Além disso, agradecemos aos cirurgiões cardíacos da Universidade de Heidelberg para a prestação de tecido atrial humano e Claudia Liebetrau, Katrin Kupser e Ramona Nagel por sua excelente suporte técnico. Um agradecimento especial também para Andy W. Trafford para suas sugestões e conselhos durante o estabelecimento dos Ca 2 + medidas transitórias. Os autores desejam expressar sua profunda gratidão aos membros do Departamento de Farmacologia e Toxicologia (cabeça: Ursula Ravens), da Universidade de Tecnologia de Dresden pela oportunidade que nos ofereceram para aprender técnicas e habilidades básicas em eletrofisiologia celular e isolamento dos cardiomiócitos. Pesquisa dos autores é apoiado pela Fundação Alemã de Pesquisa (Do769/1-1-3), o Ministério Federal Alemão de Educação e Pesquisa, através da Rede de Competência Fibrilação Atrial (01Gi0204) e do Centro Alemão de Pesquisa Cardiovascular, a União Europeia através de o EuRede ropeia para Pesquisa Translacional em Fibrilação Atrial (EUTRAF, FP7-HEALTH-2010, o projeto de integração em larga escala, Proposta n º 261057) ea Aliança de Investigação Europeia-América do Norte fibrilação atrial (ENAFRA) concessão de Fondation Leducq (07CVD03). A visão geral do esquema mostrado no arquivo de vídeo foi produzido usando Servier arte médica.

