Reperfusion-Induced Translocation of ␦PKC to Cardiac
Mitochondria Prevents Pyruvate
Dehydrogenase Reactivation
Eric N. Churchill,* Christopher L. Murriel,* Che-Hong Chen, Daria Mochly-Rosen, Luke I. Szweda
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Abstract—Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate
dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during
reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore
investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery
evident on reperfusion. Translocation of the redox-sensitive ␦-isoform of protein kinase C (PKC) to the mitochondria
occurred during reperfusion. Inhibition of this process resulted in full recovery of PDH activity. Infusion of the ␦PKC
activator H2O2 during normoxic perfusion, to mimic one aspect of cardiac reperfusion, resulted in loss in PDH activity
that was largely attributable to translocation of ␦PKC to the mitochondria. Evidence indicates that reperfusion-induced
translocation of ␦PKC is associated with phosphorylation of the ␣E1 subunit of PDH. A potential mechanism is
provided by in vitro data demonstrating that ␦PKC specifically interacts with and phosphorylates pyruvate dehydrogenase kinase (PDK)2. Importantly, this results in activation of PDK2, an enzyme capable of phosphorylating and
inhibiting PDH. Thus, translocation of ␦PKC to the mitochondria during reperfusion likely results in activation of PDK2
and phosphorylation-dependent inhibition of PDH. (Circ Res. 2005;97:78-85.)
Key Words: pyruvate dehydrogenase 䡲 ␦PKC 䡲 pyruvate dehydrogenase kinase 䡲 free radicals 䡲 mitochondria
䡲 ischemia/reperfusion
P
yruvate dehydrogenase (PDH) is responsible for the
conversion of pyruvate derived from glycolysis to acetylCoA for Krebs cycle activity. Enzyme activity is regulated, in
part, by phosphorylation- and dephosphorylation-dependent
inhibition and activation, respectively.1,2 Phosphorylation is
catalyzed by 4 PDH-associated pyruvate dehydrogenase kinases (PDK1– 4) that exhibit tissue-specific expression patterns and differences in specific activity toward 3 phosphorylation sites on the E1␣ subunit of PDH. The PDH complex
also contains 2 pyruvate dehydrogenase phosphatases (PDP 1
and PDP 2) responsible for reactivation of PDH.2– 4 PDH
therefore represents a highly regulated and critical site for the
control of glycolytic flux and ATP production.
Cardiac ischemia/reperfusion is associated with alterations in metabolism that, depending on the severity of the
ischemic insult, can progress to irreparable myocardial
damage.5 Although PDH activity in myocardial tissue has
been reported to decline during flow-induced ischemia,6
this is not universally observed.7–9 The effects of reperfusion also exhibit considerable variability, with the majority
of studies demonstrating a decrease in PDH activity.7–9
Despite the disparity in evidence regarding PDH activity,
cardiac efficiency and recovery of contractile function in
postischemic hearts can be improved by pharmacological
stimulation of PDH8,10 –16 or infusion of pyruvate.17–23
Identification of factors that regulate PDH activity during
ischemia/reperfusion may therefore enhance the potential
for therapeutic intervention.
Reperfusion of ischemic myocardium is associated with
enhanced free radical generation.5,24 Pro-oxidants have been
shown to regulate protein function either directly or indirectly
through the modulation of other regulatory molecules.25–27
One such example is the novel ␦-isoform of PKC. Exposure
of purified ␦PKC to the thiol-specific oxidant diamide and
glutathione (GSH) at concentrations that induce inactivation
of other PKC isozymes results in ␦PKC activation.28 Additionally, treatment of various cell types with H2O2, glutathione depleting agents, or the general PKC activator PMA
results in tyrosine phosphorylation and/or activation and
translocation of ␦PKC to the mitochondria where it promotes
cytochrome c release and the initiation of apoptosis.29 –35 In
contrast, inhibition of ␦ PKC translocation reduces
Original received July 7, 2004; resubmission received February 10, 2005; revised resubmission received May 18, 2005; accepted June 6, 2005.
