Download Potassium currents in ventricular myocytes from genetically diabetic

Potassium currents in ventricular myocytes
from genetically diabetic rats
KATSUHARU TSUCHIDA AND HIROSHI WATAJIMA
Research Center, Taisho Pharmaceutical Company, Ohmiya, Saitama 330, Japan
cardiomyocytes; transient outward current
that patients with diabetes
mellitus have increased risk of mortality from cardiac
failure that cannot simply be explained in terms of
atherosclerosis, hyperlipidemia, or hypertension. Many
studies have been performed to investigate the cardiac
function of diabetic animals, and a variety of mechanical, electrophysiological, and biochemical abnormalities have been identified in the myocardium of diabetic
animal models (7). Chemical-induced diabetic models,
especially the streptozotocin (STZ)-induced diabetic
model, are frequently used as a model for insulindependent diabetes mellitus (IDDM) to investigate
these abnormalities. With use of this animal model,
contractile abnormalities have been reported (7), and
this dysfunction has been related to biochemical changes
such as the altered distribution of myosin isozymes (4);
a decrease in uptake of Ca21 into sarcoplasmic reticulum (6) and sarcolemma (9); decreased activity of
Na1-K1-adenosinetriphosphatase (14), Na1-H1 exchange (15), and Na1-Ca21 exchange (16) in the sarcolemmal membrane; and Ca21 binding capacity (20).
Abnormalities of electrophysiological phenomena have
also been demonstrated in STZ-induced diabetic rats.
Ventricular and atrial action potentials were prolonged
IT HAS LONG BEEN KNOWN
when the measurement was done for a relatively longer
term after STZ injection (1, 15). The L-type calcium
current was not altered from 6 days to 2 mo (12, 17) but
was decreased from 5 to 7 mo (28), whereas the
transient outward potassium current was decreased
significantly from 6 days to 7 mo after STZ injection
(12, 17, 24, 28). Our previous study using genetically
diabetic rats (WBN/Kob) demonstrated that the ventricular action potential duration is prolonged (25).
STZ-treated rats closely resemble the IDDM model,
except in a special case (23), whereas WBN/Kob rats
show some similarities with the non-insulin-dependent
diabetes mellitus (NIDDM) model. That is, male WBN/
Kob rats have been reported to develop hyperglycemia,
glycosuria, polyuria, and glucose tolerance from ,9 mo
of age, with a gradual and moderate decrease in serum
insulin levels (19). The rats survive without the administration of insulin, in contrast to Bio-Breeding rats, a
genetic model of IDDM. The pathological process
progresses slowly in WBN/Kob rats. Much clinical
attention has been paid to NIDDM-induced cardiomyopathy (7, 23). We have previously reported that the
L-type Ca21 channel did not alter significantly, although the response of the L-type Ca21 channel to
b-adrenergic stimulation decreased in WBN/Kob rats
(25). In this study, we first examined changes in the
potassium currents, especially the transient outward
potassium current, in this strain of genetically diabetic
rats.
MATERIALS AND METHODS
Animal model of diabetes. A particular strain of Wistarderived WBN rats has been maintained at the Institute of
Pathology, Bonn University (Bonn, Germany). Several of
these animals were brought to Japan by Dr. O. Kobori in 1976
and became known as WBN/Kob rats. A colony of these rats is
currently being maintained at the Shizuoka Laboratory
Animal Center (Shizuoka, Japan). Male WBN/Kob rats of 17
to 19 mo of age and age-matched Wistar rats (purchased from
the Shizuoka Laboratory Animal Center) were used. The
main clinical sign of diabetes, glucosuria, was evidently
detected at ,14 mo of age, after a marked glucose intolerance
at 12 mo. Thereafter, some animals developed hyperlipidemia
and gradual emaciation (19, 26). After purchase at 6 mo and
until they were used in the experiment, all rats were maintained at the Taisho Pharmaceutical Animal Laboratory and
were fed standard rat chow.
