doi: 10.1161/hh0801.090537
© 2001 American Heart Association, Inc.
Editorials |
Opening of Mitochondrial KATP Channels Triggers Cardioprotection
Are Reactive Oxygen Species Involved?
From the Maryland Research Laboratories (Y.L.), Otsuka Maryland Research Institute, Rockville, Md, and Institute of Molecular Cardiobiology (B.O’R.), Johns Hopkins University, Baltimore, Md.
Correspondence to Yongge Liu, PhD, Maryland Research Laboratories, Otsuka Maryland Research Institute, 9900 Medical Center Dr, Rockville, MD 20850. E-mail yonggel{at}otsuka.com
Key Words: preconditioning • diazoxide • KATP channel • mitochondria • reactive oxygen species
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Introduction |
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 Top Introduction References |
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Ischemic preconditioning, a phenomenon in which brief episodes of ischemia and reperfusion paradoxically protect the heart against subsequent lethal ischemia,1 has been conceptually divided into triggers and mediators/effectors.2 3 The trigger, which acts before the index ischemia, is followed by the protection of mediators/effectors during the lethal ischemia. Known triggers include activation of adenosine receptors,
1-adrenegic
receptors and opioid receptors, elevated intracellular
Ca2+, and increased reactive oxygen species
(ROS).2 The mitochondrial
ATP-dependent potassium channel (mitoKATP) has
been proposed to be the mediator of this
protection.4 The link between
the trigger and effector may be the activation of protein kinases (eg,
protein kinase C, tyrosine kinase, and downstream kinases), which may
phosphorylate mitoKATP, causing the channel to
open early and/or to a greater extent to reduce injury during the
lethal ischemia. Interestingly, opening of
mitoKATP can also trigger cardioprotection.
Hearts treated with the mitoKATP opener
diazoxide for a brief period before ischemia had significantly
smaller
infarction.3 5 The
triggering effect from diazoxide can be blocked by protein kinase
C5 6 and tyrosine
kinase
inhibitors,3
suggesting that diazoxide activates protein kinases, acting
similarly to other triggers. This effect was lost when ROS scavengers
were coadministrated with
diazoxide.3 ROS are known to
activate protein kinases and act as a
trigger.2 Thus, it was
proposed by Pain et al3 that
the opening of mitoKATP by diazoxide may
increase ROS. In this issue of Circulation Research, Forbes et al7 provide a direct demonstration that opening of mitoKATP increases ROS production in isolated rat ventricular myocytes. Using a ROS-sensitive fluorescent probe 2',7'-dichlorofluorescin (DCF), they showed that diazoxide as well as pinacidil (a nonselective KATP opener) increased DCF fluorescence, implying an elevated ROS production. Furthermore, a selective mitoKATP blocker 5-hydroxydecanoate (5-HD) abolished the increase. The antioxidants N-acetylcysteine or N-mercaptopropionylglycine also blocked the fluorescence increase. Exposing hearts to diazoxide before ischemia improved functional recovery after 20 minutes of global ischemia in their isolated rat heart model, although a classical trigger effect was not tested, because there was no diazoxide washout period before the ischemia.
Although there is no question that a mild ROS stress can
trigger cardioprotection by activating protein
kinases,2 the mechanistic
links between mitoKATP opening and ROS are
unknown. Most of the knowledge about mitoKATP on
mitochondrial energetics and function has been gained from studies on
isolated mitochondria from heart and liver. A well-described
consequence of mitoKATP opening is matrix
swelling.8 Although some
studies have shown that mitoKATP openers
increase mitochondrial respiration, depolarize mitochondrial membrane
potential partially, and decrease Ca2+
uptake into mitochondria,9
others have not observed these effects. Studies from Kowaltowski et
al8 have shown no significant
changes of respiration, mitochondrial membrane potential, or
Ca2+ uptake by opening
mitoKATP. Although such studies of isolated
mitochondria have proven invaluable for assessing the direct effects of
compounds on mitochondrial function, there are several considerations
to keep in mind when comparing such results with more intact
preparations. First, the choice of medium components is often
distinctly unphysiological (eg, the presence of
high sucrose, choice of substrates, Ca2+
concentration, ionic composition, and ATP concentration), and critical
interactions with other cellular components are missing, including the
absence of a cytoskeleton (which apparently is critical for
diazoxide-induced
protection2 ) and the lack of
coupling with energy-consuming sites (eg, myofibrils and ion transport
ATPases). Furthermore, high-oxygen partial pressures (>159
mm Hg) are typically present that are orders of magnitude greater
than the in vivo mitochondrial milieu under normoxic conditions (
2
to 3 mm Hg).10 Such a
hyperoxic condition will undoubtedly increase oxidative stress. An
additional problem is introduced when comparing drug concentrations
used in isolated mitochondria with intact cells or hearts where the
intracellular concentration of the drug is unknown. Furthermore,
diazoxide has been shown to affect mitochondrial membrane potential and
energy metabolism nonspecifically in pancreatic
ß-cells.11 However, the
observations that the nonspecific effect was not blocked by
glybenclamide11 whereas the
protective effect of diazoxide was eliminated with glybenclamide and
5-HD5 7 12 13
and 5-HD also abolished the mitochondrial oxidative effect of
diazoxide13 strongly argue
against nonspecific effects of diazoxide in the observed
cardioprotection and mitochondrial oxidation.
