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.

Die Muscle-RING-finger (MuRF) Proteine sind E3-Ubiquitin-Ligasen, die im Muskelgewebe den Ubiquitin-Proteasom-System abhängigen Abbau von Proteinen vermitteln. MuRF1 wird in der Muskelatrophie verstärkt synthetisiert, was zu einem gesteigerten Proteinabbau und damit zum Verlust von Muskelmasse führt. Zudem sind Mäuse, denen MuRF1 fehlt vor Muskelatrophie geschützt. E3-Ubiquitin-Ligasen fungieren oftmals in Multiproteinkomplexen. Dies wurde für MuRF-Proteine bisher nicht gezeigt. Aufgrund dessen sollten neue MuRF-Multiproteinkomplex assoziierte Faktoren mittels Hefe-Zwei-Hybrid-System und SILAC AP-MS identifiziert und deren Einfluss auf die MuRF-Funktion charakterisiert werden. Es wurden sowohl neue als auch publizierte MuRF-Interaktionspartner (Iap) gefunden. Von den neu entdeckten MuRF-Iap wurde der Fokus auf WDR42A gelegt, da das Protein mit beiden Methoden identifiziert wurde und zudem funktionell hoch interessant ist. WDR42A homologe Proteine bilden zirkuläre β-Propeller Strukturen die Multiproteinkomplexe koordinieren. Die Interaktion zwischen MuRF-Proteinen und WDR42A wurde mittels Ko-IP Experimenten und Kolokalisationsstudien bestätigt. Cycloheximid-Abbau-Experimente deuten darauf hin, dass WDR42A kein MuRF1 Substrat-Protein ist. Da die MuRF-Proteine spezifisch im Muskel hergestellt werden, sollte überprüft werden ob WDR42A ebenfalls im Muskelgewebe synthetisiert wird. Es wurde gezeigt, dass WDR42A ubiquitär sowie im Muskelgewebe und in immortalisierten Muskelzellen hergestellt wird. Analog zu MuRF1 wird WDR42A in der Denervations-induzierten Skelettmuskelatrophie und der Muskelentwicklung verstärkt synthetisiert. Die Herunterregulation von WDR42A mittels siRNA in C2C12 Myotuben schützte diese Zellen vor dem Auftreten von Atrophie. Diese Ergebnisse zeigen, dass WDR42A wie MuRF1 an der Entstehung von Muskelatrophie beteiligt ist. Aufgrund der WDR42A Domänenstruktur wird vermutet, dass WDR42A als Scaffolding-Protein MuRF1-Multiproteinkomplexe reguliert. / The muscle-RING-finger (MuRF) proteins are E3 ubiquitin ligases which coordinate the ubiquitin-proteasome system dependent protein degradation in muscle tissue. MuRF1 is up-regulated under muscle atrophy conditions. This leads to enhanced proteolysis and thereby to loss of muscle mass and strength. Furthermore are MuRF1 knockout mice resistant to muscle atrophy. E3 ubiquitin ligases often operate in multi-protein complexes. This has not been shown for MuRF proteins. Therefore we used yeast-two-hybrid and SILAC-AP-MS to identify and subsequently characterize new MuRF multi-protein complex associated proteins. We found new and also published MuRF interaction partners (Iap) with both methods. Amongst the new Iap, we focused on WDR42A, because it was found with both techniques and his interesting functional potential. WDR42A exhibits seven consecutive arranged WD40-repeat domains. This domain arrangement leads in homologues proteins to the formation of seven-bladed β-propeller structures, which act as protein interaction platforms that coordinate multi-protein complexes. The protein interaction between the MuRFs and WDR42A was confirmed with Co-IP and co-localization experiments. Cycloheximide decay experiments indicated that WDR42A is not a MuRF1 substrate protein. The MuRF proteins are muscle specific, therefore we tested if WDR42A is also synthetized in muscle tissue. We could show that WDR42A is ubiquitously, but also in muscle tissue as well as in immortalized muscle cells produced. WDR42A is similar to MuRF1 up-regulated under denervation-induced skeletal muscle atrophy as well as in muscle development. Furthermore are C2C12 myotubes resistant to muscle atrophy after siRNA down-regulation of WDR42A. These results demonstrate that WDR42A is like MuRF1 important for the development of muscle atrophy. Due to the domain structure of WDR42A, we hypothesize that WDR42A regulates MuRF1 multi protein complexes as scaffolding protein.

