Name: high-resolution optical mapping of the mouse sino-atrial node
Uploaded: 2016-12-02T11:30:00.000+00:00
Duration: 11 min 7 s
Description: Watch this Scientific Journal Video about High-resolution Optical Mapping of the Mouse Sino-atrial Node at JoVE.com

We are supported by the University of Wisconsin-Madison Medical School start-up (A.V.G.).

Todos los experimentos se llevaron a cabo de acuerdo con los Institutos Nacionales de Salud de Guía para el Cuidado y Uso de Animales de Laboratorio (NIH Pub. No. 80-23). Todos los métodos y protocolos que se utilizan en estos estudios han sido aprobados por la Universidad de Wisconsin Cuidado de Animales y el empleo Comité de Protocolo siguiendo las directrices para el cuidado y uso de animales de laboratorio publicados por el NIH (publicación No. 85-23, revisada en 1996). Todos los animales utilizados en este estudio recibieron atención humanitaria de conformidad con la Guía para el Cuidado y Uso de Animales de Laboratorio. 1. Eliminación del corazón y la perfusión de Langendorff <ol><li> Antes del aislamiento del corazón, la solución de Tyrode caliente a 37 ° C utilizando un baño de agua y una camisa de agua. </li><li> Anestesiar al ratón con isoflurano 5% / 95% de O2. </li><li> Garantizar el nivel adecuado de anestesia mediante la comprobación de la pérdida del reflejo del dolor. </li><li> Realizar una toracotomía. Abre el cofre mediante el uso de curvas 5.5 &quot;tijeras de Mayo y 5.5&quot; hemostática para Kellyceps para hacer un corte de 1 cm en la parte frontal del tórax. <ol><li> extraer rápidamente el corazón del pecho (a menos de 30 segundos). Utilice 4 &quot;Iris fórceps curvos para agarrar el tejido pulmonar y cortar el pulmón, el timo y el corazón junto con el pericardio mediante 4.3&quot; tijeras iris. No agarre directamente el corazón. </li><li> Lavar en oxigenada (95% O 2/5% de CO 2), a temperatura constante (36,8 ± 0,4 ° C) modificado solución de Tyrode. Utilice la siguiente composición de la solución (en mM): NaCl 128,2, KCl 4,7, 1,19 NaH 2 PO 4, 1,05 MgCl 2, 1,3 CaCl 2, 20,0 NaHCO 3, y 11,1 glucosa (pH 7,35 ± 0,05). </li></ol></li><li> Mientras bañado en la misma solución, identificar el pulmón, el timo y el tejido graso. Con cuidado, diseccionar ellos desde el corazón. Utilice curva 3 &quot;tijeras Vannas-Tübingen y 4,3&quot; # 5 pinzas. </li><li> Luego identificar la aorta y canular que en un 21 G cánula a medida. Utilice dos curvas &quot;fuerza # 5B 4.3PD. </li><li> Después de la canulación, perfundir simultáneamente (a través de la cánula a una velocidad de flujo de ~ 5 ml / min, lo que debe ajustarse en base a la presión aórtica, ver más abajo) y superfuse (en el baño a una velocidad de flujo de ~ 50 ml / min) el corazón con una solución de Tyrode calentado y se filtró a través de todo el experimento. Pasar la solución de perfusión a través de un filtro de nylon de 11 micras en línea para evitar la obstrucción de la circulación coronaria. </li><li> El uso de un transductor de presión (o un indicador de presión) conectado a través de una de 3 vías de bloqueo de la llave de paso Luer a la línea de perfusión, monitorizar la presión aórtica y mantenerla entre 60 y 80 mmHg. Ajustar la velocidad de perfusión, si es necesario para mantener la presión aórtica dentro de este rango. </li></ol> 2. Mapeo óptico de la SAN de todo el corazón Langendorff perfundidos <ol><li> Instrumentación <ol><li> Colocar el corazón en posición horizontal y el pin de la punta del ventrículo a la parte inferior revestida de silicio de la cámara usando 0,1 mm de diámetro a pasadores prevenir corriente inducida por el movimiento durante el experimento. 12 </li><li> Introduzca un pequeño ( &quot;x ID 0,005&quot; 0,012) tubo de silicona en el ventrículo izquierdo (VI) a través de la vena pulmonar, la aurícula izquierda (LA) y la válvula mitral (MV). Fijar el tubo mediante una sutura de seda (4-0) en el tejido conectivo de resonancia. </li><li> Coloque el corazón con su cara posterior hacia arriba (Figura 1, panel izquierdo). </li><li> Pin el borde del apéndice RA (RAA) a la parte inferior de silicio de la cámara usando diámetro pasadores minutien 0,1 mm. Ajustar su nivel con el fin de hacer que la superficie posterior de la RA plana y permitir que se encuentra el plano focal de la cámara. Esto permitirá que las mediciones ópticas de la superficie máxima de la aurícula. </li><li> Soga superior (VCS) e inferior (IVC) de la vena cava por una sutura de seda (4-0), estirar y fijar el