The Fralin Life Science Research Institute and The Metabolic Phenotyping Core at Virginia Tech supported this work.

Os estudos em animais foram realizados sob um protocolo aprovado pelo Institutional Animal Care e usar Comité na Virginia Polytechnic Institute e State University. 1. Instalação (Time: ~ 45 min) <ol><li> Lojas de congelados descongelamento de tripsina a 0,25%, tampão de isolamento para mitocôndrias (IBM) 1 e IBM2 em um banho de água a 37 ° C. </li><li> Enxágüe copos e instrumentos de dissecação em etanol a 70%, seguido por água de alta pureza. </li><li> Preparar a solução de tripsina a 0,05% a partir de 0,25% estoque de tripsina diluindo 1 parte de tripsina em 4 partes IBM1. </li><li> Misturar protease e fosfatase cocktail inibidor com tampão de lise celular (1: 100 rácio) num tubo de microcentrífuga de 1,5 ml com tampa de rosca. </li><li> Alíquotas de 5 ml (5 ml / rato) de tripsina a 0,05% para um tubo de plástico de 15 ml. </li><li> Ajuste motorizado homogeneizador de tecidos a 80 RPM. </li></ol> 2. Isolamento de mitocôndrias Músculo Esquelético (Tempo: ~ 90 min) <ol><li> Euthanize um rato por CO 2 </sub&gt; inalação, seguido por deslocação cervical. </li><li> Remover músculo vermelho do músculo gastrocnémio e quadríceps, que inclui o sóleo (~ 75-100 mg no total) tal como descrito nas etapas a seguir: <ol><li> Descasque a pele para o mouse. </li><li> Remova a almofada de gordura sobre o ponto de origem do quadríceps. Corte o tendão do quadríceps que está ligado à patela com uma tesoura de ponta fina. Nota: O quadríceps é identificado por sua posição anatômica na porção anterior do fêmur proximal. </li><li> Lentamente cortar o aponeurosis entre o osso e os quadríceps, evitando a artéria femoral, para libertar os quadríceps do osso. Corte o tendão com uma tesoura de ponta fina no ponto de origem no fêmur para liberar o músculo quadríceps e coloque o músculo quadríceps em refrigerados PBS. </li><li> Remover o tecido adiposo visível sobre o quadríceps com uma tesoura. Virar o quadríceps de forma que a parte do músculo que se recobre o fémur é virado up. Abra o músculo quadríceps com uma pinça em um movimento de ventilação. Retire as duas porções musculares vermelhas visíveis de cada lobo com uma tesoura de ponta fina e coloque em um copo contendo 5 ml de refrigerados IBM1. Nota: músculo quadríceps aparecem como dois lobos com uma tira de músculo vermelho localizado perto da borda lateral de cada lóbulo. </li><li> Corte a pele que recobre o tendão de Aquiles com tesouras com ponta fina e descascar a pele na direção do mouse. Cortar o tendão de Aquiles exposta e descasque o músculo em direcção ao corpo do rato. </li><li> Corte o tendão no côndilos lateral e medial do fêmur com uma tesoura de ponta fina para libertar o gastrocnêmio e coloque o gastrocnêmio anexado aos seus tendões em refrigerados PBS. </li><li> Virar o gastrocnêmio sobre e descolar o músculo sóleo e coloque no copo contendo 5 ml de refrigerados IBM1. Fan abrir o gastrocnêmio. Remova os três porções visuais vermelhas musculares (duas tiras vermelhas lateral e um medial superficial) com tesoura de ponta finas e transferi-los para o copo contendo 5 ml de refrigerados IBM1. </li></ol></li><li> Remover outro ~ 75-100 mg de vermelho de músculo a outra perna do rato (conforme descrito no passo 2.2) e colocar num tubo de microcentrífuga de 1,5 ml com protease e fosfatase cocktail inibidor e tampão de lise celular (50 mM Tris-HCl, 1 mM de EDTA, 150 mM de NaCl, 1% de dodecil sulfato de sódio, 0,5% de desoxicolato de sódio, 1% de polioxietileno (9) nonilfeniléter, ramificada. Imediatamente piscar-congelar esta segunda amostra em azoto líquido para posterior utilização em transferência Western (ver passo 6.1). </li><li> Colocar uma superfície de plástico pré-gelada, plana sobre gelo em um balde grande. Pour uma gota de IBM1 sobre a superfície de plástico e utilizar uma pinça para colocar todo o músculo vermelho segmentado (passo 2.2.4 e 2.2.7) em IBM1 gota. Picar o tecido muscular durante 2 minutos usando 3 individuais lâminas de barbear borda, alterando lâminas de barbear a cada 40 seg. </li><li> Transferir o tecido moído para um novo recipiente com 5 ml de IBM1 fresco, mantendo a superfície de plástico sobre o the proveta e raspando o músculo para a proveta com uma lâmina de barbear. Tome esta solução e drená-lo através de um filtro de células 100 mm colocados em um tubo de 50 ml. </li><li> Seque o tecido com um limpador tarefa delicada e, em seguida, transferi-la para 5 ml de solução de tripsina a 0,05%. Use pinças para remover o tecido a partir da tarefa do limpador. </li><li> Incubar o tecido muscular em gelo durante 30 min em solução de tripsina a 0,05%. </li><li> Rodar as tubo de 15 ml contendo mistura de tripsina / músculo a 200 xg durante 3 min a 4 ° C. </li><li> Despeje o sobrenadante tripsina em um recipiente de resíduos e ressuspender o sedimento com 3 ml de gelo frio IBM1. Transferir o tecido para um tubo de 45 ml homogeneizador de vidro. Lavar as 15 ml tubo cônico com mais 1,5 ml IBM1 para