Feeding high levels of distillers grains increases polyunsaturated fatty acid (PUFA) levels in beef. It is well stablished that beef with higher concentrations of PUFA is more likely to have increased lipid and myoglobin oxidation. This is important because lipid and myoglobin oxidation lead to off-flavor development and discoloration of retail-displayed beef, reducing display life. In the first study, the effects of feeding different dietary fat sources with modified distillers grains plus solubles (MDGS) on beef display life were evaluated. Results suggest that feeding MDGS to cattle reduces color and lipid stability in addition to increasing C18:2 and PUFA content of beef in comparison to the corn diet. Thus, feeding MDGS to cattle has the potential to reduce beef display life. Perhaps, MDGS in feedlot diets increases PUFA concentration in the sarcoplasmic reticulum (SR) membrane, thereby altering membrane integrity, resulting in more rapid calcium leakage and improved tenderness. Therefore, the second study was dedicated to evaluate the effects of dietary fat source on the basic mechanism of beef tenderization. Feeding MDGS to cattle increased 18:2 and tended to increase PUFA concentration in the SR membrane, in addition to increase free calcium at d 2 postmortem in comparison to the corn diet. Beef from cattle finished on de-oiled MDGS and de-oiled MDGS plus oil had lower Warner-Bratzler shear force values than beef from cattle finished on corn at 2 d postmortem. No differences among dietary treatments were found for sarcomere length and troponin T degradation at 2 d postmortem. The results from this study suggest that feeding MDGS may increase tenderness, possibly by increasing free calcium in muscle early post-mortem. However, the true mechanism by which dietary fat source may accelerate the beef tenderization process is still unclear and should be further explored. / De acordo com a literatura, a adição de resíduo de destilaria com solúveis em dietas de terminação de bovinos de corte aumenta a quantidade de ácidos graxos poliinsaturados (PUFA) na carne bovina. É sabido que quanto maior a concentração de PUFA na carne, maior será a probabilidade de ocorrência da oxidação lipídica e da mioglobina. Isso é importante porque o aumento da oxidação lipídica e da mioglobina acelera o desenvolvimento de odores indesejáveis e da descoloração da carne, reduzindo assim sua vida útil. O objetivo do primeiro estudo foi avaliar os efeitos do uso de diferentes resíduos de destilaria parcialmente desidratados com solúveis (MDGS) na vida útil da carne. Os resultados deste estudo sugerem que a inclusão de 40% de MDGS na dieta de terminação de bovinos pode reduzir a estabilidade lipídica e da cor, além de aumentar a quantidade de ácido linoléico (18:2) e PUFA da carne em comparação à carne de animais terminados com milho, reduzindo a vida útil da carne. No segundo estudo, nós trabalhamos com a hipótese de que a adição de elevadas quantidades de MDGS em dietas de terminação poderia aumentar a concentração de PUFA na membrana do retículo sarcoplasmático, e assim, alterar a integridade e permeabilidade dessa membrana, antecipando a liberação de cálcio postmortem. Com mais cálcio disponível, a proteólise muscular seria favorecida, aumentando a maciez da carne nos primeiros dias após o abate. Portanto, o objetivo do segundo estudo foi avaliar os efeitos da fonte de gordura dietética contida em diferentes tipos de MDGS na maciez da carne bovina. A inclusão de 40% de MDGS na dieta de terminação de novilhos aumentou a concentração de 18:2 e tendeu a aumentar a concentração de PUFA na membrana do retículo sarcoplasmático em comparação ao gado terminado com milho (sem inclusão de MDGS). Carne de animais terminados com MDGS apresentou maior quantidade de cálcio livre no sarcoplasma 48 horas após o abate. Carne de novilhos alimentados com MDGS desengordurado apresentou menor força de cisalhamento 48 horas após o abate quando comparada à carne de animais terminados com milho. Não houve diferença significativa entre nenhum dos tratamentos dietéticos 48 horas após o abate para comprimento de sarcômero e degradação de troponina T. Os resultados deste estudo sugerem que a inclusão de 40% de MDGS desengordurado na dieta de terminação de novilhos aumenta a maciez da carne 48 horas após o abate, possivelmente por aumentar a quantidade de cálcio livre no sarcoplasma. No entanto, o mecanismo pelo qual a fonte de gordura dietética acelerou a proteólise muscular ainda não está completamente elucidado.

In this dissertation, I explored the effects of developmental hypoxia on heart contractility in three separate species of ectotherms: the common snapping turtle (Chelydra serpentina), the American alligator (Alligator mississippiensis), and the zebrafish (Danio rerio). I began with the common snapping turtle and tested whether the utilization of the sarcoplasmic reticulum was altered in response to developmental hypoxia. In the next two chapters, developmental hypoxia of the American alligator was explored studying how the cardiac tissue was affected, specifically in physiological stressors, sarcoplasmic reticulum utilization and sensitivity to pharmacological increases in contractility. The last chapter explored how zebrafish heart contractility was altered in response to chronic hypoxia from egg to adult. Findings from these chapters suggest that while developmental hypoxia did alter cardiac contractility, it did not alter the response of the heart to physiological stressors such as increased heart rate or under hypoxia. Overall, these findings contribute to increasing the current understanding of how developmental hypoxia alters the cardiovascular system but with an emphasis on the cardiac tissue level.

