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The mechanisms leading to a hypertrophic versus an endurance phenotype, the hallmarks of resistance exercise (RE) or aerobic exercise (AE), respectively, are still largely unknown. In humans, exposure of exercise naïve persons to either AE or RE results in their skeletal muscle exhibiting generic ‘exercise stress-related’ signalling, transcription, and translation responses. However, with increasing engagement in AE or RE, the responses become refined, and the phenotype typically associated with each form of exercise emerges. Phosphorylation of specific residue sites has been a dominant focus, with canonical signalling pathways (i.e. AMPK and mTOR) studied extensively in the context of AE and RE, respectively. These alone, along with protein synthesis, have only begun to elucidate key differences in AE and RE signalling. Still, key yet uncharacterized differences exist in signalling and regulation of protein synthesis that drive unique adaptation to AE and RE. Omic studies are required help in understanding the mechanisms that lead to the divergent relationship between exercise and phenotypic outcomes of training. In study 1 of this thesis, 16 young, healthy subjects (n=4 pilot study and n=12 follow-up study; 6 males [M] and 6 females [F]) performed a unilateral session of AE or RE with biopsies taken before (Pre), immediately post (0h) and 3 hours (3h) following recovery. Muscle tissue (cohort 1: n=4) was subjected to deep phosphoproteomic analyses, identifying nearly 13000 individual phosphosites and unique clusters specifically associated with AE and RE. Follow-up studies (cohort 2: n=12) were performed, and the outcomes support a thesis that prolonged activation of the MKK3/6, p38, and MK2 signalling axis is a resistance exercise-specific phenomenon and is critical in the process of muscle hypertrophy. We also demonstrated that the activation of signalling through MKK3 is robustly correlated with the increase in myofibrillar protein synthesis that occurs after RE in humans (r2= 0.60, p<0.01). In study 2, we sought to determine the divergent changes in the skeletal muscle proteome induced earlier and later in a training program using both RE and AE, in parallel, in healthy young males and females. We investigated muscle adaptations to AE and RE training during the early untrained versus trained state using novel deuterium oxide labelling and proteomic techniques. A total of 14 (8F/6M) healthy individuals completed 10-wk of thrice weekly unilateral resistance and unilateral aerobic training. Our data illustrated the common and unique networks of protein regulation following AE and RE. Specifically we highlighted the influence that both AE and RE have on mitochondrial proteins, likely aimed at improving metabolic function and possibly underpinning an increase in oxidative capacity and in supporting tissue protein remodelling. In both AE and RE, we identified changes in protein abundance that did not align with individual protein synthetic rates, suggesting that targeted degradation of certain proteins may be an adaptive feature of the shared response to aerobic and resistance training. To date, this work represents the most in-depth analysis of protein-specific fractional synthesis rates in human muscle in vivo in response to differential forms of exercise training. Together, these studies generate novel insights into training mode-specific muscle adaptations by measuring widespread phosphorylation and protein-specific changes in combination with individual protein synthesis rates. / Thesis / Doctor of Philosophy (PhD) / While some exercises help muscles become stronger, others improve the ability to resist fatigue. However, the reasons behind these different changes are not fully understood. Learning more about these processes is important because it could help develop treatments or exercise plans to improve health, especially for people at risk of losing muscle mass. We conducted two studies to explore the reason behind these changes: In the first study, we used advanced techniques to see which proteins in muscles are activated by either aerobic (endurance) or resistance (strength) exercise. We found certain proteins that were specifically turned on by resistance exercise, which helps build muscle and strength. This discovery is important because it could help us find ways to prevent or treat muscle loss, especially as people get older. In the second study, we looked at which proteins in muscles increased or decreased with both types of exercise. We found that some proteins, especially those involved in energy production, are regulated similarly in both types of exercise. However, many other proteins respond differently to aerobic and resistance exercises. Together these studies add to our knowledge on how exercise helps muscle to become more fit and mighty.

