Changes in cardiac protein kinase C activities and isozymes in
streptozotocin-induced diabetes
Xueliang
Liu1,
Jingwei
Wang1,
Nobuakira
Takeda2,
Luciano
Binaglia3,
Vincenzo
Panagia1, and
Naranjan S.
Dhalla1
1 Institute of Cardiovascular
Sciences, St. Boniface General Hospital Research Centre and Department
of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg,
Manitoba R2H 2A6, Canada; 2 Aoto
Hospital, Department of Internal Medicine, Jikei University, Tokyo 125, Japan; and 3 Institute of
Biochemistry, University of Perugia, 06100 Perugia, Italy
 |
ABSTRACT |
To understand
cardiac dysfunction in diabetes, the activity of protein kinase C (PKC)
and protein contents of its isozymes (PKC-
, -
, -
, and -
)
were examined in diabetic rats upon injection of streptozotocin (65 mg/kg iv). The hearts were removed at 1, 2, 4, and 8 wk, and some of
the 6-wk diabetic animals had been injected with insulin (3 U/day) for
2 wk. The Ca2+-dependent PKC
activity was increased by 43 and 51% in the homogenate fraction and 31 and 70% in the cytosolic fraction from the 4- and 8-wk diabetic
hearts, respectively, in comparison with control values. The
Ca2+-independent PKC activity was
increased by 24 and 32% in the homogenate fraction and 52 and 89% in
the cytosolic fraction from the 4- and 8-wk diabetic hearts,
respectively, in comparison with control values. The relative protein
contents of PKC-
, -
, -
, and -
isozymes were increased by
43, 31, 48, and 38%, respectively, in the homogenate fraction and by
126, 119, 148, and 129%, respectively, in the cytosolic fraction of
the 8-wk diabetic heart. The observed changes in heart homogenate and
cytosolic fractions were partially reversible upon treatment of the
diabetic rats with insulin. The results suggest that the increased
myocardial PKC activity and increased protein contents of the cytosolic
PKC isozymes are associated with subcellular alterations and cardiac
dysfunction in the diabetic heart.
diabetic cardiomyopathy; diabetic heart dysfunction
 |
INTRODUCTION |
SEVERAL INVESTIGATORS have suggested that chronic
diabetes mellitus in patients and experimental animals is associated
with the presence of various defects in contractile function of the heart (5). Although the exact subcellular mechanism responsible for the
impaired contractile force development in the diabetic heart is
unknown, it has been shown that the status of cardiac contractile
proteins in the diabetic heart is altered with respect to ATPase
activities in myofibrils, actomyosin, and myosin as well as myosin
isozyme composition (6, 20, 27). A depression in the sarcoplasmic
reticular Ca2+ transport has also
been reported in the diabetic heart, and this defect is considered to
account for the inability of the diabetic heart to relax fully (11, 19,
26). Because protein kinase C (PKC) has been shown to inhibit
myofibrillar ATPase (24) and sarcoplasmic reticular
Ca2+ pump (28) activities, it is
possible that subcellular changes in the diabetic heart may be due to
alterations in the PKC activity and/or PKC-mediated signal transduction
mechanism. This view is consistent with observations that diabetes is
associated with translocation of the
-isoform of PKC from cytosolic
to particulate fraction of cardiomyocytes, and this change was
prevented by the blockade of angiotensin II receptors, which are known
to stimulate the PKC activity (21). Furthermore, increased
phosphorylation of troponin-I in the diabetic heart has been considered
to be due to the activation of PKC (18, 21). In fact, the concentration of diacylglycerol, which is known to activate PKC, was increased in the
diabetic heart (25). However, the results with respect to changes in
the PKC activity in the diabetic heart are conflicting. In this regard,
Xiang and McNeill (32) have reported an increase and a decrease in
cardiac PKC activities in particulate and cytosolic fractions,
respectively, from diabetic rats. On the other hand, an increase of PKC
activity in both particulate and cytosolic fractions from the diabetic
heart was observed by Tanaka et al. (30). In view of such contradictory
findings and no information regarding changes in the PKC activity in
heart homogenate, this study was undertaken to examine PKC activities
in the homogenate, cytosolic, and particulate fractions from the 1-, 2-, 4-, and 8-wk diabetic hearts. Because PKC is expressed in rat heart
in different isoforms such as
-,
-,
-, and
-isozymes (2,
13), it was also the purpose of this investigation to examine changes in the contents of different PKC isozymes in cardiac homogenate, cytosolic, and particulate fractions in diabetes induced by
streptozotocin in rats for a period of 8 wk. The effects of insulin on
changes in cardiac PKC activities and PKC isozyme contents were studied by treating the 6-wk diabetic rats with insulin for a period of 2 wk.
