Circulation. 1998;97:1290-1297
(Circulation. 1998;97:1290-1297.)
© 1998 American Heart Association, Inc.
N-Methyl-1-Deoxynojirimycin (MOR-14), an 
-Glucosidase Inhibitor, Markedly Reduced Infarct Size in Rabbit Hearts
Masazumi Arai, MD;
Shinya Minatoguchi, MD;
Genzou Takemura, MD;
Yoshihiro Uno, MD;
Tatsuya Kariya, MD;
Hisato Takatsu, MD;
Takako Fujiwara, MD;
Masaya Higashioka, MS;
Yoshiaki Yoshikuni, PhD;
; Hisayoshi Fujiwara, MD
From the 2nd Department of Internal Medicine, Gifu University School of
Medicine; Kyoto Women's University (T.F.); and Nippon Shinyaku Co, Ltd
(M.H., Y.Y.), Kyoto, Japan.
Correspondence to Hisayoshi Fujiwara, the 2nd Department of Internal Medicine, Gifu University School of Medicine, 40 Tsukasa-Machi, Gifu 500, Japan.
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Abstract
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Background—N-methyl-1-deoxynojirimycin
(MOR-14), an -glucosidase inhibitor, reduces the
glycogenolytic rate by inhibiting the -1,6-glucosidase of
glycogen-debranching enzyme in the liver, in addition to possessing an
antihyperglycemic action by blocking -1,4-glucosidase in the
intestine. Because the reduction of the glycogenolytic rate may be one
of the mechanisms of myocardial protection in ischemic
preconditioning, the compounds inhibiting myocardial
-1,6-glucosidase may be protective against ischemic damage.
Thus, we investigated whether MOR-14 could inhibit -1,6-glucosidase
and reduce the infarct size in rabbit hearts without collateral
circulation.
Methods and Results—MOR-14 dose-dependently decreased the
-1,6-glucosidase activity in rabbit heart extract. A tracer study
demonstrated the myocardial uptake of a considerable amount of MOR-14
sufficient to fully inhibit -1,6-glucosidase. To assess the infarct
size–reducing effect of MOR-14, 54 rabbits were subjected to 30-minute
coronary occlusion followed by 48-hour reperfusion.
Preischemic treatment with 25, 50, and 100 mg/kg of MOR-14
dose-dependently reduced the infarct size (to 26±4%, 19±3%, and
14±2% of the area at risk, respectively), compared with the saline
control (45±5%) without altering the blood pressure or heart rate.
Another 40 rabbits given 100 mg of MOR-14 or saline 10 minutes before
ischemia were euthanized at 10 or 30 minutes of
ischemia for biochemical analysis. MOR-14 decreased the
-1,6-glucosidase activity to 
20% in vivo, reduced the glycogen
breakdown, and attenuated the lactate accumulation at both 10 and 30
minutes of ischemia.
Conclusions—Preischemic treatment with MOR-14
preserved glycogen, attenuated the accumulation of lactate, and reduced
the myocardial infarct size by 69%. This cardioprotective effect was
independent of changes of blood pressure and heart rate or regional
blood flow. It may be associated with -1,6-glucosidase inhibition,
because MOR-14 markedly decreased the -1,6-glucosidase activity in
the heart.
Key Words: glucose • ischemia • metabolism • myocardial infarction • pharmacology
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Introduction
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Brief episodes of
ischemia and reperfusion before prolonged ischemia,
called "ischemic preconditioning," have had a marked
cardioprotective effect against ischemia-reperfusion injury in
the hearts of rats,1
rabbits,2 dogs,3 4 and
pigs.5 Because preconditioning the heart with
brief ischemia-reperfusion is not practical for patients, it is
desirable to precondition the heart with chemicals ("pharmacological
preconditioning") that exert a beneficial effect similar to
ischemic preconditioning. Generally, adenosine
A1 receptor,6 protein
kinase C,7 free radicals,8
and the KATP channel9 have
been considered to be mediator(s) of ischemic preconditioning.
In addition, preserved intracellular pH and decreased lactate
accumulation by a reduction of anaerobic glycolysis might
have a positive beneficial effect.10 11 The
reduction of glycolysis is related to the reduction of glycogenolysis
during sustained ischemia that may have been caused mainly by a
decrease in glycogen after ischemic preconditioning. Therefore,
we hypothesized that the pharmacological inhibition of glycogenolysis
might also protect the myocardium against ischemic
injury.
