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Cardiovascular Research 2000 46(1):24-27; doi:10.1016/S0008-6363(00)00020-1
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Mitochondria — potential role in cell life and death

Elinor J. Griffiths*

Bristol Heart Institute, Department of Cardiac Surgery, Level 7, Bristol Royal Infirmary, Bristol BS2 8HW, UK

* Tel.: +44-117-928-3586; fax: +44-117-928-3581 elinor.griffiths{at}bristol.ac.uk

Received 25 January 2000; accepted 25 January 2000

KEYWORDS FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; TMRE, tetramethylrhodamine ethyl ester; TMRM, tetramethylrhodamine methyl ester; {Delta}{Psi}m, mitochondrial membrane potential

See article by Mathur et al. [10] (pages 126–138) in this issue.


   1 Mitochondria — potential role in cell life and death
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 1 Mitochondria -- potential...
2 Conclusion
 References
 
The 1990s saw a huge resurgence of interest in the role of mitochondria within cells, recognition that, as well as their traditional role as ‘powerhouses’ of the cell in generating ATP, mitochondria play an important role in other aspects of normal cell functioning, for example in cell calcium signalling. But possibly the greatest interest has been in the emerging role of mitochondria as regulators of the cell life–death transition, in both necrotic and apoptotic forms of cell death (for recent reviews see [1–3]). Changes in mitochondrial membrane potential ({Delta}{Psi}m) are integral to the cell life–death transition [4], although whether as a primary cause or secondary event is as yet not known. Thus, measurements of {Delta}{Psi}m can greatly facilitate our understanding of this process.

In physiological cell functioning, maintenance of {Delta}{Psi}m is essential for ATP synthesis, as has been known for many years. {Delta}{Psi}m is highly negative, approximately –180 mV, due to the chemiosmotic gradient of protons across the inner mitochondrial membrane, the energy of which is used to synthesise ATP by the respiratory chain. {Delta}{Psi}m also provides the driving force for Ca2+ uptake into mitochondria by the Ca2+-uniporter, and it is now generally accepted that it is this Ca2+ signal in the mitochondria which stimulates ATP production in response to an increased demand energy by the cell (reviewed in [5]). However, under certain conditions, most notably oxidative stress and ATP depletion, excessive uptake of Ca2+ by mitochondria can trigger the mitochondrial permeability transition (MPT), where a non-specific pore opens in the mitochondrial membrane (reviewed in [1,2]). The MPT causes collapse of {Delta}{Psi}m, so ATP cannot be resynthesised. Opening of the MPT pore has been demonstrated in reperfused hearts, and implicated in the pathogenesis of reperfusion injury [6]. Most damage to the heart under conditions of severe ischaemia/reperfusion occurs by necrosis, but there is evidence now for cell death by apoptosis in response to a less severe insult, or in areas surrounding an infarction (reviewed in [1,2]). The MPT has also been implicated in apoptosis, although whether as an initial or late event is under debate [3]. I would like to emphasise at this point that collapse of {Delta}{Psi}m itself does not necessarily indicate that the MPT has occurred, and this, together with use of inhibitors of the MPT such as cyclosporin A, which are not specific, may partly explain the conflicting data in this area.

A major problem in studying mitochondrial function in living cells was the lack of specific probes: for example, of {Delta}{Psi}m or intramitochondrial [Ca2+]. Although far from ‘solved’, major advances in this field came with the development of fluorescent indicators which could be localised into the mitochondrial compartment, and with increasingly sophisticated fluorescence microscopy, which enabled subcellular events to be studied. However, such developments have themselves sparked more controversy, for example whether mitochondrial Ca2+ responds rapidly enough to change during the cardiac excitation–contraction cycle [7–9]. Now the paper by Mathur et al., in this issue of Cardiovascular Research [10], has shown a similar situation with the fluorescent indicators of {Delta}{Psi}m; different dyes gave different results, and also conflicted with previous work.

I would now like to discuss the use of fluorescent indicators of {Delta}{Psi}m in living cardiomyocytes, under both physiological conditions, and under conditions mimicking ischaemia/reperfusion injury. These studies highlight how, in apparently similar models, contradictory results can be obtained.

