Esta pesquisa foi apoiada pelo Programa de Pesquisa em Ciência Básica através da Fundação Nacional de Pesquisa da Coreia (NRF), financiado pelo Ministério da Educação, Ciência e Tecnologia (2013068067); pelo Programa de Pós-Graduação Cérebro Korea 21 do Ministério da Educação, Ciência e Tecnologia, Hospital da Universidade Nacional de Seul, a sociedade coreana de Hipertensão (2013), SK Fund Research Telecom coreano (no. 3420130290) e da Fundação Nacional de Ciência Natural da China (NSFC 31460265; 81260035 NSFC).

O protocolo está de acordo com o Guia para o Cuidado e Uso de Animais de Laboratório publicados pelos Institutos Nacionais da ONU de Saúde (NIH Publication No. 85-23, revista em 1996). Foi aprovado pelo Comitê Institucional de Animal Care and Use (IACUC) da Universidade Nacional de Seul (aprovação IACUC no .: SNU-101213-1). 1. Tampão de Preparação (Materiais de mesa e equipamentos) <ol><li> Preparar 300 ml de solução de isolamento de fresco no dia da experiência (em mM: NaCl, 135; KCl, 5,4; MgCl2, 3,5; glicose, 5; HEPES, 5; Na 2 HPO 4, 0,4; taurina, 20; pH 7,4 com NaOH). </li><li> Preparação de 100 ml de solução de armazenamento de fresco no dia da experiência (em mM: NaCl 120; KCl 5,4; MgSO4 5; CaCl2 0,2; de sódio e piruvato 5; glucose 5,5; taurina 20; HEPES 10; manitol 29; pH 7,4 com NaOH). </li><li> Prepare 1 litro de solução de perfusão (em mM: NaCl, 141,4; KCI, 4; NaH 2 PO 4, 0,33; MGCL 2, 1; HEPES, 10; glicose, 5,5; CaCl2, 1,8; manitol, 14,5; pH 7,4 com NaOH). </li><li> Preparar 30 ml de uma solução de colagenase, adicionando colagenase (1 mg / ml), a protease (0,1 mg / ml), albumina de soro bovino (BSA, 1.67mg / ml), e Ca2 + (0,05 mM) a 25 ml de isolamento solução. </li><li> Preparar 20 ml de solução de colagenase 2 por adição de colagenase (1 mg / mL), BSA (1,67 mg / ml), e Ca2 + (0,05 mM) a 16,7 ml de solução de isolamento. </li><li> Preparar 40 ml de solução de BSA através da adição de 0,4 g de BSA a 40 ml de solução de isolamento. Separado 10 ml e 30 ml em duas provetas. Adicionar Ca 2+ a 30 ml de solução de BSA de modo que a concentração final de Ca2 + em solução de BSA a 1 mm. </li></ol> 2. Preparação para o isolamento de Left ventricular (LV) miócitos <ol><li> Transferir 8-12, ratos semanas de idade do sexo masculino Sprague-Dawley (SD) em gaiolas de transporte não poluentes do biotério para a sala de preparação e isolamento. </li><li> Hcomer dois banhos de água a 37 o C. Nota: Utilize banho de uma água para a água - reservatório encamisado e os tubos de perfusão do sistema de perfusão Langendorff. Use outro banho de água para agitar troncos de tecido do miocárdio, a fim de separar os miócitos individuais. </li><li> Adicionar 5 ml e 3,3 ml de solução de BSA (sem Ca2 +) para 25 ml de solução de colagenase 1 e 16,7 ml de solução de colagenase 2 para perfazer o volume para 30 ml e 20 ml, respectivamente. </li><li> Adicionar solução de isolamento (100 ml) e solução de colagenase 1 (30 ml) a uma coluna (Cl 1) e a coluna 2 (Cl 2) no sistema de perfusão de Langendorff, respectivamente (Figura 1A). </li><li> Oxigenar a solução de isolamento e uma solução de colagenase em Cl 1 e