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Circulation Research. 1997;81:1027-1033

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(Circulation Research. 1997;81:1027-1033.)
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Articles

Experimental Diabetes Is Associated With Functional Activation of Protein Kinase C{epsilon} and Phosphorylation of Troponin I in the Heart, Which Are Prevented by Angiotensin II Receptor Blockade

Ashwani Malhotra, David Reich, Daniel Reich, Antonio Nakouzi, Vinay Sanghi, David L. Geenen, , Peter M. Buttrick

From the Division of Cardiology, Albert Einstein College of Medicine, Bronx, NY.

Correspondence to Peter M. Buttrick, MD, University of Illinois at Chicago, Section of Cardiology (MC 787), Rm 929, 840 South Wood St, Chicago, IL 60612.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract A cardiomyopathy that is characterized by an impairment in diastolic relaxation and a loss of calcium sensitivity of the isolated myofibril has been described in chronic diabetic animals and humans. To explore a possible role for protein kinase C (PKC)–mediated phosphorylation of myofibrillar proteins in this process, we characterized the subcellular distribution of the major PKC isoforms seen in the adult heart in cardiocytes isolated from diabetic rats and determined patterns of phosphorylation of the major regulatory proteins, including troponin I (TnI). Rats were made diabetic with a single injection of streptozotocin, and myocardiocytes were isolated and studied 3 to 4 weeks later. In nondiabetic animals, 76% of the PKC{epsilon} isoform was located in the cytosol and 24% was particulate, whereas in diabetic animals, 55% was cytosolic and 45% was particulate (P<.05). PKC{delta}, the other major PKC isoform seen in adult cardiocytes, did not show a change in subcellular localization. In parallel, TnI phosphorylation was increased 5-fold in cardiocytes isolated from the hearts of diabetic animals relative to control animals (P<.01). The change in PKC{epsilon} distribution and in TnI phosphorylation in diabetic animals was completely prevented by rendering the animals euglycemic with insulin or by concomitant treatment with a specific angiotensin II type-1 receptor (AT1) antagonist. Since PKC phosphorylation of TnI has been associated with a loss of calcium sensitivity of intact myofibrils, these data suggest that angiotensin II receptor–mediated activation of PKC may play a role in the contractile dysfunction seen in chronic diabetes.


Key Words: diabetes mellitus • protein kinase C • angiotensin II type-1 receptor


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
A characteristic feature of diabetes in both small and large animals is the development of a progressive cardiomyopathy that is independent of intercurrent vascular pathology. Clinically, this often manifests itself as an impairment in diastolic performance.1 A spectrum of biochemical abnormalities have been described in the hearts of diabetic animals, including reduced actomyosin and actin-activated myosin ATPase activities and, in small animals, a shift from the normal V1 predominant myosin isoform to the V3 form,2,3 but to date, no simple biochemical abnormality has been sufficient to explain the mechanical defect. Likely candidates would be predicted to involve defects in calcium handling and/or in the calcium sensitivity of the sarcomere.

Activation of the PKC pathway might logically subserve this function. This enzyme has been identified as an important intermediary in the signal transduction pathways used by the adult heart to respond to a diversity of signals, including stretch–, {alpha}1-adrenergic–, endothelin–, and AT1 receptor–linked pathways (reviewed in References 4 and 54 5 ) and has been linked to phosphorylation and functional alterations of a number of key regulatory proteins, including TnI, TnT, and MLC2.6–9 It is increasingly clear that stimulation of PKC in cardiac myocytes and tissue, either by {alpha}1-adrenergic agonists or phorbol esters, results in diminished calcium sensitivity of the sarcomere and in transiently or persistently impaired muscle shortening.10–13 This, coupled with the fact that diabetic animals and patients display persistent activation of PKC in a number of organ systems, including the heart, liver, and large arteries and in the microvasculature of the retina,14–17 suggests that PKC-mediated phosphorylation may play an important role in the chronic adaptations seen in their hearts.

