Genetic Disruption of Kir6.2, the Pore-Forming Subunit
of ATP-Sensitive Kⴙ Channel, Predisposes to
Catecholamine-Induced Ventricular Dysrhythmia
Xiao-Ke Liu,1 Satsuki Yamada,1 Garvan C. Kane,1 Alexey E. Alekseev,1 Denice M. Hodgson,1
Fearghas O’Cochlain,1 Arshad Jahangir,1 Takashi Miki,2 Susumu Seino,2 and Andre Terzic1
Metabolic-sensing ATP-sensitive Kⴙ channels (KATP
channels) adjust membrane excitability to match cellular energetic demand. In the heart, KATP channel activity has been linked to homeostatic shortening of the
action potential under stress, yet the requirement of
channel function in securing cardiac electrical stability
is only partially understood. Here, upon catecholamine
challenge, disruption of KATP channels, by genetic deletion of the pore-forming Kir6.2 subunit, produced defective cardiac action potential shortening, predisposing
the myocardium to early afterdepolarizations. This deficit in repolarization reserve, demonstrated in Kir6.2knockout hearts, translated into a high risk for
induction of triggered activity and ventricular dysrhythmia. Thus, intact KATP channel function is mandatory for adequate repolarization under sympathetic
stress providing electrical tolerance against triggered
arrhythmia. Diabetes 53 (Suppl. 3):S165–S168, 2004
E
xpressed at high density in the cardiac sarcolemma, ATP-sensitive K⫹ (KATP) channels are
heteromultimers of the pore-forming Kir6.2 subunit with the regulatory sulfonylurea receptor
(1). By virtue of a unique ability to decode signals of
cellular energetic distress, KATP channels adjust membrane electrical activity in response to metabolic demand
(2,3). Disturbances in KATP channel function, either
through pharmacological blockade with sulfonylurea medication or through genetic mutation of channel proteins,
have been linked to increased susceptibility for development and progression of cardiovascular conditions (4 – 8).
In particular, in hyperadrenergic states, ranging from
physical exertion to decompensated heart failure, cardiac
KATP channels have been implicated in the maintenance of
From the 1Division of Cardiovascular Diseases, Department of Medicine,
Department of Molecular Pharmacology and Experimental Therapeutics,
Mayo Clinic College of Medicine, Rochester, Minnesota; and the 2Division of
Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan.
Address correspondence and reprint requests to Andre Terzic, MD, PhD,
Guggenheim 7, Mayo Clinic, Rochester, MN 55905. E-mail: terzic.andre
@mayo.edu.
Received for publication 12 March 2004 and accepted in revised form 18
May 2004.
This article is based on a presentation at a symposium. The symposium and
the publication of this article were made possible by an unrestricted educational grant from Servier.
APD90, action potential duration at 90% repolarization; KATP channel,
ATP-sensitive K⫹ channel; Kir6.2-KO, Kir6.2 knockout.
© 2004 by the American Diabetes Association.
DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004
cellular well-being and stress adaptation (9 –11). Specifically, under catecholamine surge, proper KATP channel
activity is required for the coordinated adjustment of
membrane-dependent cellular functions, including adequate calcium handling and sustained contractility (9,10).
Although KATP channel opening has been associated with
homeostatic shortening of the cardiac action potential
under increased metabolic demand (6,9), the precise role
of channel function in support of cardiac electrical stability is only partially understood. Thus, this study was
designed to address the contribution of KATP channels in
membrane electrical tolerance in the heart under adrenergic stress.
To this end, the electrical consequences of adrenergic
stress were tested in hearts lacking the pore-forming
subunit of KATP channels, through genetic disruption of
Kir6.2, and compared with the wild type. In KATP channel
knockout hearts, sympathomimetic challenge unmasked
an inadequate repolarization reserve predisposing to abnormal action potentials with afterdepolarizations and
inducing ventricular dysrhythmia. Hence, KATP channels
are required for electrical adaptation that protects against
triggered arrhythmia within the adrenergically stressed
myocardium.
RESEARCH DESIGN AND METHODS
Kir6.2-knockout mice. Mice deficient in KATP channels were generated by
targeted disruption of the KCNJ11 gene, which encodes the pore-forming
Kir6.2 subunit of the channel complex (12). Kir6.2-knockout mice were
backcrossed for five generations into a C57BL/6 background. This investigation was approved by the Mayo Clinic Institutional Animal Care and Use
Committee.
