Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Summary

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.

Abstract

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.

Introduction

Mitochondria are critical sites of intracellular Ca²⁺ storage and signaling. Decades of research have demonstrated that mitochondria have the ability to import and sequester Ca^{2+ 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 production^4,5. 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 death^6,7,8,9. 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 disease^13,14,15. Indeed, mitochondrial Ca²⁺ signaling has been implicated in the pathologies of a diverse array of diseases ranging from cardiac disease to neurodegeneration, and cancer^{16,17,18,19,20}.

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 dyes^21,22 and genetically-encoded Ca²⁺ sensors targeted to the mitochondria, such as cameleon and aequorin^23,24. 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.

Protocol

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

1. Make 500 mL of MS-EGTA buffer for mitochondrial isolation: 225 mM mannitol, 75 mM sucrose, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), pH adjusted to 7.4 with KOH. Sterilize it through a 0.22 μm filter and store it at 4 °C. Ensure that the MS-EGTA buffer is pre-chilled to 4 °C before use.
2. Prepare 100 mL of KCl Buffer: 125 mM KCl, 20 mM HEPES, 1 mM KH₂PO₄, 2 mM MgCl₂, 40 μM EGTA and pH adjusted to 7.2 with KOH. Store it at room temperature.
3. Prepare 1 mM calcium green-5N stock in dimethyl sulfoxide (DMSO). The calcium green-5N stock can be aliquotted and stored at -20 °C.
4. Prepare substrates for Ca²⁺ uptake.
   1. Prepare 1 M sodium pyruvate, pH 7.4. Store it in aliquots at -20 °C.
   2. Prepare 500 mM malate, pH 7.4. Store it in aliquots at -20 °C.

2. Isolation of Cardiac Mitochondria

1. Euthanize the mouse according to institutional standards.
   NOTE: For our institutionally approved method of euthanasia, mice were anesthetized by isofluorane inhalation and sacrificed by cervical dislocation.
2. Collect the heart by opening chest cavity, cutting along either side of the ribs, flanking the heart, cutting away the diaphragm, and excising the heart tissue.
   NOTE: In this protocol, mitochondrial isolation is performed from a whole heart collected from an adult mouse (approximately 120 mg), which should be sufficient for 3-4 experiments. For mitochondrial isolation from other mitochondria-rich tissues such as the liver, up to 200 mg of tissue can be used following the protocol described.
3. Rinse the tissue thoroughly in 25 mL of ice-cold 1x phosphate buffered saline (PBS) ensuring that all blood is squeezed out of the ventricles.
   NOTE: The heart will be sufficiently rinsed when the liquid squeezed out of the heart runs clear.
4. Mince the heart into small pieces in 5 mL of ice-cold 1x PBS using a pair of sharp scissors.
5. Discard the PBS and transfer minced heart tissue to a pre-chilled 7 mL glass-teflon dounce homogenizer with a 0.10 to 0.15 mm clearance.
6. Add 5 mL of ice-cold MS-EGTA buffer, and homogenize the sample until tissue pieces are no longer visible (approximately 11 strokes).
   NOTE: Do not over-homogenize the tissue as the goal is to obtain intact and functional mitochondria.
7. Transfer the homogenate to a 15 mL tube.
8. Centrifuge the homogenate at 600 x g at 4 °C for 5 min to pellet nuclei and unbroken cells.
9. Transfer the supernatant to a fresh 15 mL tube and centrifuge it at 10,000 x g at 4 °C for 10 min to pellet mitochondria.
10. Discard the supernatant and keep the mitochondrial pellet on ice.
11. Wash the mitochondrial pellet twice using ice-cold MS-EGTA buffer. For each wash, resuspend the mitochondrial pellet in 5 mL of MS-EGTA buffer, centrifuge it at 10,000 x g at 4 °C for 10 min, and discard the supernatant.
12. After the final wash, discard the supernatant and resuspend the mitochondria in 100 μL of ice cold MS-EGTA buffer. Keep the mitochondria on ice.
    NOTE: Mitochondria should be used for experimentation within 1 h.
13. Measure mitochondrial protein concentration using a Bradford Protein Assay²⁶.

3. Plate Reader-based Measurement of Mitochondrial Calcium Uptake

NOTE: Here, it is described the protocol for analyzing mitochondrial Ca²⁺ uptake using a multimode plate reader fitted with injectors. Any plate reader with the capability of reading calcium green-5N fluorescence (excitation/emission of 506/532 nm) in a kinetic mode with automated reagent injectors to keep the reaction protected from light can be used.

1. Program the plate reader to perform a kinetic read of calcium green-5N fluorescence with measurements taken every second for a total assay time of 1,000 s. Additionally, program the reagent injectors to dispense 5 μL of CaCl₂ solution at the 30 s, 150 s, 300 s, 480 s, and 690 s time points.
   NOTE: The timing of the CaCl₂ injections are user defined, and can be adjusted for according to experimental needs.
2. Prime the reagent injectors with the CaCl₂ solution to be used.
   NOTE: The concentration of the CaCl₂ solution is user defined, and can be adjusted in subsequent runs to titrate the amount of Ca²⁺ required to trigger MPTP opening.
3. Add 200 μg of mitochondria to an individual well of a 96 well plate.
4. Add the appropriate volume of KCl buffer to the well such that the total volume of mitochondria and KCl buffer comes to 197 μL.
5. Add 1 μL of 1 M pyruvate, and 1 μL of 500 mM malate to the mitochondria mixture. Pipet gently to mix, and incubate the mitochondria with the substrates for 2 min at room temperature to allow mitochondria to become energized.
6. Add 1 μL of 1 mM calcium green-5N stock. Mix gently by pipetting.
   NOTE: Protect the reaction from light following the addition of the calcium green-5N dye.
7. Start the pre-programmed kinetic protocol and monitor calcium green-5N fluorescence.