From the Department of Physiology and Biophysics (E.N.C., L.I.S.), Case Western Reserve University, Cleveland, Ohio; and the Department of
Molecular Pharmacology (C.L.M., C.-H.C., D.M.-R.), Stanford University School of Medicine, Calif.
Dr Mochly-Rosen is a founder of KAI Pharmaceuticals, whose goal it is to bring peptide regulators of protein kinase C to the clinic.
*Both authors contributed equally to this work.
Correspondence to Luke I. Szweda, Oklahoma Medical Research Foundation, 825 NE 13th St, Oklahoma City, OK 73104. E-mail Luke-Szweda@
omrf.ouhsc.edu
© 2005 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000173896.32522.6e
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Churchill et al
␦PKC Maintains PDH Inhibition During Reperfusion
reperfusion-induced myocardial dysfunction and apoptosis
and results in improved regeneration of intracellular ATP,
phosphocreatine, and pH.36 – 40
In the present study, we tested the hypothesis that ␦PKC is
involved in regulation of PDH during reperfusion. Rat hearts
were perfused in a Langendorff fashion, and a specific
peptide inhibitor of ␦PKC was used to test the contribution of
␦PKC to ischemia- and reperfusion-induced alterations in
PDH activity. In addition, hearts were infused with H2O2 to
gain insight into potential mechanisms responsible for concerted regulation of ␦PKC and PDH during ischemia/reperfusion. Finally, in vitro experiments were performed to
address potential mechanisms by which ␦PKC influences the
phosphorylation state of PDH.
Materials and Methods
Rat Heart Perfusion and Isolation of Mitochondria
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Hearts isolated from male Sprague-Dawley rats (250 to 300 g, Zivic
Miller, Pittsburgh, Pa) were perfused according to the Langendorff
technique and after each experimental procedure, mitochondria were
isolated as described.40
Measurement of PDH and Citrate
Synthase Activities
Mitochondria (100 ␮g/mL) in 20 mmol/L MOPS, 0.15% Triton, pH
7.4 were incubated with 200 ␮mol/L thiamine pyrophosphate,
40 ␮mol/L CoASH, 2.5 mmol/L pyruvate, 5.0 mmol/L MgCl2,
5.0 mmol/L CaCl2, 1.0 mmol/L NAD⫹, and ⫾0.5 mmol/L NaF. PDH
activity was measured at 25°C as the rate of NADH production at
340 nm. Citrate synthase activity was measured as described.41
Evaluation of ␦PKC Translocation
Mitochondrial protein (60 ␮g/lane) was resolved by 4% to 15%
SDS-PAGE, transferred to nitrocellulose membrane, and probed
with polyclonal anti-␦PKC (Sigma). After incubation with alkaline
phosphatase– conjugated anti-IgG rabbit antibody, binding was visualized by chemiluminescence (CSPD system, Tropix).
Analysis of PDH by 2-D Gel Electrophoresis
Mitochondria (50 ␮g from each of 3 independent experiments) were
pooled and solubilized in a buffer containing 7.0 mol/L urea, 2.0
mol/L thiourea, 4.0% CHAPS, and 0.5% IPG electrophoresis buffer.
Protein was resolved by isoelectric focusing using precast Immobiline DryStrips (pI 3 to 10, 13 cm, Amersham) followed by 10%
SDS-PAGE. On transfer to nitrocellulose membrane, Western blot
analysis was performed using monoclonal antibody to the E1␣
subunit of PDH (Molecular Probes), HRP-conjugated secondary
antibody (Amersham), and enhanced chemiluminescence (Sigma).
For samples incubated with phosphatase before analysis, mitochondria (50 ␮g) were suspended in 50 ␮L of 50 mmol/L Tris-HCl,
100 mmol/L NaCl, 0.1 mmol/L EGTA, 2 mmol/L dithiothreitol,
0.01% Brij 35, 2.0 mmol/L MnCl2, 0.05% Triton X-100 at pH 7.0,
and lysed in a water bath sonicator (Branson 1200) with three 30-s
pulses. Lambda protein phosphatase (4000 U, New England Biolabs)
was then added to the mitochondria extract and incubated at 30°C for
2 hours.
