Name: contractility measurements on isolated papillary muscles for the investigation of cardiac inotropy in mice
Uploaded: 2015-09-17T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice at JoVE.com

Este trabalho foi apoiado pelo Deutsche Forschungsgemeinschaft (KFO 196 &quot;Signaltransduktion bei adaptativen und maladaptiven kardialen Remodelação-Prozessen&quot;, FR 1638 / 1-2) e pelo (Centro Alemão DZHK for Cardiovascular Research, uma parte dos centros alemães de Pesquisa em Saúde , que é um BMBF (Ministério alemão da Educação e Investigação) iniciativa).

NOTA: As etapas básicas do processo de isolamento são mostrados na Figura 1 Todos os passos são descritos em pormenor no seguinte protocolo.. Isolamento dos músculos papilares, montagem no órgão câmara de banho, aquisição e análise é realizada em uma escala de tempo consecutivo e obrigatória. Todos os experimentos com animais foram realizados de acordo com a legislação alemã sobre a protecção dos animais e foram aprovados pelo Comitê de Ética da Universidade de Heidelberg. 1. Preparação de Instrumentação <ol><li> Para contratilidade medições usar uma configuração banho de órgãos com cavidades múltiplas. Componentes de uma contractilidade configuração banho de órgãos ex vivo são mostrados na Figura 2. NOTA: Para minimizar a quantidade de tempo entre a preparação do músculo papilar eo início do protocolo experimental, criado equipamentos e preparar buffers antes da recolha tissue.This experimentais provides a maior quantidade de tempo que o tecido permanece viável para conduzir experiências. </li><li> Inicie o computador e aquisição de dados equipamento. Ligue banho de órgãos de calor (predefinido para 32 ° C) e as unidades a temperatura predefinida permitir. </li><li> Prepare o equipamento de aquisição. Iniciar a gravação de dados, como mencionado no manual do software. </li><li> Verificar e realizar a calibração (se necessário) em cada canal e zero todos os canais para padronizar o sinal eléctrico fornecido a partir do transdutor de força respectivo (associado com aquele canal) a uma força de 2-G fornecida por um peso certificada. </li><li> Ligue o fornecimento de gás (95% de O2 / 5% de CO 2) para câmaras de banho de órgãos. Continuamente gás, todas as câmaras durante as medições de protocolo inteiras. </li></ol> 2. Preparação de tampões e soluções fisiológicas <ol><li> Prepara-se uma solução tampão de Krebs-Henseleit a alcançar concentrações finais de 119 mM de NaCl; MM NaHCO 25 3; KCl 4,6 mM; Glucose 11mm; 1 mM de Na-Pyruvate; 1,5 mM de CaCl2; 1,64 mM de MgSO4; 1,18 mM de KH 2 PO 4 e ajustar o pH para 7,4 (ver Tabela 1 solução fisiológica). </li><li> Preparar a solução tampão de Krebs-Henseleit separado para usar durante o isolamento conforme descrito no passo 2.1 com monoxime adicional 30 mM 2,3-butanodiona (BDM) e 50 IE de heparina / ml. Arrefecer a solução a 4 ° C (ver Tabela 1 Preparação de Solução). Para simulação de isquemia, isquemia preparar-solução, tal como descrito na Tabela 1. </li><li> Para o protocolo de Ca 2 + - contractilidade dependente, adicionar Ca 2+ para a solução tampão de Krebs-Henseleit a concentração final requerida. NOTA: preparar essas soluções diretamente antes de seu uso e gass com carbogênio para evitar a precipitação de Calciumcarbonate. Pré-aquecer solução a 32 ° C antes de usar. </li></ol> 3. Preparação do tubo e Dissection Dish gasificação usado durante a Excisão Processo Papillary <ol><li> Conecte um gás suavecantar tubo (diâmetro externo 2-4 mm) para a ligação do gás (oxigênio a 100%, carbogénio alternativamente) com peritos que trabalham. </li><li> Criar um anel fechado do tubo gaseamento encaixe dentro do prato dissecção. Perfurar o tubo de gaseamento várias vezes que um borbulhar contínua está assegurada. Este tubo de gaseamento pode ser utilizado várias vezes. </li><li> Prepare dissecção prato com um elastómero de silicone na parte inferior (0,5 cm de espessura), de modo que os pinos podem ser preso nele. Este prato pode ser utilizado várias vezes. </li></ol> 4. Isolamento de Anterior Esquerda Músculo Papilar de murganhos NOTA: Antes de iniciar o isolamento do músculo papilar, verifique se as linhas de gás são livre de bloqueios. <ol><li> Prepare configuração banho de órgãos tal como mencionado na secção protocolo 1-3 antes de iniciar a preparação dos músculos papilares. Preparar todas as soluções, no dia da experiência. </li><li> Sacrifique o mouse (cerca de 8-12 semanas de idade) por deslocamento