Cell preparation. Single ventricular cells were isolated
according to the methods previously reported (25). Briefly, the
rat was anesthetized with pentobarbital sodium (50 mg/kg
ip), and the heart was rapidly excised and attached to a
Langendorff perfusion apparatus. The heart was then retrogradely perfused for 2–3 min with nominally calcium-free
Krebs-Henseleit solution equilibrated with 95% O2-5% CO2 at
36°C. The Krebs-Henseleit solution contained (in mM) 130
NaCl, 4.8 KCl, 1.2 MgSO4, 1.1 NaH2PO4, 25 NaHCO3, and
0193-1849/97 $5.00 Copyright r 1997 the American Physiological Society
E695
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Tsuchida, Katsuharu, and Hiroshi Watajima. Potassium currents in ventricular myocytes from genetically diabetic rats. Am. J. Physiol. 273 (Endocrinol. Metab. 36):
E695–E700, 1997.—Our previous study demonstrated the
longer duration of action potential in ventricular myocytes
from genetically diabetic WBN/Kob rats without change in
calcium channel density compared with age-matched controls
[Tsuchida, K., H. Watajima, and S. Otamo. Am. J. Physiol.
267 (Heart Circ. Physiol. 36): H2280–H2289, 1994]. In the
present study we examined the alteration of potassium
currents, especially transient outward current, in ventricular
myocytes of genetically diabetic WBN/Kob rats. WBN/Kob
rats gradually develop hyperglycemia with aging and show
some similarity to non-insulin-dependent diabetes mellitus
models, which differ from the insulin-dependent streptozotocin-treated rat model. The density of the intracellular calcium
ion-independent transient outward current (Ito ) from 17- to
19-mo diabetic rat myocytes was significantly smaller than
that from age-matched control rat myocytes. In addition, the
density of Ito from 17- to 19-mo rat myocytes was significantly
less than that from 2-mo rat myocytes, suggesting that
aging-induced alteration of Ito was accelerated by the diabetic
state. The steady-state inactivation curves of Ito, the recovery
from Ito inactivation, and the other outward currents were not
significantly altered between diabetic myocytes and agematched control myocytes. In conclusion, the prolonged duration of action potential from genetically diabetic rat myocytes
is mainly due to the depressed Ito.
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POTASSIUM CURRENTS IN GENETICALLY DIABETIC HEARTS
Fig. 1. Representative outward currents recorded in
single ventricular myocytes isolated from diabetic (17–
19 mo) and age-matched control rat heart and in young
adult (2 mo) normal rat ventricular myocytes. Currents
were elicited by applying a 300-ms depolarizing step in
10-mV increments to 170 mV from a holding potential
of 270 mV every 5 s. Effects of 5 mM 4-aminopyridine
(4-AP) on the current elicited by a test pulse to 170 mV
from 270 mV are also represented. Six families of
current tracings are from different myocytes. Total cell
capacitances (pF) are as follows: right (R) 170, left (L)
132 in 17- to 19-mo control rats; R 153, L 186 in 17- to
19-mo diabetic rats; R 117, L 144 in 2-mo rats.
tetrodotoxin (TTX)-sensitive Na1 current, the Na1-activated
K1 current, and ICa. The composition of Na1-free solution was
(in mM) 135 choline chloride, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, 10
HEPES, 10 glucose, and 3 CoCl2 (pH 7.4 with KOH). In some
experiments, 4-aminopyridine (4-AP; Sigma Chemical, St.
Louis, MO) was used to block Ito, and TTX (Sankyo, Tokyo,
Japan) was used to block sodium ion current (INa).
Concerning the determination of cell membrane capacitance, the procedure described below was used. Just after the
patch was broken, pulses with a duration of 50 ms, from 260
to 262 mV, were applied to the cell. The exponential components of the decaying current were determined. Two time
constants were obtained corresponding to the electrode and
membrane capacitance. The time constant of the electrode
was ,0.02 ms, and the time constant of the membrane
capacitance was just under 2 ms. After the electrode capacitance was compensated for, the capacitance of the membrane
was calculated according to the equation
Cm 5 Tc · Io /Em [1 2 (I/Io)]
where Cm is the membrane capacitance, Tc is the time
constant of the membrane capacitance, Io is the maximum
capacitance current value, Em is the amplitude of the voltage
step, and I is the steady-state current. The series resistance
(Rs ) was calulated as
Rs 5 Em /Io
which ranged from ,4 to 10 MV. In some cases, membrane
capacitance and series resistance were determined electronically. Then membrane capacitance was compensated for, and
series resistance was reduced maximally for the voltageclamp experiments to examine the membrane currents.
