Thesis (MSc (Physiological Sciences))--Stellenbosch University, 2008. / Introduction: Skeletal muscle atrophy is a mitigating complication that is characterized by a reduction in muscle fibre cross-sectional area as well as protein content, reduced force, elevated fatigability and insulin resistance. It seems to be a highly ordered and regulated process and signs of this condition are often seen in inflammatory conditions such as cancer, AIDS, diabetes and chronic heart failure (CHF). It has long been understood that an imbalance between protein degradation (increase) and protein synthesis (decrease) both contribute to the overall loss of muscle protein. Although the triggers that cause atrophy are different, the loss of muscle mass in each case involves a common phenomenon that induces muscle proteolysis. It is becoming evident that interactions among known proteolytic systems (ubiquitin-proteosome) are actively involved in muscle proteolysis during atrophy. Factors such as TNF-α and ROS are elevated in a wide variety of chronic inflammatory diseases in which skeletal muscle proteolysis presents a lethal threat. There is an increasing body of evidence that implies TNF-α may play a critical role in skeletal muscle atrophy in a number of clinical settings but the mechanisms mediating its effects are not completely understood. It is also now apparent that the transcription factor NF-κB is a key intracellular signal transducer in muscle catabolic conditions. This study investigated the various proposed signalling pathways that are modulated by increasing levels of TNF-α in a skeletal muscle cell line, in order to synthesize our current understanding of the molecular regulation of muscle atrophy. Materials and Methods: L6 (rat skeletal muscle) cells were cultured under standard conditions where after reaching ± 60-65% confluency levels, differentiation was induced for a maximum of 8 days. During the last 2 days, myotubes were incubated with increasing concentrations of recombinant TNF-α (1, 3, 6 and 10 ng/ml) for a period of 40 minutes, 24 and 48 hours. The effects of TNF-α on proliferation and cell viability were measured by MTT assay and Trypan Blue exclusion technique. Morphological assessment of cell death was conducted using the Hoechst 33342 and Propidium Iodide staining method. Detection of apoptosis was assessed by DNA isolation and fragmentation assay. The HE stain was used for the measurement of cell size. In order to determine the source and amount of ROS production, MitoTracker Red CM-H2 X ROS was utilised. Ubiquitin expression was assessed by immunohistochemistry. PI3-K activity was calculated by using an ELISA assay and the expression of signalling proteins was analysed by Western Blotting using phospho-specific and total antibodies. Additionally, the antioxidant Oxiprovin was used to investigate the quantity of ROS production in TNF-α-induced muscle atrophy. Results and Discussion: Incubation of L6 myotubes with increasing concentrations of recombinant TNF-α revealed that the lower concentrations of TNF-α used were not toxic to the cells but data analysis of cell death showed that 10 ng/ml TNF-α induced apoptosis and necrosis. Long-term treatment with TNF-α resulted in an increase in the upregulation of TNF- α receptors, specifically TNF-R1. The transcription factors NF-κB and FKHR were rapidly activated thus resulting in the induction of the ubiquitin-proteosome pathway. Activation of this pathway produced significant increases in the expression of E3 ubiquitin ligases MuRF-1 and MAFbx. Muscle fibre diameter appeared to have decreased with increasing TNF-α concentrations in part due to the suppressed activity of the PI3-K/Akt pathway as well as significant reductions in differentiation markers. Western blot analysis also showed that certain MAPKs are activated in response to TNF-α. No profound changes were observed with ROS production. Finally, the use Oxiprovin significantly lowered cell viability and ROS production. These findings suggest that TNF-α may elicit strong catabolic effects on L6 myotubes in a dose and time dependent manner. Conclusion: These observations suggest that TNF-α might have beneficial effects in skeletal muscle in certain circumstances. This beneficial effect however is limited by several aspects which include the concentration of TNF-α, cell type, time of exposure, culture conditions, state of the cell (disturbed or normal) and the cells stage of differentiation. The effect of TNF-α can be positive or negative depending on the concentration and time points analysed. This action is mediated by various signal transduction pathways that are thought to cooperate with each other. More understanding of these pathways as well as their subsequent upstream and downstream constituents is obligatory to clarify the central mechanism/s that control physiological and pathophysiological effects of TNF-α in skeletal muscle.