cervical according de trabalho especializada e diretrizes institucionais. </li><li> Corrigir o animal em decúbito dorsal, fixando as patas dianteiras e traseiras patas em uma placa de dissecação, abrir a lateral do tórax de ambos os lados, cortando através das costelas com uma tesoura afiada do osso, em seguida, corte o diafragma com um corte transversal. Remover o pericárdio. Agora, o coração e a aorta são acessíveis. </li><li> Fixar o coração com uma pinça sem corte no truncus vascular perto do coração e separar o coração rapidamente com tesouras dos pulmões e do tecido circundante. </li><li> Transferir o coração batendo para uma placa de Petri com solução de preparação arrefecida gaseados com oxigénio (passo 2.2). Permitir que o coração a bater e estimular contrações do coração suavemente através de tocar o ápice cardíaco com uma pinça. </li><li> Assim o coração está completamente exsanguinous, transferi-lo para o prato dissecção preenchido com solução preparação arrefecida (passo 2.2) e gaseificada com O2. A partir deste passo sobre o uso de um microscópio estereoscópico. </li><li> Fixar o heart com um pequeno pino através do ventrículo direito, de modo que o coração é visto a partir dorsal (ventrículo direito está situado no lado direito do ponto de vista do operador). </li><li> Usando uma tesoura de microcirurgia separar ambos os átrios no nível atrioventricular, corte de ligação do tecido dos ventrículos e descarte. Realizar um corte através da parede do ventrículo-válvula de nível AV ao vértice do coração evitando qualquer pressão mecânica. </li><li> Abra o ventrículo e fixar a parede livre do lado esquerdo com uma pinça, tendo assim um olhar para ambos os músculos papilares do ventrículo esquerdo. </li><li> Ligeiramente cortar o tecido ventricular lateral, em ambos os lados do músculo papilar anterior esquerda preservando uma parte da vela valvular na preparação muscular papilar e evitar tocar ou alongamento do músculo papilar, tanto quanto possível. Dissecar o tecido da parede ventricular remanescente do músculo papilar. </li><li> Anexar fios de seda (7/0, métrica 0,5) em ambos os lados do prep músculo papilarprepara-, um na vela valvular e um na parte muscular. Fixar a preparação na câmara de banho de órgãos cheio com solução de banho de órgãos e gaseificada com carbogénio, como a temperatura de 32 ° C é sugerida. </li></ol> 5. Equilíbrio e estimulação do músculo papilar <ol><li> Estimular a preparação do músculo papilar com pulsos retangulares (duração de 2 ms e corrente de 100 mA) a freqüência de estimulação de 1 Hz imediatamente após a fixação no banho de órgãos. </li><li> Certifique-se de uma mudança frequente da solução de banho de órgãos, seja por mudança contínua ou por alteração manual freqüentes (a cada 5 min) de acordo com a configuração do tipo banho de órgãos utilizados. </li><li> Aumentar gradualmente até que a pretensão força de contração máxima em alcançado. Força de contração máxima é alcançada quando o aumento da pré-tensão não é seguido por um aumento adicional da força de contração. </li><li> Após 45-60 min de equilibração, iniciar o protocolo experimental. </li></ol> NOTA: O protocolo experimental descrito abaixo compreende manobras padrão para caracterizar a contratilidade cardíaca em condições fisiológicas e fisiopatológicas. Na seção representante descrevemos esses protocolos em detalhe também mostra resultados representativos (ver também Quadro 2). <ol><li> A gravação ea análise dos protocolos experimentais <ol><li> Para gravar, utilize um software adequado, com resolução temporal adequada (taxa de aquisição ≥ 1 kHz). </li><li> Analisar parâmetros, incluindo a amplitude da força de contração (altura), tempo para o pico de tensão (TTP) e metade do tempo de relaxamento máximo (R50 / TFall, respectivamente) como mencionado no programa de software (como exemplo 5.5 Gráfico de ADInstruments, ver Figura 4). </li></ol></li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Neste manuscrito nós descrevemos um método para investigar a contratilidade do músculo papilar murino in vitro que pode ser usada para responder a várias questões científicas relacionadas com a fisiologia do coração e patologia em ratos, bem como para apoiar a análise de linhagens transgênicas e da descoberta de novas abordagens farmacêuticas para o tratamento de disfunções cardíacas. Nós ilustram a utilização deste método para avaliar as propriedades fisiológicas, patológicas e farmacológi...

