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.

Endothelial cells (ECs) and smooth muscle cell (SMC) play a key part during development of fibrosis in the intima being partly responsible for synthesis of matrix metalloproteinase (MMPs) and various regulators and substrates of these enzymes. Omega-3 (n-3) polyunsaturated fatty acids (PUFA) consumption, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has beneficial effects on atherosclerosis but its effect on the development of fibrosis is relatively unknown. <i>Objective:</i> Determine the effects of EPA and DHA, alone or in combination, on fibrosis-related factors in aortic SMCs (AoSMCs) and human umbilical vein ECs (HUVECs) and human aortic ECs (HAECs). <i>Results:</i> Treatment of cells with/without 10 μM DHA, EPA, oleic acid (OA) or vehicle control (VC) altered expression of MMPs, regulators and substrates of MMPs and inflammatory cytokines. EPA increased the α-actin:β-actin ratio indicative of a more contractile SMC phenotype and gelatinase (MMP-2 and -9) activity in HUVECs. In aortic cells, EPA and DHA decreased uPAR mRNA and protein expressions. DHA, EPA and DHA: EPA (at 3:1 and 1:1) decreased SMC migration, this did not involve uPA/plasmin activity. <i>Conclusion:</i> EPA and DHA could decrease inflammatory cytokines and the fibrogenic environment in atherosclerotic lesions by decreasing MMP expression and activity. These fatty acids may also reduce SMC migration and proliferation, independently of uPA/plasmin activity, potentially reducing SMC build up in the intima. This could possibly prevent and/or show plaque progression and increase the stability of advanced plaques.

Estudos têm mostrado que o treinamento de força de baixa intensidade com oclusão vascular (TFOV) tem apresentado resultados similares nos ganhos de força e hipertrofia comparado ao treinamento de força (TF) de alta intensidade. O objetivo deste estudo foi comparar os efeitos de três diferentes programas de TF nos ganhos de força e hipertrofia musculares e na expressão da miostatina (MSTN) e seus antagonistas. Para isso, vinte e nove jovens do sexo masculino, sem experiência em TF, foram recrutados e divididos randomicamente nos grupos: treinamento de força de baixa intensidade sem oclusão (BI), treinamento de força de baixa intensidade com oclusão (BIO) e treinamento de força de alta intensidade sem oclusão (AI). Os grupos BIO e BI treinaram com intensidade de 20% 1RM, enquanto o grupo AI treinou com intensidade de 80% 1RM. A ANOVA one way foi utilizada para testar as diferenças percentuais nos ganhos de força (1RM) e na área de secção transversa (AST) do músculo quadríceps femoral. O modelo misto para análise das medidas repetidas foi utilizado para testar as diferenças nas variáveis miostatina (MSTN), folistatina-3 (FLST-3), SMAD-7 e GASP-1 nos grupos BI, BIO e AI nas condições pré e pós-treinamento. Os resultados mostraram que os aumentos de força e hipertrofia musculares nos grupos BIO e AI foram similares, entretanto superiores ao grupo BI. Esses resultados podem ser atribuídos a maior diminuição na expressão da MSTN nos grupos BIO (45%) e AI (41%) comparados com o grupo BI (16%) e o aumento na expressão dos genes que antagonizam sua atividade (SMAD-7, FLST-3 e GASP-1). Podemos concluir que a inibição na atividade da MSTN dos grupos BIO e AI podem responder em parte a similaridade nos ganhos de força e hipertrofia entre os grupos e a diferença para o grupo BI / It has been demonstrated that low intensity training associated to vascular occlusion (LIO) promotes similar gains in strength and muscle mass when compared to high intensity strength training (HI). The aim of the present study was to evaluate the effect of three different training programs on skeletal muscle hypertrophy and atrophy related gene expression. Twenty nine young male, with no previous experience in strength training were randomly allocated in three groups: low intensity strength training (i.e. 20% - 1-RM) (LI); low intensity strength training associated to vascular occlusion (i.e. 20% - 1-RM) (LIO); high intensity strength training (HI) (i.e. 80% - 1-RM). One-way ANOVA was used to assess differences in % delta change values of 1-RM and cross sectional area (CSA) of the quadriceps femoris. Mixed model analysis was used to compare myostatin (MSTN), folistatyn-3 (FLST-3), SMAD-7 e GASP-1 changes between groups pre and post training. Results demonstrated similar increases in strength and muscle hypertrophy for LIO and HI groups. Moreover, such increases were significantly greater when compared to LI. These results may be, at least in part, explained by a significant decrease in MSTN mRNA expression in LIO (45%) and HI (41%) when compared to LI (16%); additionally, SMAD-7; FLST-3 and GASP-1 mRNA expression were significantly increased. In conclusion, LIO training promotes similar gains than HI training. The results may be explained by changes in MSTN and related genes mRNA expression

