Ce travail a été financé par des subventions de la Deutsche Forschungsgemeinschaft (DFG), Fritz-Thyssen-Stiftung et la Charité / MDC.

1. L&#39;anesthésie des animaux et des prélèvements d&#39;organes <ol><li> Anesthésier la souris par voie intrapéritonéale (ip) d&#39;injection de kétamine (200 mg / kg de poids corporel) et de xylazine (20 mg / kg de poids corporel). </li><li> Pour anticoagulation injecter 250 UI d&#39;héparine ip pour éviter la formation de thrombus et la coagulation du sang. </li><li> Attendre jusqu&#39;à ce que la narcose profonde est atteinte, qui est caractérisé par une aréflexie. Pour vérifier les réflexes de test aréflexie, la cornée par un léger toucher la cornée ou réflexe vol d&#39;essai par pincement de la queue. </li><li> Transférer la souris sur table d&#39;opération et le fixer en position couchée. </li><li> Inciser la peau et la paroi abdominale au-dessous de l&#39;appendice xiphoïde et effectuer une thoracotomie preneuse: étendre la coupe de part et d&#39;autre le long de l&#39;arc costal et ensuite couper nervures dans la ligne axillaire médiane, dévier vers le haut la cage thoracique. </li><li> Ouvrez le péricarde, localiser gros vaisseaux. Appuyez doucement sur caudale cœur pour mieux afficher l&#39;aorte. Serrer l&#39;aorte aide d&#39;une pince. </li><li> Placer le coeur dans la concavité d&#39;une paire de s<html> ciseaux et disséquer tous les navires de connexion avec une seule coupe. Assurez-vous de conserver une partie assez grande de l&#39;aorte ascendante pour la ponction de Langendorff. </li><li> Transfert cœur excisé immédiatement à une boîte de Pétri remplie de glace froide et pré-oxygéné solution 1 (pour les solutions, voir le tableau 1). </li></ol> 2. Préparation de la perfusion cardiaque et Langendorff <ol><li> Cathétériser l&#39;aorte avec une canule en acier 1.8F. Assurez-vous d&#39;éviter une embolie gazeuse. </li><li> Fixer l&#39;aorte sur la canule avec une suture chirurgicale et coronaires rincer avec 1 ml de solution à 1. </li><li> Connectez canule avec un appareil de Langendorff. </li><li> Assurez-vous que le temps d&#39;une thoracotomie pour canulation Langendorff ne dépasse pas 120 secondes pour éviter prolongée d&#39;ischémie / reperfusion du myocarde. </li><li> Coeur perfuse avec 10 ml de solution de Ca 2 + libre 2 (4 ml / min). </li><li> Coeur perfuser avec une solution de collagénase 3 pour 8 min (4 ml / min). </li></ol>le &quot;&gt; 3. microchirurgicale dissociation des chambres cardiaques <ol><li> Coeur de transfert dans un préchauffé à 100 mm boîte de Pétri contenant une faible solution de Ca 2 + 4. </li><li> Retirez délicatement l&#39;aorte et d&#39;autres non cardiaque tissu avec des ciseaux et jetez-le. </li><li> Oreillettes et les ventricules séparés et continuent à chaque chambre séparément. Gardez les cellules immergées dans la solution 4. Utilisez de petits volumes (moins de 5 ml). </li></ol> 4. La dissociation des cardiomyocytes plus <ol><li> Cardiomyocytes auriculaires: <ol><li> Pour individualiser les