Selective Pharmacological Agents Implicate Mitochondrial
but Not Sarcolemmal KATP Channels in
Ischemic Cardioprotection
Toshiaki Sato, MD, PhD; Norihito Sasaki, MD, PhD; Jegatheesan Seharaseyon, PhD;
Brian O’Rourke, PhD; Eduardo Marbán, MD, PhD
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Background—Pharmacological evidence has implicated ATP-sensitive K⫹ (KATP) channels as the effectors of cardioprotection, but the relative roles of mitochondrial (mitoKATP) and sarcolemmal (surfaceKATP) channels remain controversial.
Methods and Results—We examined the effects of the KATP channel blocker HMR1098 and the KATP channel opener
P-1075 on surfaceKATP and mitoKATP channels in rabbit ventricular myocytes. HMR1098 (30 ␮mol/L) inhibited the
surfaceKATP current activated by metabolic inhibition, whereas the drug did not blunt diazoxide (100 ␮mol/L)-induced
flavoprotein oxidation, an index of mitoKATP channel activity. P-1075 (30 ␮mol/L) did not increase flavoprotein
oxidation but did elicit a robust surfaceKATP current that was completely inhibited by HMR1098. These results indicate
that HMR1098 selectively inhibits surfaceKATP channels, whereas P-1075 selectively activates surface KATP channels. In
a cellular model of simulated ischemia, the mitoKATP channel opener diazoxide (100 ␮mol/L), but not P-1075, blunted
cellular injury. The cardioprotection afforded by diazoxide or by preconditioning was prevented by the mitoKATP channel
blocker 5-hydroxydecanoate (500 ␮mol/L) but not by the surfaceKATP channel blocker HMR1098 (30 ␮mol/L).
Conclusions—The cellular effects of mitochondria- or surface-selective agents provide further support for the emerging
consensus that mitoKATP channels rather than surfaceKATP channels are the likely effectors of cardioprotection.
(Circulation. 2000;101:2418-2423.)
Key Words: mitochondria 䡲 potassium 䡲 ischemia 䡲 preconditioning
B
A selective opener and a blocker of mitoKATP channels,
namely diazoxide and 5HD, have been identified. A missing
link, however, has been the absence of selective agonists or
antagonists of surfaceKATP channels. In the present study, we
first examined the effects of the KATP channel blocker
HMR109815 and the KATP channel opener P-107516 on KATP
channels in rabbit ventricular myocytes. Our results show that
both HMR1098 and P-1075 target surfaceKATP but not mitoKATP channels. Using these surface-selective agents, we
investigated whether surfaceKATP channels are involved in
cardioprotection.
rief ischemic episodes protect the heart from subsequent
lethal ischemic injury (ischemic preconditioning, IPC).1
Although the precise mechanism of IPC remains elusive,
much attention has focused on the potential role of ATPsensitive K⫹ (KATP) channels as the effectors of protection.
Cardiac myocytes contain 2 distinct KATP channels: the classic
one in the surface membrane (surfaceKATP channel)2 and
another in the mitochondrial inner membrane (mitoKATP
channel).3 Although the cardioprotective effects were initially
attributed to surfaceKATP channels, the effects of surfaceKATP
channels on excitability cannot account for the protection.4 –7
Recent studies provide further evidence that mitoKATP channels rather than surfaceKATP channels are the dominant
players. Diazoxide, a selective mitoKATP channel opener in
cardiac myocytes, is cardioprotective.8,9 The mitoKATP channel blocker sodium 5-hydroxydecanoate (5HD)10,11 can prevent diazoxide-induced cardioprotection8,9 and can block
genuine IPC.12–14 Activation of protein kinase C, which
figures prominently in the signal transduction cascade of IPC,
potentiates mitoKATP channel opening.10 In light of such new
information, the role of surfaceKATP channels in cardioprotection needs to be reevaluated.
