Morphological changes, genetic modifications, and cell functional alterations are not always parallel. Therefore, assessment of skeletal muscle function is an integral part of the etiological approach. The general objective of this thesis was to look into the cellular and molecular events occurring in skeletal muscle contraction in healthy and diseased condition, using a single fiber preparation and a single fiber in vitro motility assay, in an attempt to approach the underlying mechanisms from different physiological angles. In a body size related muscle contractility study, scaling of actin filament sliding speed and its temperature sensitivity has been investigated in mammals covering a 5,500-fold difference in body mass. A profound temperature dependence of actin filament sliding speed over myosin head was demonstrated irrespective of MyHC isoform expression and species. However, the expected body size related scaling within orthologus myosin isoforms between species failed to be maintained at any temperature over 5,500-fold range in body mass, with the larger species frequently having faster in vitro motility speeds than the smaller species. This suggest that apart from the MyHC iso-form expression, other factors such as thin filament proteins and myofilament lattice spacing, may contribute to the scaling related regulation of skeletal muscle contractility. A study of a novel R133W β-tropomyosin mutation on regulation of skeletal muscle contraction in the skinned single fiber prepration and single fiber in vitro motility assay suggested that the mutation induced alteration in myosin-actin kinetics causing a reduced number of myosin molecules in the strong actin binding state, resulting in overall muscle weakness in the absence of muscle wasting. A study on a type IIa MyHC isoform missense mutation at the motor protein level demonstrated a significant negative effect on the function of the IIa MyHC isoform while other myosin isoforms had normal function. This provides evidence that the pathogenesis of the MyHC IIa E706K myopathy involves defective function of the mutated myosin as well as alterations in the structural integrity of all muscle irrespective of MyHC isoform expression.

A Disfunção temporomandibular (DTM) é um termo coletivo que abrange um largo espectro de problemas clínicos da articulação e dos músculos na área orofacial; estas disfunções são caracterizadas principalmente por dor, sons na articulação, e função irregular ou limitada da mandíbula. Os músculos da mastigação podem estar envolvidos na DTM de origem muscular, e por definição são os músculos que promovem o toque dental, portanto os elevadores da mandíbula: masseter, temporal, pterigóideos medial e lateral. A origem muscular da DTM é a mais prevalente, sendo portanto,o entendimento funcional e estrutural da composição dos músculos da mastigação essencial para a compreensão desta DTM. Este estudo tem como objetivo analisar a estrutura dos músculos da mastigação quanto a variação do calibre das fibras lentas e rápidas, densidade capilar e da expressão da miosina neonatal com a variação da idade. Foram estudadas 37 amostras dos músculos temporal e masseter (20 amostras do sexo masculino e 17 do sexo feminino) de autópsias do Serviço de Verificação de Óbitos de São Paulo com intervalo pós-mortem de até 18 horas, de ambos os gêneros e com idades divididas por décadas (1a a 9a décadas). Foram realizadas reações imunoistoquímicas com os anticorpos Ulex europaeus biotinilada aglutinina, anti-miosina neonatal, anti-miosina rápida, anti-miosina lenta para análise da expressão das proteínas. A avaliação foi feita por dois observadores, após calibração intra observador, (duas contagens no mesmo campo pelo mesmo observador em tempos diferentes) e inter observador (contagem do mesmo campo por dois observadores), até se atingir uma margem de erro menor que 10%. Em relação ao número de capilares/fibra, nos músculos masseter e temporal, da primeira a nona década, em média respectivamente foi de 1 e 0,7 respectivamente. O número de capilares por mm², nos