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Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
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Focused issue on KATP channels
KATP channels and preconditioning: A re-examination
of the role of mitochondrial KATP channels and an overview
of alternative mechanisms
Peter J. Hanley, Jürgen Daut *
Institut für Normale und Pathologische Physiologie, Universität Marburg, Deutschhausstrasse 2, 35037 Marburg, Germany
Received 23 July 2004; received in revised form 10 April 2005; accepted 11 April 2005
Available online 23 May 2005
Abstract
Preconditioning by one or several brief periods of ischemia activates an endogenous cardioprotective program that increases the resistance
of cardiomyocytes to injury by subsequent prolonged periods of ischemia. Ischemic preconditioning can be mimicked by K+ channel openers
and various other substances, a phenomenon termed pharmacological preconditioning. Initially, ischemic preconditioning has been ascribed
to the opening of ATP-sensitive K+ channels at the surface membrane of cardiomyocytes. Since 1997, numerous publications have implicated
mitochondrial ATP-sensitive K+ channels (mKATP) as a major trigger and/or end effector of preconditioning. Diazoxide has been suggested to
be a specific activator of mKATP channels, and the substituted fatty acid 5-hydroxydecanoate (5-HD) has been suggested to be a specific
inhibitor. However, diazoxide and 5-HD have multiple K+-channel-independent actions, and the experimental evidence for an obligatory role
of mKATP channels in preconditioning, or even their existence, remains inconclusive. In contrast, surface KATP channels have been well
characterized, and we summarize the evidence suggesting that they make a major contribution to preconditioning. We also discuss a number
of other factors involved in preconditioning: (1) generation of reactive oxygen species, (2) impairment of fatty acid metabolism, and (3)
opening of the mitochondrial permeability transition pore. In the light of these emerging concepts, we critically re-examine the evidence for
and against a role of mKATP channels in ischemic and pharmacological preconditioning.
© 2005 Elsevier Ltd. All rights reserved.
Keywords: Diazoxide; 5-Hydroxydecanoate; KATP channels; Ischemic preconditioning
1. Introduction
Preconditioning activates a powerful endogenous adaptive mechanism that increases the resistance of the heart to
ischemia and could potentially increase the chances of survival under pathophysiological conditions. Two different types
of ATP-sensitive potassium channels have been implicated in
preconditioning: surface membrane KATP (sKATP) channels
and mitochondrial KATP (mKATP) channels. The structure and
function of sKATP channels have been investigated extensively, whereas the subunit composition and the gene(s) coding for putative mKATP channels are still unknown. Both channels are presumed to be regulated by changes in energy
metabolism and to have cardioprotective effects.
* Corresponding author. Tel.: +49 6421 286 6494; fax: +49 6421 286
8960.
E-mail address: [email protected] (J. Daut).
0022-2828/$ - see front matter © 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.yjmcc.2005.04.002
The central question of this review is whether mKATP,
sKATP or both of these channels play a role in ischemic and/or
pharmacological preconditioning. We first provide an overview of the phenomenon of preconditioning and outline the
concept of mKATP channels as a major mediator of preconditioning (Section 2). Despite a large body of work on intact
hearts and on simplified models of preconditioning, doubts
persists as to the plausibility of current hypotheses about
mKATP channels as triggers and/or effectors of preconditioning. By nature of their localization, mKATP channels are
difficult to study. We therefore discuss the experimental
evidence for the presence of mKATP channels in the mitochondrial inner membrane in some detail and point out the merits
and pitfalls of the different methodological approaches (Section 3).
The main tools for discriminating between mKATP channels and sKATP channels are the K+ channel openers diazoxide, nicorandil and pinacidil and the blockers 5-hydroxy-
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decanoate (5-HD), HMR1098 and glibenclamide. However,
several recent studies have questioned the usefulness of these
substances for defining and characterizing mKATP channels.
To assess the validity and limitations of the pharmacological
approach to channel characterization, we provide an analysis
of the effects of diazoxide and 5-HD on mKATP channels and
on other targets (Section 4). On the basis of the available data
we summarize our view of the role of mKATP channels in
preconditioning. In addition, we discuss the possible role of
other mitochondrial K+-selective channels in regulating matrix
volume and the potential of the mitochondrial inner membrane (Section 5).
The surface KATP channel is a sensor of the metabolic state
of the cells that is modulated by a variety of metabolic intermediates. It is clear that it does contribute to the effects of
preconditioning, but the extent of its contribution is still under
debate. We discuss the mechanisms of regulation of the sKATP
channel that may be relevant under conditions of ischemia,
and we summarize the pharmacology of sKATP channels,
emphasizing similarities and differences between sKATP and
mKATP channels (Section 6). We then go on to summarize
the information available about the role of sKATP channels in
preconditioning (Section 7). Finally, we give an overview of
the most important alternative mechanisms of preconditioning. In particular, the profound alterations of fatty acid
metabolism occurring during ischemia and reperfusion, as
well as the regulation of the mitochondrial permeability transition pore by reactive oxygen species may play a role as
mediators or effectors of cardioprotection (Section 8).
The intention of this review is not to say that the current
view of the mKATP channel as the principal mediator of preconditioning is necessarily incorrect, or that mKATP channels
do not exist. In fact, there is ample evidence for the existence
of K+ channels in the mitochondrial inner membrane, and
there is growing consensus that ion channels are involved in
the regulation of matrix volume, matrix calcium and respiratory rate. Rather, we want to point out that the properties
ascribed to mKATP channels on the basis of pharmacological
experiments should not be considered as established facts. It
is not yet clear whether the mitochondrial inner membrane is
endowed with channels that bear any resemblance to surface
KATP channels, and alternative hypotheses for the cellular
defense mechanisms against ischemic damage also need to
be considered. In the summary and conclusions of this review
we try to weigh the evidence for and against a role of mKATP
and sKATP channels in preconditioning in the light of recent
findings, and we try to give an integrated picture of the mechanisms involved in protecting the heart against ischemic injury.
2. Studies of preconditioning in the intact heart
2.1. The phenomenon of preconditioning
The term ischemic preconditioning (IPC) refers to the
observation that one or several intermittent periods of ischemia
(lasting ~ 5 min) protect the myocardium against the injury
caused by a subsequent, prolonged period of ischemia (lasting ~ 30 min), which is denoted index ischemia [1–4]. There
are two windows of protection: the initial window which lasts
1–2 h after the preconditioning stimulus (“classic IPC”) and
the ‘second window of protection’ which is less effective but
covers a longer period. It occurs about 24 h after the preconditioning stimulus and lasts about 72 h. The possible mechanisms underlying the second window of protection have been
reviewed elsewhere [3,5,6] and will not be discussed here.
The cardioprotective effect of brief periods of ischemia can
be mimicked by exposing the heart to various drugs, a phenomenon termed pharmacological preconditioning (PPC).
The drugs used for PPC include K+ channel openers (diazoxide, pinacidil and nicorandil), volatile anesthetics (halothane, desflurane, isoflurane and sevoflurane) [7], various
agonists of G protein-coupled receptors (adenosine, bradykinin, catecholamines and opioids) [6,8–13], succinate dehydrogenase blockers (3-nitropropionic acid) [14], carbonmonoxide releasing molecules [15] and Na+/H+ exchange
blockers (cariporide and ethylisopropyl amiloride) [16]. In
most cases, PPC produces a similar degree of protection
against subsequent ischemic injury as IPC.
It is generally assumed that preconditioning activates
endogenous intracellular defense mechanisms that increase
the tolerance to injury [3,6]. These endogenous cell survival
programs are probably highly complex and depend, among
other factors, on mitochondrial and cytosolic energy metabolism and the electrical activity of the cells [3,17,18]. A schematic description of the protection afforded by IPC and PPC
is given in Fig. 1, which shows that the activation of cardioprotective mechanisms does not prevent, but rather delays cellular death. When the optimal preconditioning stimulus (brief
episodes of ischemia or application of cardioprotective drugs)
and the optimal duration of the index ischemia (usually
30–40 min) are chosen, infarct size after reperfusion is substantially reduced as compared to control, i.e. the infarct size
measured without preconditioning. However, with shorter or
longer duration of the index ischemia the difference between
hearts with and without preconditioning stimulus is much less
conspicuous. In other words, the effectiveness of preconditioning (reduction of infarct size compared to control) depends
critically on the duration of the index ischemia chosen. Furthermore, the effectiveness of preconditioning depends on the
timing of the index ischemia. When the interval between the
conditioning stimulus and the onset of the index ischemia is
postponed, the cardioprotective effect becomes smaller and
eventually disappears within 2 h, indicating the time course
of the decay of the defense mechanisms involved in IPC.
It is likely that preconditioning also occurs in human heart
[3,6,19], and the elucidation of the mechanisms underlying
the increased tolerance to ischemia is of considerable clinical
importance [3,20–24]. Although the most extensive studies
of preconditioning have been carried out in the heart, similar
protective phenomena induced by short periods of ischemia
have also been reported for the brain, skeletal muscle [25–27],
kidney [28] and liver [29,30].
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
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Fig. 1. Schematic diagram of the cardioprotective effect of ischemic preconditioning (IPC, red) and pharmacological preconditioning (PPC, blue). The protective effect is assumed to accumulate during the conditioning phase (trigger phase) and to delay cell death during the index ischemia. The area supplied by the
occluded coronary artery (the area at risk) is taken as 100%. The infarct size (measured after reperfusion) corresponding to a particular duration of the index
ischemia is schematically indicated by the continuous black line (without preconditioning) and by the dark blue interrupted line (with preconditioning). The
green dotted line indicates the effects of preconditioning (the reduction in infarct size or the reduction in the fraction of dead cells measured after reperfusion)
determined with an index ischemia lasting 30 min.
2.2. The receptors and intracellular messengers involved
in preconditioning
Most studies of preconditioning were carried out using
occlusion of the left coronary artery or its branches in canine
[31], porcine [32], rabbit [33–37] or rat [38–40] hearts. This
experimental model has the advantage that it mimics the
ischemic events in patients suffering from acute coronary syndromes. The readout of the cardioprotective effect is usually
infarct size, expressed as percentage of the risk zone. Reduction of infarct size to approximately 25% of control (see
Fig. 1), or to even smaller values, has been reported by many
laboratories [3,41]. A disadvantage, though, of the in vivo
approach is that the animals are (necessarily) anesthetized by
agents which by themselves may modify infarct size following prolonged ischemia. In fact, inhalational anesthetics
[13,42–44], as well as opioid anesthetics [11,45], are cardioprotective whereas various intravenous anesthetics such as
ketamine, thiopental and pentobarbital have been shown to
inhibit pharmacological preconditioning [46]. Hence, the
choice of anesthetic for preconditioning studies in vivo, which
is usually ketamine, thiopental and/or pentobarbital, may profoundly affect the measured end point (infarct size), as demonstrated by Cope et al. [42].
After nearly 20 years of intensive investigation a partial
picture of the signal transduction pathways involved in preconditioning has emerged (Fig. 2). Various agonists that are
known to accumulate in the interstitium during ischemia, such
as adenosine, bradykinin, norepinephrine and opioids, have
been shown to confer cardioprotection [33,47]. Many of the
“cardioprotective” agonists couple to phospholipase C (PLC)
[48,49]. Activation of PLC catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), generating the secondary messengers inositol 1,3,4-trisphosphate (IP3) and diacylglycerol (DAG), the latter stimulating protein kinase C
(PKC) (Fig. 2). There is convincing evidence that PKC is a
central mediator of preconditioning [34,50–53]. Liu et al. [54]
even proposed that activation of PKC, which requires its translocation from the cytosol to anchoring proteins on the membrane cytoskeleton [55], produces the preconditioned state.
This idea was supported by experiments showing that PKC
activators mimic the effects of IPC [56–59]. PKC may directly
phosphorylate the effector proteins of preconditioning (Fig. 2)
or switch on kinase cascades such as mitogen activated protein kinases [60]. Although, the role of PKC as a trigger of
preconditioning is not undisputed [3,60], it has been emphasized that specific isoforms of PKC may behave quite differently [49]. A good case, though, can be made for PKCe since
this isoform translocates from the cytosol to the surface membrane and to mitochondria during ischemia [61–68].
Activation of protein kinase a (PKA), independent of PKC,
has also been shown to be cardioprotective, possibly through
the inhibition of Rho-kinase [69]. Consistent with a role for
PKA, b-adrenergic receptor agonists and increased cAMP
have been shown to protect the heart from ischemia [70].
Hence, both the b-adrenergic/cAMP and PLC-linked signaling pathways, the latter of which includes a1-adrenergic receptors [11], can independently, and possibly synergistically, confer protection from ischemia. Other recent studies on the
mechanisms of ischemic and pharmacological precondition-
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Fig. 2. Schematic diagram of some of the mechanisms thought to be involved in the activation of mKATP and sKATP channels. The targets of diazoxide,
glibenclamide and 5-HD are indicated. Abbreviations: R, receptor; G, G-protein; PLC, phospholipase C, PKC, protein kinase C; ETC, electron transport chain.
ing have implicated mitogen activated protein kinases
(MAPKs) [60,71–76] and phosphatidylinositol-3-kinase
(PI3K) [77–80].
Reactive oxygen species (ROS), which are known to activate various kinases including PKC, also play a role in IPC
and PPC. Baines et al. [81] showed that a free radical scavenger could block preconditioning induced by one or more
short ischemic episodes. Further work in Downey’s laboratory revealed that the receptor agonists acetylcholine, bradykinin, opioids and phenylephrine, but not adenosine, triggered preconditioning by generating ROS [11]. PPC with the
K+ channel openers diazoxide and pinacidil [82] and with the
anesthetic agents halothane, isoflurane and sevoflurane
[83–87] has also been shown to increase ROS in isolated myocytes. Moreover, the protective effects of these drugs were
blocked by anti-oxidants. Hence, pharmacological and
ischemic preconditioning probably share generation of ROS
as a common signaling pathway. The downstream targets of
ROS have not been fully elucidated, but probably include PKC
[88,89] (Fig. 2) and other kinases [90–93].