A investigação de proteínas de canais iônicos, como referem o seu papel para a contratilidade cardíaca é essencial para descobrir diferentes pathomechanisms e estabelecer novas estratégias terapêuticas para doenças cardíacas como isquemia e insuficiência cardíaca. Função contrátil de cardiomiócitos de mamíferos é conhecido por ser modulada por vários canais iônicos, transportadores e outras proteínas. Potencial de ação evocava a ativação de tensão dependente sarcolemal L-tipo canais de Ca2 + leva a influxo de Ca2 + do espaço extracelular e, posteriormente, ao Ca2 + induzida por Ca2 + release (CICR) 1, o que desencadeia a contração celular 2. Ca 2+ -signaling desempenha um papel central na contratilidade cardíaca e adaptação ao estresse fisiológico ou patológico. Catecolaminas ativar receptores beta-adrenérgicos cardíacos, estimulando assim adenililciclase (AC), que sintetiza cAMP. Sendo ativado, proteiN cinase A (PKA) fosforila diferentes proteínas associadas intracelulares e de membrana de tipo L como canais de Ca2 +, e receptores de rianodina fosfolambam, resultando em modificação de transientes de Ca 2+ e 1,3,4 contractilidade cardíaca. O AMPc é degradada pela fosfodiesterase (PDE). A activação dos outros do que os receptores beta-adrenérgicos Gs-acoplado também conduz à acumulação de cAMP. A técnica das medições de contratilidade em tiras isoladas de músculo ventricular está bem estabelecida para espécies de mamíferos maiores 5-8. Com base na possibilidade de direccionamento de genes em ratinhos é importante para estabelecer métodos para analisar fisiologia cardíaca murino. No entanto, os dados existentes sobre as propriedades fisiológicas das preparações musculares isoladas em camundongos diferem dependendo das condições experimentais 9-12. O método descrito é usado para analisar a contractilidade cardíaca de pré músculo papilar do ventrículo esquerdoparações in vitro. Investigação da contractilidade cardíaca é realizada na ausência de influências que modificam a contractilidade cardíaca in vivo, como a pressão sanguínea, a estimulação neuro-humoral e stress físico ou metabólica. A taxa de batimento da preparação muscular contratação pode ser rigorosamente definido e alterado arbitrariamente. Força de contração pode ser analisado no contexto de estímulos específicos, tais como a concentração de cálcio, batendo frequência ou temperatura. Além disso, este método pode ser usado para investigar diferentes componentes da via de sinalização e para comparar o desempenho cardíaco de modelos de ratinho geneticamente modificadas controlando as condições experimentais mencionadas acima.

<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

Músculo papilar isolado a partir de corações de rato adulto pode ser utilizado para estudar a contractilidade cardíaca durante as diferentes condições patológicas / fisiológicas. As características contráteis pode ser avaliado de forma independente de influências externas, tais como tônus ​​vascular ou o estado neuro-humorais. Descreve uma abordagem científica entre as medições de células individuais com os miócitos cardíacos isolados e estudos in vivo, como a ecocardiografia. Assim, preparações de músculo papilar servir como um excelente modelo para estudar a fisiologia cardíaca / fisiopatologia e pode ser utilizado para investigações como a modulação por agentes farmacológicos ou a exploração de modelos animais transgénicos. Aqui, nós descrevemos um método de isolamento do músculo papilar anterior esquerda murino para investigar a contractilidade cardíaca num banho de órgãos de configuração. Em contraste com a preparação de uma tira de músculo isolado a partir da parede ventricular, o músculo papilar podem ser preparados in toto sem danificar o músculo tissuum e severamente. A configuração banho de órgão é composto por várias câmaras de banho equipada órgão eletrodos, gaseados e com temperatura controlada. O músculo papilar isolado é fixo na câmara de banho de órgãos e estimulado eletricamente. A força de contração evocada é gravada usando um transdutor de pressão e parâmetros, tais como contração amplitude da força e contração muscular cinética são analisados. Diferentes protocolos experimentais podem ser realizadas para investigar o cálcio e contractilidade dependente da frequência, bem como as curvas de dose-resposta de agentes contrácteis, tais como catecolaminas e outros produtos farmacêuticos. Além disso, condições patológicas, como isquemia aguda pode ser simulado.

