Además, agradecemos a los cirujanos cardiacos en la Universidad de Heidelberg para la prestación de tejido auricular humano y Claudia Liebetrau, Katrin Kupser y Ramona Nagel por su excelente apoyo técnico. Un agradecimiento especial también a Andy W. Trafford por sus útiles sugerencias y consejos durante el establecimiento de las medidas transitorias 2 + Ca. Los autores desean expresar su más profundo agradecimiento a los miembros del Departamento de Farmacología y Toxicología (cabeza: Ursula Ravens) de la Universidad Técnica de Dresde por la oportunidad que nos ofrecen para aprender las técnicas básicas y habilidades en electrofisiología celular y el aislamiento de cardiomiocitos. Investigación de los autores con el apoyo de la Fundación Alemana para la Investigación (Do769/1-1-3), el Ministerio Federal Alemán de Educación e Investigación a través de la red de competencia Fibrilación Atrial (01Gi0204) y el Centro Alemán de Investigación Cardiovascular, la Unión Europea a través de la ueRed ropeo de Investigación Traslacional en la fibrilación auricular (EUTRAF, FP7-HEALTH-2010, el proyecto de integración a gran escala, la Propuesta N º 261057) y la Alianza para la Investigación Fibrilación Atrial Europa-América del Norte (ENAFRA) concesión de Fondation Leducq (07CVD03). La visión esquemática se muestra en el archivo de vídeo se produjo usando técnica médica Servier.

