La bioénergétique systémique moléculaire des cellules cardiaques (la relation structure-fonction dans la régulation du métabolisme énergétique compartmentalisé)

An important element of metabolic regulation of cardiac and skeletal muscle energetics is the interaction of mitochondria with cytoskeleton. Mitochondria are in charge of supplying the cells with energy, adjusting its functional activity under conditions of stress or other aspects of life. Mitochondria display a tissue-specific distribution. In adult rat cardiomyocytes, mitochondria are arranged regularly in a longitudinal lattice at the level of A band between the myofibrils and located within the limits of the sarcomeres. In interaction with cytoskeleton, sarcomeres and sarcoplasmic reticulum they form the functional complexes, the intracellular energetic units (ICEUs). The ICEUs have specialized pathways of energy transfer and metabolic feedback regulation between mitochondria and ATPases, mediated by CK and AK. The central structure of ICEUs is the mitochondrial interactosome (MI) containing ATP Synthasome, respiratory chain, mitochondrial creatine kinase and VDAC, regulated by tubulins. The main role of MI is the regulation of respiration and the intracellular energy fluxes via phosophotransfer networks. The regulation of ICEUs is associated with structural proteins. The association of mitochondria with several cytoskeletal proteins described by several groups has brought to light the importance of structure-function relationship in the metabolic regulation of adult rat cardiomyocytes. To purvey a better understanding of these findings, the present work investigated the mechanism of energy fluxes control and the role of structure-function relationship in the metabolic regulation of adult rat cardiomyocytes. To show these complex associations in adult cardiac cells several proteins were visualized by confocal microscopy: a-actinin and b-tubulin isotypes. For the first time, it was showed the existence of the specific distribution of b-tubulin isotypes in adult cardiac cells. Respiratory measurements were performed to study the role of tubulins in the regulation of oxygen consumption. These results together confirmed the crucial role of cytoskeletal proteins -i.e. tubulins, a-actinin, plectin, desmin, and others- for the normal shape of cardiac cells as well as mitochondrial arrangement and regulation. In addition, in vivo - in situ mitochondrial dynamics were studied by the transfection of GFP-a-actinin, finding that fusion phenomenon does not occur as often as it is believed in healthy adult cardiac cells.Un élément important de la régulation du métabolisme énergétique des muscles cardiaque et squelettiques est l'interaction des mitochondries avec le cytosquelette. Les mitochondries sont responsables de l'approvisionnement des cellules en énergie, elles sont capables d'ajuster leur activité fonctionnelle en fonction des conditions de stress ou d'autres aspects de la vie. Les mitochondries ont une distribution spécifique selon les tissus. Dans les cardiomyocytes de rats adultes, les mitochondries sont disposées régulièrement dans un entrelacement longitudinal au niveau des bandes A, entre les myofibrilles et dans les limites des sarcomères. En interaction avec le cytosquelette, le sarcomère et le réticulum sarcoplasmique, elles forment des complexes fonctionnels appelés unités énergétiques intracellulaires (ICEUs). Les ICEUs ont des voies spécialisées de transfert d'énergie et de régulation des feedback métaboliques entre les mitochondries et les ATPases, médiée par la CK et l'AK. La structure centrale des ICEUs est l'interactosome mitochondrial (MI) qui confient l'ATP synthasome, la chaîne respiratoire, la créatine kinase mitochondriale et VDAC, qui pourrait être régulé par les tubulines. Le rôle principal du MI est la régulation de la respiration et des flux d'énergie intracellulaires via les réseaux de phosphotransfert. La régulation des ICEUs est liée aux protéines structurales. L'association des mitochondries avec plusieurs protéines du cytosquelette, décrite par plusieurs groupes, a mis en évidence l'importance de la relation structure-fonction dans la régulation métabolique des cardiomyocytes de rats adultes. Pour fournir une meilleure compréhension de ces résultats, le présent travail étudie le mécanisme de contrôle des flux d'énergie et le rôle des relations structure-fonction dans la régulation métabolique de cardiomyocytes de rats adultes. Pour montrer ces associations complexes dans les cellules cardiaques adultes, plusieurs protéines ont été visualisées par microscopie confocale: l'a-actinine et les isoformes des b-tubulines. Pour la première fois, l'existence d'une distribution spécifique des isoformes de b-tubuline dans les cellules cardiaques adultes a été montré. Des mesures respiratoires ont été réalisées pour étudier le rôle des tubulines dans la régulation de la consommation d'oxygène. Ces résultats ont confirmé le rôle déterminant des protéines du cytosquelette -tubulines, a-actinine, plectine, desmine, et autres- pour le maintien de la forme normale des cellules cardiaques, ainsi que de l'arrangement et de la régulation mitochondrial. En outre, la dynamique mitochondriale a été étudiée in vivo et in situ par la transfection de la GFP-a-actinine, ceci permettant la mise en évidence du fait que le phénomène de fusion ne se produit pas aussi souvent qu'on ne le croit pour des cellules cardiaques adultes en bonne santé.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Multi-confocal Fluorescence Correlation Spectroscopy : experimental demonstration and potential applications for living cell measurements

