Este trabajo fue apoyado por la Deutsche Forschungsgemeinschaft (de KFO 196 &quot;Signaltransduktion bei adaptativen und maladaptiven kardialen Remodelación-Prozessen&quot;, FR 1638 / 1-2) y por el (Centro DZHK Alemán de Investigaciones Cardiovasculares, una parte de los centros alemanes de Investigación en Salud , que es un BMBF (Ministerio alemán de Educación e Investigación) de iniciativa).

NOTA: Los pasos básicos del procedimiento de aislamiento se muestra en la Figura 1 Todas las etapas se describen en detalle en el siguiente protocolo.. El aislamiento del músculo papilar, de montaje en cámara de baño de órganos, la adquisición y el análisis se realiza en un plazo de tiempo consecutivo y obligatoria. Todos los experimentos con animales se realizaron de conformidad con la legislación alemana sobre protección de los animales y fueron aprobadas por la Junta de Revisión Ética de la Universidad de Heidelberg. 1. Preparación de Instrumentación <ol><li> Para la contractilidad mediciones utilizan una configuración de baño de órganos multicompartimentalizado. Componentes de una instalación de la contractilidad ex vivo baño de órganos se muestran en la Figura 2. NOTA: Para reducir al mínimo la cantidad de tiempo entre la preparación del músculo papilar y el comienzo del protocolo experimental, configurar equipos y preparar tampones antes de recoger tissue.This experimentales provides la mayor cantidad de tiempo que el tejido permanece viable para llevar a cabo experimentos. </li><li> Poner en marcha los equipos informáticos y adquisición de datos. Encienda el calor baño de órganos (preajustado a 32 ° C) y permitir que las unidades a temperatura preestablecida. </li><li> Preparar el equipo de adquisición. Iniciar la grabación de datos como se menciona en el manual del software. </li><li> Comprobar y realizar la calibración (si es necesario) en cada canal y poner a cero todos los canales de normalizar la señal eléctrica suministrada desde el transductor de fuerza respectiva (asociado a ese canal) a una fuerza de 2 g suministrado por un peso certificado. </li><li> Abra el suministro de gas (95% O 2/5% de CO 2) a las cámaras de baño de órganos. Gas continuamente todas las cámaras durante todo el protocolo de mediciones. </li></ol> 2. Preparación de tampones y soluciones fisiológicas <ol><li> Preparar una solución de tampón de Krebs-Henseleit para lograr concentraciones finales de NaCl 119 mM; 25 mM NaHCO 3; KCl 4,6 mM; Glucosa 11 mM; 1 mM Na-pyruvate; 1,5 mM CaCl 2; 1,64 mM MgSO4; 1,18 mM KH 2 PO 4 y ajustar el pH a 7,4 (véase la Tabla 1 solución fisiológica). </li><li> Prepare la solución tampón de Krebs-Henseleit separado para utilizar durante el aislamiento como se describe en el paso 2.1 con adicional monoxima 30 mM 2,3 butanodiona (BDM) y 50 IE de heparina / ml. Solución Enfriar a 4 ° C (ver Tabla 1 solución de Preparación). Para la simulación isquemia, isquemia preparar-solución como se describe en la Tabla 1. </li><li> Para el protocolo del Ca 2 + - contractilidad dependiente, añadir Ca 2 + a la solución de tampón de Krebs-Henseleit a una concentración final requerida. NOTA: preparar estas soluciones directamente antes de su uso y Gass con carbógeno para evitar la precipitación de carbonato cálcico. Precalentar la solución a 32 ° C antes de usar. </li></ol> 3. Preparación del Tubo y Disección gasificación de vajilla utilizada durante el procedimiento de escisión papilar <ol><li> Conecte un gas suavecantar tubo (diámetro externo de 2.4 mm) para la conexión de gas (oxígeno al 100%, carbógeno alternativamente) con trabajo de expertos. </li><li> Crear un anillo cerrado del tubo de gasificación apropiado en el interior del plato de disección. Perforar el tubo de gasificación varias veces que un burbujeo continuo está asegurada. Este tubo de gasificación se puede utilizar varias veces. </li><li> Preparar plato de disección con un elastómero de silicona en la parte inferior (0,5 cm de espesor), de modo que los pines se pueden pegar en ella. Este plato se puede utilizar varias veces. </li></ol> 4. Aislamiento del anterior izquierda papilar muscular de ratones NOTA: Antes de comenzar el aislamiento del músculo papilar, compruebe que las líneas de gas están libres de obstrucciones. <ol><li> Preparar configuración baño de órganos como se menciona en la sección de protocolo desde 1 hasta 3 antes de iniciar