otro extremo de la sutura a la parte inferior de una cámara revestida de silicio (véase la Figura 3A). Asegúrese de que las suturas no bloqueen elcampo de vista óptico. </li><li> Coloque el electrodo de estimulación a medida en el borde de la RAA. Para hacer electrodos, el uso de silicio recubierto alambres de plata diámetro de 0,25 mm, con 0,5 mm de distancia entre electrodos y carente de silicio para una longitud de 1 mm en el extremo de estimulación. A continuación, coloque dos ECG de 12 mm de aguja (calibre 29) electrodos monopolares cerca de la base de los ventrículos derecho e izquierdo. Coloque el ECG electrodo de tierra cerca del vértice de los ventrículos. </li></ol></li><li> tinción <ol><li> Dado que los dos tintes fluorescentes y blebbistatin desacoplador electromecánico son sensibles a la luz, realice todos los procedimientos descritos a continuación en un cuarto oscuro. En primer lugar preparar colorante sensible al voltaje RH-237 o di-4-ANNEPS como una solución madre, 1,25 mg / ml en sulfóxido de dimetilo (2,5 mM), que alícuota (30 l cada una) y almacenar las alícuotas a -20 ° C. </li><li> Diluir 5-10 l de solución de colorante de valores en 1 ml de solución de Tyrode calentado (37 ° C) y luego inyectarla en la línea de perfusión coronariadurante un período de 5-7 min usando un puerto de inyección en línea Luer. </li><li> Preparar blebbistatin como una solución madre (2 mg / ml en dimetilsulfóxido, 6,8 mM) por adelantado y almacenarlo a 4ºC. </li><li> Después de 20 min de estabilización, añadir 0,5 ml de calentado (37 ° C) blebbistatin en el perfundido y diluir 0,1 ml de blebbistatin en 1 ml de (37 ° C) solución de Tyrode calentado. A continuación, inyectar en la línea de perfusión coronaria durante un periodo de 5-7 min usando un puerto de inyección en línea Luer. </li></ol></li></ol> 3. mapeo óptico de la SAN desde el auricular aislada Preparación <ol><li> Instrumentación <ol><li> Para la preparación atrial aislado, aislar y canular el corazón como se describió anteriormente en los pasos 1.1-1.5 para la preparación de todo el corazón. </li><li> Diseccionar los ventrículos lejos de la cara anterior. Ver Figura 1A, panel derecho, cortar # 1 para más detalles. </li><li> Abra la RA cortando a través de la VA tricúspideLVE (TV) a lo largo del eje de TV-SVC. Ver Figura 1A, panel derecho, cortar # 2 para más detalles. </li><li> Corte la rama medial de la cresta terminal para abrir el RAA. Ver Figura 1A, panel derecho, cortar # 3. </li><li> Abrir la pared libre de la aurícula anterior mediante la realización de un corte de la línea media de la corte anterior # 3 hasta el borde de la esquina inferior derecha de la RAA, aplanar y el pin de la pared libre de la aurícula hasta el fondo de una cámara revestida de silicio. Ver la dirección mostrada por la flecha hueca en la Figura 1A, corte # 4. Preservar un borde de tejido ventricular para fijar la preparación para evitar daños en las aurículas. </li><li> Del mismo modo, abierta LA cortando a través de la MV a lo largo de la esquina MV-superior del apéndice LA (LAA). </li><li> Para abrir la LAA, cortado desde la mitad de la LAA abierto, a través de la pared libre de la aurícula anterior hasta cerca de un borde medio de la LAA. </li><li> Abrir la pared libre de la aurícula alo anteriorendo la misma dirección, y luego aplanar y el pin a la parte inferior de una cámara recubierta con Sylgard. </li><li> Retire parcialmente el tejido del tabique interauricular. Esto reducirá la dispersión de la señal óptica a partir de tejido que no está en el foco. Por lo tanto, tanto el LA y RA, así como la SAN y la unión aurículo-ventricular (AVJ) son accesibles en esta preparación (Figura 1B). </li><li> Levante la preparación final en alrededor de 0,5 mm desde la parte inferior de la cámara de silicio recubierto con el fin de permitir superfusión desde ambas superficies epicárdicos y endocárdicos. </li><li> Superfuse la preparación con calentado (37 ° C) solución de Tyrode a una velocidad constante de 12 ~ ml / min para un flujo de centrado situado cerca de la SVC y ~ 30 ml / min para una superfusión baño. </li><li> Coloque el Ag / AgCl 2 electrodo de medida de estimulación (0,25 mm de diámetro) en el borde de la RAA diseccionado. A continuación, coloque dos ECG de 12 mm de aguja (29G) electrodos (monopolar) cerca de la RAA y LAA, respectivamente. Coloque la planta ECG elegidosmontó cerca de la AVJ. </li></ol></li><li> tinción <ol><li> Realizar una aplicación directa del