Les statines sont des inhibiteurs de l’enzyme HMG-CoA réductase, une étape limitant la biosynthèse du cholestérol [1]. Elles sont efficaces dans la prévention primaire et secondaire des maladies cardiovasculaires [2-7]. L’utilisation de statines est associée à des effets néfastes au niveau du muscle squelettique d’environ 15% des patients, condition qui est nommée; myopathie induite par les statines (MIS) [8-15]. Les hypothèses concernant la MIS abondent mais il semble que les causes soient multifactorielles et que l’orchestration soit toujours mal comprise. Dans un premier temps, la structure générale et la compartimentalisation cellulaire seront abordées. Par la suite seront traités les acteurs et étapes du couplage excitation-contraction. Par après, les types de fibres et la plasticité phénotypique seront décrits. Puis, les mécanismes d’hypertrophie et d’atrophie seront passés en revue. Finalement, la dernière section portera sur l’utilisation des statines et la myopathie associée. Suite à cette introduction, les expériences à l’étude seront présentées sous forme d’article scientifique. Ce manuscrit en préparation élucide l’effet du traitement aux statines sur les ATP-ases calciques du réticulum sarcoplasmique. Il touche en effet les caractéristiques fonctionnelles et moléculaires de ces pompes en ce qui concerne les muscles rapides et les cellules musculaires en culture. Il contribue donc à une meilleure compréhension de la problématique multifactorielle que représente la MIS, s’insérant dans le portrait des connaissances en comblant un manque probant d’informations.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

BACKGROUND: Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is characterized by generalized skeletal muscle weakness, often from birth. To date, no therapy exists that enhances the contractile strength of muscles of NM patients. Mutations in NEB, encoding the giant protein nebulin, are the most common cause of NM. The pathophysiology of muscle weakness in NM patients with NEB mutations (NEB-NM) includes a lower calcium-sensitivity of force generation. We propose that the lower calcium-sensitivity of force generation in NEB-NM offers a therapeutic target. Levosimendan is a calcium sensitizer that is approved for use in humans and has been developed to target cardiac muscle fibers. It exerts its effect through binding to slow skeletal/cardiac troponin C. As slow skeletal/cardiac troponin C is also the dominant troponin C isoform in slow-twitch skeletal muscle fibers, we hypothesized that levosimendan improves slow-twitch muscle fiber strength at submaximal levels of activation in patients with NEB-NM. METHODS: To test whether levosimendan affects force production, permeabilized slow-twitch muscle fibers isolated from biopsies of NEB-NM patients and controls were exposed to levosimendan and the force response was measured. RESULTS: No effect of levosimendan on muscle fiber force in NEB-NM and control skeletal muscle fibers was found, both at a submaximal calcium level using incremental levosimendan concentrations, and at incremental calcium concentrations in the presence of levosimendan. In contrast, levosimendan did significantly increase the calcium-sensitivity of force in human single cardiomyocytes. Protein analysis confirmed that the slow skeletal/cardiac troponin C isoform was present in the skeletal muscle fibers tested. CONCLUSIONS: These findings indicate that levosimendan does not improve the contractility in human skeletal muscle fibers, and do not provide rationale for using levosimendan as a therapeutic to restore muscle weakness in NEB-NM patients. We stress the importance of searching for compounds that improve the calcium-sensitivity of force generation of slow-twitch muscle fibers. Such compounds provide an appealing approach to restore muscle force in patients with NEB-NM, and also in patients with other neuromuscular disorders.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The aim of the study was to investigate skeletal muscle changes induced by an acute bout of plyometric exercise before and after plyometric training. The study consisted of an acute study and training intervention study. The acute study, investigated whether direct evidence of ultrastructural damage and identification of indirect factors were more evident in subjects presenting with rhabdomyolysis. Moreover the training intervention study investigated whether plyometric training would protect the muscle from ultrastructural damage and rhabdomyolysis. During the acute intervention, twenty six healthy untrained individuals completed an acute bout of plyometric exercise (10 x 10 squat-jumps, 1 min rest). After, thirteen subjects continued with the training intervention. Eight of these subjects completed 8 weeks of