Methods
This investigation conformed to the Guide for the Care and Use of
Laboratory Animals published by the US National Institutes of
Health (NIH publication 85-23, revised 1985).
Flavoprotein Fluorescence and Electrophysiology
of Rabbit Ventricular Myocytes
Ventricular myocytes were isolated from rabbits17 and cultured on
coverslips in M199 with 5% FBS at 37°C. Experiments were performed
the next day. Mitochondrial matrix redox state, reported by the fluorescence of FAD-linked enzymes,18,19 was used to index mitoKATP channel
Received September 20, 1999; revision received November 19, 1999; accepted December 6, 1999.
From the Institute of Molecular Cardiobiology, Johns Hopkins University, Baltimore, Md. Dr Sato is now at the Department of Physiology, Oita
Medical University, Oita, Japan.
Correspondence to Eduardo Marbán, MD, PhD, Director, Institute of Molecular Cardiobiology, Johns Hopkins University, Ross 844/720 Rutland Ave,
Baltimore, MD 21205. E-mail [email protected]
© 2000 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
2418
Sato et al
activity.8 Cells were superfused with solution containing (in mmol/L)
NaCl 140, KCl 5, CaCl2 1, MgCl2 1, and HEPES 10 (pH 7.4 with
NaOH) at room temperature (⬇22°C). Endogenous flavoprotein fluorescence was excited with a xenon arc lamp with a bandpass filter
centered at 480 nm. Emitted fluorescence was recorded from 1 cell at a
time at 530 nm by a photomultiplier tube and expressed as a percentage
of the DNP-induced fluorescence. In some experiments, flavoprotein
fluorescence was measured during whole-cell patch-clamp experiments
to administer drugs through the pipette (cf Figure 3).
For whole-cell patch recordings, the internal pipette solution
contained (in mmol/L) potassium glutamate 120, KCl 25, MgCl2 0.5,
K-EGTA 10, HEPES 10, and MgATP 1 (pH 7.2 with KOH).
Currents were elicited every 6 seconds from a holding potential of
⫺80 mV by 2 consecutive steps to ⫺40 mV (for 100 ms) and 0 mV
(for 380 ms). Current amplitude at 0 mV was measured 200 ms into
the pulse to quantify surfaceKATP channel activity. In some experiments (eg, Figure 6), whole-cell currents and flavoprotein fluorescence were recorded simultaneously, and flavoprotein fluorescence
was excited during the 100-ms step to ⫺40 mV.
Functional Expression of KATP Channels
and Electrophysiology
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Details of the functional expression of KATP channels in HEK 293
cells have been described previously.11 Plasmid DNA (3 ␮g total)
containing Kir6.1 or Kir6.2 was cotransfected with either SUR2B or
SUR2A cDNA into HEK cells by use of lipofectamine (Gibco) 18
hours after the cells were split. Mouse Kir6.1, provided by Prof Y.
Kurachi (Osaka University, Japan), and rabbit Kir6.2 (GenBank
AF006262) were cloned into vector pGFP-IRES. Rat SUR2A,
supplied by Prof S. Seino (Chiba University, Japan), was expressed
in the mammalian vector pCMV6. Mouse SUR2B, supplied by Prof
Y. Kurachi, was cloned into the expression vector pCDNA3.
Electrophysiological recordings were made 48 hours after transfection with solutions identical to those used in rabbit ventricular
myocytes (see above). Voltage ramps from ⫺100 to ⫹60 mV were
applied over 100 ms every 6 seconds from a holding potential of ⫺80
mV. The current at 0 mV was measured to assay KATP channel
activity. Experiments were performed at room temperature (⬇22°C).