We report, for the first time, a multi-confocal Fluorescence Correlation Spectroscopy (mFCS) technique which allows parallel measurements at different locations, by combining a Spatial Light Modulator (SLM), with an Electron Multiplying-CCD camera (EM-CCD). The SLM is used to produce a series of laser spots, while the pixels of the EM-CCD play the roles of virtual pinholes. The phase map addressed to the SLM is calculated by using the spherical wave approximation and makes it possible to produce several diffraction limited laser spots, either aligned or spread over the field of view. To attain fast enough imaging rates, the camera has been used in different acquisition modes, the fastest of which leads to a time resolution of 100 $\mu$s. We qualified the experimental set-up by using solutions of sulforhodamine G in glycerol and demonstrated that the observation volumes are similar to that of a standard confocal set-up. To demonstrate that our mFCS method is suitable for intracellular studies, experiments have been conducted on two stable cell lines: mouse embryonic fibroblasts expressing eGFP-actin and H1299 cells expressing the heat shock factor fusion protein HSF1-eGFP. In the first case we could recover, by analyzing the auto-correlation curves, the diffusion constant of G-actin within the cytoplasm, although we were also sensitive to the complex network of interactions with F-actin. Concerning HSF1, we could clearly observe the modifications of the number of molecules and of the HSF1 dynamics during heat shock

An Analytical Fiber ODF Reconstruction in 3D Polarized Light Imaging

International audienceThree dimensional polarized light imaging (3D-PLI) utilizes the birefringence in postmortem tissue to map its spatial fiber structure at a submillimeter resolution. We propose an analytical method to compute the fiber orientation distribution function (ODF) from high-resolution vector data provided by 3D-PLI. This strategy enables the bridging of high resolution 3D-PLI to diffusion magnetic resonance imaging with relatively low spatial resolution. First, the fiber ODF is modeled as a sum of K orientations on the unit sphere and expanded with a high order spherical harmonics series. Then, the coefficients of the spherical harmonics are derived directly with the spherical Fourier transform. We quantitatively validate the accuracy of the reconstruction against synthetic data and show that we can recover complex fiber configurations in the human heart at different scales

