Department of Anesthesiology & Pain Medicine, Mitochondria and Metabolism Center, University of Washington, Seattle
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Marcu, R., Neeley, C. K., Karamanlidis, G., Hawkins, B. J. Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria. J. Vis. Exp. (67), e4131, doi:10.3791/4131 (2012).
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.
1. Isolation of Mitochondria from Mouse Heart
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.
Note: For optimal results, the pH of mitochondrial buffers should be adjusted with KOH and Acetic Acid.
Note: Trypsin during the isolation procedure increases the yield of isolated mitochondria and should be kept constant between mitochondrial preparations. We recommend testing mitochondrial functions on preparations isolated is the absence of trypsin to ensure that the enzyme does not interfere with functionality.
Note: Mitochondria Isolation Buffer can be prepared in advance and can be stored at -20 °C. Check pH upon thawing. Trypsin, trypsin inhibitor or BSA must be added to the Mitochondria Isolation Buffer on the day of the experiment.
Note: For best results during functional assays, use mitochondria within 4 hr of isolation.
2. Quality Control of Isolated Mitochondria
2.1. Measurement of respiratory control ratio (RCR)
Note: The effect of CCCP on mitochondrial respiration is dose dependent: high concentrations of CCCP can inhibit oxygen consumption and should therefore be carefully titrated.
2.2. Assessment of mitochondrial membrane integrity (cytochrome c test)
3. Measurement of the Mitochondrial Permeability Transition Pore Opening
4. Representative Results
Figure 2 shows a typical respiratory control of isolated mouse heart mitochondria. State 3 respiration is achieved by addition of ADP to mitochondria respiring on succinate, and is characterized by significantly increased oxygen consumption with respect to the substrate alone. Depletion of added ADP initiates State 4 respiration, during which oxygen consumption slows and is comparable to rates attained prior to ADP addition. RCR is obtained by dividing the oxygen consumption rate for State 3 respiration by that of State 4 respiration. The cytochrome c test is used to assay the integrity of the outer mitochondrial membrane: when cytochrome c is added to mitochondria respiring on succinate and ADP, no further increase in respiration is observed, indicating an intact outer mitochondrial membranes (Figure 3).
A representative image for the mtPTP opening in isolated heart mitochondria is shown in Figure 4. In this case mtPTP opening was triggered by the addition of 4 CaCl2 pulses (20 μM each) at 1.5 min intervals. mtPTP opening is apparent by the near simultaneous release of mitochondrial Ca2+ (increase in Fura FF ratio signal), collapse of mitochondrial membrane potential (decrease in JC-1 ratio signal) and increased mitochondrial volume (decrease in the swelling signal). When cyclosporine A is added, which binds to the cyclophilin D component of mtPTP, mtPTP opening requires 7 pulses of CaCl2 instead of 4 (Figure 5).
Figure 1. Flow chart of the multi-parameter mitochondrial permeability transition pore opening protocol for isolated heart mitochondria. Heart is harvested from mice and mitochondria are isolated through differential centrifugation. The mitochondrial preparation is then qualitatively evaluated by polarographic measurement of the respiratory control ratio and mitochondrial membrane intactness in the presence of cytochrome c. Mitochondrial permeability transition pore opening in isolated mitochondria is triggered by sequential CaCl2 additions and is measured with a spectrofluorometer by monitoring Ca2+ release from mitochondria, membrane potential collapse and swelling of the mitochondrial matrix.
Figure 2. Measurement of respiratory control ratio (RCR) of isolated heart mitochondria. Oxygen consumption by isolated heart mitochondria is measured during State 3 respiration in the presence of succinate and ADP, and during State 4 respiration after ADP consumption. The response of isolated mitochondria to uncouplers is measured by the addition of CCCP. Numbers on the graph are the rates of oxygen consumption by isolated mitochondria in nmol/min/mg protein.
Figure 3. Measurement of membrane integrity of isolated heart mitochondria. Mitochondrial membrane integrity is determined by measuring oxygen consumption in isolated mitochondria in the presence of succinate and ADP and after the addition of cytochrome c. Numbers on the graph are the rates of oxygen consumption by isolated mitochondria in nmol/min/mg protein.
