(Circulation. 2001;104:85.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Downregulation of Protein Kinase C Inhibits Activation of Mitochondrial KATP Channels by Diazoxide
From the Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio.
Correspondence to Muhammad Ashraf, PhD, Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, 231 Bethesda Ave, Cincinnati, OH 45267-0529. E-mail Muhammad.Ashraf{at}uc.edu
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Abstract |
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Background—The mitochondrial KATP (mitoKATP) channel has been shown to confer short- and long-term cardioprotection against prolonged ischemia via protein kinase C (PKC) signaling pathways. However, the exact association between PKC or its isoforms and mitoKATP channels has not yet been clarified. The present study tested the hypothesis that the activity and translocation of PKC to the mitochondria are important for cardiac protection elicited by mitoKATP channels.
Methods and Results—PKC
was downregulated by prolonged (24-hour) treatment with phorbol
12-myristate 13-acetate (4 µg/kg body weight) before
subsequent experiments in rats. Langendorff-perfused rat hearts were
subjected to 40 minutes of ischemia followed by 30 minutes of
reperfusion. Effects of PKC downregulation on the activation of
mitoKATP channels and other interventions on
hemodynamic, biochemical, and pathological changes were
assessed. Subcellular localization of PKC isoforms by Western blot
analysis and immunocytochemistry demonstrated that PKC-
and
PKC-
were translocated to the sarcolemma and that PKC- was
translocated to the mitochondria after diazoxide treatment. In hearts
treated with diazoxide (80 µmol/L), a significant improvement in
cardiac function and an attenuation of cell injury were observed. In
PKC-downregulated hearts, protection was abolished because
mitoKATP channels could not be activated
by diazoxide.
Conclusions—These data
suggest that PKC activation is required for the opening of
mitoKATP channels during protection against
ischemia and that this effect is linked to isoform-specific
translocation of PKC- to the mitochondria.
Key Words: ischemia • ion channels • protein kinase C • myocardium
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Recently we reported that activation of mitochondrial KATP (mitoKATP) channels elicits cardiac protection against ischemia via protein kinase C (PKC) signaling pathways. In that study, we demonstrated by immunocytochemistry that PKC-
was
translocated to the mitochondria and sarcolemma after treatment with
diazoxide (DZX), a relatively specific opener of
mitoKATP
channels.1 PKC is also known
to accelerate the activity of mitoKATP channels,
as reported by Sato et al.2
In the latter study, however, the effect of PKC inhibition during
DZX-induced activation of mitoKATP channels was
not studied. PKC is believed to be an important intracellular
regulatory enzyme of the myocardium under both normal and
abnormal conditions. However, the pathways by which DZX
activates PKC and mitoKATP channels are
not clear. It has also been reported that PKC activates
sarcolemmal KATP channels and plays a
significant role in ischemic
preconditioning.3 Because
inhibition of PKC abolishes the ischemic protection mediated by
mitoKATP
channels,1 it appears that
the effect of mitoKATP channels and PKC is
interdependent. Most previous studies on the role of PKC in signaling
pathways have been done with pharmacological inhibitors.
Approaches to create PKC-knockout mice to study the importance of PKC
in signaling pathways have not yet been successful. This problem is
further complicated by the presence of several PKC isoforms that have
been implicated in different functions of cardiac cells. In light of
these difficulties, studies in chemically PKC–downregulated rats may
provide some insight into the responses of
mitoKATP channel activation in cardiac
protection. In the current study, we determined whether downregulation
of PKC renders the mitoKATP channels ineffective
for cardiac protection. The data in this investigation suggest that PKC
translocation to the mitochondria is required for activation of
mitoKATP channels. |
Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Chemicals
DZX and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemical Co. Tetramethylrhodamine ethyl ester (TMRE) was obtained from Molecular Probes. Optimum cutting temperature (OCT, Tissue-Tek) compound was obtained from Miles Inc. Rabbit polyclonal isoform-specific anti-PKC antibodies were purchased from Santa Cruz Biotechnology Inc. Indocarbocyanine-conjugated anti-rabbit IgG antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. Reagents for Western blot detection by enhanced chemiluminescence were purchased from Amersham Pharmacia Biotech. Peroxidase-labeled, affinity-purified antibody was purchased from Kirkegaard & Perry Laboratories.