Los protocolos experimentales deben ser aprobados por el comité de ética local y todos los pacientes deben dar su consentimiento informado por escrito. Nuestra investigación fue aprobado por el comité de ética de la Facultad de Medicina de Mannheim, Universidad de Heidelberg (# 2011-216N-MA) y se llevó a cabo de acuerdo con todas las directrices institucionales, nacionales e internacionales para el bienestar humano. Todos los pacientes dieron su consentimiento informado por escrito. 0. Obtención de tejido auricular humano Durante los procedimientos de canulación de rutina en pacientes sometidos a cirugía a corazón abierto para injerto de bypass cardiopulmonar, la punta del apéndice auricular derecho generalmente se extirpa y se puede utilizar para el aislamiento de miocitos auriculares. Después de la escisión de la muestra de tejido se transfiere inmediatamente a un tubo de 50 ml Falcon estéril con Ca 2 + sin solución de transporte que contiene 2,3-butanodiona monoxima (Tabla I; BDM, inhibidor de la contracción, la prevención de los miocitos contracture). Con tiempos de transporte entre los 30 y 45 min, no reconocimos una clara ventaja de refrigeración u oxigenación con respecto al número y calidad de los miocitos auriculares aislados. En general de transporte al laboratorio debe ser lo más rápido posible. Sin embargo, si es más largo el tiempo de transporte no se puede evitar, de transporte a 4 ° C en solución oxigenada podría ser ventajoso. 1. Prearrangements <ol><li> Encender el thermocirculator, que controla la temperatura del vaso de precipitados con camisa (Tabla II). Asegúrese de que la temperatura dentro del recipiente es igual a 37 ° C. Cubrir el vaso con una tapa de vidrio para mantener una temperatura constante dentro del vaso de precipitados a lo largo de todo el proceso de aislamiento. </li><li> Pese la colagenasa I y XXIV proteasa en tres vasos de vidrio y guárdelo a temperatura ambiente. Las enzimas se diluyeron justo antes de su uso. </li><li> Tienda 20 ml de Ca 2 + solución libre en una placa de Petri a 4 ° C. </li><li>Tienda 250 ml de Ca 2 + solución libre en un baño de agua a 37 ° C. </li></ol> 2. Limpieza del tejido <ol><li> Transferir la muestra de tejido, junto con la solución de transporte en una placa de Petri (~ 10 cm de diámetro) y eliminar el tejido graso utilizando más o menos unas tijeras. </li><li> Pesar la muestra de tejido. Entre 200 y 600 mg se utilizan para el aislamiento de células. El tejido restante se puede congelar en nitrógeno líquido para su posterior análisis bioquímico. </li><li> Transferir la muestra de tejido en la placa de Petri que contiene 20 ml de solución de Ca 2 + libre a 4 º C (véase el paso 1.3) y cortar la muestra de tejido en trozos pequeños de aproximadamente 1 mm de tamaño 3. </li><li> Los siguientes pasos deben ser ejecutados a 37 ° C y bajo gasificación continua con O2 al 100%. Una punta de la pipeta se puede colocar sobre el tubo de oxígeno para evitar la fuerte burbujeo y generar un flujo de aire continuo. </li><li> La transferencia de los trozos de tejido junto con la solución 2 + libre de Ca enal vaso de precipitados con camisa y se agita cuidadosamente durante aproximadamente 3 minutos con una barra de agitación magnética. </li><li> Detener la agitación y permitir que los trozos de tejido para establecerse durante unos segundos. Colar con cuidado el sobrenadante a través de una malla de nylon (200 micras, Tabla II). Volver a los trozos de tejido restringidas de la malla en el vaso con unas pinzas. </li><li> Vuelva a llenar el vaso con Ca 2 + solución gratuita y repetir la etapa de lavado 2,4 y 2,5 dos veces. </li></ol> 3. En primer lugar la digestión enzimática <ol><li> Vuelva a suspender los trozos de tejido con 20 ml de solución de enzima E1 (Tabla I) que contiene colagenasa y proteasa y se agita cuidadosamente durante 10 min. </li><li> Añadir 40 l de 10 mM CaCl 2-solución para obtener una concentración final de 20 mM Ca 2 +. </li><li> Después de 35 min la cepa cuidadosamente el sobrenadante a través de una malla de nylon (200 micras, Tabla II) de una manera que la mayor parte de los trozos de tejido permanecen en el vaso de precipitados. Volver restollovió trozos de tejido de la malla en el vaso de precipitados. </li></ol> 4. En segundo lugar digestión enzimática <ol><li> Resuspender los trozos de tejido de nuevo con 20 ml de solución de enzima E2 que contiene colagenasa I sólo (Tabla I). Añadir 40 l de 10 mM CaCl 2-solución inmediatamente para obtener una concentración final de 20 mM Ca 2 +. </li><li> Durante el segundo tijeras para cortar el uso de digestión más coágulos tejido ocasionalmente. </li><li> Después de 5 minutos tomar una muestra con una pipeta Pasteur para comprobar la disociación de las células. Repita esto cada 2-3 minutos hasta que aparezcan