Figure 4. Spectrofluorometer hardware configuration for the multi-parameter measurement of mtPTP opening. The hardware configuration of the spectrofluorometer includes a light source, an excitation monochromator, a sample compartment and two detectors with fixed emission wavelengths at 525 nm and 595 nm. Detector 1 is used for the signal detection of Fura FF high and low Ca2+, JC-1 monomer, and the swelling signal. Detector 2 measures the JC-1 aggregate signal.
Figure 5. Multi-parameter measurement of mtPTP opening in isolated heart mitochondria. mtPTP opening was triggered by sequential additions of 20 μM CaCl2 and was characterized by Ca2+ release from mitochondria, mitochondrial membrane potential collapse and increased mitochondrial matrix volume (swelling). Extramitochondrial Ca2+ and membrane potential were measured with the ratiometric indicators Fura FF (green trace) and JC-1 (red trace). Swelling of mitochondria was measured by recording light scattering at 525 nm (black trace). Specificity of JC-1 and swelling signals was tested with 1 μM CCCP and 5 μg/ml alamethicin (a microbial toxin).
Figure 6. Multi-parameter measurement of mtPTP opening in isolated heart mitochondria in the presence of cyclosporine A. Cyclosporine A (1 μM) increases the number of CaCl2 pulses required to open mtPTP in isolated heart mitochondria.
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 mtPTP opening. The spectrofluorometer employed in this protocol permits rapid measurement of these three parameters simultaneously. However, the basic protocol can be employed using other commercially available instruments, albeit with decreased time resolution. Non-ratiometric dyes can also be employed, but do not permit quantitative measures of both Ca2+ and mitochondrial membrane potential5.
A good quality mitochondrial preparation is essential when assaying the mtPTP opening, since both the ability to accumulate Ca2+ and maintain a membrane potential are tightly interrelated. For instance, we found that mitochondria with a low RCR exhibit reduced JC-1 accumulation and poor Ca2+ mobilization. The procedure we are using to isolate heart mitochondria was adapted from previously published protocols6, 7 and proved to be very consistent in generating high quality mitochondria as measured by RCR and cytochrome c tests.
Opening of the mtPTP is a complex phenomenon that involves changes at several levels in the mitochondrial anatomy and physiology. The assay we describe here spectrofluorometrically measures the simultaneous changes in extramitochondrial Ca2+ levels, mitochondrial membrane potential and mitochondrial volume. The measurement of multiple parameters at the same time offers a more comprehensive picture of the phenomenon than the measurement of individual parameters alone, such as Ca2+ mobilization or swelling. Moreover, the usage of ratiometric dyes Fura FF and JC-1 for Ca2+ and membrane potential measurements limits experimental artifact related to dye concentration, uptake or photobleaching. These dyes can also be easily calibrated in order to obtain more quantitative measurements. An important observation is that all dye concentrations should be kept as low as possible to avoid interference with mitochondria respiration and membrane potential as well as sensitize mtPTP opening. In our hands, JC-1 concentrations above 1 μM reduce the number of pulses needed to open mtPTP by 1-2 pulses. Therefore appropriate controls should be performed by excluding the dyes and recording other parameters (such as swelling alone) to be sure that the number of pulses needed to open the PTP remains consistent.
Throughout this protocol we have measured mtPTP opening for mitochondria respiring on complex II substrate succinate in the presence of rotenone, which inhibits reverse electron transport from complex II to complex I. Other respiratory substrates can be used as well, such as pyruvate/malate which supports respiration on complex I. However, substrate-related differences in the number of Ca2+ pulses required to open mtPTP have been reported8. The same protocol for measuring the mtPTP opening can be employed with mitochondria isolated from different tissues or cell lines. We have successfully utilized this protocol for liver, muscle and brain mitochondria, although the number of Ca2+ pulses needed to trigger the mtPTP opening was tissue dependent as previously reported9, 10. The mitochondria isolation protocol must be adapted and optimized for each tissue or cell line due to specific requirements regarding the homogenization procedure and isolation buffers11, 12.
No conflicts of interest declared.
This work was supported by HL094536 (B.J.H.).
|Trypsin inhibitor (soybean)||Sigma-Aldrich||T9128|
|Tissue grinder Potter-Elvehjem with Teflon pestle 15 ml||Wheaton Industries|
|Overhead stirrer||Wheaton Industries||903475|
|Oxytherm (temperature controlled oxygen electrode)||Hansatech Instruments|
|QuantaMaster 80 dual emission spectrofluorometer||Photon Technology International, Inc.|