cardiomyocytes auriculaires transférer les oreillettes dans un séparé préchauffé boîte de culture de 100 mm et dissocier le tissu à travers le tirant doucement à part avec une pince fine. Veiller à une dissociation presque complète du tissu. </li><li> Utiliser une pipette de 1 ml avec un plus polie au feu embout de pipette en plastique pour suspendre les cellules dans 1 ml de solution à 5 pendant 5 min. </li><li> Séparer les cellules de débris à l&#39;aide d&#39;un filtre de cellules (200 um de maille). </li> &lt;li&gt; Ajouter 5 ml de solution 5 à la suspension de cellules et centrifuger 2 min à 16 xg à température ambiante. </li><li> Les étapes suivantes sont conduites sous une hotte de culture cellulaire. Jeter le surnageant et remettre en suspension le culot dans 5 ml de solution à 6. </li><li> Après sédimentation par gravité pendant 10 min dans un tube de 15 ml, centrifuger pendant 1 min à 16 xg à température ambiante. Enlever le surnageant. Remettre en suspension les cellules en fonction de leur quantité dans 1-5 ml de solution à 6. </li></ol></li><li> Cardiomyocytes ventriculaires: <ol><li> Disséquer la région du ventricule gauche d&#39;intérêt avec une pince fine dans 5 ml de la solution 4. </li><li> Suspendre les cellules en pipetant doucement jusqu&#39;à ce que la plupart des cellules sont séparées. Transférer la solution cellule après filtrage (200 maillage um) dans un tube de 50 ml, ajouter un volume de 25 ml. </li><li> Centrifuger pendant 2 minutes à 16 xg à température ambiante. </li><li> Les étapes suivantes sont conduites sous une hotte de culture cellulaire. Retirer le surnageant, remettre en suspension le culot dans 25 ml d&#39;6 solution et de permettre une sédimentation des cellules pendant 10 min. </li><li> Compter les cellules et éliminer le surnageant, ajouter 25 à 50 ml de solution 6 sur les cellules. </li></ol></li></ol> 5. Préparation des cardiomyocytes pour cellulaire électrophysiologie, biochimie ou si les études <ol><li> Pour les études d&#39;électrophysiologie, garder les myocytes en solution à 6 dans un tube de 50 ml et empêcher la sédimentation. </li><li> Pour les études biochimiques, d&#39;imagerie de calcium, par exemple, les myocytes plaque sur la laminine plat recouvert de culture cellulaire (concentration finale 20 pg / ml de laminine dans du PBS). </li><li> Pour immunofluorescence préparer une plaque de culture cellulaire plat avec des lamelles de verre et enduit avec une solution de laminine (concentration finale de 50 pg / ml de laminine dans du PBS). <ol><li> Retirer la solution avant de myocytes de placage. Plaque les cellules et contrôler la densité de cellules à l&#39;aide d&#39;un microscope. </li><li> Laissez les myocytes adhérer à lamelle pendant 1 heure à 37 ° C dans 2% de CO 2, enlever la solutionet à lancer immédiatement un protocole procédure de coloration standard. </li><li> Incuber avec un fixateur, par exemple 4% de PFA dans du PBS (pH 7,5) pendant 10 min à température ambiante et de suivre avec PBS trois étapes de lavage