The search for triggers/mediators of preconditioning turned
a new corner after the publication of seminal papers by Garlid et al. [94] and Liu et al. [95], who proposed that ischemic
preconditioning may be related to the opening of mitochondrial KATP channels. Liu et al. carried out measurements of
flavoprotein fluorescence in intact rat ventricular cardiomyocytes, and Garlid et al. partially purified mKATP channels and
characterized their pharmacology [94,96–98] [99]. On the
basis of these measurements (see Sections 3.2, 3.3 and 3.4) it
was proposed that mKATP channels play an obligatory and
central role in preconditioning. Moreover, it was postulated
that diazoxide selectively opens, and 5-HD specifically blocks,
mKATP channels. Numerous subsequent publications were
based on the assumption that experimental observations that
can be mimicked by diazoxide and blocked by 5-HD are
attributable to the opening of mKATP channels (reviewed in
Refs. [1–4,100–102]). However, no consensus has been
reached whether the opening of mKATP channels acts as a
trigger of the cardioprotective effect (opening during the triggering phase) or as an end effector (opening during or after
the index ischemia) [3,31,37,67,103–105]. In most earlier
studies mKATP channels were proposed to be end effectors,
and the channels were assumed to open during the index
ischemia [31,106–108]. In some recent studies, opening of
mKATP channels has been inferred to be the initial trigger of
the cardioprotective effect, promoting the generation of ROS
and inducing the activation of PKCe [37,82,91,103], whereas
Lebuffe et al. [109] proposed that generation of ROS during
the trigger phase leads to the opening of mKATP channels.
Some authors have suggested that mKATP channels act both
as a trigger and as an end effector of IPC [1,3].
In conclusion, many groups have reported that diazoxide
mimicked and 5-HD prevented preconditioning. In fact, the
effects of these drugs on infarct size have become something
like the gold standard for testing whether a phenomenon is
due to opening of mKATP channels. For a detailed coverage
of these experimental findings, and their potential shortcomings, the reader is referred to comprehensive recent reviews
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
[1–4]. Nevertheless, some doubt about the mKATP channel
hypothesis remained. Since it turned out to be difficult to pin
down the mechanisms involved in IPC and PPC using solely
studies of infarct size, more reductionist models of preconditioning have been introduced, for instance, application of global ischemia and monitoring post-ischemic recovery in isolated hearts [110–113], monitoring changes in intracellular
calcium, electrical activity and morphology in isolated cardiomyocytes [114–121], or studying the behavior of mitochondria in intact cardiomyocytes [122,123]. These simpler experimental models could indeed reproduce some of the essential
features of IPC and PPC, and this approach turned out to be
of great value for identifying discrete components of the
endogenous defense program of cardiomyocytes (see Sections 7 and 8). However, in some of the recent studies, the
assumption that preconditioning is mediated by mKATP channels has been questioned. Since most of the information on
the pharmacology and regulation of mKATP channels has in
fact been derived from studies of preconditioning, we reexamine the more direct evidence for the existence of mKATP
channels (independent of preconditioning) in the next section.
3. The search for mitochondrial KATP channels
3.1. Single-channel studies in mitoplasts
The first report suggesting the existence of mKATP channels was published in 1991 by Inoue et al. [124], who measured ATP-sensitive K+ currents with a conductance of ~10 pS
in inside-out patches excised from fused giant mitoplasts. One
concern with this experimental approach is that contaminating surface membrane could have introduced KATP channels
to the mitoplasts (mitochondria chemically divested of their
outer membrane, ruptured and reformed). The standard procedures used to isolate mitochondria, which involve tissue
homogenization, provide ample opportunity for the crosscoalescence of sarcolemmal, endoplasmic reticular and mitochondrial membranes, and Inoue et al. [124] did not employ
membrane markers to quantify the extent of contamination
of the mitoplasts with sarcolemmal proteins. Another concern is that the patches contained many different channels,
and that, apparently, inhibition of the putative KATP channel
was only observed after application of ≈0.5 mM ATP. Moreover, when more than one channel was active in the patch the
open-state was found to be unstable, which was interpreted
to indicate ‘cooperative transitions between dual or multi
states’ [124]. The authors also reported that Mg2+ was not
necessary for ATP-induced inhibition and ADP (2 mM) had
no effect. In many aspects, this study was quite preliminary
and its methodological limitations do not allow any reliable
inferences on the nature of mitochondrial K+ channels.
In another study, mitoplasts prepared from a human
T-lymphocyte cell line were investigated to detect mKATP
channels [125]. A channel with a conductance of ~80 pS at
21
positive potentials was seen in 12 out of 260 patches. It
showed strong outward rectification and was apparently not
K+ selective. Only a minor decrease in open probability was
observed upon application of 0.5 or 6 mM ATP to the matrix
side of the patch, which was irreversible and might therefore
represent rundown of the channel. At a concentration of
25 mM, ATP had no effect. Application of 1 mM 5-HD caused
a slight, irreversible, time-dependent reduction in open probability, which may also have been due to rundown. Diazoxide had no significant effect. Thus, there is actually no good
reason to denote this channel as a mKATP channel.
Er et al. [126] prepared fluorescent mitoplasts from rat cardiomyocytes preloaded with TMRE. The mitoplasts apparently had no membrane potential. In “mitoplast-attached”
measurements they observed a channel with a linear current–
voltage relation and a conductance of ~13 pS. The channel
had very slow kinetics and a very low open probability, which
could be increased after application of diazoxide (100 µM)
or testosterone (10 µM). The K+ selectivity of the channel
was not studied. The authors argued that the channel is
blocked by ATP from the matrix side and has a very low open
probability even in the absence of any blockers on the cytosolic side. This contradicts the conclusions obtained with
reconstituted mKATP channels in proteoliposomes [101,127]
(see Section 3.2). It should also be noted, that retention of
TMRE in the isolated mitochondrial preparation does not necessarily indicate that the mitochondria remained intact during the isolation procedure since the dye, in addition to electrophoretic accumulation, can bind to the inner membrane
[128].
Recently, evidence for expression of a voltage-activated
K+ channel (Kv1.3), with a conductance of about 25 pS [129],
and of a Ca2+-activated K+ channel [4,130] in the mitochondrial inner membrane has been obtained. Another mitochondrial ion channel, the classical 107 pS channel [131], is also
permeable to K+ ions. In conclusion, although several mitochondrial K+ permeable channels have been described, there
is, as yet, no convincing electrophysiological evidence for a
native K+-selective channel in the mitochondrial inner membrane that has a high sensitivity to adenine nucleotides, diazoxide and 5-HD.
3.2. Reconstitution of mKATP channels in liposomes
and lipid bilayers
Another source of evidence for the existence of mKATP
channels came from the work of Paucek et al. [96]. A protein
fraction containing a K+ channel was extracted from
detergent-solubilized membranes of isolated rat liver and beefheart mitochondria and reconstituted into liposomes or planar lipid bilayers. The dominant protein in the purified fraction had a molecular mass of 54 kDa. As in the case of the
mitoplast studies [124–126], Paucek et al. [96] did not provide evidence to rule out that the extract could have been contaminated with surface membrane proteins. Interestingly, the
molecular mass of the reconstituted protein [96] would fit the
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P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
mitochondrial Ca2+-activated K+ channel in cardiac muscle
(Mw, ~55 kDa) [132], or the adenine nucleotide translocase
(ANT; Mw , ~30 kDa), which exists as a dimer [133].
Using liposomes containing the reconstituted 54 kDa protein and loaded with the fluorescent K+ indicator PBFI, Paucek
et al. [96] tried to assess changes in the open probability of
mKATP channels by measuring CCCP-dependent “electrophoretic K+ uptake”. They found that K+ uptake into the proteoliposomes was inhibited by ‘cytosolic’ ATP or ADP, but
only when either Mg2+ or Ca2+ was present. To explain the
apparent discrepancy with the findings of Inoue et al. [124] it
was suggested [96] that the solutions used by Inoue et al.
were not really Ca2+-free but contained about 100 nM free
Ca2+. The same argument could be applied to the conflicting
data of Er et al. [126] since these authors did not include a
Ca2+ chelator in their nominally Ca2+-free solutions. Surprisingly, the inferred K+ flux into the liposomes was insensitive
to tetraethylammonium (TEA) ions [96], which would clearly
distinguish the putative mKATP channel from sKATP channels
[134] and other K+ channels [135]. The inferred K+ influx
was inhibited by glibenclamide with an IC50 value of ~50 nM
[96]. In contrast, high concentrations of glibenclamide
(>5 µM) were apparently required to inhibit the ATP-sensitive
ion channel observed electrophysiologically [124,126].
Zhang et al. [136] investigated the effects of MgATP, diazoxide, 5-HD, glibenclamide and ROS on ion channels reconstituted from submitochondrial membrane vesicles and incorporated into planar lipid bilayers. Application of 1 mM
MgATP to the trans side of the bilayer (which corresponds to
the matrix side) inhibited the channel significantly, but
MgATP had no effect on the cis (cytosolic) side, in contrast
to the conclusions drawn by Garlid and co-workers [127].
The effects of Mg2+ or ATP alone were apparently not tested.
With symmetrical 150 mM K+ the channels had a unitary
conductance of 56 pS, which is much higher than the value
reported for the mKATP channel in heart and liver mitoplasts
(10–15 pS [124,126]). The open-state probability of the channel was reduced by 5-HD (100 µM) and glibenclamide
(50 µM), whereas diazoxide and ROS increased channel activity [136]. The selective sKATP channel blocker HMR1098,
used to exclude sarcolemmal channels, had no effect.
More recently, Ardehali et al. [137] tried to reproduce some
aspects of earlier work [96] on reconstituted mKATP channels
in proteoliposomes. They suggested that their mKATP channel, partially purified from a fraction of rat mitochondrial inner
membrane, may consist of a complex of at least five proteins
including succinate dehydrogenase (SDH) and ANT [137].
The reconstituted “mKATP” channel was now described as a
non-selective cation channel [137], and the lack of K+ selectivity was attributed to the presence of low concentrations of
dithiothreitol. No evidence was presented that the channel
was impermeable to anions. The electrophoretic cation influx
into liposomes was moderately increased (by a factor of ~2.8)
in the presence of 100 µM diazoxide, and this increase could
be partially inhibited by 500 µM 5-HD, 10 µM glibenclamide
or 2 mM ATP, in the absence of Ca2+ and Mg2+. The cation
influx was also moderately increased (by a factor of ~2.3) by
the SDH inhibitor 3-nitropropionic acid. When the protein
complex was reconstituted in lipid bilayers, a channel with a
conductance of 200 pS (with symmetrical 500 mM KCl) was
found [137]. The authors interpret their findings to indicate
that “SDH regulates mKATP channels by means of its physical interaction with the ionophore”. In comparison, the reconstituted mKATP channel described by Paucek et al. [96] was
K+ selective, showed no inhibition by ATP in the absence of
divalent cations and had a conductance of 30 pS (with symmetrical 1 M KCl) [96]. In this and several other aspects, the
putative mKATP channels described by the two groups appear
to be clearly different.
In conclusion, the mKATP channels reconstituted in proteoliposomes or lipid bilayers by different groups differ in
many aspects, such as molecular mass, ion selectivity, glibenclamide sensitivity, inferred orientation in the membrane
and conductance. It is likely that the purification procedure
of the proteins and the lipid composition of the membrane
may have influenced the results. The technical difficulties of
inserting native and cloned ion channels in lipid bilayers are
well known [138,139]. In addition, validation of the PBFI
fluorescence assay in proteoliposomes with a known K+ channel protein would be desirable to define more clearly the limitations of this method. Further studies are required to define
the pore-forming constituents of the five-protein complex containing SDH and ANT [137]. It will be interesting to see if
there is any relation of this protein complex to the mitochondrial permeability transition pore (MPTP), which also contains ANT [140–142].
3.3. Measurement of matrix volume in isolated
mitochondria
There is general agreement that opening of mitochondrial
K+ channels, accompanied by the movement of permeable
anions, should lead to an increase in matrix volume
[4,96,101,143–145], and that the increase in matrix volume
will activate the respiratory chain [100,143]. Therefore, measurements of matrix volume have played an important role in
the investigation of mKATP channels. A widely used technique for determining such volume changes is to monitor the
light scattering of mitochondrial suspensions; an increase in
mitochondrial volume is associated with a decrease in light
scattering. Changes in light scattering have been used as an
indirect measurement of K+ influx, which should depend on
the potential of the mitochondrial inner membrane and on
the “cytosolic” concentration of K+ [97,146]. Garlid and
co-workers reported that the initial rate of change of light
scattering following addition of mitochondria to the cuvette
(indicating mitochondrial swelling) was inhibited by ATP,
ADP and AMP [99,146]. K+ channel openers such as diazoxide increased the change of light scattering [97], and this
effect was inhibited by the putative mKATP channel blockers
glibenclamide and 5-HD [99]. Garlid and co-workers emphasize that these changes in light scattering were not observed
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
in media in which TEA replaced potassium, arguing that this
confirms the role of potassium movements in mediating these
changes.
In contrast, Halestrap et al. [147,148] reported no changes
in light scattering induced by a range of KATP channel openers (for example, diazoxide and pinacidil) or antagonists
(5-HD and glibenclamide) and have confirmed these data
using isotopic measurements of matrix volume. They were
able to confirm the increase in light scattering induced by
ATP and ADP observed by Beavis et al. [146], but were unable
to detect any concomitant change in matrix volume measured isotopically. Furthermore, they were able to demonstrate that the effects of ATP and ADP on light scattering were
reversed by carboxyatractyloside, an inhibitor of the ANT
[148]. As a control experiment, they showed that the K+ ionophore valinomycin was able to induce a decrease in light scattering that was reflected by an increase in matrix volume measured isotopically [148]. Das et al. [148] argue that the light
scattering measurements were in fact detecting a change in
the morphology or shape of the mitochondria induced by a
conformational change of the ANT, rather than a change in
mitochondrial volume. Garlid and Paucek [101] dispute this
interpretation. However, it is generally accepted that changes
in the morphology of mitochondria upon addition of adenine
nucleotides or ANT ligands are correlated with the c–m conformation transition [149–152]. Furthermore, changes in light
scattering of heart mitochondria in K+-free media have been
reported by several groups [149–151], and this has been confirmed by A. Halestrap (personal communication), which supports the interpretation that the changes in light scattering
observed by the groups of Garlid and Halestrap were at least
partly due to morphological changes of mitochondria. Such
changes were originally described as transitions between the
orthodox and condensed conformations of mitochondria
[153]. Taken together, these methodological considerations
suggest that changes in light scattering do not necessarily
reflect changes in matrix volume, and that the effects of
adenine nucleotides on light scattering should be interpreted
with caution.