La sarcopénie est définie comme la diminution de la masse et de la force musculaires squelettiques au cours du vieillissement.Dans ce contexte, l’objectif principal de cette thèse était d’évaluer l’action régulatrice de la vitamine D sur le dialogue entre cellules immunitaires et musculaires et son implication dans les capacités de régénération du muscle âgé.Dans un premier temps, nous avons étudié in vitro la différenciation des cellules musculaires de la lignée L6 co-cultivée ou non avec des cellules immunitaires (PBMC : cellules mononuclées du sang périphérique), et en présence ou non de LPS. Ce modèle a permis d’établir que les PBMC stimulaient bien la différenciation musculaire. La réponse pro-inflammatoire induite par le LPS inhibait l’expression des marqueurs de différenciation. Même en présence de LPS, la stimulation de l’expression ce des marqueurs dans les L6 co-cultivées était conservée. De plus, l’environnement pro-inflammatoire induit par le LPS dans les co-cultures inhibait l’expression musculaire des marqueurs de la voie de la régénération, comme Notch. Nous avons ensuite utilisé le même système de co-cultures afin de déterminer si la vitamine D, ici la 25(OH)D, pouvait moduler les sécrétions cytokiniques des PBMC et ainsi modifier l’expression des marqueurs de différenciation musculaires. Le traitement des co-cultures à la 25(OH)D n’a modifié ni le profil de sécrétion cytokinique des PBMC, ni l’expression des marqueurs de différenciation des L6.Dans un deuxième temps, nous avons étudié à l’aide d’un modèle de rats âgés déplétés en vitamine D les mécanismes pouvant contribuer à l’atrophie musculaire observée. Nous avons mis en évidence que l’activité de la voie de signalisation Notch, voie clé du processus de régénération, ainsi que la prolifération musculaire étaient diminuées chez ces rats, et ceci même en absence de lésion. Nous avons ensuite évalué l’effet du statut en vitamine D sur un processus aigu de régénération au cours de vieillissement chez le rat. L’analyse de cette expérimentation, actuellement en cours, a déjà permis de mettre en évidence qu’au cours du vieillissement, la prolifération musculaire suite à une lésion est diminuée, d’autant plus si le rat âgé est carencé en vitamine D. Le même résultat a été retrouvé pour l’expression d’une cible de la voie Notch (Hes1). En outre, l’expression des marqueurs de différenciation semblaient être altérée chez les animaux âgés résultant probablement en un retard et/ou une inefficacité du processus de différenciation, en particulier chez les rats âgés déplétés en vitamine D. En revanche, la supplémentation en vitamine D ne semblait pas avoir d’effet sur la régénération musculaire du rat âgé.En conclusion, in vitro, la 25(OH)D ne modifiait pas l’expression des marqueurs de différenciation des cellules musculaires co-cultivées avec des cellules immunitaires. En revanche in vivo, la déplétion en vitamine D semblerait aggraver l’effet de l’âge sur la régénération musculaire.La diminution de la capacité de régénération musculaire est un facteur contribuant au développement de la sarcopénie. Ce travail a permis de montrer que le maintien d’un statut optimal en vitamine D serait nécessaire à la conservation des capacités de régénération musculaire. Il semble donc important de maintenir des statuts optimaux en vitamine D afin de limiter l’atrophie musculaire au cours du vieillissement. / One of the most striking effects of ageing is an involuntary loss of skeletal muscle mass known as sarcopenia. The development of sarcopenia appears to be multifactorial and includes anabolic resistance to dietary amino acids and sedentary lifestyle. The diminished ability of aged muscle to self-repair is also a key factor of sarcopenia. During the regeneration process, immune and muscle cells work in a cross-talk leading to an optimal muscle cell proliferation and differentiation. However, with aging, the immune response is impaired, possibly contributing to the reduction in the capacity of regeneration.Muscle and immune cells are both targets of vitamin D action. This vitamin modulates muscle cell proliferation and differentiation and stimulates the anti-inflammatory response of immune cells. With age, vitamin D insufficiencies or deficiencies develop.In this context, the main objective of this thesis was to evaluate the regulatory action of vitamin D on the cross-talk between immune and muscle cells and its implication in the ability of skeletl muscle to regenerate during aging.Initially, we studied in vitro the differentiation of L6 muscle cells co-cultured with or without immune cells (PBMC: peripheral blood mononuclear cells), and in with or without of LPS. From this model, PBMC stimulated muscle cell differentiation. The pro-inflammatory response induced by LPS inhibited the expression of muscle differentiation markers in muscle cells. Of note, these markers were stimulated even in presence of LPS. In addition, the LPS-associated pro-inflammatory environment inhibited the Notch signaling pathway, the key pathway of muscle regeneration process, in L6 cells co-cultured with PBMC. We then used the same system of co-cultures to determine whether vitamin D, in its 25 (OH)D form, could modulate PBMC cytokine secretion and thereby could alter the expression of markers of muscle differentiation. Unfortunately, the treatment of co-culture with 25 (OH) D has changed neither the profile of PBMC cytokine secretion nor the expression of differentiation markers in L6 cells.Secondly, we investigated in a model of old rats the mechanisms that contribute to muscle atrophy following vitamin D depletion. We have demonstrated that the activity of the Notch signaling pathway, as well as muscle proliferation were reduced in old vitamin D-depleted rats, even in the absence of lesions. Then we evaluated the effect of the vitamin D status on an acute muscle regeneration process, i.e. muscle infusion of notexin in old rats. This ongoing experiment has already highlighted that during aging, muscle proliferation is reduced after injury, especially if age is associated with a vitamin D deficiency. In addition, during aging, the expression of differentiation markers was altered resulting in delayed and/or incomplete differentiation process, in particular in vitamin D-depleted old rats. However, vitamin D supplementation seemed to have no beneficial or deleterious effects on muscle regeneration in aged rats.In conclusion, in vitro 25 (OH) D was unable to modulate the differentiation of muscle cells co-cultured with immune cells. However, in vivo, vitamin D depletion appeared to worse the effect of ageing on muscle regeneration.The diminished ability of aged muscle to self-repair is a factor of sarcopenia. Our work has demonstrated the importance of maintening optimal vitamin D status to preserve muscle regeneration capacity and thus to limit muscle atrophy during aging.