la preparación de los músculos papilares. Preparar todas las soluciones en el día de la prueba. </li><li> Sacrificar el ratón (aproximadamente 8-12 semanas de edad) por accordi dislocación cervicalng de trabajo de expertos y las directrices institucionales. </li><li> Fijar el animal en posición dorsal mediante la fijación de las patas delanteras y traseras patas en una tabla de disección, abrir el tórax lateral en ambos lados por el corte a través de las costillas con unas tijeras afiladas de hueso, luego se corta el diafragma con un corte transversal. Retire el pericardio. Ahora el corazón y la aorta son accesibles. </li><li> Fijar el corazón con unas pinzas romas en el tronco vascular cerca del corazón y separar el corazón rápidamente con las tijeras de los pulmones y el tejido circundante. </li><li> Transferir el corazón que late a un plato de Petri llena con una solución de preparación enfriado gaseados con oxígeno (paso 2.2). Deje que el corazón lata y estimular las contracciones del corazón suavemente a través de tocar el vértice cardiaco con fórceps. </li><li> Tan pronto el corazón es completamente exsanguinous, la transferencia a la placa de disección lleno de solución preparación enfriada (paso 2.2) y gaseados con O 2. A partir de este paso en el uso de un microscopio estereoscópico. </li><li> Fijar el heata con una aguja pequeña a través del ventrículo derecho para que el corazón se ve desde dorsal (ventrículo derecho se encuentra en la parte derecha de la vista del operador). </li><li> Usando tijeras de microcirugía separan ambas aurículas en el nivel auriculoventricular, corte la conexión de tejido de los ventrículos y descartan. Realizar un corte a través de la pared ventricular desde el nivel de la válvula AV a la punta del corazón evitando cualquier presión mecánica. </li><li> Abra el ventrículo y fijar la pared libre del lado izquierdo con una pinza, teniendo así un vistazo a ambos músculos papilares del ventrículo izquierdo. </li><li> Ligeramente cortar el tejido ventricular lateral en ambos lados del músculo papilar anterior izquierda conservando una parte de la vela valvular en la preparación del músculo papilar y evitar tocar o estirar el músculo papilar tanto como sea posible. Diseccionar el tejido de la pared ventricular restante del músculo papilar. </li><li> Adjuntar hilos de seda (7/0, métrica 0,5) en ambos lados de la preparación del músculo papilarprepa-, uno en la vela valvular y uno en la parte muscular. Fijar la preparación en la cámara de baño de órganos lleno de solución del baño de órganos y gaseados con carbógeno, como se sugiere la temperatura de 32 ° C. </li></ol> 5. Equilibrio y estimulación del músculo papilar <ol><li> Estimular la preparación músculo papilar con pulsos rectangulares (duración de 2 ms y corriente de 100 mA) en la frecuencia de estimulación de 1 Hz inmediatamente después de la fijación en el baño de órganos. </li><li> Asegurar un cambio frecuente de la solución del baño de órganos, ya sea por el cambio continuo o por el cambio manual de frecuente (cada 5 min) de acuerdo con la configuración del tipo de baño de órganos utilizado. </li><li> Poco a poco aumentar la pretensión hasta que la fuerza de contracción máxima en llegar. Fuerza de contracción máxima se alcanza cuando el aumento de la pretensión no es seguido por un aumento adicional en la fuerza de contracción. </li><li> Después de 45-60 min de equilibración, comenzar el protocolo experimental. </li></ol> NOTA: El protocolo experimental descrito a continuación comprende maniobras estándar para caracterizar la contractilidad cardíaca bajo condiciones fisiológicas y fisiopatológicas. En la sección representativa describimos estos protocolos en detalle que también muestra resultados representativos (véase también el cuadro 2). <ol><li> Grabación y análisis de los protocolos experimentales <ol><li> Para la grabación, utilice un software adecuado con resolución temporal adecuada (velocidad de adquisición ≥ 1 kHz). </li><li> Analizar parámetros incluyendo la fuerza de contracción de amplitud (altura), tiempo hasta el pico de tensión (TTP) y la mitad del tiempo de relajación máxima (R 50 / TFall, respectivamente) como se menciona en el programa de software (como ejemplo gráfico 5.5 de ADInstruments, véase la Figura 4). </li></ol></li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