Using isolated heart mitochondria, Korge et al. [154] simultaneously measured changes in matrix volume, oxygen consumption and mitochondrial membrane potential (Dwm) during and after addition of ADP (to stimulate respiration). Pulses
of ADP produced a transient depolarization and a transient
increase in light scattering (assumed to represent a decrease
in matrix volume). They found that, under conditions mimicking some elements of ischemia (elevated Pi, Mg2+ and
ADP, as well as low substrate concentration), the changes in
light scattering and Dwm were (weakly) dependent on the
presence of extramitochondrial (“cytosolic”) K+, and that
recovery of matrix volume and Dwm after ADP pulses were
improved after addition valinomycin or diazoxide. These findings support the concept of K+-dependent regulation of matrix
volume and ADP phosphorylation [2,143]. Unfortunately,
Korge et al. [154] did not investigate whether the effects of
diazoxide under “ischemic” conditions depended on the presence of “cytosolic” K+.
23
In conclusion, the experimental data summarized in this
section are consistent with the notion [143] that there is a
K+-dependent regulation of matrix volume. However, the published measurements of changes in matrix volume with diazoxide and 5-HD are contradictory, and the measurements
with adenine nucleotides are open to alternative interpretations, swelling versus conformational changes of the ANT.
Furthermore, the characterization of the molecular nature of
the ion channels responsible for the volume changes hinges
on the specificity of the channel openers and blockers used
(see Section 4).
3.4. Flavoprotein fluorescence as an indicator of mKATP
channel activity
In their pioneering papers on mKATP channels, Marban and
co-workers tested the effects of diazoxide and 5-HD on flavoprotein fluorescence in intact rabbit cardiac myocytes
[95,98]. The rationale for measuring flavoprotein fluorescence was that “opening of mitoKATP channels dissipates the
inner mitochondrial membrane potential established by the
proton pump. This dissipation accelerates electron transfer
by the respiratory chain and, if uncompensated by increased
production of electron donors, leads to net oxidation of the
mitochondria” [98] (note that oxidized, but not reduced, flavoproteins are fluorescent). Diazoxide produced a dosedependent increase in flavoprotein fluorescence, assumed by
the authors to signify opening of mKATP channels, which was
blocked by 5-HD. In contrast to these data obtained with rabbit ventricular myocytes, diazoxide has been shown to have
no effect on flavoprotein fluorescence measured in freshly
isolated rat [116] or guinea-pig ventricular myocytes [155].
According to a review by Garlid et al. [2], the majority of
myocytes used by Marban’s group for flavoprotein fluorescence experiments were incubated overnight in substratefree medium. However, Brian O’Rourke (personal communication) asserted that cells were in fact incubated overnight
in M199 medium, which contains ample metabolic substrate,
whereas experiments were performed in substrate-free modified Tyrode’s solution. Nevertheless, the use of substrate-free
solutions complicates the interpretation of the flavoprotein
fluorescence experiments, especially since 5-HD is known to
be metabolized, albeit poorly, by the b-oxidation pathway of
mitochondria [147,155–157], and fatty acid metabolism
induces flavoprotein reduction [158]. Thus, metabolism of
5-HD by the substrate-deprived cells could have been responsible for the observed decrease in flavoprotein fluorescence
observed after addition of 5-HD. Furthermore, at concentrations similar to that used by Sato et al. [98], diazoxide inhibits succinate dehydrogenase [147,155,159–161], which itself
can increase flavoprotein fluorescence [155]. Another problem with the flavoprotein fluorescence assays was that glibenclamide alone had an effect. Liu et al. [95] reported that,
“[They] did not observe consistent blockade of mitochondrial oxidation, probably because glibenclamide alone caused
oxidation of the flavoproteins especially at concentrations >
1 µmol/l (data not shown).”
24
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
Recently, Sato et al. [123] confirmed the oxidation of flavoproteins by diazoxide in intact cardiomyocytes in glucosefree solution. Interestingly, they found that opening of BKCa
channels with NS1619 (30 µM) oxidized flavoproteins to
about the same extent as diazoxide (100 µM). The effects of
NS1619 could be inhibited with paxilline, a blocker of BKCa
channels. These findings underscore the potential usefulness
of flavoprotein fluorescence as an indicator of the rate of electron transfer by the respiratory chain. On the other hand, the
discovery of BKCa channels in the mitochondrial inner membrane makes the interpretation of some of the earlier data more
complicated because now there is a competing K+ conductance.
3.5. Measurement of mitochondrial membrane potential
Measurements of Dwm have been very useful in the investigation of the function of mitochondrial ion channels.
Because Ca2+ uptake into mitochondria is driven primarily
by Dwm, small changes in Dwm can induce large changes in
matrix Ca2+ concentration [123,162], and thus large changes
in the functional state of mitochondria. Kowaltowski et al.
[145] argued that in fully energized mitochondria the change
in Dwm induced by the opening of mKATP channels should
be very small, and that the main result of mKATP channel
opening in the dynamic steady state is an increase in matrix
volume. This is so because the K+ influx is followed immediately by K+/H+ exchange and uptake of phosphate (Pi) via
the electroneutral Pi/OH– exchange carrier. Indeed, Ishida et
al. [162] found that 100 µM diazoxide-induced only a small,
not significant, depolarization of mitochondria in rat ventricular cardiomyocytes and did not affect matrix Ca2+, measured
as Rhod-2 fluorescence. However, when cytosolic and matrix
Ca2+ was increased by inhibiting the Na+,K+-ATPase with
ouabain, 100 µM diazoxide did produce a significant depolarization and a decrease in matrix Ca2+. This was recently
confirmed by Sato et al. [123], who went on to show that
opening BKCa channels with 30 µM NS1619 caused a similar decrease in Dwm and matrix Ca2+. From these data, and
from their flavoprotein fluorescence measurements, Sato et
al. [123] concluded that opening of either mKATP channels or
BKCa channels reduces mitochondrial Ca2+ overload by
decreasing Dwm, in accord with the work of Holmuhamedov
et al. [163].
It should be mentioned, though, that alternative interpretations of the effects of diazoxide on Dwm have been put forward. In isolated rat heart mitochondria it was found that
100 µM diazoxide or 100 µM pinacidil substantially accelerated state-4 respiration and decreased Dwm [164]. Since these
effects could be mimicked by application of 2,4-dinitrophenol
(DNP) and were maintained in K+-free medium the authors
concluded that the effects of the two K+ channel openers were
mediated by an uncoupling action, in agreement with Kowaltowski et al. [145]. Interestingly, application of DNP, like
diazoxide or pinacidil, improved functional recovery of isolated rat hearts from ischemia reperfusion, suggesting that
uncoupling per se could be cardioprotective [164–166]. Thus,
there may also be a K+-channel-independent pathway for cardioprotection by K+ channel openers, related to a decrease in
ROS production induced by uncoupling.
In a series of experiments in which isolated mitochondria
were investigated under conditions mimicking ischemia,
Korge et al. [154,167,168] demonstrated that regulation of
K+ conductance and matrix volume may be critical for cardioprotection. They suggested that when mitochondria are in
a weakened state, with reduced transport capacity of the respiratory chain (for example, during ischemia or at the onset
of reperfusion), K+ influx and the resulting increase of matrix
volume and decrease in Dwm may be specially relevant for
avoiding excessive matrix shrinkage, Ca2+ overload and ultimately cell death [2,101,154,167].
In conclusion, the measurements of matrix volume and
Dwm have considerably advanced our understanding of the
possible functions of mitochondrial K+ channels. It is likely
that in fully energized mitochondria opening of K+ channels
has relatively little effect, whereas under metabolic stress relatively small decreases in Dw m can translate into large
decreases of matrix Ca2+.
3.6. Probing for mitochondrial KATP channel subunits
A number of different laboratories have pursued the question of whether the mKATP channel may be composed of one
of the pore-forming a-subunits of sKATP channels (Kir6.1 or
Kir6.2) in combination with an unidentified b-subunit. Using
dominant-negative constructs of either Kir6.1 or Kir6.2, Seharaseyon et al. [169] observed that in rabbit cardiac myocytes
with reduced functional expression of Kir6.1 or Kir6.2 diazoxide increased flavoprotein fluorescence (used as an assay
for functional mKATP channels) to the same extent as in control cells. On this basis, the authors concluded that neither
Kir6.1 nor Kir6.2 are components of the mKATP channel.
Studies using either the Kir6.1- or Kir6.2-knockout mouse
led to the same conclusion since diazoxide increased flavoprotein fluorescence to a similar extent in wild-type and knockout mice [170,171].
In accord with the above studies, confocal fluorescence
imaging studies have shown that GFP-tagged Kir6.2, expressed in HEK cells or myocytes, is not targeted to mitochondria [172]. Using a similar method, Hambrock et al. [173]
showed that GFP-tagged Kir6.1, alone or co-expressed with
various splice forms of SUR1, is not targeted to the mitochondria of COS cells. Likewise, Seharaseyon et al. [169] showed
that an anti-Kir6.1 primary antibody labeled the sarcolemma
of myocytes but not the mitochondria. In contrast, an earlier
electron microscopy study had reported that immunogold
labeled Kir6.1 was present in mitochondria [174]. However,
Grover and Garlid [100] stated that the antibody used in this
study does not react with reconstituted and functional mKATP
channels, whereas it does bind to unidentified proteins, suggesting that the results of Suzuki et al. [174] may have been
“false-positive”.
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
Using antibodies and confocal microscopy, Singh et al.
[175] reported that Kir6.1, as well as Kir6.2, subunits were
localized to mitochondria of rat ventricular myocytes. In a
complementary study using immunoblotting and immunogold electron microscopy, Lacza et al. [176] found that both
anti-Kir6.1 and anti-Kir6.2 labeled isolated mitochondria.
However, mitochondrial proteins were enriched only by a factor of 8 in the Western blots, thus contamination with surface
membrane proteins is likely. These authors also reported that
Kir6.1 and Kir6.2 harbored mitochondrial targeting sequences. However, we could not verify this. With the most
recent versions of MITOPROT (http://mips.gsf.de/cgibin/proj/medgen/mitofilter), PREDOTAR (http://genoplanteinfo.infobiogen.fr/predotar/predotar.html) and TargetP only
low prediction scores for mitochondrial localization were
obtained. One of the problems with immunocytochemical
studies is that successful preabsorption experiments do not
rule out cross-reactivity of the antibodies. To ascertain possible mitochondrial localization of the pore-forming
Kir6.1 and Kir6.2 subunits, control experiments with Kir6.x
knockout mice are mandatory.
In the case of the regulatory sKATP channel subunit SUR,
Singh et al. [175] found that anti-SUR2A labeled mitochondria in myocytes. Similarly, Lacza et al. [176] reported that
anti-SUR2, but not anti-SUR1, labeled the mitochondrial inner
membrane. However, the anti-SUR2 labeled protein had a
much smaller molecular mass than SUR2 (~25 versus
~140 kDa; [177]) and co-immunoprecipitation experiments
did not reveal any protein-protein interaction between the SUR
candidate and Kir6.x subunits. Thus, the 25 kDa SUR-like
component described may not be associated with any K+ channel.
In conclusion, immunohistochemical and other localization studies have not contributed much to the molecular identification of mKATP channels. There is no convincing evidence that Kir6.x or SURx subunits are localized to
mitochondria.
4. The pharmacology of mitochondrial KATP channels
4.1. Openers of mKATP channels
One of the most noteworthy features of mKATP channel
pharmacology is that virtually every drug that opens these
channels also activates sKATP channels in cardiomyocytes
and/or in vascular smooth muscle. To date, the list of drugs
which activate both mitochondrial and sarcolemmal KATP
channels include: pinacidil, cromakalim, P1060, P1075,
minoxidil, KRN2391, sidenafil, desflurane, isoflurane, sevoflurane, aprikalim and levosimendan [4,178–182]. Notable
exceptions are diazoxide and nicorandil, which are thought
to activate selectively mKATP channels [183] in cardiac myocytes (but also activate sKATP in other cell types). Sato et al.
[183] reported that Nicorandil (100 µM) increased flavoprotein fluorescence, used as an index of mKATP channel activ-
25
ity, without acting on sKATP channels in myocytes, except at
high concentrations. In contrast, Tsuchida et al. [184] found
that nicorandil-induced cardioprotection was attenuated by
the selective sKATP blocker HMR1089, whereas Critz et al.
[185] found that pinacidil but not nicorandil protected isolated myocytes from simulated ischemia. Hence, nicorandil
may not be such a selective drug as it first appeared. Indeed,
[14C]-nicorandil was found to be de-nitrated by cardiac mitochondria [186], yielding the metabolite SG-86 and NO, which
complicates the interpretation of studies using this drug.
Another drug, the benzopyran derivative BMS-191095 has
been reported to have a cardioprotective effect with relatively little vasorelaxant potency and no effect on the cardiac
action potential [187,188]. Grover and Atwal [187] suggested that BMS-191095 induced the opening of mKATP channels, based on the observation that the cardioprotective effect
of the drug was not seen in the presence of glibenclamide or
5-HD. Thus, BMS-191095 may be a good substitute for diazoxide but more studies are required to establish its potential
usefulness.
The most commonly used drug for pharmacological preconditioning, diazoxide, has been reported to activate mKATP
channels in intact rat liver mitochondria with a K0.5 value of
2.3 µM under particular conditions [97]. These conditions
include the presence of 0.5 mM ATP and 1 mM Mg2+. Garlid
et al. [97] confirmed these data using partially purified mitochondrial proteins reconstituted in liposomes, which contained a fluorescent indicator to estimate K+ flux; diazoxide
activated K+ influx with a K0.5 value of about 0.4 µM in channels obtained from either liver (in the presence of 0.5 mM
ATP) or heart (in the presence of 2 mM ATP). One interpretation of the high binding affinity of diazoxide and low sensitivity to millimolar ATP is that the putative mKATP channel
is not composed of the known SURx and Kir6.x building
blocks. In support of this notion the mKATP channel is, surprisingly, inhibited by ADP and long-chain acyl-CoA esters
[189], but not by TEA [96].
The low K0.5 value for diazoxide has been used as evidence to implicate mKATP channels in the mechanism of preconditioning. The mechanism of cardioprotection by diazoxide has, in a way, simply been attributed to the site of action
with the highest binding affinity. The highly lipophilic drug
diazoxide, though, has been routinely used in preconditioning studies with myocytes and whole hearts at concentrations
of 50–100 µM. Thus, other targets of diazoxide, of which
there are many, may also come into play during such studies.