Cl 2 através do tubo de ligação de oxigénio no sistema de perfusão de Langendorff (Figura 1A). Da mesma forma, a solução de colagenase oxigenado 2 e a solução de BSA no banho de água com agitação, com 100% de O2 através datubo de ligação de oxigênio. </li></ol> 3. Isolamento de LV miócitos <ol><li> Anestesiar uma ratazana SD por meio de injecção intraperitoneal de pentobarbital sódico (30 mg / kg) e confirmar o estado anestésico por dedo do pé que comprime e falta de retirada reflexão. </li><li> Mova o rato para uma bandeja de dissecação. Na posição supina, fixe as quatro pernas para os lados do corpo com fita. </li><li> Aplicar incisões mid-axiais com tesouras cirúrgicas torácicas para abrir a caixa, garantindo a não danificar o coração. Use um outro par de tesouras limpas para dissecar o coração dos vasos comunicantes (por exemplo, superior e inferior da veia cava, vasos pulmonares e aorta) e membrana de pericárdio 13. </li><li> Deixar uma porção da aorta de um comprimento suficiente (5-8 mm), prender a aorta, com uma pinça fina e montar rapidamente a cânula do sistema de perfusão de Langendorff dentro de 1 min (Figura 1A). Amarre fio de sutura 4/0 firmemente sobre a aorta. </li><li&gt; Ligar a válvula em Col. 1 e perfundir o coração isolado com uma solução de isolamento pré-aquecido e oxigenado durante 10 min (taxa de perfusão: 12-14 mL / min). Desligue a válvula na Col. 1, acender a válvula na Col. 2 e perfundir o coração com solução de colagenase 1 para 8-10 min. </li><li> Desmontar o coração digerido por corte da aorta e transferi-lo, mantendo a aorta com uma pinça para o balão contendo uma solução de isolamento fresco (Figura 1bi). Use uma tesoura fina e pinças para cortar a maior parte de parede livre do VE (incluindo o septo) em pedaços menores (~ 22 mm, Figura 1Bii). </li><li> Transferir os pedaços para um frasco contendo uma solução de colagenase fresco pré-aquecido e oxigenado 2 (Figura 1Biii). Agitar durante 10 min. Manter a entrega de oxigênio para a solução de colagenase contendo miócitos 2. </li><li> Mova o miócitos - suspensão para um tubo de 10 ml de centrífuga usando conta-gotas com um bulbo de sucção e adicionar Ca 2+ - contendo solução de BSA (1: 1 em volume). Centrifugar a 30 g durante 2 min e desprezar o sobrenadante. Re-suspender o sedimento de miócitos em 5 ml de solução de BSA. Centrífuga e descartar o sobrenadante. </li><li> Dispersar os peletes de miócitos e manter miócitos em 10 ml de solução de armazenamento de pré-oxigenado à TA (Figura 1Biv-VI). </li><li> Repita os passos processuais (3,7-3,9) com o tecido do LV restante no frasco. Nota: Manter repetindo os procedimentos (passos 3,7-3,9) novamente até que a maior parte do tecido do LV desaparece para se obter um bom rendimento de miócitos. </li><li> Manter os miócitos em solução de armazenamento para 6-8 horas a temperatura ambiente, até ao fim das experiências. </li></ol> 4. As medições simultâneas de Ca2 + intracelular Transitórios e miócitos Contração <ol><li> miócitos carga LV com o éster de acetoximetilo de Fura-2 AM (2 | iM). Nota: Execute todos os procedimentos de carga e experiências com miócitos carregados