En este manuscrito se describe un método para investigar la contractilidad del músculo papilar murino in vitro que puede ser usado para responder a varias preguntas científicas relacionadas con la fisiología del corazón y de la patología en ratones así como para apoyar el análisis de las líneas transgénicas y el descubrimiento de nuevos enfoques farmacéuticos para el tratamiento de disfunciones cardíacas. Se ilustra el uso de este método para evaluar las propiedades fisiológicas, patológicas y fa...

La investigación de las proteínas como los canales iónicos referencia su papel de la contractilidad cardíaca es esencial para descubrir diferentes mecanismos patogénicos y establecer nuevas estrategias terapéuticas para las enfermedades cardiacas como la isquemia y la insuficiencia cardíaca. La función contráctil de los cardiomiocitos de mamífero se sabe que está modulada por diversos canales iónicos, transportadores y otras proteínas. Potencial de acción evocados activación de tensión depende del sarcolema de tipo L de Ca 2 + canales conduce a Ca 2 + afluencia desde el espacio extracelular y posteriormente a Ca 2 + inducida por Ca 2 + liberación (CICR) 1, lo que desencadena la contracción celular 2. Ca 2 + -señalización juega un papel central en la contractilidad cardíaca y la adaptación al estrés fisiológico o patológico. Las catecolaminas activan los receptores beta-adrenérgicos cardíacos, estimulando así la adenilciclasa (AC) que sintetiza cAMP. Al estar activada, protein quinasa A (PKA) fosforila diferentes intracelulares y de membrana proteínas asociadas como de tipo L de Ca 2 + canales, fosfolamban y receptores de rianodina que resulta en la modificación de Ca 2 + transitorios y 1,3,4 contractilidad cardíaca. cAMP se degrada por la fosfodiesterasa (PDE). La activación de los receptores acoplados a Gs distintos de beta-adrenoceptores también conduce a la acumulación de cAMP. La técnica de las mediciones de contractilidad en tiras de músculo ventricular aislado está bien establecido para las especies de mamíferos más grandes 5-8. En base a la posibilidad de orientación de genes en ratones es importante establecer métodos para analizar la fisiología cardiaca murino. Sin embargo, los datos existentes sobre las propiedades fisiológicas de las preparaciones musculares aisladas en ratones difieren dependiendo de las condiciones experimentales 9-12. El método descrito se utiliza para analizar la contractilidad cardíaca de pre músculo papilar ventricular izquierdoprepara- in vitro. Investigación de la contractilidad cardiaca se lleva a cabo en ausencia de influencias que modifican la contractilidad cardiaca in vivo, como la presión arterial, la estimulación neurohumoral y estrés físico o metabólico. La tasa de latidos de la preparación muscular contratación puede ser rigurosamente definido y cambió arbitrariamente. Twitch fuerza puede ser analizado en el contexto de estímulos específicos tales como la concentración de calcio, superando la frecuencia o la temperatura. Además, este método puede ser utilizado para investigar diferentes componentes de la vía de señalización y para comparar el rendimiento cardíaco de modelos de ratones modificados genéticamente mediante el control de las condiciones experimentales mencionadas anteriormente.

