Pax3 protein is initially expressed in the dermomyotome of embryonic somites, which gives rise to skeletal muscle. Following myogenesis, Pax3 expression is mostly down-regulated and becomes restricted to a few satellite cells (SCs) of select mature muscles. SCs are activated to form new myonuclei during muscle hypertrophy, regeneration and repair. Intrafusal fibers of muscle spindles are thought to persist in a comparatively immature state as, unlike extrafusal fibers, they maintain small diameters, developmental myosins, Myf5 expression and high SC concentrations. This thesis tests the hypotheses that Pax3 expression is preferentially maintained in SCs of adult skeletal muscle spindles and can be augmented under conditions of SC activation. To study Pax3 through development, immunohistochemical techniques were used to identify SCs by their Pax7 expression, and analyze the proportion of SCs and myonuclei (MN) expressing Pax3 in chicken anterior latissimus dorsi (ALD) muscle excised at 9, 30, 62, and 145 days post-hatch. To induce SC activation, tenotomy was performed on the right ALD muscle of 138-day post-hatch chicks to induce compensatory hypertrophy of the ipsilateral synergistic posterior latissimus dorsi (PLD) muscle. The PLD was analyzed seven days after ALD tenotomy using similar immunohistochemical techniques. This is the first study to show Pax3 expressing SCs within intrafusal fibers of muscle spindles. This thesis demonstrates that throughout development there is a higher percentage of Pax3 expressing SCs within intrafusal fibers of muscle spindles than the surrounding extrafusal fibers that compose the bulk of the muscle. It is also revealed that the proportion of the SC population expressing Pax3 declines with age in both intrafusal and extrafusal fibers. Compensatory hypertrophy of the PLD resulted in a greater percentage of Pax3 expressing SCs in intrafusal and extrafusal fibers than under control conditions. The percentage of SCs expressing Pax3 after PLD overload was similar to that seen in young control muscle. The percentage of Pax3 expressing MN also increased after muscle overload to levels seen in young muscle. A disproportionate decrease in the proportion of SCs expressing Pax3 during development, and a disproportionate increase in the percentage of Pax3 positive SCs as a result of experimentally induced muscle hypertrophy, suggests that Pax3 expression in maturing muscle may be more than just a developmental vestige. Pax3 may be a factor in the activation and differentiation of SCs in maturing muscle.

Šio darbo tikslas buvo nustatyti ar NAD(P)H fluorescencija, registruojama neinvaziniu fluorescenciniu metodu, gali būti naudojama kaip miogeninių ląstelių diferenciacijos žymuo, ištiriant fluorescencijos pokyčius miogeninių ląstelių diferenciacijos metu bei išaiškinant pagrindines šių pokyčių priežastis. Tyrimai parodė, kad 95 % mioblastų fluorescencijos, išmatuotos prie 460 nm emisijos, priklauso nuo NAD(P)H kiekio. Ląstelėms diferencijuojantis fluorescencija ženkliai didėja, tačiau susiejus fluorescenciją su didėjančiu baltymų kiekiu, ženklus fluorescencijos padidėjimas nustatytas tik po 10 diferenciacijos dienų. Panaudojus ląstelių metabolizmą ir NAD(P)H kiekį veikiančius veiksnius (skyriklį CCCP ir slopiklį KCN) buvo įvertinti santykiniai NADH ir NAD+ kiekiai bei NAD+/NADH santykis, kuris diferenciacijos metu mažėjo. 535 nm fluorescencijos pokyčiai diferenciacijos metu atskleidžia didėjantį FAD kiekį. Tiek NAD+/NADH santykis tiek 535 ir 460 nm fluorescencijos emisijų santykis yra patikimesni diferenciacijos rodikliai nei pati NAD(P)H fluorescencija. Šio darbo rezultatai patvirtina, kad tokie greiti, tikslūs ir neinvaziniai fluorescencijos matavimai gali būti naudingi nustatant mioblastų diferencacijos pradžią ir vyksmą bei taikytini įvairiems bandymams siekiant nustatyti vaistų ir kitų faktorių įtaką ląstelių metabolizmui. / The aim of this study is to check hypothesis that increase of fluorescence at 460 nm emission upon myoblast differentiation display alteration of NADH quantity that change NAD+/NADH ratio and could be a myoblast marker of entering into differentiation process. To evaluate NAD(P)H influence on total cell fluorescence emission intensity at 460 nm wavelenght were used metabolism effectors: carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to obtain minimal NADH fluorescence; and KCN to obtain maximal NADH fluorescence. Assuming, that CCCP caused maximal oxidation and that KCN caused maximum reduction, estimated that NAD+/NADH ratio decreased upon myoblast differentiation. CCCP effect showed that about 95 % of myoblast fluorescence at 460 nm emission is dependent on NAD(P)H. The fluorescence intensity at 460 nm was increasing with myoblast differentiation. Using fluorescence measuring estimated average NADH concentration significantly increased on 10 day of differentiation compared with control cells. Changes of fluorescence intensity at 535 nm reveal accumulation of FAD upon myoblast differentiation. Therefore, the best indicator of myoblast differentiation stage is ratio of fluorescence emission at 535 and 460 nm. Our results indicate that quick, quantitative and real-time fluorescence method may be useful for non-invasive detection and evaluation of myoblast differentiation stage.