Transforming growth factor beta 1 (TGF-beta1) ist ein multifunktionales Zytokin, welches eine Rolle in der Sepsis und in der Sepsis-induzierten Myopathie spielen könnte. Weiterhin könnten erhöhte TGF-beta1-Level zur Muskelschwäche, die mit der Intensivpflege assoziiert ist (engl. intensiv care unit-acquired weakness, ICUAW), beitragen. Der TGF-beta1- Signalweg wurde in Skelettmuskelbiopsien von ICUAW-Patienten heraufreguliert. Secreted frizzled related protein 2 (SFRP2) wurde in einer Gen-Set-Anreicherungsanalyse als das am höchsten regulierte Gen identifiziert. Im Mausmodell führten Sepsis und Hunger zu einer verringerten Sfrp2-Expression, während dies in der Denervation-induzierten Skelettmuskelatrophie nicht festzustellen war. In differenzierten C2C12-Myotuben führte TGF-beta1 zu einer verringerten Sfrp2-mRNA- und Proteinexpression. Luciferase-Assays deuteten auf eine TGF-beta1-abhängige Herunterregulation von Sfrp2 hin, welche auf Promoterebene durch mögliche negative regulatorische Elemente im Sfrp2-Promoter vermittelt wurde. Weiterhin wurde eine TGF-beta1 induzierte Muskelatrophie durch transkriptionelle Repression der myosin heavy chain Gene beobachtet. Im Gegensatz dazu veränderte TGF-beta1 nicht den proteasomalen Abbau muskulärer Proteine. Die Genexpression von Tripartite motif containing 63 und F-box only protein 32 war hingegen leicht herunterreguliert. TGF-beta1-induzierte Atrophie in differenzierten C2C12-Myotuben wurde teilweise durch rekombinantes Sfrp2 aufgehoben. Weiterhin wurde eine direkte physikalische Interaktion zwischen Sfrp2 und TGF-beta1 gefunden, welche diesen Effekt verursacht haben könnte. Zusammengefasst lässt sich feststellen, dass der TGF-beta1- Signalweg eine wichtige Rolle in der ICUAW durch Inhibition der myosin heavy chain Expression spielt. TGF-beta1-abhängige Herunterregulation von Sfrp2 könnte zu einer Feedback-Antwort, die das Ausmaß der Atrophie durch TGF-beta1 verstärkt, führen. / Transforming growth factor beta 1 (TGF-beta1) is a multifunctional cytokine that may play a role in sepsis and in sepsis-induced myopathy. Our group speculated that increased TGF-beta1 could contribute to intensive care (ICU)-acquired weakness (ICUAW), a catastrophic muscle disease in critically ill patients. We found that TGF-beta1 signaling in skeletal muscle biopsies of ICUAW patients was upregulated. Secreted frizzled related protein 2 (SFRP2) was the most regulated gene identified by gene set enrichment analysis (GSEA). I then studied the regulation and function of SFRP2 in different skeletal muscle atrophy models. In three mouse models, downregulated Sfrp2 expression was observed in sepsis and starvation, but not in denervation-induced skeletal muscle atrophy. In differentiated C2C12 myotubes, TGF-beta1 downregulated Sfrp2 expression on both mRNA and protein levels. Luciferase assays suggested that TGF-beta1-dependent downregulation of Sfrp2 was mediated at the promoter level through possible negative regulatory elements in the Sfrp2 promoter. I also observed that TGF-beta1-induced muscle atrophy was accompanied by transcriptional repression of myosin heavy chain genes. In contrast, TGF-beta1 did not increase proteasomal degradation of muscular proteins since gene expression of Tripartite motif containing 63 (Trim63) and F-box only protein (Fbxo32) was not upregulated; instead, they were slightly downregulated. TGF- beta1-induced differentiated C2C12 myotube atrophy was partially reversed by recombinant Sfrp2. This inhibitory effect could have resulted from direct interaction between Sfrp2 and TGF-beta1, since I found a physical interaction between these two proteins. Taken together, TGF-beta1 signaling pathway could play an important role in ICUAW via inhibition of myosin heavy chain expression. TGF-beta1-dependent downregulation of Sfrp2 may establish a feedback loop augmenting the atrophic effect of TGF-beta1.