<table><tbody><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761&amp;nbsp;</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>D9434&amp;nbsp;</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P5280&amp;nbsp;</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>230391</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P 5655</td><td></td></tr><tr><td>2,3-Butanedione monoxime</td><td>Sigma-Aldrich</td><td>B0753</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I5879</td><td>Hazard statement H 302, solve in DMSO</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Isoprenaline hydrochloride</td><td>Sigma-Aldrich</td><td>I5627</td><td>Hazard statement H 315-H319-H335</td></tr><tr><td>Sodium Heparine 250.000 IE/10 ml</td><td>ratiopharm</td><td>PZN 3874685</td><td></td></tr><tr><td>Histamine dihydrochloride</td><td>Sigma-Aldrich</td><td>H7250</td><td>Hazard statement H 315-H 317-H319- H334-H335</td></tr></tbody></table>

contractility measurements on isolated papillary muscles for the investigation of cardiac inotropy in mice

Del músculo papilar aislado a partir de corazones de ratón adulto se puede utilizar para estudiar la contractilidad cardíaca durante las diferentes fisiológicas / condiciones patológicas. Las características contráctiles se pueden evaluar de manera independiente de las influencias externas, como el tono vascular o el estado neurohormonal. Representa un enfoque científico entre las mediciones de células individuales con miocitos cardíacos aislados y los estudios in vivo, como la ecocardiografía. Por lo tanto, preparaciones de músculo papilar sirven como un excelente modelo para estudiar la fisiología cardiaca / fisiopatología y puede ser utilizado para investigaciones como la modulación por agentes farmacológicos o la exploración de modelos de animales transgénicos. Aquí se describe un método para aislar el músculo papilar anterior izquierda murino para investigar la contractilidad cardíaca en una configuración de baño de órganos. En contraste con una preparación tira de músculo aislado de la pared ventricular, el músculo papilar se puede preparar in toto sin dañar el músculo tissue severamente. La configuración de baño de órganos consiste en varias cámaras de baño de órganos de electrodos equipado con control de temperatura, gaseados y. El músculo papilar aislado se fija en la cámara de baño de órganos y estimuló eléctricamente. La fuerza de contracción evocada se registra utilizando un transductor de presión y parámetros tales como contracción fuerza de la amplitud y la contracción se analizan cinética. Diferentes protocolos experimentales se pueden realizar para investigar el calcio y la contractilidad dependiente de la frecuencia, así como curvas dosis-respuesta de los agentes contráctiles tales como catecolaminas u otros productos farmacéuticos. Además, las condiciones patológicas como la isquemia aguda pueden ser simulados.

El protocolo de este manuscrito para las mediciones de contractilidad del músculo papilar murinos preparaciones aisladas se sintoniza en condiciones óptimas para lograr resultados experimentales reproducibles en condiciones fisiológicas. Para definir las condiciones experimentales óptimas hemos realizado experimentos piloto variables temperatura del baño de órganos y la concentración de calcio extracelular (véase también 12). El protocolo aquí descrito se realizó con una concentración de calcio ex...

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Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Músculo papilar ventricular izquierdo murino se puede utilizar para investigar la contractilidad cardíaca in vitro. En este artículo se describe en detalle el aislamiento y los protocolos experimentales para estudiar las características contráctiles cardiacas.

Contractilidad Mediciones en músculos papilares aislados para la Investigación de Cardíaca inotropía en ratones

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

contractility-measurements-on-isolated-papillary-muscles-for

Research

JoVE Journal

Biology

14.9K vistas.  Universität Heidelberg.  Músculo papilar ventricular izquierdo murino se puede utilizar para investigar la contractilidad cardíaca in vitro. En este artículo se describe en detalle el aislamiento y los protocolos experimentales para estudiar las características contráctiles cardiacas. 

Video: Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

Mediciones in vitro de la constricción de la tráquea Utilizando ratones

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

Investigación

Medicina

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Cateterismo cardíaco en ratones para medir la relación de volumen Presión: Investigar el efecto Bowditch

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

Imágenes ópticas de miocitos ventriculares murinos aislados

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Bioingeniería

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

Aislamiento, cultivo y caracterización funcional de ratón adulto Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Biología

Isolation and Physiological Analysis of Mouse Cardiomyocytes

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

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

Aislamiento y fisiológica Análisis de Ratón cardiomiocitos

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

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

Medición dinámica e imagen de capilares, arterias y pericitos en el corazón del ratón

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Revealing Subtle Changes in Cardiac Function using Transthoracic Dobutamine Stress Echocardiography in Mice