Le muscle strié squelettique dispose de la capacité de régénérer à la suite d’un dommage, qu’il soit traumatique, chimique, pathologique ou encore associé à l’exercice. La régénération musculaire est un phénomène complexe faisant appel à de nombreux types cellulaires tels que les cellules souches musculaires, les cellules vasculaires ou encore les cellules immunitaires. Parmi ces cellules immunitaires, les macrophages jouent un rôle majeur, en sécrétant des facteurs trophiques notamment. En effet, en fonction de leur état d’activation, pro ou anti-inflammatoire, les macrophages exercent des effets distincts sur le comportement des cellules souches musculaires et la restauration du tissu musculaire. Parmi les régulateurs des macrophages et des cellules souches musculaires émergent les espèces réactives de l’oxygène.Ainsi, ce travail de doctorat pluridisciplinaire en sciences du sport a pour ambition d’identifier et de déterminer l’implication des espèces réactives de l’oxygène et des molécules sécrétées par les macrophages ainsi leurs effets fonctionnels respectifs au cours de la régénération musculaire chez la souris et l’Homme. En outre, un suivi des marqueurs biologiques associés à la balance pro/antioxydante réalisé chez des footballeurs de haut niveau durant une saison permet de renseigner l’évolution d’un facteur associé à l’étiologie des dommages induits par l’exercice. / Skeletal muscle has the remarkable ability to regenerate following injury. Skeletal muscle regeneration is a complex process that requires different cell types to restore the tissue. Among these cells are found muscle stem cells, vascular cells and immune cells. Among immune cells, macrophages are play a key role by releasing trophic factors. Depending on their activation states, pro or antiinflammatory, they exert different effects on muscle stem cells and regeneration process. Interestingly, reactive oxygen species emerge as important regulators of muscle stem cells and macrophage biology.Consequently, this pluridisciplinary PhD thesis in sport sciences aims to identify and determine the involvement of macrophage derived-reactive oxygen species and secreted molecule and their functional effects on skeletal muscle regeneration both in mice and human. Furthermore, a one-season follow-up of pro/antioxidant balance in high level soccer players contributes to knowledge regarding the evolution of a factor involved in the etiology of exercise-induced muscle damages

Adult skeletal muscles have remarkable regenerative capacity. Muscle regeneration occurs when muscle tissue experiences injury, causing a population of normally quiescent muscle-resident stem cells, called satellite cells, to become activated. The CCAAT/enhancer binding proteins known as C/EBPs are transcription factors belonging to the bZIP family. Previous work from our lab has identified C/EBPβ as an important negative regulator of myogenesis. C/EBPβ expression is localized to muscle satellite cells and is downregulated upon induction to differentiate, mirroring the loss of Pax7 expression in early myogenesis. C/EBPβ expression also negatively regulates MyoD protein expression. Leaky ribosomal scanning of the Cebpb mRNA produces three C/EBPβ isoforms: LAP*, LAP and LIP, though the individual role of each of these isoforms has not been investigated in myoblasts. This thesis focuses on determining the role of each of the C/EBPβ isoforms during skeletal muscle differentiation. Forced expression of the C/EBPβ-LIP isoform in myoblasts led to a decrease in Myf5, MyoD, and myogenin expression under differentiation conditions when compared to empty vector controls. Further, the fusion of cells was greatly reduced following differentiation. C/EBPβ-LIP expressing cells also demonstrated a growth defect, with pronounced G1 arrest and features of senescence. In contrast, myoblasts expressing the C/EBPβ-LAP isoform has impaired differentiation, though this was not as pronounced as in C/EBPβ-LIP expressing cells and proliferated normally. While LIP is not normally expressed in primary myoblasts from healthy muscle, the ratio of LIP:LAP was increased in primary myoblasts isolated from mdx mice, an animal model for Duchenne muscular dystrophy. These findings suggest that the regulation of C/EBPβ isoform expression could regulate stem cell stamina and may contribute to defects in muscle regeneration in disease.