en forma de barra, cardiomiocitos estriados. </li><li> Detener la agitación y permitir que los trozos de tejido que se establecen por unos 20 a 30 segundos. </li><li> Colar el líquido sobrenadante con cuidado a través de una malla de nylon (200 m, Tabla II) en un tubo de 50 ml Falcon (Tubo A). Volver a los trozos de tejido restringidas de la malla en el vaso. </li><li> Vuelva a suspender los trozos de tejido en el vaso con 20 ml Storasolución de GE (Tabla I) 10 y disociar aún más las células por trituración mecánica suave con una pipeta serológica de 20 ml con dispensador. Trate de evitar la formación de burbujas ya que las burbujas son perjudiciales para la supervivencia celular y la calidad. </li><li> Colar el líquido sobrenadante con cuidado a través de una malla de nylon (200 m, Tabla II) en un tubo de 50 ml Falcon (Tubo B). </li></ol> 5. Preparación final y ajuste final de Ca 2 + de concentración <ol><li> Centrifugar tanto en tubos Falcon de A y B (véase el paso 4.5 y 4.7) a 95 xg durante 10 min. </li><li> Eliminar el sobrenadante de ambos tubos cuidadosamente por aspiración. Asegúrese de no perturbar el sedimento. Descartar el sobrenadante. </li><li> Vuelva a suspender las dos pastillas de 1,5 ml solución de almacenamiento (Tabla I) cada uno (temperatura ambiente). </li><li> Agregue dos veces 7,5 l de solución de 10 mM CaCl 2 a cada Falcon y remover con cuidado. Incubar durante 10 min después de cada paso. </li><li>Añadir 15 l de solución 10 mM de CaCl2 para obtener una final concentración de Ca2 + de 0,2 mM. </li></ol> 6. Carga de los miocitos con el Ca 2 +-fluorescente indicador Fluo-3 AM (Figura 1) <ol><li> Transferir 1,5 ml de suspensión celular (tubo A y / o el tubo B) en un tubo de microcentrífuga de 2 ml (tubo Eppendorf). </li><li> Los siguientes pasos deben ser ejecutados bajo la consideración de la sensibilidad a la luz del fluorescente de Ca 2 + Indicador Fluo-3. </li><li> Disolver 50 g de la membrana permeable derivado de éster de acetoximetilo de Fluo-3 (Fluo-3 AM, Tabla III) en 44 l de Pluronic F-127 (Tabla III) solución madre (20% w / v en DMSO anhidro) para conseguir un 1 mM de Fluo-3 a.m. solución madre, que puede ser almacenado a -20 ° C durante un máximo de 1 semana. </li><li> Añadir 15 l de la Fluo-3 a.m. solución madre al tubo de microcentrífuga que contiene 1,5 ml de suspensión de células (véase el paso 6.1) y agitarcuidadosamente. </li><li> Incubar la suspensión de células durante 10 min en una caja ópticamente opaca. </li><li> Centrifugar brevemente a aproximadamente 6.000 rpm. </li><li> Descartar el sobrenadante y resuspender el precipitado en 1,5 ml de solución de baño (Tabla IV). </li><li> Agregar la suspensión de células durante aproximadamente 30 min para la de-esterificación antes de comenzar con los experimentos. </li></ol> 7. Patch-clamp y epifluorescencia Ca 2 + mediciones simultáneas Dado que las mediciones de patch-clamp no son el tema principal de esta revisión, remitimos al lector interesado a otras publicaciones que proporcionan una descripción más profunda de esta técnica. 11-14 En aras de la exhaustividad, se proporciona un breve resumen de un protocolo para medir potenciales de acción o L-tipo Ca 2 + corrientes, ambos junto con grabaciones simultáneas Ca 2 +-transitorios. Durante los experimentos miocitos se superfundieron a 37 ° C con bañera solutien (Tabla IV) usando un sistema de perfusión rápida (Octaflow IITM, ALA Scientific Instruments, NY). Para los experimentos de fijación de voltaje, corrientes de K + se bloquean mediante la adición de 4-aminopiridina (5 mmol / L) y BaCl 2 (0,1 mmol / L) a la solución del baño. Microelectrodos de vidrio de borosilicato se utilizan y deben inclinar han resistencias de 2-5 mW cuando se llena con la solución de la pipeta (Tabla V). Además de la Fluo-3 a.m. carga de los miocitos (vea el paso 6), Fluo-3 también se incluye en la solución de la pipeta (Tabla V). Fluorescencia se excita a 488 nm y la luz emitida (&lt;520 nm) convierte en [Ca 2 +] i suponiendo <img alt="Ecuación 1" fo:content-width="1.540in" fo:src="/files/ftp_upload/50235/50235eq1highres.jpg" src="/files/ftp_upload/50235/50235eq1.jpg" /> donde k d = constante de disociación de Fluo-3 (864 nM), F = Fluo-3 fluorescencia;. F max = Ca 2 +-saturada de fluorescencia obtenido al final de cada experimento 12 Ambas señales eléctricas y señales de Ca 2 + epifluorescencia se registran simultáneamente. Los potenciales de acción son estimulados a 0,5 Hz en modo de corriente-clamp con 1 ms pulsos de corriente de fuerza umbral de 1,2 x. De tipo L Ca 2 +-corrientes se miden en el modo de fijación de voltaje con un potencial de mantenimiento de -80 mV y una rampa de 100 mseg-pulso a -40 mV para inactivar el ayuno de Na +-corriente, seguido de un 100-mseg prueba de pulso a 10 mV a 0,5 Hz.