-Glucosidases are classified into -1,1-, -1,2-, -1,4-, and
-1,6-glucosidases. Because some -glucosidase
inhibitors, such as acarbose and voglibose, have an
antihyperglycemic action by inhibiting the -1,4-glucosidase that
induces the breakdown of oligosaccharides into absorbable
monosaccharides in the intestine, the inhibitors
have been universally used for the treatment of patients with diabetes
mellitus.12 It is reported that
1-deoxynojirimycin, an -glucosidase inhibitor, inhibits
-1,6-glucosidase of glycogen-debranching enzymes in the
liver13 14 and reduces the glycogenolytic rate,
in addition to inhibiting -1,4-glucosidase in the
intestine.15 However, it is unknown whether this
effect is also present in the heart. An -glucosidase
inhibitor, N-methyl-1-deoxynojirimycin (MOR-14,
synthesized by Nippon Shinyaku Co, Ltd) (Fig 1
) inhibits -1,4-glucosidase in the
intestine.15 Thus, in the present study, we
examined whether MOR-14 could block the -1,6-glucosidase activity in
the rabbit heart without collateral circulation to inhibit
glycogenolysis and reduce infarct size.
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Methods
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Materials
MOR-14 and [14C]MOR-14 were prepared by
Nippon Shinyaku Co, Ltd. [14C]glucose,
[14C]glucose-1-phosphate,
UDP-[14C]glucose, and
[3H]sucrose were purchased from Amersham Ltd.
BSA (fraction V) was obtained from Nacalai Tesque Inc. The
spectrophotometer was from Shimadzu Co, and the liquid scintillation
counter was from Packard Instrument Co. All other chemicals were
purchased from Sigma Chemical Co or Wako Pure Chemical Industries Ltd
and were of reagent grade.
In Vitro Study
The inhibitory action of MOR-14 against myocardial
-1,6-glucosidase was first examined in rabbit heart
extracts. For the preparation of -1,6-glucosidase, rabbit heart was
homogenized in a fivefold volume (wt/vol) of ice-cold
buffer containing 100 mmol/L glycylglycine (pH 7.4), 10
mmol/L EDTA, 10 mmol/L mercaptoethanol, and 100 mmol/L NaF.
-1,6-Glucosidase activity was determined by measurement of
[14C]glucose incorporation into glycogen,
according to the procedure of Nelson and
Larner.16 The substrate mixture contained 44
mmol/L glycylglycine (pH 6.5), 12.5% rabbit liver glycogen, 2.5
mmol/L [14C]glucose (20
µCi/µmol), 2.1 mmol/L EDTA, 4.1 mmol/L
mercaptoethanol, 0.02% gelatin, and MOR-14 (0, 0.01, 0.03, 0.1, 0.3,
or 1.0 µmol/L). This solution (16 µL) was warmed at 30°C for
2 minutes, and the reaction was then initiated by the addition of 4
µL of the rabbit heart homogenate. The reaction was
stopped 60 minutes later by the addition of 20 µL of 0.2N HCl. An
aliquot (30 µL) was spotted onto a Whatman GF/A glass fiber disk. The
disk was immediately washed in 66% ethanol for 20 minutes three times
each and dipped in 15 mL of acetone for 10 minutes. Then the disk was
dried, and the [14C] activity incorporated into
glycogen was measured with a liquid scintillation counter.
In Vivo Study
Surgical Preparation
Male Japanese White rabbits, each weighing 1.7 to 2.3 kg, were
anesthetized with 30 mg/kg sodium pentobarbital and
mechanically ventilated with room air. For rabbits receiving the
48-hour reperfusion, all surgical procedures were performed
aseptically. A polyethylene catheter (0.9-mm lumen diameter) was
inserted into the internal carotid artery and was advanced 1 cm
toward the heart for the drug and saline administration and blood
pressure monitoring. Blood pressure was measured with a fluid-filled
pressure transducer connected to the end of the cannula. After a left
thoracotomy was performed in the third intercostal space, the heart was
exposed and 4–0 silk string was placed beneath the large
arterial branch coursing down the middle of the
anterolateral surface of the left ventricle. Coronary
arterial occlusion and reperfusion were performed by
pushing or releasing the snare made with this string.