To explore this hypothesis, we measured the cytosolic and particulate distribution of two major cardiac isoforms of PKC ({epsilon} and {delta}) in myocardiocytes isolated from the hearts of rats with established diabetes. Phosphorylation patterns of potential target proteins were also determined in these cells. PKC{epsilon} activation and TnI phosphorylation were a primary focus because PKC{epsilon} is the predominant isoform in adult cardiocytes,18,19 associates with the sarcomere on activation,20,21 and has been functionally linked to phosphorylation of TnI and to reduced myofibrillar ATPase activity in vitro.8,9,22 In addition, because of the central role of Ang II in the pathological adaptation in the heart in general (as well as in diabetes) and also in the activation of PKC, we studied the effect of a specific angiotensin receptor (AT1) blocker on the PKC response in diabetes.

Our data suggest that specific alterations in the subcellular localization of PKC{epsilon} and phosphorylation of TnI are characteristic features of chronic diabetes and that these phenomena are mediated both by hyperglycemia and by activation of AT1 receptors.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Animal Models
All animals were female Wistar rats weighing from 200 to 225 g at the outset. Animals were maintained on a 12-hour light/dark cycle and were allowed access to food and water ad libitum. Two separate animal protocols were used. In the first protocol, three groups of animals were studied: controls (C), animals made diabetic with a single intravenous injection of 60 mg/kg streptozotocin (D), and animals injected with streptozotocin and then treated daily with 5 U of long-acting insulin (ultra-lente, Eli Lilly) (D+I). We have previously used this experimental design to study diabetes-associated cardiac adaptations.1 In the second protocol, animals were made diabetic with a single injection of 60 mg/kg streptozotocin or were untreated, and then animals in both the control (C) and the diabetic (D) groups were randomized to either treatment with the specific AT1 blocker L-158,809 (Dupont)23,24 or vehicle alone. The AT1 blocker was administered on Monday and Thursday as a subcutaneous injection (10 mg/kg in dimethyl sulfoxide). We have used this agent and administration protocol previously and have shown that the drug is specific for cardiac AT1 receptors, that it does not block cardiac {alpha}-adrenergic responsiveness, that its duration of action is such as to completely block Ang II effects when administered every 72 to 96 hours, and that it is not associated with tachyphylaxis in rats.25,26

Heart rates and blood pressures were measured in awake animals after 3 weeks of hyperglycemia and/or AT1 treatment via direct intra-arterial cannulation. Blood glucose was determined on plasma samples using a commercially available kit (Sigma, No. 115-A), and only animals with a nonfasting serum glucose level of >300 mg/dL were considered diabetic. The cotreatment of animals with L-158,809 did not have any effect on the development of diabetes, since all of the streptozotocin-treated animals became diabetic to an equivalent degree regardless of whether they were treated with L-158,809 or vehicle.

Cell Isolation
Animals were killed 3 to 4 weeks after randomization in both experimental protocols, and their hearts were perfused with collagenase for the isolation of cardiocytes. Whenever possible, one animal from each group was killed per day. The procedure used to isolate cardiocytes has been described previously.27 Briefly, hearts were cannulated and perfused at 37°C at physiological pressure and flow with oxygenated (95% O2/5% CO2) calcium-free MEM (Sigma) for 5 minutes. After this procedure, the perfusate was changed to one containing collagenase (0.5 mg/mL, Worthington Biochemical), and perfusion was continued with recirculation for an additional 30 minutes. The heart was then removed from the perfusion apparatus, and the left ventricle was dissected away from the other chambers, minced in MEM containing 0.1 mg/mL collagenase at 37°C, and dispersed into single cells by gentle agitation through a serological pipette. The resultant cell suspension was centrifuged at 500g, resuspended in MEM without collagenase, and subsequently centrifuged through a Percoll cushion at 1500g. The cells were >80% cardiocytes, as determined by their characteristic morphological shape and striations. Phosphorylation studies were performed immediately after cell isolation (see below), whereas immunoelectrophoretic analyses were performed on cell pellets that were fast-frozen in liquid nitrogen and stored at -70°C.