colorante sensible al voltaje (RH-237 o di-4-ANNEPS). Para esto, diluir 1-2 l de la solución de colorante de valores de 1 mm de solución de Tyrode calentado (37 ° C) y liberar lentamente el colorante diluido en la superficie de la preparación mediante el uso de un 1 mm de la pipeta. </li><li> Alternativamente, lleve a cabo la tinción de la aurícula con el colorante sensible al voltaje a través de una perfusión coronaria del corazón perfundido Langendorff similar a la descrita anteriormente para la preparación de todo el corazón (pasos 2.2.1-2.2.2). <ol><li> Después de la tinción de la perfusión coronaria, aislar la preparación atrial como se describe. Realizar tinción de la superficie adicional cuando sea necesario para alcanzar un nivel de fluorescencia satisfactorio. aislamiento auricular rápido no blanquear el tinte y no afecta a la calidad de la tinción. </li></ol></li><li> Inmovilizar la preparación con el electro-mecánico de desenganche, blebbistatin. Diluir 3-5 l blebbistatin solución madre en la solución de Tyrode calentado (37 ° C) y liberan lentamente el blebbistatin diluida sobre la superficie de la preparación y la solución circundante. Desde blebbistatin ha demostrado ser sensible a la luz, de 13,14 a evitar una larga exposición a la luz durante la preparación y tinción. </li><li> Realizar la aplicación blebbistatin adicional tanto como sea necesario para suprimir la contracción. Normalmente hasta 3 aplicaciones adicionales (3-5 l por cada aplicación) se puede utilizar para suprimir el artefacto de movimiento suficientemente con el fin de reconstruir con precisión SAN y activación auricular. </li><li> Añadir 0,5 ml adicionales de blebbistatin calentado en el perfundido. Durante este procedimiento, una pausa en la superfusión durante 30 segundos para permitir que el tinte / blebbistatin para teñir el tejido auricular. </li></ol></li></ol> 4. Mapeo óptico configurar NOTA:. Una descripción detallada del sistema de mapeo óptico se proporciona en otra parte 12 <ol><li> Utilizar una cámara con una temresolución poral de 2.000 cuadros / seg o superior, y una serie de lentes de condensador para llegar a una resolución espacial de 100 m / pixel o superior. Esto es necesario para reconstruir la activación y propagación SAN intranodal. </li><li> Para reducir los artefactos de movimiento de la solución de vibración, fijar un pequeño vidrio de cubierta en la superficie de la solución sobre el corazón / aislados preparación auricular. </li><li> Utilice una luz de excitación (520 ± 45 nm de longitud de onda) proporcionado por una corriente constante, lámpara halógena de bajo ruido. Dirigir el haz de luz que se filtra en la preparación mediante el uso de una guía de luz bifurcada flexible. </li><li> Se filtra la fluorescencia emitida por un filtro de paso largo (&gt; 715 nm). Recoger, digitalizar y guardar la señal fluorescente adquirida en un software de ordenador utilizando proporcionada por el fabricante de la cámara. </li></ol> 5. Procesamiento de Datos Nota: Los datos de mapeo óptico se recoge y se almacena como una serie de matrices de intensidad de fluorescencia en diferentes puntos temporales. Cada píxel represeNTS una medida de la intensidad de fluorescencia recogida de una ubicación específica en la preparación del tejido en un punto de tiempo específico. La fluorescencia de fondo se elimina automáticamente por píxel para permitir una mejor visualización de los cambios de fluorescencia producidos por cambios de voltaje de membrana y de arriba abajo para que se corresponda con los potenciales de acción (AP) medidos por los sistemas de microelectrodos. Los detalles de los diferentes pasos en el procesamiento de datos de imágenes ópticas, incluyendo la segmentación de imágenes, filtrado espacial, filtrado temporal, y la eliminación deriva de referencia, se proporcionan en la revisión enfocada. 15 <ol><li> Manualmente o mediante el algoritmo especial (por ejemplo, un umbral o el borde de detección) 15 para determinar los límites del tejido, crear una máscara binaria de 1 (píxeles incluidas) y 0 (píxeles excluidos) y aplicar la máscara para cada trama de la secuencia. Este proceso crea una imagen binaria que pone de relieve las áreas de interés para su posterior procesamiento. </li><li> Para mejorar la relación señal-ruido de la opciónlos datos de iCal, filtran las señales dentro de las áreas de interés seleccionadas. Para ello, utilice espacial (es decir, un promedio de píxeles fluorescentes vecinos según la definición de un contenedor de convolución deseado o kernel, por ejemplo 3 x 3 o, para los datos ruidosos, 5 x 5) y / o filtrado temporal (por ejemplo, Butterworth, tipo Chebyshev 1, Chebyshev tipo 2, elíptica etc.) (Figura 2). Tenga en cuenta la posibilidad de artefactos inducidos filtrados-hora de interpretar los datos. Para más detalles, véase la revisión. 