La contraction du muscle squelettique est initiée par une libération de Ca2+ du réticulum sarcoplasmique (RS) en réponse à une dépolarisation du sarcolemme. Celle-ci induit un changement de conformation du récepteur des dihydropyridines (DHPR) localisé dans les tubules T entraînant l'ouverture du récepteur de la ryanodine de type 1 (RyR1), canal calcique du RS, et la libération du Ca2+ accumulé dans le RS. Au repos, RyR1 serait maintenu fermé par une action répressive du DHPR. Néanmoins, un efflux de Ca2+ continu se développe à travers la membrane du RS, constamment compensé par l'activité des pompes Ca2+-ATPases. Des études suggèrent que cet efflux pourrait être impliqué dans la perturbation de l'homéostasie calcique dans une des pathologies musculaires des plus fréquentes et sévères, la myopathie de Duchenne. Le travail présenté vise à caractériser l'efflux de Ca2+ du RS dans les fibres musculaires squelettiques de souris normales et mdx, modèle murin de la myopathie de Duchenne, en couplant la technique de potentiel imposé et la mesure fluorimétrique du Ca2+ intracellulaire. La mise au point d'une mesure directe des variations de Ca2+ du RS à l'aide du Fluo-5N a permis de révéler dans les fibres mdx une fuite calcique du RS exacerbée. Cette approche a permis de démontrer que l'efflux calcique du RS dans la fibre musculaire squelettique au repos n'est pas un phénomène incontrôlé à travers RyR1 mais un efflux étroitement contrôlé par le DHPR. Enfin, on s'est intéressée à l'efflux de Ca2+ du RS lors d'une stimulation musculaire prolongée. Nos résultats montrent que le déclin du signal calcique cytosolique dans ces conditions résulterait de la déplétion calcique du RS / Contraction of skeletal muscle is triggered by the release of Ca2+ from the sarcoplasmic reticulum (SR) in response to depolarization of the sarcolemma. Depolarization elicits a conformational change of the dihydropyridine receptor (DHPR) localized in the tubular membrane that controls the opening of the type 1 ryanodine receptor (RyR1), the SR Ca2+ release channel. At rest, RyR1s are kept in a closed state imposed by the repressive action of DHPRs. Yet, a resting Ca2+ efflux occurs across the SR membrane, constantly balanced by the pumping activity of SR Ca2+-ATPases. Several studies suggest that this SR Ca2+ efflux, considered as purely passive, may contribute to the alteration of Ca2+ homeostasis in one of the most common and severe skeletal muscle disease, namely the Duchenne Muscular Dystrophy. The present work aims at characterizing the SR Ca2+ efflux in skeletal muscle fiber from normal and mdx mice, the murine model of Duchenne Muscular Dystrophy, by combining voltage-clamp and intracellular Ca2+ measurements. The development of a methodology allowing direct monitoring of Ca2+ changes in the SR using the Fluo-5N led us to reveal an elevated SR Ca2+ leak in mdx fibers, which may contribute to the alteration of Ca2+ homeostasis. Still using this approach, we demonstrate that the resting SR Ca2+ efflux in normal skeletal muscle fiber is not, an uncontrolled process through RyR1 but is tightly controlled by DHPR. Finally, we investigates the SR Ca2+ efflux during long-lasting stimulation. Our data indicate that the decline of SR Ca2+ release in these conditions results from SR Ca2+depletion and does not involve voltage-dependent inactivation of SR Ca2+ release

Made available in DSpace on 2016-06-02T19:22:10Z (GMT). No. of bitstreams: 1 5716.pdf: 1902370 bytes, checksum: 9a5b25a55be97bb37553eeb1f8130b09 (MD5) Previous issue date: 2014-01-28 / Financiadora de Estudos e Projetos / Phenomena as feeding and thermoregulation can challenge both the cardiac output and heart rate (fH). Postprandial changes in many physiological patterns are typically recorded for ectotherms, although predominantly studied in snakes. Furthermore, thermoregulation is a critical process to ensure different survival and reproduction strategies from reptiles. Ventricular preparations in vitro were used to investigate the inotropic plasticity at 30 ºC and 25 ºC and during fasting (30 days fasting group) and after feeding (2 days digesting group). No significant differences were verified in the relative ventricular mass (RVM) after feeding. The peak tension (Fc) was significantly higher and rates (dFc/dt) of contraction (TC) and relaxation (TR) were faster at 25° C than at 30° C for both experimental groups. However, feeding caused significant increase in peak tension (PT) at both temperatures (396.7 % at 30 ºC and 416.7 % at 25 ºC). A significant post-rest potentiation was recorded for digesting group at 25 ºC (121.0 ± 6.4 %), which was inhibited by ryanodine (86.0 ± 2.9 %). At 30 ºC, myocytes from fasting group were able to support higher frequencies (up to 1.6 Hz) than at 25 ºC (up to 1.0 Hz), even with the sarcoplasmic reticulum (SR) blocked by 10 μM ryanodine (RYA). Data from digestion group showed greater values of the Fc and reached higher frequencies than fasting group, at both temperatures. However, pre-treatment with RYA decreased PT in frequencies between 1.4 and 1.8 Hz for digesting group at 30 