Musculoskeletal injuries are commonly associated with muscle atrophy as a function of immobilization or change of normal function. For example, injuries to the anterior cruciate ligament (ACL) which may involve ligament reconstruction, results in the "quadriceps avoidance" gait which leads to atrophy of the knee extensormuscles. In these situations it is not clear whether or not the atrophy is associated with loss of specific muscle fibre types with accompanying functional deficits. Such knowledge would be helpful in implementing exercise regimes designed to compensate for loss of particular fibre types. It is believed that isokinetic exercise performed at speeds below 180° per second strengthens type I muscle fibres, and type II fibres at fast speeds. However, there is no evidence to indicate the specific muscle fibre response to different rates of muscle contraction. Identification of muscle fibre type is most directly determined by biopsy technique but is too invasive for a routine measurement. Electromyography median frequency has been used as a non-invasive measure to infer muscle fibre composition in various studies. However, the reliability and accuracy of this technique has been questioned and improvement is necessary. This research was designed to provide a more accurate and reliable protocol for the determination of EMG median frequency which may be used, after validation against more direct biopsy techniques, as a routine method for inferring muscle fibre composition. The investigation also explored the muscular response as measured by EMG median frequency to varying speeds of muscle contraction, fatiguing exercise and atrophy following ACL reconstruction. The ultimate aim of this research was to improve the reliability of the determination of EMG median frequency to enhance its application as a predictor of muscle fibre composition. This provides information which may improve ACL rehabilitation programs designed to restore and prevent specific muscle fibre types loss that have not previously been targeted by current rehabilitation programs. This research was conducted in three studies. Study one determined the stability of the EMG median frequency bilaterally for the quadriceps and hamstrings muscles and identified the mode of contraction associated with the greatest reliability. The strength and EMG median frequency of the vastus lateralis, medial hamstrings and vastus medialis of 55 subjects was determined across 5 speeds from 0° to 240° per second using a Kin-Com isokinetic dynamometer and an EMG data acquisition system. Isometric contraction was found to have the least bilateral discrepancy (4.01% ±3.06) and between trials standard deviation (4.50) in the vastus lateralis, medial hamstrings and vastus medialis. Study two investigated the EMG median frequency changes in the vastus lateralis which occur immediately following different speeds of isokinetic exercise to the point of fatigue in normal subjects. Thirty-four subjects participated in the study, and performed a 90-second period of isokinetic exercise to activate the knee extensors at either 30° or 300° per second. EMG median frequency of the vastus lateralis was determined before, immediately after and 7 minutes after the fatiguing exercise. The percentage drop in EMG median frequency of the vastus medialis was gnificantly (p<0.05) greater after slow speed (27.9%) than fast speed (20.25%) exercise, while no significant difference was found for the percentage drop in extension torque. Full recovery was found 7 minutes after the fatiguing exercise. By reference to previous research showing a relationship between EMG median frequency and muscle fibre type, an increase in activation of type I muscle fibres with slow speed exercise and an increase in type II muscle fibres with fast speed exercise was observed. Study three identified the changes in EMG median frequency following ACL reconstruction and evaluated the bilateral differences in EMG median frequency of the knee muscles. The relationships between EMG median frequency and the measures of knee functional ability, knee muscle strength, age and time since surgery were also investigated. Twelve subjects who had undergone ACL reconstruction using a semitendinosus and gracilis graft 6 to 12 months earlier, participated in the study. EMG median frequency was determined from an 8-second isometric contraction and knee functional ability was assessed using the Cincinnati Rating Scale. Bilateral EMG median frequency shifts were inconsistent among subjects. On the basis of previous research which indicated a relationship between EMG median frequency and fibre type, no consistent pattern of muscular fibre type atrophy subsequent to ACL reconstruction occurred within 6 to 12 months (ranged from -43 to 57 Hz). Additionally, no significant correlations were found between the EMG median frequency and the knee functional score and knee extension and flexion torques, age, time since operation and the bilateral differences in EMG median frequency. The results of this investigation will serve to improve the reliability of EMG median frequency across a range of conditions in which it has been evaluated. Further research is needed to confirm the relationship between EMG median frequency and direct observations of muscle fibre composition to improve the predictive value of this measure. Following this validation it will be possible to evaluate the bilateral EMG median frequency shift to infer the type of muscle fibre atrophy, and use this measure in determining the efficacy of specific rehabilitation programs. In conclusion * An 8-second isometric contraction is recommended for determining EMG median frequency. * EMG median frequency of a muscle decreases significantly more after slow fatiguing exercise than after fast speed fatiguing exercise. * There was no generalised bilateral EMG median frequency shift found in a group of subjects 6 to 12 months following semitendinosus and gracilis graft ACL reconstruction. * The results of this study will serve to improve the reliability of procedures used to determine EMG median frequency under a range of different contractile conditions. The EMG median frequency changes in response to these conditions require further validations with muscle biopsy in future.