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12.5 glucose. Enzymatic digestion was achieved by recirculating the perfusion apparatus with the calcium-free KrebsHenseleit solution containing 40–170 U/ml collagenase
(Yakult, Tokyo, Japan). The perfusion pressure was maintained at ,80 mmHg. Enzymatic perfusion was stopped after
15–30 min. The heart was then washed with Kraftbrühe (KB)
solution containing (in mM) 70 L-glutamic acid, 5 KCl, 20
taurine, 5 KH2PO4, 11 glucose, 5 N-2-hydroxyethylpiperazineN8-2-ethanesulfonic acid (HEPES), and 0.5 ethylene glycolbis (b-aminoethyl ether)-N,N,N8,N8-tetraacetic acid (EGTA)
free acid. The ventricle was separated from the whole heart
and minced into small pieces. Cells were filtered and stored in
KB solution at 4°C before being used in the electrophysiological experiment. After storage for 3–6 h in KB solution, the
cells were taken into the experimental chamber, which was
perfused with Ca21-containing Tyrode solution when the
electrophysiological experiment was performed.
Electrophysiological recording. Some cells were transferred to a recording chamber (0.3 ml vol) placed on the stage
of an inverted microscope (Diaphot TMD, Nikon, Tokyo,
Japan), and the chamber was perfused at a constant rate of
1–2 ml/min. Membrane currents were recorded using the
whole cell patch-clamp method described by Hamill et al. (8)
by use of a patch-clamp amplifier (CEZ-2300, Nihon Kohden)
connected to pClamp software program (Axon Instruments,
Burlingame, CA) or an Atari Mega ST4-operated EPC-9
patch-clamp system (Heka, Lambrecht, Germany). Patch
pipettes (2–4 MV) were fabricated using a puller (PP-83,
Narishige, Tokyo) and were heat polished (MF-83, Narishige).
To record transient outward current (Ito ), we filled the pipettes with solution containing (in mM) 140 KCl, 2 MgCl2, 1
CaCl2, 10 HEPES, 11 EGTA, and 5 Na2ATP (pH ajusted to 7.2
with KOH). The extracellular mediums were standard Tyrode
solution containing Co21, to eliminate a calcium ion current
(ICa ), or a Na1-free Co21-containing solution to eliminate the
POTASSIUM CURRENTS IN GENETICALLY DIABETIC HEARTS
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Data analysis. Statistical significance was determined
with the Student’s t-test for unpaired data or with the
repeated-measures analysis of variance test for multiple
comparison. A value of P , 0.05 was considered significant.
RESULTS AND DISCUSSION
Fig. 2. Current-voltage relationships of transient outward current
(Ito ) and terminal components of the outward current in ventricular
myocytes from diabetic (17–19 mo) and age-matched control rats,
and from young adult (2 mo) rats. Amplitude of Ito was measured as
the difference in current amplitude between the peak current amplitude and the terminal current amplitude at the end of 300-ms test
pulses. A: Ito; B: terminal current. r, Diabetic myocytes (n 5 23); s,
age-matched control myocytes (n 5 22); k, young adult myocytes (n 5
14). Inset a: voltage-clamp protocol; inset b: 4-AP-sensitive current
induced by a 300-ms test pulse to 170 mV from 270 mV. Control, n 5
16; WBN/Kob, n 5 16. Values are means 6 SE. * P , 0.05 vs.
age-matched control (17–19 mo) for diabetic rat myocytes, and 1 P ,
0.05 vs. young adult (2 mo) rat for aged (17 – 19 mo) rat myocytes at
each membrane potential (Student’s t-test). A supplemental explanation: repeated-measures analysis of variance test showed that current (Ito )-voltage relationships were significantly (P , 0.05) different
between diabetic rats and age-matched control rats. Current (Ito )voltage relationships were also significantly (P , 0.05) different
between 2-mo rats and 17- to 19-mo rats.