PKC Analysis
The method for extraction of PKC is as described previously.18 Briefly, cells were homogenized in buffer I, which consisted of 2.5 mL of 20 mmol/L Tris-HCl, 0.33 mol/L sucrose, 2 mmol/L EGTA, and 2 mmol/L EDTA (pH 7.5) containing 0.1 mmol/L sodium vanadate, 20 mmol/L NaF, 20 µmol/L leupeptin, 10 µmol/L trans-epoxysuccinyl-L-leucyl-amido(4-guanidino)-butane (E64), 200 µmol/L phenylmethylsulfonyl fluoride, and 5 mmol/L dithiothreitol. The cytosolic fraction was separated by centrifugation of the crude homogenate (2 to 5 mg) at 25 000g for 30 minutes. The particulate fraction was collected by resuspending and shaking the pellets in homogenization buffer without sucrose containing 0.1% Triton X-100 for 15 minutes, centrifuging at 25 000g for 30 minutes, and collecting the supernatant; this process was repeated using homogenization buffer with Triton X-100 and centrifugation at 25 000g for 15 minutes. Myofilament proteins localize to the particulate fraction, and Triton X-100 treatment dissociates PKC from the contractile protein fraction in the pellet. The two supernatants were pooled and collected as the particulate fraction. No PKC{epsilon} was detected in the remaining pellet by immunoblotting, confirming that all of the enzyme was extracted using this procedure. Protein from both fractions was precipitated in 5% trichloroacetic acid and centrifuged at 2000g for 15 minutes, and the pellets were resuspended in SDS buffer. To quantify total immunoreactive PKC{epsilon}, myocytes were homogenized in buffer I (1 mL) and shaken for 30 minutes in the presence of 0.1% Triton X-100. The supernatants obtained after centrifugation were then equivalent amounts of total protein subjected to immunoelectrophoresis, and the amount of PKC was quantified by densitometry as described below.

Fifteen to 20 µL (2 to 3 mg/mL) of the protein samples were separated by SDS-PAGE gel using an 8% (wt/vol) acrylamide gel and 4.5% stacking gel. Protein was then electrophoretically transferred to nitrocellulose using a Semi-Dry Transfer Cell (Bio-Rad). Nonspecific sites were blocked with 5% (wt/vol) nonfat milk and 0.05% (vol/vol) Tween 20 in phosphate-buffered saline overnight at 4°C. The nitrocellulose was incubated with primary antibodies against PKC{delta} or PKC{epsilon} isoforms (1/1000 to 1/2500 dilution in the blocking solution, GIBCO-BRL, Life Technologies) for 2 hours and then with the horseradish peroxidase–linked secondary antibodies (1/5000 dilution, Accurate Chemical and Scientific Corp) for 1 hour at room temperature. The bound antibody was detected by the enhanced chemiluminescence method (Amersham Intl). The specificity of the antibody was confirmed by incubation with the appropriate blocking peptide. Quantification of PKC isoform translocation was performed by densitometric analysis of suitable autoradiographs. Exposure was for 10 to 180 seconds (Kodak hyperfilm), and only appropriate autoradiographs with exposures within the linear range were analyzed and quantified using a computerized image analyzer (Molecular Analyst software and Bio-Rad Gel Doc 1000 image analyzer).