recolher qualquer amostra restante e adicione ao tubo de homogeneizador de vidro. </li><li> Colocar o tubo homogeneizador de vidro para um copo ou recipiente de plástico a meio caminho cheio de gelo de modo que o homogeneizador de vidro move-se minimamente no interior da taça. </li><li&gt; Homogeneizar a mistura tecido / IMB1 com o pilão de PTFE ligados a um homogeneizador de tecido accionada por motor com 10 passagens a 80 rpm. Segure a parte inferior de cada passagem para ~ 2 seg. </li><li> Transferir o homogeneizado de tecido para um novo tubo de 15 mL cónico e enxaguar o tubo homogeneizador de vidro com 6,5 ml de IBM1. Verter a IBM1 para dentro do tubo cónico de 15 ml contendo homogeneizado de tecido. </li><li> Girar o tubo de 15 ml a 700 g durante 10 min a 4 ° C. Delicadamente despeje o sobrenadante para um tubo de centrífuga de alta resistência vidro e descartar o pellet. </li><li> Girar o sobrenadante do passo anterior a 8000 xg durante 10 min a 4 ° C. </li><li> Remover o sobrenadante e re-IBM1 suspender o sedimento, adicionando-se lentamente 500 ul de IBM2. Cortar a extremidade de uma ponta de pipeta e homogeneizar suavemente o sedimento em IBM2 com mistura e agitação movimentos. Adicionar mais 4,5 ml de IBM2 à mistura após a pelete é totalmente suspenso. Nota: Evite trituração excessiva ao quebrar pedaços maiores de pelotas como tHis pode danificar as membranas mitocondriais. </li><li> Girar o homogenato / tecido IBM2 a 8000 xg durante 10 min a 4 ° C. Nota: Esta etapa pode ser repetida num tubo de centrífuga limpo de alta resistência, para a quantificação mais precisa da proteína de mitocôndrias isoladas no Passo 4, uma vez de BSA residual pode contribuir para o conteúdo de proteína total. </li><li> Remover o sobrenadante e suavemente, mas completamente re-suspender o sedimento, adicionando dois incrementos de 25 ul de IBM2 com uma ponta de pipeta com a ponta cortada. Agitar suavemente o sedimento e misturar após cada adição de 25 ul de IBM2. Coloque este estoque mitocondrial no gelo. </li></ol> 3. A homogeneização da Whole Tissue Lysate (Tempo: ~ 45 min; pode ser feito durante Passo 7) <ol><li> Remover o músculo do que foi colocada em azoto líquido a partir do passo 2.3 e incubar à temperatura ambiente até estarem completamente descongelados. </li><li> Picar o tecido para ~ 30 segundos em gelo com uma tesoura de ponta fina. </li><li> Adicione duas colheres de 100 ul de ZircoÓxido nio Beads para o tubo de microcentrífuga de 1,5 ml com tampa de rosca a partir do passo 3.2 que contém o tecido picada. Homogeneizar o tecido em um homogeneizador de tecidos moinho de esferas usando dois a três ciclos de 5 min a uma velocidade de 4-6 configuração (ajuste médio). </li><li> Girar-se a mistura a partir do passo 3.3 a 12.000 xg durante 10 min a 4 ° C. Remover o sobrenadante utilizando uma ponta de pipeta de 1.000 ul, após a centrifugação e transferência para um tubo de microcentrífuga de 1,5 ml. Desprezar o pelete e colocar o sobrenadante em gelo. </li></ol> 4. Determinação da proteína (Tempo: ~ 30 min) <ol><li> Determinar a concentração de proteína de lisado de todo o tecido (passo 3.4) e da mitocondrial (passo 2.17) utilizando o kit de ensaio de proteínas BCA de acordo com as especificações do fabricante. </li></ol> 5. As mitocôndrias Membrane Integridade; Citrato sintase (CS) Atividade (Tempo: ~ 15 min) <ol><li> Faça dois separados 1: 20 diluições do estoque mitocondrial em água de alta pureza. Adicionar0,1% de Triton X-100 para uma amostra e sonicate-lo em um nível baixo (765 Watts, amplitude de 2). Deixe a outra diluição no gelo. Recolocar a amostra de gelo após sonicação. </li><li> Realizar um ensaio de citrato sintase tanto com a não-sonicada e sonicada diluições mitocondriais utilizando um ensaio espectrofotométrico conforme anteriormente descrito 11,12. </li><li> Determinar a percentagem de membranas mitocôndrias intactas, utilizando as seguintes equações: (CS atividade não-sonicado ÷ sonicado atividade CS) * 100 </li></ol> 100- N- (percentagem obtida a partir de cima) 6. As mitocôndrias Qualidade de isolamento; Western Blotting (Horário: De acordo com Lab Protocol) <ol><li> Realizar Western blotting em todo o lisado de tecido e mitocôndrias isoladas como anteriormente descrito 12. Nota: O uso de anticorpos primários para GAPDH (1: 1000 de diluição em 5% de BSA / TBS-T) e COXIV (1: 1000 de diluição em 5% de gordura não-leite / TBS-T), seguido por anti-cabra, e anticorpos secundários de coelho, respectivamente (1: 10.000). </li></ol> 7. respirométrico Ensaio Run (Time: ~ 90 min) <ol><li> Realizar ensaios respirométricos desejados utilizando baseado O 2 tecnologia de medição do consumo de microplaca, como descrito anteriormente (ver artigo do companheiro e Rogers et al 8). </li><li> Determinar a relação de controle respiratório (RCR) dividindo Estado 3 por Estado 4o (RCR) ou dividindo 3U Estado por Estado 4o. Use o (média) ponto médio, ou o mais elevado (estado 3) e taxas mais baixas (4o Estado) a partir de vestígios ponto-a-ponto ao determinar RCR. </li></ol>