In the whole heart, for example, diazoxide potently activates
endothelial and smooth muscle KATP channel isoforms. It also
weakly activates the cardiac muscle type of sKATP channels,
an effect which would be greatly augmented by ischemia due
to the concomitant increase in the ADP/ATP ratio [190]. Aside
from these KATP channel-related actions, diazoxide is known
to inhibit succinate dehydrogenase [147,155,160,161,191],
to act as a protonophoric uncoupler [164], to promote the
inhibition of ATP-synthase [192] and to inhibit other
nucleotide-requiring enzymes [161].
26
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
Another pharmacological approach used to characterize
the role of mKATP channels in preconditioning was to compare the relative potency of different K+ channel openers in
cardioprotection and in activation of sKATP channels [94]. For
example, Garlid et al. [94] reported that application of diazoxide or cromakalim improved post-ischemic functional
recovery in isolated perfused rat and rabbit hearts subjected
to global ischemia with approximately equal potency. Since
they found diazoxide to be significantly less potent than cromakalim in increasing sarcolemmal K+ currents they concluded that the effects were due to opening of mKATP channels. In contrast, Haruna et al. [41], analyzing IPC in rabbits
by measuring infarct size, found that cromakalim was much
more potent than diazoxide in limiting infarct size and concluded that sKATP channels also play an important role in IPC.
Thus, it is important to note that the potency of drugs influencing cardiac metabolism (such as K+ channel openers) may
be critically dependent on the metabolic state of the cells and
on the precise experimental conditions chosen.
4.2. Blockers of mKATP channels
5-HD is generally regarded as a specific inhibitor of mKATP
channels, and in most of the studies carried out so far 5-HD
has been used as a tool to test for the involvement of mKATP
channels. In nearly all studies using infarct size as a readout,
5-HD impaired the recovery of the preconditioned heart after
the index ischemia. In isolated rat hearts, however, hemodynamic recovery after IPC was found to be unaffected by 100 or
300 µM 5-HD [112,147]. The initial evidence that 5-HD
blocks mKATP channels was obtained from indirect measurements of channel activity. Using isolated rat heart mitochondria, Jaburek et al. [99] observed that 5-HD inhibited
diazoxide-induced K+ influx, in the presence of 0.2 mM ATP,
with a K0.5 value of 45 µM. In these experiments light scattering was used as an indicator of mitochondrial matrix swelling, and matrix swelling was used as a measure of K+ influx.
As mentioned earlier (Section 3.5), the authors proposed that
the main effect of K+ channel openers in intact mitochondria,
with K+/H+ exchange and Pi/OH exchange in place, was an
increase in matrix volume.
In contrast, Das et al. [148], who also used rat heart mitochondria and employed very similar experimental conditions, reported that neither diazoxide (50 µM) nor 5-HD
(50 µM) had any effect on light scattering or matrix volume
measured using mitochondrial entrapment of radioactive
markers. They were “unable to offer an explanation for why
[their] data differ from those of Garlid and colleagues” [148].
Interestingly, 5-HD alone, applied at higher concentrations
(100 or 300 µM), was found to elicit an increase in matrix
volume before and during ischemia, and a decrease in respiration rate during reperfusion [147]. The increase in matrix
volume is contrary to what would be expected for a mKATP
channel blocker, and the decrease in respiration rate may be
related to metabolic effects of 5-HD [156,157]. Taken
together, the findings summarized here strongly suggest that
5-HD has effects on mitochondrial energy metabolism independent of its action on mKATP channels [147,148,155–157].
In support of the hypothesis that 5-HD is a specific blocker
of mKATP channels, Liu et al. [95] reported that 5-HD reversed
the increase in flavoprotein fluorescence induced by diazoxide in rabbit myocytes deprived of metabolic substrate. However, these supportive data rely on the assumption that flavoprotein fluorescence is an unequivocal indicator of mKATP
channel activity, which clearly is not the case [155]. Using
analytical HPLC and mass spectrometry, Hanley et al. [155]
showed that 5-HD, like physiological fatty acids, is activated
via acyl-CoA synthetase. Using intact heart and liver mitochondria, Lim et al. [147] confirmed that 5-HD is activated to
5-HD-CoA. Moreover, these authors showed that liver, but
not heart, mitochondria can metabolize 5-HD, whereas neither heart nor liver mitochondria appeared to be able to
metabolize 5-HD-CoA. Lim et al. [147] suggested that extramitochondrially activated 5-HD (5-HD-CoA) cannot enter the
matrix via carnitine palmitoyltransferases (CPTI/CPTII). In
further work, Hanley et al. [156] demonstrated that 5-HDCoA, once in the matrix, serves as substrate for at least the
first two enzymes of the b-oxidation pathway. Up to this point,
it appeared that heart mitochondria cannot take up 5-HDCoA synthesized on the outer membrane. However, we have
recently observed that purified 5-HD-CoA is indeed a substrate for intact heart mitochondria, albeit it is metabolized
much more slowly than decanoyl-CoA, probably due to slow
kinetics at the penultimate step of b-oxidation [157]. The slow
rate of metabolism, and confounding reactions such as acylCoA oxidase activity, may have masked the metabolism of
5-HD-CoA in the study of Lim et al. [147].
The notion that 5-HD selectively blocks mKATP but not
sKATP channels (Kir6.2/SUR2A) in cardiomyocytes is disputable. Although some groups have reported that 5-HD does
not inhibit sKATP channels activated by K+ channel openers
such as cromakalim and pinacidil [98,193], others have shown
that 5-HD blocks various surface KATP channels, including
Kir6.1/SUR1 and Kir6.2/SUR1, indicating that it has poor
mitochondrial specificity [194]. Moreover, Notsu et al. [195]
found that 5-HD blocked sKATP channels in cardiomyocytes
subjected to metabolic inhibition. This suggests that 5-HD
may be able to block sKATP channels under ischemic conditions. Examination of the effects of 5-HD on single-channel
activity of sKATP channels may help to clarify this issue.
Glibenclamide has been widely assumed to block both
sKATP channels and mKATP channels. However, this drug has
KATP channel-independent actions as well. Glibenclamide has
been shown to reduce the rate of fatty acid oxidation by inhibiting carnitine palmitoyltransferase [196–199]. At higher concentrations, it has also been shown to block ABC transporters [200,201] and chloride channels [202,203]. Whether such
inhibitory actions play a role in the ability of glibenclamide
to block preconditioning remains to be tested. In addition,
glibenclamide has been reported to uncouple rat liver mitochondria [204], which may explain the increase in flavoprotein fluorescence reported by Liu et al. [95] (see Section 3.4).
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
The uncoupling induced by glibenclamide may, in fact, not
be due to enhanced proton permeation but, instead, to permeabilization of the mitochondrial inner membrane to Cl–, which
promotes net Cl–/K+ influx and dissipates the mitochondrial
membrane potential [205].
On balance, the findings summarized in this section
strongly suggest that neither diazoxide nor 5-HD are ‘selective’ drugs in the sense that they only activate or block mKATP
channels. Since both substances have KATP channelindependent actions in the range of concentrations commonly used for preconditioning they can no longer be considered as arbitrators of the involvement of mKATP channels
in cardioprotection. Therefore, alternative mechanisms should
be taken into consideration when interpreting the effects of
diazoxide and 5-HD on cell survival or functional recovery
after ischemia, hypoxia or metabolic stress (see Section 8).
5. Do mitochondrial K+ channels play a role in
preconditioning?
5.1. Three candidate K+ channels in the mitochondrial
inner membrane
Are mKATP channels involved in preconditioning? Due to
the lack of specificity of the most important openers, diazoxide and pinacidil, and the most important blockers, 5-HD and
glibenclamide, we must admit that we just do not know. The
two K+ channel openers can induce uncoupling in the relevant concentration range [164], and uncoupling per se, for
example by DNP, has been shown to confer cardioprotection
[164]. 5-HD has been shown to be activated to 5-HD-CoA
and to be metabolized by b-oxidation, with a bottleneck at
the penultimate step [157]. Thus, it can interfere with the
b-oxidation of endogenous fatty acids or be used, under certain conditions, as a weak metabolic substrate. Therefore, the
notion that a process that is activated by diazoxide and inhibited by 5-HD is necessarily related to mKATP channels is no
longer tenable.
The case is made more difficult by the fact that we do not
know the gene(s) that may code for the channel. It is unlikely
that Kir6.x or SURx channels are involved, and the new concept of the mKATP channel as a protein complex consisting of
at least five proteins including ANT and SDH [137] is associated with many imponderables. One problem is the lack of
K+ selectivity. If the channel is not K+-selective and is only
moderately influenced by ATP, and by the presumed mKATP
channel openers and blockers, why should we call it a KATP
channel? Another problem is the possible relation to the mitochondrial permeability transition pore, of which ANT may
be an essential component (Section 8.3). Furthermore, the
measurements of single-channel activity in mitoplasts ascribed
to mKATP channels are not at all convincing (Section 3.1).
Thus, as long as we do not know the gene and the characteristics of the single channels we cannot be certain that mKATP
channels exist.
27
On the other hand, the case of the mKATP channel is, paradoxically, strengthened by the discovery of BKCa channels in
cardiac mitochondria [130,132,143]. The similarity of the
effects of NS1619 and diazoxide on flavoprotein fluorescence [123] lends some support to the mechanistic concept
that flavoprotein fluorescence, under certain conditions, can
be used as an indicator of the rate of electron transfer by the
respiratory chain (Section 3.4). Although we do not know the
targeting sequence directing the channel to the mitochondria,
the evidence for the expression of BKCa channels in the mitochondrial inner membrane is stronger than the evidence for
expression of mKATP channels. Firstly, because there is a candidate gene, and, secondly, because two groups have obtained
plausible single-channel measurements [130,132]. Pharmacological preconditioning of the isolated perfused rat heart
with NS1619, an opener of BKCa channels, produced the same
extent of cardioprotection as PPC with diazoxide in the same
model. Furthermore, ischemic preconditioning was blunted
[123,206] or abolished [207] by blockers of BKCa channels.
The blockers were effective only when they were present during the trigger phase of preconditioning, they were ineffective when applied only during the index ischemia [206,207].
These findings support the idea that an increase in the K+
conductance of the mitochondrial inner membrane is an essential element of ischemic preconditioning.
Another complication, relevant for the interpretation of previous data, was introduced by the recent identification of
Kv1.3 as a mitochondrial K+ channel by Gulbins and coworkers [129]. Using patch-clamp measurements in mitoplasts and an array of molecular genetic techniques, it was
clearly shown that the same channel is expressed both at the
cell surface and in the mitochondrial inner membrane [129].
The channel can be inhibited by TEA, margatoxin (MgTx),
and the sea anemone (Stichodactyla helianthus) toxin, ShK.
Application of MgTx and ShK increased Dwm in isolated
mitochondria [129]. Furthermore, mitochondria of cells lacking Kv1.3 showed no decrease in Dwm and no release of cytochrome c after application of the cytostatic drug actinomycin
D [208], in contrast to wild-type cells. Thus, we now have
three K+ channels to consider that might be involved in preconditioning (Fig. 3).
5.2. The possible functional role of mitochondrial K+
channels
The classical K+ flux studies in isolated mitochondria
[2,101,143,209], together with recent studies using the K+
channel modulators diazoxide, NS1619 and MgTx
[4,123,154,162,168,210], give us some clues about the possible function of K+ channels in the mitochondrial inner membrane. Under physiological conditions, the K+ conductance
of the mitochondrial inner membrane is probably small, but
not negligible, and is tightly regulated. In fully energized mitochondria, K+ influx is compensated by H+ extrusion, so that
moderate activation of K+ channels probably has little effect
on Dwm [145,162] and matrix Ca2+. In contrast, under meta-
28
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
Fig. 3. Schematic diagram of some of the elements thought to be involved in the regulation of mitochondrial matrix volume. The targets of diazoxide, NS1619 and
paxilline are indicated. Abbreviations: ROS, reactive oxygen species; ETC, electron transport chain; MPTP, mitochondrial permeability transition pore; mKCa,
mitochondrial calcium-activated K+ channel; mKv, mitochondrial voltage-activated K+ channel.
bolic stress activation of a K+ conductance may lead to a substantial depolarization. This could occur (i) when the energy
supply is compromised [154], (ii) when the rate of electron
transfer by the respiratory chain is inhibited, or (iii) when the
mitochondrial matrix is loaded with Ca2+ [123,162]. Since
matrix Ca2+ is very sensitive to Dwm, depolarization will lead
to a reduction in matrix Ca2+.
In Fig. 4 we have summarized our current view of the role
of K+ channels in a qualitative scheme of the events occurring in cardiac mitochondria during preconditioning. The
model is based on the recent findings obtained with BKCa
channels expressed in the mitochondrial inner membrane,
denoted as mKCa channels here, and has been (speculatively)
extended to include Kv1.3 channels, denoted mKv channels,
as well as mKATP channels. The blue lines represent the time
course of K+ influx, Dwm, matrix volume, matrix Ca2+, and
opening of the MPTP in the absence of preconditioning. The
dotted red lines represent the possible time course of events
under conditions of IPC. Please note that, for simplicity of
presentation, the duration of trigger ischemia and index
ischemia (both shaded in gray), respectively, is not drawn to
scale. The uppermost tracing represents the time course of
the K+ conductance provided by opening of mKCa, mKATP
and mKv channels. Their open probability is assumed to be
regulated by PKA and PKC, which are activated during the
transient period(s) of ischemia and the subsequent brief reperfusion phase. PKC [211,212] and PKA [69,70] have long
been known to play a role in preconditioning (see Section
2.2). Sato and co-workers have provided evidence that the
putative mKATP may be regulated by PKC [98] and that the
mKCa channel may be regulated by PKA [123]. Kv1.3 channels are activated by depolarization, and also via PI3K [213–
215].
We assume that the three K+ channels act in concert to
regulate the volume of the matrix, and this may have very
profound effects on mitochondrial function [2,101,216,217].
However, in order to induce swelling, the influx of K+ ions
requires a counterion, most likely chloride, although the structure of mitochondrial Cl– channels is not yet clear [218–
222]. Recently, Juhaszova et al. [122] found that many preconditioning stimuli, including IPC and diazoxide, d-opioid
and bradykinin B2 receptors, converge upon a common
mechanism: slight swelling (2.5–4%) of mitochondria. Both
swelling and cardioprotection were abolished by the selective Cl– channel inhibitor IAA94. Cl– flux dependent swelling of mitochondria caused enhanced substrate oxidation.