 |
MATERIALS AND METHODS |
Experimental model. Male
Sprague-Dawley rats weighing ~175-200 g were randomly separated
into control and experimental groups. The experimental animals received
an intravenous injection of 0.1 M citrate-buffered streptozotocin (pH
4.5) at a dosage of 65 mg/kg body wt. Control animals received a
similar injection of the vehicle alone. These animals were maintained
on normal rat chow and water ad libitum and then killed by decapitation at 1, 2, 4, and 8 wk. In some experiments, randomly selected diabetic animals at 6 wk after streptozotocin injection were given subcutaneous injections of 3 U Humulin U/day for 2 wk and were labeled as the insulin-treated group. It is pointed out that Humulin U (Eli Lilly Canada, Toronto, ON) is a long-acting insulin with a slower onset of
action in comparison with the regular insulin. To assess the control of
diabetic state by insulin, the blood samples were obtained from the
tail vein twice a week in the morning before insulin was injected, and
the glucose concentration was measured with a blood-glucose meter
(Lifescan Canada, Burnaby, BC, Canada). The blood from the decapitated
animals was collected in heparinized tubes and centrifuged at 1,000 g for 10 min in order to obtain plasma. The heart was dissected out immediately and perfused for ~2
min with ice-cold Krebs-Ringer phosphate buffer containing 5 mM glucose
to remove blood. The atria and large vessels were carefully trimmed,
and the ventricles were weighed and cut into small pieces (~50 mg
each) for biochemical analysis. The pieces of tissue were frozen in
liquid N2 and stored at
80°C for 7-12 days. The freezing and storage of the
small pieces of tissue had no effect on the enzyme activity or isozyme
composition. Plasma samples were analyzed for glucose and insulin
levels with the Sigma diagnostic kit (Sigma-Aldrich Canada, Oakville,
ON) and the standard radioimmunoassay technique (Amersham, Arlington
Heights, IL), respectively. Some of the animals were used to assess the status of cardiac contractile activity in terms of the rate of pressure
development (+dP/dt) and the rate of
pressure fall (
dP/dt) in the
left ventricle according to the method described previously (8). The
experimental model employed in this study is similar to that used
previously for establishing the presence of diabetic cardiomyopathy as
indicated by alterations in cardiac function, metabolism, subcellular
organelles, and ultrastructure (5, 11, 18, 27).
Preparations of tissue extract for PKC
determination. The preparations of tissue extract for
PKC was carried out by the method described by other investigators (3).
All procedures were carried out at 4°C. The ventricular tissue (50 mg) was minced in 1 ml of buffer
A (50 mM Tris · HCl,
0.25 M sucrose, 10 mM EGTA, 4 mM EDTA, 20 µg/ml leupeptin, and 200 U/ml aprotinin, pH 7.5) and homogenized in a Polytron (Brinkmann
PT3000, Mississauga, Canada) at a setting of eight for 2 × 30 s
and sonicated for 2 × 15 s. In one set of experiments, the
homogenate was incubated with 1% Triton X-100 on ice for 60 min to
solubilize the PKC enzyme that is bound with subcellular structures.
This Triton X-100-treated homogenate was then centrifuged at 105,000 g for 60 min in a Beckman ultracentrifuge (Beckman L70); the supernatant thus obtained was labeled as the homogenate fraction. In another set of experiments, the
homogenate without Triton X-100 treatment was centrifuged at 105,000 g for 60 min to separate the soluble
and particulate-bound enzyme. The resulting supernatant was labeled as
the cytosolic fraction, whereas the pellet was resuspended in 1 ml of
buffer A with 1% Triton X-100 and incubated
on ice for 60 min. The resuspended pellet was centrifuged at 105,000 g for 60 min, and this supernatant was
labeled as the particulate fraction. It should be mentioned that the
above treatments of homogenate and pellet from control and diabetic
hearts with 1% Triton X-100 were found to solubilize the PKC enzyme
completely, as no PKC activity was detected in the supernatant upon
further treatment with 1 or 2% Triton X-100. To rule out the
possibility of artifacts that may interfere with PKC activities,
purified preparations according to the method described by others (30,
32) were employed in some experiments. For this purpose, the
homogenate, cytosolic, and particulate fractions were passed through
diethylaminoethyl cellulose columns, which were prewashed with 50 mM
Tris · HCl (pH 7.5). Each of these columns was washed
twice with 5 ml of buffer
B containing 50 mM
Tris · HCl, 10 mM EGTA, 5 mM EDTA, 0.3%
2-mercaptoethanol (wt/vol), and 200 U/ml aprotinin (pH 7.5). The enzyme
was finally eluted with 1 ml of buffer
B containing 200 mM NaCl.
