The aim of this study was to investigate the mode of action of pavetamine on rat cardiomyocytes. Pavetamine is the causative agent of gousiekte (“quick-disease”), a disease of ruminants characterized by acute heart failure following ingestion of certain rubiaceous plants. Two in vitro rat cardiomyocyte models were utilized in this study, namely the rat embryonic cardiac cell line, H9c2, and primary neonatal rat cardiomyocytes. Cytotoxicity of pavetamine was evaluated in H9c2 cells using the MTT and LDH release assays. The eventual cell death of H9c2 cells was due to necrosis, with LDH release into the culture medium after exposure to pavetamine for 72 h. Pavetamine did not induce apoptosis, as the typical features of apoptosis were not observed. Electron microscopy was employed to study ultrastructural alterations caused by pavetamine in H9c2 cells. The mitochondria and sarcoplasmic reticula showed abnormalities after 48 h exposure of the cells to pavetamine. Abundant secondary lysosomes with electron dense material were present in treated cells. Numerous vacuoles were also present in treated cells, indicative of autophagy. During this exposure time, the nuclei appeared normal, with no chromatin condensation as would be expected for apoptosis. Abnormalities in the morphology of the nuclei were only evident after 72 h exposure. The nuclei became fragmented and plasma membrane blebbing occurred. The mitochondrial membrane potential was investigated with a fluorescent probe, which demonstrated that pavetamine caused significant hyperpolarization of the mitochondrial membrane, in contrast to the depolarization caused by apoptotic inducers. Pavetamine did not cause opening of the mitochondrial permeability transition pore, because cyclosporine A, which is an inhibitor of the mitochondrial permeability transition pore, did not reduce the cytotoxicity of pavetamine significantly. Fluorescent probes were used to investigate subcellular changes induced by pavetamine in H9c2 cells. The mitochondria and sarcoplasmic reticula showed abnormal features compared to the control cells, which is consistent with the electron microscopy studies. The lysosomes of treated cells were more abundant and enlarged. The activity of cytosolic hexosaminidase was nearly three times higher in the treated cells than in the control cells, which suggested increased lysosomal membrane permeability. The activity of acid phosphatase was also increased in comparison to the control cells. In addition, the organization of the cytoskeletal F-actin of treated cells was severely affected by pavetamine. Rat neonatal cardiomyocytes were labelled with antibodies to detect the three major contractile proteins (titin, actin and myosin) and cytoskeletal proteins (F-actin, desmin and β-tubulin). Cells treated with pavetamine had degraded myosin and titin, with altered morphology of sarcomeric actin. Vacuoles appeared in the β-tubulin network, but the appearance of desmin was normal. F-actin was severely disrupted in cardiomyocytes treated with pavetamine and was degraded or even absent in treated cells. Ultrastructurally, the sarcomeres of rat neonatal cardiomyocytes exposed to pavetamine were disorganized and disengaged from the Z-lines, which can also be observed in the hearts of ruminants that have died of gousiekte. It is concluded that the pathological alteration to the major contractile and cytoskeleton proteins caused by pavetamine could explain the cardiac dysfunction that characterizes gousiekte. F-actin is involved in protein synthesis and therefore can play a role in the inhibition of protein synthesis in the myocardium of ruminants suffering from gousiekte. Apart from inhibition of protein synthesis in the heart, there is also increased degradation of cardiac proteins in an animal with gousiekte. The mitochondrial damage will lead to an energy deficiency and possibly to generation of reactive oxygen species. The sarcoplasmic reticula are involved in protein synthesis and any damage to them will affect protein synthesis, folding and post-translational modifications. This will activate the unfolded protein response (UPR) and sarcoplasmic reticula-associated protein degradation (ERAD). If the oxidizing environment of the sarcoplasmic reticula is disturbed, it will activate the ubiquitin-proteasome pathway (UPP) to clear aggregated and misfolded proteins. Lastly, the mitochondria, sarcoplasmic reticula and F-actin are involved in calcium homeostasis. Any damage to these organelles will have a profound influence on calcium flux in the heart and will further contribute to the contractile dysfunction that characterizes gousiekte. / Thesis (PhD)--University of Pretoria, 2010. / Paraclinical Sciences / unrestricted

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.

No description available.