ºC. Sarcolemma mechanisms were more representative in the fasting group at 25 ºC, but the SR must contribute to increased speed of contractility under those conditions. During SDA, the SR seem representative at 25 ºC, however the positive inotropic effect caused by SDA enlarged sarcolemma mechanisms representativeness. The SR significance at 30 °C seems critical during high frequencies. At this temperature, the SDA increases the contractility efficiency, even with the RS blocked, despite the relaxation and Fc development at high frequencies been present impaired. Our results indicate that Caiman latirostris myocytes show large inotropic plasticity at different temperatures, frequencies and metabolic states. / A digestão e a termorregulação são processos que podem desafiar tanto o débito quanto a frequência cardíaca (fH). Alterações pós-prandiais em diferentes padrões fisiológicos são comumente identificadas para ectotérmicos, embora sejam predominantemente estudados em serpentes. Além disso, a termorregulação é um processo crítico para garantir diferentes estratégias de sobrevivência e reprodução nos répteis. Preparações ventriculares in vitro foram utilizadas para investigar a plasticidade inotrópica dos miócitos ventriculares do Caiman latirostris a 25 e 30 °C, durante o período pós- absortivo (30 dias - após a alimentação) e após a alimentação (2 dias - grupo digestão). A massa ventricular relativa (MVR) após 2 dias da alimentação não apresentou diferenças significativas em relação ao grupo pós-absortivo. A força de contração (Fc) foi significativamente maior e as taxas (dFc/dt) de contração (TC) e relaxamento (TR) foram mais rápidas a 25 °C do que a 30 °C, para ambos os grupos experimentais. No entanto, a alimentação causou aumento significativo na força de contração (Fc) em ambas as temperaturas (316.7% a 30 °C e a 296.7% 25 °C). Uma significativa potenciação pós-pausa foi identificada no grupo digestão a 25 °C (121.0 ± 6.4 %), que foi inibida pela rianodina (86.0 ± 2.9 %). No grupo pós-absortivo a 30 °C, os miócitos foram capazes de suportar frequências de estimulação superiores (até 1.6 Hz) às suportadas a 25 °C (até 1.0 Hz), mesmo com a função do retículo sarcoplasmático (RS) bloqueada pela adição de 10 μM de rianodina (RYA). As preparações ventriculares do grupo digestão apresentaram valores maiores de Fc e alcançaram frequências superiores do que o grupo pós-absortivo, em ambas as temperaturas. Contudo, o pré-tratamento com RYA reduziu a Fc em frequências entre 1,4 e 1,8Hz no grupo digestão a 30 °C. Os mecanismos sarcolemais são mais representativos no grupo pós-absortivo a 25 °C, porém o RS pode contribuir com o aumento da velocidade da contratilidade nessas condições. Durante a SDA, o RS parece representativo nessa condição de temperatura, porém a SDA causou efeito inotrópico positivo e ampliou a representatividade dos mecanismos sarcolemais. A importância do RS a 30 °C parece crítica apenas em altas frequências de estimulação. Nessa temperatura, a SDA garante a eficiência da contratilidade mesmo com o RS bloqueado, apesar do prejuízo significativo no desenvolvimento da Fc e do relaxamento em altas frequências. Nossos resultados indicam que os miócitos ventriculares do Caiman latirostris apresentam ampla plasticidade inotrópica, capaz de assegurar o débito cardíaco em diferentes temperaturas, frequências e estados metabólicos.

Abstract Skeletal muscle cells (myofibers) are huge multinucleated cells responsible for muscle contraction and hence for the everyday movements of the joints. The structure of these voluminous cells differs greatly from that of the mononucleated cells – the characteristic features of the myofibers include dozens of peripherally located nuclei, tightly packed contractile apparatus and a sophisticatedly organized endomembrane system. The basic physiology involving myofibers is quite well known, but scarce data exist on the membrane biology of the myofibers. The purpose of this study was to examine the localization of mRNA and the site of protein synthesis in the myofibers. The characterization of the membrane dynamics in muscle cells was also performed. In this study we utilized a primary cell culture model obtained from the rat flexor digitorum brevis (FDB) muscle. Also frozen sections from the rat extensor digitorum longus muscle were used. The precursor cells of the myofibers – myoblasts and myotubes – were also utilized in some experiments. Furthermore, methods of immunohistochemistry and molecular biology were applied extensively in this study. We found that in FDB myofibers the mRNA lies just under the plasma membrane. Protein synthesis seemed to be concentrated in the vicinity of nuclei locating beneath the plasma membrane but also in interfibrillar dot-like structures. Protein products moved hundreds of micrometers away from the nuclei of origin. Moreover, there were no barriers for protein movement into the core regions of the myofibers. Movement of proteins was found to be rapid in the cytosol and in the endomembrane system, too. Interestingly, when examining exocytic trafficking we observed that ER-to-Golgi