Protocol for Measurement of Oxygen Consumption Rate In Situ in Permeabilized Cardiomyocytes

Summary

Analysis of mitochondrial respiration function represented by the oxygen consumption rate is necessary for assessing mitochondrial respiration function. This protocol describes steps to evaluate the respiration function of mitochondria in situ in saponin-permeabilized cardiomyocytes. In permeabilized cells, mitochondria are in a relatively integrated cellular system, and mitochondrial respiration is more physiologically relevant than isolated mitochondria.

For complete details on the use and execution of this protocol, please refer to Gong et al. (2015a) and Gong et al. (2015b).

Graphical Abstract

Highlights

• •Mitochondria are retained in a relatively integrated cellular system
• •Mitochondrial respiration in situ is more physiologically relevant
• •Saponin concentration is critical for mitochondrial integrity

Analysis of mitochondrial respiration function represented by the oxygen consumption rate is necessary for assessing mitochondrial respiration function. This protocol describes steps to evaluate the respiration function of mitochondria in situ in saponin-permeabilized cardiomyocytes. In permeabilized cells, mitochondria are in a relatively integrated cellular system, and mitochondrial respiration is more physiologically relevant than isolated mitochondria.

Before You Begin

Timing: 0.2–3 days

• 1.Prepare freshly isolated or cultured cardiomyocytes according to our step-by-step STAR protocol (Tian et al., 2020) or other protocols before you start the diagnostic study.

Adult cardiomyocytes were cultured in serum-free M199 medium, supplemented with 10 mM glutathione, 26.2 mM sodium bicarbonate, 5 mM creatine, 2 mM L-carnitine, 5 mM taurine, 0.1% insulin-transferrin-selenium-X, 0.02% bovine serum albumin, 50 U/mL penicillin-streptomycin, and 5% fetal bovine serum, for up to 3 days (depend on experiment). Change medium every 2 days.

Note: The protocol is primarily for adult cardiomyocytes. Cardiomyocyte cell lines and other cell types can also be assessed following this protocol, but the concentration of saponin needs to be rigorously screened.

• 2.Prepare necessary solutions before the respirometric measurements. Refer to Key Resources Table and Materials and Equipment sections for a complete list of materials and equipment.
• 3.Prepare several microsyringes by cutting off the long needle according to the length of the Transparent polycarbonate plunger (Figure 1).

Note: The length of the needle is 0.5–1 mm longer than the plunger. Too long will reach to the electrode and damage it.

Figure 1.

Prepared Microsyringes

Key Resources Table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, Peptides, and Recombinant Proteins
M199	Sigma-Aldrich	Cat# M2520
Glutathione	Sigma-Aldrich	Cat# G6013
NaHCO3	Sigma-Aldrich	Cat# V900182
Creatine	Sigma-Aldrich	Cat# C3630
L-carnitine	Sigma-Aldrich	Cat# C0158
Insulin-transferrin-selenium-X	Thermo Fisher Scientific	Cat# 51500056
Fetal bovine serum	Thermo Fisher Scientific	Cat# 12483020
Pen/Strep (100×)	Thermo Fisher Scientific	Cat# 10378016
Glutamic acid (Glutamate)	Sigma-Aldrich	Cat# C27647
L-Malic acid (Malate)	Sigma-Aldrich	Cat# M1000
ADP	Sigma-Aldrich	Cat# A5285
TMPD	Sigma-Aldrich	Cat# T3134
L-Ascorbic acid (Ascorbate)	Sigma-Aldrich	Cat# A4034
Oligomycin A	Sigma-Aldrich	Cat# O4876
FCCP	Sigma-Aldrich	Cat# C2920
Cytochrome c	Sigma-Aldrich	Cat# C2506
Pyruvate	Sigma-Aldrich	Cat# P2256
EGTA	Sigma-Aldrich	Cat# E3889
MgCl2.6H2O	Sigma-Aldrich	Cat# V900020
Taurine	Sigma-Aldrich	Cat# T8691
KH2PO4	Sigma-Aldrich	Cat# V900041
HEPES	Sigma-Aldrich	Cat# V900477
BSA	Sigma-Aldrich	Cat# A6003
Potassium lactobionate	Bio-sugars	Cat# 69313
Mannitol	Sigma-Aldrich	Cat# M9546
Dithiothreitol	Sigma-Aldrich	Cat# V900830
Saponin	Sigma-Aldrich	Cat# 47036
Ethanol Absolute	Sigma-Aldrich	Cat# 51976
Na2SO3	Sigma-Aldrich	Cat# 71988
KOH	Sigma-Aldrich	Cat# 5958
trypsin-EDTA	Gibico	Cat# 25200056
PBS	ThermoFisher	Cat# 10010001
Experimental Models: Organisms/Strains
SD rat	Shanghai SLAC	Cat# SlacSD
Software and Algorithms
782 Oxygen system	Strathkelvin Instruments	https://www.mendeley.com/catalogue/b4bf87c2-4afe-379f-b85d-52b1ca2bc3f4/
Other
Plastic forceps	zmkam	Cat# 93305
Microsyringe	Sh-Gaoge	Cat# G025
Rinse Bottle	ThermoFisher	Cat# N10809
Bottle Top Filter	ThermoFisher	Cat# 2903345
Cell counter	Countstar	IC1000
Inverted microscope	Leica	DMi8
Refrigerated centrifuge	Hettich	Universal 320R0
Vacuum pump	Qilinbeier	GL-802
pH meter	Mettler Toledo	FE20 plus
Mitocell respirometry system	Strathkelvin Instruments	MS200