pour 5 min chacun. </li><li> Pour perméabiliser les cellules et d&#39;inhiber la liaison non spécifique d&#39;anticorps incuber les myocytes avec 10% de sérum, Triton 0,3%, BSA 0,2% dans du PBS pendant 30 min à température ambiante. </li><li> Incuber avec l&#39;anticorps primaire pendant 1 heure à 37 ° C et laver comme indiqué auparavant. </li><li> Incuber avec l&#39;anticorps secondaire pendant 1 heure à température ambiante. Pour contre-coloration des noyaux et l&#39;utilisation α-actinine fluorochrome DAPI et phalloïdine conjugué (Alexa Fluor 488, Invitrogen). </li><li> Après le lavage, transférer le couvercle en verre glisse prudemment sur des lames de microscope silane traitées et les cellules embed dans un milieu de montage fluorescence. </li></ol></li></ol> 6. Electrophysiologie cellulaire <ol><li> Effectuer cellules entières patch-clamp sur la fréquenceshly isolé cardiomyocytes auriculaires et ventriculaires à la température ambiante. </li><li> Transférer en bonne santé cardiomyocytes apparaissant dans la chambre de perfusion remplie d&#39;un volume défini de solution de bain extracellulaire. 117 mM de NaCl, 4 mM de KCl, 1 mM KH 2 PO 4, 4 mM NaHCO 3, 1,7 mM MgCl 2, 3 mM de CoCl 2, 10 mM HEPES, 10 mM de glucose et 0,02 mM tétrodotoxine (TTX), (pH 7,4) . Utilisez NaOH pour ajuster le pH. </li><li> Utiliser un matériel de patch-clamp (c.-à-IX71 microscope inversé, un amplificateur Multiclamp 700B, un système d&#39;acquisition Digidata 1440A et PC avec le logiciel pClamp10.3 (Molecular Devices)). </li><li> N&#39;utiliser que des pipettes de patch avec des résistances de 3-5 MQ quand il est rempli avec une solution intracellulaire contenant 130 mM de KCl, 2 mM de MgCl2, 11 mM HEPES, 11 mM d&#39;EGTA 5 mM Na 2 ATP, 0,4 mM Na 2 GTP, 5 mM Na 2 CP et 4,9 mM de CaCl 2 (pH 7,2). Utilisez KOH pour l&#39;ajustement du pH. </li><li> Evoke courants K + vers l&#39;extérieur duescaliers du ring tension de 4,5 sec pour tester des potentiels compris entre -60 et +50 mV à 10 mV par incréments de-un potentiel de maintien de -70 mV après un intervalle de 20 ms à -40 mV prepulse. </li><li> Assurez-vous que les courants de fuite sont toujours &lt;100 pA. </li><li> Pour la dissection de K + différents courants utiliser des inhibiteurs spécifiques tels que le 4-aminopyridine (4-AP, ICN Biomedicals, Irvine, CA, USA), Heteropodatoxin 2 (HpTx2; Alomone) ou tétraéthylammonium (TEA; Sigma). Les solutions mères doivent être préparés en solution de bain extracellulaire, et appliqué directement sur le voisinage le plus proche possible de la cellule via une micropointe après «de base» des enregistrements. L&#39;équilibrage devraient être autorisés pendant 2-3 min avant «drogue» des enregistrements. </li><li> Pour l&#39;analyse de normaliser les amplitudes de courant dans des cellules individuelles à la taille des cellules (capacitance de la membrane de cellule entière). Analyser les données hors connexion en utilisant le logiciel pClamp10.3 (Molecular Devices) ou un logiciel comparable. </li></ol>