Assay of PKC activity. Unless
otherwise mentioned in the text, okadaic acid was used in PKC activity
assay in small samples of nonpurified homogenate, cytosolic, and
particulate fractions from ventricular tissue (3). It should be
mentioned that okadaic acid is a specific inhibitor of type 1 and type
2A phosphatases and is thus valuable in measuring protein
phosphorylation due to protein kinases (1). The
Ca2+-dependent PKC activity was
determined with a PKC assay kit (Upstate Biotechnology, Lake Placid,
NY) in the reaction buffer
C containing 20 mM MOPS, pH 7.2, 25 mM
-glycerol phosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol,
and 4 mM CaCl2. Substrate cocktail
containing 500 µM PKC substrate peptide in
buffer
C, inhibitor cocktail containing 2 µM protein kinase A inhibitor peptide in
buffer
C, and lipid activator containing 0.5 mg/ml phosphatidyl serine and 0.05 mg/ml diglyceride in
buffer
C was used. Each assay
tube contained substrate (10 µl), inhibitor (10 µl), lipid
activator (10 µl), enzyme preparation (10 µl), and 26 µM okadaic
acid (1 µl); the final concentration of
Ca2+ was 1 mM. The
Ca2+-independent PKC activity was
determined in a reaction buffer D containing 20 mM MOPS, pH 7.2, 25 mM
-glycerol phosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol,
and 1.25 mM EGTA; for this purpose, substrate cocktail (specific for
PKC
- and
-isozymes; Quality Controlled Biochemicals, Hopkinton,
MA) containing 500 µM substrate peptide in
buffer
D, inhibitor cocktail containing 2 µM protein kinase A inhibitor peptide in
buffer
D, and lipid activator containing 0.5 mg/ml phosphatidyl serine and 0.05 mg/ml diglyceride in
buffer
D was used. Each assay
tube contained substrate (10 µl), inhibitor (10 µl), lipid
activator (10 µl), enzyme preparation (10 µl), and 26 µM okadaic
acid (1 µl). The reactions for both Ca2+-dependent and
Ca2+-independent PKC activities
were initiated by the addition of [
-32P]ATP (10 µl)
and allowed to proceed at 30°C for 10 min.
[
-32P]ATP was
prepared by mixing one part of ~3,000 Ci/mmol
[
-32P]ATP and nine
parts of MgATP (75 mM MgCl2 and
500 µM ATP either in buffer
C or in
buffer
D). The reaction
mixture (25 µl) was placed onto the P81 phosphocellulose paper for 30 s, and this paper was then washed three times (5 min each time) with
0.75% phosphoric acid and once with acetone. The papers were placed in
scintillation vials, and the radioactivity was counted in a scintillation counter.
The incorporation of 32P from
-32P into the synthesized
substrates, which are specific substrates with respect to
Ca2+-dependent and -independent
PKC, was measured (17, 23).
Ca2+-dependent PKC activities in
the partially purified homogenate and cytosolic and particulate samples
were measured in the absence of okadaic acid in the assay medium.