Intact isolated myocyte studies are one step closer to the
intact system but are still subject to some of the limitations
mentioned above, including choice of external solution, substrate
selection, hyperoxia, and an unphysiologically
low workload. Nevertheless, the ability to control some of these
factors and to monitor cellular ion concentrations and mitochondrial
redox balance has enabled us to determine if
mitoKATP opening occurs in intact cells. Our
group has used the native autofluorescence of mitochondrial
flavoproteins to monitor the redox state of the mitochondria and showed
that diazoxide (as well as pinacidil and nicorandil) causes a
significant partial oxidation of mitochondrial flavoproteins, which can
be blocked in a competitive and isoform-selective manner by 5-HD but
not the surface KATP-selective
antagonist HMR1098, indicating that the response is
attributable to mitoKATP
opening.13 14 The
concentration dependence
(K1/2 of
27 µmol/L) and pharmacology of this redox response correlates well
with cardioprotection in both perfused
hearts12 and cellular models
of
ischemia.6 13
In the study by Forbes et
al,7 the effect of diazoxide
on the cellular autofluorescence was almost undetectable under
their recording conditions, but a clear change on diazoxide
addition (120 seconds in their Figure 1) is still evident.
Unfortunately, the low gain of the recording, the high
background noise, and a very narrow emission bandpass did not permit a
detailed comparison between the ROS response and flavoprotein
oxidation. Also not tested were the effects of inhibitors
of specific mitochondrial sites on the ROS production. It is
worth noting that the partial oxidation of flavoproteins has sometimes
been misinterpreted to indicate that the mitochondrial membrane
potential is highly depolarized by diazoxide. This is, in fact, not the
case. The mitochondrial membrane depolarization associated with
diazoxide exposure is likely to be <15
mV9 and difficult to detect
in intact myocytes. Whether such a small change in mitochondrial
membrane potential could be a factor limiting
Ca2+ accumulation during or after
ischemia, as previously
proposed,9 13
remains to be determined; however, even small changes in the
electrochemical driving force could potentially lead to large
differences in total mitochondrial Ca2+
accumulation over long periods of time.
How mitoKATP opening might be linked to ROS production is unclear. It is known that the mitochondrial electron transport chain can be an important source of ROS in isolated myocytes, most likely originating from the cytochrome b-c1 segment of complex III in the respiratory chain.15 However, it is not clear whether ROS will be produced from an increase or decrease in flux through the electron transfer chain. Vanden Hoek et al15 demonstrated that ROS increased during a period of hypoxia, when electron transfer chain activity was presumably lower because of a lack of oxygen. They attributed this to downstream block of electron transfer leading to shunting of electrons to ROS production. Reperfusion after a brief hypoxia, when mitochondrial respiration should return to normal or even higher, actually reduced ROS production. Other studies have also shown that mitochondrial depolarization and stimulated electron transport chain have no effect or reduce mitochondrial ROS generation in intact cells.16 17 Thus, it is difficult to predict whether the opening of the mitoKATP, presumably associated with partial uncoupling, should elevate ROS production from mitochondria. There are also no data available on the possible effect of mitochondrial matrix volume expansion on ROS generation, nor is there a clear relationship between changes in mitochondrial redox balance and the state of thiol oxidation in the cell, the site targeted by the antioxidants used in the study by Forbes et al.7 The effects of opening of mitoKATP are likely to be complex, and these effects may not only affect ROS production from mitochondria but also from nonmitochondrial sources. Additionally complicating the issue is the observation that exogenously generated ROS may trigger a larger release of ROS derived from mitochondria.18
Although it has only recently emerged that mitoKATP opening may trigger cardioprotection and such an effect may involve ROS,3 7 it has been long known that KATP openers reduce ROS production during reperfusion. Pinacidil applied during reperfusion reduced ROS production and limited cell death, and these effects were eliminated in the presence of 5-HD, indicating a role of mitoKATP.19 Diazoxide also reduced ROS production during reperfusion.20 Possible interactions between ROS and mitoKATP have been demonstrated in plant mitochondria.21 Opening of plant mitoKATP reduced superoxide anion generation, and superoxide anion formation stimulated plant mitoKATP, suggesting a possible feedback mechanism to protect against ROS in plants. Whether a similar feedback mechanism of ROS and mitoKATP also exists in mammalian mitochondria is unknown. If elevation of ROS by mitoKATP opening is additionally confirmed, it is intriguing and puzzling that mitoKATP opening could either enhance or attenuate ROS production, depending on the preconditioning phase, ischemia, or reperfusion, and the effect could also be tissue-type specific.
