Name: analyses of mitochondrial calcium influx in isolated mitochondria and cultured cells
Uploaded: 2018-04-27T13:30:00
Description: Watch this Scientific Journal Video about Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells at JoVE.com

This work was supported by grant funding from the American Heart Association (J.Q.K.).

All methods described in this protocol have been approved by the Institutional Animal Care and Use Committee of Emory University.
NOTE: The first part is the experimental procedure for measuring mitochondrial Ca2+ influx in isolated cardiac mitochondria using a plate reader.
1. Reagents and Solutions
<ol>
	<li>Make 500 mL of MS-EGTA buffer for mitochondrial isolation: 225 mM mannitol, 75 mM sucrose, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic aci...

<ol>
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Here, we describe two different approaches to measure mitochondrial Ca2+ influx. The plate reader-based calcium green-5N method monitors extramitochondrial Ca2+ levels and is a Ca2+ uptake assay that is well suited for measurements in isolated mitochondria. While we have shown representative results from isolated murine cardiac mitochondria, this assay can be readily adapted for mitochondria isolated from tissues with high mitochondrial abundance including the liver, skeletal muscle, and ...

Mitochondria are critical sites of intracellular Ca2+ storage and signaling. Decades of research have demonstrated that mitochondria have the ability to import and sequester Ca2+ 1,2. Mitochondria, however, are not merely passive sites of Ca2+ storage. Ca2+ at the mitochondrial compartment performs fundamental signaling functions including regulation of metabolic output and activation of mitochondrial-mediated cell death pathways, which has been reviewed previously3. For metabolic regulation, Ca2+ enhances the activity of three matrix-localized dehydrogenases of the tricarboxylic acid cycle as well as respiratory chain complexes, to increase mitochondrial energy production4,5. With mitochondrial Ca2+ overload and dysregulated mitochondrial Ca2+ handling, Ca2+ triggers mitochondrial permeability transition pore (MPTP) opening, leading to mitochondrial inner membrane permeabilization, membrane potential loss, mitochondrial dysfunction, swelling, rupture and ultimately, cell death6,7,8,9. Thus, mitochondrial Ca2+ signaling directly impacts both cellular life and death pathways through metabolic control and MPTP-death axis.
In recent years, there has been rapidly expanding interest in the study of mitochondrial Ca2+ dynamics due in large part to the identification of the molecular constituents of the mitochondrial Ca2+ uniporter complex, a mitochondrial inner membrane transporter that is a primary mode of Ca2+ import into the mitochondrial matrix 10,11,12. Identification of these structural and regulatory subunits of the uniporter has brought forth the possibility of genetically targeting mitochondrial Ca2+ influx to modulate mitochondrial function and dysfunction and facilitated the study of the contribution of the uniporter complex and mitochondrial Ca2+ influx to disease13,14,15. Indeed, mitochondrial Ca2+ signaling has been implicated in the pathologies of a diverse array of diseases ranging from cardiac disease to neurodegeneration, and cancer16,17,18,19,20.
Given the fundamental importance of mitochondrial Ca2+ signaling in metabolism and cell death, and combined with the broad reach of biological systems that mitochondrial Ca2+ signaling impacts, methods to assess mitochondrial Ca2+ influx are of great interest. Not surprisingly, a variety of techniques and tools to measure mitochondrial Ca2+ have been developed. These include methods that utilize tools such as fluorescent Ca2+-sensitive dyes21,22 and genetically-encoded Ca2+ sensors targeted to the mitochondria, such as cameleon and aequorin23,24. The goal of this article is to highlight different methods and model systems in which mitochondrial Ca2+ uptake can be measured. We present two experimental methods to assess mitochondrial Ca2+ influx capacity. Using cardiac mitochondria as an example, we detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N that is ideally suited for isolated tissue mitochondria14. Using cultured NIH 3T3 cells, we also describe a confocal microscopy imaging-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM25.

<table>
	<tbody>
		<tr>
			<td>Olympus FV1000 Laser Scanning confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy Neo2 Multimode microplate reader with injectors</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Homogenizer</td>
			<td>Kimble</td>
			<td>886000-0022</td>
			<td></td>
		</tr>
		<tr>
			<td>22 x 22 mm coverslips</td>
			<td>Corning</td>
			<td>2850-22</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Corning</td>
			<td>3628</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Green-5N</td>
			<td>Invitrogen</td>
			<td>C3737</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker green FM</td>
			<td>Invitrogen</td>
			<td>M7514</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhod-2, AM</td>
			<td>Invitrogen</td>
			<td>R1244</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Invitrogen</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Invitrogen</td>
			<td>P3000MP</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma</td>
			<td>M9546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>EMD Millipore</td>
			<td>8510</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E8145</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
			<td></td>
		</tr>
		<tr>
			<td>L-malic acid</td>
			<td>Sigma</td>
			<td>M1125</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Fisher</td>
			<td>BP364-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate magnesium salt</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphocreatine disodium salt</td>
			<td>Sigma</td>
			<td>P7936</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>S7900</td>
			<td></td>
		</tr>
		<tr>
			<td>Ru360</td>
			<td>Calbiochem</td>
			<td>557440</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyses of mitochondrial calcium influx in isolated mitochondria and cultured cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Figure 1 shows mitochondrial Ca2+ uptake measurements in isolated cardiac mitochondria using the plate reader-based platform and the Ca2+ dye calcium green-5N. Under control conditions (Figure 1A), cardiac mitochondria were suspended in KCl buffer containing calcium green-5N and then challenged with sequential pulses of CaCl2 (5 &#956;L of a 0.6 mM CaCl2 solution) added at the 30 s, 150...

Watch this Scientific Journal Video about Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells at JoVE.com

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

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