plyometric jump training, while five subjects were instructed to rest from physical activity for 8 weeks. Seven days after the final training session the training and rest group repeated a second acute bout of plyometric exercise. Acute Study: Creatine kinase (CK) activity increased significantly following the single bout of plyometric exercise in all subjects (baseline: 129 to day 4: 5348 U/l). This was accompanied by an increase in perceived pain, C-reactive protein (CRP) a marker of inflammation as well as white blood cells (WBCs). Electron micrographs of muscle biopsies taken 3 days post exercise showed evidence of ultrasructural damage and membrane damage was apparent by immunofluorescence by the loss of dystrophin staining. A stretch of the c-terminus of titin was observed by immunogold, and western blot analysis indicated an increase in calpain-3 autolysis. Based on individual CK responses (CK range: 153-71,024 U/L at 4days after exercise) the twenty six subjects were divided into two groups, namely the high (n=10) and low responders (n=16). Training intervention: Following training the trained group did not experience: a rise of CK activity (110.0 U/l), perceived pain, CRP, WBCs, Z-line streaming, a stretch of titin or calpain-3 activation; while in the control group only two subjects presented with Z-line streaming. The results indicate that high responders have a more pronounced inflammatory response compared to low responders after eccentric exercise, therefore more WBCs and more specifically neutrophils are recruited to damaged areas resulting in greater membrane damage by respiratory burst in high responders. This damage can be limited with training by remodelling sarcomeric proteins via calpain activation resulting in the stable assembly of proteins in the sarcomere preventing the release of proteins. / AFRIKAANSE OPSOMMING: Die doel van die studie was om skeletspier veranderinge wat teweeggebring is deur voor en na afloop van akute pleometriese oefening, te ondersoek. Die studie bestaan uit ‘n akute intervensie en ‘n oefeningsintervensie gedeelte. Die akute intervensie het ondersoek ingestel na die direkte bewyse van ultrastrukturele skade en identifikasie van indirekte faktore meer sigbaar is in proefpersone wat met rhabdomiolose presenteer. Meerso het die oefningsintervensie die moontlikheid dat pleometriese oefening die spier van ultrastrukturele skade en rhabdomiolose beskerm, ondersoek. Tydens die akute intervensie is 26 gesonde ongeoefende individue die akute pleometriese oefeningsessie (10 x 10 hurkspronge, 1 min rus) voltooi. Hierna het 13 proefpersone voortgegaan met die oefeningsintervensie. Agt van hierdie proefpersone het agt weke pleometriese sprongsessie oefeninge voltooi, terwyl vyf proefpersone gevra is om vir 8 weke geen oefeninge te doen nie. Sewe dae na afloop van die finale oefeningssessie het die oefening en kontrole groep in ‘n tweede herhaalde akute pleometriese oefeningsessie deelgeneem. Akute intervensie: kreatienkinase (KK) aktiwiteit het betekenisvol verhoog na die enkel pleometriese oefeningsessie in all proefpersone (basislyn: 129 tot op dag vier: 5348 U/l). Hierdie is vergesel met ‘n toename in die persepsie van pyn, c-reaktiewe proteïen (CRP) ‘n merker van inflammasie sowel as witbloedselle (WBS). Elektronmikrograwe van spierbiopsies wat geneem is drie dae na afloop van die oefeninge, het tekens van ultrastrukturele skade en membraanskade getoon wat ook deur immunofluoresensie duidelik warneembaar was deur die verlies van distrofienverkleuring. ‘n Verrekking van die c-terminus van titin is ook waargeneem deur middel van immunogold. Westernblot analyse het ‘n toename in calpain-3 outolise getoon. Gegrond op individuele KK response (KK grense: 153-71,024 U/L na vier dae post oefening) is 26 proefpersone verdeel in twee groepe naamlik ‘n hoë (n=10) en lae responders (n=16). Oefeningintervensie:: Na oefening het die geoefende groep nie ‘n toename in KK aktiwiteit getoon nie (KK aktiwiteit (110.0 U/l)), pynervaring, CRP, WBS, Z-lynstroming, ‘n strekking van titin of calpain-3 aktivering; terwyl in die kontrole groep daar slegs twee proefpersone met Z-lynstroming geïdentifiseer is. Die resultate wyse daarop dat hoë responders ‘n meer uitgesproke inflammatoriese reaksie toon vergeleke met die lae responders na afloop van essentriese oefening. Daar word dus meer WBS en spesifiek meer neutrofiele na beskadigde areas gelokaliseer wat in grootter membraanskade deur respiratoriese inspanning in die hoë responders. Hierdie skade kan beperk word deur oefening waardeur hermodulering van sarkomeriese proteïene via calpain aktivering tot stabiele rangskiking van proteïene in die sarcomere lei en daardeur proteïen vrystelling verhinder. / The NRF for financial assistance

No description available.