consistent with findings reported in STZ-treated diabetic rats (12, 17, 28). We used aged rats of 17–19 mo as
controls, because WBN/Kob rats gradually develop
hyperglycemia with aging. We examined the influence
of aging on Ito as well. Wei et al. (29) demonstrated that
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Plasma glucose level and clinical state. Plasma glucose levels were 558 6 16 (SE) mg/dl with 17- to 19-mo
WBN/Kob rats (n 5 34), whereas plasma glucose levels
of age-matched control Wistar rats were within the
range of ,110–140 mg/dl. Almost all WBN/Kob rats
showed glucosuria and ruffled hair including transient
alopecia and seemed sluggish at 17–19 mo.
Effects of genetic diabetes on Ito. Outward currents
were recorded after blocking ICa with 3 mM Co21 or INa
with 30–50 mM TTX, or changing extracellular NaCl
with choline chloride and the intracellular Ca21activated current with 11 mM EGTA added in the
pipettes. From a holding potential of 270 mV, depolarizing pulses in 10-mV increments were applied from
260 mV up to 170 mV. Ito was elicited by depolarizing
pulses more positive than 220 mV in both normal and
diabetic ventricular myocytes. Application of 5 mM
4-AP suppressed Ito, and the 4-AP-insensitive component of the outward current remained. Figure 1 shows
typical currents obtained from diabetic (17–19 mo),
age-matched control (17–19 mo), and young (2-mo) rat
ventricular myocytes.
Ito consists of 4-AP-sensitive and -insensitive components. The 4-AP-insensitive Ito is reported to be activated by increased intracellular calcium concentration
([Ca21]i ). Because we used a pipette solution containing
a relatively high concentration of EGTA (11 mM), the
[Ca21]i-dependent activation of Ito could be ruled out,
thereby leaving only one component of Ito, namely, the
4-AP-sensitive component shown in Fig. 1. Hereafter,
we will refer only to this component of Ito. The amplitude of Ito was expressed as the difference in current
between the peak current amplitude and the terminal
component of the current at the end of depolarizing test
pulses of 300-ms duration. The amplitude of Ito is
shown in Fig. 2A. The current density of the difference
in current between peak and 300-ms terminal currents
was significantly less in diabetic ventricular myocytes
than in the age-matched control myocytes. Apkon and
Nerbonne (2) described a slowly activating and inactivating K1 current (IK in their terminology) in rat
ventricular myocytes, which was sensitive to external
tetraethylammonium but not to 4-AP. Thus the terminal component at the end of 300-ms pulses is considered to consist of two different currents. One is the
residual component of the slowly inactivating Ito at the
end of 300-ms pulses, and the other is the 4-APinsensitive outward current. To determine the total Ito
as the 4-AP sensitive current, the difference in current
between the absence and the presence of 5 mM 4-AP
was obtained and shown in the inset of Fig. 2A. The
density of the 4-AP-sensitive current was also significantly less in the diabetic ventricular myocytes than in
the age-matched control myocytes. The decrease of Ito
density in the genetically diabetic WBN/Kob rats is
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POTASSIUM CURRENTS IN GENETICALLY DIABETIC HEARTS
the action potential duration in the senescent (24-mo)
rat ventricular myocytes was longer than that in the
young adult (7-mo) rat myocytes. Other authors have
reported similar changes of action potential duration
(3). Walker et al. (27) demonstrated that Ito was depressed more markedly in the cardiac cells of the aged
(24- to 25-mo) rat than in those of young adult (2- to
3-mo) rats. The current density of Ito from 17- to 19-mo
rat myocytes was significantly less than that from 2-mo
rat myocytes (Fig. 2A). The decay process of Ito seemed
to be fitted with biexponential curves with fast and slow
time constants (tf and ts, respectively). Because the
pulse duration was too short to determine ts correctly,
we have presented only tf. At 170 mV, tf was 32.8 6 2.9
ms (n 5 21) in 17- to 19-mo control, 32.3 6 1.2 ms (n 5
19) in 17- to 19-mo diabetic, and 33.4 6 1.5 ms (n 5 14)
in 2-mo normal rats. All values were not significantly
different. Possible values of ts seemed roughly ten times
longer than those of tf. The terminal current amplitude
at the end of 300-ms pulses is shown in Fig. 2B. The
terminal component was not altered in WBN/Kob rat
myocytes compared with that in the age-matched control rat myocytes. Jourdon and Feuvray (12) and Wang
et al. (28) described the significant decrease in this
time-independent outward current in the STZ-treated
rat myocytes. The present result is different from their
results. In addition, the current amplitude of the
terminal component was not altered between examinations of 17- to 19-mo rats and 2-mo rat myocytes (Fig.
