(Circulation. 1998;98:1479-1480.)
© 1998 American Heart Association, Inc.
Recombinant Cardiac ATP-Sensitive Potassium Channels and Cardioprotection
Garrett J. Gross, PhD
From the Department of Pharmacology and Toxicology, Medical College of
Wisconsin, Milwaukee.
Correspondence to Garrett J. Gross, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail ggross{at}mcw.edu
Key Words: Editorials • potassium • calcium • hypoxia • ischemia • molecular biology
The ATP-dependent potassium
channels (KATP channels) were originally
identified in isolated membrane patches prepared from guinea pig
ventricular myocytes by Noma1 in
1983. Since their discovery in cardiac cells,
KATP channels have also been discovered in many
other tissues, such as smooth muscle, skeletal muscle, pancreas, and
brain, in which they have been shown to couple cellular
metabolism to membrane electrical
activity.2 Primarily on the basis of studies
using pharmacological tools, openers of KATP
channels have been shown to elicit cardioprotective effects, whereas
KATP channel antagonists have been
shown to block the cardioprotective effects of
KATP channel openers and the powerful protective
effect produced by single or multiple brief episodes of
ischemia to reduce myocardial infarct size, a phenomenon called
ischemic preconditioning.3 Because the
results of these previous studies were obtained indirectly by the use
of pharmacological agonists and antagonists, the results of
the present study published by Jovanovic and
colleagues4 in this issue of
Circulation are particularly exciting and are relevant for
helping to clearly define an important role for the
endogenous KATP channel protein
subunits in conferring the cardioprotective effects of
KATP channel openers and ischemic
preconditioning. In this elegant study by Jovanovic and coworkers, the
authors transfected KATP-deficient COS-7 cells
with the Kir 6.2/SUR 2A genes, which Okuyama et
al5 recently showed to form functional
KATP channels in HEK 293T cells and to possess
the main properties of native KATP channels in
terms of activation by pinacidil and nicorandil but not diazoxide,
channel rundown, and regulation by intracellular
nucleotides such as ADP and UDP. In
KATP-deficient COS-7 cells, Jovanovic et al found
that when these cells were exposed to 3 minutes of chemical
hypoxia (dinitrophenol, DNP) and subsequently
reoxygenated, significant calcium loading occurred. In
these COS-7 cells deficient in KATP channels, the
KATP channel opener pinacidil had no significant
effect on calcium loading produced by chemical
hypoxia-reoxygenation injury. However, when
both subunits of the KATP channel Kir
6.2/SUR 2A were cotransfected in COS-7 cells, a phenotype
was produced in which pinacidil was capable of markedly attenuating the
calcium loading produced by
hypoxia-reoxygenation. That this effect was the
result of opening KATP channels was confirmed by
demonstration that glyburide (1 µmol/L) was capable of
abolishing the protective effect of pinacidil. Interestingly, opening
of the channel by chemical hypoxia with DNP produced only a
marginally protective effect; however, this may have been the result of
the short period (3 minutes) of exposure to DNP or the need to
sensitize the KATP channel before the main
hypoxic insult, such as occurs in ischemic preconditioning.
Moreover, in COS-7 cells transfected with either Kir 6.2
or SUR 2A alone, pinacidil had no significant
cytoprotective effect. These results suggest that the cardiac
KATP channel protein possesses
endogenous cytoprotective properties when transfected into
a noncardiac cell type. Cardiac myocytes expressing the native
endogenous KATP channel were also
exposed to the same chemical
hypoxia-reoxygenation protocol and demonstrated
a marked increase in cellular calcium that was significantly attenuated
by pinacidil, an effect that was abolished by glyburide.