em um tubo escuro (Figura 1Bvii). <ol><li> Centrifuge a suspensão de miócitos (1 ml) a 2000 xg durante 10 seg. Descartar o sobrenadante e re-suspender o pellet de miócitos em 1 ml de solução de Tyrode com uma baixa concentração de Ca2 + (250? M, Materiais de mesa e equipamentos). </li><li> Adicionar Fura-02:00 e poloxamer 407 (2 mL), suavemente dispersar a suspensão de miócitos, e manter a mistura temperatura ambiente (20-24 ° C) durante 15 minutos (Figura 1Bvii). </li><li> Centrífuga-se a mistura durante 5 seg, descartar o sobrenadante e dispersar o sedimento de miócitos em 1 ml de solução de perfusão contendo 500 uM de Ca 2+. Depois de 10 minutos, centrifugar a mistura durante 5 seg e desprezar o sobrenadante. </li><li> Adicionar solução de perfusão fresco (500 | iM de Ca2 +, 1 ml) e manter o Fura-02:00 - sedimento miócito carregado nesta solução de gravação. </li></ol></li><li> A medição da contracção dos miócitos e Ca2 + intracelular <ol><li> Antes de gravar, encher o tubo de perfusão que é executado através de umacamisa de água com a solução de Tyrode, que é pré-aquecido a 36 o C. </li><li> Coloque algumas gotas do Fura 02:00 - carregado suspensão de miócitos LV na câmara de um microscópio de fluorescência invertido para 5-8 min. Lentamente perfundir a solução de Tyrode (2,2 ml / min). </li><li> Pressione o botão &quot;start&quot; no painel frontal do estimulador digital para iniciar a estimulação de campo (2 Hz). Nota: A tensão de saída (10 V, 5 mseg de duração) é aplicado aos miócitos na câmara por meio de fios de platina posicionados em ambos os lados da câmara. <ol><li> Seleccione miócitos que se contraem de forma estável (não aqueles que apresentam hiper ou hipo-contração) para a gravação. </li></ol></li><li> Ajuste do miócito de escolha na posição horizontal do sistema de detecção à base de sarcómero vídeo e ajustar a focagem do microscópio, para obter imagens óptimas de sarcómeros. Posicionar a caixa retangular roxo na área em que sarcômeros claras são claramente observados até a médiade comprimentos de sarcômero (pico vermelho) mostra um pico singular afiada (Figura 2A, imagem inferior). </li><li> Gravar as mudanças de comprimento do sarcómero em resposta à estimulação do campo (Figura 2A e B). Nota: Use os miócitos carregados dentro de 1 - 2 horas. </li><li> Ajustar a abertura da câmara de modo a que o campo no sistema de detecção à base de sarcómero vídeo é o tamanho do miócito (Figura 2A). Estimular a miócitos com excitação a 360 nm / 380 nm e emissão a 510 nm (freqüência de aquisição de 1.000 Hz). Encurtamento do sarcômero Record e a relação Fura2 AM com estimulação de campo (Figura 2B). </li></ol></li></ol> 5. Avaliação de miofilamentos Ca2 + Sensibilidade <ol><li> Calcular a média dos comprimentos de sarcômero e Ca 2 + transientes no estado estacionário (10 - 20 traços) e traçar o ciclo do plano de fase da relação de Fura-2 vs. comprimento do sarcômero do mesmo miócitos (ambos com valor medidos e delta mudanças; Figura 2c). </li><li> Em cada parcela, definir rácio Fura -2 a 50% de relaxamento (EC50, Figura 2C). Compare tanto o loop e EC 50 de cada intervenção. </li></ol>