Heart Preparation
Hearts from anesthetized male Sprague-Dawley
rats (Harlan Sprague-Dawley, Indianapolis, Ind) were removed and
retrogradely perfused in a noncirculating Langendorff
apparatus with Krebs-Henseleit buffer, which
consisted of the following (in mmol/L): NaCl 118, KCl 4.7,
MgSO4 1.2,
KH2PO4 1.2,
CaCl2 1.8, NaHCO3 25, and
glucose 11. The buffer was saturated with 95%
O2/5% CO2 (pH 7.4,
37°C) for 50 minutes. Hearts were perfused at a constant pressure of
80 mm Hg. A water-filled latex balloon–tipped catheter was
inserted into the left ventricle through the left atrium and was
adjusted to a left ventricular end-diastolic
pressure (LVEDP) of 4 to 6 mm Hg during initial equilibration.
Thereafter, the balloon volume was not changed. The distal end of the
catheter was connected to a Digi-Med heart performance
analyzer (model 210, version 1.01, Micro-Med) by way of a
pressure transducer (Case).4
Hearts were paced at 350 beats/min except during ischemia.
Pacing was reinitiated after 3 minutes of reperfusion in all groups.
The indexes of myocardial function were determined as previously
described.5 The present
study conformed to the protocols approved by the institutional Animal
Care Committee.
Experimental Protocol
After equilibration, hearts were randomly divided
into the following experimental groups.
Group 1: Normal Control and Vehicle
Control
Hearts (n=6) were perfused for 95 minutes with
Krebs-Henseleit buffer as a normal control for the different
experimental groups. Hearts were treated for 6 minutes with vehicle
(0.04% dimethyl sulfoxide, n=6), after which they were subjected to
ischemia/reperfusion. The amount of dimethyl sulfoxide used in
the vehicle control experiments did not affect any
hemodynamic parameters, lactate
dehydrogenase (LDH) release, ATP content, or cell
morphology.6
Group 2: Ischemia/Reperfusion
After equilibration, hearts (n=6) were subjected to
ischemia for 40 minutes followed by reperfusion for 30
minutes.
Group 3: Role of
MitoKATP Channels in Cardiac Protection
A potent activator of
mitoKATP channels, DZX was used before
ischemia. DZX opens sarcolemmal KATP
channels at higher concentration (855
µmol=K1/2), whereas it opens
mitoKATP channels at very low concentration (0.4
µmol=K1/2).7
Activation of this channel is blocked by a specific
inhibitor, 5-hydroxydecanoic acid, as previously
reported.1 2 7
This group tested the hypothesis that direct activation of
mitoKATP channels induces cardiac protection.
For this purpose, hearts (n=6) were perfused with Krebs-Henseleit
buffer containing DZX (80
µmol/L)1 for 6 minutes.
Then the hearts were subjected to
ischemia/reperfusion.
Group 4: Effect of PKC Downregulation on
MitoKATP Channel Activation by DZX
The purpose of these experiments was to determine
whether activation of mitoKATP channels after
PKC downregulation could protect the heart against ischemia.
PKC was downregulated by prolonged treatment with 4 µg/kg body weight
PMA injected into the tail vein of rats every 4 to 6 hours for 24
hours, a procedure known to downregulate PKC
activity.8 After 24 hours,
hearts (n=6) were perfused in a manner similar to that for group
3.
Group 5: Direct Influence of PKC on Cardiac
Protection
To determine whether direct activation of PKC
protects the hearts in a manner similar to that of
mitoKATP channel activation, hearts were treated
for 6 minutes with PMA (100
nmol/L4 ) before
ischemia/reperfusion.