Simulated Ischemia and Cellular Injury
A cell-pelleting model of ischemia modified from Vander Heide et
al4 was used to quantify myocyte injury. In brief, adult rabbit
ventricular cells were washed with incubation buffer: (in mmol/L)
NaCl 119, NaHCO3 25, KH2PO4 1.2, KCl 4.8, MgSO4 1.2, CaCl2 1,
HEPES 10, glucose 11, and taurine 58.5, supplemented with 1%
BME amino acids and 1% MEM nonessential amino acids (pH 7.4
with NaOH). Aliquots (0.5 mL) of suspended cells were placed into
a microcentrifuge tube and centrifuged for 10 seconds. Approximately 0.25 mL of excess supernatant was removed to leave a thin
fluid layer above the pellet, and 0.2 mL of mineral oil was layered on
the top to prevent gaseous diffusion. After 60 minutes or 120
minutes, 5 ␮L of cell pellet was sampled through the oil layer and
mixed with 75 ␮L of 85 mOsm hypotonic staining solution:
(in mmol/L) NaHCO3 11.9, KH2PO4 0.4, KCl 2.7, MgSO4 0.8, and
CaCl2 1, with 0.5% glutaraldehyde and 0.5% trypan blue. Cells
permeable to trypan blue were counted and expressed as a percentage
of the total cells counted (⬎300 for each sample).
In the control group, cells were pelleted and sampled at 60 or 120
minutes. For the diazoxide-treated or P-1075–treated groups, diazoxide (100 ␮mol/L) or P-1075 (30 ␮mol/L) was added to the solution
15 minutes before the pelleting. Cells treated with diazoxide in the
presence of 500 ␮mol/L 5HD or in the presence of 30 ␮mol/L
HMR1098 were likewise pelleted and sampled at 60 minutes. Once
applied, drugs were not washed out and thus were present throughout
the simulated ischemia.
For the IPC group, the cells pelleted were incubated under oil for
10 minutes and then removed from beneath the oil with a pipette and
resuspended in fresh buffer for 30 minutes. Subsequently, the cells
were pelleted again and subjected to a prolonged period of simulated
ischemia. In the control group, cells were subjected only to the
Mitochondrial KATP Channels in Cardioprotection
2419
Figure 1. Effect of HMR1098 on DNP-activated surfaceKATP current. A, Time course of current measured at 0 mV. Lines indicate
periods when cell was exposed to DNP (100 ␮mol/L) and
HMR1098 (30 ␮mol/L). B, Summarized data from 5 cells. *P⬍0.05
vs current before HMR1098. CONT indicates control group.
prolonged period of simulated ischemia without IPC. For the
5HD-treated and HMR1098-treated groups, either 5HD
(500 ␮mol/L) or HMR1098 (30 ␮mol/L) was added to the incubation
buffer 10 minutes before the IPC. All 4 conditions were tested
simultaneously in each of 6 replications.
The small percentage of cells (⬇18%) that were nonviable at the
beginning of the experiment were mostly rounded and had been
damaged as a consequence of the enzymatic isolation process. The
osmotic fragility of cells induced by ischemia was quantified as
percentage of the vital cells at the beginning of each experiment. In
nonpelleted control cells suspended in oxygenated buffer with or
without drugs, there was no change in the percentage of stained cells
measured after 120 minutes of incubation. Pelleting experiments
were performed at 37°C.
Chemicals
Diazoxide and DNP were purchased from Sigma Chemical Co. 5HD
was purchased from Research Biochemicals International.
HMR1098 was a gift from Hoechst Marion Roussel (now Aventis
Pharmaceuticals), and P-1075 was a gift from Leo Pharmaceutical
Products. Diazoxide and P-1075 were dissolved in DMSO before
being added into the experimental solution. The final concentration
of DMSO was ⬍0.1%.
Statistical Analysis
All data are presented as mean⫾SEM, and the number of cells or
experiments is shown as n. Statistical analysis was performed with
ANOVA combined with the Fisher post hoc test. Values of P⬍0.05
were considered significant.