trafficking significantly differed from that of mononucleated cells. Finally, myofibers were found to be able to generate lipid bodies under stress conditions. The dynamics of lipid bodies seemed to deviate from the dynamics found in other cells types. Nowadays not much muscle research with primary myofibers is done worldwide, and therefore dilemmas involving myofibers such as insulin resistance and myotoxicity of statins are mostly unresolved. The knowledge gained from this study may be used in the future to solve clinical problems related to the cell biology of the myofibers. / Tiivistelmä Luurankolihassolut eli myofiiberit ovat jättimäisiä monitumaisia soluja, jotka vastaavat lihassupistuksen aikaansaamisesta ja siten mahdollistavat jokapäiväisen liikkumisemme. Näiden suurten solujen rakenne poikkeaa selkeästi yksitumaisten solujen rakenteesta: myofiiberien tunnusomaisia piirteitä ovat kymmenet solun reunoille sijoittuneet tumat, tiiviisti pakkautunut supistumiskoneisto ja monimutkaisesti järjestynyt solukalvostojärjestelmä. Vaikka myofiiberien perusfysiologia tunnetaankin hyvin, niin tiedetään itse myofiiberien kalvostobiologiasta sangen vähän. Kokonaisuutena tämän tutkimuksen tarkoituksena oli tarkastella mRNA:n ja proteiinisynteesin sijaintia myofiibereissä. Lisäksi selvitimme lihassolujen kalvostodynamiikkaa. Tässä tutkimuksessa käytimme rotan flexor digitorum brevis (FDB) -lihaksesta saatua primääristä soluviljelymallia. Lisäksi hyödynsimme rotan extensor digitorum longus -lihaksesta hankittuja jääleikkeitä. Joissakin kokeissa käytimme myös myofiiberien esiastesoluja (myoblasteja ja myotuubeja). Immunohistokemian ja molekyylibiologian menetelmiä sovellettiin tutkimuksessa laajasti. Havaitsimme, että FDB –myofiibereissä mRNA sijaitsee aivan solukalvon alla. Proteiinisynteesi vaikutti olevan keskittynyt solukalvon alla sijaitsevien tumien ympärille, mutta myös solusisäisiin pistemäisiin rakenteisiin. Proteiinituotteet ylsivät satojen mikrometrien päähän alkuperäisestä tumastaan. Lisäksi proteiineille ei ilmennyt leviämisestettä myofiiberin sisäosiin. Leviämisen havaittiin olevan nopeaa sekä solulimassa että solulimakalvostoissa. Tutkiessamme solun eritystoimintaa huomasimme, että kuljetus ER:stä Golgin laitteeseen eroaa huomattavasti yksitumaisten solujen vastaavasta kuljetuksesta. Lopuksi havaitsimme myofiiberien pystyvän muodostamaan rasvapisaroita rasitusolosuhteissa. Rasvapisaroiden käyttäytyminen näytti myös poikkeavan siitä, mitä muissa soluissa on havaittu. Nykyään lihastutkimusta primäärisoluilla ei juuri tehdä maailmalla, minkä vuoksi myofiibereihin liittyvät lääketieteelliset pulmat kuten insuliiniresistenssi ja statiinien lihashaitat ovat suurelta osin ratkaisematta. Tästä tutkimuksesta saatuja tuloksia voitaneen jatkossa käyttää myofiiberien solubiologiaan liittyvien kliinisten ongelmien selvittämiseen.

Abstract The function of skeletal muscle is to generate force and produce movement. These tasks are carried out by long multinucleated cells, the skeletal myofibres. The membrane system and the cytoskeleton of these cells are uniquely organized to respond rapidly to neuronal stimuli and to achieve efficient contraction. In the present study the organization and distribution of selected protein/lipid based microdomains that reside in the plasma membrane and sarcoplasmic reticulum of isolated rat skeletal myofibres, were investigated. Aquaporin 4 (AQP4) water channels are arranged as higher order oligomers of several sizes in the sarcolemma and in the T tubules. These oligomers, however, were absent from many specialized micro- and- macrodomains. The distribution of AQP4 coincided with that of a highly organized protein assembly, the dystrophin glycoprotein complex (DGC), in the sarcolemma. A chimaeric venus-AQP4 was equally mobile in the T tubules and sarcolemma, but the anchoring mechanisms of the protein appeared to be different. In contrast to AQP4, the proteins resident in cholesterol and sphingolipid-based microdomains, known as rafts, also occupied DGC deficient areas, which surround the T tubule openings. Indeed, flotillin-1 rafts were located in the neck portions of the T tubules. The rafts defined by the influenza haemagglutinin (HA) also resided in DGC deficient areas, but at the borders of the DGC area. Importantly, of the raft proteins, only the localization of caveolin 3 (CAV3) was dependent on the cholesterol enriched lipid environment, as evidenced by cholesterol depletion experiments and localization studies on a non-raft associated variant of HA. The organization and distribution of membrane associated rough ER (RER) proteins were also analysed. Biochemical detergent extraction analyses and immunofluorescence staining indicated that the ER proteins were assembled as microdomains within the sarcoplasmic reticulum (SR). The microdomains were distributed throughout the SR network and they were capable of protein translocation. Taken together, skeletal myofibres comprise visually distinct microdomains both in the plasma membrane and in the SR. In the plasma membrane, different types of microdomains are not homogenously