Juhaszova et al. [122] speculate that the characteristic
‘memory’ of preconditioning is encoded by the mitochondrial swelling, but the upstream mechanisms of mitochondrial swelling are still unknown. An obvious way in which
this swelling can be brought about is by activation of K+ channels, followed by K+ influx driven by the steep electrical gradient, and subsequent Cl– influx through anion-selective channels and osmotic influx of water. Therefore it may be that the
upstream mechanisms mentioned above, PKC and PKA, con-
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
Fig. 4. Schematic diagram of the hypothetical changes of some mitochondrial parameters during preconditioning. The trigger ischemia (left) and the
index ischemia (right) are marked by gray shading. Only qualitative changes
are shown, and, for simplicity of presentation, the time axis is not drawn to
scale (the index ischemia is actually much longer than the trigger ischemia).
Production of ROS under control conditions (blue) and after preconditioning (red) is indicated by circles.
verge on the activation of K+ conductances in the mitochondrial inner membrane [123].
Once activated by kinases, the three K+ channels are regulated by [Ca2+]m , Dwm and, possibly cytosolic and/or matrix
ATP, respectively (Fig. 4). During the initial phase of ischemia,
Dwm is expected to fall. It may transiently rise again during
the brief reperfusion phase, eliciting the release of ROS [211].
Simultaneously, [Ca2+]m is expected to rise, thus activating
mKCa. During the index ischemia, Dwm decreases, [Ca2+]m
rises slowly and ATP falls, thus activating mKv, mKCa and
mKATP channels. The resulting K+ influx will produce a higher
matrix volume during the index ischemia and during the initial phase of reperfusion [147,223]. In the absence of preconditioning, a strong hyperpolarization of the mitochondrial
inner membrane is expected to occur immediately after reperfusion (blue line in Fig. 4), associated with a large Ca2+
influx and matrix alkalinization, giving rise to the generation
of ROS [224,225]. After IPC, when the mitochondrial K+
channels are activated, the post-ischemic increase in [Ca2+]m
will trigger a large K+ influx through mKCa channels, counteracting the increase in Dwm. The compensatory K+ influx
may prevent excessive matrix contraction, and this may represent an important cardioprotective mechanism.
Excessive shrinkage distorts the architecture of the intermembrane space and leads to disruption of contact sites
between the inner and outer membranes [2,101,216,217]. The
contact sites play a key role in energy coupling with the cyto-
29
sol via creatine kinase [226] and in the uptake of fatty acids
[227–229]. We hypothesize that the K+ influx activated by
preconditioning leads to a higher matrix volume during most
phases of preconditioning. The increase in matrix volume is
followed by an increase in the rate of oxidative phosphorylation, especially b-oxidation of fatty acids [122,143]. Opening of mKCa channels may be particularly important in preventing the sudden buildup of protonic potential immediately
after reperfusion, as illustrated in Fig. 4, which mitigates the
generation of ROS. The price for this cardioprotection is a
mild uncoupling of the mitochondria.
In conclusion, as a result of the collective effort of the scientific community to understand preconditioning, some hypothetical mechanisms emerged by which K+ channels in the
mitochondrial inner membrane might have a cardioprotective effect. Preconditioning may activate PKC and PKA, and
this may increase the open probability of mKCa, mKv and
mKATP channels. Although the complex metabolic changes
occurring during ischemia and reperfusion are incompletely
understood, it appears functionally plausible to have three different K+ channels in the mitochondrial inner membrane that
collectively regulate matrix volume during ischemia and reperfusion: (i) a channel that is activated by depolarization
(Kv1.3), (ii) a channel that is activated by a rise in matrix
Ca2+ (BKCa), and (iii) a channel that is activated by a fall in
cytosolic and/or matrix ATP (the putative mKATP channel).
K+ release from the matrix via the K+/H+ antiporter is also
controlled in a very complex way [101]. Thus, regulated
uptake of K+ and release from the matrix may allow the fine
tuning of matrix volume, without requiring too much energy
expenditure.
6. Metabolic regulation of surface KATP channels
in relation to preconditioning
6.1. History of the role of sKATP channels in
preconditioning
In initial studies of preconditioning, sKATP channels were
assumed to be the major effectors of preconditioning
[31,106,230]. The shortening of the action potential and the
resulting reduction in Ca2+ influx and energy expenditure were
thought to readjust the balance between energy supply and
energy expenditure and thus to protect the heart from ischemic
damage [231]. This notion was questioned when it was shown
that low concentrations of K+ channel openers that did not
shorten the action potential were still able to induce cardioprotection [232–234]. Later experiments using flavoprotein
fluorescence and light scattering of mitochondria led to the
suggestion that mKATP channels play a more central role in
cardioprotection [95,100,104,235]. However, recent findings
brought about some kind of renaissance regarding the role of
sKATP channels as mediators of cardioprotection [120,236–
241]. Since sKATP channels are now known to be extremely
complex metabolic sensors, it is often difficult to distinguish
30
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
metabolic effects on sKATP from metabolic effects on mKATP.
Thus, a thorough understanding of the factors that control
opening of sKATP channels in vivo and an analysis of the pharmacology of sKATP channels under various experimental conditions is a prerequisite for the identification and characterization of the putative mKATP channels. In the following brief
summary of the metabolic regulation of sKATP channels we
emphasize those aspects that may be relevant for our understanding of preconditioning and thus potentially overlap with
the metabolic control of mKATP channels.
thought to form vascular sKATP channels [261–265], which
play an important role in the metabolic regulation of blood
flow. In smooth muscle cells the activation of sKATP channels
leads to hyperpolarization and subsequent vasodilatation. In
microvascular endothelial cells, increased sKATP channel
activity may promote the synthesis of NO by increasing transmembrane Ca2+ influx [264].
6.2. Structure and functional roles of sKATP channels
The defining property of sKATP channels is their inhibition by intracellular ATP. However, it is now clear that sKATP
channels are regulated by a host of different mechanisms and
that the other mechanisms may in fact be of greater functional importance than regulation by ATP. Even after more
than 20 years of research on the function of sKATP channels,
it is not quite clear which factors control the open probability
of sKATP channels in vivo. In excised membrane patches from
cardiomyocytes the ATP sensitivity is very high. The ATP
concentration required for half-maximal inhibition (IC50) is
in the range 10–50 µM, whereas intracellular ATP does not
fall below millimolar levels except under very extreme conditions. If the IC50 value observed in inside-out patches of
sKATP channels would also apply for intact cardiomyocytes,
one would not normally expect these channels to open at all
in vivo. This apparent discrepancy was resolved when it was
recognized that the ATP sensitivity of sKATP channels can be
decreased by ADP, membrane-associated phosphatidylinositol phosphates, acyl-CoA esters and many other factors, which
brings the IC50 of sKATP channels much closer to the physiological and pathophysiological range of intracellular ATP.
The most important nucleotide for modulation of sKATP
channels in vivo is probably ADP [266]. SUR1 and SUR2 possess two nucleotide-binding domains (NBDs), which cooperatively regulate the kinetics of sKATP channel opening [267–
269]. Binding of MgADP or MgGDP at NBD2, which is
facilitated by binding of ATP at NBD1, promotes opening of
sKATP channels in the presence of otherwise inhibitory concentrations of ATP [270]. Hydrolysis of ATP at NBD2 additionally regulates the gating of sKATP channels by inducing
conformational changes [269]. Thus, the kinetics of ATP
hydrolysis by SUR subunits and the local cytosolic ADP concentration are important factors in determining the function
of sKATP channels [271].
Phosphatidylinositol phosphates are not only components
of the cell membrane but also important second messengers
[272]. PIP2 activates sKATP channels, probably by binding to
a domain at the C-terminus that is in close proximity to or
overlaps with the ATP-binding domain [273–277]. The competition between the two ligands results in a shift of the IC50
for ATP binding, adding to the shift induced by intracellular
ADP. Since the concentration of PIP2 at the inner leaflet of
the cell membrane is modulated by G protein-coupled receptors, for example, via activation of PLC or PI3K, the regulation of sKATP channel activity may play an important role in
The octameric structure of sKATP channels, consisting of
four pore-forming a-subunits (Kir6.1 or Kir6.2) and four regulatory b-subunits (SUR1 or SUR2), is well known [242–
244]. The a-subunits are members of the inward rectifier K+
channel (Kir) family, whereas the b-subunits are members of
the superfamily of ATP-binding cassette (ABC) proteins. The
a-subunits determine the permeation properties of the K+selective pore and contain the ATP-binding sites, whereas the
interaction between a- and b-subunits regulates the ATP sensitivity of sKATP channels. Heteromeric sKATP channels containing both Kir6.1 and Kir6.2 subunits can form functional
KATP channels in expression systems [245], but there are conflicting data concerning the possibility that such heteromultimers exist in vivo [245–247]. However, Yoshida et al. [248]
have now shown that Kir6.1, Kir6.2 and SUR2B can form
heteromultimeric channels in coronary endothelial cells. The
ability of Kir6.x subunits to form heteromeric channels
implies that a large number of KATP channel proteins with
different functional characteristics may exist, as is the case
for Kir2.x channels [249]. The number of possible sKATP
channels is further increased by alternative splicing of the
b-subunits. SUR2A and SUR2B, the main splice variants of
the SUR2 gene, show different pharmacological profiles. Various other splice variants of SUR1 and SUR2 have been found,
some of which form functional channels when co-expressed
with Kir6.x subunits [173,250,251]. Little is known about the
pharmacology of these splice variants.
The primary function of sKATP channels is to link cellular
metabolism and membrane excitability [252]. Cardiac sKATP
channels probably consist of Kir6.2 and SUR2A [252] and
provide for a decrease in energy expenditure of the heart by
shortening the cardiac action potential and reducing Ca2+
influx through L-type Ca2+ channels [31,104,231,253–255]
as well as through the Na+/Ca2+ exchanger [256]. The reduction of Ca2+ influx is particularly important during the initial
phase of reoxygenation and is thought to prevent Ca2+ overload of the mitochondria [241,256–258]. Opening of sKATP
channels also prevents arrhythmias and sudden death during
vigorous sympathetic stimulation [259].
Pancreatic sKATP channels consist of Kir6.2 and SUR1
[260] and regulate insulin secretion in pancreatic b-cells as
well as the excitability of certain types of neurons in the central nervous system. Complexes of Kir6.1 and SUR2B are
6.3. Regulation of sKATP channels by ATP, ADP
and phosphatidylinositol phosphates
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
signal transduction in cardiac muscle [278]. This may be particularly relevant during ischemia, which leads to activation
of PLC by various agonists.
6.4. Regulation of sKATP channels by metabolic
intermediates
The local submembrane concentrations of ATP may differ
substantially from the ‘bulk concentration’ of ATP in the cytosol [279–281], despite the fact that under physiological conditions the submembrane ATP and ADP concentrations are
buffered by the phosphotransfer enzymes creatine kinase and
adenylate kinase [18,282]. Inhibition of the sarcolemmal
Na+,K+-ATPase can lead to closure of sKATP channels by
increasing the submembrane concentration of ATP [281,283].
Conversely, the strong activation of the Na+,K+-ATPase during reperfusion can lead to the opening of sKATP channels by
depleting submembrane ATP [41].
Recent studies indicate that, in cardiomyocytes and in other
cell types, sKATP channels, like other cardiac ion channels
[284], may in fact exist as a protein complex [285–287]. The
sKATP channel complex includes creatine kinase, adenylate
kinase and the muscle form of lactate dehydrogenase (MLDH). It was found that the physical association of the sKATP
channel with M-LDH modulates ATP sensitivity [286]. The
sKATP channel is also regulated by the catalytic activity of
glyceraldehyde-3-phosphate dehydrogenase; 1,3-bisphosphoglycerate, an intermediate product of glycolysis, directly regulates the sKATP channel, even in the presence of high cytosolic ATP [288].
Long-chain fatty acids are the most important metabolic
substrate of the heart under physiological conditions. The activated form of these fatty acids, long-chain acyl-CoA esters,
have been shown to decrease the ATP sensitivity of sKATP
channels in cardiomyocytes with a similar potency as PIP2
[289], and it has been suggested that these soluble cytosolic
molecules interact with sKATP channels by the same mechanism as PIP2 [277]. Furthermore, medium- and short-chain
acyl-CoA esters have also been shown to reduce the ATP sensitivity of sKATP channels [290].
Intracellular acidification also decreases the sensitivity of
sKATP channels to ATP [291–295]. The stimulatory effect of
intracellular acidification on the open probability of sKATP
channels is particularly large at low cytosolic ATP concentrations, which facilitates channel opening during metabolic inhibition [295]. The ATP sensitivity of sKATP channels can also
be decreased directly by intracellular lactate [296]. Thus, PIP2,
acyl-CoA esters, intracellular protons and lactate, all of which
are increased during ischemia, relieve the inhibition of KATP
channels and thus may have a cardioprotective effect.
31
several studies it was found that application of HMR1098 at
concentrations that can inhibit sKATP channels did not significantly attenuate IPC or PPC [38,298,299]. However, some
forms of IPC and PPC were sensitive to HMR1098
[171,180,184,300,301].
The K+ channel opener P1075, a pinacidil analogue, binds
with high affinity to SUR2A and to SUR2B , but not to SUR1,
in the presence of MgATP [302–305]. This drug has been
proposed to be a selective opener of sKATP channels and has
been used as a tool to clarify the relative contributions of
sKATP and mKATP channels to preconditioning [121,235,
238,241,256]. Baczko et al. [121] showed in a cellular model
of cardioprotection that addition of P1075 to the reoxygenation solution reduced the contractile dysfunction and Ca2+
overload in rat ventricular myocytes after metabolic inhibition. This effect was blocked by HMR1098 and did not occur
in myocytes expressing a dominant-negative Kir6.2 construct, indicating that it was indeed related to opening of sKATP
channels. Similar cardioprotective effects were observed in
quiescent cardiomyocytes [241,256]. The authors propose that
improved Ca2+ homeostasis, due to reduction of reverse
Na+/Ca2+ exchange, is responsible for the beneficial effect of
sKATP channel activation. It should be noted, however, that
some researchers questioned the selectivity of P1075 and proposed that the drug also opens mKATP channels [181,306–
308].
Diazoxide is generally considered not to activate cardiac
sKATP channels under normal conditions [232–234]. However, recent experiments on isolated cardiomyocytes exposed
to metabolic inhibition have shown that pretreatment with diazoxide led to earlier opening of sKATP channels, earlier failure of action potentials and earlier contractile failure, which
suggests that diazoxide can indeed enhance the open probability of sKATP channels during metabolic inhibition [239].
