Name: multi-parameter measurement of the permeability transition pore opening in isolated mouse heart mitochondria
Uploaded: 2012-09-07T12:00:00
Description: Watch this Scientific Journal Video about Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria at JoVE.com

Dette arbejde blev støttet af HL094536 (BJH).

1. Isolering af Mitokondrier fra mus Heart <ol><li> For at isolere hjerte mitokondrier, bedøver og ofre mus i overensstemmelse med de procedurer, der er godkendt af din lokale Institutional Animal Care og Use Udvalg. </li></ol> Bemærk: Alle trin i mitokondrier isoleret protokollen skal udføres på is. Brug is kolde buffere og præ-kølet Petriskaale Falcon rør og Eppendorf rør. De mængder der er angivet i protokollen er for en prøve indeholdende 2 mus hjerter. <...

<ol>
	<li>Kroemer, G., Galluzzi, L., Brenner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Membrane+Permeabilization+in+Cell+Death.">Mitochondrial Membrane Permeabilization in Cell Death.</a> Physiol. Rev. 87, 99-163 (2007).</li><li>Elrod, J., Wong, R., Mishra, S., Vagnozzi, R. J., Sakthievel, B., Goonasekera, S. A., Karch, J., Gabel, S., Farber, J., Force, T., Brown, J. H., Murphy, E., Molkentin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclophilin+D+controls+mitochondrial+pore-dependent+Ca2++exchange,+metabolic+flexibility,+and+propensity+for+heart+failure+in+mice.">Cyclophilin D controls mitochondrial pore-dependent Ca2+ exchange, metabolic flexibility, and propensity for heart failure in mice.</a> J. Clin. Invest. 120, 3680-3687 (2010).</li><li>Hom, J. R., Quintanilla, R. A., Hoffman, D. L., de Mesy Bentley, K. L., Molkentin, J. D., Sheu, S. S., Porter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+permeability+transition+pore+controls+cardiac+mitochondrial+maturation+and+myocyte.">The permeability transition pore controls cardiac mitochondrial maturation and myocyte.</a> 21, 469-478 (2011).</li><li>Halestrap, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+mitochondrial+permeability+transition+pore.">What is the mitochondrial permeability transition pore.</a> Journal of Molecular and Cellular Cardiology. 46, 821-831 (2009).</li><li>Wei, A. C., Liu, T., Cortassa, S., Winslow, R. L., O'Rourke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Ca2++influx+and+efflux+rates+in+guinea+pig+cardiac+mitochondria:+low+and+high+affinity+effects+of+cyclosporine+A.">Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A.</a> Biochim. Biophys. Acta. 1813, 1373-1381 (2011).</li><li>Saks, V. A., Kuznetsov, A. V., Kupriyanov, V. V., Miceli, M. V., Jacobus, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creatine+kinase+of+rat+heart+mitochondria.+The+demonstration+of+functional+coupling+to+oxidative+phosphorylation+in+an+inner+membrane-matrix+preparation.">Creatine kinase of rat heart mitochondria. The demonstration of functional coupling to oxidative phosphorylation in an inner membrane-matrix preparation.</a> J. Biol. Chem. 260, 7757-7764 (1985).</li><li>Boehm, E. A., Jones, B. E., Radda, G. K., Veech, R. L., Clarke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+uncoupling+proteins+and+decreased+efficiency+in+palmitate-perfused+hyperthyroid+rat+heart.">Increased uncoupling proteins and decreased efficiency in palmitate-perfused hyperthyroid rat heart.</a> AJP - Heart. 280, 977-983 (2001).</li><li>Fontaine, E., Eriksson, O., Ichas, F., Bernardi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+Permeability+Transition+Pore+in+Skeletal+Muscle+Mitochondria.">Regulation of the Permeability Transition Pore in Skeletal Muscle Mitochondria.</a> J. Biol. Chem. 273, 12662-12668 (1998).</li><li>Berman, S. B., Watkins, S. C., Hastings, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+biochemical+and+ultrastructural+comparison+of+mitochondrial+permeability+transition+in+isolated+brain+and+liver+mitochondria:+evidence+for+reduced+sensitivity+of+brain+mitochondria.">Quantitative biochemical and ultrastructural comparison of mitochondrial permeability transition in isolated brain and liver mitochondria: evidence for reduced sensitivity of brain mitochondria.</a> Exp. Neurol. 164, 415-425 (2000).</li><li>Panov, A., Dikalov, S., Shalbuyeva, N., Hemendinger, R., Greenamyre, J. T., Rosenfeld, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-+and+tissue-specific+relationships+between+mitochondrial+permeability+transition+and+generation+of+ROS+in+brain+and+liver+mitochondria+of+rats+and+mice.">Species- and tissue-specific relationships between mitochondrial permeability transition and generation of ROS in brain and liver mitochondria of rats and mice.</a> Am. J. Physiol. Cell Physiol. 292, 708-718 (2007).</li><li>Frezza, C., Cipolat, S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+isolation:+functional+mitochondria+from+mouse+liver,+muscle+and+cultured+filroblasts.">Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts.</a> Nature Protocols. 2, 287-295 (2007).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 80, 3-44 (2007).</li></ol>