<ol>
	<li>Abbott, G. W., Goldstein, S. A., Sesti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+all+voltage-gated+potassium+channels+use+MiRPs?.">Do all voltage-gated potassium channels use MiRPs?.</a> Circ. Res. 88, 981-983 (2001).</li><li>Abbott, G. W., Sesti, F., Splawski, I., Buck, M. E., Lehmann, M. H., Timothy, K. W., Keating, M. T., Goldstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRP1+forms+IKr+potassium+channels+with+HERG+and+is+associated+with+cardiac+arrhythmia.">MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia.</a> Cell. 97, 175-187 (1999).</li><li>Barry, D. M., Trimmer, J. S., Merlie, J. P., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+voltage-gated+K++channel+subunits+in+adult+rat+heart.+Relation+to+functional+K++channels?.">Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels?.</a> Circ. Res. 77, 361-369 (1995).</li><li>Benjamin, E. J., Wolf, P. A., D'Agostino, R. B., Silbershatz, H., Kannel, W. B., Levy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+atrial+fibrillation+on+the+risk+of+death:+the+Framingham+Heart+Study.">Impact of atrial fibrillation on the risk of death: the Framingham Heart Study.</a> Circulation. 98, 946-952 (1998).</li><li>Bou-Abboud, E., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+correlates+of+the+calcium-independent,+depolarization-activated+K++currents+in+rat+atrial+myocytes.">Molecular correlates of the calcium-independent, depolarization-activated K+ currents in rat atrial myocytes.</a> J. Physiol. 517 (Pt 2), 407-420 (1999).</li><li>Gaborit, N., Steenman, M., Lamirault, G., Le, M. N., Le, B. S., Lande, G., Leger, J., Charpentier, F., Christ, T., Dobrev, D., Escande, D., Nattel, S., Demolombe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+atrial+ion+channel+and+transporter+subunit+gene-expression+remodeling+associated+with+valvular+heart+disease+and+atrial+fibrillation.">Human atrial ion channel and transporter subunit gene-expression remodeling associated with valvular heart disease and atrial fibrillation.</a> Circulation. 112, 471-481 (2005).</li><li>Guo, W., Xu, H., London, B., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+transient+outward+K++current+diversity+in+mouse+ventricular+myocytes.">Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes.</a> J. Physiol. 521 (Pt 3), 587-599 (1999).</li><li>Lundby, A., Ravn, L. S., Svendsen, J. H., Hauns, S., Olesen, S. P., Schmitt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCNE3+mutation+V17M+identified+in+a+patient+with+lone+atrial+fibrillation.">KCNE3 mutation V17M identified in a patient with lone atrial fibrillation.</a> Cell Physiol. Biochem. 21, 47-54 (2008).</li><li>Ma, K. J., Li, N., Teng, S. Y., Zhang, Y. H., Sun, Q., Gu, D. F., Pu, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+KCNQ1+current+by+atrial+fibrillation-associated+KCNE4+(145E/D)+gene+polymorphism.">Modulation of KCNQ1 current by atrial fibrillation-associated KCNE4 (145E/D) gene polymorphism.</a> Chin Med. J. (Engl.). 120, 150-154 (2007).</li><li>McCrossan, Z. A., Abbott, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MinK-related+peptides.">The MinK-related peptides.</a> Neuropharmacology. 47, 787-821 (2004).</li><li>Pongs, O., Schwarz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancillary+subunits+associated+with+voltage-dependent+K++channels.">Ancillary subunits associated with voltage-dependent K+ channels.</a> Physiol. Rev. 90, 755-796 (2010).