Este trabajo fue financiado por becas de la Deutsche Forschungsgemeinschaft (DFG), Fritz Thyssen-Stiftung y Charité / MDC.

1. Animal Anestesia y sustracción de órganos <ol><li> Anestesiar el ratón por vía intraperitoneal (ip) de inyección de ketamina (200 mg / kg de peso corporal) y xilazina (20 mg / kg BW). </li><li> Para anticoagular inyectar ip 250 UI de heparina para evitar la formación de trombos y coágulos sanguíneos. </li><li> Esperar hasta que la narcosis se alcanza, que se caracteriza por arreflexia. Para comprobar si hay prueba de reflejo arreflexia corneal tocando suavemente la córnea o el reflejo vuelo de prueba de pellizco cola. </li><li> Transferir el ratón sobre mesa de operaciones y fijarlo en posición supina. </li><li> Incisión de la piel y la pared abdominal por debajo del apéndice xifoides y realizar una toracotomía clamshell: Extender el corte a lo largo de ambos lados del arco costal y posteriormente cortar las costillas en la línea axilar media, desviar hacia arriba la caja torácica. </li><li> Abra pericardio, localizar los grandes vasos. Presione suavemente caudal corazón para mostrar mejor la aorta. Sujete la aorta utilizando fórceps. </li><li> Colocar el corazón en la concavidad de un par de s<html> cissors y diseccionar todos los barcos que conectan con un solo corte. Asegúrese de preservar una parte suficientemente grande de la aorta ascendente para la canulación de Langendorff. </li><li> Transferir corazón extirpados inmediatamente a una placa de Petri llena de hielo frío y pre-oxigenada solución 1 (para soluciones, véase la Tabla 1). </li></ol> 2. Preparación de perfusión del corazón y Langendorff <ol><li> Canular la aorta con una cánula de acero 1.8F. Asegúrese de evitar una embolia gaseosa. </li><li> Fije aorta en la cánula con una sutura quirúrgica y coronarias ras con 1 ml de solución 1. </li><li> Conectar la cánula con un aparato Langendorff. </li><li> Asegúrese de que el tiempo de toracotomía de la canulación Langendorff no exceda de 120 segundos para evitar extendida por isquemia / reperfusión en el miocardio. </li><li> Corazón perfundir con 10 ml de Ca 2 + libre solución 2 (4 ml / min). </li><li> Corazón perfundir con solución de colagenasa 3 de 8 min (4 ml / min). </li></ol>le &quot;&gt; 3. Microsurgical disociación de Cardiac Cámaras <ol><li> Transferencia de corazón en una pre-calentado 100-mm placa de Petri que contiene bajo Ca 2 + solución 4. </li><li> Retire con cuidado la aorta y otros no-tejido cardíaco con unas tijeras y lo descarta. </li><li> Aurículas y los ventrículos separados y continuar con cada cámara por separado. Mantener las células sumergidas en solución 4. Utilizar volúmenes pequeños (menos de 5 ml). </li></ol> 4. Disociación Además de los cardiomiocitos <ol><li> Cardiomiocitos auriculares: <ol><li> Para individualizar miocitos auriculares transferir las aurículas en un aparte precalentado placa de cultivo de 100 mm y disociar el tejido a través de él con cuidado separando con unas pinzas finas. Asegurar una disociación casi completa del tejido. </li><li> Usar una pipeta de 1 ml con una vista ampliada pulida al fuego punta de pipeta de plástico para suspender las células en 1 ml de solución de 5 por 5 min. </li><li> Se separan las células de los desechos mediante el uso de un filtro celular (200 micras de malla). </li> &lt;li&gt; Añadir 5 ml de solución 5 a la suspensión de células y se centrifuga durante 2 min a 16 xg a la temperatura ambiente. </li><li> Los siguientes pasos se hacen funcionar bajo una campana de cultivo de células. Eliminar el sobrenadante y se resuspendió el sedimento en 5 ml de solución 6. </li><li> Después de la sedimentación por gravedad durante 10 min en un tubo de 15 ml, centrifugar durante 1 min a 16 x g a temperatura ambiente. Eliminar el sobrenadante. Volver a suspender las células en función de su cantidad en 1-5 ml de solución 6. </li></ol></li><li> Cardiomiocitos ventriculares: <ol><li> Diseccionar la región ventricular izquierda de interés con unas pinzas finas en 5 ml de solución 4. </li><li> Suspender las células pipeteando suavemente hasta que la mayoría de las células se separan. Transferir la solución de células después de la filtración (200 micras de malla) en un tubo de 50 ml, añadir un volumen de 25 ml. </li><li> Centrifugar durante 2 min a 16 xg a la temperatura ambiente. </li><li> Los