Results
Effect of HMR1098 on SurfaceKATP and
MitoKATP Channels
We first verified the inhibitory effect of HMR1098 on
surfaceKATP channels by whole-cell patch clamp. Figure 1A
shows surfaceKATP current (IK,ATP) elicited by exposure to
2,4-dinitrophenol (DNP, 100 ␮mol/L). Although DNP eventually increased IK,ATP, subsequent application of 30 ␮mol/L
HMR1098 suppressed IK,ATP. As summarized in Figure 1B,
HMR1098 (30 ␮mol/L) inhibited DNP-induced IK,ATP from
2.22⫾0.89 to 0.52⫾0.14 nA (P⬍0.05, n⫽5).
The effects of HMR1098 on mitoKATP channels were
examined by measuring mitochondrial matrix redox potential.
Figure 2A shows the time course of flavoprotein fluorescence
in a cell exposed twice to diazoxide, a selective mitoKATP
channel opener in heart cells.8,20 Diazoxide (100 ␮mol/L)
induced reversible oxidation of the flavoproteins. A second
application of diazoxide in the presence of HMR1098
2420
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May 23, 2000
Figure 2. Effect of HMR1098 on diazoxide (DIAZO)-induced flavoprotein oxidation. A, DIAZO (100 ␮mol/L) reversibly oxidized
flavoprotein. HMR1098 (30 ␮mol/L) did not affect oxidative
effect of DIAZO. Lines indicate periods when cell was exposed
to drug. B, Summarized data for diazoxide-induced flavoprotein
oxidation measured in absence and presence of HMR1098.
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(30 ␮mol/L) once again increased the flavoprotein fluorescence, and the degree of oxidation was identical to that
achieved during the first exposure to diazoxide. As summarized in Figure 2B, diazoxide (100 ␮mol/L) reversibly increased flavoprotein oxidation to 41⫾8% of the DNP value
(n⫽4). HMR1098 (30 ␮mol/L) did not alter the effect of
diazoxide (42⫾9% of the DNP value, n⫽4).
To verify that the lack of effect of HMR1098 on diazoxideinduced flavoprotein oxidation did not result from inadequate
diffusion of the drug to mitochondria, we measured flavoprotein
fluorescence after including HMR1098 (30 ␮mol/L) in the patch
pipette. Figure 3A shows that after 10 minutes in the whole-cell
configuration, exposure to diazoxide (100 ␮mol/L) still induced
flavoprotein oxidation. This effect of diazoxide could be blocked
by 5HD (500 ␮mol/L), a specific mitoKATP channel inhibitor.10,11 Subsequent reapplication of diazoxide in the absence of
5HD once again increased flavoprotein oxidation. Despite the
presence of HMR1098 in the pipette, diazoxide increased
flavoprotein oxidation to 46⫾6% of the DNP value (n⫽5,
Figure 3B). This degree of oxidation is comparable to that
observed in the absence of HMR1098 (cf Figure 2). The results
indicate that HMR1098 selectively inhibits surfaceKATP channels
but not mitoKATP channels.
Figure 3. Effect of diazoxide (DIAZO) on flavoprotein fluorescence measured in whole-cell patch-clamp configuration. Patch
pipette contained 30 ␮mol/L HMR1098. A, DIAZO (100 ␮mol/L)
reversibly increased flavoprotein oxidation. Oxidative effect of
DIAZO was inhibited by 5HD (500 ␮mol/L). B, Summarized
pooled data for DIAZO-induced flavoprotein oxidation when
HMR1098 was administered via pipette. Intracellular HMR1098
did not alter oxidative effect of diazoxide.
Figure 4. Effects of P-1075 on surfaceKATP current. A, Time
course of current measured at 0 mV. Lines indicate periods
when cell was exposed to P-1075 (30 ␮mol/L) and HMR1098
(30 ␮mol/L). B, Summarized data from 4 cells. **P⬍0.01 vs control (CONT) and P-1075⫹HMR1098. C, Dose-response curve for
P-1075 in rabbit ventricular myocytes. Each point constitutes
measurements from 4 to 5 cells. D, Dose-dependent effects of
P-1075 on Kir6.2⫹SUR2A (cardiac-type) and Kir6.1⫹SUR2B
KATP (vascular smooth muscle–type) channels expressed in HEK
cells. Each point constitutes measurements from 5 to 10 cells.