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

Eccentric overload in training settings utilizes loads higher than concentric one repetition maximum (1RM). There is no clear definition of eccentric “failure” or 1RM using conventional weights, so eccentric 1RM is estimated to be between 145-190% concentric 1RM. Historically, the highest intensity used for eccentric overload is typically 120% of concentric 1RM despite little research using conventional weights with higher eccentric intensities. The purpose of this study was to conduct an exploratory neuro-mechanical analysis of different intensities of elbow flexors eccentric overload using free weights by examining angular kinematics during contraction. Twenty male participants with weight training experience had unilateral concentration curl isometric peak torque assessed on a Humac Norm Dynamometer and concentric 1RM assessed with dumbbells while biceps brachii electromyography (EMG) and elbow joint angle were recorded. Angles were recorded using a custom made electrogoniometer and elbow joint torque was estimated using inverse dynamics. Participants were randomly assigned in counter balanced order to perform eccentric actions at 120%, 140%, 150%, 160% and 170% concentric 1RM with 4 minutes rest between. Variables included peak torque, angular velocity at peak torque, impulse, power, mean EMG, and EMG normalized to peak. Data were analyzed using repeated measures ANOVA or a Friedman test. Angular velocity at peak torque was significantly lower for 120% (65.3 ± 40.8°/s) compared to all other conditions (range: 65.3 ± 40.8 to 162.1 ± 75.2°/s; p<0.01). Peak torque for all conditions (range: 98.2 ± 16.2 to 108.2 ± 21.6 Nm) was significantly higher than isometric peak torque (77.4 ± 16.8Nm; p<0.05). Peak torque at 160% (108.2 ± 21.6Nm) was significantly higher than at 120% (98.2 ± 16.2Nm; p<0.05). Power for 140-170% (range: 166.2 ± 85.7W to 265.8 ± 111.3W) was significantly higher than power at 120% (79.9 ± 66.8W; p<0.05). Impulse was highest at 120% (56.1 ± 54.6Nms) compared to all other conditions (range: 56.2 ± 54.6 to 9.6 ± 3.8Nms; p≤0.05). Impulse at 140% (20.6 ± 11.8Nms) was significantly higher than 170% (9.6 ± 3.8Nms; p<0.05). Isometric mean EMG (0.792 ± 0.285 mV) was significantly higher than all eccentric conditions (range: 0.654 ± 0.313 to 0.533 ± 0.259mV; p<0.05) with no difference between eccentric conditions for mean EMG or EMG normalized to peak. It was concluded that compared to 120%, eccentric overload with intensity ranging from 140-170% concentric 1RM involves minimal increases in peak torque and no change in EMG activation. Intensities above 120% enhance power and decrease impulse. This research has implications on future training prescription of eccentric exercise.

The aim of this Thesis was to further characterise and utilise an in vitro skeletal muscle (SkM) model, to investigate its potential use in further understanding the cellular and molecular adaptations to exercise in vivo. Candidate genes and proteins have been identified using in vivo, ex vivo and targeted in vitro experiments, however the complete picture of these molecular mechanisms are far from understood. Furthermore, the extent to which mechanical signals contribute to the intra-cellular mechanisms associated with exercise is also underinvesitgated. To this end, developing an in vitro model of SkM that can recapitulate in vivo SkM and respond to mechanical stimulation in a similar way to exercise will provide a means to begin to delineate the complex cellular and molecular regulation of SkM. The initial investigation (Chapter 3) characterised an optimal seeding density and culture period of C2C12 myoblasts within a 3 ml collagen gel. These data provided support for the use of collagen constructs seeded at 4 x 106 cells/ml, with no statistical differences observed in peak force, rate of force development and relative force compared to other seeding densities examined (table 3-2, all p > 0.05). However the use of 4 x 106 cells/ml supports previous data in a larger construct volume model, whilst the highest cell density possible in the system increases cell-cell contact required for fusion. Immunohistochemical and gene expression analyses provided evidence for the fusion of single seeded myoblasts into multinucleate myotubes, demonstrating an in vivo-like architecture. Chapter 4 presented data towards the characterisation and use of two distinct cyclical stretch regimens with respect to the acute biochemical and transcriptional responses. Data revealed increases in peak media lactate and reductions in peak media glucose, following cyclical stetch compared to control (p = 0.000 and p = 0.001 respectively, Fig. 4-2). Changes in mtDNA (Fig. 4-5) and associated mRNA transcriptional signals (Fig. 4-7) were mode dependent.