O dano muscular induzido pelo exercício (DMIE) e os diferentes elementos envolvidos em seu processo têm sido amplamente estudados. A doença periodontal (DP), por sua vez, tem sido indicada como um possível fator de risco para várias condições sistêmicas, como diabetes, doenças cardiovasculares, partos prematuros, obesidade, entre outros. Tais associações têm sido atribuídas à possibilidade de que a DP possa induzir um processo de inflamação sistêmica de baixa intensidade, caracterizado pela elevação de biomarcadores sanguíneos que também estão envolvidos no mecanismo de dano muscular induzido pelo exercício (DMIE). O objetivo do presente estudo é investigar se a doença periodontal pode atuar como um modificador do DMIE em homens saudáveis. Foram avaliados 40 indivíduos, com idade entre 25 e 45 anos, que buscaram atendimento na faculdade de odontologia da UFRGS ou eram praticantes de atividades físicas. Questionário estruturado para obtenção de dados demográficos e comportamentais e o IPAQ (International Physical Activity Questionnaire) foram aplicados. Dois periodontistas examinaram perda de inserção (PI), profundidade de sondagem, sangramento à sondagem e índices de placa e sangramento gengival no exame basal, juntamente com avaliações antropométricas. Os participantes então realizaram um protocolo de indução de dano muscular que incluiu cinco séries de 15 contrações excêntricas máximas dos quadríceps de uma perna, em um dinamômetro isocinético. Força muscular (contrações isométricas voluntárias máximas - CIVM), espessura e ecogenicidade muscular (ultrassonografia) e dor (escala visual analógica) foram avaliadas em diferentes momentos em relação ao protocolo. Modelos de regressão logística multivariados foram ajustados para idade, educação, índice de massa corporal, fumo, consumo de álcool, proteína C reativa e nível de atividade física. Nessa amostra, PI esteve associada a maiores reduções de força muscular após o protocolo, com um aumento de 1 mm na média de PI representando 7% a mais de redução na CIVM. Pode-se concluir, a partir dessa análise preliminar, que a doença periodontal pode ser considerada um modificador do processo de dano muscular, aumentando a deterioração da força muscular. / Exercise-induced muscle damage (EIMD) and the different elements involved in it´s process has been largely studied. Periodontal diseases (PD) has been pointed out as a possible risk fator for a number of systemic conditions, like diabetes, cardiovascular diseases, preterm birth, obesity, and others. Such associations has been atribbuted to the fact that PD can lead to a low-grade inflammatory process, characterized by elevated blood concentrations of biomarkers that are also involved in the EIMD mechanisms. The aim of this study is to assess whether PD can act as a EIMD modifier in healthy men. This study included 40 healthy males with 25-45 yrs that seek for treatment at Dentistry Faculty or are physical activiy practitioners. A structured questionnaire to obtain demographic and comportamental data and the IPAQ (International Physical Activity Questionnaire) were applied. Two periodontists assessed attachment loss (AL), probing depth (PD), bleeding on probing (BOP), plaque and bleeding index in the baseline exam, together with anthropometrical evaluation. The participants then performed a muscle damage protocol comprising five sets of 15 maximum eccentric contractions of the quadriceps muscles of one leg in a isokinetic dynamometer. Evaluations of muscle strength (maximal voluntary isometric contraction), muscle thickness and echo intensity (ultrasonography images) and soreness (visual analogue scale) were made at different periods in relation to the protocol. Multivariable logistic models were fitted adjusting for age, education, body mass index (BMI), smoking, alcohol consumption, C-reactive protein (CRP) and physical activiy level. In this sample, AL was associated with higher reductions of muscle force, with a 1- mm increment in AL mean significantly decreased CIVM by 7%. It can be concuded, based on this preliminar analysis, that PD may be considered as a modifier of EIMD, increasing muscle strenght deterioration.