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic dobutamine stress echocardiography in humans, a reductionist investigation approach to unmask subtle changes in cardiac function has become possible. Here, we provide a protocol for using this technique in mice to facilitate expanded analysis of LV architecture and function in physiology and pathology enabling the observation of alterations in models of cardiac disease hidden in unstressed hearts. This investigation can be performed in one and the same animal and allows both, basal and pharmacologically stress-induced measurements. We outline detailed criteria for appropriate anesthesia, imaging-based LV analysis, consideration of intra- and interobserver variability, and obtaining positive inotrope response that can be attained in mice after intraperitoneal injection of dobutamine under near physiological conditions. To recapitulate the characteristics of human physiology and disease in small animal models, we highlight critical pitfalls in evaluation, e.g., a pronounced Bowditch effect in mice. To further meet translational objectives, we compare stress-induced effects in humans and mice. When used in translational studies, attention must be paid to physiological differences between mice and human. Experimental rigor dictates that some parameters assessed in patients can only be used with caution due to restrictions in spatial and temporal resolution in mouse models.

Left ventricular dysfunction constitutes the final common pathway for a host of cardiac disorders. We here provide a detailed protocol of transthoracic dobutamine stress echocardiography approach for comprehensive evaluation of the left ventricular function of mouse models of cardiac disease as well as cardiac phenotyping.

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic ...

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

Análisis de disfunción contráctil cardíaca y transitorios de Ca2+ en miocitos de roedores

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury ...

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This paper describes a protocol for assessing changes in contractility in adult human primary ventricular cardiomyocytes utilizing the MyoBLAZER, a non-invasive optical method that preserves the normal physiology and pharmacology of the cells. This optical recording method continuously measures contractility transients from multiple cells in parallel, providing both medium-throughput and valuable information for each individual cell in the field of view, enabling the real-time tracking of drug effects. The cardiomyocyte contractions are induced by paced electrical field stimulation, and the acquired bright field images are fed to an image-processing software that measures the sarcomere shortening across multiple cardiomyocytes. This method rapidly generates different endpoints related to the kinetics of contraction and relaxation phases, and the resulting data can then be interpreted in relation to different concentrations of a test article. This method is also employed in the late stages of preclinical development to perform follow-up mechanistic studies to support ongoing clinical studies. Thus, the adult human primary cardiomyocyte-based model combined with the optical system for continuous contractility monitoring has the potential to contribute to a new era of in vitro cardiac data translatability in preclinical medical therapy development.

This protocol describes how to measure contractility in adult human primary cardiomyocytes from donor hearts with the MyoBLAZER system, a reliable platform for assessing drug-induced changes in contractility during preclinical development.

Medición de la contractilidad cardíaca en cardiomiocitos primarios humanos adultos aislados

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This ...

Mechanical Control of Relaxation Using Intact Cardiac Trabeculae

Diastolic dysfunction is a common phenotype across cardiovascular disease presentations. In addition to elevated cardiac stiffness (elevated left ventricular end-diastolic pressure), impaired cardiac relaxation is a key diagnostic indicator of diastolic dysfunction. While relaxation requires the removal of cytosolic calcium and deactivation of sarcomeric thin filaments, targeting such mechanisms has yet to provide effective treatments. Mechanical mechanisms, such as blood pressure (i.e., afterload), have been theorized to modify relaxation. Recently, we showed that modifying the strain rate of a stretch, not afterload, was both necessary and sufficient to modify the subsequent relaxation rate of myocardial tissue. The strain rate dependence of relaxation, called the mechanical control of relaxation (MCR), can be assessed using intact cardiac trabeculae. This protocol describes the preparation of a small animal model, experimental system and chamber, isolation of the heart and subsequent isolation of a trabecula, preparation of the experimental chamber, and experimental and analysis protocols. Evidence for lengthening strains in the intact heart suggests that MCR might provide new arenas for better characterization of pharmacological treatments, along with a method to assess myofilament kinetics in intact muscles. Therefore, studying the MCR may elucidate a path to novel approaches and new frontiers in the treatment of heart failure.

Rapid myocardial and cardiac relaxation is essential for normal physiology. Mechanical relaxation mechanisms are now known to be dependent on strain rate. This protocol provides an overview of the acquisition and analysis of experiments to further study the mechanical control of relaxation.

Contractilidad Mediciones en músculos papilares aislados para la Investigación de Cardíaca inotropía en ratones

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