Infarct Size
To investigate the infarct size–reducing effect of MOR-14, 54
rabbits were assigned randomly into drug treatment or saline control
groups. There were four drug treatment groups, ie, three
preischemic treatment groups given 100 mg/kg (100/0 group,
n=10), 50 mg/kg (50/0 group, n=11), or 25 mg/kg (25/0 group, n=11) of
MOR-14 10 minutes before ischemia, and one prereperfusion
treatment group given 100 mg/kg of the drug 5 minutes before
reperfusion (0/100 group, n=11). In all treatments, the injected volume
was <1 mL/kg body wt. In the control group (n=11), an equivalent
volume of saline was injected 10 minutes before ischemia. After
the treatment, the coronary artery was occluded for 30 minutes
and reperfused. The blood pressure and heart rate were monitored
throughout the experiment until 20 minutes after reperfusion and were
recorded at baseline, at 0, 1, 3, 5, 10, 20, and 30 minutes of
ischemia, and at 5, 10, and 20 minutes of reperfusion. The
rabbits were then allowed to recover from anesthesia.
Forty-eight hours after reperfusion, the rabbits were
reanesthetized, and the hearts were excised. After the
coronary artery was reoccluded at the identical position,
monastral blue dye was retrogradely infused into the ascending aorta at
80 mm Hg to determine the area at risk. Because we left the
string beneath the coronary artery at the occluded site after
reperfusion and closed the chest, it was easy to identify the location
of the previous coronary ligation. The left ventricle was
sectioned into five slices parallel to the
atrioventricular ring. Each slice was weighed,
incubated in a 1% solution of triphenyl tetrazolium chloride at 37°C
to visualize the infarct area,17 and
photographed. The areas of the ischemic region and the
infarcted myocardium were traced on each slice of left
ventricle and multiplied by the weight of the slice, then expressed as
a fraction of the risk region or left ventricle for each heart.
Biochemical Determinations
Forty additional rabbits were used to assess the in vivo effect
of MOR-14 on myocardial glycogen metabolism and lactate
accumulation in the ischemic myocardium. The
rabbits were randomized to receive either 100 mg/kg of MOR-14 or saline
10 minutes before ischemia and were killed at 10 or 30 minutes
of ischemia. Hearts were excised, and transmural samples, each
weighing 200 mg, were taken from the center of the ischemic
region and the opposite nonischemic region. The border of the
ischemic region was defined by the distribution of cyanosis and
marked on the epicardium in ink. The samples were frozen immediately
and stored at -80°C until measurement. Samples were weighed,
homogenized, and used for the following measurements.
Myocardial glycogen and lactate
For myocardial glycogen measurement, the homogenate
was subsequently hydrolyzed with amyloglucosidase. The resulting
glucose residue was then measured by an NADP-linked spectrophotometric
method using hexokinase and glucose-6-phosphate
dehydrogenase.18 Lactate in the extracts was
measured spectrophotometrically by monitoring the hydrogen peroxide
formation resulting from the enzymatic reaction with lactate
oxidase.19 For precise analysis, we
estimated the glycogen reduction and the lactate accumulation in
ischemic myocardium as a difference in the
concentration of glycogen and lactate between ischemic and
nonischemic areas in each heart.
-1,6-Glucosidase activity
As already mentioned, the -1,6-glucosidase activities of the
frozen samples were determined by measurement of the
[14C]glucose incorporation into glycogen. The
homogenate was incubated with the substrate mixture
containing 47 mmol/L glycylglycine (pH 6.5), 13.3% rabbit liver
glycogen, 2.7 mmol/L [14C]glucose (20
µCi/µmol), 2.2 mmol/L EDTA, 4.4 mmol/L
mercaptoethanol, and 0.02% gelatin. The -1,6-glucosidase activity
is expressed as pmol glucose incorporated per minute per mg protein.