Measurement of LDH and Tissue ATP
LDH, an indicator of myocardial tissue injury, was
determined in the coronary effluent by a coupled
enzyme-spectrometric technique,1,4 and
tissue ATP was analyzed by a spectrophotometric method as
described earlier.5
Separation of Membrane, Cytosolic, and
Mitochondrial Fractions for PKC Localization
Membrane and cytosolic fractions were prepared as
previously described.9 The
purity of the cytosolic extracts was examined by using LDH as a
marker.10 The sarcolemmal
fraction was confirmed with a 5'-nucleotidase assay as
described.11 Mitochondria
were isolated by differential
centrifugation.12
Succinate cytochrome c
reductase activity was used to determine the purity of the
mitochondrial pellet as described
previously.13
Western Blots
The subcellular localization of PKC isoforms was
examined by quantitative
immunoblotting.14
Equal amounts of proteins were loaded on each lane of a 10%
SDS-polyacrylamide gel. Proteins were separated by
electrophoresis and transferred from the gel to nitrocellulose
membranes (Bio-Rad catalog No. 162-0095) by using an electroblotting
apparatus. The membrane was incubated for 60 minutes in 5%
dry milk and Tris-buffered saline (20 mmol/L Tris HCl [pH 7.4]
and 137 mmol/L NaCl) to block nonspecific binding sites. Western
blots were probed with affinity-purified, PKC isoform–specific primary
antibodies at dilutions of 1:500 to 1:1000 for 1 hour. After being
washed, blots were incubated with a 1:10 000 dilution of horseradish
peroxidase–labeled anti-rabbit IgG for 1 hour at room temperature. PKC
isoforms were detected by the enhanced chemiluminescence method. The
amounts of PKC isoforms on the immunoblots were quantified
by use of a computer program (ImageQuant
Solution).
Subcellular Localization of PKC Isoforms by
Immunocytochemistry
Immunocytochemical localization of PKC isoforms after
various interventions was performed as previously
described.4 5
Sections (5 µm thick) were fixed for 10 minutes in a 70%
acetone/30% methanol mixture at -20°C, incubated in 10% normal
goat serum in phosphate-buffered saline for 30 minutes to block
nonspecific binding, and then incubated with primary antibodies.
Confocal images were also obtained with a Leitz DMRBE
fluorescence microscope equipped with a TCS 4D confocal
scanning attachment (Leica, Inc). Fluorescence was excited by
the 568-nm wavelength from a krypton/argon laser, and emission at 568
to 580 nm was recorded. For mitochondrial imaging, additional
hearts were perfused with either 200 nmol/L TMRE, a mitochondrial
marker alone, or with 200 nmol/L TMRE plus 80 µmol/L DZX for 6
minutes.1 Tissue was frozen
and sectioned for microscopy. TMRE excitation was induced by using the
488-nm wavelength of a krypton/argon laser and was recorded at
>530 nm with a fluorescein isothiocyanate
filter1 4
Statistical Analysis
All values are expressed as mean±SEM. Group
comparisons were analyzed by 1-way ANOVA (Statview 4.0). All
groups were analyzed simultaneously with a
Bonferroni/Dunn test. A difference of
P<0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Ischemic Control Hearts
Heart function was significantly decreased after ischemia. At the end of reperfusion, left ventricular developed pressure (LVDP) and coronary flow (CF) were decreased, whereas LVEDP was increased (Figures 1A through 1C
). ATP levels were reduced to 4.62±0.9
µmol/g dry weight compared with the normal control value of 22.3±0.8
µmol/g dry weight
(Figure 2), and LDH leakage was increased on reperfusion
compared with the control value
(Figure 2).
|
) or DZX-(
) preconditioned hearts compared with ischemic control hearts (
). PKC downregulation (DOWN) completely blocked protection by DZX. Values are mean±SE, n=6 for each group. *P<0.05 vs ischemic control. I/R indicates ischemia/reperfusion; DZX+I/R, DZX pretreatment before I/R; DOWN+I/R, PKC-downregulated hearts treated with DZX and then subjected to I/R.
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Effect of DZX and PMA on Ischemic
Injury
A significant increase in LVDP and CF or a decrease in
LVEDP was observed on reperfusion of hearts pretreated with DZX and PMA
(Figures 1A through 1C
). ATP contents were markedly preserved
after ischemia/reperfusion in hearts pretreated with both DZX
and PMA, and LDH release was also significantly decreased
(Figure 2).
Effect of DZX on the Heart After PKC
Downregulation
In hearts subjected to repetitive injection of PMA, DZX
was totally ineffective. LVDP, LVEDP, and CF values were
similar to those for ischemia/reperfusion
(Figure 1). No significant differences in ATP content and LDH
release were observed in ischemic control and treated hearts
(Figure 2).