Submitted by André Krug (andre.krug@bol.com.br) on 2018-05-28T21:21:29Z No. of bitstreams: 2 TESE_DOUTORADO_ANDRE_KRUG.pdf: 1523685 bytes, checksum: eaa1e414ce0fea68ddb7c3b618d037b6 (MD5) Carta_biblioteca_Andre.pdf: 2380256 bytes, checksum: d398258e7a2f5c73594cf8230b4b1957 (MD5) / Approved for entry into archive by Ronildo Prado (ri.bco@ufscar.br) on 2018-06-06T13:33:50Z (GMT) No. of bitstreams: 2 TESE_DOUTORADO_ANDRE_KRUG.pdf: 1523685 bytes, checksum: eaa1e414ce0fea68ddb7c3b618d037b6 (MD5) Carta_biblioteca_Andre.pdf: 2380256 bytes, checksum: d398258e7a2f5c73594cf8230b4b1957 (MD5) / Approved for entry into archive by Ronildo Prado (ri.bco@ufscar.br) on 2018-06-06T13:34:02Z (GMT) No. of bitstreams: 2 TESE_DOUTORADO_ANDRE_KRUG.pdf: 1523685 bytes, checksum: eaa1e414ce0fea68ddb7c3b618d037b6 (MD5) Carta_biblioteca_Andre.pdf: 2380256 bytes, checksum: d398258e7a2f5c73594cf8230b4b1957 (MD5) / Made available in DSpace on 2018-06-06T13:40:53Z (GMT). No. of bitstreams: 2 TESE_DOUTORADO_ANDRE_KRUG.pdf: 1523685 bytes, checksum: eaa1e414ce0fea68ddb7c3b618d037b6 (MD5) Carta_biblioteca_Andre.pdf: 2380256 bytes, checksum: d398258e7a2f5c73594cf8230b4b1957 (MD5) Previous issue date: 2018-03-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Synthetic glucocorticoids have potent anti-inflammatory and immunosuppressive action, though its chronic usage can trigger muscle atrophy. On the other hand the resistance training (RT) acts in opposition to atrophic situations, although its effects on muscle atrophy induced by treatment with dexamethasone (DEX) are poorly known.The purpose of this study was to determine how long RT is required to promote preventive effects in flexor halluces longus (FHL) muscle atrophy induced by dexamethasone (DEX). After maximal voluntary carrying capacity (MVCC), 100 rats were separated in sedentary (SED) or resistance trained for 10 (RT10), 40 (RT40), 70 (RT70) and 100 (RT100) days. Groups were divided as control (CTRL) or treated with DEX. RT was performed with 80% of maximal voluntary carrying capacity (MVCC). During the last 10 days, the animals either received DEX (0.5 mg/kg/day, i.p.) or vehicle (saline, same volume as DEX treatment, i.p.). The FHL muscle was removed, cleaned, weighed and stored for determining the cross-sectional area, proteassomal activity 26s, and total p70S6K, p-p70S6KThr389, MuRF1, REDD1 and GAPDH protein level. The results arepresented as mean ± SEM. The repeated measures two-way analysis of variance (ANOVA) were used for food intake and, for further analysis,it was used two-way ANOVA, both with Tukey post hoc test and significance levelset as α<0.05. DEX reduced FHL mass (-26%), but RT70 and RT100 DEX groups presented atrophy attenuation. DEX reduced proteasome activity in SED (-33%) and RT70 (-44%) DEX. RT70 CTRL had increased proteasome activity when compared with RT10 and RT40 CTRL (+48% and +51%, respectively) groups and RT100 CTRL had reduced activity (-56%). DEX reduced phospho-p70S6KThr389/total p70S6k ratio in SED DEX (-24%), but it was reverted in RT10 (+48%) and RT70 DEX(+70%). RT70 CTRL presented higher values of this ratio than SED, RT40 and RT100 CTRL groups. DEX increased REDD1 (+47%) protein level only in SED DEX. MuRF-1 protein level increased in SED(+50%), RT10 (+45%) and RT40 (+46%)DEX groups, but it was blocked in RT70 and RT100 DEX groups. In summary, we suggest that DEX-induced FHL muscle atrophy requires at least 70 days of RT to be attenuated and this response involves a complete blockade of MuRF-1 and REDD1 protein level increase and the blockade phospho-p70S6KThr389/total p70S6k ratio reduction. Also, 100 days of RT did not promote any additional effects. It is interesting to note that only 10 days of RT evoked improvements in the synthesis pathway, which suggest that some molecular adjustments are required in early stages of skeletal muscle mass maintenance. / Os glicocorticoides sintéticos possuem potente ação anti-inflamatória e imunossupressora, entretanto seu uso crônico pode desencadear atrofia muscular. Por outro lado o treinamento resistido (TR) contrapõe-se a situações atróficas, embora seus efeitos sobre a atrofia muscular induzida pelo tratamento com dexametasona (DEX) são pouco conhecidos.O presente trabalho teve como objetivo verificar qual é o momento em que o efeito preventivo do TR (80% do carregamento máximo) é mais efetivo sobre a redução peso corporal e atrofia muscular induzidas pelo tratamento com DEX. Separamos 100 ratos