Alternatives: You can use Chlorolab2+ from Hansatech Instruments instead of Mitocell respirometry system.

Materials and Equipment

Solution Preparation

Note: Prepare all solutions using 18.2 Ω MilliQ sterilized H₂O or absolute ethanol. Aliquot these solutions and store at −20°C or −70°C.

• •Mitochondrial respiration buffer (MRB)

Reagent	Final Concentration	Amount
EGTA	0.5 mM	0.19 g
MgCl2·6H2O	3 mM	0.61 g
Taurine	20 mM	2.502 g
KH2PO4	10 mM	1.361 g
HEPES	20 mM	4.77 g
BSA	0.1%	1.0 g
Potassium lactobionate	60 mM	23.93 g
Mannitol	110 mM	20.04 g
Dithiothreitol	0.3 mM	0.046 g
ddH2O	n/a	∼1,000 mL
Total	n/a	1,000 mL

Note: Adjust the pH to 7.4 with 5M KOH, filter with a 0.45 μm bottle top filter, dispense into 50 mL aliquots and store at −20°C.

CRITICAL: Ca²⁺ overload can cause the dysfunction of mitochondrial. EGTA is used to chelate Ca²⁺.

• •5 M KOH: Dissolve 14.02 g KOH in 50 mL ddH₂O. Store at room temperature (20°C–26°C).
• •0.05% trypsin-EDTA: Dilute 0.25% trypsin-EDTA with PBS to 0.05%. Store at 4°C.
• •0.6 M MgCl₂: Dissolve 2.44 g MgCl₂·6H₂O in 20 mL ddH₂O. Store at room temperature (20°C–26°C).
• •5 mM Oligmycin A: Dissolve 5 mg Oligmycin A in 1.26 mL absolute ethanol. Dispense into aliquots and store at −20°C. Dilute to 100 μM with ddH₂O before use.
• •10 mM FCCP: Dissolve 5 mg FCCP in 1.967 mL absolute ethanol. Dispense into aliquots and store at −20°C. Dilute to 20 μM with ddH₂O before use.
• •400 mM Glutamate: Dissolve 0.748 g glutamate in 10 mL ddH₂O. Neutralize with 5 M KOH and check pH. Dispense into aliquots, store at −20°C.
• •200 mM Malate: Dissolve 0.268 g malate in 10 mL ddH₂O. Neutralize with 5 M KOH and check pH. Dispense into aliquots, store at −20°C.
• •80 mM Ascorbate: Dissolve 0.158 g in 10 mL ddH₂O.
• •1 mg/mL Saponin: Dissolve 10 mg in 10 mL ddH₂O.

CRITICAL: Saponin needs to be freshly prepared each day.

• •20 mM TMPD solution

Reagent	Final Concentration	Amount
TMPD	20 mM	4.74 mg
80 mM Ascorbate	1 mM	12.5 μL
ddH2O	n/a	∼987.5 μL
Total	n/a	1 mL

Note: Ascorbate is used to prevent the oxidation of TMPD.