1.1 Detection of {Delta}{Psi}m in living cardiomyocytes
The most commonly used fluorescent indicators of {Delta}{Psi}m are rhodamine 123 (Rh123) and its derivatives TMRM and TMRE, and the carbocyanine JC-1. These dyes rely on the highly negative {Delta}{Psi}m to be taken up into mitochondria, and should partition between the cytosol and mitochondria according to the Nernst equation. However, binding of the fluorescent probes to both inner and outer mitochondrial membranes may cause deviations from the predicted theoretical behaviour, and result in enhanced apparent uptake [11] which makes calculations of exact {Delta}{Psi}m unreliable. So results are usually compared to those obtained in the presence of a mitochondrial uncoupler, such as FCCP, to obtain a maximal depolarisation. The rhodamines are single-excitation, single-emission dyes, whereas JC-1 emits light at red and green wavelengths according to its concentration: at high concentrations, J-aggregates form and emit red light, whereas at low concentrations the monomer form emits green light. So in respiring mitochondria, which have a highly negative membrane potential, JC-1 will accumulate in the mitochondria and largely red fluorescence will be detected [12]. In fact, the ratio of the green/red (530/590 nm) light has been used as an indicator of {Delta}{Psi}m in an attempt to overcome artefacts due to cell motion [13,14].

Mathur et al. [10], have compared and re-evaluated the use of the dyes in neonatal cardiomyocytes using confocal microscopy of individual cells, and, for the first time, flow cytometry of cell cultures. This latter technique has highlighted the heterogeneous response of an apparently homogeneous cell population.

They concluded that JC-1 is the best dye for detecting changes in {Delta}{Psi}m, agreeing with conclusions of others [13,14]; for example, Minezaki et al. [13] found, in guinea-pig myocytes, that Rh123 increased cell membrane permeability, and, at high concentrations, the rhodamine derivatives may also inhibit mitochondrial respiration [11]. However, Mathur et al. [10] questioned the use of JC-1 as a ratiometric indicator, and found that much information could be gained by studying changes in the fluorescence of the individual wavelengths. Originally, the JC-1 monomer was thought to be independent of {Delta}{Psi}m, but it has now been shown that this is not the case, and that both green and red fluorescence can provide valuable information (see also [14]). An increase in green fluorescence appears to represent complete mitochondrial depolarisation, whereas a decrease in red fluorescence, with no change in green fluorescence, may reveal smaller changes in {Delta}{Psi}m [10,14]. It is therefore important to present data from both wavelengths, rather than just as the ratio.

The rhodamines can also undergo self-quenching in mitochondria, and uncouplers, such as FCCP, may therefore produce an increase in fluorescence when {Delta}{Psi}m is depolarised. This has been observed in studies using concentrations of 1–5 µM RH123 or TMRE [13,15,16]. Other studies, using lower concentrations of the indicator, 100–300 nM, have reported a loss of fluorescence of in response to depolarisation [17,18]. But the study by Mathur et al. [10] has now shown that, even using a range of indicator concentrations and different loading conditions, Rh123 fluorescence did not change in response to FCCP. So what are we to make of these results? At present, only that they highlight the danger of indiscriminate use of these indicators, and how, ideally, another indicator should probably be used in conjunction, to avoid either false-positive, or false-negative, results.