This study investigates the skeletal muscle repair and regeneration process following blunt trauma injury in murine models. Skeletal muscle injury is recorded most often in sports injuries and include strains and sprains, contusions, and bruising, however, there is growing consensus about the role skeletal muscle plays in the reparative process of bone fractures. Skeletal muscle stem cells or satellite cells are mesenchymal stem cell derived cells that exist between the basal lamina and cell membrane of muscle fibers usually in close proximity to capillary beds. After a traumatic injury, satellite cells respond to the influx of signaling from immune cells, oxygen tension, and myogenic proteins which influence differentiation into myoblasts for repair of tissue damage. Research continues to elucidate the relationship between bone and skeletal muscle following trauma injuries. Skeletal muscle stem cells play a vital role in fracture healing, and in certain conditions, are even induced into the osteogenic pathway. The goals of this study are to characterize the temporal progression of myogenesis during muscle repair that will be used with future studies of muscle and bone injury. And to identify potential crosstalk mechanisms between muscle and bone repair during trauma. In our experiment model trauma was introduced to mice with a modified muscle contusion device where a weight was dropped onto the femoral quadriceps muscles and the quadriceps and biceps muscle tissues were harvested at post-operative days (POD) 2, 4, 12, 16, and 24. Reverse-Transcriptase Quantitative Polymerase Chain Reaction was used to analyze gene expression profiles for satellite/stem cells (Pax7 and Prx1), muscle regeneration (MyoD, Myf5, Myl2, and Myh1), angiogenesis (VegfA, VegfR2), myokine (Myostatin and IL6), and BMP signaling (ID1). Our findings indicate that both Pax7 and Prx1 expression slightly decreased after injury but showed a significant (p<0.05) increase and peak of expression at POD 16 in the femoral quadriceps muscles. The early myogenic genes, MyoD and Myf5 peaked early at POD 4 while the adult myofiber markers, Myl2 and Myh2, peaked later at POD 16 in the femoral quadriceps muscles. Only slight changes were observed in the femoral biceps muscles. The angiogenic genes peaked at POD16 in the femoral quadriceps muscles and POD 12 in the femoral biceps muscles. The expression of Myostatin, an inhibitor of muscle mass, decreased early (POD 4 and 12) however showed a non-significant increase at POD 16 in the femoral quadriceps muscles. Lastly, the expression of ID1, which is downstream target of BMP signaling peaked early at POD 4 in the femoral quadriceps muscles. These data indicates that stem/satellite cells decrease in response to muscle injury but by POD 4, myogenic commitment and programming occurs. While early myogensis occurs, BMP signaling peaks and Myostatin expression decreases suggesting a coordinated event. Adult myofiber regeneration occurs in parallel to angiogenesis. The myogenic events were primarily isolated to the injured femoral quadriceps muscles. This model of muscle injury can be used to study muscle regeneration within context to bone injury.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22116号 / 医博第4529号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸口田 淳也, 教授 妻木 範行, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background: Cellular cardiomyoplasty is the method of transplanting myogenic cells into injured myocardium to restore the lost heart muscle cells and to improve ventricular function. Method: Three patients, all with a history of coronary heart disease, underwent coronary artery bypass grafting and implantation of autologous satellite cells. A muscle biopsy of 2-4 g from the right vastus lateralis muscle was obtained for satellite cell (myogenic stem cell from skeletal muscle) isolation and proliferation before implanted into the donor's heart. The cells were suspended in serum-free medium and injected into 30-40 sites at and around the ischemic areas just before reversing the hypothermic cardioplegia to eliminate arrhythmia and to improve retention. After recovery, each patient was maintained at the intensive care unit for 3-4 days with ECG monitoring before transferring to the patient floor. Results: All patients survived the procedure with an uneventful recovery and were discharged from the hospital. At 3-4 months follow-up examination, increased left ventricular ejection fraction of 11% (35-46%), 5.4% (40-45.4%) and 1% (40-41%) and decreased left ventricular diastolic diameter of 4, 2 and 9 mm were observed for the patients, respectively. Arrhythmia was not detected during the follow-up evaluation by ECG. Improved perfusion (99mTC-MIBI) and increased metabolic activity (18F-deoxyglucose) were found at the sites of satellite cell implantation. Significant increase of wall thickness and movement at the areas of cell injection was also observed using 2D-echo. Conclusion: Cellular cardiomyoplasty using autologous satellite cells is a safe procedure with encouraging beneficial outcomes in patients.

Skeletal muscle satellite cells (myoblasts) are the primary stem cells of skeletal muscle which contribute to growth, maintenance, and repair of the muscles. Satellite cells are the first stem cells used for cellular cardiomyoplasty more than 20 years ago. The isolation, culture, labeling, and identification of satellite cells are described in detail here. The implantation and outcomes of cellular cardiomyoplasty using satellite cells have been summarized in the previous chapter (Chapter 1).

Skeletal muscle satellite cells (myoblasts) are the primary stem cells of skeletal muscle which contribute to growth, maintenance, and repair of the muscles. Satellite cells are the first stem cells used for cellular cardiomyoplasty more than 20 years ago. The isolation, culture, labeling, and identification of satellite cells are described in detail here. The implantation and outcomes of cellular cardiomyoplasty using satellite cells have been summarized in the previous chapter (Chapter 1).

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