15 </li><li> Retire la deriva de la línea de base en las grabaciones ópticas cuando sea necesario (mediante el uso de filtración de paso alto o ajuste polinómico a la señal original) y luego normalizar cada señal de píxel desde 0 (fluorescencia mínimo) a 1 (fluorescencia máxima). </li><li> Seleccionar una ventana de tiempo que abarca los tiempos de activación de todos los píxeles para una única propagación AP. Asignar a cada pixel de su tiempo de activación como el tiempo de derivado de máxima carrera ascendente (dF / dt max, donde F es fluorescenintensidad ce). El uso de todos los tiempos de activación de píxeles, reconstruir el mapa de activación isócrono. Cada isócrona será por lo tanto mostrar los píxeles activados al mismo tiempo. </li><li> Para reconstruir el mapa de distribución de la duración del PA (APD), para cada pixel calcular la duración entre el tiempo de activación y el tiempo en un nivel específico de la repolarización (por ejemplo, al 80% de la repolarización, APD 80). El uso de todos los valores de píxel de APD, reconstruir la distribución Mapa isócrono APD. Cada isócrona será por lo tanto mostrar los píxeles con la misma APD. </li><li> Para el cálculo de la velocidad de conducción para la propagación AP, adaptarse a una superficie de la preparación de los datos de tiempo de activación calculados en 5.4. Para ello, utilice polinomio superficie de ajuste o local suavizado superficie del núcleo y luego calcular los vectores de velocidad de conducción locales a partir del gradiente de la superficie ajustada. </li><li> Para crear el mapa de la repolarización, asignar a cada pixel de su tiempo de repolarización, definida como la segunda derivada máxima (D 2 F / dt2) de la señal óptica en el extremo de la OAP, o el tiempo de 90% de repolarización. </li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+cardiac+pacemaker+circuits:+methodological+puzzles+of+the+sinoatrial+node+optical+mapping.">Mapping cardiac pacemaker circuits: methodological puzzles of the sinoatrial node optical mapping.</a> Circ Res. 106 (2), 255-271 (2010).</li><li>Aschar-Sobbi, R., 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=Increased+atrial+arrhythmia+susceptibility+induced+by+intense+endurance+exercise+in+mice+requires+TNFalpha.">Increased atrial arrhythmia susceptibility induced by intense endurance exercise in mice requires TNFalpha.</a> Nat Commun. 6, 6018 (2015).</li><li>Hansen, B. J., 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+driven+by+micro-anatomic+intramural+re-entry+revealed+by+simultaneous+sub-epicardial+and+sub-endocardial+optical+mapping+in+explanted+human+hearts.">Atrial fibrillation driven by micro-anatomic intramural re-entry revealed by simultaneous sub-epicardial and sub-endocardial optical mapping in explanted human hearts.</a> Eur Heart J. 36 (35), 2390-2401 (2015).</li><li>Lang, D., Petrov, V., Lou, Q., Osipov, G., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+heart+rate+in+a+rabbit+heart.">Spatiotemporal control of heart rate in a rabbit heart.</a> J Electrocardiol. 44 (6), 626-634 (2011).</li><li>Kanlop, N., Sakai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+study+of+blebbistatin-induced+chaotic+electrical+activities+in+isolated+rat+atrium+preparations.">Optical mapping study of blebbistatin-induced chaotic electrical activities in isolated rat atrium preparations.</a> J Physiol Sci. 60 (2), 109-117 (2010).</li><li>Glukhov, A. V., Flagg, T. P., Fedorov, V. V., Efimov, I. R., Nichols, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+K(ATP)+channel+pharmacology+in+intact+mouse+heart.">Differential K(ATP) channel pharmacology in intact mouse heart.</a> J Mol Cell Cardiol. 48 (1), 152-160 (2010).</li><li>Wu, J., 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=Altered+sinoatrial+node+function+and+intra-atrial+conduction+in+murine+gain-of-function+Scn5a+/DeltaKPQ+hearts+suggest+an+overlap+syndrome.">Altered sinoatrial node function and intra-atrial conduction in murine gain-of-function Scn5a+/DeltaKPQ hearts suggest an overlap syndrome.</a> Am J Physiol Heart Circ Physiol. 302 (7), H1510-H1523 (2012).</li></ol>