The amount of protein was determined by a dye-binding assay method with
BSA.20
Glycogen phosphorylase activity
Like -1,6-glucosidase, the glycogen phosphorylase
activities were measured in the direction of glycogen synthesis
according to the procedure of Stalmans and
Hers.21 For the measurement of glycogen
phosphorylase a, an active form of glycogen
phosphorylase, 133 mmol/L maleate (pH 6.5) and
1.3 mmol/L caffeine were added to the substrate mixture containing
1.3% rabbit liver glycogen, 13 mmol/L
[14C]glucose-1-phosphate (0.2 µCi/µmol),
267 mmol/L NaF, 6.7 mmol/L EDTA, and 6.7 mmol/L
mercaptoethanol to specifically inhibit phosphorylase
b, an inactive form of glycogen phosphorylase,
and allow the determination of phosphorylase a
without the interference of phosphorylase b. The
total phosphorylase (a+b) activity was measured
after the addition of 33 mmol/L glycylglycine (pH
6.5) and 6.7 mmol/L AMP, which increases the activity of
phosphorylase b with little effect on
phosphorylase a, instead of maleate and
caffeine. For both assays, the homogenate of the myocardial
samples was incubated for 20 minutes. The reaction was stopped by the
addition of 0.2N HCl. The enzyme activity was measured by
[14C] radioactivity counted in the glycogen
with a liquid scintillation counter and is expressed as nmol
glucose-1-phosphate incorporated per minute per milligram protein.
Cellular Uptake of MOR-14
To assess the myocardial cellular uptake of MOR-14, we performed
a tracer study. Six isolated rabbit hearts were perfused with a buffer
containing [14C]MOR-14 and
[3H]sucrose (specific activity, 223 mCi/mol for
both). After constant perfusion, the radioactivities of
[14C] and [3H] in the
myocardium and the perfusate were measured with a
liquid scintillation counter. The cellular uptake of MOR-14 was
determined by the method of Angello et al22 and
Rattigan et al.23
[14C]MOR-14 was prepared as described by Faber
et al.24 The specific activity of
[14C]MOR-14 was calculated to be 55.8
mCi/mmol.
Myocardial Ultrastructure
Eight rabbits were given 100 mg/kg of MOR-14 or an equivalent
volume of saline 10 minutes before ischemia. Thirty minutes
after coronary occlusion, the hearts were excised, and
myocardial samples were obtained from the ischemic region and
the nonischemic region. Each sample was fixed with 2.5%
glutaraldehyde in 0.1 mol/L phosphate buffer and
postfixed with 1% osmium tetroxide. For each heart, four
representative blocks from the ischemic and
nonischemic region were thin-sectioned, mounted on plain copper
grids, stained with uranyl acetate and lead citrate, and examined under
a transmission electron microscope.25
Statistical Analysis
All data are presented as mean±SEM. Risk and infarct
sizes were compared among the groups by one-way ANOVA combined with
Bonferroni's post hoc test for multiple comparisons. The difference in
blood pressure and heart rate over the time course between the control
and the drug-treated groups was assessed by two-way repeated-measures
ANOVA. Student's t test was used to assess the differences
in glycogen, lactate, and enzyme activities between pairs of groups.
Differences with P<.05 were considered significant.
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Results
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In Vitro Study
The inhibitory action of MOR-14 against
-1,6-glucosidase was examined in vitro. In rabbit heart extracts,
MOR-14 dose-dependently inhibited -1,6-glucosidase (Fig 2). The IC50 value
of MOR-14 for rabbit -1,6-glucosidase activity was 0.03
µmol/L.
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Figure 2. Effect of MOR-14 on activity of
-1,6-glucosidase in rabbit heart extracts. Results are mean±SEM of
six observations.
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In Vivo Study
Infarct Size
Of the 54 rabbits used for the infarct-size study, 1 rabbit in the
25/0 group died of ventricular fibrillation. One rabbit in
the 0/100 group died before completion of the experiment of an
undefined cause. An additional 2 rabbits with areas at risk <15% of
the left ventricle were excluded to avoid an inaccurate infarct size
definition as the percent of area at risk. The areas at risk were not
significantly different among the groups (Fig 3A). The mean infarct size, assessed by
tetrazolium staining and expressed as a percentage of the area at risk,
was significantly and dose-dependently reduced in the
preischemic treatment groups of 100 mg/kg (14.4±2.2%,
n=10), 50 mg/kg (18.7±2.6%, n=10), and 25 mg/kg (25.7±4.4%, n=10)
compared with the saline control group (44.6±5.2%, n=10) or the
prereperfusion treatment group of 100 mg/kg (39.5±4.2%, n=10) (Fig 3B). There were no significant differences in infarct size between the
saline control group and the prereperfusion treatment group, indicating
that only preischemic treatment with MOR-14 reduced infarct
size. The administration of MOR-14 had no effect on blood pressure or
heart rate (Table). The five groups
showed similar blood pressures and heart rates during the
experiments.