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Res. 66, 1277-1286 (1990).</li><li>Hove-Madsen, L., 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=Atrial+fibrillation+is+associated+with+increased+spontaneous+calcium+release+from+the+sarcoplasmic+reticulum+in+human+atrial+myocytes.">Atrial fibrillation is associated with increased spontaneous calcium release from the sarcoplasmic reticulum in human atrial myocytes.</a> Circulation. 110, 1358-1363 (2004).</li><li>Molina, C. E., 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=Cyclic+adenosine+monophosphate+phosphodiesterase+type+4+protects+against+atrial+arrhythmias.">Cyclic adenosine monophosphate phosphodiesterase type 4 protects against atrial arrhythmias.</a> J. Am. Coll. Cardiol. 59, 2182-2190 (2012).</li></ol>

Worthington Biochemical Corp. apoyó esta publicación cubriendo los costos de producción del archivo de vídeo. Los financiadores no tenía papel en el diseño del estudio, recogida y análisis de datos, decisión a publicar, o la preparación del manuscrito.

Aquí se describe un método para el aislamiento de miocitos auriculares humanos de apéndices auriculares adecuados obtenidos a partir de pacientes sometidos a cirugía de corazón abierto. Para utilizar estos miocitos para las mediciones de citosólica de Ca 2 + se adaptó un método descrito previamente 4-11 omitiendo EGTA de la solución de almacenamiento. Ya en 1970 se observó que a pesar de los miocitos se disocian en presencia de Ca 2 + durante la digestión, todos ellos estaban en la contractura y no viables. 16,17 Por lo tanto, el aislamiento de células se realiza en solución de Ca 2 + libre. Sin embargo, la re-introducción de concentraciones fisiológicas de Ca 2 + se tradujo en un rápido Ca 2 + afluencia y la muerte celular. Esto ha sido descrito como el fenómeno de Ca 2 + paradoja que se observó originalmente en corazones perfundidos por Zimmerman y Hulsman. 18 Las modificaciones de los medios de aislamiento, incluyendo la reducción del pH a 7,0, &lt;sup&gt; 19 adición de taurina 20 o de pequeñas cantidades de Ca 2 + (véase el paso 3.2 y 4.1), 21 así como almacenamiento de miocitos aislados en almacenamiento que contienen EGTA-solución 22 se han sugerido para evitar que el Ca 2 + paradoja. 17 Sin embargo, es bien sabido que el Ca 2 + tampón a través de EGTA reduce la amplitud de la L-tipo Ca 2 + Ca 2 + inducida por la corriente amplitudes de transitorios y los resultados en un decaimiento bifásica de los transitorios de Ca 2 +. 23 Por lo tanto, hemos omitido EGTA lo largo de todo el proceso de aislamiento con el fin de obtener Ca 2 + transitorios con las propiedades típicas y decae monofásicos. Para proteger las células de la Ca 2 + paradoja aumentamos el final de concentración de Ca2 + de la solución de almacenamiento de una manera escalonada hasta 0,2 mM. La elección de la colagenasa es probablemente el paso más crítico para el aislamiento de miocitos éxito. Coll convencionalagenases son preparaciones crudas obtenidas a partir de Clostridium histolyticum y contienen colagenasa, además de un número de otras proteinasas, polisacaridasas y lipasas. Con base en sus colagenasas generales de composición se subdividen en distintos tipos. 24 Worthington colagenasa tipo I y II se han utilizado con éxito para el aislamiento de los miocitos auriculares humanos. 4-10,15,25-30 En el protocolo descrito actualmente se recomienda el uso de colagenasa Tipo I, aunque también hemos sido capaces de obtener cantidades aceptables de células viables utilizando colagenasa tipo II. Sin embargo, incluso dentro de un solo tipo colagenasa hay una variación de lote a lote significativa con respecto a las actividades enzimáticas. Estas variaciones requieren la selección de lote y comprobar cuidadosamente los diversos lotes para optimizar el procedimiento de aislamiento. La herramienta dispone de lotes de selección en línea de Worthington Biochemical Corp. ( <a href="http://www.worthington-biochem.com/cls/match.php" target="_blank">http://www.worthington-bio</a>chem.com / cls / match.php) puede ser usado para encontrar los lotes disponibles con una composición que ha demostrado ser adecuado para el aislamiento de miocitos auriculares humanos. Actualmente utilizamos colagenasa tipo I con 250 U / mg de actividad de la colagenasa, U / mg de actividad caseinase 345 U / mg de actividad clostripaína 2,16 y 0,48 U / mg de actividad tríptico (lote # 49H11338). Las células obtenidas usando el procedimiento descrito en este manuscrito se pueden utilizar dentro de 8 horas para los estudios de patch-clamp, Ca 2 + mediciones de transitorios y una combinación de ambos. 15 Además, estas células permiten mediciones de la contracción celular en respuesta a la estimulación de campo eléctrico o la estimulación eléctrica utilizando la pipeta de patch-clamp (observaciones no publicadas).