Die Komposition der Skelettmuskulatur resultiert aus der fein abgestimmten Balance von Proteinauf- und Abbaumechanismen. Die Skelettmuskelatrophie kann in verschiedenen Situationen entstehen bzw. von diversen Krankheiten ausgelöst werden (Altern, Hunger, Krebs, Nervenschädigung, Kachexie) und ist meist die Folge von gesteigertem Proteinabbau, der die Proteinsynthese überwiegt. Der Muskelabbau ist physiologisch teilweise sinnvoll und dient der Notversorgung von lebenswichtigen Organen mit Lipiden, Aminosäuren und Glukose. Insgesamt ist eine funktionsfähige Muskulatur sehr wichtig, sowohl für Gesunde als auch Erkrankte, da bei Muskelatrophie auslösenden Erkrankungen das Gesamtüberleben wesentlich verringert ist und die Lebensqualität der Patienten enorm reduziert ist. Der Abbau von strukturellen Muskelproteinen wurde hauptsächlich dem Ubiquitin-Proteasom System zugeschrieben, dessen Regulation und von seinen einzelnen Enzymen muss genauestens verstanden sein, um in der Zukunft zielgerichtete Therapien entwickeln zu können. Eines der zentralen Enzyme in der Skelett- und Herzmuskelatrophie ist die E3 Ubiquitin Ligase MuRF1. In nahezu allen Modellen für Muskelatrophie wurde eine starke Zunahme der Expression von MuRF1 beschrieben. Betrachtet man die sehr zentrale Rolle von MuRF1 im UPS, dort vermittelt MuRF1 den Abbau von strukturellen Proteinen des Sarkomers, und der beobachteten starken Regulation bei diversen Atrophie-Modellen, wird klar, wie wichtig das Verständnis der transkriptionellen Regulation von MuRF1 selbst ist. In den letzten Jahren wurden bereits einige Transkriptionsfaktoren identifiziert, die an der Regulation von MuRF1 bei verschiedenen Atrophie-Modellen beteiligt sind, die Studien zeigten aber auch, dass noch nicht alle Modelle erklärt werden konnten. Um die verbleibenden Wissenslücken zu füllen, wurde in dieser Studie nach neuen transkriptionellen Regulatoren von MuRF1 gesucht und deren Beteiligung an bereits bekannten Signalwegen analysiert. / Skeletal muscle mass is permanently balanced as a result of fine tuned protein synthesis and degradation mechanisms. Skeletal muscle atrophy occurs when protein degradation exceeds protein synthesis, which happens in a variety of conditions, such as aging, starvation, cancer, cachexia or denervation. Degradation of muscle mass can sometimes be useful, e.g. as source for lipids, amino acids and glucose in case of critical malnutrition as well as several other physiological conditions. But a solid composition and thereby functional maintenance of muscles is necessary for healthy individuals as well as individuals suffering from atrophy releasing diseases as to retain their mobility and to preserve full heart functions. Since degradation of structural proteins in muscle tissue has been addressed mainly to the ubiquitin-proteasome-system, the regulation of the participating components needs to be understood in detail to develop constructive treatments and therapies for atrophy prevention. One of the key enzymes in skeletal and heart muscle atrophy is the E3 ubiquitin ligase MuRF1. Its expression levels and protein content was found to be elevated in almost every know atrophy model. MuRF1 is very critical for the muscles composition and thus their functional integrity, as it marks and initiates degradation of structural and contractile proteins via the UPS. Since MuRF1 plays a prominent role in muscle atrophy, its transcriptional regulation needs to be well understood to develop effective therapies for all the different atrophy models MuRF1 has been linked to. Several transcription factors have been identified to regulate MuRF1 at different ratios and in diverse atrophy models. Importantly, they do not explain all MuRF1 inducing events observed. To fill some of the remaining knowledge gaps, the studies aims were to find new transcriptional regulators for MuRF1 and to analyze potential involvements of the obtained candidates in pathways affecting skeletal muscle atrophy.