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Figure 3. Comparison of area at risk as percentage of left
ventricle (A) and of infarct area as percentage of area at risk (B).
100/0, 50/0, 25/0, and 0/100 represent doses (mg/kg) of MOR-14
injected before ischemia/before reperfusion. One-way ANOVA
combined with Bonferroni's post hoc test for multiple comparisons was
used. *P<.05 vs saline group, #P<.05 vs
0/100 treated group. Bars represent mean±SEM.
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Myocardial Glycogen and Lactate
The myocardial glycogen content (mg/g wet tissue) was
significantly reduced in the ischemic region compared with the
nonischemic region both in the saline and the pretreatment (100
mg/kg of MOR-14) groups at 10 minutes of ischemia (saline,
n=10, 1.1±0.1 versus 2.5±0.2; and MOR-14, n=10, 1.5±0.2 versus
2.5±0.2) and at 30 minutes of ischemia (saline, n=10,
0.4±0.03 versus 2.7±0.3; and MOR-14, n=10, 1.3±0.1 versus 2.6±0.2)
(Fig 4A). The lactate level (mg/g wet
tissue) was significantly higher in the ischemic than in
the nonischemic myocardium in both the groups at 10
minutes of ischemia (saline, 3.6±0.2 versus 1.7±0.1; and
MOR-14, 2.6±0.1 versus 1.5±0.2) and at 30 minutes of ischemia
(saline, 3.9±0.2 versus 1.3±0.1; and MOR-14, 2.6±0.2 versus
1.4±0.1) (Fig 4B). In the saline group, myocardial glycogen
consumption and lactate accumulation in the ischemic area,
estimated as a difference in the concentration of glycogen and lactate
between nonischemic and ischemic areas, progressively
increased between 10 and 30 minutes of ischemia. Glycogen
reduction was 1.5±0.1 and 2.2±0.3 mg/g wet wt (P<.05) and
lactate accumulation, 1.9±0.2 and 2.6±0.2 mg/g wet wt
(P<.05) at 10 and 30 minutes of ischemia,
respectively (Fig 4C and 4D). However, in the drug-treated group, there
was no further significant consumption of glycogen or accumulation of
lactate in the ischemic area during this period. In this group,
glycogen reduction was 1.0±0.2 and 1.3±0.2 mg/g wet wt
(P=NS) and lactate accumulation, 1.1±0.1 and 1.2±0.2 mg/g
wet wt (P=NS) at 10 and 30 minutes of ischemia,
respectively (Fig 4C and 4D). The glycogen reduction and the lactate
accumulation were significantly lower in the pretreatment group than
the saline group at both 10 and 30 minutes of ischemia.
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Figure 4. Myocardial glycogen (A) and lactate (B)
concentrations in ischemic and nonischemic areas at 10
and 30 minutes of ischemia, and glycogen consumption (C) and
lactate accumulation (D) in ischemic area for 10 and 30 minutes
of ischemia. Both glycogen and lactate concentrations in
transmural myocardial samples were measured spectrophotometrically by
use of enzyme reactions. Glycogen reduction and lactate accumulation in
ischemic myocardium were assessed by difference in
content of glycogen and lactate between ischemic and
nonischemic areas. *P<.05 for each comparison.
Bars represent mean±SEM.
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To assess further effects of MOR-14 on carbohydrate
metabolism, the amounts of lactate accumulation and
glycogen breakdown were converted from the value of mg/g wet wt shown
above to µmol/g wet wt. Then we calculated the ratio of lactate
accumulation (µmol/g wet wt) to the breakdown of glycogen measured as
glycosyl units (µmol/g wet wt). Their ratios (lactate accumulation to
glycogen breakdown) were 2.1±0.2 (17.3±1.6 to 8.2±0.7) at 10 minutes
of ischemia and 2.0±0.2 (23.5±2.1 to 12.4±1.4) at 30 minutes
of ischemia in the saline group, and 1.8±0.3 (9.8±1.2 to
5.4±0.8) at 10 minutes of ischemia and 1.5±0.2 (10.8±2.1 to
7.3±1.0) at 30 minutes of ischemia in the MOR-14–treated
group. The ratio of lactate accumulation to glycogen breakdown at 30
minutes of ischemia was significantly lower in the
MOR-14–treated group than in the saline group.