El estudio de las propiedades electrofisiológicas de los canales iónicos cardíacos con la técnica de patch-clamp y la exploración de Ca2 + celular anomalías de manejo requieren cardiomiocitos aislados. Estos se obtienen normalmente después de la exposición in vitro de muestras de tejidos cardiacos a las enzimas digestivas (colagenasa, hialuronidasa, peptidasa, etc). Desde el primer informe de aislamiento de los miocitos cardíacos viables en 1955 2 una gran cantidad de protocolos ha sido desarrollado con el fin de cosechar individuales cardiomiocitos auriculares y ventriculares a partir de diferentes especies, incluyendo ratón, rata, conejo, perro, conejillo de indias y humanos. En esta revisión, nos centramos en el aislamiento de los miocitos auriculares humanos. En cuanto a los procedimientos para el aislamiento de los miocitos de otras especies nos referimos a la &quot;Worthington Tissue disociación guía&quot; proporcionada por Worthington Biochemical Corp., EE.UU. ( <a href="http://www.tissuedissociation.com" target="_blank">www.tissuedissociation.com</a> ). et al. 3 A continuación presentamos una descripción paso a paso de una técnica, la cual está adaptada a partir de un método publicado previamente, con el fin de obtener miocitos auriculares adecuados no sólo para los experimentos de patch-clamp, sino también por Ca 2 + intracelular mediciones simultáneas. 4-11