␦PKC Overlay Interaction Screen
Approximately 20 000 lambda phage plaques were screened from a
Sprague-Dawley rat heart cDNA library (Stratagene) as previously
described.42,43 Recombinant bacterially expressed ␦PKC and partially purified rat brain PKC, in the presence of PKC activators (12
␮g/mL phoshatidylserine, 2 ␮g/mL diacylglycerol) (Avanti Polar
Lipids, Inc), were used as bait proteins. PKC binding was detected
using rabbit polyclonal antibodies against ␣, ␤II, ⑀, or ␦PKC (Santa
Cruz Biotechnology).
79
PKC and PDK2 Enzymes
Recombinant rat ␦PKC was cloned into the pET28 vector, transformed into Escherichia coli BL21(DE3)pLysS cells, and expressed
as a His-tagged fusion protein (Dirk Bossemeyer, Heidelberg,
Germany). Recombinant rat His-tagged PDK2 protein was obtained
from Paresh Sanghani (Indiana University School of Medicine,
Indianapolis, Ind). Rat brain PKC enzymes were purified as previously described.44 Human recombinant ⑀ and ␦PKC were purchased
from Invitrogen (Carlsbad, Calif).
Column Overlay Affinity Binding Assay
〈 partially purified rat brain PKC preparation (1.0 ␮g/mL in TBS)
was incubated with 2.0 ␮g of polyclonal IgG antibodies against the
␣, ␤II, ⑀, or ␦PKC (Santa Cruz) and Protein G agarose beads (Santa
Cruz) overnight at 4°C. Beads were washed (20 mmol/L Tris-HCl,
pH 7.5, 150 mmol/L NaCl, 0.1% Triton X-100) and recombinant
PDK2 protein (30 ␮g) was added and incubated for 1 hour at 4°C.
Agarose-immobilized protein complexes were then washed and
eluted by boiling the samples in Laemmli buffer. Protein was
resolved (12.5% SDS-PAGE), transferred to nitrocellulose membranes, and probed with anti-His conjugated to HRP (Clontech) or
with antibodies against ␣, ␤II, ⑀, or ␦PKC followed by anti-rabbit
IgG antibodies liked to HRP (Amersham).
Assessment of Interactions Between PDK2 and
PKC by ELISA
Recombinant PDK2 (20 ng/␮L) in carbonate buffer (4.0 mmol/L
Na2CO3, 3.6 mmol/L NaHCO3, pH 9.6) was placed in a 96-well (100
␮L/well) flat bottom high binding Costar EIA/RIA plate (Corning)
and incubated overnight at 4°C. Wells were washed and treated with
10 ␮g of partially purified brain PKC (diluted in 20 mmol/L
Tris-HCl, pH 7.5) in the presence of activators (1 hour, 25°C).
Binding was assessed using antibodies against ⑀ or ␦PKC (Santa
Cruz), alkaline phosphatase (AP)-conjugated secondary antibodies
(Boehringer Mannheim), and AP substrate (Pierce).
Assay of PKC-Dependent Phosphorylation
of PDK2
Phosphorylation of recombinant PDK2 by purified brain ⑀ and ␦PKC
was determined by detecting the incorporation of ␥-32P from
[␥-32P]ATP (Amersham) in the presence of PKC activators but in the
absence of Ca2⫹. The reactions were conducted at room temperature
(20 minutes) and terminated by boiling samples in Laemmli buffer.
Proteins were then resolved by 12.5% SDS-PAGE and transferred to
nitrocellulose membranes. Densitometric analyses of autoradiograms
were performed using NIH ImageJ software program.
PDK2 (40 ␮g/mL) was incubated with recombinant human ⑀ or
␦PKC (4.0 ␮g/mL) in 20 mmol/L Tris-HCl, 10 mmol/L EGTA,
20 mmol/L ATP, 20 mmol/L MgCl2, pH 7.5 in the presence of PKC
activators for 20 minutes at 37°C. Reactions were terminated by
boiling in Laemmli buffer and proteins resolved by 10% SDSPAGE. After transfer to nitrocellulose, blots were probed with
anti-PDK2 antibody (Abgent), anti-␦PKC (Santa Cruz), or a mixture
of anti-phosphorylated serine PKC substrate, anti-phosphorylated
threonine, and anti-phosphorylated threonine-X-arginine antibodies
(Cell Signaling). Binding was detected using HRP-conjugated antirabbit IgG antibodies (Amersham).