Protocolos experimentais precisa ser aprovado pelo comitê de ética local e todos os pacientes precisam dar consentimento informado por escrito. Nossa pesquisa foi aprovado pelo comitê de ética da Faculdade de Medicina de Mannheim, da Universidade de Heidelberg (# 2011-216N-MA) e foi realizado em conformidade com todas as diretrizes institucionais, nacionais e internacionais para o bem-estar humano. Todos os pacientes assinaram termo de consentimento. 0. A obtenção de tecido atrial humano Durante os procedimentos de rotina de punção, em pacientes submetidos a cirurgia de coração aberto de circulação extracorpórea, a ponta da aurícula direita é normalmente removida e pode ser usada para o isolamento dos cardiomiócitos atriais. Após a excisão da amostra de tecido é transferido imediatamente para um tubo Falcon de 50 ml estéril com Ca2 + livre transporte solução contendo 2,3-butanodiona monoxime (Tabela I; BDM, inibidor contráctil, impedindo miócitos contracture). Com o tempo de transporte entre 30 e 45 min, que não reconhecemos uma clara vantagem de arrefecimento ou oxigenação em relação ao número e qualidade dos cardiomiócitos atriais isoladas. Nos transportes em geral para o laboratório deve ser tão rápido quanto possível. No entanto, se for mais longo o tempo de transporte não pode ser evitada, o transporte, a 4 ° C em solução oxigenada pode ser vantajosa. 1. Prearrangements <ol><li> Ligue o thermocirculator, que controla a temperatura do recipiente encamisado (Tabela II). Certifique-se de que a temperatura dentro do recipiente é igual a 37 ° C. Cobrir a proveta com uma tampa de vidro para manter uma temperatura constante no interior da taça ao longo de todo o processo de isolamento. </li><li> Pesa-se a colagenase I e protease XXIV em três copos de vidro e armazená-lo a temperatura ambiente. As enzimas serão diluídos imediatamente antes da utilização. </li><li> Loja 20 ml de uma solução de Ca2 + livre numa placa de Petri, a 4 ° C. </li><li>Loja 250 ml de Ca2 + livre em solução num banho de água a 37 ° C. </li></ol> 2. Limpeza do Tecido <ol><li> Transfira a amostra de tecido em conjunto com a solução de transporte numa placa de Petri (~ 10 cm de diâmetro) e remover o tecido adiposo aproximadamente usando uma tesoura. </li><li> Pesa-se a amostra de tecido. Entre 200 e 600 mg são utilizados para isolamento de células. O tecido restante pode ser congelada em azoto líquido para posterior análise bioquímica. </li><li> Transfira a amostra de tecido para a placa de Petri contendo 20 ml de uma solução de Ca2 + livre, a 4 ° C (ver passo 1.3) e cortar a amostra de tecido em pequenos pedaços de aproximadamente 1 mm 3 de tamanho. </li><li> Os passos seguintes devem ser executadas a 37 ° C e sob gaseificação contínua com 100% de O 2. Uma ponta de pipeta pode ser colocado no tubo de oxigénio para evitar a forte ebulição e gerar um fluxo de ar contínuo. </li><li> Transfira os pedaços de tecido juntamente com a solução de 2 Ca + livre emao béquer com camisa e agitou-se cuidadosamente durante cerca de 3 min, com uma barra de agitação magnética. </li><li> Pare de mexer e deixe os pedaços de tecido para se acalmar por alguns segundos. Estirpe cuidadosamente o sobrenadante através de uma malha de nylon (200 um, Tabela II). Retorne os pedaços de tecido contido da malha no copo usando uma pinça. </li><li> Encher o copo com Ca 2 + solução gratuita e repita a etapa de lavagem 2.4 e 2.5, duas vezes. </li></ol> 3. Primeiro digestão enzimática <ol><li> Re-suspender os pedaços de tecidos com 20 ml de solução de enzima E1 (Quadro I) contendo colagenase e protease e agita-se cuidadosamente durante 10 min. </li><li> Adicionar 40 ul de 10 mM de CaCl 2-solução para obter uma concentração final de 20 mM de Ca 2 +. </li><li> Após 35 min coe o sobrenadante cuidadosamente através de uma malha de nylon (200 um, Tabela II) de uma maneira que a maioria dos pedaços de tecido permanecem no copo. Resto retornarchover pedaços de tecido a partir da malha para o copo. </li></ol> 4. Segundo digestão enzimática <ol><li> Ressuspender os pedaços de tecido de novo com 20 ml de solução de enzima E2 colagenase I contendo apenas (Tabela I). Adicionar 40 ul de 10 mM de CaCl 2-solução imediatamente para se obter uma concentração final de 20 mM de Ca 2 +. </li><li> Durante o segundo tesoura para cortar o uso de digestão mais coágulos tecido ocasionalmente. </li><li> Após 5 min tomar uma amostra, utilizando uma pipeta de Pasteur, para procurar a dissociação de células. Repita isso a cada 2-3 min até em forma de bastonete, cardiomiócitos estriados aparecer. </li><li> Pare de