distributed and function in diverse locations. This may have important physiological implications concerning, among other things, local regulation of ion concentrations and cell signalling cascades. Different constraints ranging from protein-protein interactions to the surrounding lipid environment are important for dictating the observed distribution patterns. / Tiivistelmä Luustolihaksen toimintojen perustana ovat supistumiskykyiset lihassolut, joiden kalvorakenne on järjestynyt erityisellä tavalla ohjaamaan supistusta. Tässä tutkimuksessa analysoitiin proteiini- ja lipidiperustaisten mikroalueiden järjestäytymistä ja tähän vaikuttavia tekijöitä luustolihassolun solukalvolla sekä lihassolun sisäisessä kalvojärjestelmässä, sarkoplasmisessa verkossa (SR). Ensin analysoitiin vesikanavatyyppiä 4 (AQP4), joka oligomerisoituessaan muodostaa erikokoisia mikroalueita. Havaittiin, että AQP4-mikroalueita esiintyy kaikkialla solukalvolla lukuun ottamatta eräitä erilaistuneita mikro- ja makroalueita. AQP4-oligomeerien jakauma solukalvon lateraalisessa osassa, sarkolemmalla, noudatti dystrofiini-glykoproteiinikompleksin jakaumaa. Fluoresoivan venus-AQP4-proteiinin avulla osoitettiin, että proteiinin liikkuvuus oli samanlainen solun sisään ulottuvissa poikkiputkistoissa ja sarkolemmalla, mutta liikkuvuutta rajoittavat tekijät olivat erilaisia näissä solukalvon osissa. Toiseksi analysoitiin kolesteroli- ja sfingolipidipitoisia mikroalueita, kalvolauttoja. Flotilliini-1- ja influenssaviruksen hemagglutiniini (HA) -proteiinia sisältäviä lauttoja esiintyi vain poikkiputkien suuaukkojen alueella, mutta lauttojen jakauma oli erilainen. Lauttojen lipidiympäristöllä ei ollut vaikutusta proteiinien sijaintiin. Tämä osoitettiin kolesterolin poistokokeilla sekä kokeilla, joissa käytettiin mutatoitua HA-proteiinia, joka ei hakeudu kolesteroliympäristöön. Kaveoliini-3-proteiinin sijainti poikkeaa edellä mainituista, ja kolesterolin poisto vaikutti merkittävästi sijainnin määräytymiseen. Kolmanneksi analysoitiin, miten karkean endoplasmakalvoston proteiinit ovat järjestäytyneet SR:ssä. Havaittiin, että endoplasmiset kalvoproteiinit eivät ole homogeenisesti levittäytyneet SR-kalvostoon vaan muodostavat pieniä mikroalueita. Detergenttiuuttoanalyysit osoittivat lisäksi, että näissä mikroalueissa on erilainen lipidikoostumus kuin SR:ssä yleensä. Huomattavaa oli myös, että mikroalueet olivat toiminnallisia kaikkialla SR-kalvostossa. Tulosten perusteella luustolihassolujen kalvojärjestelmä sisältää mikroalueita, joiden jakautuminen vaikuttaa hyvin organisoituneelta. Erityisesti solukalvon mikroalueet esiintyvät tietyillä spesifeillä alueilla, joissa niiden voidaan olettaa toimivan mm. erilaisissa solusignalointitapahtumissa ja paikallisessa ionipitoisuuksien säätelyssä. Eräissä tapauksissa lipidiympäristöllä on merkitystä mikroalueiden sijainnin määräytymisessä, mutta proteiinien sitoutuminen solukalvo- tai solukalvon alaisiin rakenteisiin saattaa myös olla määräävä tekijä.

La cascade d’évènements permettant la contraction de la fibre musculaire striée squelettique en réponse à l’activité électrique de sa membrane plasmique est regroupée sous le terme de couplage excitation-contraction (EC). Le couplage EC a lieu au niveau des triades, domaines nanoscopiques au niveau desquels les invaginations transversales de la membrane plasmique (tubules-T) sont en contact étroit avec deux citernes terminales adjacentes de réticulum sarcoplasmique (RS). Plus précisément, lors de l’excitation d’une fibre musculaire, un potentiel d’action se propage dans toute la surface de la membrane plasmique et en profondeur de la cellule via les tubules-T. Cette dépolarisation y est détectée par les protéines membranaires sensibles au potentiel Cav1.1 qui en retour, par couplage mécanique, déclenchent l’ouverture des canaux calciques du RS que sont les récepteurs de la ryanodine de type 1 (RYR1s). Ceci est à l’origine de l’augmentation massive de Ca2+ intracellulaire qui déclenche l’activation des myofilaments et donc la contraction. La compréhension des mécanismes de contrôle et de régulation des canaux RYR1s reste encore aujourd’hui limitée. En particulier, la mesure de l’activité physiologique de ces canaux dans la fibre musculaire intacte est toujours réalisée de manière très indirecte. Par ailleurs le rôle éventuel de variations de potentiel de la membrane du RS pendant l’activité musculaire n’a jamais été révélé. Une connaissance approfondie de ces phénomènes est pourtant essentielle à la compréhension de la fonction musculaire squelettique normale et pathologique. Dans ce contexte, l’objectif général de mon projet de thèse a été de mettre au point et utiliser des biosenseurs fluorescents localisés spécifiquement à la membrane des citernes terminales du RS de fibres musculaires différenciées – par leur fusion à une séquence d’adressage appropriée. Grâce à la combinaison des techniques d’électrophysiologie et d’imagerie de la fluorescence des biosenseurs sur fibres musculaires isolées, nous avons pu étudier l’activité du RS au cours de la fonction musculaire. Plus particulièrement, mon travail de thèse