PDK2 Peptide Activity Assay
The activity of purified recombinant rat PDK2 was determined by
measuring phosphorylation of the PDH E1␣ subunit tetradecapeptide
substrate of PDK2 (YHGHSMSNPGVSYR, SynPep Corporation).45– 47 Phosphorylation was initiated by incubation of
[␥-32P]ATP (Amersham) with 1.0 ␮g of PDK2, 100 ␮mol/L peptide,
and 100 ng of ⑀ or ␦PKC (Invitrogen) in 20 mmol/L Tris-HCl,
10 mmol/L EGTA, 20 mmol/L ATP, 20 mmol/L MgCl2, pH 7.5.
Reactions were conducted at 25°C for 20 minutes and terminated on
addition of 25 ␮L of 200 mmol/L ATP/EDTA. The solution was
applied to chromatograph paper, dried for 15 minutes at 25°C, rinsed
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Figure 1. Reactivation of PDH during
cardiac reperfusion is prevented by
translocation of ␦PKC to the mitochondria. A, Hearts were equilibrated (10
minutes) and then subjected to 150 minutes of perfusion (P150), 30 minutes of
no-flow ischemia (I30), or 30 minutes of
ischemia followed by 120 minutes of
reperfusion (I30R120), in the presence or
absence of the ␦PKC specific peptide
inhibitor, Tat-␦V1-1 (1.0 ␮mol/L), infused
for the first 10 minutes of reperfusion. B,
The activity of PDH was measured in the
presence of NaF (5.0 mmol/L). Values
are presented as the mean⫾SD of 4 to 6
individual experiments. P values (2-tailed
t test) where like symbols indicate values
compared: * ⱕ0.002, † ⱕ0.001, ‡ ⱕ0.02,
§ ⱕ0.001. Native lipoic acid on the E2
subunit of PDH was examined by Western blot analyses using polyclonal antilipoic acid antibodies.54 C, Mitochondrial
protein was subjected to Western blot
analyses (60 ␮g protein per lane) using
antibodies specific to ␦PKC, phospho␦PKC (serine/threonine), and adenine
nucleotide translocase (ANT). Results of
densitometric analyses of Western blots
for ␦PKC (n⫽4) are represented as mean⫾SD with the mean for I30R120 intensity assigned a value of 100. P values: * ⱕ0.0005, †
ⱕ0.00005, ‡ ⱕ0.0002. Citrate synthase (CS) activity was measured as described in Materials and Methods. D, Protein from total homogenate (T) (10 ␮g per lane) and isolated mitochondria (M) (10 ␮g per lane) were subjected to Western blot analysis using antibodies
that recognize BiP (endoplasmic reticulum), GAPDH (cytosolic), the ␤-subunit of NaKATPase (plasma membrane),55 and ANT
(mitochondria).
with H2O2 and 70% ethanol, and then dried for 10 minutes. Peptide
phosphorylation was measured using a scintillation counter.
Results
Translocation of ␦PKC to Mitochondria During
Reperfusion Prevents Recovery of Pyruvate
Dehydrogenase Activity
No-flow ischemia (30 minutes) resulted in a 65% loss in PDH
activity relative to activity measured in mitochondria isolated
from perfused hearts (Figure 1B). PDH activity remained
depressed during reperfusion (120 minutes), with only partial
recovery in activity relative to ischemic values. The level of
native lipoic acid on the E2 subunit of PDH was unaffected
by ischemia or reperfusion indicating no alterations in protein
content (Figure 1B). As shown in Figure 1C, ␦PKC translocated to the mitochondria during reperfusion. The relative
level of total ␦PKC associated with the mitochondria is
reflected by the appearance of phospho-␦PKC (Figure 1C).