No conflicts of interest are declared.

A continuación, presentamos dos tipos de preparaciones de ratón SAN: 1) SAN intacto en el corazón entero perfundido Langendorff, y 2) en el SAN aislado, abrimos la preparación de la aurícula. Estos dos tipos de preparación sirven para diferentes propósitos experimentales. En la preparación todo el corazón perfundido Langendorff, la estructura de la aurícula intacto se conserva lo que hace posible el estudio de arritmias complejas auriculares como la fibrilación auricular, así como las interacciones entre el ...

Novel scientific breakthroughs that lead to leaps in the understanding of human physiology are often preceded by technological advances. Fluorescent optical mapping, for example, enables investigation of multiple physiological parameters in both cells and tissues.1,2 It significantly improved our understanding of how the anatomical structure is associated with electrophysiological functions and dysfunctions. In the heart, the natural pacemaker and conduction systems such as the sinoatrial node (SAN) and the atrioventricular junction consist of nodal myocytes that are insulated by the surrounding atrial myocytes.3-5 Such organization creates complex three-dimensional structures with specialized electrical properties. Both structural and functional remodeling of these pacemaker structures has been recognized to form significant electrophysiological heterogeneities.6,7 Understanding the mechanism underlying how such heterogeneities result in SAN dysfunctions and atrial arrhythmogenesis will dramatically benefit the clinical treatment of these diseases. It requires a technique to visualize the propagation of electrical signals at the tissue level, such as optical mapping. 
Recently, accumulating evidence has proven the advance of optical mapping in studies of atrial electrophysiology and pathology.2 However, novel and rigorous studies are dependent on the accurate interpretation of experimental data, whose validity and stability rely on careful experiment protocols. Genetically modified mice are extensively used for research as animal models of human diseases including sick sinus syndrome, pacemaker abnormalities and atrial arrhythmias.6,8-11 Thus, a combination of fluorescent optical mapping with transgenic mouse models provides a powerful tool to study cardiac electrical abnormalities associated with various pathologies. In this paper, we present a protocol for high-resolution optical mapping of the mouse SAN and atrium. Specifically, we discuss and compare different dye loading approaches, time effects on dye bleaching and heart rate stability during the experiment.