Since the original study by Noma in
1983,1 there has been a great deal of interest in
the mechanism by which opening the KATP channel
by drugs or brief periods of ischemia produces a
cardioprotective effect. Until recently, the prevailing theory was that
either KATP openers or ischemia resulted
in the enhanced shortening of action potential duration (APD), which
shortened phases 2 and 3 of the action potential and resulted in
a blockade of L-type calcium channels and a reduction in calcium
overload during ischemia and/or
reperfusion.6 Membrane
hyperpolarization or a slowing of membrane
depolarization would also be an expected consequence of
KATP channel opening, and this would also be
expected to slow calcium entry through L-type channels and prevent the
reversal of the sodium-calcium exchanger, which normally extrudes
calcium for sodium. Indeed, the studies by Cole et
al6 in an isolated perfused guinea pig right
ventricular wall preparation seemed to support this theory,
because pinacidil enhanced APD shortening during ischemia and
enhanced functional recovery of muscle exposed to
ischemia-reperfusion, whereas glyburide blocked APD shortening
and worsened functional recovery. The first study to suggest that the
cardioprotective effect of a KATP opener was not
related to enhanced APD shortening was published by Yao and
Gross7 in 1994 and showed that a small dose of
the KATP opener bimakalim produced a marked
reduction in infarct size in dogs in the absence of enhanced APD
shortening. Similar results were published in 1995 by Grover and
colleagues,8 who also showed no correlation
between APD shortening and cardioprotection in dogs treated with
cromakalim. The present data of Jovanovic et
al4 directly support the idea that APD shortening
is not an important component of the cardioprotective effect resulting
from opening the cardiac KATP channel, because
COS-7 cells do not generate an action potential yet showed protection
against calcium loading when the recombinant channel was opened by
pinacidil. Unfortunately, membrane potential was not measured in these
COS-7 cells, so one cannot rule out an effect of pinacidil to
hyperpolarize these cells and slow calcium entry by this mechanism.
Another site of action that has been proposed for
KATP channel openers and possibly
ischemic preconditioning that is distinct from the cardiac
sarcolemmal channel is the recently identified mitochondrial
KATP channel.9 Garlid et
al10 showed that diazoxide, a
KATP opener, which has no effect on the
sarcolemmal KATP channel but opens the
mitochondrial KATP
channel,11 produced a cardioprotective effect
similar to that of cromakalim in isolated rat hearts subjected
to ischemia and reperfusion at low micromolar concentrations.
That this protective effect of diazoxide was the result of activation
of a KATP channel was confirmed by demonstration
that glibenclamide and 5-hydroxydecanoic acid both blocked its
protective effect. Similar results were recently published by Liu et
al,12 who also showed that diazoxide selectively
opened a cardiac mitochondrial KATP channel and
produced a cardioprotective effect at low micromolar concentrations in
an isolated cell model of preconditioning. These results and previous
ones that suggest no correlation between APD shortening and
cardioprotection strongly suggest that the mitochondrial
KATP channel may be the major site of action for
the cardioprotective effects of KATP channel
openers and ischemic preconditioning. Conversely, the results
of the present study by Jovanovic et al4
suggest that the sarcolemmal KATP channel may
also be an important site of action for the cardioprotective effect of
compounds such as pinacidil and that the channel protein subunits may
confer a protective effect themselves when they combine to form a
functional channel that is independent of APD shortening. It would be
interesting to test the effect of diazoxide in these cotransfected
COS-7 cells in future experiments and to test the effect of several
KATP channel openers in COS-7 cells transfected
with the appropriate Kir 6.x and SUR
subunits from cardiac mitochondria once they are clearly identified to
help better define the roles of these 2 channels in attenuating injury
due to ischemia and reperfusion.
Molecular cloning of the KATP channel
subunit proteins Kir 6.x and SUR have
shown that this channel consists of a number of subtypes and will allow
investigators to study the regulation and function of this channel in
different organs as well as under different
pathophysiological situations, as the present
study by Jovanovic et al did.4 In this regard,
several studies have recently been published in which the expression
and regulation of the KATP channel subunits were
studied in isolated cardiomyocytes and intact animals. Lu
and Halvorsen,13 using chick
cardiomyocytes, showed that the mRNA for Kir
6.1 was upregulated by 1.8- to 2.4-fold by either ATP
depletion or prolonged exposure (12 hours) to the
KATP channel openers pinacidil, cromakalim, and
diazoxide. Glibenclamide completely abolished the increase in
Kir 6.1 levels produced by pinacidil. Similarly, Akao et
al14 demonstrated that mRNA for Kir
6.1 but not Kir 6.2 was upregulated by 2.7- to
3.1-fold in the ischemic region of intact rat hearts subjected
to 60 minutes of coronary artery occlusion and 24 to 72 hours
of reperfusion or by 24 hours of continuous ischemia. Similar
results were observed with Western blots for the Kir 6.1
protein. These results, combined with those of the present study by
Jovanovic et al,4 clearly suggest an important
role for KATP channel regulation in the response
to acute and prolonged ischemic or hypoxic insults and
pharmacological interventions. Interestingly, because it is thought
that Kir 6.1 may be localized primarily to mitochondria
in heart and other organs11 and that Kir
6.2 is localized to the sarcolemma, it appears that these 2
channels are differentially regulated under acute and chronic
conditions. It is interesting to speculate that these 2 channels may
also play different roles in the response to acute or chronic
ischemia. In this regard, evidence is rapidly accumulating that
the KATP channel may be the end effector molecule
responsible for the cardioprotective effects of both the early and
delayed phases of ischemic
preconditioning15 ; therefore, future studies like
the present one by Jovanovic et al4 using
recombinant KATP subunits should allow us to
better understand the role and function of KATP
channel subtypes in producing cardioprotection and lead to the
development of site-specific pharmacological agents that may have
potential therapeutic uses.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
References
1.