Infusion of the ␦PKC specific inhibitor Tat-␦V1-136,48 during
the first 10 minutes of reperfusion (Figure 1A) reduced
translocation of ␦PKC to the mitochondria during reperfusion
(Figure 1C). Importantly, inhibition of ␦PKC translocation
resulted in recovery of PDH activity to near control values
during reperfusion (Figure 1B). Infusion of ␦V1-1 before
ischemia failed to diminish ischemia-induced inhibition of
PDH indicating that this decrease in activity is ␦PKCindependent. Alterations in PDH activity did not appear to be
caused by global changes in mitochondrial function given
that citrate synthase activity remained unchanged (Figure
1C). In addition, isolation of mitochondria did not result in
significant copurification of contaminating fractions (Figure
1D), and infusion of the Tat carrier alone had no effect on
PDH activity or ␦PKC translocation (not shown). Finally, the
PKC inhibitor rottlerin, at a concentration (10 ␮mol/L)
specific to ␦PKC, exhibited effects similar to those observed
for Tat-␦V1-1 (Figure 2). Therefore, whereas ␦PKC does not
appear to be involved in inhibition of PDH activity during
ischemia, translocation of the kinase to the mitochondria
during reperfusion prevents complete reactivation of PDH.
H2O2 Induces ␦PKC Translocation and Inhibition
of PDH
Pro-oxidants have been shown to activate ␦PKC, whereas
other isoforms of PKC are inactivated.28,31,32,35 To further test
whether ␦PKC translocation to the mitochondria is responsible for inhibition of PDH and to determine whether alterations in redox status may act as the stimulus for ␦PKC
translocation in the intact heart, hearts were perfused in the
absence and presence of H2O2 (250 ␮mol/L; Figure 3A). This
resulted in translocation of ␦PKC to the mitochondria (Figure
3B) and an ⬇50% loss in PDH activity relative to controls
(Figure 3C). Treatment of isolated respiring mitochondria
with H2O2 had no effect on PDH activity suggesting the
requirement for cytosolic factors (not shown). Coinfusion of
the ␦PKC inhibitor Tat-␦V1-1 with H2O2 resulted in inhibition
of ␦PKC translocation to the mitochondria (Figure 3B) and
significant protection of PDH from H2O2-induced inhibition
(Figure 3C). Thus, H2O2-induced inhibition of PDH is due, in
large part, to ␦PKC translocation.
Phosphorylation-Dependent Inhibition of PDH
during Ischemia and Reperfusion
To determine whether reperfusion induces phosphorylationdependent inhibition of PDH, enzyme activity was measured
Churchill et al
␦PKC Maintains PDH Inhibition During Reperfusion
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Figure 2. Rottlerin prevents ␦PKC translocation and PDH inhibition during cardiac reperfusion. Hearts were subjected to 150
minutes of perfusion (P) or 30 minutes of ischemia followed by
120 minutes of reperfusion (I30R120), in the presence or absence
of rottlerin (10 ␮mol/L), infused for the first 10 minutes of reperfusion. The activity of PDH and the relative level of ␦PKC associated with the mitochondria were measured as described in the
Figure 1 legend. Values for PDH activity are presented as the
mean⫾SD of 4 individual experiments. P values: * ⱕ0.0003, †
ⱕ0.001.
in the presence and absence of the general phosphatase
inhibitor NaF. In mitochondria isolated from reperfused
tissue, PDH activity was ⬇25% higher when measured in the
absence of NaF (Figure 4A), indicating that ⬇50% of the
enzyme activity lost during ischemia/reperfusion may be
attributable to phosphorylation. In contrast, NaF had no
significant effect on activity in mitochondria isolated from
perfused tissue (⫹NaF, 84.9⫾12.1 nmol/min/mg; ⫺NaF,
79.8⫾5.4 nmol/min/mg). Thus, whereas PDH activity remained significantly below control values indicating inhibition/dissociation of enzyme associated phosphatase(s) or
alternative mechanisms of inhibition, reperfusion-induced
loss in PDH activity appears due, in part, to phosphorylation
of the enzyme. PDH can be inhibited to varying degrees by
phosphorylation of 3 serine residues on the E1 subunit of the
enzyme.2 Two-dimensional Western blot analysis using
anti-E1 antibody indicates that PDH migrates at 4 distinct
isoelectric points consistent with 4 phosphorylation states of
the protein (Figure 4B). In mitochondria isolated from perfused control hearts, the relative abundance of the E1 subunit
increased with increasing pI (Figure 4B). On ischemia/
reperfusion, this distribution shifted in the acidic direction
consistent with an increase in phosphorylation. Infusion of
␦PKC inhibitor Tat-␦V1-1 during ischemia/reperfusion prevented this shift (Figure 4B). Preincubation of mitochondria
from reperfused hearts with phosphatase collapsed the distribution of the E1 subunit consistent with near complete
dephosphorylation (⬇90% by densitometry; Figure 4B).
