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

This work was supported by HL094536 (B.J.H.).

1. Isolation of Mitochondria from Mouse Heart
<ol>
 <li>To isolate heart mitochondria, anesthetize and sacrifice mice according to the procedures approved by your local Institutional Animal Care and Use Committee.</li>
</ol>
Note: All steps of the mitochondria isolation protocol must be performed on ice. Use ice cold buffers and pre-chilled Petri dishes, Falcon tubes and Eppendorf tubes. The volumes given in the protocol are for a sample containing 2 mouse hearts.
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<ol>
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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. 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The protocol presented here describes the necessary experimental steps to assess permeability transition pore opening in isolated heart mitochondria (Figure 1 and Figure 4): the procedure for isolating mouse heart mitochondria, the respiratory controls that ensure their integrity and functionality, the mitochondrial parameters monitored during mtPTP opening and the dyes employed for their measurement, the setting up of the spectrofluorometric instrumentation, and the characterization of ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T3030</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor (soybean)</td>
			<td>Sigma-Aldrich</td>
			<td>T9128</td>
		</tr>
		<tr>
			<td>Sodium hydrosulfite</td>
			<td>Sigma-Aldrich</td>
			<td>71699</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
		</tr>
		<tr>
			<td>Cytochrome 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>239835</td>
		</tr>
		<tr>
			<td>Fura FF</td>
			<td>Invitrogen</td>
			<td>F14180</td>
		</tr>
		<tr>
			<td>JC-1</td>
			<td>Invitrogen</td>
			<td>T3168</td>
		</tr>
		<tr>
			<td>Tissue grinder Potter-Elvehjem with Teflon pestle 15 ml</td>
			<td>Wheaton Industries</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Overhead stirrer</td>
			<td>Wheaton Industries</td>
			<td>903475</td>
		</tr>
		<tr>
			<td>Oxytherm (temperature controlled oxygen electrode)</td>
			<td>Hansatech Instruments</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>QuantaMaster 80 dual emission spectrofluorometer</td>
			<td>Photon Technology International, Inc.</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

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

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.

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

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