Phosphorylation Studies
Phosphorylation of myofibrillar proteins in cardiac myocytes using [32P]orthophosphate was performed as previously described.28 Briefly, freshly isolated myocytes were suspended in 2 mL phosphate-free basic MEM buffer ({approx}5x105 cells/mL) and incubated with 50 µCi of [32P]orthophosphate and gently agitated for 90 minutes at 37°C. After incubation, myocytes were washed three times in 3 mL of Tris-Cl–buffered physiological saline (pH 7.5) at 4°C in the presence of protease inhibitors and the protein phosphatase inhibitor calyculin A (Sigma No. C5552). Subsequently, cells were sonicated in 0.5 mL of 20 mmol/L Tris-Cl (pH 7.5), 2 mmol/L EDTA, 2 mmol/L EGTA, 6 mmol/L ß-mercaptoethanol, 0.1 mmol/L sodium vanadate, and 20 mmol/L NaF as well as the other protease inhibitors.32P incorporation was determined by SDS-gel electrophoresis and subsequent autoradiography as previously described.28 Analysis of samples from each of the three groups was performed on matched samples, and data are expressed in arbitrary units relative to their individual internal control values. Exposure of the autoradiographs was from 24 to 40 hours, and only films within the linear range of detection were quantified (Bio-Rad Gel Doc 1000 image analyzer and Molecular Analyst software).

Statistical Analysis
Data are expressed as mean±SE. Statistical differences were determined by two-factor ANOVA using diabetes and drug or insulin treatment as the two variables in order to determine if there were any interactive effects. Post hoc Newman-Keuls analysis subsequently established differences between individual groups.29 Significance was defined as P<.05.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Two separate protocols were used (see "Materials and Methods"). In the first protocol, three groups of animals were studied: control (C), diabetics (D), and diabetics treated with insulin (D+I). In the second, four groups were compared: control (C), diabetic animals (D), control animals treated with the specific AT1 receptor antagonist, L-158,809 (AT1), and diabetic animals treated with the antagonist (D+AT1). Relevant group characteristics of the animals studied are shown in the Table. In the initial study, only animals in which insulin treatment completely prevented both the rise in serum blood sugar and the decrease in body weight associated with chronic diabetes were included in the D+I group (5 of 7 were included). Glucose values for diabetics in the second study were lower than in the first study (mean, 384 versus 634 mg/dL), although the range of plasma glucose values in the two diabetic groups overlapped. The diabetic animals treated with the AT1 antagonist L-158,809 had blood sugars that were identical to the matched untreated diabetic group. In the second study, AT1 alone did not reduce systolic blood pressure, but the D+AT1 group had a slight decrease in systolic blood pressure relative to the other groups. Heart rates were similar in all cohorts.

Fig 1Down shows both compiled data as well as representative Western blots from the animals in the Table illustrating the effect of diabetes and intercurrent insulin treatment on PKC{epsilon} distribution. Cardiocytes isolated from control animals showed a normal predominantly cytosolic distribution of the enzyme (78% cytosol:22% particulate). With diabetes, this ratio shifted so that 41% of the enzyme was associated with the particulate fraction (P<.05 versus control), consistent with chronic activation of the enzyme. The magnitude of this shift was not a reflection of the absolute level of plasma glucose, since the group of diabetic animals with relatively low serum glucose levels of 384 mg/dL from the second experimental protocol (see above) had 49% of the PKC{epsilon} associated with the particulate fraction (P=NS). Treatment of the diabetics with insulin, which normalized plasma glucose, completely prevented this shift. In cardiocytes from the D+I group, 76% of the enzyme was detected in the cytosolic fraction (P<.05 versus diabetic animals and P=NS versus control animals). To establish whether diabetes was associated with an increase in total immunoreactive PKC{epsilon} as well as with an increase in the particulate fraction, total PKC was directly measured in lysates from cardiocytes from control and diabetic hearts, and no difference in the total immunoreactive PKC{epsilon} was seen between control and diabetic hearts using semiquantitative Western blotting (16.8±.2 versus 16.6±4.6 arbitrary units, P=NS [n=3]).