Taken together, these results provide further evidence that the
4 isoelectric points represent different phosphorylation states
Figure 3. H2O2-induced translocation of ␦PKC to mitochondria
and inactivation of PDH. A, Hearts were perfused for 60 minutes
and then infused ⫹/⫺ H2O2 (250 ␮mol/L) for 10 minutes followed by 10 minutes of perfusion in the absence of H2O2. To
inhibit ␦PKC translocation, Tat-␦V1-1 (1.0 ␮mol/L) was infused for
20 minutes (5 minutes before H2O2 until 5 minutes after H2O2).
B, Mitochondria were subjected to Western blot analyses (60 ␮g
protein per lane) using antibodies specific to ␦PKC and ANT.
Results of densitometric analyses of Western blots for ␦PKC
(n⫽6) are represented as mean⫾SD with the mean for H2O2
intensity assigned a value of 100. P values: * ⱕ0.0001, †
ⱕ0.002. C, The activity of PDH was measured in the presence
of NaF (5.0 mmol/L). The content of the PDH E2 subunit was
assessed by Western blot analyses using antibodies specific to
lipoic acid. Values are presented as the mean⫾SD of 8 individual experiments. P values: * ⱕ0.000003, † ⱕ0.005, ‡ ⱕ0.0006.
of the E1 subunit, and that ␦PKC plays a role in phosphorylation and inhibition of PDH.
␦PKC Phosphorylation and Activation of Pyruvate
Dehydrogenase Kinase 2
A potential mechanism for ␦PKC-dependent inhibition of
PDH is through the activation of specific kinases that phosphorylate and inhibit PDH. An unbiased screen of ⬇20 000 ␭
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Figure 4. Phosphorylation-dependent inhibition of PDH during
ischemia/reperfusion. A, The activity of PDH was measured in
the presence or absence of NaF (5.0 mmol/L) using mitochondria prepared after perfusion (P120) or ischemia/reperfusion (I30/
R120) in the absence or presence of Tat-␦V1-1. Values are presented as the mean⫾SD of 4 to 6 individual experiments. P
values: * ⱕ0.03, † ⱕ0.02. B, Mitochondrial protein was resolved
by 2-D gel electrophoresis followed by Western blot analysis
using polyclonal antibodies to the E1␣ subunit of PDH. Where
indicated, mitochondria were preincubated with phosphatase as
described in Materials and Methods. Blots shown represent
pooled mitochondrial samples from 3 separate hearts for each
experimental protocol.
phage clones from a rat heart cDNA expression library was
conducted using ␦PKC as the bait protein. After secondary
and tertiary screens to enrich and validate ␦PKC protein/
protein interactions, 2 clones were isolated, sequenced, and
identified as the rat form of pyruvate dehydrogenase kinase 2.