The opening of sKATP channels induced by diazoxide is not
necessarily due to a direct effect on the channel but may be
partially attributable to uncoupling of mitochondria [164] or
inhibition of succinate dehydrogenase [147,155,159,161].
Furthermore, D’Hahan et al. [190] found that the sensitivity
of cardiac sKATP channels to diazoxide increased in the presence of MgADP and during simulated ischemia. Thus, especially during ischemia, where mKATP channel are assumed to
be most important, diazoxide may not be selective enough to
serve as a useful tool for identifying mKATP channels.
7. Do surface KATP channels play a role
in preconditioning?
7.1. Changes in action potential duration during metabolic
inhibition
6.5. Blockers and openers and of sKATP channels
KATP channel blockers selective for sKATP channels, such
as the sulfonylthiourea HMR1098 [297], have been used as
tools to explore the role these channel in preconditioning. In
Opening of sKATP channels is expected to be cardioprotective by shortening the action potential and reducing Ca2+
influx during ischemia and reperfusion (see Sections 6.1 and
6.2). Gross and Auchampach [31] showed that the protection
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P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
in dog heart induced by IPC could be blocked by glibenclamide and mimicked by the K+ channel opener aprikalim.
Similarly, experiments in guinea-pig ventricular muscle
showed that application of glibenclamide inhibited shortening of the action potential during ischemia and impaired recovery of ventricular function, whereas the opposite effects were
obtained with the K+ channel opener pinacidil [309]. In swine
heart, IPC also induced shortening of the action potential during the index ischemia and this was associated with cardioprotection [310]. Both effects were not observed after application of glibenclamide.
Using cell-attached patch-clamp recording in guinea-pig
cardiomyocytes, Zhu et al. [114] showed that anoxic preconditioning shortened the time interval between metabolic inhibition and opening of sKATP channels. The effect was associated with decreased ATP sensitivity of sKATP channels and
could be abrogated by blocking PKC. Thus, phosphorylation
of the channel protein by PKC during hypoxia may modulate
its open probability during subsequent metabolic inhibition.
As mentioned in Section 6.5, Rodrigo et al. [239] found that
diazoxide improved functional recovery of rat cardiomyocytes subjected to metabolic inhibition. The improved recovery was associated with earlier activation of sKATP channels
during metabolic inhibition and improved recovery of contractile function after removal of metabolic inhibition. Extending this work, Rainbow et al. [240] have used isolated rat
cardiomyocytes transfected with a C-terminal fragment of
SUR2A (residues 1294–1358). After 2 days in culture, the
SUR2A fragment had reduced sarcolemmal KATP currents to
15% of control. In transfected cells, action potential failure
during metabolic inhibition was delayed, Ca2+ loading of the
cells was increased and the cardioprotective effect of PPC
with DNP was abolished. These findings are consistent with
a major role of sKATP channels in PPC in the rat.
7.2. Evidence from experiments with transgenic mice
The possible involvement of sKATP channels in cardioprotection was also tested with Kir6.2 knockout mice. It was
found that knockout of Kir6.2 abolished the protective effects
of IPC [171,311,312]. The recovery of contractile function
of isolated perfused hearts from Kir6.2 knockout mice was
significantly impaired in comparison to control animals. Interestingly, the cardioprotective effect of pharmacological preconditioning with diazoxide was also absent in Kir6.2 knockout mice [300]. In wild-type mice, PPC with diazoxide and
IPC could be prevented by blocking sKATP channels with the
surface-selective KATP channel blocker HMR1098, but the
putative mK ATP channel blocker 5-HD was ineffective
[171,300].
Gumina et al. [312] studied the adaptation of energy
metabolism to IPC in wild-type and in Kir6.2 knockout mice.
They found that in wild-type mice the rates of ATP synthesis
and ATP hydrolysis were better preserved after preconditioning, resulting in improved ionic homeostasis and contractility, confirming earlier reports [104,313,314]. The beneficial
effects of preconditioning on energy metabolism were absent
in the Kir6.2 knockout mice [312]. These findings suggest
that that the sensing of metabolic deficits and the adaptation
of electrical and mechanical activity to the overall metabolic
situation, which is normally provided by sKATP channels, is
an essential component of preconditioning. Taken together,
the experiments with Kir6.2 knockout mice provide strong
evidence for involvement of sKATP channels in IPC and PPC,
at least in the mouse [315–317]. Since the transfer of energetic signals between different intracellular compartments,
to which sKATP channels contribute [18], is probably similar
in all mammals, it is likely that the sKATP channels contribute
to stress tolerance also in the heart of larger mammals, including humans [271]. It should be mentioned, however, that some
studies came to the conclusion that the sKATP channels do
not contribute to preconditioning in larger mammals. This
view was mainly based on the observation that the sKATPselective K+ channel blocker HMR1098 was ineffective at
inhibiting IPC or PPC with larger mammals [38,166,298,299].
However, in these studies the inhibitor was not present during index ischemia and reperfusion, when sKATP channel
activity most likely effects cardioprotection.
Kir6.1 channels are also expressed in cardiac muscle
[264,318]. However, the sKATP channel activated by pinacidil in mouse ventricular myocytes does not seem to be affected
by deletion of the gene coding for Kir6.1 channels [170]. Thus,
Kir6.1 channels are unlikely to be a constituent of sKATP channels. In addition, the channel is unlikely to be involved in the
formation of mKATP channels, since in Kir6.1 knockout mice
a similar increase in flavoprotein fluorescence was elicited
by diazoxide and inhibited by 5-HD as in wild-type mice
[170]. In contrast, knockout of Kir6.1 abolished
glibenclamide-sensitive KATP channel current in vascular
smooth muscle. In conclusion, the physiological role of
Kir6.1 in cardiac muscle remains unclear [319], but the
Kir6.2 knockout experiments provide strong support for a role
of surface KATP channels in preconditioning.
7.3. Effects of adenosine on sKATP channels
Adenosine is a special mediator of preconditioning because, unlike acetylcholine, bradykinin, opioids and phenylephrine, it affords preconditioning without stimulating the
release of ROS [11,40] and without causing mitochondrial
swelling [122]. Extracellular adenosine accumulates in the
heart during ischemia, inducing cardioprotection [320–322].
One of the most important mediators of adenosinergic cardioprotection is PKC [52]. Activation of PKC in cardiomyocytes increases the open probability of sKATP channels, most
likely by phosphorylating Kir6.2 at threonine 180 [257,323–
327]. Thus, opening of sKATP channels may contribute both
to PPC and to IPC [257,322,328].
It has now become clear that the surface expression of
receptors, channels and transporters is subject to regulation
of trafficking, although in many cases the signal transduction
pathways modulating biosynthetic forward trafficking or
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
endocytosis are still unclear [329]. In a pioneering study, Hu
et al. [330] have shown that incubation of cardiomyocytes
with adenosine for 30 min downregulates the surface expression of sKATP channels in a PKC-dependent manner. This
effect was due to enhanced endocytosis mediated by PKC,
and it may serve to limit the activating effect of adenosine on
sKATP currents [330]. Modulation of the number of functional KATP channels at the surface of cardiomyocytes via
PKC or via other mechanisms [120] may also play a role in
preconditioning, although it would antagonize the early effects
of PKC on the open probability of sKATP channels and would
increase action potential duration.
7.4. Effects of preconditioning on surface expression
of sKATP channels
Immunoprecipitation, Western blot and immunofluorescence experiments with guinea-pig cardiomyocytes showed
that preconditioning with a 5 min episode of hypoxia induced
recruitment of additional sKATP channels to the sarcolemma
[120]. At the beginning of the sustained (index) hypoxia the
number of functional sKATP channels at the cell surface was
increased by a factor of ~6 as compared to control cardiomyocytes. The authors suggested that the cardioprotective effect
observed after the 5 min episode of hypoxia was due to opening of sKATP channels at the beginning of the sustained
hypoxia. Cardioprotection was abrogated in the presence of
glibenclamide or 5-HD, as well as in the presence of the selective sKATP channel blocker HMR1098. It may be concluded
that, at least in the model of reperfusion damage after a brief
conditioning period of hypoxia, sKATP channels may play a
major role in extending the period that cardiomyocytes can
survive during hypoxia in the guinea pig.
7.5. The contribution of other channels to K+ efflux during
ischemia
The massive K+ efflux from cardiomyocytes observed during ischemia or hypoxia [331,332] cannot be explained on
the basis of KATP channel opening alone but requires an equal
flow of a counter-ion [333–335]. One possibility is that K+
efflux is at least partially balanced by Na+ influx [334,336].
Another possibility is that the K+ efflux observed during myocardial ischemia is accompanied by a substantial efflux of
anions through volume-activated Cl– channels [337]. An interesting hypothesis concerning the role of K+ channels in preconditioning has recently been put forward by Diaz et al.
[338]. They suggested that the beneficial effect of K+ channel opening may be related to the fact that regulatory volume
decrease mediated by Cl– efflux requires concomitant K+
efflux to be effective. They found that blockade of Cl– channels impaired both IPC and PPC [339,340], and they postulated that this was due to inhibition of regulatory volume
decrease. Furthermore, they found that IPC diminished cell
swelling induced by ischemia and proposed that improved
cell volume regulation plays a significant role in the protec-
33
tion afforded by IPC. In agreement with this hypothesis, they
observed that inhibition of inward rectifier K+ channels in
cardiomyocytes abolished cardioprotection induced by IPC
[338]. Furthermore, genetic or pharmacological inactivation
of Cl– channels also prevented ischemic preconditioning
[341]. Thus, it appears possible that one of the functions of
sKATP channel opening is to facilitate volume-activated Cl–
efflux.
In conclusion, there is no doubt that sKATP channels make
a substantial contribution to preconditioning, in agreement
with the original ideas put forward by Gross and Auchampach [31]. The most convincing pieces of evidence come from
the results obtained with Kir6.2 knockout mice [171,300,312]
and from the recent studies on the regulation of surface expression of sKATP channels in guinea-pigs by IPC [120]. The role
of sKATP channels can be explained by the fact that these channels are integrative sensors of metabolic intermediates and
adapt the electrical activity to the energy status of the cardiomyocytes. This adaptive response is particularly important
under conditions of stress, such as hypoxia and reperfusion,
but it may also play a role in human disease [271]. Enhanced
cell volume regulation during index ischemia, mediated by
the opening of sKATP channels and volume-activated Cl– channels, may be one of the key downstream mechanisms for cardioprotection through preconditioning [340,341].
8. Alternative mechanisms of preconditioning
It is now clear that several mechanisms cooperate to induce
cardioprotection. The most important mechanisms, apart from
the opening of K+ channels, are generation of ROS, changes
in fatty acid metabolism and opening of the mitochondrial
permeability transition pore (MPTP). The different mechanisms are most probably all interdependent. For clarity, we
discuss the three alternative mechanisms separately (Sections 8.1–8.3) and then try to present an integrated picture of
the mechanisms underlying preconditioning (Section 8.4).
8.1. The role of ROS production in preconditioning
Reactive oxygen species (ROS) have been incorporated in
most conceptual schemes of preconditioning. During normal
respiration, approximately 2–4% of electron flow through the
respiratory chain results in only partial reduction of O2, generating superoxide anions [342,343], most likely at complexes I and III [226,344]. Increased ROS production takes
place during ischemia [345–348], causing damage to the electron transport chain [348–350]. During reperfusion there is a
burst of ROS production that may inflict massive cardiac damage [224,226,351]. The loss of function of the respiratory
chain and the production of ROS progress with increasing
duration of the ischemia. As a result, recovery of mitochondrial function during reperfusion, a prerequisite of cell survival, critically depends on the duration of the preceding
ischemia.
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P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
ROS play both a “good” and a “bad” role in relation to
preconditioning [2–4,352,353]. On one hand, preconditioning is thought to reduce the massive ROS production which
accompanies reperfusion after index ischemia. Thus, inhibition of ROS production may represent an end effector of IPC
and PPC. On the other hand, low concentrations of ROS are
well-recognized intracellular messengers [351,354–357], and
have been proposed to represent a trigger for IPC, and even
PPC, by switching on cardioprotective mechanisms. However, exactly when, where and how ROS increase under physiological and pathophysiological conditions is still a controversial issue. This is mainly due to, first, the inherent
limitations of the fluorescent dyes used to monitor ROS in
intact cells and, second, the potentially misleading results of
experiments with isolated mitochondria, submitochondrial
particles or purified enzyme systems [358], in which ROS
can be more directly measured.
A high electrochemical proton gradient across the mitochondrial inner membrane has been reported to favor the generation of ROS [359–363]. Based on these observations, it
has been hypothesized that ROS production can be decreased
by uncoupling and subsequent depolarization of the mitochondrial inner membrane [363,364]. Uncoupling can be provided by uncoupling proteins or long-chain fatty acids
[168,365,366], and this may represent a protective mechanism preventing excessive production of ROS. Many of the
commonly used mKATP channel openers are highly lipophilic and capable of transferring protons across the mitochondrial inner membrane [145,159,164]. Specifically, diazoxide and pinacidil have been shown to promote an H+selective current across lipid bilayers, and both agents
increased state-4 respiration in isolated heart mitochondria
to a similar extent when K+ was substituted with Na+ [164].
Interestingly, the classic uncoupler DNP, similar to diazoxide and pinacidil, also protected hearts from ischemia,
assessed by measuring mechanical recovery after 45 min of
global ischemia [164,165]. These data suggest that protonophoric uncoupling itself can precondition the heart, and that
diazoxide could in principle limit ROS production by its
uncoupling action during reperfusion after index ischemia or
hypoxia [212,224,367,368]. Consistent with this idea, diazoxide has been shown to prevent fatty acid induced, ROSmediated loss of cyctochrome c from isolated mitochondria
[168]. However, the molecular mechanism by which enhanced
ROS production might trigger cyctochrome c loss is still under
debate [40,122,168,350,369,370].