O protocolo deste manuscrito para medições de contratilidade de murino isolados preparações de músculos papilares está sintonizado com as condições ideais para atingir resultados experimentais reprodutíveis em condições fisiológicas. Para definir as condições experimentais ótimas, realizamos experimentos-piloto variando temperatura do banho de órgãos e concentração de cálcio extracelular (ver também 12). O protocolo aqui descrito foi realizado com uma concentração extracelular de cálci...

Watch this Scientific Journal Video about Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice at JoVE.com

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

Músculo papilar do ventrículo esquerdo de murino pode ser usado para investigar in vitro a contractilidade cardíaca. Este artigo descreve em detalhes o isolamento e protocolos experimentais para estudar características contráteis cardíacas.

Medidas de contratilidade no isolado músculos papilares de Investigação de Cardiac inotropia em Ratos

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.9K visualizações.  Universität Heidelberg.  Músculo papilar do ventrículo esquerdo de murino pode ser usado para investigar in vitro a contractilidade cardíaca. Este artigo descreve em detalhes o isolamento e protocolos experimentais para estudar características contráteis cardíacas. 

Vídeo: 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.

Em medições in vitro de constrição traqueal Utilizando camundongos

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

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.

O cateterismo cardíaco em camundongos para medir o volume Pressão do relacionamento: Investigando o efeito Bowditch

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

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

Abordagens ecocardiograma e protocolos de Comprehensive Caracterização fenotípica de doença cardíaca valvular em ratos

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

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.

Isolamento, cultura e caracterização funcional de rato 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 ...

Biologia

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.

Isolamento e Fisiológica Análise de rato Cardiomyocytes

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.

Medição dinâmica e imagem de capilares, arterioles e pericias em coração de rato

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 ...

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial malfunction in the diseased heart remains to be fully clarified. Recent compelling evidence demonstrates that changes in the myofilament Ca2+ sensitivity affect intracellular Ca2+ homeostasis and ion channel activities in cardiac myocytes, the essential mechanisms responsible for the cardiac action potential and contraction in healthy and diseased hearts. Indeed, activities of ion channels and transporters underlying cardiac action potentials (e.g., Na+, Ca2+ and K+ channels and the Na+-Ca2+ exchanger) and intracellular Ca2+ handling proteins (e.g., ryanodine receptors and Ca2+-ATPase in sarcoplasmic reticulum (SERCA2a) or phospholamban and its phosphorylation) are conventionally measured to evaluate the fundamental mechanisms of cardiac excitation-contraction (E-C) coupling. Both electrical activities in the membrane and intracellular Ca2+ changes are the trigger signals of E-C coupling, whereas myofilament is the functional unit of contraction and relaxation, and myofilament Ca2+ sensitivity is imperative in the implementation of myofibril performance. Nevertheless, few studies incorporate myofilament Ca2+ sensitivity into the functional analysis of the myocardium unless it is the focus of the study. Here, we describe a protocol that measures sarcomere shortening/re-lengthening and the intracellular Ca2+ level using Fura-2 AM (ratiometric detection) and evaluate the changes of myofilament Ca2+ sensitivity in cardiac myocytes from rat hearts. The main aim is to emphasize that myofilament Ca2+ sensitivity should be taken into consideration in E-C coupling for mechanistic analysis. Comprehensive investigation of ion channels, ion transporters, intracellular Ca2+ handling, and myofilament Ca2+ sensitivity that underlie myocyte contractility in healthy and diseased hearts will provide valuable information for designing more effective strategies of translational and therapeutic value.

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

Avaliação de miofilamentos Ca<sup&gt; 2+</sup&gt; Sensibilidade Subjacente Cardiac excitação-contração acoplamento

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial ...

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.

Análise da disfunção contrátil cardíaca e dos transientes de Ca2+ em miócitos 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.

Medida da Contratilidade Cardíaca em Cardiomiócitos Primários Humanos Isolados

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

Medidas de contratilidade no isolado músculos papilares de Investigação de Cardiac inotropia em Ratos

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