<table><tbody><tr><td>water jacket</td><td>Radnoti</td><td>1660 Series Tissue Bath for Large Organ or Single Cell Isolation Procedures</td><td></td></tr><tr><td>water bath / circulator</td><td>Fisher Scientific</td><td>1016S</td><td></td></tr><tr><td>pressure amplifier</td><td>AD Instruments</td><td>MLT0670</td><td></td></tr><tr><td>EMD Millipore Nylon Net Filters</td><td>Fisher Scientific</td><td>NY1102500</td><td></td></tr><tr><td>Pressure transducer</td><td>AD Instruments</td><td>MLT0670</td><td></td></tr><tr><td>Stainless Steel Minutien Pins - 0.1 mm Diameter</td><td>Fine Science Tools</td><td>26002-10&amp;nbsp;</td><td></td></tr><tr><td>Perfusion pump</td><td>World Precision Instruments</td><td>PERIPRO-4LS</td><td></td></tr><tr><td>Superfusion pump</td><td>World Precision Instruments</td><td>PERIPRO-4HS</td><td></td></tr><tr><td>Vannas Tubingen scissors&amp;nbsp;</td><td>World Precision Instruments</td><td>503379</td><td></td></tr><tr><td>Dumont forceps</td><td>World Precision Instruments</td><td>501201, 500085</td><td></td></tr><tr><td>Mayo scissors</td><td>World Precision Instruments</td><td>501750</td><td></td></tr><tr><td>Kelly hemostatic forceps</td><td>World Precision Instruments</td><td>501241</td><td></td></tr><tr><td>Iris forceps</td><td>World Precision Instruments</td><td>15917</td><td></td></tr><tr><td>Iris scissors</td><td>World Precision Instruments</td><td>501263</td><td></td></tr><tr><td>ECG 12 mm needle (29-gauge) electrodes (monopolar)&amp;nbsp;</td><td>AD Instruments</td><td>MLA1203</td><td></td></tr><tr><td>in-line Luer injection port</td><td>Ibidi</td><td>10820</td><td></td></tr><tr><td>Ultima-L CMOS camera&amp;nbsp;</td><td>SciMedia</td><td>MiCAM-05&amp;nbsp;</td><td></td></tr><tr><td>halogen lamp</td><td>Moritex USA Inc</td><td>MHAB-150W</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-1</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>S217-500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; (6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>M33-500</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>S369-500</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233-3</td><td></td></tr><tr><td>D-Glucose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Blebbistatin</td><td>Tocris Bioscience</td><td>1760</td><td></td></tr><tr><td>RH237</td><td>ThermoFisher Scientific</td><td>S1109</td><td></td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr></tbody></table>