<table border="1">
 <tbody>
 <tr>
 <td> &nbsp;</td>
 <td> &nbsp;</td>
 <td> &nbsp;</td>
 <td>Transport solution</td>
 </tr>
 <tr>
 <td>Albumin</td>
 <td>Sigma-Aldrich</td>
 <td>A3059</td>
 <td>Storage solution: 1%</td>
 </tr>
 <tr>
 <td>BDM</td>
 <td>Sigma-Aldrich</td>
 <td>31550</td>
 <td>Transport solution: 30</td>
 </tr>
 <tr>
 <td>DL-b-Hydroxy-butyric acid</td>
 <td>Sigma-Aldrich</td>
 <td>H6501</td>
 <td>Storage solution: 10</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td>Transport solution: 20 
 Ca2+-free solution: 20 
 Enzyme solution E1 and E2: 20 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>L-Glutamic acid</td>
 <td>Sigma-Aldrich</td>
 <td>G1251</td>
 <td>Storage solution: 70</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Transport solution: 10 
 Ca2+-free solution: 10 
 Enzyme solution E1 and E2: 10 
 Storage solution: 20</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>Sigma-Aldrich</td>
 <td>P5655</td>
 <td>Transport solution: 1.2 
 Ca2+-free solution: 1.2 
 Enzyme solution E1 and E2: 1.2 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>MgSO4</td>
 <td>Sigma-Aldrich</td>
 <td>M9397</td>
 <td>Transport solution: 5 
 Ca2+-free solution: 5 
 Enzyme solution E1 and E2: 5</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Sigma-Aldrich</td>
 <td>M1254</td>
 <td>Transport solution: 5 
 Ca2+-free solution: 5 
 Enzyme solution E1 and E2: 5</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S3014</td>
 <td>Transport solution: 100 
 Ca2+-free solution: 100 
 Enzyme solution E1 and E2: 100</td>
 </tr>
 <tr>
 <td>Taurin</td>
 <td>Sigma-Aldrich</td>
 <td>86330</td>
 <td>Transport solution: 50 
 Ca2+-free solution: 50 
 Enzyme solution E1 and E2: 50 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>Collagenase I</td>
 <td>Worthington</td>
 <td>4196</td>
 <td>Enzyme solution E1 and E2: 286 U/ml</td>
 </tr>
 <tr>
 <td>Protease XXIV</td>
 <td>Sigma-Aldrich</td>
 <td>P8038</td>
 <td>Enzyme solution E1 and E2: 5 U/ml*</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Transport solution: 7.00 
 Ca2+-free solution: 7.00 
 Enzyme solution E1 and E2: 7.00 
 Storage solution: 7.40</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Transport solution: 1 M NaOH 
 Ca2+-free solution: 1 M NaOH 
 Enzyme solution E1 and E2: 1 M NaOH 
 Storage solution: 1 M KOH</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Concentrations in mM unless otherwise stated. BDM, 2,3-Butanedione monoxime. *Protease XXIV is included in Enzyme solution E1 only.</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table I. Solutions.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nylon mesh (200 &mu;m)</td>
 <td>VWR-Germany</td>
 <td>510-9527</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Jacketed reaction beaker</td>
 <td>VWR</td>
 <td>KT317000-0050</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table II. Specific equipment.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dimethyl-sulphoxide</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluo-3 AM (special packaging)</td>
 <td>Invitrogen</td>
 <td>F-1242</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pluronic F-127</td>
 <td>Invitrogen</td>
 <td>P6867</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table III. Substances for loading of myocytes with Fluo-3 AM.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Bath solution</td>
 </tr>
 <tr>
 <td>4-aminopyridine*</td>
 <td>Sigma-Aldrich</td>
 <td>A78403</td>
 <td>Bath solution: 5</td>
 </tr>
 <tr>
 <td>BaCl2*</td>
 <td>Sigma-Aldrich</td>
 <td>342920</td>
 <td>Bath solution: 0.1</td>
 </tr>
 <tr>
 <td>CaCl2 &times; 2H2O</td>
 <td>Sigma-Aldrich</td>
 <td>C5080</td>
 <td>Bath solution: 2</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td>Bath solution: 10</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H9136</td>
 <td>Bath solution: 10</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Bath solution: 4</td>
 </tr>
 <tr>
 <td>MgCl &times; 6H2O</td>
 <td>Sigma-Aldrich</td>
 <td>M0250</td>
 <td>Bath solution: 1</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S3014</td>
 <td>Bath solution: 140</td>
 </tr>
 <tr>
 <td>Probenecid</td>
 <td>Sigma-Aldrich</td>
 <td>P8761</td>
 <td>Bath solution: 2</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bath solution: 7.35</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bath solution: 1 M HCl</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table IV. Bath solution for patch-clamp. *4-aminopyridine and BaCl were included for voltage-clamp experiments only.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Pipette solution</td>
 </tr>
 <tr>
 <td>DL-aspartat K+-salt</td>
 <td>Sigma-Aldrich</td>
 <td>A2025</td>
 <td>Pipette solution: 92</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma-Aldrich</td>
 <td>E4378</td>
 <td>Pipette solution: 0.02</td>
 </tr>
 <tr>
 <td>GTP-Tris</td>
 <td>Sigma-Aldrich</td>
 <td>G9002</td>
 <td>Pipette solution: 0.1</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H9136</td>
 <td>Pipette solution: 10</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Pipette solution: 48</td>
 </tr>
 <tr>
 <td>MgATP</td>
 <td>Sigma-Aldrich</td>
 <td>A9187</td>
 <td>Pipette solution: 1</td>
 </tr>
 <tr>
 <td>Na2ATP</td>
 <td>Sigma-Aldrich</td>
 <td>A2383</td>
 <td>Pipette solution: 4</td>
 </tr>
 <tr>
 <td>Fluo-3**</td>
 <td>Invitrogen</td>
 <td>F3715</td>
 <td>Pipette solution: 0.1</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>7.20</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>1 M KOH</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table V. Pipette solution for patch-clamp*. *On experimental days pipette solution is stored on ice until use. **Fluo-3 is added from a 1 mM stock solution on experimental days.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of human atrial myocytes for simultaneous measurements of ca2+ transients and membrane currents