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The authors declare that they have no competing financial interests.

Aqui, descrevemos os protocolos para avaliar as alterações de sensibilidade dos miofilamentos Ca 2+ em um único miócitos cardíacos isolados e enfatizar a importância de se medir este parâmetro juntamente com propriedades eletrofisiológicas, intracelulares de Ca2 + transientes, ea dinâmica dos miofilamentos. Isto é porque as gravações de um ou dois dos parâmetros não podem explicar os mecanismos subjacentes a contracção cardíaca e relaxamento. Ao contrário dos métodos convencionai...

Acoplamento de excitação-contracção cardíaca (CE) é o esquema fundamental para analisar as propriedades mecânicas do miocárdio, ou seja, a função contráctil do coração 1,2. CE de acoplamento é iniciado pela despolarização da membrana secundária para as actividades dos canais de iões de sarcolema (por exemplo, a voltagem-canal de Na +, que pode ser medido através de técnicas de patch-clamp). Activação subsequente do tipo L dependentes da voltagem canais de Ca2 + (LTCCs) e influxo de Ca2 + através LTCCs desencadear a grandes quantidades de libertação de Ca2 + através dos receptores de rianodina (RyRs), aumentando a concentração citosólica de Ca2 + a partir do nanomolar (nM ) a micromolar nível (^ M). Um tal aumento no Ca 2+ citosólica de Ca2 + promove a ligação à troponina C (TNC) em filamentos finos e induz alterações conformacionais do complexo filamento para facilitar a interacção actina-miosina e alcança myocardial contração 3. Por outro lado, o Ca2 + citosólico é re-captado volta para o retículo sarcoplasmático (RS) através da Ca2 + -ATPase em SR (SERCA2a) ou é extrudada para fora do miócito através do Na + / Ca2 + e o permutador de plasmalemmal Ca2 + ATPase 1,2. Consequentemente, o declínio no Ca 2+ citosólico instiga mudanças conformacionais de filamentos finos de volta ao estado original, resultando na dissociação da actina-miosina e miócitos relaxamento 1-3. Neste esquema, a actividade da SERCA2a é geralmente considerada para determinar a velocidade de relaxamento do miocárdio porque é responsável por 70 - 90% de remoção citosólica de Ca2 + na maioria das células de mamífero do coração 1. Como tal, anormal manipulação de Ca 2+ por LTCC, RyR e SERCA2a, etc. tem sido considerado os mecanismos principais para comprometimento da contratilidade e relaxamento no coração doente 1-4. de Ca2 +, que funciona como o mensageiro em contas de acoplamento CE para cerca de 1% do total de Ca 2+ e a maioria dos Ca 2+ é obrigado a intracelulares de Ca2 + buffers 5,6. Isto é devido ao facto de vários tampões Ca 2+ são abundantes em miócitos cardíacos, por exemplo, fosfolípidos de membrana, ATP, fosfocreatina, a calmodulina, a parvalbumina, miofibrila TnC, miosina, SERCA2a, e calsequestrin no SR. 5,6,7. Entre eles, SERCA2a e TnC são as predominantes Ca 2+ buffers 5,6,7. Além disso, o Ca 2+ ligação aos seus tampões é um processo dinâmico durante tique (por exemplo, 30-50% de Ca 2+ se liga a TnC e dissociam-se que durante os transientes de Ca2 + 7) e a alteração no Ca 2+ causa adicional de ligação &quot;libertação&quot; de Ca2 + livre para o citosol, os resultados nas alterações do Ca2 + intracelular concentration. Consequentemente, a perturbação do nível intracelular de Ca 2+ induz movimentos miofilamentos anormais, que são os precursores da disfunção contráctil e arritmias 8,9. Muitos factores (tanto fisiológicos e patológicos) podem ser as fontes de modificações pós-transcricionais de proteínas miofilamentos, que influenciam miofilamentos Ca 2+ e de tamponamento miofilamentos Ca 2+ sensibilidade 8-10. Recentemente, foi relatado que mutações em proteínas miofilamentos aumentar o Ca2 + intracelular e a afinidade de ligação a manipulação de Ca2 +, desencadeando a potenciação dependente de pausa de transientes de Ca2 +, Ca2 +, libertação anormal e arritmias 8. De acordo com este conceito, mostrámos também que miofilamentos Ca 2+ dessensibilização em corações de ratos hipertensos secundárias à sobre-regulação da sintase do óxido nítrico neuronal está associada com elevada diastólica e sistólica Ca 2+ níveis11, que por sua vez, aumenta a vulnerabilidade do LTCC ao Ca2 + inactivação dependente 12. Assim, miofilamentos Ca 2+ sensibilidade é um regulador &quot;ativo&quot; da função contrátil intracelular de Ca2 + homeostase e miócitos. Tornou-se necessário analisar as interacções entre miofilamentos e Ca 2+ manipulação proteínas para investigação aprofundada sobre miócitos acoplamento CE e função cardíaca. Aqui, descrevemos um protocolo que avalia as mudanças de miofilamentos Ca 2+ sensibilidade em miócitos cardíacos isolados. Análise abrangente do intracelular perfil Ca 2+, miofilamentos Ca 2+ sensibilidade e contração vai descobrir novos mecanismos mecânicos do miocárdio subjacentes.