Analysis of PKC isozyme contents. The
relative contents of
-,
-,
-, and
- isozymes in the
homogenate, cytosolic, and particulate fractions were measured by 8%
mini-SDS-PAGE and Western blot analysis. The concentration of protein
in each fraction was adjusted to 1 mg/ml with the homogenizing buffer
(buffer
A or
buffer
A with 1% Triton-100). The sample
loads for control, experimental, and insulin-treated groups were of the
same volumes (10 µl in each well). The proteins thus separated by
SDS-PAGE were electroblotted to the Immobilon-P transfer membrane
(Millipore, Bedford, MA) for the determination of relative protein
contents by immunoblotting. Primary binding of PKC isozyme-specific
antibody was detected by with anti-rabbit IgG (1:1,000 for PKC-
,
-
, -
, and -
isozymes; Life Technologies, Burlington, ON)
conjugated with horseradish peroxidase (1: 5,000, Amersham). For
chemiluminescent detection, the electrochemiluminescence system
(Amersham) was employed according to the instructions of the
manufacturer. The relative contents of PKC isozymes were determined by
the model GS-670 imaging densitometer (Bio-Rad, Hercules, CA) with the
image analysis software version 1.0. The contents of PKC isozymes were
also measured by the employment of the partially purified homogenate,
cytosolic, and particulate samples.
Data analysis. Data are expressed as
means ± SE. The differences among different groups were evaluated
statistically by one-way ANOVA followed by the Newman-Keuls test. A
P value < 0.05 was taken to
represent a significant difference.
 |
RESULTS |
General characteristics of diabetic and
insulin-treated animals. In comparison with the control
animals, the body weight and ventricular growth were significantly
decreased, whereas the ventricular-to-body weight ratio was increased
in rats 8 wk after the streptozotocin injection (Table
1). The plasma glucose concentration was
increased markedly, and the plasma insulin level in diabetic animals
was depressed compared with control values. Daily injections of insulin to the 6-wk diabetic animals for 2 wk normalized the plasma glucose and
insulin concentrations as well as the ventricular weight and the ratio
of ventricular to body weight; however, the insulin-treated diabetic
rats still had lower body weight. Diabetic rats exhibited a depression
in cardiac +dP/dt and
dP/dt when compared with
control animals; these changes in diabetic animals were reversed by
insulin treatment (Table 1). These results with respect to general
characteristics are similar to those reported for diabetic animals from
this laboratory (11, 27).
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Table 1.
General characteristics and cardiac contractile function of control,
diabetic, and insulin-treated diabetic animals
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Cardiac PKC activities. PKC activities
were measured in the homogenate, cytosolic, and particulate fractions
of control, diabetic, and insulin-treated diabetic hearts (Fig.
1). As shown in Fig. 1A, the
Ca2+-dependent PKC activities were
increased by 43 and 51% in the homogenate fraction and 31 and 70% in
the cytosolic fraction from the 4- and 8-wk diabetic hearts,
respectively, in comparison with control values. There were no
significant changes in the
Ca2+-dependent PKC activity in all
fractions from the 1- and 2-wk diabetic hearts and particulate
fractions from all diabetic hearts. The
Ca2+-independent PKC activity was
increased by 32% in homogenate and 89% in the cytosolic fraction from
the 4- and 8-wk diabetic hearts in comparison with control values,
respectively (Fig. 1B).
No significant changes in
Ca2+-independent PKC activity were
found in all fractions from 1- and 2-wk diabetic hearts and the
particulate fractions from all diabetic hearts (Fig.
1B). Compared with 8-wk diabetic
hearts, the Ca2+-dependent PKC
activity in the insulin-treated group was decreased by 19 and 18%,
whereas the Ca2+-independent PKC
activity in the insulin-treated group was decreased by 15 and 33%, in
the homogenate and cytosolic fractions, respectively. However, these
values were still higher (by 23% in the homogenate fraction and 39%
in the cytosolic fraction for the
Ca2+-dependent PKC activity and by
14% in the homogenate fraction and 28% in cytosolic fraction for the
Ca2+-independent PKC activity)
than the control values (Fig. 1). To examine if the changes in PKC
activities observed in the diabetic homogenate and cytosolic fractions
are due to any alterations in the protein concentrations of these
fractions, the yield of proteins in different fractions was determined.
The results shown in Table 2 indicate no
difference in protein concentrations in the homogenate, particulate, or
cytosolic fractions obtained from control, diabetic, and
insulin-treated diabetic hearts.

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Fig. 1.
Cardiac protein kinase C (PKC) activities in homogenate, cytosolic, and
particulate fractions from control, diabetic, and insulin-treated
diabetic rats at different times of diabetes induction.
A:
Ca2+-dependent PKC activity.