</li><li>Ravn, L. S., Aizawa, Y., Pollevick, G. D., Hofman-Bang, J., Cordeiro, J. M., Dixen, U., Jensen, G., Wu, Y., Burashnikov, E., Haunso, S., Guerchicoff, A., Hu, D., Svendsen, J. H., Christiansen, M., Antzelevitch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain+of+function+in+IKs+secondary+to+a+mutation+in+KCNE5+associated+with+atrial+fibrillation.">Gain of function in IKs secondary to a mutation in KCNE5 associated with atrial fibrillation.</a> Heart Rhythm. 5, 427-435 (2008).</li><li>Ravn, L. S., Hofman-Bang, J., Dixen, U., Larsen, S. O., Jensen, G., Haunso, S., Svendsen, J. H., Christiansen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+97T+polymorphism+in+KCNE5+to+risk+of+atrial+fibrillation.">Relation of 97T polymorphism in KCNE5 to risk of atrial fibrillation.</a> Am. J. Cardiol. 96, 405-407 (2005).</li><li>Roepke, T. K., Abbott, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacogenetics+and+cardiac+ion+channels.">Pharmacogenetics and cardiac ion channels.</a> Vascul. Pharmacol. 44, 90-106 (2006).</li><li>Roepke, T. K., Kontogeorgis, A., Ovanez, C., Xu, X., Young, J. B., Purtell, K., Goldstein, P. A., Christini, D. J., Peters, N. S., Akar, F. G., Gutstein, D. E., Lerner, D. J., Abbott, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+deletion+of+kcne2+impairs+ventricular+repolarization+via+disruption+of+I(K,slow1)+and+I(to,f).">Targeted deletion of kcne2 impairs ventricular repolarization via disruption of I(K,slow1) and I(to,f).</a> FASEB J. 22, 3648-3660 (2008).</li><li>Sesti, F., Abbott, G. W., Wei, J., Murray, K. T., Saksena, S., Schwartz, P. J., Priori, S. G., Roden, D. M., George, A. L., Goldstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+polymorphism+associated+with+antibiotic-induced+cardiac+arrhythmia.">A common polymorphism associated with antibiotic-induced cardiac arrhythmia.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 10613-10618 (2000).</li><li>Temple, J., Frias, P., Rottman, J., Yang, T., Wu, Y., Verheijck, E. E., Zhang, W., Siprachanh, C., Kanki, H., Atkinson, J. B., King, P., Anderson, M. E., Kupershmidt, S., Roden, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+fibrillation+in+KCNE1-null+mice.">Atrial fibrillation in KCNE1-null mice.</a> Circ. Res. 97, 62-69 (2005).</li><li>Yang, Y., Xia, M., Jin, Q., Bendahhou, S., Shi, J., Chen, Y., Liang, B., Lin, J., Liu, Y., Liu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+KCNE2+gain-of-function+mutation+in+patients+with+familial+atrial+fibrillation.">Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation.</a> Am. J. Hum. Genet. 75, 899-905 (2004).</li><li>Zeng, Z. Y., Pu, J. L., Tan, C., Teng, S. Y., Chen, J. H., Su, S. Y., Zhou, X. Y., Zhang, S., Li, Y. S., Wang, F. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[The+association+of+single+nucleotide+polymorphism+of+slow+delayed+rectifier+K++channel+genes+with+atrial+fibrillation+in+Han+nationality+Chinese].">[The association of single nucleotide polymorphism of slow delayed rectifier K+ channel genes with atrial fibrillation in Han nationality Chinese].</a> Zhonghua Xin. Xue. Guan. Bing. Za Zhi. 33, 987-991 (2005).</li><li>Zhang, M., Jiang, M., Tseng, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=minK-related+peptide+1+associates+with+Kv4.2+and+modulates+its+gating+function:+potential+role+as+beta+subunit+of+cardiac+transient+outward+channel?+Circ.">minK-related peptide 1 associates with Kv4.2 and modulates its gating function: potential role as beta subunit of cardiac transient outward channel? Circ.</a> Circ. Res. 88, 1012-1019 (2001).</li></ol>