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  		Figure 1. Panel A shows lanes from a representative Western blot demonstrating the cytosol (lanes 1, 3, and 5) versus particulate (lanes 2, 4, and 6) distribution of PKC{epsilon} in myocardiocytes from untreated control animals (group C, lanes 1 and 2), diabetic animals (group D, lanes 5 and 6), or diabetic animals treated with insulin (group D+I, lanes 3 and 4). Also seen in lane 2 is a nonspecific high molecular weight band (probably a nondenatured protein) that reacts with the PKC{epsilon} antibody. Molecular weights (kD) are indicated to the right. Panel B shows composite data (mean±SEM) indicating the percent distribution (cytosol versus particulate) of PKC{epsilon} in hearts of animals in these three groups. The numbers within the columns (1 to 6) correspond to the lane numbers in panel A. A translocation of PKC{epsilon} from the cytosol to the particulate fraction was observed in group D that was prevented by insulin treatment (C, n=6; D, n=7; and D+I, n=5; *P<.05 vs matched control).

To define a functional role for this PKC{epsilon} translocation, the phosphorylation patterns of some relevant target proteins were investigated. Figs 2Down and 3Down show a set of representative autoradiographs of the myofibrillar protein fraction (with a companion Coomassie-stained SDS gel) and compiled TnI data, respectively. A significant (almost 5-fold) increase in 32P incorporation of TnI was seen in diabetic animals that was completely prevented by intercurrent insulin treatment (P<.05). No similar consistent increase in phosphorylation was seen in association with any other protein species, including MLC2, although the level of resolution of the technique might not have been sufficient to detect small increases in incorporation or phosphorylation of other relatively low-abundance proteins. Western blotting using a specific cardiac TnI antibody confirmed that the labeled band was identical in size to TnI and, importantly, that there was no degradation of this protein.



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  		Figure 2. Lanes from a Coomassie-stained SDS gel (panel A) and the companion autoradiographs of the crude protein homogenate from cardiocytes isolated from control (group C), diabetic (group D), and diabetic plus insulin (group DI) hearts that had been subjected to [32P]orthophosphate labeling in vitro (panel B; see text for details). Three different sets of gels all run at different times are shown in panels A and B; however, each experimental sample is shown with a separate matched control sample that was run at the same time. The locations of myofibrillar proteins and potential PKC phosphorylation targets are indicated on each panel, as are molecular weight markers (kD): myosin heavy chain (MHC), C protein (C), actin, TnT, TnI, MLC1, and MLC2. Significant increases in phosphorylation relative to controls (panel B) are seen only in TnI from group D.



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  		Figure 3. Compiled data (mean±SEM) showing relative phosphorylation of the cardiac TnI (cTnI) band in cardiocytes isolated from control (group C), diabetic (group D), and diabetic plus insulin (group D+I) hearts. Data are expressed in arbitrary units relative to the level of incorporation seen in control preparations that were matched to each experimental sample (C, n=5; D, n=5; and D+I, n=6). See "Materials and Methods" for details.

Since diabetes is associated with an increase in the number (and possibly the affinity) of cardiocyte AT1 receptors30,31 and angiotensin II causes an acute translocation of PKC{epsilon} in intact hearts and in isolated cardiocytes,32,33 we next established whether AT1 receptor blockade would prevent the activation of PKC{epsilon} in cardiocytes isolated from diabetic animals. Data from this experiment are shown in Fig 4Down. Cardiocytes isolated from both control animals and animals treated with the AT1 blocker showed a normal predominantly cytosolic distribution of PKC{epsilon}: 76% in the control condition and 79% with AT1 blockade (P=NS). As previously demonstrated, with diabetes this value decreased so that {approx}50% of the enzyme was particulate; however, when the diabetic animals were exposed to AT1 blockade (which did not normalize plasma glucose), the PKC enzyme distribution was identical to that in control animals (71% was cytosolic). The translocation of PKC with diabetes appeared to be specific for the PKC{epsilon} isoform, since no change in the distribution of PKC{delta} (from particulate to cytosol) was seen with either diabetes or AT1 treatment. PKCß was not detectable by immunoblot (using commercially available antibodies) in either control or diabetic cardiocyte lysates.