aborde deux problèmes biologiques principaux : le potentiel de membrane du RS et la signalisation calcique du RS au cours du couplage EC. Le premier objectif a visé à caractériser les changements de potentiel de la membrane du RS pendant l’activation du couplage EC. Pour cela, nous avons utilisé des biosenseurs de FRET de la famille Mermaid. Nos résultats montrent qu’il n’y a pas de changement substantiel du potentiel transmembranaire du RS pendant l’activation du couplage EC. Ces données confirment – pour la première fois en condition physiologique – que le flux de Ca2+ à travers les canaux RYR1s est équilibré par des contre-flux ioniques compensatoires qui permettent le maintien du potentiel de membrane du RS. Ceci assure la pérennité du flux de Ca2+ et contribue à l’efficacité du couplage EC. Le deuxième objectif a visé à détecter les variations de concentration en Ca2+ à proximité immédiate des canaux RYR1s. Pour cela, nous avons utilisé le biosenseur fluorescent sensible au Ca2+ GCamP6f. Le biosenseur adressé à la membrane du RS fournit un accès unique à l’activité individuelle de populations distinctes de canaux RYR1s au sein de différentes triades d’une même fibre musculaire. Au-delà de la caractérisation détaillée des propriétés des sondes GCaMP6f dans cette préparation, nos résultats montrent la stupéfiante synchronisation de l’activité de libération de Ca2+ des triades d’une même fibre musculaire au cours du couplage EC. Les résultats ouvrent des perspectives particulièrement intéressantes pour les études de situations pathologiques d’altération de l’activité des canaux RYR1s / Excitation-contraction (EC) coupling in skeletal muscle corresponds to the sequence of events through which muscle fiber contraction is triggered in response to plasma membrane electrical activity. EC coupling takes place at the triads; these are nanoscopic domains in which the transverse invaginations (t-tubules) of the surface membrane are in closed apposition with two adjacent terminal cisternae of the sarcoplasmic reticulum membrane (SR). More precisely, EC coupling starts with action potentials fired at the endplate, propagating throughout the surface membrane and in depth into the muscle fiber through the t-tubules network. When reaching the triadic region, action potentials activate the voltage-sensing protein Cav1.1. In turns, Cav1.1 directly open up the type 1 ryanodine receptor (RYR1) in the immediately adjacent SR membrane, through intermolecular conformational coupling. This triggers RYR1-mediated SR Ca2+ release which produces an increase in cytosolic Ca2+ triggering contraction. Current understanding of the mechanisms involved in the control and regulation of RYR1 channels function is still limited. One reason is related to the fact that detection of RYR1 channel activity in intact muscle fibers is only achieved with indirect methods. Also, whether SR the membrane voltage experiences changes during muscle activity has so far never been experimentally assessed. Yet, deeper knowledge of these processes is essential for our understanding of muscle function in normal and disease conditions. In this context, the general aim of my PhD project was to design and use fluorescent protein biosensors specifically localized at the SR membrane of differentiated muscle fibers, by fusing them to an appropriate targeting sequence. Thanks to a combination of single cell physiology and biophysics techniques based on electrophysiology and biosensor fluorescence detection, we were able to study the SR activity during muscle fiber function. Specifically, my PhD work focused on two major issues: SR membrane voltage and SR calcium signaling during EC coupling. The first aim of my work was to characterize SR membrane voltage changes during muscle fiber activity. For this, we used voltage sensitive FRET-biosensors of the Mermaid family. Results show that the SR trans-membrane voltage experiences no substantial change during EC coupling. This provides the first experimental evidence, in physiological conditions, for the existence of ion counter-fluxes that balance the charge deficit associated with RYR1-mediated SR Ca2+ release. Indeed, this process is essential for maintaining the SR Ca2+ flux upon RYR1 channels opening and thus critically important for EC coupling efficiency. The second objective of my work aimed at detecting the changes in Ca2+ concentration occurring in the immediate vicinity of the RYR1 Ca2+ release channels during muscle fiber activation. For this, we took advantage of one member of the recent generation of genetically encoded Ca2+ biosensor: GCaMP6f. The SR-targeted biosensor provides a unique access to the individual activity of RYR1 channels populations within distinct triads of a same muscle fiber. Beyond allowing a detailed characterization of the biosensor properties in this preparation, results highlight the remarkable uniformity of SR Ca2+ release activation from one triad to another, during EC coupling. These results open up stimulating perspectives for the investigation of disease conditions associated with defective behavior of RYR1 channels.