• •20 mM ADP solution

Reagent	Final Concentration	Amount
ADP	20 mM	20.04 mg
0.6 M MgCl2	6 mM	20 μL
5 M KOH	∼ 1.14 M	∼18 μL
ddH2O	n/a	∼1,962 μL
Total	n/a	2,000 μL

Note: ADP is the crucial compound to trigger mitochondria respiration. Use MgCl₂ stock solution instead of powder because MgCl₂ dissolves in water will release heat, which will decrease the stability of ADP. Aliquot and store at −70°C.

Step-By-Step Method Details

Calibrating the Oxygen Electrodes

Timing: 10 min

• 1.Turn on the Mitocell respirometry system (Figure 2).

Note: The MT200 mitocell respirometer only needs 100 μL of sample each time, which is far less than the traditional Clark oxygen electrode meter.

• 2.Turn on the 782 Oxygen system software and check the connection of oxygen meter.

Note: The software cannot connect to the oxygen meter if you turn on the software before step 1.

• 3.Fill the chamber with MilliQ water using a rinse bottle, and discard the water with a vacuum pump, rinse 3 times (Figures 3A and 3B).

Note: The tip must be inserted against the wall of the oxygen electrode chamber, otherwise it can easily damage the membrane of the electrode.

• 4.Fill air-saturated water (MilliQ water in contact with air) into the oxygen electrode chamber.
• 5.Put a micro-magnetic stirrer into the oxygen electrode chamber of MT200 respirometer with a plastic forceps, and start stirring (Figure 3C).

Note: Keep eyes on the tiny magnetic stirrer because it is easily aspirated on the tip. Prepare a big magnetic to find the lost magnetic stirrer.

• 6.Click on “Calibration high” of calibration panel to start calibrating the high value of the electrode. Please wait until the process is completed.
• 7.Add a small amount (3–5 mg) of sodium sulfite into the air-saturated water in the oxygen electrode chamber and click on “Calibration zero” to calibrate the zero value of the electrode (Figure 3D).
• 8.Rinse the chamber 6–10 times to wash out the sodium sulfite. Now, the oxygen meter is ready for use.

Note: Please rinse more times if the sloped basal line occurred.

Pause Point: The calibrated oxygen meter could wait for a break of 2–3 h.

Figure 2.

The Mitocell Respirometry System

The system comprising a 782 oxygen meter and a MT200 respirometer with a 1302 electrode.

Figure 3.

The Process of Calibration

(A) Rinse the oxygen electrode chamber.

(B) The tip site for sucking out the solution in the oxygen electrode chamber.

(C) Put the magnetic stirrer into the oxygen electrode chamber.

(D) Add 3–5 mg sodium sulfite to calibrate the zero of the electrode.

Counting Cardiomyocytes

Timing: 10 min

• 9.Wash cardiomyocytes twice with 5 mL PBS, detach cells using 1 mL 0.05% trypsin digesting 2–3 min and pipette 5–8 times, and collected cardiomyocytes through 80 g, 2 min centrifugation, and discard the supernatant.

Note: The ability of attachment of adult cardiomyocytes is weaker than the cell lines.

• 10.Resuspend cells in 1–2 mL mitochondrial respiration medium B (MRB).
• 11.Pipette the cells well and use one drop to count the number of cells with an automatic cell counter.
• 12.Dilute the cells to 5 × 10⁵ cells per mL with MRB and keep on ice.

CRITICAL: Appropriate cell number is very important for measurement. Too many cells will cause the quick decline of the basal line without enough time to treat the cells with substrates and inhibitors. Also, few cells will lead to a weak response.

Measuring Mitochondrial Respiration

Timing: 15–20 min

• 13.Add 100 μL cell suspension (5 × 10⁴ cells) using a pipette to oxygen electrode chambers. Close the oxygen electrode chamber with the Transparent polycarbonate plunger, equilibrate again for 3–5 min.

Note: keep the cardiomyocytes, mitochondrial electron transport chain substrates and inhibitors on the ice during measurement.

• 14.Start recording oxygen consumption. After 2–3 min of basal line recording, add 5 μL saponin (final concentration is ∼50 μg/mL) using a 25 μL microsyringe for cell membrane permeabilization, and continue recording (Figure 4).

Note: Add the solution of saponin, substrates, or inhibitors slowly; otherwise, it will cause bubbles to disrupt the trace.