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  		Figure 4. Panels A and B show representative Western blots indicating the relative distribution (cytosol versus particulate) of PKC{epsilon} (panel A) and PKC{delta} (panel B) in cardiocytes from hearts of animals that were either untreated control animals (group C, lanes 1 and 5), AT1 blocker–treated animals (group C+AT1, lanes 2 and 6), diabetic animals (group D, lanes 3 and 7), or diabetic animals treated with the AT1 blocker (group D+AT1, lanes 4 and 8). In both panels, lanes 1 to 4 are cytosolic and 5 to 8 are particulate. Molecular weight markers are indicated to the right. A translocation of PKC{epsilon} from the cytosol to the particulate fraction was observed in group D, and this was prevented by treatment with the AT1 blocker, whereas no significant change in PKC{delta} distribution was seen in any group. Panel C shows composite data (mean±SEM) indicating the distribution (cytosol versus particulate) of PKC{epsilon} in cardiocytes from hearts of animals from each of these groups. As illustrated in panel A, a translocation of PKC{epsilon} from the cytosol to the particulate fraction was observed in group D, and this was prevented by treatment with the AT1 blocker (C, n=12; C+AT1, n=5; D, n=7; and D+AT1, n=7). Panel D shows similar data for PKC{delta} and indicates no change in distribution in any treatment group (C, n=3; C+AT1, n=2; D, n=3; and D+AT1, n=4). Numbers within the columns in panels C and D correspond to the lanes indicated in the matched Western blots in panels A and B.

To establish that AT1 blockade also prevented the increase in TnI phosphorylation seen in diabetic animals, phosphorylation studies analogous to those shown in Figs 2Up and 3Up were performed on cardiocytes from control and diabetic animals treated with the AT1 antagonist. The composite data are shown in Fig 5Down and demonstrate that the increased phosphorylation seen in diabetic animals was prevented by intercurrent AT1 blockade.



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  		Figure 5. Incorporation of [32P]orthophosphate into cardiac TnI. As in Fig 4Up, data are expressed in arbitrary units relative to the level of incorporation in hearts from control animals (group C, n=4). AT1 indicates AT1 blocker–treated animals (n=3); D, diabetic animals (n=2); and D+AT1, diabetic animals treated with the AT1 blocker (n=4).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The major findings of the present study are that (1) diabetes is associated with a persistent alteration in subcellular localization (and presumably in activation) of PKC{epsilon} in adult cardiocytes and an increase in phosphorylation of troponin I, and (2) these phenomena are prevented either by normalization of plasma glucose by use of insulin or by concomitant treatment of diabetic animals with a specific AT1 receptor antagonist. Chronic activation of PKCß, a calcium-dependent isoform that is prominent in large blood vessels, has previously been seen in the aorta and the epicardial vessels in diabetic animals, where it is postulated to mediate an increase in vasomotor tone17 and where an increase in the translocation of several PKC isoforms, {alpha}, {epsilon}, and {zeta}, has been described in the liver and is postulated to contribute to insulin resistance.15 Our study is the first to look at the calcium-independent isoform, PKC{epsilon}, specifically in cardiomyocytes from diabetic animals, where activation might influence the contractile behavior of the intact muscle, independent of its vasculature.

The fact that isolated cardiocytes were used in the present study rather than intact heart muscle is important, as is the fact that specific alterations were defined for activation of PKC{epsilon} and not for PKC{delta}. The expression of PKC isoforms is both developmentally regulated and tissue specific.18,19 In the heart, different cell types express different isoforms,5,19 leading to some confusion about the relative role of each, especially in studies performed on whole-heart preparations.34 The functional importance of the calcium-independent isoform, PKC{epsilon}, in neonatal cardiocytes has been suggested by a number of immunohistochemical studies that have shown translocation of the isoform from the cytosol to the region of the myofilament after treatment of cells with phorbol esters or {alpha}1-adrenergic agonists.20,21,35 Although the catalytic domain of all the PKC isoforms is similar, implying some functional redundancy, the fact that this particular isoform is abundant in cardiocytes and translocates to the region of the sarcomere suggests a specific functional role for it in the modification of sarcomeric proteins, such as TnI, TnT, and MLC2, which has been demonstrated in vitro.8,9,36 This, in turn, could contribute to the regulation of contraction and relaxation of the intact muscle fibers.