<table border="0" cellpadding="0" cellspacing="0" width="1136">
	<tbody>
		<tr height="66">
			<td height="66" style="height:66px;width:315px;">Sprague Dawley rat</td>
			<td style="width:131px;">Koatech</td>
			<td style="width:125px;"></td>
			<td style="width:565px;">8-12 weeks</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:315px;">Pentobarbital Sodium</td>
			<td style="width:131px;">Hanlim Pharmaceutical (Korea)</td>
			<td style="width:125px;">AHN901</td>
			<td style="width:565px;">Insurance code:645301220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">NaH2PO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S8282</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">MgCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">M2001</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Mannitol</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M4125</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:315px;">MgSO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M5921</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium Pyruvate</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P2256</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Taurine</td>
			<td style="width:131px;">Merck</td>
			<td style="width:125px;">8.08616.1000</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Na2HPO4&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">71649</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Bovine Fetal Albumin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">A7906</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Collagenase Type 2</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:125px;">LS004177</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Protease</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P6911</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fura-2 (AM)</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:125px;">F1221</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Pluronic F127 20% solution in DMSO</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:125px;">P3000MP</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking Water Bath</td>
			<td style="width:131px;">Chang Shin Scientific</td>
			<td style="width:125px;">Model: C-108</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:315px;">IonWizard Softwae Suite</td>
			<td style="width:131px;">IonOptix Ltd</td>
			<td style="width:125px;">Experimental Builder</td>
			<td style="width:565px;">Acquisition and Analysis of EC Coupling Data in Myocytes</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:315px;">Myocyte Calcium &#38; Contractility Recording System</td>
			<td style="width:131px;">IonOptix Ltd</td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Circulating Water Bath</td>
			<td style="width:131px;">BS-Tech</td>
			<td style="width:125px;">BW2-8</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:315px;">Myocyte Fluorescence Microscope</td>
			<td style="width:131px;">Nikon</td>
			<td style="width:125px;">DIATPHOTO 200</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:315px;">MyoCam-S Power</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="36">
			<td height="119" rowspan="3" style="height:119px;width:315px;">Fluorescence &#38; Video Detection</td>
			<td rowspan="3" style="width:131px;">IonOptix</td>
			<td style="width:125px;">MyoCam-S</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:125px;">CFA300</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:125px;">PMT400</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">Fluorescence &#38; System Interface</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;">FSI700</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">Excitation Light Source</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;">mSTEP</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">High intensity ARC Lamp Power supply</td>
			<td style="width:131px;">Cairn Reseach</td>
			<td></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">Filter wheel controller</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;">GB/MUS200</td>
			<td></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;">Digital Stimulator</td>
			<td style="width:131px;">Medical Systems Corportion</td>
			<td>S-98 Mutimode</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Compositions of Experimental Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Name</td>
			<td style="width:131px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:565px;">Comments</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:315px;">Isolation Solution (pH: 7.4, NaOH)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;">Concentration (mmol) 135</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;">Concentration (mmol) 5.4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">MgCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">M2001</td>
			<td style="width:565px;">Concentration (mmol) 3.5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Taurine</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">CB2742654</td>
			<td style="width:565px;">Concentration (mmol) 20</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Na2HPO4&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">71649</td>
			<td style="width:565px;">Concentration (mmol) 0.4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Storage Solution (pH: 7.4, NaOH)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;">Concentration (mmol) 120</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;">Concentration (mmol) 5.4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;">Concentration (mmol) 10</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;">Concentration (mmol) 5.5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 0.2</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Mannitol</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M4125</td>
			<td style="width:565px;">Concentration (mmol) 29</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">MgSO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M5921</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Sodium Pyruvate</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P2256</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Taurine</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">CB2742654</td>
			<td style="width:565px;">Concentration (mmol) 20</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:315px;">Perfusion Solution (Tyrode solution, pH: 7.4, NaOH)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;">Concentration (mmol) 141.4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;">Concentration (mmol) 4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaH2PO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S8282</td>
			<td style="width:565px;">Concentration (mmol) 0.33</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;">Concentration (mmol) 10</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;">Concentration (mmol) 5.5</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td>Concentration (mmol) 1.8&#160;&#160;&#160;&#160;&#160; For Fura 2AM loading, CaCl2 concentrations are 0.25 mM and 0.5 mM</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">MgCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">M2001</td>
			<td style="width:565px;">Concentration (mmol) 1</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Mannitol</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M4125</td>
			<td style="width:565px;">Concentration (mmol) 14.5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Collangenase Solution 1</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Isolation Solution (30mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Bovine Fetal Albumin (BSA solution 5 ml)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;">Concentration (mmol) 1.67 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Collagenase Type 2</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:125px;">LS004177</td>
			<td style="width:565px;">Concentration (mmol) 1 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Protease</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P6911</td>
			<td style="width:565px;">Concentration (mmol) 0.1 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 0.05 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Collangenase Solution 2</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Isolation Solution (20mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Bovine Fetal Albumin (BSA solution 3.3 mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;">Concentration (mmol) 1.67 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Collagenase Type 2</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:125px;">LS004177</td>
			<td style="width:565px;">Concentration (mmol) 1 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 0.05 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">BSA solution</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Isolation Solution (40mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Bovine Fetal Albumin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">A7906</td>
			<td style="width:565px;">Concentration (mmol) 400 mg</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 1mM</td>
		</tr>
	</tbody>
</table>