Glycogenolytic Enzymes
The -1,6-glucosidase activities (pmol ·
min-1 · mg
protein-1) in the saline and the drug-treated
groups were, respectively, 9.4±0.9 and 2.0±0.2 at 10 minutes of
ischemia and 7.8±1.4 and 1.7±0.2 at 30 minutes of
ischemia in the ischemic area, and 9.7±0.8 and
1.9±0.1 at 10 minutes of ischemia and 8.9±1.5 and 1.9±0.2 at
30 minutes of ischemia in the nonischemic area (Fig 5). Thus, consistent with
glycogen preservation, myocardial -1,6-glucosidase activity in the
drug-treated hearts was strongly inhibited in vivo in both the
ischemic and nonischemic myocardium over 30
minutes of ischemia.
In both the saline and the drug-treated groups, the myocardial glycogen
phosphorylase a activity (nmol ·
min-1 · mg
protein-1) in the ischemic area was
significantly suppressed compared with that in the nonischemic
area (saline, 13.3±1.4 versus 20.5±2.0; and MOR-14, 13.8±2.4 versus
21.2±2.4) at 30 minutes of ischemia, presumably by higher
tissue levels of anaerobic glycolytic products such as
lactate26 (Fig 6A).
The total phosphorylase activities were not different
between the ischemic and the nonischemic areas (Fig 6B). There was no difference in glycogen phosphorylase
a and a+b activities between the
saline and the drug-treated groups in both the ischemic and the
nonischemic regions at given time points, indicating that the
treatment with MOR-14 did not alter the glycogen
phosphorylase activities.
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Figure 6. Myocardial glycogen phosphorylase
a (A) and a+b (B) activity (nmol ·
min-1 · mg protein-1) at 10 and 30
minutes of ischemia. MOR-14 had no effect on
phosphorylase activities. *P<.05 for
comparison. Bars represent mean±SEM.
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Cellular Uptake of MOR-14
The amount of MOR-14 trapped by the cells after 10 minutes of
perfusion was 10.3±4.4 µmol/kg tissue. This concentration is
much higher than the IC50 value of MOR-14 for
rabbit -1,6-glucosidase (Fig 2
).
Myocardial Ultrastructure
Thirty minutes after the coronary arterial
occlusion, the ischemic myocardium pretreated with
100 mg/kg of MOR-14, compared with untreated ischemic
myocardium, showed considerable glycogen preservation, rare
myofibril disruption, less margination of chromatin, and clearly less
intramitochondrial amorphous densities (Fig 7).
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Figure 7. Representative transmission
electron micrographs. Bar=1 µm. A, Nonischemic
myocardium. B and C, Ischemic region of untreated
and drug-treated heart. Note that glycogen granules (arrows) in
pretreated heart (C) are considerably preserved compared with control
heart (B), although less than those in nonischemic heart (A).
Ultrastructural damage, indicated by disruption of myofibrils, markedly
clumped and marginated chromatin in nucleus (N), and amorphous
densities (arrowheads) in mitochondria (M), is conspicuous in untreated
myocardium (B) but only mild in pretreated
myocardium (C).
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Discussion
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The present data demonstrated that (1) MOR-14 dose-dependently
inhibited myocardial -1,6-glucosidase activity in vitro and (2)
preischemic treatment with MOR-14 markedly inhibited
-1,6-glucosidase activity during 30-minute ischemia in vivo
and reduced the infarct size after 48-hour reperfusion. We believe that
this is the first report of the inhibition of -1,6-glucosidase in
the heart and the reduction of myocardial infarct size by an
-glucosidase inhibitor.