Anaemia, which is caused by iron deficiency, is a global nutritional disorder of utmost concern. It has been assumed that meat, which contains haem as well as non-haem iron, enhances non-haem iron absorption due to the presence of the “meat factor”. In the experiment reported here, 24 semi-anaemic, 3-week-old piglets were utilised as human nutrition models to assess the effects of dietary lactoferrin, meat and LSF (Low Molecular Weight Sarcoplasmic Fraction) on iron bioavailability during a 4-week feeding period. The parameters that were used as measurements of iron bioavailability were changes in haematological indices, haemoglobin iron repletion efficiency, intestinal morphology and mineral balances. Non-significant (p > 0.05) dietary effects were observed for growth performance and for all the haematological and some histological parameters (small intestine villi height, crypt depth and mucosal thickness). Haemoglobin iron repletion efficiency was highest for the control group and was not significantly different between the other 3 diets. The superiority of the control diet in this respect was not consistent with previous trials and can not be explained. Results suggested that increased retention of calcium, magnesium, phosphorous and manganese tended to inhibit iron absorption. However, the LSF and meat diets significantly (p = 0.003) increased the number of goblet cells/100 µm suggesting that mucin secretion was favoured by these two diets. Additionally, all immunological parameters were significantly (p < 0.05) improved by the LSF diet. As such, the LSF diet can be a potential immunobooster feed ingredient for weanling piglets. Overall, the level of LSF in the LSF diet was insufficient to exert a desirable enhancement of iron bioavailability and betterment of 3 iron status of the semi-anaemic piglets relative to the control group. However, the diet containing LSF was as effective as the meat diet with respect to these characteristics.

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.

La Myotubularine (MTM1) est une 3-phosphatase à phosphoinositides (PI) mutée dans la myopathie centronucléaire liée au chromosome X (XLCNM), caractérisée par une faiblesse musculaire et un positionnement anormal des noyaux dans les fibres musculaires. MTM1 définit une grande famille de phosphatases, exprimées dans tous les tissus, et qui englobent des phosphatases catalytiquement actives et inactives. Les myotubularines actives dephosphorylent le phosphatidylinositol 3 monophosphate [PtdIns3P] et le 3,5-bisphosphate [PtdIns(3,5)P2] en PtdIns et PtdIns5P, respectivement. Le rôle de MTM1 et son activité phosphatase à lipide dans le muscle restaient peu connus. L’étude approfondie de la protéine a révélé une association de MTM1 au réticulum sarcoplasmique des triades, un sous-compartiment impliqué dans la régulation calcique. La caractérisation de la souris Mtm1 KO, qui reproduit la XLCNM, a témoigné d’une anomalie de l’organisation et de la forme du réticulum sarcoplasmique. Afin d’explorer l’implicationde l’activité phosphatase de MTM1 dans l’organisation de réticulum sarcoplasmique, j’ai utilisé une approche in vivo avec des virus adéno-associé (AAV) pour moduler l’activité phosphatase en sur-exprimant MTM1 et sa forme phosphatase-inactive (MTM1-C375S) dans un muscle sauvage. L’observation des muscles transduits a dévoilé une implication de MTM1 dans le remodelage du réticulum sarcoplasmique et un rôle potentiel de PtdIns3P avec MTM1 dans la courbure des membranes du réticulum sarcoplasmique. Afin de comprendre l’importance de l’activité phosphatase dans le maintien du phénotype XLCNM, les muscles de souris Mtm1 KO ont été injectés avec ces AAVs contenant la forme active et inactive de MTM1 au moment de l’apparition des premiers signes de XLCNM. Étonnamment, la forme phosphatase-inactive(MTM1-C375S) a sauvé le phénotype de la souris Mtm1 KO de la même façon que la forme active, suggérant que l'activité de phosphatase de MTM1 n’est pas nécessaire pour le maintien de la structure intracellulaire des fibres du muscle adulte. Ces données suggèrent que MTM1 exerce une fonction phosphatase-indépendante dans le maintien de la structure musculaire, certainement via des interactions protéine-protéine, et une fonction phosphatase-dépendente dans le remodelage de la forme du réticulum sarcoplasmique dans le muscle squelettique. / Myotubularin (MTM1) is a phosphoinositide (PI) 3-phosphatase mutated in X-linked centronuclear myopathy (XLCNM), a rare congenital myopathy characterized by muscle weakness and abnormal positioning of nuclei in muscle fibers. MTM1 defines a large family of ubiquitously expressed catalytically active and inactive phosphatases. Active myotubularins dephosphorylate both phosphatidylinositol 3-phosphate [PtdIns3P] and 3,5-bisphosphate [PtdIns(3,5)P2] to PtdIns andPtdIns5P, respectively. The specific role of MTM1 and its PI phosphatase activity in muscle remains unknown. Comprehensive analysis of the protein unveiled the association of MTM1 with the sarcoplasmic reticulum (SR) at the triads. Characterization of Mtm1-KO mouse, which reproduce the XLCNM phenotype, revealed a defect of SR organization and shape. In order to gain insight into the involvement of MTM1 phosphatase activity on SR shape and organization, we employed an in vivo approach using Adeno-Associated Virus (AAV) to modulate the phosphatase activity by overexpressingMTM1 and its phosphatase inactive mutant in wild type muscle. The analysis of transduced muscle revealed the involvement of MTM1 in the SR remodeling and its potential role together with PtdIns3P in modulating membrane curvature. In order to understand the importance of the phosphatase activity in the generation of the XLCNM phenotype, Mtm1 KO mice were injected with AAV expressing the active form and the phosphatase inactive form. Surprisingly, both, the phosphatase active and the phosphatase inactive mutant corrected the Mtm1-KO mouse phenotype to a similar extent, thus suggesting that the PI-phosphatase activity of MTM1 is not essential for adult skeletal muscle maintenance. Our data indicates that MTM1 has a phosphatase-independent function in adult muscle structure maintenance and a phosphatase-dependent function in sarcoplasmic reticulum remodeling and shape in skeletal muscle.