músculos masseter e temporal, não variou ao longo das nove décadas estudadas, e o número de capilares por mm², foi significantemente maior no músculo masseter quando comparado ao temporal, (p=0,025). A miosina neonatal manteve-se presente embora com decréscimo em todas as décadas dos músculos masseter e temporal. Observou-se o diâmetro das fibras do tipo II menores que as fibras do tipo I / Temporomandibular disfunction (TMD) is a colletive term that refers to different clinical problems of the temporomandibular joint and the jaw muscles. These disfunctions are characterizied meanly by pain, joint sounds and irregular or limited mandibular function. The jaw muscles can be involved in the TMD of muscle etiology, and by definition they are the ones which provide the teeth touch, then the jaw elevators: masseter, temporalis, medial pterygoid and lateral pterygoid. As the muscular etiology is the most prevalent cause of TMD, the detailed understanding of structural and functional composition of the masticatory muscles is paramount to better comprehend TMD due to muscle disorder. This study has the aim to analyze the jaw muscle structure concerning capillarie density, neonatal myosin expression, and the cross sectional area of the fast and slow fibers in temporalis and masseter muscles in autopsy samples from 1st to 9th decades of age. Thirty seven temporalis and masseter muscles samples were studied (20 from male and 17 from female) from Serviço de Verificação de Óbitos of São Paulo. The specimens were divided by gender and ages. The samples were collected up to 18 hours post-mortem. Imunohistochemistry stainning were made with antibodies to analize the protein expression. The evaluations were made by two observers, after intra observer calibration (two evaluations on the same field by the same observer in different times) and inter observer (evaluation of the same field by two observers), unti getting less than 10% of error. The number of capillaries per fiber in the masseter and temporalis muscle was in average 1 and 0,7 respectively. The number of capillaries per mm² was significantly higher in the masseter when compared to temporalis muscle (p=0.025). The neonatal myosin was present in all decades in both muscles, and it was observed that the cross sectional area of the type II fibers was smaller than the type I fibers

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23060号 / 医博第4687号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 湊谷 謙司, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Das Ziel dieser Studie war es, die Beteiligung der nichtmuskulären schweren Myosinketten an der Kontraktion der glatten Muskeln unter physiologischen Bedingungen zu untersuchen. Als Versuchsmodell wurde die Harnblase von neugeborenen Wildtyp und transgenen Mäusen verwendet, bei denen das Gen für die glattmuskelspezifischen schweren Myosinketten durch "Gene Targeting" funktionell eliminiert wurde (Knock-Out). Das Fehlen der Expression der glattmuskelspezifischen schweren Myosinketten wurde durch Elektrophorese und Immunfärbung bestätigt. Im Gegensatz dazu blieb die Expression der nichtmuskulären schweren Myosinketten unverändert. Die mechanische Analyse des glatten Muskels wurde mit intakten Muskelpräparaten aus der Harnblase durchgeführt. Das Muskelpräparat wurde in KCl-Lösung oder mit Phorbolester stimuliert. Die Aktivierung mittels depolarisierender KCl-Lösung führte bei neugeborenen Wildtyp Mäusen zuerst zu einer transienten Kontraktion (Phase 1) mit hoher Kraftentwicklung und maximaler Verkürzungsgeschwindigkeit, und danach zu einer tonischen Kontraktion (Phase 2) mit niedrigerer Kraftentwicklung und maximaler Verkürzungsgeschwindigkeit. Blasenpräparate neugeborener Knock-Out Mäuse dagegen zeigten keine Phase 1, sondern nur eine tonische Kontraktion, die mit Wildtyp Mäusen vergleichbar war. Daher scheint nichtmuskuläres Myosin an der tonischen Kontraktion des glatten Muskels beteiligt zu sein. Durch Stimulierung mit Phorbolester waren ähnliche tonische Muskelkontraktionen der Blasenpräparate sowohl bei Wildtyp als auch bei