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George Rogers is an employee of Seahorse Bioscience that produces the instrument for which this protocol was modified.

Os métodos aqui apresentados proporcionam uma descrição detalhada de um procedimento de isolamento de quantidades mínimas mitocondrial (~ 75-100 mg) de rato músculo esquelético. Este procedimento de isolamento é capaz de produzir alto funcionamento, as mitocôndrias puro (~ 450 mg) como evidenciado pela O2 taxas de consumo, valores RCR, atividade da citrato sintase máxima e expressão de proteína de immunoblotting. Importante, as mitocôndrias isoladas a partir deste processo pode ser usado para múl...

The primary function of mitochondria is to produce ATP from oxidative phosphorylation. However, mitochondria have many other important cellular functions including but not limited to: the production and detoxification of reactive oxygen species, the regulation of cytoplasmic and mitochondrial calcium, organelle trafficking, ionic homeostasis, and involvement in apoptosis1,2. Therefore, it is not surprising that dysfunctional mitochondria play a role in many disease pathologies, such as aging, neurodegenerative diseases, cardiovascular disease, cancer, obesity, and diabetes3,4. Importantly, skeletal muscle mitochondria specifically are involved in many of these pathologies3-5.
Mitochondrial respiration assays using isolated mitochondria allow for the assessment of electron transport chain and oxidative phosphorylation function, and the determination of mechanism(s) of action of drugs and proteins that modulate metabolism. Mitochondrial isolation procedures exist for multiple tissue and cell types for a variety of species6,7. However, these procedures often require large quantities of tissue/cells for a high quality mitochondria yield necessary for classic respirometric assays. 
Microplate based respirometirc assays allow for high throughput measurements using minimal quantities of isolated mitochondria, often just several &#181;g per well8. Therefore, we present a modification of previously published methods7 to allow for high quality mitochondria to be isolated from smaller quantities of mouse skeletal muscle for use in microplate based respirometirc assays. In addition, methods are provided to establish the quality of the mitochondrial isolation preparation and the integrity of the mitochondrial membranes. Given that skeletal muscle mitochondria are involved in many pathological conditions, the measurement of O2 consumption in mechanistically driven studies is becoming more prevalent in biomedical research9,10.