Um die Kontraktion einer Herzmuskelzelle durch den Kalziumeinstrom zu ermöglichen, ist die Kopplung von Erregung und Kontraktion (ECC) von zentraler Bedeutung. Durch das elektrische Signal einer Nachbarzelle wird die Depolarisation des Sarkolemmas verursacht, wodurch sich die L-Typ-Kalziumkanäale (LKK) öffnen und der Amplifizierungsprozess eingeleitet wird. Letzterer ist bekannt als Kalzium induzierte Kalzium Freisetzung (CICR). Durch die LKK wird ein Kalziumeinstrom in die Zelle ermöglicht, welcher zur Öffnung der Ryanodinrezeptoren (RyR) des Sarkoplasmatischen Retikulums (SR) führt. Durch die Kalziumfreisetzung des SR wird dieses im Cytoplasma akkumuliert. Modelle für diese Prozesse werden seit mehreren Jahrzenten entwickelt. Bisher fehlte jedoch die Kombination aus räumlich aufgelösten Kalziumkonzentrationen der dyadischen Spalte mit stochastischen Simulationen der einzelnen Kalziumkanäle und die Kalziumdynamiken in der ganzen Zelle mit einem Elektrophysiologiemodell einer ganzen Herzmuskelzelle. In dieser Arbeit entwickleten wir ein neues Modell, in welchem die Konzentrationsgradienten von einzelnen Kanälen bis zum Ganzzelllevel räumlich aufgelöst werden. Es wurde der quasistatische Ansatz und die Finite-Elemente-Methode zur Integration partieller Differentialgleichungen verwendet. Es wurden Simulationen mit unterschiedlichen RyR Markow-Kette-Modellen, verschiedenen Parametern für die Bestandteile des SR, verschiedenen Konditionen des Natrium-Kalzium-Austauschers und unter Einbindung der Mitochondrien durchgeführt. Ziel war es, das physiologische Verhalten einer Kaninchen-Herzmuskelzelle zu simulieren. In dem neu entwickelten Multiskalenmodell wurden Hochleistungsrechner verwendet, um detaillierte Informationen über die Verteilung, die Regulation und die Relevanz von den im ECC involvierten Komponenten aufzuzeigen. Zukünftig soll das entwickelte Modell Anwendung bei der Untersuchung von Herzkontraktionen und Herzmuskelversagen finden. / Excitation contraction coupling (ECC) is of central importance to enable the contraction of the cardiac myocyte via calcium in ux. The electrical signal of a neighbouring cell causes the membrane depolarization of the sarcolemma and L-type Ca2+ channels (LCCs) open. The amplifcation process is initiated. This process is known as calcium-induced calcium release (CICR). The calcium in ux through the LCCs activates the ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR). The Ca2+ release of the SR accumulates calcium in the cytoplasm. For many decades models for these processes were developed. However, previous models have not combined the spatially resolved concentration dynamics of the dyadic cleft including the stochastic simulation of individual calcium channels and the whole cell calcium dynamics with a whole cardiac myocyte electrophysiology model. In this study, we developed a novel approach to resolve concentration gradients from single channel to whole cell level by using quasistatic approximation and finite element method for integrating partial differential equations. We ran a series of simulations with different RyR Markov chain models, different parameters for the SR components, sodium-calcium exchanger conditions, and included mitochondria to approximate physiological behaviour of a rabbit ventricular cardiac myocyte. The new multi-scale simulation tool which we developed makes use of high performance computing to reveal detailed information about the distribution, regulation, and importance of components involved in ECC. This tool will find application in investigation of heart contraction and heart failure.

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