la MEC musculaire avec une élimination de la majorité des antigènes cellulaires afin d'éviter une réponse immunitaire délétère après implantation. Néanmoins, le protocole retenu ne permet de conserver certaines molécules trophiques d’intérêt comme les GAGs. Les « ReGeneRaTing Agent®» (RGTA®) sont des mimétiques fonctionnels de ces GAGs, utilisés en clinique pour améliorer la cicatrisation cutanée et cornéenne. Ces mimétiques conservent une capacité de liaison aux facteurs de croissance avec une résistance aux dégradations enzymatiques. Nous avons évalué l’utilisation de ces molécules au cours de la réparation musculaire, dans un modèle in vivo chez le rongeur. Nous avons réalisé une analyse histologique précoce (8e jour de régénération) mettant en évidence une augmentation du nombre de noyaux par myofibre en faveur d’une augmentation de la fusion, validée également in vitro sur des progéniteurs musculaires. Nous avons également observé une augmentation du nombre de vaisseaux, suggérant une amélioration de l’angiogenèse. Le nombre de gouttelettes lipidiques, marqueur d’une mauvaise régénération, était en diminution. L’exploration histologique plus tardive (28e jour de régénération) n’a retrouvé que l’augmentation du nombre de vaisseaux en faveur d’un effet durable sur l’angiogenèse. Ces RGTA® peuvent être couplés aux biomatériaux et sont particulièrement résistants dans un environnement inflammatoire pouvant être rencontré dans les lésions avec perte de substance musculaire. Des chimiokines et des facteurs de croissance pourront également être ajoutés au biomatériau matriciel afin de favoriser la migration des différents progéniteurs nécessaires à une néoformation musculaire. L’efficacité thérapeutique de ces biomatériaux optimisés nécessitera d’être évaluée dans un modèle in vivo de perte de substance
Skeletal muscle exhibits high capacity for regeneration after an injury that relies on resident stem cells. Muscle regeneration is tightly regulated by both the immune response and other resident cells, as well as by cues from the local extracellular matrix (ECM), contributing to a coordinated repair process. Muscle ECM is a network of structural macromolecules with a large majority of collagens and trophic molecules such as glycosaminoglycans (GAGs). In the skeletal muscle tissue, ECM was overlooked due to its complex organization making investigations difficult. Muscle regenerative ability can be overtaken in large muscle wasting, such as in volumetric muscle loss (VML), leading to fibrosis formation and chronic inflammation. This type of injury predominantly occurs in traumatology and in war-wounded patients, with functional disability despite an optimal treatment. The use of biomaterials could provide the biochemical and physical cues that are missing in this pathologic repair. In this work we have focused on obtaining a biomaterial composed of skeletal muscle ECM. We have tested several decellularization protocols both to preserve the three-dimensional architecture of the muscle ECM and to completely remove cell components in order to avoid a deleterious immune response after implantation. However, the protocol did not allow the preservation of trophic molecules such as GAGs, in the scaffold.“ReGenerating Agents” (RGTA®) are functionally analogous of GAGs with a crucial property to resist enzymatic degradation. They function to restore a proper microenvironment for tissue healing with already a clinical application in skin and corneal repair. We have explored the effects of RGTA® in muscle regeneration using an in vivo model in mouse. At early time of regeneration (day 8), we performed histologic analysis. We showed that regenerating myofibers contained more nuclei in the treated animals, in favor of an increase of progenitor fusion, which has been validated in vitro in myogenic cultures. The number of capillaries was higher in favor of a better angiogenesis. Lipid droplets, a marker of impaired regeneration, were reduced by RGTA® administration. At later time of regeneration (day 28), capillary number was still improved in favor of a durable effect of RGTA® on angiogenesis. RGTA® could be incorporated into biomaterials and are particularly resistant in an inflammatory environment, such as that occurring after a VML injury. Chemokines and growth factors could also be added in ECM-based scaffolds to promote the migration of progenitors that are essential for myofiber neoformation. Therapeutic efficacy of these optimized biomaterials will require to be evaluated in an in vivo model of VML