high-resolution optical mapping of the mouse sino-atrial node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Mapeo óptico de la avería SAN desde el corazón Langendorff perfundidos Un ejemplo típico de un mapa de contornos activación de la AD reconstruida por el ritmo sinusal espontánea se muestra en la Figura 3 para un corazón de ratón Langendorff perfundidos. El punto de activación temprana se encuentra dentro de la región intercaval cerca del SVC en la red SAN se define anatómicamente. 3,16 Dos RA mapas de contorno de activación ...

Watch this Scientific Journal Video about High-resolution Optical Mapping of the Mouse Sino-atrial Node at JoVE.com

High-resolution Optical Mapping of the Mouse Sino-atrial Node

A continuación, se presenta un protocolo para el mapeo óptico de la actividad eléctrica de la aurícula derecha del ratón y, especialmente, el nodo sinoauricular, con una resolución espacial y temporal alta.

-Alta resolución de mapeo óptico del nodo sinoauricular Ratón

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The ...

high-resolution-optical-mapping-of-the-mouse-sino-atrial-node

Research

JoVE Journal

Biology

15.6K vistas.  University of Wisconsin-Madison School of Medicine and Public Health.  A continuación, se presenta un protocolo para el mapeo óptico de la actividad eléctrica de la aurícula derecha del ratón y, especialmente, el nodo sinoauricular, con una resolución espacial y temporal alta. 

Video: High-resolution Optical Mapping of the Mouse Sino-atrial Node

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparamétrico óptico Cartografía del corazón de conejo Langendorff perfundidos

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Investigación

Bioingeniería

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

Mapeo óptico de potenciales de acción y transitorios de calcio en el corazón de ratón

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

Cartografía de alta resolución óptica endocárdicos y epicárdicos en un modelo de ovejas inducidas por el estiramiento de la fibrilación auricular

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

Medicina

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Mapeo óptico de corazones de ratas Langendorff perfundidos

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Biología

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Evaluación electrofisiológica de Atria murino con mapeo óptico alta resolución

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Microscopía de lámina de luz y limpieza de tejidos para obtener imágenes cardíacas completas

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights into fundamental cardiac physiology and pathologic responses. We hereby delve into the development and implementation of the LSM technique to elucidate the micro-architecture of the heart in mouse models. The methodology integrates a customized LSM system with tissue clearing techniques, mitigating light scattering within cardiac tissues for volumetric imaging. The combination of conventional LSM with image stitching and multiview deconvolution approaches allows for the capture of the entire heart. To address the inherent trade-off between axial resolution and field of view (FOV), we further introduce an axially swept light-sheet microscopy (ASLM) method to minimize out-of-focus light and uniformly illuminate the heart across the propagation direction. In the meanwhile, tissue clearing methods such as iDISCO enhance light penetration, facilitating the visualization of deep structures and ensuring a comprehensive examination of the myocardium throughout the entire heart. The combination of the proposed LSM and tissue clearing methods presents a promising platform for researchers in resolving cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

Autor destacado: Imágenes personalizadas de lámina de luz para investigar estructuras miocárdicas en corazones de roedores

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

Imágenes con lámina de luz para revelar la estructura cardíaca en corazones de roedores

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights ...

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.
The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.
Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.
Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

Tinción de inmunofluorescencia de montaje completo, imágenes confocales y reconstrucción 3D del nódulo sinoauricular y auriculoventricular en el ratón

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

Registro de la matriz de microelectrodos de la tasa de disparo del nódulo sinoauricular para identificar defectos intrínsecos de marcapasos cardíacos en ratones

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

-Alta resolución de mapeo óptico del nodo sinoauricular Ratón

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Registro de la matriz de microelectrodos de la tasa de disparo del nódulo sinoauricular para identificar defectos intrínsecos de marcapasos cardíacos en ratones