Protokollen præsenteres her beskriver de nødvendige eksperimentelle procedure for at vurdere permeabilitetsovergang pore åbning i isolerede hjerte mitokondrier (figur 1 og figur 4): fremgangsmåden til isolering af muse-hjerte mitokondrier, de respiratoriske kontrol, der sikrer deres integritet og funktionalitet, de mitochondriale parametre overvåges under mtPTP åbning og farvestofferne anvendt for deres måling, oprettelsen af ​​den spektrofluorometrisk instrumentering og kara...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> Trypsin </td><td> Sigma-Aldrich </td><td> T3030 </td></tr><tr><td> Trypsininhibitor (sojabønne) </td><td> Sigma-Aldrich </td><td> T9128 </td></tr><tr><td> Natriumhydrosulfit </td><td> Sigma-Aldrich </td><td> 71.699 </td></tr><tr><td> Rotenon </td><td> Sigma-Aldrich </td><td> R8875 </td></tr><tr><td> Cytochrom c </td><td> Sigma-Aldrich </td><td> C7752 </td></tr><tr><td> Alamethicin </td><td> Sigma-Aldrich </td><td> A4665 </td></tr><tr><td> CCCP </td><td> Sigma-Aldrich </td><td> C2759 </td></tr><tr><td> Cyclosporin A </td><td> Calbiochem </td><td> 239.835 </td></tr><tr><td> Fura FF </td><td> Invitrogen </td><td> F14180 </td></tr><tr> &lt;td&gt; JC-1 </td><td> Invitrogen </td><td> T3168 </td></tr><tr><td> Vævsformaler Potter-Elvehjem med teflonstøder 15 ml </td><td> Wheaton Industries </td><td></td></tr><tr><td> Overliggende omrører </td><td> Wheaton Industries </td><td> 903.475 </td></tr><tr><td> Oxytherm (temperaturreguleret oxygenelektrode) </td><td> Hansatech Instruments </td><td></td></tr><tr><td> QuantaMaster 80 dual emission spektrofluorometer </td><td> Photon Technology International, Inc. </td><td></td></tr></tbody></table>

multi-parameter measurement of the permeability transition pore opening in isolated mouse heart mitochondria