2B).
Modification of inactivation kinetics of Ito. In this
series of experiments, Ito was also expressed as the
change in current between the peak amplitude and the
terminal component of the current. The steady-state
inactivation kinetics of Ito were compared between the
diabetic myocytes and the age-matched control myocytes. A test pulse to 170 mV (duration 300 ms) was
preceded by 1-s conditioning prepulses to various potentials (from 280 mV to 0 mV). The relative amplitude of
Ito (normalized by taking the value at 280 mV as unity)
was plotted against conditioning potentials, and the
data were fitted by the following Boltzmann distribution function with a least squares method
I/Imax 5 1 / [1 1 e(Vm2V0.5)/k]
where I/Imax is the relative amplitude of Ito, Vm is the
conditioning voltage, V0.5 is the voltage of half-inactivation, and k is the slope factor. The values of V0.5 and k
were 243.9 6 2.1 and 7.7 6 0.54 mV for age-matched
control myocytes, and 247.3 6 3.1 and 6.9 6 0.38 mV
for diabetic myocytes, respectively (Fig. 3). The value of
V0.5 was not altered significantly between the two
groups. The value of k was not significantly different
between the two groups, either. Furthermore, the values of V0.5 and k were 251.6 6 2.9 and 6.6 6 0.65 mV for
2-mo rat myocytes. The V0.5 value in 2-mo rat myocytes
Fig. 5. Inward rectifying potassium current density in ventricular
myocytes from diabetic and age-matched (17–19 mo) control and
young adult (2 mo) rats. Steady-state inward current measured at
end of 1-s duration of hyperpolarizing test pulses at 10 mV increased
voltage steps from a holding potential of 270 mV to 210 up to 2110
mV. r, Diabetic (n 5 15); s, age-matched (17–19 mo) control (n 5 13);
k, young adult (2 mo) (n 5 10) myocytes. Values are means 6 SE.
Inset: voltage-clamp protocol.
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Fig. 3. Steady-state inactivation curves of Ito in diabetic (17–19 mo)
and age-matched controls and young adult (2 mo) rat myocytes.
I/Imax, relative amplitude of Ito. r, Diabetic (n 5 18) myocytes; s,
controls (n 5 16); k, young adult myocytes (n 5 10). Values are
means 6 SE. Inset: voltage-clamp protocol.
Fig. 4. Time course of recovery from inactivation of Ito in ventricular
myocytes from diabetic (n 5 18), age-matched (17–19 mo) controls
(n 5 17), and young adult (2 mo) myocytes (n 5 12). Double pulses
(each duration 300 ms) from 270 mV to 170 mV were applied at
varying interpulse intervals (from 10 ms to 1 s) every 10 s. r, Diabetic
myocytes; s, age-matched control myocytes; k, young adult myocytes. Values are means 6 SE. Inset: voltage-clamp protocol.
POTASSIUM CURRENTS IN GENETICALLY DIABETIC HEARTS
understanding the pathophysiology of the diabetic heart
more thoroughly. We previously demonstrated that the
L-type Ca21 current was not altered in 19-mo WBN/Kob
rat ventricular myocytes in vitro under the condition of
the inhibition of the potassium current by use of the
whole-cell patch-clamp technique (25). However, the in
situ ventricular myocytes exist in circumstances without any artificial restriction of the potassium current.