<table><tbody><tr><td>Essentially Fatty</td><td>Sigma Aldrich</td><td>A6003</td><td>N/A</td></tr><tr><td>Acid Free- BSA</td><td></td><td></td><td></td></tr><tr><td>Tris/HCl</td><td>Promega</td><td>H5123</td><td>N/A</td></tr><tr><td>KCL</td><td>Sigma Aldrich</td><td>P9541</td><td>N/A</td></tr><tr><td>Tris Base</td><td>Promega</td><td>H5135</td><td>N/A</td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E6511</td><td>N/A</td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td>N/A</td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S7903</td><td>N/A</td></tr><tr><td>D-Mannitol</td><td>Sigma Aldrich</td><td>63559</td><td>N/A</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Thermo Scientific</td><td>25200-056</td><td>N/A</td></tr><tr><td>Sodium Chloride&lt;br/&gt; White Crystals or Crystalline Powder&lt;br/&gt; &amp;ge;99.0 %</td><td>Fisher Scientific</td><td>BP3581</td><td>N/A</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma Aldrich</td><td>L3771&amp;nbsp;</td><td>N/A</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma Aldrich</td><td>D6750&amp;nbsp;</td><td>N/A</td></tr><tr><td>Polyoxyethylene (12) nonylphenyl ether, branched</td><td>Sigma Aldrich</td><td>238651</td><td>N/A</td></tr><tr><td>Single Edge Razor Blades</td><td>Fisher Scientific</td><td>12-640</td><td>N/A</td></tr><tr><td>Falcon- 100 uM Nylon Cell Strainers</td><td>Fisher Scientific</td><td>352360</td><td>N/A</td></tr><tr><td>Halt Protease &amp;amp; Phosphatse Inhibitor Cocktail</td><td>Thermo Scientific</td><td>1861284</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes with screw cap</td><td>Thermo Scientific</td><td>3474</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Zirconium Oxide beads</td><td>Fisher Scientific</td><td>C9012112</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>GAPDH antibody (1D4)</td><td>Santa Cruz Biotechnology</td><td>sc-59540</td><td>N/A</td></tr><tr><td>Anti- COXIV antibody</td><td>Cell Signaling</td><td>4844s</td><td>Any mitochondrial inner membrane protein will suffice</td></tr><tr><td>Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)</td><td>Jackson ImmunoResearh</td><td>711-035-152</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)</td><td>Jackson ImmunoResearh</td><td>115-001-003</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Triton-X100</td><td>Sigma Aldrich</td><td>X100</td><td>N/A</td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Scientific</td><td>23225</td><td>N/A</td></tr><tr><td>Pyruvic Acid, 98%</td><td>Sigma Aldrich</td><td>107360</td><td>Store at 4 &amp;deg;C,pH to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>Succinic Acid</td><td>Sigma Aldrich</td><td>S9512</td><td>Store at room temperature, pH to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>L(-) Malic Acid, BioXtra, &amp;ge;95%</td><td>Sigma Aldrich</td><td>M6413</td><td>Store at room temperature, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>L-Glutamic acid</td><td>Sigma Aldrich</td><td>G1251</td><td>Store at room temperature, to 7.4 with KOH prior to use in respirometric assay, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Palmitoyl L-carnitine chloride</td><td>Sigma Aldrich</td><td>P1645</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Oligomycin A, &amp;ge; 95% (HPLC)</td><td>Sigma Aldrich</td><td>75351</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)</td><td>Sigma Aldrich</td><td>C2920</td><td>Store at 2-8 &amp;deg;C</td></tr><tr><td>phenylhydrazone</td><td></td><td></td><td></td></tr><tr><td>&amp;ge;98% (TLC), powder [FCCP]</td><td></td><td></td><td></td></tr><tr><td>Antimycin A from streptomyces sp.</td><td>Sigma Aldrich</td><td>A8674</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Adenosine 5&amp;prime;-diphosphate monopotassium salt dehydrate [ADP]</td><td>Sigma Aldrich</td><td>A5285</td><td>Store at -20 &amp;deg;C, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Rotenone</td><td>Sigma Aldrich</td><td>R8875</td><td>Store at room temperature</td></tr></tbody></table>