Subcellular Distribution of PKC
Isoforms
Figure 3 shows representative Western blots
and their quantitative estimates of PKC isoforms in membrane and
mitochondrial fractions after various treatments. No significant
differences in the distribution of PKC isoforms were observed in
cytosolic fractions (data not shown). DZX treatment before
ischemia significantly increased the content of PKC-, -
,
and - isoforms in the membrane fractions, but only PKC- was
increased in the mitochondria compared with the other isoforms
(Figure 3). PKC-, -, and - were not
activated after DZX treatment of PKC-downregulated hearts, and
similarly, translocation of PKC- to the mitochondria did not occur
in downregulated hearts.
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Immunohistochemical Localization of PKC
Isoforms in Myocytes
Representative photomicrographs
from the immunohistochemical study are shown in
Figure 4. PKC-, which was diffusely distributed in the
cytoplasm of control cells, was distinctly localized in the sarcolemma
of DZX-pretreated hearts. DZX pretreatment further resulted in
translocation of PKC- to intercalated discs and mitochondria;
however, its translocation to the mitochondria was weaker than that of
PKC-, of PKC-ß1 to the nuclear region, and
of PKC- to the mitochondria. No specific distribution of
PKC-ß1, -, and - isoforms was observed
in PKC-downregulated hearts after DZX treatment. Staining with PKC-
was less intense and weaker than with PKC-,
-ß1, -, and - isoforms after DZX
treatment (data not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the current study, we have demonstrated that PKC is an integral component of the mitoKATP channel–induced protection against ischemic injury. First, isoform PKC-
is translocated to the mitochondria at the time of
mitoKATP channel activation. Second,
mitoKATP channels could not be opened by DZX in
PKC-downregulated hearts, resulting in no protection against
ischemia. These results suggest that PKC activity is required
for the mitoKATP channels to be effective
against ischemia. Previous studies have shown that PKC is an
important component of signal transduction pathways leading to
protection by ischemic
preconditioning.4 15
How PKC regulates mitoKATP channels is not yet
known. PKC is known to upregulate KATP channels
at physiological levels of
ATP.16 Recently Sato et
al2 have demonstrated that
mitochondrial flavoprotein oxidation by a selective
mitoKATP channel opener, DZX, was potentiated by
PMA, suggesting that the activity of mitoKATP
channels is upregulated by PKC in isolated myocytes. PKC inhibition
before activation of mitoKATP channels by DZX
resulted in the loss of protection against ischemic injury in
the rat
myocardium.1 17
Thus, it appears that PKC-catalyzed phosphorylation of
KATP channels is vital in
preconditioning-induced protection. PKC regulates a number of functions, and because of the lack of a PKC-knockout animal model and the complexity of PKC isoforms, it is rather difficult to assess the exact role of PKC in ischemic preconditioning. Long-term exposure of cells to phorbol esters is known to cause downregulation of PKC and loss of responsiveness to phorbol esters.8 Young et al18 reported that downregulation of PKC by phorbol ester is a consequence of either an increased rate of degradation of the polypeptide or multiple proteases may be involved in PKC downregulation.19 The mechanism whereby phorbol esters induce downregulation of PKC is still unclear. In this study, downregulation of PKC totally blocked the protection by DZX, suggesting a crucial role for PKC in the regulation of mitoKATP channels. This study also strongly showed that PKC translocation to the mitochondria was essential for activation of mitoKATP channels. The importance of PKC was further emphasized by Sato et al,2 who suggested that PKC primes the mitoKATP channel to open earlier and more intensely during prolonged ischemia. It is well established that DZX selectively opens mitoKATP channels1 2 7 and is responsible for both early and late preconditioning.1 17 20 Activation of mitoKATP channels retards calcium accumulation by the mitochondria and either preserves ATP1 4 or increases ATP synthesis.21 Szewczyk22 pointed out that the opening of mitoKATP channels partially compensates the membrane potential, thereby enabling additional protons to be pumped out to form a substantial proton electrochemical gradient for both ATP synthesis and Ca2+ transport. Janczewski et al23 reported that the Ca2+ transient in the mitochondrial matrix is important for ATP synthesis. Therefore, the increase in ATP retention or synthesis as a result of the opening of mitoKATP channels is important for improving postischemic cardiac functional recovery and maintaining Ca2+ homeostasis. The loss of mitoKATP channel–mediated effects by downregulation of PKC further reinforces the notion that signal transduction cascades of a large number of receptors are coupled to PKC activation, which may phosphorylate different channels leading to preconditioning.