siguientes pasos se hacen funcionar bajo una campana de cultivo de células. Remover el sobrenadante, resuspender el precipitado en 25 ml desolución de 6 y permitir la sedimentación de las células durante 10 min. </li><li> Contar las células y eliminar el sobrenadante, añadir 25 a 50 ml de solución 6 en las células. </li></ol></li></ol> 5. Preparación de cardiomiocitos de Electrofisiología Celular, Bioquímica o si los estudios <ol><li> Para los estudios de electrofisiología, mantener los miocitos en solución en un tubo de 6 ml y 50 inhiben la sedimentación. </li><li> Para los estudios bioquímicos, formación de imágenes por ejemplo, calcio, miocitos placa sobre laminina plato recubierto cultivo de células (concentración final 20 mg / ml de laminina en PBS). </li><li> Para la tinción de inmunofluorescencia preparar un cultivo de células placa plato con cubreobjetos de vidrio y recubierto con una solución de laminina (concentración final 50 mg / ml de laminina en PBS). <ol><li> Retire la solución antes de miocitos galvanoplastia. Placa de las células y controlar la densidad de las células utilizando un microscopio. </li><li> Deje que los miocitos se adhieren a cubreobjetos durante 1 hora a 37 ° C en 2% de CO 2, eliminar la solucióny comenzar inmediatamente con un protocolo de tinción procedimiento estándar. </li><li> Incubar con fijador, por ejemplo, 4% de PFA en PBS (pH 7,5) durante 10 min a temperatura ambiente y sigue con tres pasos de lavado con PBS durante 5 min cada uno. </li><li> Para permeabilizar las células y de inhibir la unión de anticuerpos inespecíficos incubar los miocitos con 10% de suero, 0,3% de Triton, 0,2% de BSA en PBS durante 30 min a temperatura ambiente. </li><li> Incubar con el anticuerpo primario durante 1 hora a 37 ° C y se lavan como se ha descrito antes. </li><li> Se incuba con el anticuerpo secundario durante 1 hr a temperatura ambiente. Para contrateñir los núcleos y α-actinina uso DAPI y phalloidin fluorocromo conjugado (Alexa Fluor 488, Invitrogen). </li><li> Después del lavado, transferir la cubierta de cristal se desliza con cuidado en silano portaobjetos tratados y las células incrustar en medio de fluorescencia de montaje. </li></ol></li></ol> 6. Electrofisiología Celular <ol><li> Realizar Plenario de células patch-clamp grabaciones en frecuenciashly aislado cardiomiocitos auriculares y ventriculares a temperatura ambiente. </li><li> Transferir sanos cardiomiocitos que aparecen en la cámara de perfusión llena con un volumen definido de la solución de baño extracelular. 117 mM NaCl, 4 mM KCl, 1 mM KH 2 PO 4, 4 mM NaHCO 3, 1,7 mM de MgCl 2, 3 mM CoCl 2, HEPES 10 mM, glucosa 10 mM, y 0,02 mM de tetrodotoxina (TTX), (pH 7,4) . Utilice NaOH para ajustar el pH de valor. </li><li> Utilice el equipo de patch clamp (es decir IX71 microscopio invertido, un amplificador 700B MultiClamp, un sistema Digidata 1440A adquisición y PC con pClamp10.3 software (Molecular Devices)). </li><li> Usar pipetas de patch con resistencias de 3-5 mW cuando se llena con una solución intracelular contenía 130 mM KCl, 2 mM de MgCl 2, 11 mM HEPES, 11 mM EGTA, 5 mM Na 2 ATP, 0,4 mM Na 2 GTP, 5 mM Na 2 CP y 4,9 mM CaCl 2 (pH 7,2). Use KOH para el ajuste del valor de pH. </li><li> Evocar exterior K + corrientes duanillo 4,5-sec pasos de voltaje para probar potenciales entre -60 y +50 mV en incrementos de 10-mV desde un potencial de mantenimiento de -70 mV después de un 20-mseg prepulso a -40 mV. </li><li> Asegúrese de que las corrientes de fuga son siempre &lt;100 pA. </li><li> Para la disección de K + corrientes utilizar diferentes inhibidores específicos tales como 4-aminopiridina (4-AP; ICN Biomedicals, Irvine, CA, EE.UU.), Heteropodatoxin 2 (HpTx2; Alomone) o tetraetilamonio (TEA, Sigma). Las soluciones madre se preparan en solución extracelular baño, y se aplica directamente a la zona más cercana posible de la célula a través de una micropunta después de &quot;base&quot; grabaciones. El equilibrio se debe permitir durante 2-3 min antes de &quot;medicamentos&quot; grabaciones. </li><li> Para el análisis de normalizar las amplitudes de corriente en las células individuales a tamaño celular (células enteras capacitancia de la membrana). Analizar los datos sin conexión mediante pClamp10.3 software (Molecular Devices) o software similar. </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>