As mentioned above, ROS generated during IPC appear to
represent an important upstream mechanism that is required
for the cardioprotective effect of the trigger ischemia
[56,102,371]. One of the principal downstream effectors of
ROS is PKCe [58,62,63,66,109,211,212,372,373], which may
be translocated to the mitochondria [68]. Cardioprotection
conferred by IPC, as well as PPC, can be prevented by application of ROS scavengers before the index ischemia
[37,82,84,347] or by the inhibition of PKC [34]. Oldenburg
et al. [102] and others [44,102,374] have proposed that open-
ing of mKATP channels is the primary mechanism, and that
generation of ROS is downstream of mKATP. This hypothesis
was based on the observation that diazoxide [181,375], volatile anesthetics [13,44,87] and acetylcholine can increase ROS
production. In contrast, using malondialdehyde as an indicator of ROS, Dzeja et al. [161] reported that brief exposure to
30 µM diazoxide reduced rather than increased ROS production, whereas an increase in ROS production occurred after
washout of diazoxide. The inferences of Oldenburg et al. [102]
were based on the assumption that diazoxide and 5-HD act
selectively on mKATP channels, which we no longer share. In
view of the difficulty of measuring ROS in intact cells, we
think it is also possible that generation of ROS is the primary
event during the trigger phase of preconditioning and that
cardioprotection results from subsequent activation of PKCe
and other downstream enzymes.
When one examines the expanding list of drugs that afford
cardioprotection, a common mechanism of action emerges:
diazoxide and 3-nitroproprionic acid inhibit succinate dehydrogenase (complex II), volatile anesthetics and pinacidil
inhibit complex I and, finally, nicorandil can inhibit complex
IV. Most of the cardioprotective drugs inhibit the respiratory
chain to some extent. This might explain why such structurally diverse drugs share the ability to induce cardioprotection. Although the ability of these drugs to block partially the
respiratory chain is not disputed, and may even be dismissed
as a (predictable) toxic side effect [2,144], the possibility that
pharmacological inhibition of the respiratory chain, like
ischemic inhibition, can trigger cardioprotective mechanisms should be further pursued. The mechanism, by which
partial inhibition of the respiratory chain may afford preconditioning has not yet been resolved. It has been proposed,
that inhibition of the respiratory chain at more distal sites
may enhance the basal rate of ROS generation, because
decreased flux through the electron transport chain increases
the reduction of proximal sites, enhancing electron leak and
superoxide generation [161,226,342,376–378]. This mechanism would be consistent with the hypothesis that ROS act as
initial triggers of preconditioning [56].
In conclusion, the double-edged role of ROS in regulating
cell death and cell survival is potentially important for understanding preconditioning. There is good evidence that application ROS scavengers during the trigger phase prevents preconditioning and that excessive production of ROS during
the reperfusion phase causes cell injury. There are indications that a moderate increase in ROS production during the
trigger phase may initiate preconditioning, but the underlying molecular mechanisms are not yet clear. The targets of
ROS during the trigger phase (including PKC, but perhaps
also other kinases) require further investigation. One of the
most plausible downstream targets of ROS is the mitochondrial permeability transition pore (see Sections 8.3 and 8.4).
8.2. The role of fatty acid metabolism in preconditioning
In normoxic cardiac muscle, over 70% of cardiac energy
expenditure is related to b-oxidation of fatty acids [379], and
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
uptake and utilization of fatty acids are finely tuned, presumably to prevent intracellular accumulation of fatty acids, as
these are toxic at high concentrations [380]. During ischemia,
the myocardial content of lipids strongly increases
[379,381,382], glycloysis rates are high and the rates of glucose oxidation are low [383–385]. Thus, during initial
ischemia, before the oxygen supply becomes too low, fatty
acid oxidation dominates even more than during normoxia
[379,386]. The shift away from glucose oxidation to fatty acid
oxidation is mainly due to a decrease in malonyl-CoA concentration [387,388], mediated by the rapid rise in AMP during ischemia and the resulting inhibition of acetyl-CoA carboxylase [386,389]. The decrease in malonyl-CoA promotes
the uptake of acyl-CoA esters into mitochondria via carnitine
palmitoyl transferase I (Fig. 5) or carnitine octanoyltransferase. During prolonged ischemia the paradoxical situation
arises that acyl-CoA esters continue to be delivered to the
mitochondrial matrix in the absence of a sufficient oxygen
supply for b-oxidation [389].
During reperfusion, the levels of malonyl-CoA fall even
further [390,386]. Moreover, reperfusion is associated with
enhanced degradation of membrane phospholipids, probably
mediated by phospholipase A2 [379], which leads to further
accumulation of fatty acids [391]. The combination of fatty
acid accumulation and low malonyl-CoA levels leads to strong
enhancement of fatty acid oxidation at the expense of glucose metabolism, and it has been suggested that the excessive use of fatty acids by the heart during and after ischemia
contributes to ischemic injury [168,386,392,393].
35
Lim et al. [147] have shown that ischemia induces swelling of mitochondria, and that matrix volume is increased even
further after 30 min of reperfusion. In earlier work, Halestrap
had shown that matrix swelling is associated with an enhancement of fatty acid oxidation [143,394,395]. These results were
recently confirmed by Juhaszova et al. [122] who found that
mitochondrial swelling was associated with enhanced substrate oxidation (palmitate ≥ octanoate > glucose) and production of ROS. Thus, matrix swelling is expected to further
accentuate the dominance of fatty acid metabolism during
ischemia and reperfusion.
5-Hydroxydecanoate, a substituted fatty acid, has also been
shown to be metabolized by b-oxidation [147,155–157]. Since
5-HD has been widely used as an inhibitor of preconditioning it may be useful to pursue the question to what extent the
effects of 5-HD may be related to metabolism of the drug
(Fig. 5). For example, it may be speculated that the inhibitory
effect of 5-HD on PPC may be related to its b-oxidation,
which could act to bypass partial inhibition at either complex
I or both complex II [155,156]. This mechanism may be relevant in experimental models in which the PPC is performed
in the absence of metabolic substrates.
Since 5-HD has been shown to be activated to 5-HD-CoA
[147,155] and metabolized by the b-oxidation pathway [157],
it may interfere with the increase in fatty acid oxidation
observed during reperfusion. Recent experiments suggest that
5-HD is metabolized much less efficiently than decanoate,
and that metabolic intermediates of 5-HD can indeed inhibit
fatty acid oxidation [157]. Thus, metabolism of 5-HD could
undermine the beneficial consequences of ischemic or phar-
Fig. 5. Schematic diagram of the mechanisms thought to be involved in IPC and PPC in the light of recent results. The possible targets for diazoxide (orange),
5-HD (red) and glibenclamide (green) are indicated as dotted lines. Abbreviations: ACC, acetyl-CoA carboxylase; MCD, malonyl-CoA dehydrogenase; GSK3b, glycogen synthase kinase-3b; MPTP, mitochondrial permeability transition pore; CPTI and II, carnitine palmitoyl transferase I and II; PI3K, phosphoinositide3-kinase; PDK, phosphoinositide-dependent kinase; MAPK, mitogen activated protein kinase.
36
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
macological preconditioning. Interestingly, Juhaszova et al.
[122] found that diazoxide caused an increase in substrate
oxidation (associated with matrix swelling) and induced cardioprotection, and that application of 5-HD or the partial fatty
acid oxidation inhibitor trimetazidine prevented these effects.
The inhibitory effect of 5-HD was most pronounced when
palmitate or octanoate were used as substrates. Thus, in view
of the partial metabolism of 5-HD via b-oxidation [156,396]
an obvious possibility is that 5-HD does not act by blocking
the putative mKATP channel but that one of its metabolites,
most likely L3,D5-dihydroxydecanoyl-CoA, provides a
bottleneck for b-oxidation [147,155,157]. There is another
metabolic effect that cytosolic 5-HD-CoA might share with
endogenous acyl-CoA esters: inhibition of the ANT [379],
which would reduce the rate of oxidative phosphorylation.
Lim et al. [147] observed that application of 5-HD increased
mitochondrial matrix volume and suggested that this might
have been mediated by inhibition of the ANT by the CoA
ester of 5-HD [147].
Glibenclamide, another presumed inhibitor of mKATP channels, has been shown to reduce the rate of fatty acid oxidation
by inhibiting carnitine palmitoyl transferases [197–199]. At
a free concentration of 5 µM, glibenclamide inhibited
CPTI/CPTII by about 50%. Hence, both glibenclamide and
5-HD may share the common property of inhibiting fatty acid
metabolism. Could such an action block preconditioning? This
may indeed be the case, since aside from the study of Juhaszova et al. [122] mentioned above, the b-oxidation inhibitor
trimetazidine has been shown to abolish both IPC and PPC,
indexed by measuring infarct size relative to area at risk in rat
hearts [165]. It may be speculated, therefore, that the substantial changes in fatty acid metabolism induced by ischemia, or
even by 5-HD and glibenclamide, may play a pivotal role in
preconditioning.
In conclusion, the ability of mitochondria to cope with the
massive alterations in fatty acid metabolism after ischemia is
a crucial factor for survival of the cardiomyocytes; the excessive use of fatty acids is detrimental to both the ischemic and
the reperfused heart [397,398]. Mitochondrial matrix volume modulates fatty acid metabolism even more strongly than
carbohydrate metabolism [122,143]. The ‘universal’ blocker
of IPC, 5-HD, is a substituted fatty acid that may interfere
with fatty acid metabolism at various points, and this is also
the case for glibenclamide. Therefore it may be worthwhile
to study the modulation of fatty acid metabolism by ischemic
and pharmacological preconditioning in more detail.
8.3. The role of the mitochondrial permeability transition
pore in preconditioning
The mitochondrial permeability transition pore (MPTP) is
a protein complex consisting of the ANT, cyclophilin D, the
voltage-dependent anion channel (VDAC) and other proteins
[140,142,399–403]. It represents a Ca2+-, Mg2+-, voltage-,
pH- and redox-gated channel in the mitochondrial inner membrane [404]. In addition, the MPTP is regulated by adenine
nucleotides [403], similar to KATP channels. The conditions
for opening the MPTP are optimal during reperfusion after
prolonged ischemia [403], and various groups have shown
that block of the MPTP with cyclosporin-A (CsA) or
sanglifehrin-A (SfA) during reperfusion can reduce the number of necrotic cells [223,405–408]. It is now generally
accepted that prolonged opening of the MPTP during the first
few minutes of reperfusion is a critical determinant of myocardial cell injury (reviewed in refs. [141] and [403]).
A brief report by Hausenloy et al. [40] provides some evidence that, in addition, the MPTP may be transiently opened
during the preconditioning phase (before the index ischemia)
and that this may play a role in fostering cell survival. These
authors showed that blockade of the MPTP during the preconditioning phase with either CsA or SfA abrogated (i) IPC
in isolated rat heart, (ii) PPC with diazoxide, (iii) PPC with
the uncoupler DNP, and (iv) PPC with the selective adenosine A1-receptor agonist CCPA. These data suggest that all of
these variants of preconditioning are in fact related to transient opening of the MPTP. In the presence of a ROS scavenger the protection afforded by all variants of preconditioning
was abolished, except for the protection induced by CCPA
[166], in agreement with an earlier study by Cohen et al. [11].
In addition, Hausenloy et al. showed that diazoxide caused
loss of the fluorescent probe calcein from mitochondria, which
they interpreted to indicate transient opening of the MPTP
[40,409,410] because calcein can only cross the mitochondrial inner membrane when the pore opens [369,411,412].
Moreover, the diazoxide-induced reduction of calcein fluorescence was abolished in the presence of CsA or 5-HD [40].
The authors postulate that mitochondrial uncoupling or an
increase in matrix pH can induce transient opening of the
MPTP, which then promotes the release of ROS [40]. The
exact order in which these events occur is not yet clear since,
in the converse situation, ROS have been shown to facilitate
the opening of the MPTP [142,413], possibly resulting in a
positive feedback [369]. The findings of Hausenloy et al. [40]
extend previous work showing that transient MPTP opening
mediates depolarization and Ca2+ efflux from mitochondria
[404], and possibly induces mitochondrial ROS release [369].
Phosphoinositide-3-kinase (PI3K) also appears to be
involved in cardioprotection [77–80,414–416]; the downstream mediators of PI3K are PKB/Akt and glycogen synthase kinase-3b (GSK-3b) [78]. Recently, GSK-3b has come
into focus as an potential mediator of preconditioning
[78,416,417]. The non-phosphorylated form of GSK-3b is
active in unstimulated cells, where it phosphorylates several
targets (in addition to glycogen synthase), usually resulting
in inhibition of these targets [418,419]. GSK-3b can be phosphorylated by various kinases, including PKB/Akt, which
inhibits its activity (often stimulating downstream targets).
Interestingly, GSK-3b is also localized in mitochondria,
together with PKB/Akt [420]. A recent study by Juhaszova et
al. [122] on the role of GSK-3b in preconditioning provides
some insights into the mechanisms underlying preconditioning. Analyzing changes in mitochondrial permeability transition (MPT), Dwm and mitochondrial swelling, they found
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
that various preconditioning stimuli converge upon GSK-3b
[122]. They propose that phosphorylation of GSK-3b leads
to an increase in the threshold for MPT activation by ROS
during reperfusion, which may account for cardioprotection
(Fig. 5). In essence, the authors proposed that multiple protective pathways, including PKA, PKB/Akt and PKC, converge on GSK-3b and thereby induce inhibition of the MPTP,
a key mediator of cell death.
Based on Fourier analysis of line-scan images, Juhaszova
et al. [122] report that exposure to a variety of cardioprotective agents, including diazoxide, was associated with mitochondrial matrix swelling and that this swelling could be
reversed or prevented by the Cl– channel blocker IAA94. They
also found that mitochondrial swelling was associated with
enhanced substrate oxidation (measured as oxygen consumption) and production of ROS (measured with a fluorescent
indicator) [122]. The increased ROS production was assumed
to induce translocation of PKC to mitochondria [58,66,68],
followed by inhibition of GSK-3b [122]. GSK-3b was found
to be localized in mitochondria, and experiments with transgenic mice as well as gene silencing with siRNA showed that
GSK-3b but not GSK-3a was involved in cardioprotection.
The most significant finding of this study is the characterization of the shift in the threshold for ROS activation of the
MPTP that can be elicited by various forms of preconditioning [122]. The conclusions of Juhaszova et al. are consistent
with the observation that IPC and a variety of cardioprotective drugs increase the delay between ischemic insult and
myocardial damage [223,405,421,422] or ROS-induced loss
of Dw [166].
Multiple studies have shown that activation of PKCe and
translocation of the enzyme to mitochondria is critical for
preconditioning [54,62,372]. Using co-immunoprecipitation
from mouse cardiac mitochondria, Baines et al. [68] have now
shown that PKCe can interact with and phosphorylate the
VDAC, a component of the MPTP. Furthermore, using measurements of mitochondrial swelling and transgenic mice
overexpressing PKCe, they obtained indirect evidence that
phosphorylation via PKCe inhibits opening of the MPTP.