Infarct Size–Reducing Effect of MOR-14
The infarct size–reducing effect was observed in the
preischemic treatment groups but not in the prereperfusion
treatment group. In addition, the electron microscopic analysis
clearly revealed the protective effect of MOR-14 against myocardial
cellular damage during ischemia. Therefore, this compound
protects the myocardium against ischemic injury but
not reperfusion injury. MOR-14 did not have any effect on blood
pressure and heart rate, which might alter the infarct size. In
addition, the infarct size–reducing effect was also independent of the
regional blood flow, because the rabbit heart has no collateral
circulation.27
The MOR-14 treatment preserved glycogen content and attenuated lactate
accumulation in the ischemic myocardium. Although
glycogenolysis followed by anaerobic glycolysis is the
major metabolic pathway for the energy production
during ischemia, accumulated anaerobic glycolytic
products, such as lactate, NADH, and H+, may
contribute to intracellular acidosis and osmotic
load28 and cause irreversible cell
damage.29 This anaerobic
glycolysis-mediated mechanism of ischemic injury has been
supported by several observations. Ischemic preconditioning
decreases myocardial glycogen content after preconditioning, attenuates
lactate accumulation and intracellular acidosis during the sustained
ischemia, and reduces the infarct
size.10 30 31 In addition, the time course of
glycogen repletion after a brief ischemic episode
paralleled the loss of the protection from ischemic
injury.32 These findings suggest that the
attenuation of glycolytic flux and lactate accumulation is an important
mechanism of protection in the ischemic preconditioned heart.
In addition, preischemic glycogen reduction and glycolytic
inhibition have each been reported to improve the functional recovery
of isolated ischemic-reperfused rat
hearts.33 34 35 More recently, Hadour et
al36 demonstrated that the myocardial infarct
size was limited in diabetic rabbits, in which the glycogenolytic
activity might be suppressed by the low insulin level. Thus, the
cardioprotective effects of MOR-14 appear to be associated with the
inhibition of glycogenolysis during ischemia.
We found that MOR-14 markedly inhibited the -1,6-glucosidase
activity in the rabbit hearts. This suggests that the preservation of
glycogen and the attenuation of lactate accumulation by this compound
are related to the inhibition of -1,6-glucosidase. In the
present study, however, the inhibitory effect of MOR-14
on -1,6-glucosidase was observed in an extract of
myocardium in which the cell membranes were disrupted.
Therefore, we performed the experiment to assess the myocardial
cellular uptake of MOR-14. The amount of MOR-14 trapped by the cells
after 10 minutes of perfusion was 10.3±4.4 µmol/kg tissue,
which is much higher than the IC50 value
(0.03 µmol/L) of MOR-14 for rabbit -1,6-glucosidase.
Conversely, the concentration of MOR-14 in the perfusate was
200 µg/mL, which is lower than the plasma concentration of MOR-14 at
5 (470±32) and 10 (392±29) minutes after injection in our infarct
study. Thus, the amount of MOR-14 taken up by the cells of the perfused
rabbit heart was sufficient to completely block myocardial
-1,6-glucosidase activity. Because it is reasonable to assume
that membrane function in isolated heart preparations is not
fundamentally different from membrane function in vivo, a considerable
amount of MOR-14 may also be incorporated and inhibit
-1,6-glucosidase in vivo.
One mole of glycogen measured as glycosyl units gives rise to 2 mol
lactate. In the present study, the accumulation of lactate
(µmol/g wet wt) during ischemia was actually nearly twice as
high as the amount of glucose (µmol/g wet wt) originated from
glycogen in the saline group. However, the ratio of lactate
accumulation to the amount of mobilized glycogen in the MOR-14–treated
group was significantly lower than that in the saline group at 30
minutes of ischemia (1.5±0.2 versus 2.0±0.2,
P<.05). This suggests other beneficial effects of MOR-14 on
glucose metabolism during ischemia in addition to
the glycogenolytic inhibition through the inhibitory effect
of -1,6-glucosidase. That is, MOR-14 might decrease the accumulation
of products of anaerobic glycolysis through inhibition
of glycolysis itself and/or might stimulate glucose oxidation and
divert lactate to be used as a substrate. The stimulation of glucose
oxidation also may be cardioprotective, because it allows the
production of more ATP per quantity of O2
and reduction in the buildup of
H+.37 Further studies are
warranted to clarify whether these metabolic alterations
are also involved in the pharmacological effects of MOR-14 and
contribute to cardioprotection.