B:
Ca2+-independent PKC activity. D,
diabetes; I, insulin-treated; w, week; Pi, inorganic
phosphate. Values are means ± SE of 6 animals in each diabetic
group and 24 animals in control group. Because values for the PKC
activities at 1, 2, 4, and 8 wk of injection of vehicle did not differ
from each other (P > 0.05), these
values were grouped together. * Significantly different from
control (P < 0.05).
# Significantly different from diabetic
(P < 0.05).
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Table 2.
Protein concentration per unit of heart tissue in homogenate,
particulate and cytosolic fractions isolated from control, diabetic,
and insulin-treated diabetic rat hearts
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Relative protein content of PKC
isozymes. The relative protein contents of PKC-
,
-
, -
, and -
isozymes in homogenate, cytosolic, and particulate
fractions of the cardiac muscle from control, 8-wk diabetic, and
insulin-treated diabetic rats were identified by Western blotting. The
typical bands representing of PKC-
, -
, -
, and -
isozymes in
these fractions of rat hearts are shown in Figs.
2 and 3.
Polyclonal antibodies to PKC isozymes detected proteins at 76 kDa for
-isozyme, 77 kDa for
-isozyme, 83 kDa for
-isozyme, and 67 kDa
for
-isozyme. There was a nonspecific band at ~80 kDa below the
bands for
-isozyme in Figs. 2 and 3; however, in view of the fact
that its identity is unknown, this band was not included in the
densitometric analysis. The densitometric analysis of bands for
PKC-
, -
, -
, and -
isozymes revealed a significant increase
in relative protein contents in the diabetic homogenate fraction by 43, 31, 48, and 38%, respectively, and in the cytosolic fraction by 126, 119, 148, and 129%, respectively, in comparison with the control
values, respectively (Figs. 4 and 5). Insulin administration to diabetic rats
decreased protein contents for PKC-
, -
, -
, and -
isozymes
by 19, 23, 14, and 19%, respectively, in the homogenate fraction (Fig.
4) and by 29, 35, 48, and 51%, respectively, in the cytosolic fraction
when compared with diabetic hearts, respectively (Fig. 5). There were no significant alterations in PKC isozyme protein contents of the
particulate fractions from the 8-wk diabetic rat heart compared with
values from the control or insulin-treated diabetic hearts (Figs. 4 and
5). In preliminary experiments with two preparations from 4-wk diabetic
animals, PKC-
, -
, -
, and -
isozymes in both homogenate and
cytosolic fractions, unlike the particulate fraction, were increased by
15-50% of the respective control values.

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Fig. 2.
Typical immunoblots for PKC- , - , - , and - isozymes in
homogenate fractions from control, diabetic, and insulin-treated
diabetic rat hearts at 8 wk after induction of diabetes.
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Fig. 3.
Typical immunoblots for PKC- , - , - , and - isozymes in
cytosolic (C) and particulate fractions (P) from control, diabetic, and
insulin-treated diabetic rat hearts at 8 wk after induction of
diabetes.
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Fig. 4.
Densitometric analysis of results for relative protein contents of
protein kinase C- (A), -
(B), -
(C), and -
(D) isozymes in homogenate fraction
of control, diabetic, and insulin-treated diabetic rat hearts at 8 wk
after induction of diabetes. Values are means ± SE of 6 animals in
each group. * Significantly different from control
(P < 0.05). # Significantly
different from diabetic (P < 0.05).
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Fig. 5.
Densitometric analysis of results for relative protein contents of
protein kinase C- (A), -
(B), -
(C), and -
(D) isozymes in cytosolic and
particulate fractions from control, diabetic, and insulin-treated
diabetic rat hearts at 8 wk after induction of diabetes. Values are
means ± SE of 6 animals in each group. * Significantly
different from control (P < 0.05).
# Significantly different from diabetic
(P < 0.05).
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PKC activities and isozyme contents in partially
purified preparations.
Ca2+-dependent PKC activities (in
the absence of okadaic acid) and isozyme contents were also measured in
the partially purified homogenate, cytosolic, and particulate
preparations. The results in Fig. 6 show an
increase in PKC activities in the homogenate and cytosolic fractions
from the 8-wk diabetic heart without any significant change in the
particulate fraction. Furthermore, treatment of diabetic animals with
insulin significantly reduced the PKC activities in both homogenate and
cytosolic fractions toward the control levels. Increases in protein
contents of PKC-
, -
, -
, and -
isozymes in the purified
homogenate and cytosolic fractions, unlike the particulate fraction,
from the diabetic heart were significantly attenuated upon treatment of
the animals with insulin (Table 3). The
pattern of changes in PKC isozymes observed in partially purified
fractions from the diabetic heart was essentially similar to that
observed with unpurified fractions (Figs. 4 and 5).