Thus, the MPTP may be a downstream target of a variety of
cardioprotective stimuli that inhibit GSK-3b or stimulate
PKCe.
In conclusion, the experimental data summarized in this
section suggest that the MPTP is a crucial element of the
endogenous cardioprotective program. Indeed, the MPTP may
be one of the elusive “end effectors” of PPC and IPC. Whether
transient opening of the MPTP is, in addition, a “trigger” for
ischemic preconditioning remains to be elucidated.
8.4. Bringing it all together
As we have shown in the preceding sections, several
mechanisms contribute to the endogenous cardioprotective
program: (i) It is likely that K+ channels in the mitochondrial
inner membrane, perhaps including mKATP, contribute to preconditioning (see Section 5). (ii) There is no doubt that sKATP
37
channels contribute to preconditioning (see Section 7). (iii)
Since IPC and PPC are prevented by ROS scavengers, the
generation of ROS appears to be essential for preconditioning. (iv) The ability of mitochondria to cope with the massive
alterations in fatty acid metabolism after ischemia is certainly also a crucial factor for the survival of cardiomyocytes
(see Section 8.2). (v) The MPTP probably plays a decisive
role in mediating myocardial injury during reperfusion (Section 8.3).
How can these mechanisms be integrated into a coordinated response of the cell to ischemia and reperfusion, or
metabolic stress in general? A plausible hypothesis for the
mechanism of action of ROS is that they activate PKC
[109,211,212], and possibly other kinases, which then leads
to an increased open probability of K+ channels in the mitochondrial inner membrane lasting for 1 h or more (see Section 5.2). The opening of the K+ channels is cardioprotective,
because, in essence, it produces a mild uncoupling that mitigates hyperpolarization and ROS release during a subsequent sequence of ischemia and reperfusion. Put in a very
simple way, the generation of ROS in cardiomyocytes activates an adaptive response that diminishes ROS generation
in the next few hours. In that sense, IPC confers a ‘memory’
that could be encoded, for example, as matrix volume [122]
or as the phosphorylated state of matrix K+ channels.
Since opening of the MPTP is such a critical factor for
reperfusion damage, it is reasonable to ask how it may be
connected to the other mechanisms. Experiments using inhibitors of the MPTP suggested that reduced opening of the MPTP
may indeed represent one of the major factors in preconditioning [24,166,167,223,403]. Inhibition of GSK-3b is a hypothetical upstream event leading to an increase in the threshold for MPTP opening [122]. But there are other plausible
mechanisms for decreasing the open probability of the MPTP.
As outlined in Section 5.2, opening of K+ channels in the
mitochondrial inner membrane is expected to reduce Ca2+
accumulation as well as ROS production during the reperfusion phase, two important factors mediating MPT. Furthermore, there is some experimental evidence that both activation of the mKATP channel and activation of the BKCa channel
confer cardioprotection by inhibiting the opening of the MPTP
during reperfusion [24,207]. Thus, it appears possible that
the MPTP is a downstream target of mitochondrial K+ channel opening. This would bring together the three important
factors involved in preconditioning, K+ channels, ROS and
the MPTP, in one coordinated adaptive response of the heart
to transient ischemia or hypoxia. Furthermore, PKCe (activated by ROS during the trigger phase) has been reported to
physically interact with VDAC1, a component of the MPTP,
and it was shown that PKCe-mediated inhibition of the MPTP
contributes to cardioprotection [68]. These findings create
another link between preconditioning and downstream signaling to the MPTP. Considering the massive alterations in
fatty acid metabolism after ischemia and the universal inhibitory effects of 5-HD, a fourth factor, adaptive changes in fatty
acid metabolism, may also be involved in the complex
38
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
sequence of events leading to cardioprotection. In this context it is interesting to note that acetyl-CoA carboxylase (see
Fig. 5) is one of the downstream targets of GSK-3b ([419].
Surprisingly, Halestrap et al. obtained evidence that a relatively short period of ischemia is sufficient to produce opening of the MPTP [406,421,423], and they concluded that the
MPTP was able to close again after reoxygenation [223].
Hausenloy et al. [40] showed that the beneficial effects of
IPC were abolished when either a MPTP blocker or a ROS
scavenger was present during the trigger phase. They hypothesized that it was in fact the transient opening of the MPTP
during the triggering phase and the concomitant release of
ROS that induced the cardioprotective effect. They speculate
that the preconditioning stimulus may induce transient MPTP
opening by mediating uncoupling.
Javadov et al. [223] measured opening of the MPTP with
the [3H]DOG entrapment technique [403] and concluded from
their study that the blockers of MPTP opening were less effective than IPC in preventing MPTP opening. This important
conclusion, and also the results of Hausenloy et al. [40] are
consistent with the hypothesis that the primary event may be
release of ROS after the brief trigger ischemia, followed by
activation of PKC. The subsequent adaptive response, which
involves K+ channels, matrix swelling and perhaps also transient opening of the MPTP or other mechanisms, inhibits
MPTP opening after the index ischemia (see Section 5.2).
Thus, assuming that transient opening of the MPTP during
the trigger phase does in fact take place, it remains to be determined whether this opening is elicited by uncoupling [40],
by ROS (Fig. 5) or by some other mechanism.
In conclusion, the results summarized in this review suggest that short periods of ischemia may activate an endogenous cardioprotective program that reduces the cell injury
during subsequent periods of ischemia for several hours, or
even for days, if the second window of protection is considered [3] (which has not been discussed here). Many factors
are involved in this protective program, including activation
of sKATP channels, activation of K+ channels in the mitochondrial inner membrane (possibly including mKATP channels),
changes in fatty acid metabolism, release of ROS and opening the MPTP. Although inhibiting one of these factors may
impair preconditioning, probably only the combination of
these factors improves the resistance of the heart to further
ischemic injury. Furthermore, recent findings suggest that
regulation of cell metabolism and the regulation of cell
survival/apoptosis may be functionally interconnected
[80,416,424–428]. Thus, by modulating the MPTP and various other mitochondrial targets [415,416,420,429,430] the
signal transduction pathways activated during preconditioning may enhance cell survival and antagonize apoptosis.
9. Summary and conclusions
9.1. Do mitochondrial KATP channels exist?
Ischemic or pharmacological preconditioning initiates a
complex signaling cascade which extends the duration of
ischemia that cells can tolerate without activating the cell
death pathway upon reperfusion (Section 2). Mitochondrial
KATP channels have been implicated in most studies of preconditioning, but the evidence for their structure and functional properties, or even their existence, has remained inconclusive. Several approaches have been used to characterize
mKATP channels: (i) Single-channel recording in mitoplasts,
(ii) partial purification of the mKATP channel protein and
reconstitution into liposomes or lipid bilayers, (iii) measurement of light scattering of isolated mitochondria, (iv) measurement of flavoprotein fluorescence, and (v) measurement
of Dwm with fluorescent dyes. None of the approaches, by
itself, provided sufficient evidence to prove the existence of
the channels (Section 3). It might be argued that, collectively,
the studies on the properties of mKATP channels and their
role in preconditioning make their existence more likely. However, the different aspects of the channels studied do not provide a unifying picture, and many of the basic characteristics
of the putative mKATP channels, such as single-channel properties, ion selectivity, polarity of insertion into the membrane, sensitivity to inhibition by ATP, and structural similarities with sKATP channels or the MPTP remain unclear
(Section 3).
The pharmacological approach has been extensively used
to prove the existence of a distinct population of mKATP channels that shares some essential properties with sKATP channels (Section 4). Diazoxide, the most widely used preconditioning drug, is often assumed to activate selectively mKATP
channels, but at preconditioning concentrations it exerts welldocumented other actions, such as inhibition of SDH and protonophoric uncoupling, which independently confer cardioprotection. These actions also independently increase
flavoprotein fluorescence, which has been used as an assay of
mKATP channel activity in isolated myocytes. Thus diazoxide, and other mKATP channel openers (Section 4.1), have
alternative targets, which limits their usefulness in identifying mKATP channels. The substituted fatty acid 5-HD is often
assumed to be a selective mKATP channel inhibitor. However,
5-HD can be activated to 5-HD-CoA and metabolized in mitochondria, and one of its metabolic intermediates can inhibit
fatty acid metabolism. Both 5-HD and the other presumed
blocker of mKATP channels, glibenclamide, have multiple
other actions that invalidate their use as selective blockers to
identify the mechanisms underlying preconditioning (Section 4.2).
In conclusion, the hypothesis of mKATP channels as the
initial trigger or as the end effector of preconditioning (or
both) has been extremely fruitful and stimulated many sophisticated studies, the results of which remain valid. However,
14 years after the first description of mKATP channels [124]
and 8 years after the proposal that mKATP channels may play
a role in preconditioning [94,95], the existence of mKATP
channels is still an open question, and it turned out that it is
nearly impossible to prove the existence of a channel in the
absence of selective activators or blockers, in particular if the
gene coding for the channel is not known. Should we then
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
“throw away the ladder after we have climbed up upon it”
[431]? We do not think so. It is not at all our intention to
argue that K+-selective channels with high sensitivity to ATP
(these are the defining properties of KATP channels) do not
exist in the mitochondrial inner membrane. We just do not
have sufficient experimental evidence either for or against,
and the targets of diazoxide and 5-HD, very efficient modulators of preconditioning, remain unclear.
Several recent studies on preconditioning discuss the role
of the putative mKATP channels and the possible mechanisms
of action of 5-HD and diazoxide as unresolved issues. We are
therefore inclined to think that it may be time to end the
obligatory ‘liaison’between preconditioning and mKATP channels. On one hand, it would be useful to devise further experiments on the mechanisms of cardioprotection that do not rely
on the assumption that diazoxide opens and 5-HD blocks
mKATP channels. On the other hand, more direct evidence,
independent of preconditioning, should be sought to prove or
disprove the existence of mKATP channels. Hopefully, further single-channel recordings in mitoplasts prepared from
isolated cardiomyocytes will provide the answer.
9.2. An integrative view of preconditioning
Studies of ischemic preconditioning have helped to reveal
a general adaptive response of the cells to metabolic stress. A
brief period of ischemia represents a strong metabolic stimulus and elicits a coordinated response that makes the cardiomyocytes more resistant to injury during subsequent periods of ischemia. In fact, the adaptive response to short-term
perturbation is slightly overcompensatory, and the overcompensated state is sustained for more than 1 h, representing the
“memory” of preconditioning. We propose that at least five
factors are involved in the response of the heart to metabolic
stress: (i) opening of surface KATP channels (Sections 6 and
7), (ii) release of reactive oxygen species (Section 8.1), (iii)
opening of K+ channels in the mitochondrial inner membrane (Section 5.2), (iv) changes in fatty acid metabolism
(Section 8.2), and (v) modulation of the mitochondrial permeability transition pore (Section 8.3).
The studies with Kir6.2 knockout mice have clearly shown
that sKATP channels contribute to the cardioprotective effect
of preconditioning. The sKATP channel is a complex metabolic sensor that is integrated in a metabolic network that
regulates energy transfer from mitochondria to the contractile proteins and to the surface membrane. This metabolic network, which includes creatine kinase, adenylate kinase and
several metabolic intermediates, has an important function in
adapting action potential duration, preventing mitochondrial
Ca2+ overload and regulating cellular energy demand. sKATP
channels play only a minor role during normal cardiac function but become very important during disturbances of energetic homeostasis such as hypoxia, ischemia and reperfusion
(Sections 6 and 7).
39
There is general agreement that ROS scavengers applied
during the trigger phase abolish preconditioning, and release
of ROS is a strong candidate for the primary event that elicits
the complex adaptive response of preconditioning (Section
8.1). The main sources of ROS are probably complexes I and
III of the electron transport chain, but the mechanisms that
cause the generation of ROS during the trigger phase are not
yet clear. We hypothesize that ROS are released during reperfusion after the brief initial trigger period(s) of ischemia
and then activate the cascade of events that ultimately leads
to cardioprotection (Sections 5.2 and 8.4).
The mitochondrial inner membrane probably contains at
least two or three K+-selective channels: Ca2+-activated K+
channels (mKCa), voltage-activated K+ channels (mKv) and,
perhaps, ATP-sensitive K+ channels (mKATP). These channels may be activated via PKC, PKA and possibly other
kinases during the trigger phase of preconditioning, during
ischemia and during reperfusion. The opening of these channels is expected to produce mild uncoupling, depolarization
and matrix swelling, depending on the metabolic state of the
mitochondria. The combination of these effects is likely to
reduce the production of ROS, especially during reperfusion,
and this may be one of the major mechanisms responsible for
preconditioning (Section 5.2). Thus, K+ channels in the mitochondrial inner membrane are likely to be mediators of cardioprotection. Considering the massive alterations in fatty acid
metabolism after ischemia and the universal inhibitory effects
of the substituted fatty acid 5-HD, the fourth factor, adaptive
changes in fatty acid metabolism, may also be involved in the
complex sequence of events leading to cardioprotection (Section 8.2).
The mitochondrial permeability transition pore appears to
be one of the key “end effectors” of preconditioning. Many
studies have shown that inhibition of the MPTP during reperfusion confers cardioprotection. Various preconditioning
signals appear to converge on the MPTP and prevent its opening, and initiation of cell death, in response to matrix Ca2+
overload and oxidative stress. Indeed, receptor- or druginduced activation of various signal kinases, such as PKA,
PKCe, PI3K, and survival kinases such as Akt and ERKs, all
implicated in cardioprotection, may effect protection by
preventing/delaying opening of the MPTP. Furthermore, brief
episodes of ischemia or various cardioprotective drugs may
trigger preconditioning by transiently opening, directly or
indirectly via ROS, the MPTP. While the latter hypothesis
requires further testing, it is clear that regulation of the open
probability of the MPTP, the fifth factor, plays a decisive role
in preconditioning (Section 8.3).
Finally, we conclude that preconditioning activates a cellular survival program that requires the integration of several
processes (Section 8.4) including opening of surface KATP
channels, regulation of fatty acid metabolism, ROS production, regulation of the mitochondrial permeability transition
and opening of K+ channels in the mitochondrial inner membrane.
40
P.J. Hanley, J. Daut / Journal of Molecular and Cellular Cardiology 39 (2005) 17–50
Acknowledgements
We thank the Deutsche Forschungsgemeinschaft (grants
Da 177/8-1 and SFB593) and the P.E. Kempkes Stiftung for
financial support. We also thank Heimo Ehmke and Jochen
Roeper for thoughtful comments on the first version of the
manuscript.
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