Effect of MOR-14 on Glycogen Metabolism
In the saline group, the glycogen reduction and lactate
accumulation in the ischemic area increased progressively
between 10 and 30 minutes of ischemia. In the pretreatment
group, they occurred during the first 10 minutes of ischemia
but disappeared between 10 and 30 minutes of ischemia. The
difference in glycogen reduction and lactate accumulation between the
saline group and the pretreatment group became larger at 30 minutes
than at 10 minutes of ischemia. This special feature of the
time course of glycogen depletion in drug-treated hearts could be
explained by the present observation that pretreatment with the
drug markedly decreased the myocardial -1,6-glucosidase activity but
did not affect the glycogen phosphorylase activity during
ischemia. The breakdown of glycogen proceeds through the
interaction of glycogen phosphorylase and the
-1,6-glucosidase and glucanotransferase activities of the
glycogen-debranching enzyme. The external side chains of a glycogen
molecule attach through an -1,6 bond to an adjacent -1,4–linked
chain. Glycogen phosphorylase first releases
glucose-1-phosphate until the external chains have been shortened to
form maltotriosyl units. Subsequently, glucanotransferase removes the
maltotriosyl unit from the -1,6–linked stub and attaches it to the
free C4 of the main chain. The single remaining
-1,6–linked glucosyl unit is then removed as free glucose by
-1,6-glucosidase, while additional -1,4–linked glucosyl residues
become available for phosphorylase. Therefore, even when
-1,6-glucosidase activity is blocked, some glucose-1-phosphates are
released by the action of unaffected phosphorylase, and the
glycogen concentration decreases. However, glycogen may be decreased
slowly thereafter, because glycogen phosphorylase alone
cannot trim the four glucosyl residues located distal to the glucose,
which has an -1,6–linked glucosyl unit.
In the present study, the total glycogen content (mg/g wet wt) in
the nonischemic area in the MOR-14–treated group (2.6±0.2)
was similar to that in the saline-treated group (2.7±0.3) at 30
minutes of ischemia (ie, 40 minutes after injection of MOR-14).
Myocardial glycogen content is regulated by both synthesis and
degradation (ie, glycogen turnover). Therefore, when glycogenolysis is
inhibited by MOR-14, one would expect an increase in net glycogen.
However, Henning et al38 recently reported that
glycogen synthesis ranged from 0.33 to 0.51 µmol glucose
· min-1 · g dry
wt-1 in the aerobic, isolated working rat heart,
corresponding to 0.3% to 0.5% of initial glycogen content per minute.
These rates of synthesis agree with observations in a rat model
reported by Goodwin et al39 (0.17 to 0.62
µmol glucose · min-1 · g dry
wt-1). This indicates that, even when the
baseline rate of synthesis is completely maintained and degradation is
totally stopped, glycogen content increases by only 12% to 20% over
40 minutes, although data from a rabbit in vivo model are unknown.
MOR-14 specifically inhibited -1,6-glucosidase but did not affect
phosphorylase activity. In fact, glycogen content in the
ischemic region decreased by 50% after 30-minute
ischemia despite the blockade of -1,6-glucosidase by MOR-14
in the treated group. This suggests that the glycogenolytic rate
mediated by phosphorylase activity is quite high, although
the precise rate is unknown in nonischemic rabbit
myocardium. In addition, glycogen itself may inhibit
glycogen synthesis when degradation of glycogen is blocked. That is,
MOR-14 may shut off the glycogen turnover altogether. These
suppositions may explain the absence of increase in glycogen levels in
the nonischemic area of the MOR-14–treated group. At
present, the baseline rate of glycogen turnover is unknown, and how
it is affected by MOR-14 in rabbit hearts remains to be clarified.
The reported glycogen concentrations in nonischemic
myocardium were quite variable in both
rabbits40 41 and rats,32 33
although the glycogen levels of 2.7 and 2.5 mg/g wet wt in the
present study were within the range of reported values. The reason
for the variability remains obscure, because the measurement was done
by the same standard method. However, the myocardial glycogen levels in
rats have recently been reported to be approximately two times higher
in the fasted than in the fed state.42 43
Therefore, diet milieus before experiment may be one factor causing
this variability.
Clinical Benefits of MOR-14
In the present study, MOR-14 clearly protected
myocardium against the ischemic induction of
infarcts when given just before ischemia but not when given
before reperfusion. MOR-14, like other N-substituted
derivatives of 1-deoxynojirimycin, is completely absorbed from the
intestines.14 In addition, when taken orally,
MOR-14 develops an antihyperglycemic action through its
-1,4-glucosidase inhibitory action in the
intestine.15 Further studies should be conducted
to assess the suitability of this drug for clinical use.
|
Acknowledgments
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This study was supported in part by a research grant,
"Mechanism and Clinical Application of Ischemic
Preconditioning," from the Japanese Circulation Society.
Received July 9, 1997;
revision received October 16, 1997;
accepted November 3, 1997.
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Top
Abstract
Introduction