assessment of myofilament ca2+ sensitivity underlying cardiac excitation-contraction coupling

insuficiência cardíaca e arritmias cardíacas são as principais causas de mortalidade e morbidade em todo o mundo. No entanto, o mecanismo de patogênese e mau funcionamento do miocárdio no coração doente continua a ser totalmente esclarecido. Provas convincentes recente demonstra que as mudanças na miofilamentos Ca 2+ sensibilidade afetam Ca 2+ intracelular atividades homeostase e canais iônicos em miócitos cardíacos, os mecanismos essenciais responsáveis ​​pelo potencial de ação cardíaco e contração em corações saudáveis ​​e doentes. De facto, as actividades dos canais de iões e transportadores subjacentes potenciais de acção cardíaca (por exemplo, Na +, Ca 2+ e os canais de K + e Na + -Ca 2+) e proteínas de permutador de Ca 2+ intracelular de manuseamento (por exemplo., Os receptores de rianodina e Ca 2 + -ATPase no retículo sarcoplasmático (SERCA2a) ou fosfolambam e a sua fosforilação) são convencionalmente medido para avacomeu os mecanismos fundamentais do acoplamento excitação-contração cardíaca (CE). Ambas as atividades elétricas na membrana e Ca 2+ alterações intracelulares são os sinais de disparo de acoplamento CE, enquanto miofilamentos é a unidade funcional de contração e relaxamento, e miofilamentos Ca 2+ sensibilidade é imperativo na implementação de desempenho miofibrila. No entanto, poucos estudos incorporar miofilamentos Ca 2+ sensibilidade para a análise funcional do miocárdio, a menos que é o foco do estudo. Aqui, descrevemos um protocolo que mede sarcomere encurtamento / re-alongamento eo nível de Ca2 + intracelular usando Fura-2 AM (detecção ratiometric) e avaliar as alterações de sensibilidade dos miofilamentos Ca 2+ em miócitos cardíacos de corações de ratos. O principal objetivo é enfatizar que miofilamentos Ca 2+ sensibilidade deve ser levado em consideração no acoplamento CE para análise mecanicista. investigação detalhada do iem canais, transportadores iônicos, intracelular de manipulação de Ca2 +, e miofilamentos Ca 2+ sensibilidade que fundamentam a contratilidade dos miócitos em corações saudáveis ​​e doentes irá fornecer informações valiosas para a concepção de estratégias mais eficazes de translação e valor terapêutico.

miócitos do VE são isolados de corações normais e hipertensos ratos. Miócitos em forma de haste com estrias claras (representando sarcómeros) e contracções estáveis ​​em resposta à estimulação do campo são considerados os miócitos óptimos e são seleccionados para gravações (Figura 2A). No exemplo mostrado na F igura 2A, uma Fura 02:00 -loaded miócitos está posicionado horizontalmente e a abertura da câmara é ajustado de modo que...

Watch this Scientific Journal Video about Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling at JoVE.com

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 ...

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Research

JoVE Journal

Biology

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

9.4K visualizações.  Seoul National University College of Medicine. 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. 

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biologia

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

Mecanismos imunológicos investigando a rejeição do transplante de órgãos Subjacente

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Focal Ca 2 + Detecção de transitórios no músculo liso

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

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 ...

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

Avaliação fosforilação oxidativa respirométrico em saponina-fibras cardíacas permeabilizadas

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Visualização dos Vascular Ca 2 + Sinalização Disparado por parácrina ROS Derivado

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Microscopia intravital da Microcirculação no músculo Rato Cremaster de Análise de Migração Stem Peripheral celular

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Automated Análise de Ca dinâmico<sup&gt; 2 +</sup&gt; Sinais em seqüências de imagens

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Medindo espacial e temporal Ca<sup&gt; 2 +</sup&gt; Sinais em Arabidopsis Plantas

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagem local Ca<sup&gt; 2+</sup&gt; Sinais em células de mamíferos em cultura

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...