No tenemos nada que revelar.

Con el desarrollo creciente de cepas de ratón genéticamente modificados para el estudio de la función cardiaca y la patología cardíaca relacionada con una deleción del gen también hay un creciente interés en métodos especializados para estudiar los efectos de la deleción del gen específico in vitro. En nuestro laboratorio se estudia el papel de una familia de canales Kv subunidades accesorias sobre la repolarización cardiaca. Los genes KCNE comprenden una familia de 5 genes (KCNE1-5)</e...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td></tr><tr><td> La tetrodotoxina </td><td> Alomone </td></tr><tr><td> 4-aminopiridina </td><td> ICN Biomedicals </td></tr><tr><td> Heteropodatoxin 2 </td><td> Alomone </td></tr><tr><td> Tetraetilamonio </td><td> Sigma Chemicals </td></tr><tr><td> Colagenasa de tipo 2 </td><td> Worthington </td></tr></tbody></table>

isolation and kv channel recordings in murine atrial and ventricular cardiomyocytes

KCNE genes codifican para una pequeña familia de canales Kv subunidades auxiliares que forman complejos con heteroméricos Kv canal subunidades alfa para modificar sus propiedades funcionales. Las mutaciones en los genes de KCNE se han encontrado en pacientes con arritmias cardiacas tales como el síndrome de QT largo y / o la fibrilación auricular. Sin embargo, la fisiopatología precisa molecular que conduce a estas enfermedades sigue siendo difícil de alcanzar. En estudios previos de las propiedades electrofisiológicas de la enfermedad que causa mutaciones en estos genes han sido estudiados principalmente en sistemas de expresión heterólogos y no podemos estar seguros de si los efectos reportados directamente se puede traducir en cardiomiocitos nativos. En nuestro laboratorio, por lo tanto, utilizar un enfoque diferente. Nuestros estudiar los efectos de la supresión de genes KCNE en cardiomiocitos aislados de los ratones knockout de electrofisiología celular - una técnica única que se describe en este número de la Revista de experimentos visualizados. Los corazones de la geneticaLLY diseñaron ratones KCNE se extirparon rápidamente y se montaron en un aparato Langendorff por canulación aórtica. Gratis Ca 2 + en el miocardio está obligado por EGTA, y la disociación de los miocitos cardíacos se consigue después de una perfusión retrógrada de las arterias coronarias con un especializado bajo Ca 2 + que contiene tampón de colagenasa. Atria, la pared libre del ventrículo derecho y el ventrículo izquierdo se puede separar por técnicas de microcirugía. El calcio se añade entonces lentamente de nuevo a cardiomiocitos aislados en un paso múltiple que comprende procedimiento de lavado. Cardiomiocitos auriculares y ventriculares de apariencia sana sin contracciones espontáneas son entonces inmediatamente se sometió a análisis electrofisiológicos mediante la técnica de patch clamp o otros análisis bioquímicos dentro de las primeras 6 horas después del aislamiento.

Aislamiento de cardiomiocitos adultos murinos procedentes de ratones modificados genéticamente para estudiar la función de genes específicos de interés in vitro se ha convertido en una herramienta poderosa para entender mejor la fisiopatología cardíaca. Este método es utilizado actualmente por sólo un número pequeño pero creciente de laboratorios de ciencias básicas en todo el mundo. Sin embargo, el aislamiento de cardiomiocitos ventriculares adultos murinos puede ser complicado y necesita ser hecho ...

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

Kv canal disfunción se asocia con arritmias cardíacas. Con el fin de estudiar los mecanismos moleculares que conducen a tales arritmias que utilizan un protocolo sistemático para el aislamiento de cardiomiocitos auriculares y ventriculares de Kv de canal ratones knockout auxiliares de subunidades. Cardiomiocitos aislados a continuación, puede ser utilizado inmediatamente para estudios electrofisiológicos celulares, bioquímicos o inmunofluorescencia (IF) ensayos.

Aislamiento y grabaciones Kv Canal murinos en miocitos auriculares y ventriculares

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

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13.3K vistas.  Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC). Kv canal disfunción se asocia con arritmias cardíacas. Con el fin de estudiar los mecanismos moleculares que conducen a tales arritmias que utilizan un protocolo sistemático para el aislamiento de cardiomiocitos auriculares y ventriculares de Kv de canal ratones knockout auxiliares de subunidades. Cardiomiocitos aislados a continuación, puede ser utilizado inmediatamente para estudios electrofisiológicos celulares, bioquímicos o inmunofluorescencia (IF) ensayos.

Video: 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.

El análisis fenotípico y aislamiento de células madre hematopoyéticas murinas y progenitores comprometidos Lineage

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

Investigación

Biología

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.

Aislamiento y cultivo de cardiomiocitos neonatales Ratón

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.

La reconstitución de un canal Kv en las membranas lipídicas de Estudios estructurales y funcionales

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.

Aislamiento y fisiológica Análisis de Ratón cardiomiocitos

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.

Aislamiento y cultivo de ratón adulto cardiomiocitos de Señalización Celular y<em&gt; In vitro</em&gt; Hipertrofia Cardíaca

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 celular conectado a Patch-clamp grabación

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.

Reconstitución funcional y la actividad del canal Las mediciones de Purificada Wildtype y mutante CFTR Proteína

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.

Tensión y calcio Dual Channel Mapping óptico de Cultivadas HL-1 auricular miocitos monocapa

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.

Aislamiento, cultivo y la transducción de cardiomiocitos de ratones adultos

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.

Potencial de acción óptica específica de subtipo grabaciones en humanos inducida por cardiomiocitos ventriculares derivadas de células madre pluripotentes