Effect of P-1075 on SurfaceKATP and
MitoKATP Channels
We then examined the effects of the KATP channel opener
P-107516 on surfaceKATP and mitoKATP channels. P-1075 is a
derivative of the cyanoguanidine KATP channel agonist pinacidil,
which is known to open both mitoKATP and surfaceKATP channels.8 Figure 4, A and B, shows that P-1075 (30 ␮mol/L)
significantly increased IK,ATP (P⬍0.01, n⫽4). Subsequent application of 30 ␮mol/L HMR1098, which we have found to be a
selective surfaceKATP channel blocker, suppressed IK,ATP completely. The EC50 for P-1075 to activate IK,ATP in rabbit ventricular myocytes was 13.4 ␮mol/L (Figure 4C), but cardiovascular
effects have been described at much lower concentrations.21 To
investigate the molecular basis of P-1075 action, we expressed
the cardiac and vascular smooth muscle isoforms of surfaceKATP
channels heterologously in human embryonic kidney (HEK) 293
cells. Figure 4D shows dose-response curves for the agonist
effect of P-1075 on Kir6.2⫹SUR2A (cardiac type)22 and
Sato et al
Mitochondrial KATP Channels in Cardioprotection
2421
Figure 5. Effect of P-1075 on mitochondrial matrix oxidation. A,
Diazoxide (DIAZO, 100 ␮mol/L) reversibly oxidized flavoproteins,
whereas P-1075 (30 ␮mol/L) did not oxidize flavoproteins. Lines
indicate periods when cell was exposed to drugs. B, Summarized data for flavoprotein oxidation. DIAZO(1) and DIAZO(2)
indicate first and second exposures to diazoxide (100 ␮mol/L),
respectively.
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Kir6.1⫹SUR2B (vascular smooth muscle type)23 KATP channels.
In cells expressing the cardiac-type Kir6.2⫹SUR2A channels,
P-1075 effectively increased IK,ATP in a concentration-dependent
manner (EC50 for P-1075⫽2.5 ␮mol/L). Conversely, P-1075
activated Kir6.1⫹SUR2B KATP channels at nanomolar concentrations (EC50⫽102 nmol/L). Thus, the low-dose effects previously described21 are unlikely to reflect activation of cardiac
surface KATP channels.
The effects of P-1075 on mitoKATP channels were examined
by measurement of mitochondrial redox potential. Figure 5
shows that diazoxide (100 ␮mol/L) induced reversible oxidation
of the mitochondrial matrix to 44⫾4% of the DNP value (n⫽5).
Subsequent exposure to P-1075 (30 ␮mol/L) had no effect
(2⫾1% of the DNP value, n⫽5), whereas diazoxide once again
increased flavoprotein oxidation (39⫾5% of the DNP value,
n⫽4). Even when very high (100 ␮mol/L) or low (100 nmol/L)
concentrations of P-1075 were applied, the drug failed to elicit
any flavoprotein response (not shown). These results indicate
that P-1075 selectively activates surfaceKATP channels without
affecting mitoKATP channels.
To verify further the specificity of diazoxide and P-1075
for mitoKATP and surfaceKATP channels, respectively, we
measured flavoprotein fluorescence and membrane current
simultaneously. Figure 6A and 6B shows the effects of
diazoxide and P-1075 in a representative experiment. Diazoxide (100 ␮mol/L) induced reversible oxidation of flavoproteins but did not affect IK,ATP. In contrast, exposure to
P-1075 (30 ␮mol/L) failed to increase flavoprotein oxidation
but did elicit IK,ATP. As summarized in Figure 6C and 6D,
unlike diazoxide, P-1075 activated only IK,ATP.