The fact that we demonstrated changes in subcellular localizations of the enzyme and did not directly measure activity should be taken as a caveat. The literature is replete with studies making the assumption that translocation is tantamount to activation,37–39 including a recent study40 confirming that PKC activity and enzyme translocation are altered in parallel in hypertrophic rabbit cardiac muscle; our data suggest that TnI is the PKC{epsilon} phosphorylation target. However, in the absence of formal proof, this must still be regarded as an assumption.

Several additional lines of evidence suggest an important role for PKC activation in chronic cardiac adaptations. These include studies in intact cardiac muscle preparations subjected to stimulation by long-acting PKC activators, such as phorbol esters, which demonstrate sustained negative inotropic responses.10–13 In addition, phosphorylation of TnI and/or TnT by PKC in vitro is clearly associated with inhibition of the calcium-stimulated MgATPase activity both of myofibrils and of the reconstituted actomyosin complex,22,41 an effect that is likely mediated by altered interactions among the contractile protein components. Important additional supportive evidence for this general hypothesis comes from the work of Kuo and colleagues8,9,42 showing that the PKC{epsilon} phosphorylation site on TnI identified in vitro is identical to that defined in situ in cardiocyte preparations. Using skinned rat trabeculae, we have recently shown that the pCa-force relationship is right-shifted and maximal force generation is depressed by treatment with the catalytic subunit of PKC and that this is associated with phosphorylation of TnI (authors' unpublished data, 1997). Recent studies have shown an increase in TnI phosphorylation in cardiac homogenates from diabetic animals43 and have also shown abnormalities of the regulatory proteins in myofibrils and myocytes from diabetic animals that are associated with diminished calcium sensitivity and impaired contractile performance.44,45 Strikingly, the PKC-dependent changes in calcium sensitivity of myofibrillar ATPase, which have been defined in vitro, are identical to those seen in myofibrils isolated from diabetic animals (reviewed in Reference 4646 ).

Despite this, the precise influence of PKC activation on sarcomeric contraction is not so clear. Both phenylephrine and endothelin, agents that are felt to act at least in part via PKC activation, have been reported to induce sustained positive inotropic effects in cardiac muscle preparations.11,47,48 Certainly, alternate PKC phosphorylation targets exist, and in particular, phosphorylation of MLC2 by PKC (or by MLC kinase) has been shown to cause increases in calcium-stimulated MgATPase in thick-filament reconstituted myofibrils.49,50 In light of this, a reasonable hypothesis might be that the ultimate impact of PKC phosphorylation in vivo reflects a balance between opposing effects, each of which might be influenced by the relative abundance of the individual target protein isoforms within the sarcomere and/or by the relative susceptibility of these proteins to phosphorylation by PKC in vivo. The fact that MLC2 does not appear to be selectively phosphorylated in myocardiocytes from the diabetic animals in the present study (or at any event that TnI is disproportionately targeted) coupled with the fact that diabetic hearts have impaired mechanical performance strongly supports this conclusion.

Of note is the fact that we did not directly address the role of PKA in diabetic hearts. Activation of PKA has been implicated both in the phosphorylation of cardiac TnI (at a different site) and in the regulation of cardiac contraction, and this might additionally influence cardiac performance in diabetic animals.