mexer e deixe os pedaços de tecido para estabelecer-se para cerca de 20-30 segundos. </li><li> Coe o sobrenadante cuidadosamente através de uma malha de nylon (200 um, Tabela II) num tubo Falcon de 50 ml (um tubo). Retorne os pedaços de tecido contido da malha para o copo. </li><li> Re-suspender os pedaços de tecido no copo com 20 ml storasolução ge (Tabela I) e após 10 dissociar as células por trituração mecânica suave usando uma pipeta de 20 ml serológica com doseador. Tente evitar a formação de bolhas, desde bolhas são prejudiciais para a sobrevivência e qualidade celular. </li><li> Coe o sobrenadante cuidadosamente através de uma malha de nylon (200 mM, Tabela II) em um Falcon 50 ml (tubo B). </li></ol> 5. Preparação final e Ajuste de Final Ca 2 + Concentração <ol><li> Centrifugar os tubos Falcon ambos A e B (ver passo 4.5 e 4.7) a 95 xg durante 10 min. </li><li> Remove-se o sobrenadante de ambos os tubos cuidadosamente por aspiração. Certifique-se de não perturbar o sedimento. Descartar o sobrenadante. </li><li> Re-suspender em ambos os peletes de 1,5 ml de solução de armazenamento (Tabela I), cada uma (temperatura ambiente). </li><li> Adicione duas vezes 7,5 mL de solução 10 mM CaCl 2 para cada Falcon e mexa com cuidado. Incubar durante 10 minutos depois de cada passo. </li><li>Adicionar 15 ul de solução de CaCl2 10 mM para se obter uma última concentração de Ca2 + de 0,2 mM. </li></ol> 6. O carregamento dos miócitos com o fluorescente de Ca 2 + Fluo-indicador-3 AM (Figura 1) <ol><li> Transferir 1,5 ml de suspensão de células (tubo A e / ou o tubo B) para um tubo de microcentrifugação de 2 ml (tubo Eppendorf). </li><li> Os seguintes passos devem ser executados tendo em consideração a sensibilidade à luz fluorescente do Ca 2 + Indicador de Fluo-3. </li><li> Dissolve-se 50 mg do derivado do éster de acetoximetilo permeável membrana de Fluo-3 (Fluo-3 AM, Tabela III), em 44 mL do Pluronic F-127 (Tabela III), solução de reserva (20% w / v em DMSO anidro) para obter de 1 mm de Fluo-03:00 solução de stock, o que pode ser armazenada a -20 ° C durante, no máximo, de 1 semana. </li><li> Adicionam-se 15 ul do Fluo-03:00 solução estoque para o tubo de microcentrífuga contendo 1,5 ml de suspensão de células (ver passo 6.1) e agitecuidado. </li><li> Incubar a suspensão de células durante 10 minutos numa caixa opticamente opaca. </li><li> Resumidamente, centrifugar a cerca de 6.000 rpm. </li><li> Descartar o sobrenadante e ressuspender o sedimento em 1,5 ml de solução de banho (Tabela IV). </li><li> Deixe a suspensão celular por cerca de 30 min para de-esterificação, antes de iniciar os experimentos. </li></ol> 7. Simultâneas de patch-clamp e epifluorescência Ca 2 + Medições Desde que as medições de patch-clamp não são o principal tema desta revisão, referem-se ao leitor interessado em outras publicações fornecendo uma descrição mais aprofundada desta técnica. 11-14 Por uma questão de exaustividade nós fornecemos um breve resumo de um protocolo para medir potenciais de ação ou L-tipo Ca 2 + correntes, ambos com gravações simultâneas Ca 2 +-transitória. Durante as experiências de miócitos são superfused a 37 ° C com banho de solução quena (Tabela IV), utilizando um sistema de perfusão rápida (Octaflow IITM, ALA Scientific Instruments, NY). Para as experiências de tensão-grampo, as correntes de K + são bloqueados pela adição de 4-aminopiridina (5 mmol / L) e BaCl 2 (0,1 mmol / L) à solução de banho. Microeléctrodos de vidro de borosilicato e são utilizados devem ter resistências de ponta de 2-5 mohms quando cheio com a solução da pipeta (Tabela V). Além do Fluo-3 AM carregando dos miócitos (ver passo 6), Fluo-3 também está incluído na solução de pipeta (Tabela V). Fluorescência está animado a 488 nm e luz emitida (&lt;520 nm) convertido em [Ca 2 +] i assumindo <img alt="Equação 1" fo:content-width="1.540in" fo:src="/files/ftp_upload/50235/50235eq1highres.jpg" src="/files/ftp_upload/50235/50235eq1.jpg" /> d, em que k = constante de dissociação de Fluo-3 (864 nM), F = fluorescência de Fluo-3,. Fmax = fluorescência de Ca2 +-saturada obtida no final de cada experiência de 12 Ambos os sinais eléctricos e Ca 2 + sinais epifluorescência são gravados simultaneamente. Os potenciais de ação são estimulados a 0,5 Hz no modo atual-clamp usando um ms pulsos de corrente de força limiar 1.2x. Tipo-L de Ca 2 +-correntes são medidas no modo de voltagem-grampo com um potencial de manutenção de -80 mV e 100 mseg de rampa de pulso para -40 mV para inactivar o jejum de Na +-corrente, seguido por uma de 100 mseg ensaio de pulso para 10 mV a 0,5 Hz.