Although DCF is a commonly used probe for ROS, it is not without potential problems. Its reduced acetate form, dichlorodihydrofluorescein (DCFH), can be loaded into mitochondria to monitor locally generated ROS, particularly hydroxyl radical. However, changes of mitochondrial membrane potential or membrane permeability could alter the subcellular dye distribution independent of ROS, thus causing misinterpretation of the results when the compartmentation of the dye cannot be determined, as in the study by Forbes et al.7 This is particularly problematic when changes in the leakage rate of the dye are undetermined and can even contribute to a decline in total signal in some cases. These problems can be minimized by using less-permeant (eg, carboxy or chloromethyl) derivatives of DCF. DCF can also be auto-oxidized in the presence of light and may sensitize cells to light-induced radical generation.18 An important control missing in the study by Forbes et al7 is a DCF recording in which only beginning and endpoints of the time course are taken with no illumination in between. This would definitively rule out DCF-mediated effects on cellular or mitochondrial ROS production.
The role of mitoKATP as a trigger of preconditioning has been demonstrated in isolated as well as in vivo hearts.22 However, the role of ROS in this trigger effect has only been shown in isolated rabbit3 and rat7 hearts. ROS scavengers blocked the protection from diazoxide in both studies, thus strongly implicating a role of ROS in isolated heart model. Again, isolated hearts were perfused with O2-saturated buffer with a PO2 of >500 mm Hg, which may prime the organ for ROS production. It will be interesting to see if the ROS involvement can be confirmed in an in vivo setting. Obata et al23 reported that in in vivo rat hearts, cromakalim and nicorandil both increased ROS, measured with a microdialysis probe perfused with sodium salicylate, which reacts with hydroxyl radicals produced in the muscle to yield a colored product 2,3-dihydroxybenzoic acid. Glybenclamide and 5-HD both reduced ROS production by KATP openers during ischemia and reperfusion. However, KATP modulators were infused through microdialysis probes along with the ROS probe, making it hard to assess the local concentrations of the compounds throughout the tissue. Furthermore, the observation that KATP inhibitors attenuated ROS production from ischemia and reperfusion is contradictory to other studies.19 20
Because ROS scavengers blocked diazoxide-induced protection, it is a logical explanation that mitoKATP opening in cardiac ventricular myocytes increases ROS production.3 7 However, two other possible explanations also exist. First, a mild oxidant environment may be required for the mitoKATP to open or be effective; ie, ROS may have a permissive role, because ROS are known to modulate surface KATP,24 and a complete elimination of ROS by scavengers could prevent channel opening and thus indirectly abrogate protection. Second, it is also possible that opening of KATP in the cardiac nerve terminal facilitates the release of catecholamines, which then increase ROS production to trigger the protection. Consistent with this idea, pinacidil, a KATP opener, has been shown to increase catecholamine release in isolated guinea pig hearts.25 Studies in isolated single myocytes without the presence of neurons may help resolve this issue.
Emerging evidence supports a new paradigm for the roles of mitoKATP in cardioprotection. MitoKATP plays a dual role both as a trigger and mediator of cardioprotection. Protein kinase activation is important during the trigger phase. The link from mitoKATP opening and kinase activation may be ROS, as suggested by two studies.3 7 Another link was suggested through an elevation of intracellular Ca2+.5 The mitoKATP also acts as a mediator; the opening of mitoKATP during the index ischemia is required for protection.22 Although the true mechanism of mitoKATP opening as a mediator of protection has not been elucidated, several mechanisms have been proposed, including improvement of cellular energetics, prevention of mitochondrial Ca2+ overloading, and an alteration of ROS production during ischemia and reperfusion.26 In the future, a full range of studies from the intact animal to the molecular level is needed to uncover and verify the mechanisms.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
See related article, pages 802-809
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References |
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