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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Indiana University-Purdue University Indianapolis (IUPUI) / Type 2 diabetes manifests from peripheral insulin resistance and a loss of functional beta cell mass due to decreased beta cell function, survival, and/or proliferation. Beta cell stressors impair each of these factors by activating stress response mechanisms, including endoplasmic reticulum (ER) stress. The glucolipotoxic environment of the diabetic milieu also activates a stress response in beta cells, resulting in death and decreased survival. Whereas the cell cycle machinery (comprised of cyclins, kinases, and inhibitors) regulates proliferation, its involvement during beta cell stress in the development of diabetes is not well understood. Interestingly, in a screen of multiple cell cycle inhibitors, p21 was dramatically upregulated in INS-1-derived 832/13 cells and rodent islets by two independent pharmacologic inducers of beta cell stress - dexamethasone and thapsigargin. In addition, glucolipotoxic stress mimicking the diabetic milieu also induced p21. To further investigate p21’s role in the beta cell, p21 was adenovirally overexpressed in 832/13 cells and rat islets. As expected given p21’s role as a cell cycle inhibitor, p21 overexpression decreased [3H]-thymidine incorporation and blocked the G1/S and G2/M transitions as quantified by flow cytometry. Interestingly, p21 overexpression activated apoptosis, demonstrated by increased annexin- and propidium iodide-double-positive cells and cleaved caspase-3 protein. p21-mediated caspase-3 cleavage was inhibited by either overexpression of the anti-apoptotic mitochondrial protein Bcl-2 or siRNA-mediated suppression of the pro-apoptotic proteins Bax and Bak. Therefore, the intrinsic apoptotic pathway is central for p21-mediated cell death. Like glucolipotoxicity, p21 overexpression inhibited the insulin cell survival signaling pathway while also impairing glucose-stimulated insulin secretion, an index of beta cell function. Under both conditions, phosphorylation of insulin receptor substrate-1, Akt, and Forkhead box protein-O1 was reduced. p21 overexpression increased Bim and c-Jun N-terminal Kinase, however, siRNA-mediated reduction or inhibition of either protein, respectively, did not alter p21-mediated cell death. Importantly, islets of p21-knockout mice treated with the ER stress inducer thapsigargin displayed a blunted apoptotic response. In summary, our findings indicate that p21 decreases proliferation, activates apoptosis, and impairs beta cell function, thus being a potential target to inhibit for the protection of functional beta cell mass.

Indiana University-Purdue University Indianapolis (IUPUI) / Diabetes mellitus affects an estimated 285 million people worldwide, and a central component of diabetes pathophysiology is diminished pancreatic islet beta cell function resulting in the inability to manage blood glucose effectively. The beta cell is a highly specialized metabolic factory that possesses a number of specialized characteristics, chief among these a highly developed endoplasmic reticulum (ER). The sarco endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ gradient between the cytosol and ER lumen, and while the Pancreatic and duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in beta cell development and function, recent data also implicate Pdx-1 in the maintenance of ER health. Our data demonstrates that a decrease of beta cell Pdx-1 occurs in parallel with decreased SERCA2b expression in models of diabetes, while in silico analysis of the SERCA2b promoter reveals multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. Results revealed reduced SERCA2b expression and decreased ER Ca2+, which was measured using an ER-targeted D4ER adenovirus and fluorescence lifetime imaging microscopy. Co-transfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity three-fold relative to the empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1+/- mice were fed high fat diet for 8 weeks. Isolated islets from these mice demonstrated increased expression of spliced Xbp1, signifying ER stress, while subsequent SERCA2b overexpression in isolated islets reduced spliced Xbp1 levels to that of wild-type controls. These results identify SERCA2b as a direct transcriptional target of Pdx-1 and define a novel role for altered ER Ca2+ regulation in Pdx-1 deficient states.