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Res. 66, 1277-1286 (1990).</li><li>Hove-Madsen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+fibrillation+is+associated+with+increased+spontaneous+calcium+release+from+the+sarcoplasmic+reticulum+in+human+atrial+myocytes.">Atrial fibrillation is associated with increased spontaneous calcium release from the sarcoplasmic reticulum in human atrial myocytes.</a> Circulation. 110, 1358-1363 (2004).</li><li>Molina, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+adenosine+monophosphate+phosphodiesterase+type+4+protects+against+atrial+arrhythmias.">Cyclic adenosine monophosphate phosphodiesterase type 4 protects against atrial arrhythmias.</a> J. Am. Coll. Cardiol. 59, 2182-2190 (2012).</li></ol>

Worthington Biochemical Corp apoiaram esta publicação, cobrindo os custos de produção do arquivo de vídeo. Os financiadores não tiveram nenhum papel no desenho do estudo, coleta de dados e análise, decisão de publicar ou preparação do manuscrito.

Aqui, nós descrevemos um método para o isolamento de miócitos atriais humanos de apêndices atriais direito obtidas de pacientes submetidos a cirurgia de coração aberto. De forma a utilizar estas miócitos para medições de Ca 2 + citosólico que adaptado com um método previamente descrito 4-11 omitindo EGTA a partir da solução de armazenamento. Já em 1970, verificou-se que, embora os miócitos dissociar na presença de Ca2 + durante a digestão, todos eles foram em contractura e não viáveis. 16,17 Assim, o isolamento de células é realizada em solução de Ca2 + livre. Contudo, a re-introdução de concentrações fisiológicas de Ca 2 + resultou numa rápida influxo de Ca2 + e na morte celular. Isto foi descrito como o 2 + Ca paradoxo fenómeno que foi inicialmente observada em corações perfundidos por Zimmerman e Hulsman. 18 modificações dos meios de isolamento, incluindo a redução do pH para 7,0, &lt;sup&gt; 19 20 adição de taurina ou de pequenas quantidades de Ca 2 + (veja passo 3.2 e 4.1), 21, bem como o armazenamento de miócitos isolados em EGTA, contendo solução de armazenagem-22, têm sido sugeridos para impedir o paradoxo de Ca 2 + 17. No entanto, é bem conhecido que o Ca 2 + tamponamento através EGTA reduz a amplitude de tipo-L de Ca 2 + corrente induzida por Ca 2 + amplitudes transientes e resulta numa deterioração bifásica dos transientes de Ca 2 + 23. Portanto, é omitida EGTA ao longo de todo o processo de isolamento, a fim de obter os transientes de Ca 2 + com propriedades típicas e decai monofásicas. Para proteger as células do Ca 2 + paradoxo que aumentou a última concentração de Ca2 + da solução de armazenamento de uma forma gradual até 0,2 mM. A escolha da colagenase é provavelmente o passo mais crítico para o sucesso do isolamento dos miócitos. Coll convencionalagenases são preparações brutas obtidas a partir de Clostridium histolyticum e contêm colagenase, além de uma série de outras proteases, polissacaridases e lipases. Com base nas suas colagenases composição geral são subdivididas em tipos diferentes. Worthington 24 Tipos colagenase I e II têm sido utilizados com sucesso no isolamento de miócitos atriais humanos 4-10,15,25-30. Em nosso protocolo atualmente descrito recomendamos o uso de colagenase O tipo I, apesar de, também foram capazes de obter quantidades aceitáveis ​​de células viáveis ​​utilizando colagenase de Tipo II. No entanto, mesmo dentro de um único tipo de colagenase há uma variação significativa de lote para lote em relação às actividades enzimáticas. Estas variações exigir a selecção cuidadosa e lote de teste de vários lotes para optimizar procedimento de isolamento. A ferramenta disponível batch-seleção on-line a partir de Worthington Biochemical Corp ( <a href="http://www.worthington-biochem.com/cls/match.php" target="_blank">http://www.worthington-bio</a>chem.com / cls / match.php) pode ser usada para encontrar os lotes disponíveis com uma composição que tem sido mostrado para ser adequado para o isolamento de miócitos atriais humanos. Atualmente nós usamos colagenase tipo I com 250 U / mg atividade da colagenase, 345 U / mg atividade caseinase, 2,16 U / mg atividade clostripaína e 0,48 U / mg atividade tríptica (lote # 49H11338). As células obtidas utilizando o processo descrito neste manuscrito podem ser utilizadas dentro de 8 horas para os estudos de patch-clamp, Ca 2 + medições transientes e uma combinação de ambos. 15 Além disso, estas células permitem a medição de contracção celular, em resposta à estimulação do campo eléctrico ou estimulação elétrica usando a pipeta de patch-clamp (observações não publicadas).

Estudar propriedades eletrofisiológicas de canais iônicos cardíacos com a técnica de patch-clamp ea exploração de celular Ca 2 + anormalidades manuseio requer cardiomiócitos isolados. Estes são geralmente obtidos após a exposição in vitro de amostras de tecidos cardíacos de enzimas digestivas (colagenase, hialuronidase, peptidase, etc.) Desde o primeiro relato de isolamento de miócitos cardíacos viáveis ​​em 1955 2 uma grande quantidade de protocolos foi desenvolvido a fim de colher cardiomiócitos atriais e ventriculares individuais a partir de diferentes espécies, incluindo ratinho, rato, coelho, cão, cobaia e humano. Nesta revisão, vamos nos concentrar no isolamento de miócitos atriais humanos. Em relação aos procedimentos para o isolamento de miócitos de outras espécies que se referem ao &quot;Worthington Tissue dissociação guia&quot; fornecido pelo Worthington Biochemical Corp, EUA ( <a href="http://www.tissuedissociation.com" target="_blank">www.tissuedissociation.com</a> ). et ai. 3 Aqui nós fornecemos uma descrição passo a passo de uma técnica, que é adaptado de um método previamente publicado, a fim de obter miócitos atriais adequadas não só para os experimentos de patch-clamp, mas também para Ca 2 + intracelular medições simultâneas 4-11.