Knock-Out Mäusen zu beobachten. Vermutlich wird also das nichtmuskuläre Myosin durch Stimulierung mit Phorbolester aktiviert. Intrazelluläre Filamente wurden durch Immunfluoreszenz mit einem spezifischen Antikörper gegen nichtmuskuläre schwere Myosinketten in kultivierten primären glatten Muskelzellen untersucht. Dabei zeigten die Muskelzellen sowohl von Wildtyp als auch von Knock-Out Mäusen intrazelluläre dicke Myosinfilamente, was für die Beteiligung des nichtmuskulären Myosins an der glatten Muskelkontraktion spricht. Entsprechend wurden intrazelluläre Filamente mit einem Antikörper gegen glattmuskelspezifische schwere Myosinketten in kultivierten primären glatten Muskelzellen untersucht. Wie erwartet, konnten nur in glatten Muskelzellen von Wildtyp Mäusen intrazelluläre Filamente nachgewiesen werden, nicht aber in denen von Knock-Out Mäusen. In dieser Arbeit konnte zum ersten Mal gezeigt werden, dass nichtmuskuläres Myosin zumindest an der tonischen Kontraktion glatter Muskelzellen beteiligt sein kann. / The aim of the present study was to investigate the involvement of non-muscle myosin heavy chain in smooth muscle contraction under physiological conditions. As an experimental model urinary bladder from neonatal wild-type mice as well as from neonatal mice with disrupted smooth muscle myosin heavy chain expression was used. This animal model was established through gene targeting technology, resulting in complete elimination of the expression of smooth muscle myosin heavy chains. The lack of expression of smooth muscle myosin heavy chains was confirmed by electrophoresis and immunoblotting. On the other hand, non-muscle myosin heavy chain expression remained normal, as verified by Western blot analysis. The mechanical analysis of smooth muscle was performed with intact urinary bladder preparations, stimulated using prolonged KCl depolarization or with phorbol ester. Prolonged activation by KCl depolarization of intact bladder preparations from wild-type neonatal mice produced an initial transient state (phase 1) of high force generation and maximal shortening velocity, followed by a sustained state (phase 2) with lower force generation and maximal shortening velocity. In contrast, bladder preparations from homozygous knockout neonatal mice did not exhibit phase 1, but phase 2 could be observed, i.e. a similar isometric force and maximal shortening velocity, compared to wild-type phase 2. Thus, non-muscle myosin appears to be recruited in the sustained phase of smooth muscle contraction during prolonged KCl depolarization in the animal model used. Upon stimulation with phorbol ester a similar sustained contraction was observed in both wild-type and knockout smooth muscle preparations. Therefore, non-muscle myosin may also be recruited during phorbol ester stimulation in both wild-type and knockout muscle preparations. The participation of non-muscle myosin in smooth muscle contraction was further supported by the finding of longitudinally arranged intracellular filaments in cultivated smooth muscle cells from both wild-type and knockout mice by immunofluorescence microscopy, using a specific antibody raised against non-muscle myosin heavy chain. In a similar way, smooth muscle myosin heavy chain structures were investigated in cultivated smooth muscle cells. As expected, longitudinally arranged intracellular filamentous structures of smooth muscle myosin were observed in wild-type smooth muscle cells, but not in smooth muscle cells from knockout mice. In conclusion, in neonatal smooth muscle the initial phase of contraction elicited by KCl depolarization is generated by smooth muscle myosin heavy chain recruitment. Upon prolonged KCl depolarization non-muscle myosin is recruited in the sustained phase of contraction, as well as upon stimulation with phorbol ester. Thus, it was possible, for the first time, to verify the involvement of the non-muscle myosin in smooth muscle contraction in vivo. The results of the present study contribute to the understanding of the regulatory mechanisms of smooth muscle contraction.