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Made available in DSpace on 2016-06-02T19:22:07Z (GMT). No. of bitstreams: 1 4217.pdf: 3961717 bytes, checksum: 3053793c39a626c9cf9d5fb8f9c0fa21 (MD5) Previous issue date: 2011-02-18 / Universidade Federal de Sao Carlos / The ventricular myocardial contractility of the matrinxã teleost, Brycon amazonicus, was analysed in previous experimentation with isometric multicellular preparations, in time effect and force-frequency relationship experiments, with and without exposure to the alkaloid ryanodine, a sarcoplasmic reticulum (SR) Ca2+ release blocker. In this study, different methodological approaches were used, such as isometric stress (EI, mN.mm-2), that permitted to identify the sensitivity of the myocardium to ryanodine, with the majority contribution of Ca2+ from the SR. The use of time to peak tension and time to half relaxation parameters (TPT and THR, ms) were ineffective to evaluate the contraction and relaxation performances during time effect experiments. New approaches such as contraction rates and initial relaxation rates (TC and TIR, mN.mm-2.s-1) demonstrated directly the impairment of the myocardium exposed to ryanodine. The negative staircase effect, characteristic of the teleost s forcefrequency relationship was evidenced by EI. The maximum rates of contraction and maximum rates of relaxation (TMC and TMR, mN.mm-2.s-1) and the average rates of contraction and average rates of relaxation (TMedC and TMedR, mN.mm-2.s-1) showed the impairment of the myocardium contractility exposed to ryanodine as well as the low sensitivity of frequency increments on the contractility when considered the values of TPT and THR. The TMedC and TMedR values indicated a greater possibility of heart rate regulation than the TMC and TMR values, probably due to these estimates consider the amount of instantaneous rate changes of contraction wave on calculation instead of just one point on the curve. The cardiac pumping capacity (CBC, mN.mm2.min-1) showed that the optimal range of frequency for isometric contraction is narrow and that the myocardium of the species should be working on the limit at rest. The analyses of integral of isometric tension (ITI, mN.mm- 2.s), and integral of isometric tension per minute (ITIPM, mN.mm-2.min), showed that these were unsuitable indexes for the assessment of cardiac contractility in the way they were calculated, as inconsistent interpretations were generated, probably by using information from the curve of contraction irrelevant to the isometric preparation. The contractility index (IC, mN.mm-2) created in this work, suggested that the myocardium contractility of the species is more sensitive to increases in frequency. On the other hand, the contractility index per minute (ICPM, mN.mm-2) showed that the optimum range of frequencies for the B. amazonicus myocardial contraction can be much wider and could allow performance reserve, such as reported in other studies of cardiac function in teleosts. The protein expression of SERCA2a and phospholamban (PLB) were analysed by Western Blot technique and their expression supported the findings of the SR functionality. The comparative analysis of these proteins, using amino acid sequences available in public databases (GenBank and UniProt), revealed levels of similarity between the SERCA2a and PLB in fish and other vertebrates, strengthened the findings of studies with Western Blot experiments. Taken together, the results suggest that B. amazonicus myocardium is dependent on SR Ca2+ stores under physiological frequencies and, despite the negative staircase pattern, must possess a performance reserve at supraphysiologic frequencies. / A contratilidade do miocárdio ventricular do teleósteo matrinxã, Brycon amazonicus, foi analisada com dados de experimentos prévios, realizados com preparações isométricas multicelulares e protocolos de efeito do tempo e de relação forçafrequência, com e sem exposição ao alcalóide rianodina, bloqueador dos canais de liberação de Ca2+ do retículo sarcoplasmático (RS). Nesse estudo foram utilizadas diferentes abordagens metodológicas de tratamento de dados, como o estresse isométrico (EI, mN.mm-2), a partir do qual foi possível identificar a sensibilidade do miocárdio à rianodina, com contribuição majoritária do Ca2+ proveniente do RS. A utilização dos tempos para o pico de tensão e para metade do relaxamento (TPT e THR, ms), se mostraram ineficazes para avaliar o desempenho da contração e relaxamento no experimento do efeito do tempo. Novas abordagens na análise dos dados, como a taxa de contração e taxa inicial de relaxamento (TC e TIR, mN.mm- 2.s-1) demonstraram explicitamente o comprometimento do miocárdio durante a exposição à rianodina. O efeito escada negativo, característico da relação forçafrequência de teleósteos foi evidenciado pelo EI. As taxas máximas de contração e de relaxamento (TMC e TMR, mN.mm-2.s-1) e as taxas médias de contração e de relaxamento (TMedC e TMedR, mN.mm-2.s-1) além de demonstrarem o comprometimento da contratilidade durante exposição à rianodina, indicaram que existe um menor comprometimento da tensão durante elevação da frequência, quando comparados aos valores de TPT e THR. A TMedC e a TMedR apresentaram resultados relacionados a uma frequência cardíaca com maior possibilidade de ajustes do que a TMC e a TMR, provavelmente por considerarem o conjunto de variação das taxas instantâneas da contração e não somente um único ponto da curva. A capacidade de bombeamento cardíaco (CBC, mN.mm-2.min-1) mostrou que a faixa ótima de frequência para contração isométrica é estreita e que o miocárdio da espécie deve estar trabalhando no limiar na condição de repouso. As análises da integral da tensão isométrica (ITI, mN.mm-2.s), e da integral da tensão isométrica por minuto (ITIPM, mN.mm-2.min) mostraram-se inapropriadas para a avaliação da contratilidade cardíaca pelo modo como foram calculadas, uma vez que geraram interpretações incoerentes, provavelmente por utilizarem informações da curva de contração irrelevantes para a preparação isométrica. O índice de contratilidade (IC, mN.mm-2) criado nesse trabalho, sugere que o miocárdio da espécie é mais sensível às elevações de frequência. Por outro lado, o índice de contratilidade por minuto (ICPM, mN.mm-2) mostrou que a faixa ótima de frequências para a contração cardíaca de B. amazonicus pode ser mais ampla, o que permitiria uma reserva de desempenho, assim como observado para outros teleósteos. A expressão das proteínas SERCA2a e fosfolambano (PLB) foram analisadas pela técnica de Western Blot e sua expressão reforçaram os achados de funcionalidade do RS. A análise comparativa dessas proteínas utilizando sequências de aminoácidos disponíveis em bancos de dados públicos (GenBank e UniProt) revelou os níveis de similaridade entre a SERCA2a e PLB de peixes e mamíferos, reforçando os achados dos estudos com Western Blot. Em conjunto, os dados sugerem que o miocárdio do B. amazonicus, apresenta uma nítida dependência do RS em frequências fisiológicas e, apesar de exibir um claro padrão escada negativo, deve apresentar uma reserva de desempenho para frequências suprafisiológicas.