4. Reagents and Solutions

NOTE: The second part is experimental procedure for confocal imaging of mitochondrial Ca²⁺ in cultured cells

1. Make Tyrode's solution (130 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.2 with KOH). Store it at 4 °C. Warm it to room temperature before use.
2. Prepare Wash Solution (100 mM potassium acetate, 15 mM KCl, 5 mM KH₂PO₄, 5 mM Mg-ATP, 0.35 mM EGTA, 0.12 mM CaCl₂, 0.75 mM MgCl₂, 10 mM phosphocreatine, 10 mM HEPES, pH 7.2 with KOH). Store it at 4 °C. Warm it to room temperature before use.
3. Prepare Permeabilization Solution, which contains 0.005% saponin in Wash Solution. Prepare it fresh daily.
4. Prepare 0 Ca²⁺ Internal Solution (100 mM potassium acetate, 15 mM KCl, 0.35 mM EGTA, 0.75 mM MgCl₂, 10 mM HEPES, pH adjusted to 7.2 with KOH). Store it at 4 °C. Warm it to room temperature before use.
5. Prepare internal solution containing Ca²⁺. Add CaCl₂ to the internal solution above to reach the desired concentration of free Ca²⁺. The amount of CaCl₂ to add is calculated using the MaxChelator program (maxchelator.stanford.edu).
6. Prepare 1 mM Rhod-2/AM stock in DMSO and store it at -20 °C until use.
7. Prepare 1 mM MitoTracker green stock in DMSO and store it at -20 °C until use.

5. Plating Cells for Imaging

1. Wash 22 x 22 cm² glass coverslips with 100% ethanol and allow the coverslips to air dry.
2. Place the glass coverslips into individual wells of a 6-well tissue culture dish.
   NOTE: Coverslips can be coated with laminin, poly-L-lysine, or similar matrix to promote cell attachment.
3. Trypsinize and plate the cells onto the coverslips aiming for approximately 70% confluence on the day of imaging.

6. Loading Cells with the Rhod-2/AM and MitoTracker Green

1. Prepare Rhod-2/AM-MitoTracker green working solution. Add 20 μL of Rhod-2/AM 1 mM stock, 0.2 μL of MitoTracker green 1 mM stock, and 2.5 μL of 20% pluronic F-127 to 1 mL of Tyrode's solution. The final concentration of Rhod-2 in the working solution is 20 µM and the final concentration of MitoTracker green is 200 nM.
2. Gently remove the growth media from the coverslip.
   NOTE: A wash step is not necessary prior to dye solution addition.
3. Add the Rhod-2/AM-MitoTracker green solution in a dropwise fashion onto the coverslip until the coverslip is just covered (approximately 3-4 drops per coverslip).
4. Incubate it for 30 min at room temperature protected from light to allow the cells to load with the dyes.
5. De-esterify the Rhod-2/AM. Gently remove the Rhod-2/AM-MitoTracker green solution, replace it with fresh room temperature Tyrode's solution, and incubate it for 30 min at room temperature protected from light.

7. Confocal Imaging of the Mitochondrial Rhod-2/AM and MitoTracker Green Fluorescence

1. Transfer the coverslip to the microscope imaging chamber and fill the chamber with wash solution.
2. Under settings for observing cells in phase contrast at 40X, adjust focus until cells are visible and in focus.
3. Permeabilize the plasma membrane of Rhod-2/AM-MitoTracker green loaded cells to remove cytosol-localized Rhod-2, while retaining mitochondria-localized dye.
   1. Remove the Wash Solution from the coverslip.
   2. Replace it with Permeabilization Solution for approximately 1 min.
   3. Visually monitor the plasma membrane morphology throughout the permeabilization process. Permeabilized cells will develop a roughened surface.
   4. When permeabilization is complete, immediately remove the Permeabilization Solution and replace it with 0 Ca²⁺ Internal Solution.
4. Simultaneously image Rhod-2 fluorescence (excitation using the 559 nm laser line and emission collected at wavelengths between 575-675 nm) and MitoTracker green fluorescence (excitation using the 488nm laser line and emission collected at wavelengths between 505-525 nm). Focus on permeabilized cells displaying a clear colocalization of Rhod-2 and MitoTracker green.
5. Decrease the microscope laser and gain settings such that the mitochondrial Rhod-2 fluorescence is dim and just visible.
6. Select microscope settings to acquire 2-dimensional scans at an appropriate frame rate and time course for the specific application. A frame rate of at least 30 frames/s is recommended to accurately capture the kinetics of changes in mitochondrial Ca²⁺.
7. Remove the 0 Ca²⁺ Internal Solution without disturbing the cells or microscope focus.
8. Start image acquisition.
9. Add Ca²⁺-replete Internal Solution at the 10 s time point either manually or with a perfusion system.
10. Select regions of interest (ROIs) to encompass regions of colocalization between mitochondrial Rhod-2 and MitoTracker green signal in the image acquisition software.
    NOTE: Arbitrary fluorescence (F) values for each time point of the recording are obtained for each ROI. These values are background subtracted and normalized to the initial fluorescence (prior to Ca²⁺ addition; F₀) and plotted as F/F₀ over time for data presentation and quantification of the amplitude of the change of mitochondrial Ca²⁺.

Results

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 μL of a 0.6 mM CaCl₂ solution) added at the 30 s, 150...

Discussion

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 ...

Materials

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