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

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

O estudo das propriedades eletrofisiológicas de canais iônicos cardíacos com a técnica de patch-clamp ea exploração de celular cardíaca Ca 2 + anormalidades manuseio requer cardiomiócitos isolados. Além disso, a possibilidade de investigar miócitos de pacientes que utilizam estas técnicas é um requisito de valor inestimável para elucidar a base molecular das doenças cardíacas, tais como fibrilação atrial (FA). Uma Descrevemos aqui um método para o isolamento de miócitos atriais humanos, que são apropriados tanto estudos de patch-clamp e medições simultâneas de Ca 2 + concentrações intracelulares. Primeiro, apêndices atriais direita obtidas de pacientes submetidos a cirurgia de coração aberto são cortadas em pedaços pequenos de tecido (&quot;método pedaço&quot;) e lavados em solução de Ca 2 +-free. Em seguida, os pedaços de tecido são digeridos em colagenase e protease contendo soluções com 20 mM de Ca 2 +. Posteriormente, os miócitos isolados são harvested por filtração e centrifugação da suspensão de tecido. Finalmente, a concentração de Ca2 + na solução de armazenamento de células é ajustada gradualmente para 0,2 mM. Brevemente discutidos o significado de Ca 2 + e Ca 2 + tamponamento durante o processo de isolamento e também proporcionar gravações representativos de potenciais de acção e as correntes de membrana, juntamente com a simultânea tanto de Ca 2 + medições transientes, realizada nesses miócitos isolados.

Figura 2A mostra três exemplos representativos de miócitos atriais direitos humanos isolados. Para quantificar o rendimento das células que pipetar com 10 ul de suspensão de células (passo 5.5) numa lâmina de microscópio CellFinder ( <a href="http://www.antenna.nl/microlab/index-uk.html" target="_blank">http://www.antenna.nl/microlab/index-uk.html</a> ). Colheitas de células em média, na Figura 2B indicam claramente que há uma tendência para os rendimentos mais baixos em células crónica AF (CAF) de amostras do doente (tubo A: 16,5 ± 3,1 células/10 ul (n = 29) versus 5,1 ± 2,3 células/10 ul (n = 10), em SR e CAF, respectivamente, p &lt;0,05; tubo B: 17,9 ± 3,9 células/10 mL (n = 29) versus 5,9 ± 2,0 células/10 mL (n = 9) em SR e CAF, respectivamente, p = 0,107). Os exemplos representativos de acção potenciais medições simultâneas e gravações de Ca 2 + citosólico transientes são apresentados na Figura 3. Em cerca de 90% das células estudadas, tele acção potencial disparado Ca 2 + liberação provoca contrações de células claras e regular. Como relatado anteriormente, o potencial de repouso da membrana, que é um indicador aceite para a integridade das células, em média, cerca de -73,9 ± 2,7 mV (n = 23/10 / miócitos pacientes) e -77,7 ± 1,8 mV (n = 19/8 / miócitos pacientes ) em SR e CAF, respectivamente (p&gt; 0,05). 15 A Figura 4 mostra registos representativos de simultâneas por voltagem tipo-L de Ca 2 + e de correntes de Ca 2 + citosólico transientes. Aplicação da isoprenalina agonista β-adrenérgico não selectivos (1 uM), aumenta as amplitudes de ambos I Ca, L e citosólica de Ca 2 + transientes, sugerindo cascata de transdução de sinal β-adrenérgico intacta. <img alt="Figura 1" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig1highres.jpg" src="/files/ftp_upload/50235/50235fig1.jpg" /> Figura 1. Fluxograma dos miócitos Fluo-03:00 carregamento protocolo (veja o passo 6,1-6,5). m / v, massa / volume. <img alt="Figura 2" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig2highres.jpg" src="/files/ftp_upload/50235/50235fig2.jpg" /> Figura 2. A, miócitos atriais direito humano isolado depois de uma hora em solução de armazenamento. B, média ± SEM de rendimento celular contados em 10 ul de células em solução de armazenamento (ver passo 5.5). n significa o número de preparações em cada grupo. * P &lt;0,05. <img alt="Figura 3" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig3highres.jpg" src="/files/ftp_upload/50235/50235fig3.jpg" /> Figura 3. Gravações representativas de ação e potencial acionado Ca 2 + transientes (CAT), em um dos miócitos atrial do ritmo sinusal e um chronic paciente fibrilação atrial. Top: Injetado atual membrana (I M), usado para a estimulação (0,5 Hz). Abaixo: gravação simultânea do potencial de membrana (V M), e gato acionado (baixo). (Traçado de novo com a permissão de Voigt et al. 2.012) 15 <img alt="Figura 4" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig4highres.jpg" src="/files/ftp_upload/50235/50235fig4.jpg" /> Figura 4. Gravações representativos da (1 uM) no efeito isoprenalina tipo L de Ca 2 + desencadeada corrente Ca2 +-transientes (CAT), em um dos miócitos atrial de um ritmo sinusal e um doente de fibrilação atrial crónica. Top: Voltage-clamp protocolo (0,5 Hz). Abaixo: gravação simultânea de membrana líquida total de corrente (I M), reflectindo predominantemente do tipo L Ca 2 + atual (meio) e provocou Gato (baixo). (Traçado de novo com a permissão de Voigt,et ai. de 2012) 15 <img src="/files/ftp_upload/50235/50235table1.jpg" alt="Tabela 1" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235table1highres.jpg" /> Tabela I. Solutions. <img src="/files/ftp_upload/50235/50235table2.jpg" alt="Tabela 2" fo:content-width="2in" fo:src="/files/ftp_upload/50235/50235table2highres.jpg"/> Tabela II. Equipamento específico. <img src="/files/ftp_upload/50235/50235table3.jpg" alt="Tabela 3" fo:content-width="2in" fo:src="/files/ftp_upload/50235/50235table3highres.jpg"/> Tabela III. Substâncias para o carregamento de miócitos com Fluo-3 AM. <img src="/files/ftp_upload/50235/50235table4.jpg" alt="Tabela 4" fo:content-width = &quot;2.5in&quot; fo: src = &quot;/ files/ftp_upload/50235/50235table4highres.jpg&quot; /&gt; Tabela IV. Solução do banho de patch-clamp. <img src="/files/ftp_upload/50235/50235table5.jpg" alt="Tabela 5" fo:content-width="2.5in" fo:src="/files/ftp_upload/50235/50235table5highres.jpg"/> Tabela solução Pipeta V. de patch-clamp *.