Nous n&#39;avons rien à déclarer.

Avec le développement croissant de souches de souris génétiquement modifiées pour étudier la fonction cardiaque et une pathologie cardiaque liée à la délétion du gène il ya aussi un intérêt croissant pour les méthodes spécialisées pour étudier les effets de la délétion du gène spécifique in vitro. Dans notre laboratoire, nous étudions le rôle d&#39;une famille de sous-unités de canaux Kv auxiliaires sur la repolarisation cardiaque. Les gènes KCNE comprennent une famille...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td></tr><tr><td> La tétrodotoxine </td><td> Alomone </td></tr><tr><td> 4-aminopyridine </td><td> ICN Biomedicals </td></tr><tr><td> Heteropodatoxin 2 </td><td> Alomone </td></tr><tr><td> Tetraethylammonium </td><td> Sigma Chemicals </td></tr><tr><td> Type de collagénase 2 </td><td> Worthington </td></tr></tbody></table>

isolation and kv channel recordings in murine atrial and ventricular cardiomyocytes

Gènes codent KCNE pour une petite famille de sous-unités de canaux Kv auxiliaires qui forment des complexes avec des sous-unités hétéromériques Kv canal alpha de modifier leurs propriétés fonctionnelles. Des mutations dans les gènes KCNE ont été trouvés chez les patients souffrant d&#39;arythmies cardiaques telles que le syndrome du QT long et / ou la fibrillation auriculaire. Cependant, la physiopathologie moléculaire précise qui mène à ces maladies reste insaisissable. Dans des études précédentes, les propriétés électrophysiologiques de la maladie provoquant des mutations dans ces gènes ont surtout été étudiée dans des systèmes d&#39;expression hétérologues et nous ne pouvons pas être sûr si les effets rapportés peuvent être directement traduits en cardiomyocytes natifs. Dans notre laboratoire, nous utilisons donc une approche différente. Nous étudier directement les effets de la délétion du gène KCNE dans les cardiomyocytes isolés de souris knock-out par électrophysiologie cellulaire - une technique unique que nous décrivons dans ce numéro de la Revue des expériences visualisées. Les cœurs de geneticasouris KCNE lly modifiées sont rapidement excisé et monté sur un appareil de Langendorff par canulation aortique. Ca 2 + libre dans le myocarde est lié par l&#39;EGTA, et la dissociation des myocytes cardiaques est alors réalisée par perfusion rétrograde des artères coronaires avec un faible spécialisée Ca 2 + tampon contenant de la collagénase. Atria, paroi libre du ventricule droit et le ventricule gauche peut alors être séparés par des techniques de microchirurgie. Le calcium est ensuite ajouté lentement vers les cardiomyocytes isolés dans une étape multiple comprenant procédure de lavage. Cardiomyocytes auriculaires et ventriculaires d&#39;apparence saine sans contractions spontanées sont alors immédiatement soumis à des analyses électrophysiologiques par la technique de patch-clamp ou d&#39;autres analyses biochimiques au cours des 6 premières heures qui suivent l&#39;isolement.

Isolement des adultes cardiomyocytes murins de souris génétiquement modifiées pour étudier la fonction des gènes spécifiques d&#39;intérêt in vitro est devenue un outil puissant pour mieux comprendre la physiopathologie cardiaque. Cette méthode est actuellement utilisée par un nombre faible mais croissant de laboratoires de sciences fondamentales à travers le monde. Cependant, l&#39;isolement des cardiomyocytes ventriculaires adultes murines peut être difficile et doit être fait avec soin ...

Watch this Scientific Journal Video about Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes at JoVE.com

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Dysfonctionnement des canaux Kv est associée à des arythmies cardiaques. Afin d&#39;étudier les mécanismes moléculaires qui conduisent à des arythmies telles que nous utilisons un protocole systématique pour l&#39;isolement des cardiomyocytes auriculaires et ventriculaires chez des souris knock-out canaux Kv auxiliaires sous-unités. Cardiomyocytes isolés peuvent alors être immédiatement utilisé pour les études électrophysiologiques cellulaires, biochimiques ou immunofluorescence (IF) essais.

Isolement et enregistrements Chaîne Kv en cardiomyocytes murins auriculaire et ventriculaire

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

isolation-kv-channel-recordings-murine-atrial-ventricular

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13.3K vues.  Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC). Dysfonctionnement des canaux Kv est associée à des arythmies cardiaques. Afin d'étudier les mécanismes moléculaires qui conduisent à des arythmies telles que nous utilisons un protocole systématique pour l'isolement des cardiomyocytes auriculaires et ventriculaires chez des souris knock-out canaux Kv auxiliaires sous-unités. Cardiomyocytes isolés peuvent alors être immédiatement utilisé pour les études électrophysiologiques cellulaires, biochimiques ...

Vidéo: Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Analyse phénotypique et isolement de cellules souches hématopoïétiques murines et Lineage-engagés progéniteurs

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

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Biologie

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolement et culture de cardiomyocytes de souris néonatales

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

Reconstitution d&#39;un canal Kv dans les membranes lipidiques des études structurales et fonctionnelles

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

L&#39;isolement et l&#39;analyse physiologique de cardiomyocytes de souris

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Isolement et culture de cardiomyocytes adultes de souris pour Signalisation cellulaire et<em&gt; In vitro</em&gt; Hypertrophie cardiaque

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Un canal cellulaire attaché patch-clamp enregistrement

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Reconstitution fonctionnelle et Activité du Chan mesures de type sauvage purifiée et Mutant protéine CFTR

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Tension et calcium double cartographie de canal optique de Cultured HL-1 auriculaire myocytes monocouche

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

L&#39;isolement, la culture et la transduction des adultes cardiomyocytes souris

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

Enregistrements de potentiel d’Action optique sous-type spécifique dans l’homme induit pluripotentes cellules souches dérivées des Cardiomyocytes ventriculaires