Effects of HMR1098 and P-1075 on Simulated
Ischemia and Cellular Injury
Using the mitochondria- or surface-selective agents, we
examined the role of mitoKATP and surfaceKATP channels for
ischemic cardioprotection. The mitoKATP channel opener
diazoxide (100 ␮mol/L) significantly decreased the percentage of cells stained after 60 minutes of simulated ischemia
(from 32⫾3% to 17⫾3%, P⬍0.001, n⫽5), and this protection was completely prevented by the mitoKATP channel
blocker 5HD (500 ␮mol/L) (Figure 7A). In contrast, the
surfaceKATP channel blocker HMR1098 (30 ␮mol/L) did not
prevent the cardioprotection by diazoxide (from 38⫾4% to
Figure 6. Effects of diazoxide and P-1075 on flavoprotein fluorescence and IK,ATP. A and B, Simultaneous measurements of
flavoprotein fluorescence and IK,ATP. Diazoxide (DIAZO,
100 ␮mol/L) reversibly oxidized flavoprotein without affecting
membrane current. P-1075 increased IK,ATP but not flavoprotein
fluorescence. C and D, Summarized data for flavoprotein oxidation and IK,ATP from 5 cells.
18⫾1%, P⬍0.001, n⫽4) (Figure 7B). In a separate series of
experiments (Figure 7C), simulated ischemia for 60 and 120
minutes stained 35⫾2% (n⫽5) and 42⫾2% (n⫽5) of cells,
respectively. Inclusion of diazoxide (100 ␮mol/L) significantly decreased the percentage of cells stained, to 18⫾2%
Figure 7. Summarized data for percentage of cells stained after 60
minutes or 120 minutes of simulated ischemia. Cells stained after
simulated ischemia were plotted as a percentage of total viable
cells before ischemia. A, Effect of 5HD on diazoxide-induced cardioprotection. CONT indicates control group; DIAZO, diazoxide
(100 ␮mol/L)-treated group; and DIAZO⫹5HD, diazoxide plus 5HD
(500 ␮mol/L)–treated group. B, Effects of HMR1098 on diazoxideinduced cardioprotection. DIAZO⫹HMR1098 indicates diazoxide
plus HMR1098 (30 ␮mol/L)–treated group. C, Comparative effects
of diazoxide and P-1075 on ischemic cardioprotection. P1075 indicates P-1075 (30 ␮mol/L)–treated group. Data obtained from parallel experiments are summarized in each panel. **P⬍0.001 vs
CONT group.
2422
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May 23, 2000
Figure 8. Effects of 5HD and HMR1098 on ischemically preconditioned cells. Cells stained after 60 and 120 minutes of simulated ischemia were plotted as percentage of total viable cells
before ischemia. CONT indicates nonpreconditioned control
group; IPC, preconditioned group; IPC⫹5HD, 5HD (500 ␮mol/L)
added 10 minutes before preconditioning; and IPC⫹HMR1098,
HMR1098 (30 ␮mol/L) added 10 minutes before preconditioning. **P⬍0.005 vs CONT group.
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(n⫽5) after 60 minutes and 24⫾2% (n⫽5) after 120 minutes
of simulated ischemia (P⬍0.001 versus control group). In
contrast, the selective surfaceKATP channel opener P-1075
(30 ␮mol/L) did not alter the extent of stained cells as a
consequence of ischemia (34⫾4% after 60 minutes, 38⫾4%
after 120 minutes). These results indicate that mitoKATP but
not surfaceKATP channels are involved in pharmacological
cardioprotection.