The second major finding of the present study is that AT1 blockade in addition to insulin treatment prevented PKC{epsilon} translocation. Hyperglycemia per se has been postulated to activate various isoforms of PKC in other tissues, presumably by generation of diacylglycerol17; therefore, it is not surprising that normalization of plasma glucose via insulin restores normal enzyme distribution and activity. However, the fact that AT1 blockade in the presence of persistent hyperglycemia also normalizes enzyme distribution is a novel result and suggests that activation of this receptor-mediated pathway (which is linked to PKC activation) is both necessary and sufficient for PKC{epsilon} activation by hyperglycemia in myocardiocytes. It should be taken as a caveat that systolic blood pressure was slightly decreased by the use of the AT1 blocker in diabetic animals (although not in control animals); thus, it is theoretically possible, though unlikely, that the AT1 effect seen in diabetics was a reflection of the decreased blood pressure. A significant role for activation of the renin-angiotensin system in diabetes has been demonstrated in a number of organ systems, most strikingly, in the kidney, where angiotensin-converting enzyme inhibition has clearly been shown to prevent/delay the onset of diabetic nephropathy.51 Several investigators30,31 have reported an increase in the steady-state mRNA levels of the AT1 receptor in the heart associated with an increase in myocardial Ang II receptor density. This would seem to make the heart particularly responsive and perhaps vulnerable to the effects of Ang II. Multiple effects of Ang II on the heart, which conceivably could involve PKC-mediated signal transduction, have been proposed beyond the posttranslational modification of sarcomeric proteins, which seems to be relevant in the present study, including stimulation of cardiocyte growth,52,53 induction of a fetal pattern of gene expression,52,54 and proliferation of fibroblasts with a secondary elaboration of matrix proteins.55

The AT1 antagonist used in the present study is highly specific and does not interact with other membrane-coupled receptor pathways, such as those linked to {alpha}-adrenergic agonists, insulin-like growth factors, or endothelin.23–25 This specificity, in the absence of an increase in blood pressure in any group and in the presence of persistent hyperglycemia and hypoinsulinemia (which is a characteristic feature of the streptozotocin model), is important and suggests that the effect is specific to this receptor-linked signaling pathway and not a manifestation of intercurrent metabolic abnormalities associated with diabetes, as has been suggested for other cardiac manifestations of the disease. Whether chronic AT1 blockade can translate into improvement in cardiac muscle performance in diabetic animals is not directly addressed in the present study. Future studies using isoform-specific PKC inhibitors36 could conceivably establish the contribution of PKC activation and TnI phosphorylation to the contractile defect seen in diabetic cardiomyopathy.


*    Selected Abbreviations and Acronyms
 
Ang II = angiotensin II
AT1 = Ang II type 1
MLC = myosin light chain
PKA, PKC = protein kinase A and C
TnI, TnT = troponin I and T


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  		Table 1. Group Characteristics


*    Acknowledgments
 
This study was supported by National Institutes of Health grant R01–15498 (Dr Buttrick). Expert technical assistance was provided by Rendi Cheng. The authors also wish to thank Dr James Scheuer for his thoughtful comments.

Received July 28, 1997; accepted September 4, 1997.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Fein FS, Sonnenblick EH. Diabetic cardiomyopathy. Prog Cardiovasc Dis. 1985;27:255–270.[Medline] [Order article via Infotrieve]
  2. Malhotra A, Penpargkul S, Fein FS, Sonnenblick EH, Scheuer J. The effect of streptozotocin induced diabetes in rats on cardiac contractile proteins. Circ Res. 1981;49:1243–1250.[Abstract/Free Full Text]
  3. Pierce GN, Dhalla NS. Cardiac myofibrillar ATPase activity in diabetic rats. J Mol Cell Cardiol. 1981;13:1063–1069.[Medline] [Order article via Infotrieve]
  4. Sugden P, Bogoyevitch MA. Intracellular signalling through protein kinases in the heart. Cardiovasc Res. 1995;30:478–492.[Medline] [Order article via Infotrieve]
  5. Steinberg SF, Goldberg M, Rybin V. Protein kinase C isoform diversity in the heart. J Mol Cell Cardiol. 1995;271:141–153.
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