3D-Voronoi Diagramme zur quantitativen Bildanalyse in der Interphase-Cytogenetik

Um die Anordnung von Chromosomen in Zellkernen der Interphase zu untersuchen, wurde ein Verfahren aus der Computergeometrie adaptiert. Dieser Ansatz basiert auf der Zerlegung von dreidimensionalen Bildvolumen mithilfe des Voronoi-Diagramms in konvexe Polyeder. Die graphenorientierte, geometrische Struktur dieses Verfahrens ermöglicht sowohl eine schnelle Extraktion von Objekten im Bildraum als auch die Berechnung morphologischer Parameter wie Volumina, Oberflächen und Rundheitsfaktoren. In diesem Beitrag wird exemplarisch die dreidimensionale Morphologie von XChromosomen in weiblichen Interphasezellkernen mithilfe dieser drei Parameter untersucht. Um diese Zellkerne mit lichtoptischen Methoden zu untersuchen, wurden die Territorien der X-Chromosomen mit einem molekularcytogenetischen Verfahren fluoreszierend dargestellt. Zur Unterscheidung des aktiven und inaktiven X-Chromosoms wurde das Barr-Körperchen zusätzlich markiert und mithilfe eines Epifluoreszenzmikroskops, ausgerüstet mit einer CCD-Kamera, aufgenommen. Anschließend wurden 1 2 - 2 5 äquidistante, lichtoptische Schnitte der X-Chromosomenterritorien mit einem konfokalen Laser Scanning Mikroskop (CLSM) aufgenommen. Diese lichtoptischen Schnitte wurden mithilfe des Voronoi-Verfahrens segmentiert und analysiert. Methoden aus der Computergraphik wurden zur Visualisierung der Ergebnisse eingesetzt. Es konnte gezeigt werden, daß mithilfe des Voronoi-Verfahrens Chromosomen- Territorien anhand der morphologischen Parameter zuverlässig beschrieben werden können

The Myosin Myocardial Mesh Interpreted as a Biological Analogous of Nematic Chiral Liquid Crystals

International audienceThis article is an open access article distributed under the terms and conditions of the Creative Commons Attribution .There are still grey areas in the understanding of the myoarchitecture of the ventricularmass. This is despite the progress of investigation methods since the beginning of the 21st century(diffusion tensor magnetic resonance imaging, microcomputed tomography, and polarised lightimaging). The objective of this article is to highlight the specificities and the limitations of polarisedlight imaging (PLI) of the unstained myocardium embedded in methyl methacrylate (MMA). Thus,to better differentiate our method from other PLI modes, we will refer to it by the acronym PLI-MMA.PLI-MMA shows that the myosin mesh of the compact left ventricular wall behaves like a biologicalanalogous of a nematic chiral liquid crystal. Results obtained by PLI-MMA are: the main directionof the myosin molecules contained in an imaged voxel, the crystal liquid director n, and a regionalisotropy index RI that is an orientation tensor, the equivalent of the crystal liquid order parameter.The vector n is collinear with the first eigenvector of diffusion tensor imaging (DTI-MRI). The RIhas not been confounded with the diffusion tensor of DTI that gives information about the threeeigenvectors of the ellipsoid of diffusion. PLI-MMA gives no information about the collagen network.The physics of soft matter has allowed the revisiting of Streeter’s conjecture on the myoarchitectureof the compact left ventricular wall: “geodesics on a nested set of toroidal surfaces”. Once thetorus topology is understood, this characterisation of the myoarchitecture is more accurate andparsimonious than former descriptions. Finally, this article aims to be an enthusiastic invitationto a transdisciplinary approach between physicists of liquid crystals, anatomists, and specialistsof imaging

The Nematic Chiral Liquid Crystal Structure of the Cardiac Myoarchitecture: Disclinations and Topological Singularities

International audienceThis is our second article devoted to the cardiac myoarchitecture considered as a nematic chiral liquid crystal (NCLC). While the first article focused on the myoarchitecture of the left ventricle (LV), this new article extends to the whole ventricular mass and introduces the concept of disclinations and topological singularities, which characterize the differences and relationships between the left and right ventricles (RV). At the level of the ventricular apices, we constantly observed a vortex shape at the LV apex, corresponding, in the terminology of liquid crystals, to a "+1 disclination"; we never observed this at the RV apex. At the level of the interventricular septum (IVS), we identified "−1/2 disclinations" at the anterior and posterior parts. During the perinatal period, there was a significant difference in their distribution, with more "−1/2 disclinations" in the posterior part of the IVS. After birth, concomitant to major physiological changes, the number of "−1/2 disclinations" significantly decreased, both in the anterior and posterior parts of the IVS. Finally, the description of the disclinations must be considered in any attempt to segment the whole ventricular mass, in biomechanical studies, and, more generally, for the characterization of myocardial remodeling