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Fig. 6.
Protein kinase C activities in partially purified homogenate,
cytosolic, and particulate fractions from control, diabetic, and
insulin-treated diabetic hearts. Values are means ± SE of 6 separate experiments. * Significantly different from control
(P < 0.05). # Significantly
different from diabetic (P < 0.05).
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Table 3.
Relative protein content of PKC isozymes in partially purified
homogenate, cytosolic, and particulate fractions of the 8-wk diabetic
and insulin-treated diabetic hearts
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 |
DISCUSSION |
In this study, we observed an increase in the
Ca2+-dependent and -independent
PKC activities in the ventricular homogenate fraction obtained from the
4- and 8-wk diabetic rats. Such an increase in the enzyme activity
seems to be dependent on the duration of diabetes because no change in
the Ca2+-dependent and
Ca2+-independent PKC activities
was evident in hearts from 1- and 2-wk diabetic rats. Because the
observed increase in the PKC activities in the diabetic heart
homogenate fraction was associated with an increase in the PKC activity
in the cytosolic fraction without any change in the particulate
fraction, it is unlikely that these changes in cardiac PKC activities
are due to translocation of the enzyme from the particulate to the
cytosolic fractions. This view is supported by the observation that the
protein concentrations of cytosolic fraction or particulate fraction
from the diabetic heart were not different from those from the control
hearts. Although an increase in cardiac PKC activity in both cytosolic
and particulate fractions in 10-wk diabetic rats has been observed by
some investigators (30), others (32) have reported a decrease and an
increase in the PKC activity in cardiac cytosolic and particulate
fractions, respectively, from 6-wk diabetic animals. These differences
in the results for the PKC activities in the cytosolic and particulate fractions of the diabetic heart as seen in the present and previous studies may be due to differences in the intensity and duration of
diabetes, methods for the preparation of cytosolic and particulate fractions, methods for the extraction of the enzyme, and procedures employed for the assay of PKC activities. In this regard, it should be
noted that Xiang and McNeill (32), unlike the present and other studies
(30), employed histone H-1 protein, a nonspecific substrate for
assaying the PKC activity. Because previous investigators (30, 32) did
not measure the PKC activity in the homogenate fraction, it is
difficult to evaluate their results in terms of the status of PKC
activities in the diabetic heart. Furthermore, a decrease in the PKC
activity in the cytosolic fraction without any significant alterations
in the particulate fraction and an impaired translocation of PKC were
observed in mononuclear cells from poorly controlled diabetic patients
(22). Nonetheless, the observed changes in cardiac PKC activities in
diabetes were not due to any artifact because a similar pattern of
changes in the enzyme activity was seen in a separate set of
experiments in which partially purified samples were employed.
Furthermore, treatment of 6-wk diabetic animals for 2 wk with insulin
was found to partially reverse the elevated levels of PKC activity in
both homogenate and cytosolic fractions without any change in the
particulate fraction of the diabetic heart.
The increased PKC activities in the homogenate and cytosolic fractions
were associated with corresponding increases in the contents of
PKC-
, -
, -
, and -
isoforms, whereas neither the PKC
activities nor the content of PKC isozymes was altered in the
particulate fraction of the diabetic heart. It thus appears that the
observed increase in cardiac PKC activities may be due to an increase
in protein contents of different PKC isozymes in the cytosolic
fraction. Whether such an increase in different PKC isozymes in the
diabetic heart is a consequence of an increase in gene expression or
some other molecular mechanism remains to be investigated. Because the
increases in different PKC isozyme contents varied from 30 to 59% in
homogenate fraction and from 127 to 168% in the cytosolic fraction
from the diabetic heart, it is possible that insulin deficiency may
affect the genes specific for these isozymes in a differential manner.