Binding was specific to the ␦-isoform of PKC with no
binding evident for ␣, ␤II, or ⑀PKC (Figure 5A). The
interaction between PDK2 and ␦PKC and specificity of this
interaction were confirmed using affinity chromatography
with immobilized PKC isoforms (Figure 5B) and ELISA with
immobilized PDK2 (Figure 5C). As shown in Figure 6A,
purified ␦ and ⑀PKC catalyzed the in vitro phosphorylation of
PDK2, with greater levels of phosphorylation evident for
␦PKC. Phosphorylation of PDK2 occurred on a serine/
threonine residue(s), consistent with the catalytic properties
of ␦PKC (Figure 6B). To determine whether phosphorylation
of PDK2 activates the kinase, a peptide analog of the
phosphorylation site on PDH was used as substrate. As shown
in Figure 6C, ␦PKC-dependent phosphorylation resulted in
Figure 5. In vitro interactions between PDK2 and ␦PKC. A, Purified recombinant rat heart ␦PKC was used as bait to screen a
rat heart ␭ phage cDNA expression library in the presence of 12
␮g/mL phosphatidylserine (PS) and 2.0 ␮g/mL diacylglycerol
(DAG). Two positive clones were isolated and identified as
PDK2. The specificity of PDK2/PKC interaction(s) were further
evaluated using indicated isoforms of PKC purified from rat
brain. B, Partially purified rat brain PKC isoforms (␣, ␤II, ⑀, ␦)
were incubated with polyclonal IgG antibodies against the ␣, ␤II,
⑀, or ␦PKC and Protein G agarose beads. Immobilized protein
was then incubated with purified recombinant His-tagged PDK2
(rat heart) in the presence of PS and DAG. PDK2 binding to
PKC was detected by subjecting the eluted proteins to Western
blot analysis with anti–His-HRP antibody. PKC immunoprecipitates were also probed with anti-PKC polyclonal antibodies.
Blots shown are representative of 3 independent experiments.
C, PDK2 was immobilized onto 96-well plates and incubated
with either ␦ or ⑀PKC in the presence of PS and DAG. Protein
complexes were probed with polyclonal antibodies against ⑀
and ␦PKC. Values are presented as the mean⫾SD of 6 individual experiments. P values: * ⱕ0.002, † ⱕ0.003.
activation of PDK2. Activation appears specific to ␦PKC in
that no appreciable phosphorylation of the peptide analog was
observed in the absence of ␦PKC or PDK2 or in the presence
of ⑀PKC. It is well documented that PDK2 catalyzes the
phosphorylation and inhibition of PDH.2 These results provide a plausible mechanism for ␦PKC-dependent inhibition
of PDH during cardiac reperfusion.
Churchill et al
␦PKC Maintains PDH Inhibition During Reperfusion
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Figure 6. ␦PKC-dependent phosphorylation and activation of
PDK2. A, Purified ␦ and ⑀PKC were incubated with purified
recombinant PDK2 in the presence of PS and DAG but in the
absence of Ca2⫹. Phosphorylation of PDK2 was measured by
incorporation of 32P on incubation with [␥-32P]ATP. Values are
presented as the mean⫾SD of 4 individual experiments. P values: *Pⱕ0.02, † ⱕ0.0005. B, Recombinant rat heart PDK2 and
human ␦PKC were incubated in the presence of ATP, PS, and
DAG for 20 minutes at 37°C. Western blot analysis was then
performed using antibodies raised to PDK2, phospho-serine/
threonine, and ␦PKC. C, 32P incorporation into the E1␣ subunit
substrate peptide of PDK2 was determined in the presence
PDK2, PDK2⫹␦PKC, or PDK2⫹⑀PKC. Values are presented as
the mean⫾SD for 4 separate experiments conducted in duplicate. P values: * ⱕ0.001, † ⱕ0.002.
Discussion
PDH was shown to decline in activity during cardiac ischemia. Although a fractional regain in PDH activity occurred on
reperfusion, the activity of the enzyme remained depressed
relative to control values. ␦PKC translocated to the mitochondria during reperfusion. Prevention of ␦PKC translocation
resulted in complete recovery in PDH activity. Thus, ␦PKC
prevents reactivation or promotes continued inhibition of
PDH in response to cardiac reperfusion. Activation of PDH or
inclusion of pyruvate during cardiac ischemia/reperfusion
improves recovery of hemodynamic function.10 –14,16 –18,21–23
83
Recovery of PDH activity on inhibition of ␦PKC translocation may therefore, in part, provide an explanation for the
previously observed cardioprotective role of the ␦PKC specific peptide inhibitor.36 – 40
Reperfusion of myocardial tissue is associated with a rapid
increase in the level of various pro-oxidant species.5,24 Purified ␦PKC is sensitive to redox status, increasing in catalytic
activity under oxidative conditions that induce inactivation of
other PKC isoforms.28 In addition, treatment of cells in
culture with H2O2 results in the translocation of ␦PKC to the
mitochondria.33 We have demonstrated that perfusion of rat
hearts with H2O2 induces the translocation of ␦PKC to the
mitochondria and reduction in PDH activity. H2O2-dependent
loss of PDH activity was largely prevented by inhibition of
␦PKC translocation. Treatment of mitochondria with H2O2
did not have an effect on PDH activity, indicating that
cytosolic component(s) are necessary for inhibition of PDH.