Wistar machos em 10 grupos: sedentário controle (SED CTRL); sedentário tratado com DEX (SED DEX); treinado controle 10, 40, 70 e 100 dias (TR10 CTRL, TR40 CTRL, TR70 CTRL e TR100 CTRL) e treinado tratado com DEX 10, 40, 70 e 100 dias (TR10 DEX, TR40 DEX, TR70 DEX e TR100 DEX). Utilizamos o TR em escada (80% TCM). Nos 10 últimos dias os animais receberam DEX (0,5 mg/kg por dia, i.p.) ou o mesmo volume de salina. O músculo flexor longo do hálux (FHL) foi removido, limpo, pesado e armazenado para determinação da área de secção transversa, atividade do proteassoma 26s, e produção proteica dep70S6K total, p-p70S6KThr389, MuRF1, REDD1 e GAPDH. Os resultados são apresentados como média±EPM. Foi utilizada aanálise de variância de dois caminhos (ANOVA) para medidas repetidas para ingestão alimentar e para as variáveis restantes foi utilizada a ANOVA de dois caminhos. Na presença de interação, foi utilizado o posthoc de Tukey,com significância de α<0,05. A DEX reduziu 26% a massa muscular do FHL, mas o grupo TR70 e TR100 DEX apresentaram essa atrofia atenuada. O tratamento com DEX reduziu a atividade do proteassoma nos grupos SED (-33%) e TR70 DEX (-44%). O grupo TR70 CTRL teve sua atividade do proteassoma aumentada em relação aos grupos TR10 e TR40 CTRL (+48% e +51%, respectivamente), além do mais, o grupo TR100 CTRL teve sua atividade reduzida (-56%). A DEX reduziu a razão p-p70S6KThr389/p70S6k total no grupo SED DEX (-24%), mas essa resposta foi revertida no grupo TR10 (+48%) e TR70 (+70%) DEX. O grupo TR70 CTRL apresentou valores superiores dessa razão em relação aos grupos SED, TR40 e TR100 CTRL. A DEX aumentou a produção proteica de REDD1 (+47%) somente no grupo SED DEX. A produção proteica de MuRF1 foi aumentada nos grupos SED (+50%), TR10 (+45%) e TR40 (+46%) DEX, mas essa resposta foi completamente bloqueada nos grupos TR70 TR100 DEX. Com base nos resultados do presente estudo, pode-se sugerir que a atrofia muscular induzida por DEX no músculo FHL necessita de pelo menos 70 dias de TR para ser atenuada e essa resposta parece envolver o completo bloqueio dos aumentos de MuRF1 e REDD1, somados ao bloqueio da redução da razão p-p70S6KThr389/p70S6k. Além disso, 100 dias de TR não provocaram nenhum efeito preventivo adicional. É interessante notar que o TR, mesmo realizado por curto período (10 dias), promove melhorias na via de síntese de proteínas, oque sugere que alguns ajustes moleculares são necessários em estágios iniciais da manutenção da massa muscular. / CAPES: 1452526

Patients admitted to intensive care unit (ICU) are known to develop significant impairments in physical function. Patients with critical illness suffer up to 30% reductions in muscle size within the first ten days of admission to the ICU. Muscle strength testing, Medical Research Council-sum score, is current gold-standard to diagnosis ICU-acquired weakness and predicts risk of mortality and long-term physical function. Muscle power different from muscle strength in that it accounts for velocity of movement, is potentially a better independent predictor of function that has not been studied in this population. In addition, we hypothesize that muscle size and quality measured through ultrasound imaging has better applicability and prediction that strength testing. Therefore, we prospectively collected data surrounding these muscle parameters in patients admitted to the medicine ICU at University of Kentucky. Primary outcomes included physical function, muscle power with a novel assessment tool for the critically ill population, muscle strength, and muscle size and quality assess through ultrasound imaging. 36 patients admitted to ICU and 18 aged-matched controlled were enrolled. Patients had significantly lower scores on muscle power assessment at ICU discharge (33.6 ±19.0 W; t= 4.01, p < 0.001) and at hospital discharge (40.9 ±16.5 W; t= 4.81, p < 0.001) in comparison to controls (59.3± 14.7 W). Patients with better scores on muscle power assessment had significantly better scores on physical function measures (Six-minute walk test; rs = 0.548, p = 0.0001). Muscle size (cross-sectional area of rectus femoris muscle) and muscle power were strongly correlated (rs = 0.66, p < 0.0001). These data suggest that patients with critical illness have significantly reduced muscle power which directly related to deficits in physical function.

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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.