Noma A. ATP-regulated K+
channels in cardiac muscle. Nature. 1983;305:147–148.[Medline]
[Order article via Infotrieve]
2.
Kersten JR, Gross GJ, Pagel PS, Warltier DC.
Activation of adenosine triphosphate-regulated potassium
channels: mediator of cellular and organ protection.
Anesthesiology. 1998;88:495–513.[Medline]
[Order article via Infotrieve]
3.
Murry CE, Jennings RB, Reimer KA. Preconditioning with
ischemia: a delay of lethal cell injury in ischemic
myocardium. Circulation. 1986;74:1124–1136.[Abstract/Free Full Text]
4.
Jovanovic A, Jovanovic S, Lorenz E, Terzic A.
Recombinant cardiac ATP-sensitive K+ channel
subunits confer resistance to chemical
hypoxia-reoxygenation injury.
Circulation. 1998;98:1548–1555.[Abstract/Free Full Text]
5.
Okuyama Y, Yamada M, Kondo C, Satoh E, Isomoto S,
Shindo T, Horio Y, Kitakaze M, Hori M, Kurachi Y. The effects of
nucleotides and potassium channel openers on the
SUA2A/Kir6.2 complex K+ channel
expressed in a mammalian cell line, HEK293T cells. Pflugers
Arch. 1998;435:595–603.[Medline]
[Order article via Infotrieve]
6.
Cole WC, McPherson CD, Sontag D. ATP-regulated
K+ channels protect the myocardium
against ischemia/reperfusion damage. Circ Res. 1991;69:571–581.[Abstract/Free Full Text]
7.
Yao Z, Gross GJ. Effects of the
KATP channel opener bimakalim on coronary
blood flow, monophasic action potential duration, and infarct size in
dogs. Circulation. 1994;89:1769–1775.[Abstract/Free Full Text]
8.
Grover GJ, Dalonza AJ, Parham CS, Darbenzio RB.
Cardioprotection with the KATP opener cromakalim
is not correlated with ischemic myocardial action potential
duration. J Cardiovasc Pharmacol. 1995;26:145–152.[Medline]
[Order article via Infotrieve]
9.
Paucek P, Mironova G, Mahdi F, Beavis AD, Woldegiorgis
G, Garlid KD. Reconstitution and partial purification of the
glibenclamide-sensitive, ATP-dependent K+ channel
from rat liver and beef heart mitochondria. J Biol
Chem. 1992;267:26062–26069.[Abstract/Free Full Text]
10.
Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN,
Darbenzio RB, D'Alonzo AJ, Lodge NJ, Smith MA, Grover GJ.
Cardioprotective effect of diazoxide and its interaction with
mitochondrial ATP-sensitive K+ channels: possible
mechanism of cardioprotection. Circ Res. 1997;81:1072–1082.[Abstract/Free Full Text]
11.
Suzuki M, Kotake K, Fujikura K, Imagaki N, Suzuki
T, Gonoi T, Seino S, Takata K. Kir 6.1: a possible
subunit of ATP-sensitive K+ channels in
mitochondria. Biochem Biophys Res Commun. 1997;241:693–697.[Medline]
[Order article via Infotrieve]
12.
Liu Y, Sato T, O'Rourke B, Marban E. Mitochondrial
ATP-dependent potassium channels: novel effectors of cardioprotection?
Circulation. 1998;97:2463–2469.[Abstract/Free Full Text]
13.
Lu C, Halvorsen SW. Channel activators
regulate ATP-sensitive potassium channel (KIR 6.1)
expression in chick cardiomyocytes. FEBS Lett. 1997;412:121–125.[Medline]
[Order article via Infotrieve]
14.
Akao M, Otani H, Horie M, Takano M, Kuniyasu A,
Nakayama H, Kouchi I, Sasayamo S, Murakami T. Myocardial
ischemia induces differential regulation of
KATP channel gene expression in rat hearts.
J Clin Invest. 1997;100:3053–3059.[Medline]
[Order article via Infotrieve]
15.
Meldrum DR. Mechanisms of cardiac preconditioning: ten
years after the discovery of ischemic preconditioning.
J Surg Res. 1997;73:1–13.[Medline]
[Order article via Infotrieve]