CRITICAL: 1–2 min is enough for complete cell membrane permeabilization. Excess of saponin (more than 100 μg/mL) can damage the mitochondrial membrane (Kuznetsov et al., 2008). The permeabilization conditions should be strictly optimized for different cell types to preserve mitochondrial intactness. To check whether the mitochondrial outer membrane is damaged, we can assess the oxygen consumption of TMPD/Ascorbate/ADP and cytochrome c (Please refer to the section of Troubleshooting 2)

• 15.1–2 min later, add 5 μL Glutamate/Malate solution (final concentration is ∼10 mM glutamate, ∼5 mM malate) to the chamber and record 2 min of resting complex I-supported respiration.

Note: Make a mixed Glutamate/Malate substrate solution by mixing 400 mM Glutamate and 200 mM malate in a 1:1 ratio. Alternatively, you can use 10 mM Pyruvate instead of Glutamate.

• 16.Add 5 μL 20 mM ADP (final concentration is ∼1 mM) to the chamber, incubate for 3–5 min and record the respiration.

Note: If the slope of trace (state 3) only slightly increases (the ratio of respiration rate of control cells before and after the addition of ADP is less than 2), it indicates that the mitochondria may be damaged by permeabilization. Please perform the oxygen consumption of TMPD/Ascorbate/ADP and cytochrome c (Please refer to the section of Troubleshooting 2)

• 17.Add 5 μL 100 μM Oligmycin A (final concentration is ∼ 5 μM ) to the chamber, to inhibit ATP synthase and reduce oxygen consumption rate. Incubate for 2–3 min, and record the respiration.
• 18.Add 5 μL 20 μM FCCP (final concentration is ∼ 1 μM) to the chamber to uncouple oxygen consumption from ATP production, and trigger the maximal oxygen consumption, record the respiration until it reaches zero.

Figure 4.

Add Saponin, Substrates or Inhibitors with a Microsyringe

Cleaning the Oxygen Electrode Chambers

Timing: 35 min

• 19.Rinse the chamber with MilliQ H₂O for several times. Then sterilize with 70% ethanol for 30 min after each experiment.

Expected Outcomes

The goal of the method is for analyzing mitochondrial respiration function in permeabilized cardiomyocytes. In our protocol, we evaluate mitochondrial respiration function in permeabilized cardiomyocytes using an MT200 mitocell respirometer. Only 100 μL of the sample (5 × 10⁴ myocytes) is needed each time, which can save precious samples. The mitochondrial respiration is more physiologically relevant than isolated mitochondria due to preserving their essential interactions with other intracellular systems. At the same time, the mitochondria in situ are usually more stable than isolated mitochondria. A typical oxygen concentration traces for successfully permeabilized cardiomyocytes are shown in figure (Figure 5).

Figure 5.

A Representative Trace of Mitochondrial Respiration in Permeabilized Cardiomyocytes

Compounds were added as indicated. The slope of trace represent the different state of mitochondrial respiration. State 2: Mitochondrial respiration stimulated by the substrate in the absence of and added ADP; State 3: Mitochondrial respiration initiated by adding ADP in the presence of substrate; State 4: Mitochondrial respiration in the absence of ATP synthesis, which is uncoupled with the Oligmycin A. Maximal respiration: Mitochondrial respiration triggered by FCCP.

Limitations

The permeabilization method, not specific target to cytoplasm membrane, may also cause damage to the mitochondrial membrane. The permeabilization conditions must be strictly optimized for different cell types to preserve mitochondrial intactness. The cell number is also need be optimized for different cell types. This method is not able to separately investigate distinct mitochondrial subpopulations.

Troubleshooting

Problem 1

The rates of initial respiration before the addition of ADP is high

Potential Solutions

Wash glassware with RMB supplemented with EGTA to remove the calcium. Decrease the cell number.

Problem 2

ADP-stimulated respiration is too low.

Potential Solutions

First, perform the oxygen consumption of TMPD/Ascorbate/ADP followed with cytochrome c addition (Figure 6). If cytochrome c markedly increases the oxygen consumption triggered by TMPD/Ascorbate/ADP, please decrease the concentration of saponin to avoid damaging mitochondrial outer membrane. Slightly increase the concentration of saponin if cytochrome c does not affect oxygen consumption.

Figure 6.

A Representative Trace of Mitochondrial Oxygen Consumption Triggered by TMPD/Ascorbate and Cytochrome c in Permeabilized Cardiomyocytes

Compounds were added as indicated.

Resource Availability

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Guohua Gong (guohgong@tongji.edu.cn).

Materials Availability

This study did not generate new unique reagents.

Data and Code Availability

This study did not generate any unique datasets or code.