It should be noted that PKC-
, -
, -
, and -
isoforms have
also been reported to increase in the diabetic liver (4), whereas
PKC-
, -
, -
, and -
isoforms have been shown to increase in
both the aorta and heart from diabetic rats (12, 15, 21). Although
Malhotra et al. (21) observed translocation of
-isoform of PKC from the cytosolic to the particulate fraction without any change in the
-isoform in the diabetic cardiomyocytes, these findings should not
be compared with the observations reported in this study. The reasons
for this view are based on the fact that these investigators (21)
employed 3- to 4-wk diabetic rats, whereas we have used rats at 4 and 8 wk after the induction of diabetes. Furthermore, Malhotra et al.
separated the cytosolic fraction and particulate fractions by
centrifugation at 25,000 g for 30 min,
whereas the present experiments were carried out by employing
centrifugation at 105,000 g for 60 min.
Because the increase in the contents of cardiac PKC isozymes as well as
the elevated plasma levels of glucose were partially or fully
reversible upon the treatment of diabetic animals with insulin, the
observed increase in PKC isozymes may be due to an increase in the
plasma glucose level. In this regard, it should be noted that glucose
has been shown to modulate the PKC isozymes and PKC activities in
different types of cells (14, 15, 31); however, it is unlikely that the
increased PKC activity and contents of PKC isozymes in the diabetic
heart are entirely due to alterations in plasma glucose in diabetes.
This contention is substantiated by the fact that treatments of
diabetic animals with verapamil or an angiotensin II receptor blocker,
L-158,809, which does not affect the plasma level of glucose, reversed
the increased cardiac PKC activities in both cytosolic and particulate
fractions (30) and changes in the contents of cardiac PKC
-isoform
in the particulate and cytosolic fractions (21), respectively. Although
a number of receptor systems including angiotensin II and
1-adrenergic receptors have
been reported to be coupled with PKC enzyme (9), the exact role of
these receptor mechanisms in increasing the contents of PKC isozymes in
the cytosolic fraction of the diabetic heart remains to be investigated.
It has been reported that the activated PKC exerts a negative inotropic
effect in the heart at the level of the contractile apparatus by
phosphorylation of troponin I and troponin T and subsequent inhibition
of the myofibrillar ATPase activities (24). Another mechanism, which
has been suggested to account for this cardiodepression, concerns the
phosphorylation of phospholamban by PKC and subsequent inhibition of
Ca2+ transport by the sarcoplasmic
reticulum (28). In view of the inhibitory effects of PKC on
myofibrillar ATPase and sarcoplasmic reticular
Ca2+ transport, the increased
cardiac PKC activity as well as contents of different PKC isozymes in
the cytosolic fraction can be seen to contribute toward the depression
in the rate of contraction as well as the rate of relaxation of the
diabetic heart. It should be noted that heart dysfunction in this
experimental model has been reported to occur at 4 and 8 wk of
induction of diabetes (11); this time coincides with the time of PKC
changes observed here. PKC
-isozyme, a predominant isoform in
cardiomyocytes (2, 29), has been shown to be associated with sarcomeres
upon activation (7) and to be responsible for the phosphorylation of
troponin I (21, 24). On the other hand, PKC
-isoform has been shown to stimulate the promoter of
-myosin heavy chain in the myocardium (16). However, further evidence is needed to demonstrate whether increased PKC isoforms are associated with increased phosphorylation of
troponin I and marked changes in the myosin isozyme composition in the
diabetic heart (6, 20). Because the role of
- and
-isoforms of
PKC in changing the characteristics of any specific subcellular or
metabolic site has not yet been established, it is difficult to
speculate the exact functional significance of the increased contents
of these PKC isozymes in the diabetic heart. However, sustained
increase in the contents of different PKC isozymes in the cytosolic
fraction and subsequent increase in PKC activity in the diabetic heart
may reflect signal transduction abnormalities and
Ca2+ handling defects in
cardiomyocytes during the development of heart dysfunction in chronic
diabetes (5,10).
 |
ACKNOWLEDGEMENTS |
The work reported in this paper was supported by a grant from
Medical Research Council of Canada (MRC Group in Experimental Cardiology). V. Panagia is a senior scientist supported by the Medical
Research Council of Canada, whereas N. S. Dhalla holds the
MRC/Pharmaceutical Research and Development Chair in Cardiovascular Research supported by Merck Frosst, Canada.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. S. Dhalla,
Institute of Cardiovascular Sciences, St. Boniface General Hospital
Research Centre, 351 Tache Ave., Winnipeg, Manitoba R2H 2A6, Canada
(E-mail: cvso{at}sbrc.umanitoba.ca).
Received 26 January 1999; accepted in final form 10 June 1999.
 |
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