Made available in DSpace on 2016-06-02T19:22:58Z (GMT). No. of bitstreams: 1 5111.pdf: 6171719 bytes, checksum: 0356f236abaa5b549156bc72fde00f70 (MD5) Previous issue date: 2012-09-14 / Universidade Federal de Minas Gerais / Some snakes have the ability to survive long periods without food and are capable to ingest large meal size. The ingestion of proportionally large preys triggers an expressive increase on the oxidative metabolic demand (SDA Specific Dinamic Action) which can become several times higher than the resting metabolic rate. The two described extreme situations might lead to cardiac changes to adapt structure and function in order to afford these two opposite physiological demands. During long food deprivation (Phase III), the depletion of the body structure may affect the heart muscle. That should be reverted to the SDA and prevent an overload on cardiovascular system. The standard cardiac function of South-american Rattlesnake, Crotalus durissus terrificus, was described as well as the changes caused by long term food deprivation and SDA. The importance of sodium-calcium exchanger (NCX), functionality of sarcoplasmic reticulum, extracellular calcium dependence and the effect of adrenergic stimulation were tested in adult animals at 30°C, under three different metabolic states: postabsorptive, SDA peak and food deprivation. The ventricular mass does not change after food deprivation and SDA. The force of contraction was higher in the base of the heart if compared to the apex, but there is no difference among the experimental groups and it is probably reflex of the fiber orientation in each region of the ventricle. The sarcoplasmatic reticulum is functional in all groups, but the dependence of reticular calcium is lower during starvation compared to the other groups. Muscle contraction is mostly supported by the extracellular Ca2+. The NCX have minor contribution to force generation (20%) but has a major role pumping calcium out of the cell (faster than SERCA). The increase in extracellular Ca2+ concentration during digestion can augment twitch force and would represent a contractile advantage to support the increased cardiac work without the development of hypertrophy. The adrenergic stimulation produced sustained increase in Fc for a wide range of stimulation frequencies in all the tested groups. / O coração possui a importante capacidade de se remodelar diante de alterações nas demandas funcionais. Serpentes apresentam a capacidade de ingestão de grandes presas e a capacidade de sobreviver a grandes períodos de privação alimentar. A ingestão de grandes massas de alimento demanda uma elevação metabólica e leva a uma compensação na massa ventricular e um aumento na força de contração do miocárdio, evitando uma sobrecarga no sistema cardiovascular. Durante o jejum ocorre depleção da estrutura corpórea podendo também atingir o músculo cardíaco. Para descrever a função cardíaca da Cascavél Sul-americana, Crotalus durissus terrificus, e as possíveis alterações diante dos extremos metabólicos, foi testada a importância do trocador Na+/Ca2+ (NCX), a funcionalidade do retículo sarcoplasmático (RS), a dependência do Ca2+ extracelular e o efeito da estimulação adrenérgica em animais adultos à temperatura constante de 30°C em período pós-absortivo, pico de SDA e após jejum prolongado. Observou-se que a massa ventricular se mantém estável mesmo durante os extremos metabólicos. Há uma diferença na geração de tensão entre as tiras da base e ápice ventriculares, presente nos 3 grupos alimentares e que pode se dar em função da orientação das fibras nas duas regiões do ventrículo. O teste com rianodina mostrou que o RS é funcional nos 3 grupos porém a dependência do cálcio proveniente do RS é menor após o jejum prolongado quando comparado aos demais grupos. O cálcio extracelular é o principal responsável pela contração do miócito cardíaco e o NCX, que fornece uma pequena parte de cálcio para a contração, é o principal bombeador de cálcio para fora da célula e é mais rápido do que a Ca2+- ATPase do RS. O meio extracelular é a principal fonte de cálcio ativador da contração sendo que o aumento de cálcio circulante disponível para a contração nos animais durante a digestão ofereceria uma vantagem contrátil para este grupo sem necessidade de hipertrofia. A adrenalina produziu um aumento substancial na Fc capaz e ser mantido por diferentes frequências de estimulação nos 3 grupos testados.