El estudio de las propiedades electrofisiológicas de los canales iónicos cardíacos con la técnica de patch-clamp y la exploración de celular cardiaca Ca 2 + anomalías manejo requiere cardiomiocitos aislados. Además, la posibilidad de investigar los miocitos de los pacientes que utilizan estas técnicas es un requisito muy valiosa para dilucidar la base molecular de las enfermedades cardiacas tales como la fibrilación auricular (FA). 1 Aquí se describe un método para el aislamiento de miocitos auriculares humanos que son adecuados para ambos estudios de patch-clamp y mediciones simultáneas de 2 concentraciones de Ca + intracelular. En primer lugar, apéndices auriculares adecuados obtenidos de pacientes sometidos a cirugía a corazón abierto se cortan en trozos pequeños de tejido (&quot;método trozo&quot;) y se lavaron en solución de Ca 2 + libre. A continuación, los trozos de tejidos se digieren en colagenasa y proteasa que contiene soluciones con 20 mM de Ca 2 +. A partir de entonces, los miocitos aislados son harvested por filtración y la centrifugación de la suspensión de tejido. Por último, la concentración de Ca2 + en la solución de almacenamiento celular se ajusta paso a paso a 0,2 mM. Se discute brevemente el significado de Ca 2 + y Ca 2 + amortiguación durante el proceso de aislamiento y también proporcionar grabaciones representativas de los potenciales de acción y las corrientes de membrana, tanto con simultánea Ca 2 + transitorios mediciones, realizadas en estos miocitos aislados.

Figura 2A muestra tres ejemplos representativos de miocitos aislados de aurícula derecha. Para cuantificar el rendimiento de células que pipeta de 10 l de suspensión celular (paso 5.5) en un portaobjetos CellFinder ( <a href="http://www.antenna.nl/microlab/index-uk.html" target="_blank">http://www.antenna.nl/microlab/index-uk.html</a> ). Rendimientos celulares promediados en la Figura 2B indican claramente que hay una tendencia a la baja en los rendimientos celulares FA crónica (CAF) muestras de pacientes (tubo A: 16,5 ± 3,1 células/10 l (n = 29) vs 5.1 ± 2.3 l células/10 (n = 10) en la SR y la CAF, respectivamente, p &lt;0,05; tubo B: 17,9 ± 3,9 células/10 l (n = 29) frente a 5,9 ± 2,0 células/10 l (n = 9) en SR y la CAF, respectivamente, p = 0,107). Los ejemplos representativos de las mediciones del potencial de acción y grabaciones simultáneas de citosólica de Ca 2 + transitorios se dan en la Figura 3. En aproximadamente el 90% de las células investigadas, tque la acción potencial desencadenado Ca 2 + en libertad hace que las contracciones de células claras y regulares. Como se informó anteriormente, el potencial de membrana en reposo, que es un indicador aceptado de la integridad celular, promedio de alrededor de -73,9 ± 2,7 mV (n = 23/10 / miocitos pacientes) y -77,7 ± 1,8 mV (n = 19/8 / miocitos pacientes ) en la SR y la CAF, respectivamente (p&gt; 0,05). 15 Figura 4 muestra grabaciones simultáneas representativas de voltaje de tipo L de Ca 2 + corrientes y citosólica de Ca 2 + transitorios. Aplicación de la isoprenalina agonista β-adrenérgico no selectivo (1 M) aumenta amplitudes de ambas Ca I, L y citosólica de Ca 2 + transitorios, lo que sugiere intacta β-adrenérgico cascada de transducción de señal. <img alt="Figura 1" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig1highres.jpg" src="/files/ftp_upload/50235/50235fig1.jpg" /> Figura 1. Diagrama de flujo de los miocitos Fluo-03 a.m. cargando protocolo (véase el paso 6.1 a 6.5). m / v, masa / volumen. <img alt="La figura 2" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig2highres.jpg" src="/files/ftp_upload/50235/50235fig2.jpg" /> Figura 2. A, miocitos aislados de aurícula derecha después de una hora en una solución de almacenamiento. B, Media ± SEM del rendimiento de células contadas en 10 l de células en solución de almacenamiento (véase el paso 5.5). n se refiere al número de preparaciones dentro de cada grupo. * P &lt;0,05. <img alt="Figura 3" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig3highres.jpg" src="/files/ftp_upload/50235/50235fig3.jpg" /> Figura 3. Grabaciones representativas del potencial de acción activada por Ca 2 +-transitorios (CAT) en un miocito auricular de un ritmo sinusal y la Comisión de Derechos Humanoslos pacientes de fibrilación auricular ónico. Top: corriente inyectada membrana (I M) que se utiliza para la estimulación (0,5 Hz). Abajo: Grabación simultánea de potencial de membrana (V M), y el gato activado (abajo). (Dibujado de nuevo con el permiso de Voigt et al. 2012) 15 <img alt="Figura 4" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig4highres.jpg" src="/files/ftp_upload/50235/50235fig4.jpg" /> Figura 4. Grabaciones representativas de la (1 M) efecto de isoprenalina en L-tipo Ca 2 + corriente activada por Ca 2 +-transitorios (CAT) en un miocito auricular de un ritmo sinusal y un paciente de la fibrilación auricular crónica. Top: Voltage-clamp protocolo (0,5 Hz). Abajo: Grabación simultánea de la membrana total neto actual (I M), principalmente como resultado del tipo L Ca 2 + CaT actual (centro) y provocó (abajo). (Dibujado de nuevo con el permiso de Voigt,et al. 2012) 15 <img src="/files/ftp_upload/50235/50235table1.jpg" alt="Tabla 1" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235table1highres.jpg" /> Tabla I. Solutions. <img src="/files/ftp_upload/50235/50235table2.jpg" alt="Tabla 2" fo:content-width="2in" fo:src="/files/ftp_upload/50235/50235table2highres.jpg"/> Tabla II. Equipos. <img src="/files/ftp_upload/50235/50235table3.jpg" alt="Tabla 3" fo:content-width="2in" fo:src="/files/ftp_upload/50235/50235table3highres.jpg"/> Tabla III. Sustancias para la carga de los miocitos con Fluo-3 PM. <img src="/files/ftp_upload/50235/50235table4.jpg" alt="Tabla 4" fo:content-width = &quot;2,5 pulgadas&quot; fo: src = &quot;/ files/ftp_upload/50235/50235table4highres.jpg&quot; /&gt; Tabla IV. Solución de baño para patch-clamp. <img src="/files/ftp_upload/50235/50235table5.jpg" alt="Tabla 5" fo:content-width="2.5in" fo:src="/files/ftp_upload/50235/50235table5highres.jpg"/> Tabla V. solución de pipeta de patch-clamp *.