We examined the effects of 5HD and HMR1098 on simulated
IPC. As summarized in Figure 8, IPC significantly decreased the
percentage of cells stained during 60 minutes (from 26⫾4% to
13⫾2%, P⬍0.005, n⫽6) and 120 minutes (from 35⫾3% to
20⫾3%, P⬍0.005, n⫽6) of simulated ischemia. 5HD
(500 ␮mol/L) added 10 minutes before preconditioning abolished the protection (25⫾3% after 60 minutes and 34⫾3% after
120 minutes ischemia, respectively). In contrast, HMR1098
(30 ␮mol/L) did not interfere with IPC after 60 minutes
(15⫾2%, P⬍0.005, n⫽6) or 120 minutes (20⫾3%, P⬍0.005,
n⫽6) of simulated ischemia. These results indicate that the
cardioprotection afforded by IPC is mediated by mitoKATP
channels, not surfaceKATP channels.
Discussion
The cardioprotective effects of IPC are abolished by antagonists of KATP channels and are mimicked by agonists of KATP
channels.24 –26 On the basis of these studies, an early hypothesis
proposed that opening of surfaceKATP channels abbreviates
action potential duration, thereby reducing cellular calcium
overload and preserving viability in ischemic myocardium.
However, this hypothesis cannot account for IPC, because
abbreviation of action potential duration is not necessary for
protection. Grover et al5 showed that dofetilide attenuated the
shortening of action potential duration but did not abolish IPC in
dogs. Yao and Gross6 demonstrated that low-dose bimakalim
produced cardioprotection without any effect on monophasic
action potential duration. Furthermore, IPC occurs even in
unstimulated (electrically quiescent) cardiac myocytes, in which
action potential abbreviation cannot be a factor.4,7
MitoKATP channels share some pharmacological properties
with surfaceKATP channels, while possessing a distinct pharmacological profile. Garlid et al20 demonstrated that diazox-
ide opens mitoKATP channels ⬎2000-fold more potently than
surfaceKATP channels in cardiac myocytes. Although direct
effects of diazoxide on mitochondrial energy metabolism in
pancreatic ␤-cells have been proposed,27 previous studies in
our laboratory demonstrated that diazoxide oxidizes the
mitochondrial matrix redox potential via opening of mitoKATP
channels in rabbit hearts, whereas surfaceKATP channels are
quite resistant to this drug8; the specificity of diazoxide for
mitoKATP channels is verified in Figure 6 of the present
article. Moreover, we reported that 5HD completely and
reversibly blocks the oxidative effect of diazoxide without
affecting surfaceKATP channels, indicating that 5HD is an
effective and specific blocker of mitoKATP channels8,10,11
(although this may not be the case in other species28,29). When
these drugs are used as pharmacological tools to activate or to
inhibit mitoKATP channels, growing evidence supports the
idea that mitoKATP channels rather than surfaceKATP channels
are the dominant players for cardioprotection.8,9 Moreover, in
animal models in vivo, diazoxide mimics,30 whereas 5HD
abolishes, the infarct size–limiting effect of IPC.12–14 These
findings motivated us to reevaluate the role of surfaceKATP
channels in cardioprotection.
The sulfonylthiourea HMR1098 has been reported to be a
cardioselective KATP channel blocker, blocking KATP channels
in cardiac muscle cells with 10- to 50-fold higher potency
than in pancreatic ␤-cells with little effect on the coronary
vasculature.15 We demonstrated that HMR1098 inhibited
surfaceKATP channels activated by metabolic inhibition (Figure 1) and by the surfaceKATP channel opener P-1075 (Figure
4). However, HMR1098 did not affect diazoxide-induced
flavoprotein oxidation (Figure 2). Furthermore, direct application of HMR1098 to the cytoplasm by inclusion in the
pipette failed to prevent the oxidizing effect of diazoxide
(Figure 3). These results, taken together, indicate that
HMR1098 selectively inhibits surfaceKATP channels without
affecting mitoKATP channels.
