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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To understand cardiac dysfunction in diabetes, the activity of protein kinase C (PKC) and protein contents of its isozymes (PKC-alpha , -beta , -epsilon , and -zeta ) 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-alpha , -beta , -epsilon , and -zeta 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 epsilon -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 alpha -, beta -, epsilon -, and zeta -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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 beta -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 beta -glycerol phosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, and 1.25 mM EGTA; for this purpose, substrate cocktail (specific for PKC epsilon - and zeta -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 [gamma -32P]ATP (10 µl) and allowed to proceed at 30°C for 10 min. [gamma -32P]ATP was prepared by mixing one part of ~3,000 Ci/mmol [gamma -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 gamma -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 alpha -, beta -, epsilon -, and zeta - 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-alpha , -beta , -epsilon , and -zeta 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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

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

Relative protein content of PKC isozymes. The relative protein contents of PKC-alpha , -beta , -epsilon , and -zeta 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-alpha , -beta , -epsilon , and -zeta 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 alpha -isozyme, 77 kDa for beta -isozyme, 83 kDa for epsilon -isozyme, and 67 kDa for zeta -isozyme. There was a nonspecific band at ~80 kDa below the bands for epsilon -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-alpha , -beta , -epsilon , and -zeta 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-alpha , -beta , -epsilon , and -zeta 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-alpha , -beta , -epsilon , and -zeta 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-alpha , -beta , -epsilon , and -zeta 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-alpha , -beta , -epsilon , and -zeta 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-alpha (A), -beta (B), -epsilon (C), and -zeta (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-alpha (A), -beta (B), -epsilon (C), and -zeta (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).

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-alpha , -beta , -epsilon , and -zeta 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


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha , -beta , -epsilon , and -zeta 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-alpha , -beta , -epsilon , and -zeta isoforms have also been reported to increase in the diabetic liver (4), whereas PKC-alpha , -beta , -epsilon , and -zeta 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 epsilon -isoform of PKC from the cytosolic to the particulate fraction without any change in the beta -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 epsilon -isoform in the particulate and cytosolic fractions (21), respectively. Although a number of receptor systems including angiotensin II and alpha 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 epsilon -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 beta -isoform has been shown to stimulate the promoter of beta -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 alpha - and zeta -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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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