1.2 Measurements of {Delta}{Psi}m in physiological and pathological myocyte function
{Delta}{Psi}m has been monitored in single-cell models of ischaemia/reperfusion injury in an attempt to determine whether changes in {Delta}{Psi}m can be correlated with cell death. For example, Chacon et al. [17] found that prolonged metabolic inhibition induced {Delta}{Psi}m depolarisation, but only after myocytes had undergone rigor-contracture. This is in contrast to other studies using either anoxia [14] or metabolic inhibition [15] which found that {Delta}{Psi}m partially depolarised before rigor-contracture, and peaked shortly after rigor development was complete. In both these latter studies, {Delta}{Psi}m repolarised when cells were reoxygenated (or inhibitors washed out), regardless of whether cells recovered or hypercontracted. This might initially suggest that cell recovery is independent of {Delta}{Psi}m, but in both studies only whole cell fluorescence measurements were taken, and so it is impossible to determine the behaviour of individual mitochondria within cells. However, the results are consistent with the idea that some resynthesis of ATP is in fact necessary for hypercontraction. Thus, restoration of {Delta}{Psi}m on reoxygenation can be considered a double-edged sword: resynthesis of ATP is obviously necessary for cell recovery, but insufficient recovery, in the presence of a high [Ca2+] [19,20], will cause myocyte hypercontracture. The full extent of the interplay between {Delta}{Psi}m and Ca2+ in contributing to cell hypercontracture remains to be elucidated. A recent study by Delcamp et al. [21] has added more controversy by concluding that cells only hypercontracted upon reoxygenation if mitochondria remained depolarised. The extent of depolarisation compared to that produced by an uncoupler, was not given in this study, and it is possible that mitochondria were only partially depolarised during this time, allowing some ATP resynthesis.

In all these studies, a complete collapse of {Delta}{Psi}m would be detected only if all mitochondria depolarised simultaneously, a situation which seems unlikely [20]. A more probable scenario is that of a ‘steady-state’ of mitochondrial depolarisation and repolarisation, and evidence for such oscillations of {Delta}{Psi}m has recently been found in myocytes under physiological conditions: Duchen et al. [16] noticed spontaneous depolarisations of {Delta}{Psi}m in unstimulated rat myocytes. They suggested that this was due to rapid Ca2+ uptake by mitochondria situated in microdomains of high [Ca2+], near the sarcoplasmic reticular Ca2+-release channel. The depolarisation was found to be about 10% of that produced upon exposure to uncoupler, suggesting a partial depolarisation only, and was also reversible and repeatable within the same microdomain. Romashko et al. [18] found similar waves mitochondrial depolarisation, in guinea-pig myocytes, and this was also associated with flavoprotein oxidation, or ‘redox waves’, in these cells.

Finally, to briefly mention another ‘hot’ area of current research — that of the role of mitochondrial KATP channels in mediating the protective effect of ischaemic preconditioning. At present we have no ‘assay’ for these in whole cells. Implications for a role in preconditioning came from pharmacological data, which relied on ‘specific’ openers of the channels, notably diazoxide and 5-hydroxydecanoate [22,23]. Exposure of myocytes to diazoxide can affect parameters of mitochondrial function, such as increasing flavoprotein [23] and NADH (Griffiths, unpubl. data) oxidation. But diazoxide was also reported to decrease {Delta}{Psi}m in both isolated mitochondria and in isolated myocytes [24], and this was suggested to promote the MPT. This latter observation appears inconsistent with a protective role of diazoxide, since the MPT is associated with, and may cause, cell death. This brings us back to the cautionary nature of interpreting results with the indicators of {Delta}{Psi}m, and also ‘specific’ inhibitors of mitochondrial channels: although KATP channel openers have been suggested to depolarise mitochondria, this was estimated to be only a small depolarisation of 10–15 mV [23,24]. This could be due to factors other than opening of KATP channels, for example rapid uptake of Ca2+ by mitochondria situated in microdomains of high [Ca2+] [16].


   2 Conclusion
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 1 Mitochondria -- potential...
 2 Conclusion
References
 
Measurements of {Delta}{Psi}m are currently, and no doubt will in the future, tell us much about the role of mitochondria in normal cell function, and in processes leading to cell death. But we must be aware of the limitations of using the fluorescent indicators of {Delta}{Psi}m. It is also becoming apparent that, as well as populations of cells behaving in a heterogeneous fashion, so may sub-populations, or even single, mitochondria, within a cell. In some cases this may remain a discrete or random event, but in other cases may trigger a signal across the whole cell [16,18]. Finally, we must take care that {Delta}{Psi}m changes are not misinterpreted either as direct evidence of the MPT, or of opening of KATP channels. Other methods must be used in conjunction, and in some areas these have yet to be developed. Clearly, there is much exciting work ahead!


   References
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 1 Mitochondria -- potential...
 2 Conclusion
 References
 

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