The KATP channel opener P-1075 activated IK,ATP in rabbit
ventricular myocytes; IK,ATP was blocked by HMR1098 (Figure
4). Moreover, the molecularly defined cardiac surfaceKATP channel (Kir6.2⫹SUR2A)22 expressed in HEK cells was effectively
activated by P-1075. Indeed, P-1075 showed similar potencies in
activating native surfaceKATP channels and expressed
Kir6.2⫹SUR2A channels (EC50s of 13 ␮mol/L and 2.5 ␮mol/L,
respectively). P-1075 activated Kir6.1⫹SUR2B (vascular
smooth muscle type)23 channels ⬇100-fold more potently than
Kir6.2⫹SUR2A channels (EC50 of 102 nmol/L). Conversely,
P-1075 did not affect mitochondrial oxidation state measured
with or without invasion by patch pipettes or their contents
(Figures 5 and 6). These results indicate that, unlike diazoxide,
P-1075 selectively activates surfaceKATP but not mitoKATP
channels.
We previously reported that diazoxide (50 ␮mol/L) decreased
myocyte death in a cellular model of simulated ischemia.8 In the
present study, we verified the cardioprotective effects of diazoxide (Figure 7). Diazoxide-induced cardioprotection was prevented by the mitoKATP channel blocker 5HD. In contrast, the
surfaceKATP channel blocker HMR1098 did not abolish the
cardioprotective effect of diazoxide (Figure 7B). Furthermore,
we found that the surfaceKATP channel opener P-1075 did not
Sato et al
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produce cardioprotection (Figure 7C). Sargent et al21 reported
that, in Langendorff-perfused rat hearts, P-1075 increases coronary flow and protects ischemic myocardium at nanomolar
concentrations. Consistent with their findings, P-1075 does
activate the vascular smooth muscle type (Kir6.1⫹SUR2B) KATP
channel at nanomolar concentrations, whereas the cardiac-type
(Kir6.2⫹SUR2A) KATP channel is affected only in the micromolar range (Figure 4D). However, we found that the surfaceKATP channel opener P-1075 (30 ␮mol/L) did not produce cardioprotection (Figure 7C). Although the reason for this discrepancy
is unknown, our results imply that the direct activation of
surfaceKATP channels in rabbit myocytes does not underlie
myocyte cardioprotection.
IPC is present in all species examined, including humans.31
Compelling evidence suggests that mitoKATP channels rather
than surfaceKATP channels may serve as end effectors of IPC.
However, another study reported that digoxin preserves the
subsarcolemmal ATP and inhibits surfaceKATP channels,
thereby abolishing IPC in rabbit hearts.32 To clarify these
discordant data, further studies using the selective agonist and
antagonist of surfaceKATP or mitoKATP channels are necessary.
Our present results demonstrate that the mitoKATP channel
blocker 5HD but not the surfaceKATP channel blocker
HMR1098 abolished the cardioprotection in a cellular model
of preconditioning. These results divorce the cardioprotective
effect of IPC from activation of surfaceKATP channels.
In conclusion, our observations with diazoxide, P-1075,
5HD, and HMR1098 provide the first definitive pharmacological evidence that surfaceKATP channels are not involved in
cardioprotection in isolated rabbit myocytes. By extension,
the present data provide further support for the emerging
consensus that mitoKATP channels are the end effectors of
preconditioning specifically in rabbit heart.
Mitochondrial KATP Channels in Cardioprotection
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
Acknowledgments
This study was supported by the National Institutes of Health
(R37-HL-36957 to Dr Marbán) and by a Banyu Fellowship in Lipid
Metabolism and Atherosclerosis (to Dr Sato). Dr Marbán holds the
Michel Mirowski, MD, Professorship of Cardiology of the Johns
Hopkins University.
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Selective Pharmacological Agents Implicate Mitochondrial but Not Sarcolemmal KATP
Channels in Ischemic Cardioprotection
Toshiaki Sato, Norihito Sasaki, Jegatheesan Seharaseyon, Brian O'Rourke and Eduardo Marbán
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Circulation. 2000;101:2418-2423
doi: 10.1161/01.CIR.101.20.2418
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