Watch this Scientific Journal Video about Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents at JoVE.com

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

Se describe el aislamiento de miocitos auriculares humanos que pueden ser utilizados para el Ca intracelular<sup&gt; 2 +</sup&gt; Mediciones en combinación con estudios de patch-clamp electrofisiología.

El aislamiento de miocitos auriculares humanos para la medición simultánea de Ca<sup&gt; 2 +</sup&gt; Transitorios y corrientes de membrana

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

isolation-human-atrial-myocytes-for-simultaneous-measurements-ca2

Research

JoVE Journal

Biology

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

20.2K vistas.  University of Duisburg-Essen. Se describe el aislamiento de miocitos auriculares humanos que pueden ser utilizados para el Ca intracelular 2 + Mediciones en combinación con estudios de patch-clamp electrofisiología.

Video: Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultivo primario de miocitos adultos de corazón de rata

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Investigación

Biología

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

El aislamiento y la manipulación genética de los miocitos cardiacos adultos de microscopía confocal

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Técnicas de Imagen Ca 2 + Señalización en el esperma humano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

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 ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Aislamiento de mitocondrias a partir de cantidades mínimas de ratón músculo esquelético para mediciones de alto rendimiento de microplacas Respiratorias

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Desarrollo y caracterización de<em&gt; In Vitro</em&gt; Red de microvasos y mediciones cuantitativas de endotelial [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; Y la producción de óxido nítrico

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Los métodos para el aislamiento, cultivo y caracterización funcional de los miocitos del nodo sinusal ratones adultos

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Medición simultánea de calcio mitocondrial y mitocondrial potencial de membrana en células vivas por microscopía fluorescente

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

Asignación simultánea y la cuantificación de ribonucleótidos en el ADN mitocondrial humano

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Medidas simultáneas de calcio intracelular y el potencial de membrana en el endotelio Cerebral de ratón recién aislado e intacto

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...