In situ aortic cannulation and Langendorff perfusion. Mice were anesthetized with intraperitoneal injection of 2,2,2-tribromoethanol (0.375 mg/g
body wt; Sigma), intubated, and ventilated, and the aortic root was cannulated
in situ (10). Perfusion was sustained ex vivo on a Langendorff system, at 90 cm
H2O with 37°C-prewarmed and 100% O2-bubbled Tyrode solution (in mmol/l:
NaCl 137, KCl 5.4, CaCl2 2, MgCl2 1, HEPES 10, and glucose 10, pH 7.4 with
NaOH). After a 10-min equilibration, KCl was reduced to 2.7 mmol/l and MgCl2
to 0.5 mmol/l, with the atrioventricular node cauterized to allow ventricular
pacing (13). Coronary flow was monitored with a T106 blood flow meter
(Transonic Systems).
Electrogram and monophasic action potential recordings. Orthogonal
electrogram signals were simultaneously recorded using four silver-silver
chloride electrodes surrounding the perfused heart in a simulated “Einthoven”
configuration, and signals were amplified by an electrocardiographic amplifier
(Gould Electronics). A catheter (NuMed) was placed in the left ventricular
endocardium to pace the heart at twice diastolic threshold intensity with 2-ms
pulse width and 100-ms cycle length using a pulse generator (A310 Accupulser; World Precision Instruments). Monophasic action potentials were
continuously recorded from the left ventricle by a probe (EP Technologies)
positioned on the epicardial surface, and amplified signals (IsoDam; World
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KATP CHANNELS AND TRIGGERED ARRHYTHMIA
FIG. 1. Isoproterenol challenge induced
action potential shortening (APD90) in
wild-type (WT) (A) but not Kir6.2-KO
(B) hearts, which developed early afterdepolarizations (EAD) (C). D: Incidence of EAD in the initial 50 action
potentials after 5 min of isoproterenol
infusion.
Precision Instruments) were acquired at 11.8 kHz and stored for off-line digital
analysis (9).
Whole-cell patch clamp recording from isolated cardiomyocytes. Cardiomyocytes were enzymatically dissociated from the ventricular myocardium
(10). Action potentials were recorded at 30 ⫾ 1°C from current-clamped
isolated cells paced at 1 Hz, and were superfused with Tyrode solution (pH 7.2
adjusted with KOH) using the whole-cell patch clamp technique with 5–10
mol/l⍀ pipettes containing (in mmol/l) KCl 120, MgCl2 1, Na2ATP 5, HEPES 10,
EGTA 0.5, and CaCl2 0.01 (14).
Statistics. Comparisons were made using the Student’s t test. A significance
level of 0.05 was preselected. Data are reported as means ⫾ SE.
RESULTS AND DISCUSSION
Whereas at baseline the action potentials were similar, the
metabolic challenge of adrenergic stimulation induced
distinct outcomes depending on the presence of functional
KATP channels, with significant shortening of the action
potential duration observed in wild-type hearts but not in
age- and sex-matched counterparts lacking the Kir6.2
pore-forming channel subunit (Kir6.2-KO) (Fig. 1A and B).
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After a 10-min perfusion with the sympathomimetic isoproterenol (1 ␮mol/l), monophasic action potential duration at 90% repolarization (APD90) shortened from 82 ⫾ 2
to 74 ⫾ 2 ms in wild-type hearts (P ⬍ 0.01, n ⫽ 6; Fig. 1A).
In contrast, APD90 remained at 79 ⫾ 3 and 80 ⫾ 3 ms
before and following isoproterenol treatment, respectively, in Kir6.2-KO hearts (n ⫽ 6) (Fig. 1B). This deficit in
repolarization led to distorted action potential profiles
with characteristic phase 3 early afterdepolarizations manifested as distinct humps in hearts lacking functional KATP
channels (Fig. 1B and C). In all Kir6.2-KO hearts (n ⫽ 8),
adrenergic challenge induced early afterdepolarizations,
which occurred in 97 ⫾ 2% of the action potentials
examined (Fig. 1D). This is in contrast to the action
potential profile of the wild type (n ⫽ 6) that maintained a
smooth repolarization contour following isoproterenol
challenge (Fig. 1A) without significant afterdepolarizations (1 ⫾ 1%; P ⬍ 0.01 vs. Kir6.2-KO) (Fig. 1D).
DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004
X.-K. LIU AND ASSOCIATES
FIG. 2. A: Similar coronary flow in the absence and presence of isoproterenol (1
␮mol/l) in wild-type (WT) and Kir6.2-KO
hearts. B: Isoproterenol-induced abnormal repolarization with early afterdepolarization in
an isolated current-clamped Kir6.2-KO cardiomyocyte.
Abnormal electrical response of Kir6.2-KO hearts under
adrenergic challenge was not associated with an isoproterenol-induced deficit in coronary perfusion (Fig. 2A). In
fact, abnormal electrical activity during repolarization
observed at the whole-heart level was reproduced at the
single-cell level using action potential recording in isoproterenol-stressed current-clamped Kir6.2-KO cardiomyocytes (Fig. 2B).
Afterdepolarizations in isoproterenol-challenged Kir6.2KO hearts translated into increased electrical vulnerability
(Fig. 3). In the absence of functional KATP channels,
afterdepolarizations induced triggered activity, disrupting
regular rhythm and manifesting as premature ventricular
complexes on the electrogram (Fig. 3A). On average,
isoproterenol-induced afterdepolarizations complicated
by triggered activity were observed in six of eight
Kir6.2-KO (75%) compared with one of six wild-type (16%)
hearts, translating into a 16-fold higher risk (P ⬍ 0.05) of
developing premature ventricular complexes (Fig. 3B).
Hence, absence of KATP channels produces a deficit in the
repolarization reserve leading to a pronounced susceptibility of Kir6.2-KO hearts to isoproterenol-induced ventricular dysrhythmia. Thus, sarcolemmal KATP channels
provide for membrane electrical stability that reduces the
risk for arrhythmia under hyperadrenergic conditions.
Imposed catecholamine stress is a well-established precipitator of triggered activity and arrhythmia (15,16). Anal-
ogous to hearts with genetic and/or environmental
compromise of repolarizing currents, as observed in congenital or acquired long QT syndrome (17), here isoproterenol challenge was proarrhythmic in the KATP channel–
deficient myocardium provoking afterdepolarizations and
triggered activity. This is in line with pharmacological
studies that demonstrate that KATP channel activation with
potassium channel openers prevents—whereas channel
blockade with sulfonylurea drugs enhances—afterdepolarizations and triggered activity (18 –20). In fact, in a recent
randomized clinical trial in patients with type 2 diabetes,
the sulfonylurea glyburide, but not the alternative oral
hypoglycemic agent metformin, caused an increase in QT
prolongation with QT dispersion on the electrocardiogram
(21). Moreover, mutations in the cardiac sulfonylurea
receptor inducing deficits in KATP channel function have
been identified in patients with cardiomyopathy and ventricular arrhythmia (8).
Suppression of KATP channel activity, whether by genetic deletion of channel subunits or through use of
channel antagonists, predisposes to inadequate calcium
handling and intracellular calcium overload (5,9). In turn,
excessive cytosolic calcium acts as a trigger for early
depolarizations (22,23). Conversely, in the intact heart,
where KATP channel opening is promoted by ␤-adrenergic–
mediated phosphorylation of channel proteins (24,25) or
subsarcolemmal ATP depletion (26), shortening of cardiac
FIG. 3. A: Kir6.2-KO hearts with early
afterdepolarizations (EAD) demonstrated triggered activity on monophasic
action potentials (MAP) and premature
ventricular complexes (PVC) on electrograms (EG). For comparison, an EG is
shown from wild-type hearts that are not
prone to EAD-triggered activity/PVC. B:
Incidence of triggered activity with accompanying PVC in the initial 50 action
potentials following 5 min of isoproterenol infusion.
DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004
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KATP CHANNELS AND TRIGGERED ARRHYTHMIA
action potentials would balance catecholamine-induced
increase in calcium influx protecting against triggered
arrhythmia.
In summary, the present study underscores the homeostatic requirement for functional KATP channels in the
adaptation of cardiac repolarization under adrenergic
stress. In this regard, a deficit in KATP channel function is
here identified as a previously unrecognized risk factor for
the development of catecholamine-induced afterdepolarizations and triggered arrhythmia.
ACKNOWLEDGMENTS
Supported by the National Institutes of Health (HL64822,
GM08685, HL07111, and AG21201), American Heart Association, Miami Heart Research Institute, Marriott Foundation, Mayo Foundation Clinician-Investigator Program,
Japan Heart Foundation, Mayo-Dubai Healthcare City
Research Project, and Japanese Ministry of Education,
Science, Sports, Culture and Technology. A.T. is an
Established Investigator of the American Heart Association.
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