Den mitokondriel permeabilitetsovergang pore (mtPTP) er en ikke specifik kanal, der dannes i den indre mitokondriemembran at transportere opløste stoffer med en molekylvægt mindre end 1,5 kDa. Selv om den endelige molekylære identitet af pore er stadig under debat, proteiner, såsom cyclophilin D, VDAC og ANT bidrage til mtPTP formation. Mens inddragelsen af mtPTP åbning i celledød er veletableret 1, akkumulere beviser tyder på, at mtPTP serverer en fysiologisk rolle under mitochondrial Ca 2 + homeostase 2, bioenergetik og redox signalering 3. mtPTP åbning udløses af matrix Ca2 +, men dets aktivitet kan moduleres ved en række andre faktorer, såsom oxidativt stress, adeninnukleotid udtømning, høje koncentrationer af Pi, mitokondriel membrandepolarisering eller frakobling, og langkædede fedtsyrer 4. in vitro mtPTP åbningen kan være achieved ved at forøge Ca2 +-koncentration inden for mitochondrial matrix via exogene tilsætninger af Ca2 + (calcium tilbageholdelseskapacitet). Når Ca2 + niveauer inde mitokondrier når en bestemt tærskel, mtPTP åbnes og fremmer Ca2 +-frigivelse, dissipation af proton motorkraft, membranpotentiale kollaps og en stigning i mitochondrial matrix volumen (hævelse), som i sidste ende fører til brud på ydre mitochondriemembran og uopretteligt tab af organel funktion. Her beskriver vi et fluorometrisk assay, som tillader en omfattende karakterisering af mtPTP åbning i isolerede mus hjerte mitokondrier. Assayet involverer samtidig måling af 3 mitokondrielle parametre, der ændres, når mtPTP åbning opstår: mitochondrial Ca2 + håndtering (optagelse og frigivelse som målt ved Ca2 +-koncentration i assaymediet), mitokondriemembranspændingen, og mitochondrial volumen. Farvestofferne anvendes til Ca2 +-målingen i assaymediet og mitokondriemembranspændingen er Fura FF, en membran impermeabel, ratiometrisk indikator, der undergår en ændring i excitations-bølgelængden i nærvær af Ca2 +, og JC-1, et kationisk, ratiometrisk indikator, som danner grønne monomerer eller røde aggregater med lav og høj membranpotentiale hhv. Ændringer i mitochondrie volumen måles ved en registrering lysspredning af mitochondrial suspension. Da høj kvalitet, funktionelle mitokondrier er nødvendige for mtPTP åbning assay, vi beskriver også de trin, der er nødvendige for at opnå intakt, stærkt koblet og funktionelle isolerede hjerte mitokondrier.

Watch this Scientific Journal Video about Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria at JoVE.com

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

Et spektrofluorometrisk protokol for målingen af ​​mitokondriel permeabilitetsovergang pore åbning i isoleret muse hjerte mitokondrier er præsenteret her. Assayet involverer samtidig måling af mitokondrier Ca<sup&gt; 2 +</sup&gt; Håndtering, mitokondriemembranspændingen og mitokondrie volumen. Proceduren for at indhente høj kvalitet og funktionelle hjerte mitokondrier er også beskrevet.

Multi-parameter Måling af permeabilitetsovergang pore åbning i isolerede Mouse Heart Mitokondrier

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

Research

JoVE Journal

Biology

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Konfokal Imaging af Single Mitochondriale Superoxide Blinker i intakte hjerte eller<em&gt; In Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

Kvantificering Single mikrokardannelse Permeability i Isoleret Blood-perfunderet rottelunge Forberedelse

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Måling af Osmotisk Vand permeabilitetskoefficienten (P<sub&gt; F</sub&gt;) Af sfæriske celler: Isolerede planteprotoplaster som et eksempel

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Immunodetektion af ydre membranproteiner ved flowcytometri Isoleret Mitokondrier

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Måling af Glat muskel funktion i den isolerede vævsbad-ansøgninger til Farmakologi Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

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

Isolering af mitokondrier fra minimale mængder af Mouse Skeletmuskel for High Throughput Microplate Respiratory Målinger

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

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

Metoder til bestemmelse af priser for glucose og fedtsyreoxidation i Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

The Mouse Isolated Perfused Kidney Technique

Mus isoleret perfunderet Nyre Teknik

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

MitoCeption: Overførsel isoleret humant MSC Mitokondrier til Glioblastoma Stamceller

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found ...