La cytokine TRAIL est un candidat anticancéreux qui induit la mort spécifique de cellules tumorales. La liaison de TRAIL à ses récepteurs (DR) permet de former le complexe DISC qui induit la mort cellulaire. La raison de la mort sélective des cellules tumorales induite par TRAIL est inconnue. Nous avons découvert des partenaires de DR: chaînes lourdes de myosine IIa, IIb (NMHCIIa, NMHCIIb), chaîne légère régulatrice de myosine (MLC2) et ß-actine. Dans les cellules tumorales, la liaison de TRAIL abroge l'interaction NMHCII/DR, et DISC est activé. Au contraire, dans les cellules normales, l'interaction NMHCII/DR persiste et l'activation de DISC est incomplète. Affaiblir l'interaction NMHCII/DR par des inhibiteurs chimiques ou diminuer NMHCIIa permet d'augmenter l'apoptose liée à TRAIL. L'interaction réduite NMHCII/DR induit des niveaux altérés de phospho-MLC2 et de kinases régulant MLC2. Nous proposons que la résistance de cellules normales à TRAIL soit basée sur l'interaction DR/cytosquelette, déficiente dans des tumeurs. NMHCII étant aussi impliqué dans l'adhésion/migration cellulaire, il serait intéressant d'étudier les fonctions de NMHCII/DISC dans le détachement cellulaire, afin de mieux comprendre la résistance à TRAIL de certains cancers. / The cytokine TRAIL is a promising cancer therapeutic candidate as it induces apoptosis selectively in transformed cells. TRAIL-induced clustering of its receptors (DR) is essential for the DISC complex formation, which induces cell death. The mechanism for TRAIL’s tumour selective effect is largely unknown. We identified the cytoskeleton proteins non-muscle myosin heavy chain IIa, IIb (NMHCIIa, NMHCIIb), myosin regulatory light chain (MLC2) and ß-actin as novel DR-interactors. An initially weak and TRAIL-induced abrogation of NMHCII/DR interaction correlated with efficient DISC formation in tumour cells. In contrast, a robust NMHCII/DR interaction that was sustained upon TRAIL stimulus was accompanied by incomplete DISC arrangement. Weakening the NMHCII/DR interaction in normal cells using chemical inhibitors enhanced TRAIL-induced apoptosis. Intriguingly, siRNA-mediated NMHCIIa- but not NMHCIIb depletion potently released TRAIL resistance in normal cells and influenced DISC composition. Reduced NMHCII/DR interaction in transformed cells was characterised by diminished MLC2 phosphorylation and altered protein expression of upstream regulatory kinases. Our results suggest that normal cell resistance to TRAIL-apoptosis is based on the interaction of cytoskeleton components with DR that is impaired upon transformation. Since NMHCII function in cell adhesion and migration, it will be interesting to study possible roles of the interaction in cell detachment and altered TRAIL sensitivity; moreover this link may provide clues as to the cause of TRAIL resistance in some cancers.

Skeletal muscle dysfunction in chronic obstructive pulmonary disease (COPD) is a condition in which peripheral skeletal muscle undergoes myopathic changes which impair muscle function, limit physical performance, and can lead to significant disability. While the etiology of the dysfunction is unknown, this study was conducted to test the hypothesis that chronic hypoxemia leads to alterations in oxygen transport and muscle function. A primary objective was to validate elastase-induced emphysema in rats as an animal model of skeletal muscle dysfunction in COPD.Arterial blood gases were used to determine the severity of hypoxemia and sodium dodecyl sulfate- polyacrylamide gel electrophoresis was used to determine the proportions of myosin heavy chain isoforms I, IIa, IIx, and IIb. Measures of microvascular oxygenation and blood flow in the spinotrapezius muscle allowed for determination of both convective and diffusive oxygen supply to the muscle, as well as calculation of muscle oxygen consumption at rest and during electrically stimulated three-minute muscle contractions. Muscle performance measures included peak force, force-time integral, and fatigue index. Due to a presumed rat respiratory virus, which likely resulted in the control group being nearly as hypoxemic as the elastase-induced emphysema group, this study was not able to definitively test the hypothesis that chronic hypoxemia leads to both a diminished supply and demand of oxygen in skeletal muscle. Although many of the results of the present study were not statistically significant, they exhibited consistent trends over time and are likely of physiological significance. All measures of muscle performance were lower in the emphysema group. In addition, spinotrapezius muscle oxygen consumption and blood flow were lower in the emphysema group. The addition of supplemental oxygen during isolated, small-muscle mass exercise did increase the force-time integral by ~18% in both groups, suggesting that muscle work in these hypoxemic animals may be limited by oxygen supply. Thus, the data on muscle fiber type, oxygen consumption and muscle performance suggest that elastase-induced emphysema in rats leads to a similar skeletal muscle dysfunction that is observed in humans with COPD, and indicates that it is a valid animal model of skeletal muscle dysfunction in COPD.