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

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

Descrevemos o isolamento de miócitos atriais humanos, que podem ser utilizados para a intracelular de Ca<sup&gt; 2 +</sup&gt; Medidas, em combinação com estudos de patch-clamp eletrofisiológico.

Isolamento de miócitos atriais humanos para Medições Simultâneas de Ca<sup&gt; 2 +</sup&gt; Transitórios e correntes de membrana

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

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

Research

JoVE Journal

Biology

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

20.3K visualizações.  University of Duisburg-Essen. Descrevemos o isolamento de miócitos atriais humanos, que podem ser utilizados para a intracelular de Ca 2 + Medidas, em combinação com estudos de patch-clamp eletrofisiológico.

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

Primary Culture of Adult Rat Heart Myocytes

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

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

Cultura primária de Miócitos Coração Adulto Rat

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

Biologia

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

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

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

Isolamento e manipulação genética de adultos miócitos cardíacos for Imaging Confocal

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

Techniques for Imaging Ca2+ Signaling in Human Sperm

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

Techniques for Imaging Ca2+ Signaling in Human Sperm

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

Técnicas de imagem Ca 2 + Sinalização no esperma humano

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

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

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

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

Isolamento e gravações de canais Kv em Murino cardiomiócitos atriais e ventriculares

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

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

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

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

Isolamento de mitocôndrias de quantidades mínimas de Rato Músculo Esquelético para medições High Throughput Microplate respiratórias

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

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

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

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

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

Desenvolvimento e Caracterização de<em&gt; In Vitro</em&gt; Rede de microvasos e medições quantitativas de endotelial [ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; E óxido nítrico Produção

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

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

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

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

Métodos para o isolamento, Cultura e caracterização funcional de SINOATRIAL Nó miócitos de ratos adultos

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

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

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

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

Medição simultânea de mitocondrial de cálcio e mitocondrial potencial de membrana em células vivas por Microscopia fluorescente

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

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

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

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

Mapeamento simultâneo e quantificação de ribonucleotídeos no DNA mitocondrial humano

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

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

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

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

Medições simultâneas de cálcio intracelular e potencial de membrana no endotélio Cerebral rato recém isoladas e intacta

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