isolation of mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements

Disfuncionais mitocôndrias do músculo esquelético desempenha um papel no metabolismo alterado observado com o envelhecimento, obesidade e diabetes tipo II. Ensaios respirométricos mitocondriais isoladas a partir de preparações mitocondriais permitir a avaliação da função mitocondrial, bem como a determinação do mecanismo (s) de acção de drogas e proteínas que modulam o metabolismo. Procedimentos de isolamento atuais requerem muitas vezes grandes quantidades de tecido para produzir mitocôndrias de alta qualidade necessários para ensaios respirométricos. Os métodos aqui apresentados descrevem como de alta qualidade mitocôndrias purificadas (~ 450 mg) pode ser isolado a partir de quantidades mínimas (~ 75-100 mg) de rato músculo esquelético para utilização em medições respiratórias alto rendimento. Nós determinamos que nosso método de isolamento produz 92,5 ± 2,0% mitocôndrias intacto medindo atividade da citrato sintase espectrofotometria. Além disso, a análise de Western blot em mitocôndrias isoladas resultou na expressão fraca da cytosoproteína lic, GAPDH, e a expressão robusta da proteína mitocondrial, COXIV. A ausência de uma banda proeminente GAPDH nas mitocôndrias isoladas é indicativo de pouca contaminação a partir de fontes não-mitocondriais durante o procedimento de isolamento. Mais importante ainda, a medição da taxa de consumo de O2 com a tecnologia de micro-placa de base e a determinação do índice de controlo respiratório (RCR) para ensaios respirométricos acoplados mostra altamente acopladas (RCR;&gt; 6 para todos os ensaios) e mitocôndrias funcionais. Em conclusão, a adição de um passo de picagem separado e reduzindo significativamente accionada por motor de velocidade de homogeneização de um método previamente relatado que permitiu o isolamento de alta qualidade e de mitocôndrias purificadas a partir de pequenas quantidades de rato músculo esquelético que resulta em mitocôndrias altamente acopladas que respiram com elevada função durante microplacas ensaios respirometirc.

Atividade da citrato sintase serve como uma medida para a integridade da membrana, uma vez sintase de citrato é localizado na membrana mitocondrial interna, e, portanto, não devem estar presentes nas suspensões de mitocôndrias com membranas intactas. A Figura 1 representa a actividade da sintase de citrato em amostras não sonicada mitocondriais em comparação com sonicada amostras do mesmo isolamento. Sonicando os resultados mitocôndria em um aumento estatisticamente significativo na atividade da...

Watch this Scientific Journal Video about Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements at JoVE.com

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

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 ...

isolation-mitochondria-from-minimal-quantities-mouse-skeletal-muscle

Research

JoVE Journal

Biology

12.7K visualizações.  Virginia Tech. 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.

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

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Usando mitocôndrias isoladas de quantidades mínimas de Rato Músculo Esquelético para alta taxa de transferência de microplacas respiratórias Medições

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Biologia do Desenvolvimento

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

Medição da Capacidade de Importação de Proteínas das Mitocôndrias Musculares Esqueléticas

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

Bioquímica

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Preparação e Avaliação respirométrico de mitocôndrias isoladas de tecido muscular esquelético Obtido por meio de agulha de biópsia percutânea

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Medicina

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultas e embrionárias Cultura Microexplant Músculo Esquelético e isolamento de células-tronco musculares

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Biologia

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Isolamento mitocondrial do músculo esquelético

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purificação de Progenitores do músculo esquelético

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

<meta charset="utf8"></head><body>Isolamento mitocondrial de roedores para estudos bioenergéticos de alta qualidade

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

<meta charset="utf8"></head><body>Autor em destaque: Descobrindo o papel do fosfato de cálcio mitocondrial na insuficiência cardíaca e bioenergética

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

Isolamento mitocondrial robusto de tecido cardíaco de roedores 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Parâmetro Multi-Medição da abertura dos poros transição de permeabilidade em mitocôndrias isoladas de coração Rato

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

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

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