The action potential duration of rat ventricular cells is
short enough to allow maximum Ca21 influx from
extracellular medium, so that the lengthening of the
action potential duration due to the inhibition of Ito may
lead to increased Ca21 influx and subsequent enhancement of Ca21 release from intracellular stores, resulting in some compensatory effects on the decreased
contractile force of diabetic myocardium (10, 21). Our
previous study (25) indicated that WBN/Kob rats demonstrated decreased contractile force in situ. Thus the
compensation resulting from lengthening the action
potential duration was not considered to be sufficient
for many reasons (see the introductory section of this
paper) other than the decreased ICa being responsible
for the decreased contractile force in WBN/Kob rats.
Recently, Xu et al. (30) demonstrated that the decreased Ito density may be caused by the decrease in
cellular glucose metabolism in ventricular myocytes of
short-term (14 days to 1 mo) STZ-treated rats (30).
Such biochemical changes may be involved in downregulating Ito in genetic NIDDM myocytes. The lower density of the Ito observed in the diabetic ventricular
myocytes may result from a decrease in the number of
channels. Concerning a molecular basis of the decreased Ito in diabetic myocytes, because Dixon et al. (5)
demonstrated that both the ventricular potassium (Kv)
4.2 and Kv 4.3 channels were likely to contribute to the
Ito in rat heart, the alteration of the expression of these
genes may account for the altered Ito density in WBN/
Kob rats. The molecular alterations in the channel
proteins remain to be elucidated as an interesting
problem.
Address for reprint requests: K. Tsuchida, Research Center,
Taisho Pharmaceutical Co., Ltd., 1–403 Yoshino-cho, Ohmiya, Saitama
330, Japan.
Received 25 February 1997; accepted in final form 4 June 1997.
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shifted several mV negatively, and significantly, compared with 17- to 19-mo normal rat myocytes, without
any change in k.
The time course of recovery from inactivation of Ito
was also compared between the diabetic and agematched control rat myocytes by use of a double-pulse
method. Double pulses to 170 mV from a holding
potential of 270 mV (each pulse duration 300 ms) were
applied every 10 s, while the interpulse interval was
increased from 10 ms to 1 s. The relative amplitude of
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induced by the first pulse was plotted against the
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Effects of diabetes on inward rectifying current. The
current amplitudes, elicited by hyper- and depolarizing
steps from 270 mV to up to 210 to about 2110 mV,
were not different between diabetic rat myocytes and
age-matched control rat myocytes (Fig. 5). Furthermore, the terminal component of the outward current
elicited by depolarizing test pulses up to ,0 mV from a
holding potential of 270 mV was not altered in diabetic
rat myocytes compared with age-matched control myocytes (Fig. 2B). These results suggest that the inward
rectifying potassium current (IK1 ) was not altered in
the diabetic state. No alteration in the IK1 has also been
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rats (12, 17, 20), whereas reduced IK1 was reported in
other diseased cardiac myocytes (13).
Summary of results and significance of the decrease
in Ito. The present study has, first of all, demonstrated
that the current density of Ito in ventricular myocytes
isolated from genetically diabetic rats sharing the
characteristics with NIDDM was significantly less than
that from 17- to 19-mo-old matched control rats. The
current density of Ito from aged (17- to 19-mo) rats was
significantly less than that from young adult (2-mo)
rats. Thus the Ito density was reduced by aging, and the
reduction was further accelerated by a diabetic state. Ito
is considered to be one of the most important repolarizing currents in rat myocytes (11). The present study
indicates that the lower Ito density, but not kinetic
changes of the Ito, may be responsible for the longer
duration of action potential in WBN/Kob rats, as already shown in our previous study (25). Shimoni et al.
(24) demonstrated that the diabetic state exerted differential effects on the Ito in epicardial and endocardial
myocytes from the left ventricle of short-term (6- to
7-day) STZ-treated rats. Such regional differences were
not a focus of the present study but may be important in
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POTASSIUM CURRENTS IN GENETICALLY DIABETIC HEARTS
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