© 1997 American Heart Association, Inc.
Articles |
Experimental Diabetes Is Associated With Functional Activation of Protein Kinase C
and Phosphorylation of Troponin I in the Heart, Which Are Prevented by Angiotensin II Receptor Blockade
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.
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Abstract |
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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
isoform was located in the cytosol and 24% was particulate,
whereas in diabetic animals, 55% was cytosolic and 45% was
particulate (P<.05). PKC
, 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 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
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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–,
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 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 ( and ) in
myocardiocytes isolated from the hearts of rats with
established diabetes. Phosphorylation patterns of
potential target proteins were also determined in these cells. PKC
activation and TnI phosphorylation were a primary focus
because PKC 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 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
-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 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, 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 or PKC 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
(
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.
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Results |
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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 1
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 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 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 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 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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To define a functional role for this PKC translocation, the
phosphorylation patterns of some relevant target
proteins were investigated. Figs 2 and 3 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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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 in intact hearts and in isolated
cardiocytes,32,33 we next established whether
AT1 receptor blockade would prevent the activation of
PKC in cardiocytes isolated from diabetic animals. Data from
this experiment are shown in Fig 4.
Cardiocytes isolated from both control animals and animals
treated with the AT1 blocker showed a normal predominantly
cytosolic distribution of PKC: 76% in the control condition and
79% with AT1 blockade (P=NS). As previously
demonstrated, with diabetes this value decreased so that 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 isoform, since no change in the
distribution of PKC (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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To establish that AT1 blockade also prevented the increase
in TnI phosphorylation seen in diabetic animals,
phosphorylation studies analogous to those shown in
Figs 2
and 3 were performed on cardiocytes from control and
diabetic animals treated with the AT1
antagonist. The composite data are shown in Fig 5 and demonstrate that the increased
phosphorylation seen in diabetic animals was prevented
by intercurrent AT1 blockade.
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Discussion |
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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
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, , , and 
, 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, 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 and not for
PKC. 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, 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 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
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 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 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 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
-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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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.
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