Therefore, pro-oxidants produced during cardiac reperfusion
may provide the stimulus for ␦PKC translocation and PDH
inhibition.
PDH is regulated by specific kinases and phosphatases
associated with the PDH complex.2– 4,49 In mitochondria
isolated from reperfused tissue, PDH activity was partially
recovered when assayed in the absence of the phosphatase
inhibitor NaF. In addition, reperfusion-induced declines in
PDH activity were associated with an acidic shift in the
isoelectric point of the ␣E1 subunit of pyruvate dehydrogenase that was prevented on inhibition of ␦PKC translocation.
Thus, ␦PKC appears to promote phosphorylation-dependent
inhibition of PDH. In vitro data indicates that ␦PKC specifically interacts with PDK2, a kinase that can phosphorylate
and inhibit PDH. Importantly, the interaction between ␦PKC
and PDK2 leads to the phosphorylation and activation PDK2.
Endogenous dephosphorylation of PDH (assayed in the
absence of NaF) partially restored the ischemia/reperfusioninduced loss in PDH activity, suggesting additional modes of
␦PKC-dependent inhibition. One potential mechanism is
through the inhibition of PDP1 and/or PDP2. However, when
mitochondrial samples isolated from reperfused tissue were
incubated with alkaline phosphatase to promote dephosphorylation, no further regain in enzyme activity was observed
(not shown). In addition, it has been demonstrated that
treatment of L6 skeletal muscle cells and immortalized
hepatocytes with insulin results in ␦PKC activation and
interaction with PDP1/2. This leads to activation of PDP1/2
and stimulation of PDH activity.49 Nevertheless, the effects of
␦PKC are likely to be tissue specific.49
An alternative mechanism of PDH inhibition may involve
oxidative modification of PDP1/2 or PDH. Precedence for
this possibility is provided by evidence that the phosphatase
PTP1B is readily inhibited on glutathionylation in response to
receptor stimulated pro-oxidant production50 and that PDH is
susceptible to oxidative inhibition.51–53 In the present study,
H2O2-dependent loss of PDH activity was partially prevented
by inhibition of ␦PKC translocation. In contrast, prevention
of ␦PKC translocation to the mitochondria during reperfusion
resulted in full reactivation of PDH. This difference may be
explained by previous findings that translocation of ␦PKC to
the mitochondria results in release of cytochrome c that could
84
Circulation Research
July 8, 2005
in turn amplify mitochondrial free radical production.29 –35
Thus, prevention of ␦PKC translocation to the mitochondria
during reperfusion would be expected to prevent ␦PKC- and
redox-dependent inhibition of PDH.
Acknowledgments
This work was supported by grants from the National Institutes of
Health (R01 AG-19357 and R01 AG-16339 to L.I.S and 2RO1
HL-52141 to D.M.-R.). The authors thank Robert Harris, Paresh C.
Sanghani and the Indiana Genomics Initiative (INGEN) Protein
Expression Core of Indiana University (supported in part by Lilly
Endowment Inc) for providing PDK-2.
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Reperfusion-Induced Translocation of δPKC to Cardiac Mitochondria Prevents Pyruvate
Dehydrogenase Reactivation
Eric N. Churchill, Christopher L. Murriel, Che-Hong Chen, Daria Mochly-Rosen and Luke I.
Szweda
Circ Res. 2005;97:78-85; originally published online June 16, 2005;
doi: 10.1161/01.RES.0000173896.32522.6e
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