It has previously been reported that in rat myocardial
cells, PKC-, -, -, and -ß are the dominant isoforms
expressed.9 These isoforms
have specific functions. For example, activation of PKC-ß and - in
hypertrophied
hearts9 24 and
translocation to cell junctions of cardiac myocytes have been
reported.5 PKC- is growth
inhibitor.25
Mitchell et al15 reported
translocation of PKC-, -, or
both26 to the membrane after
preconditioning ischemia. However, in the former study, PKC-
was translocated in the perinuclear zone. In other studies, it has been
shown that PKC- but not - is associated with the early phase of
ischemic preconditioning in rabbit cardiomyocytes
and reduction of ischemic injury in rat
myocytes.27 28
Our present study demonstrates that DZX pretreatment caused
translocation of PKC- to the mitochondria, which may
phosphorylate mitoKATP
channels29 or accelerate
their opening.2 On the other
hand, overexpression of PKC- could confer beneficial effects against
ischemic injury.28
It is also noteworthy that DZX pretreatment, besides PKC-, also
caused the translocation of PKC- to the sarcolemma, of
-ß1 to the nucleus, and of - to
intercalated discs. PKC- may have a role in phosphorylating membrane
proteins, which have been implicated in ischemic
preconditioning.30
Translocation of PKC- to the intercalated disc of myocytes may
facilitate intercellular communication through nexuses located in the
intercalated disc, or PKC- may participate in late
preconditioning.31
Translocation of PKC isoforms to the nucleus may have implications for
late preconditioning induced by DZX
treatment.17 It is still a
matter of debate which PKC isoform mediates the effect of
preconditioning. To further implicate that downregulation of PKC is
responsible for the loss of protection, DZX was unable to open the
channels in PKC-downregulated hearts, as evidenced by the extent of
cell injury. Two approaches were taken to assess the modulation of
mitoKATP channels by PKC. First, PKC was
downregulated in the rat heart by prolonged treatment with PMA, and
second, the mitoKATP channel was
activated pharmacologically by a selective opener, DZX. To
assess the effectiveness of PKC downregulation, Western blot
analysis and immunocytochemistry of PKC isoforms were carried
out. As expected, no isoforms could be detected in PKC-downregulated
hearts after 24 hours. DZX was totally ineffective on the
mitoKATP channels in PKC-downregulated
hearts.
This is the first study to demonstrate a PKC isoform–specific role in
cardiac protection. PKC- was significantly translocated to the
mitochondrial fraction after DZX treatment, and no other isoforms were
observed in the mitochondria, suggesting a role for PKC- in
mitoKATP channel–mediated protection. Thus, it
appears that PKC-dependent phosphorylation of
mitoKATP channels may indeed be an important
link in ischemic preconditioning. In summary, these data
demonstrate that PKC downregulation renders the
mitoKATP channel ineffective, resulting in
increased ischemic injury. The data further suggest that
PKC- may be important for mitoKATP
channel–mediated protection.
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Acknowledgments |
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This study was supported by grants HL 23597 and HL 55678 from the National Institutes of Health, Bethesda, Md.
Received January 3, 2001; revision received March 6, 2001; accepted March 9, 2001.
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References |
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M. T. Jiang, M. Ljubkovic, Y. Nakae, Y. Shi, W.-M. Kwok, D. F. Stowe, and Z. J. Bosnjak Characterization of human cardiac mitochondrial ATP-sensitive potassium channel and its regulation by phorbol ester in vitro Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1770 - H1776. [Abstract] [Full Text] [PDF]
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A. D.T. Costa, K. D. Garlid, I. C. West, T. M. Lincoln, J. M. Downey, M. V. Cohen, and S. D. Critz Protein Kinase G Transmits the Cardioprotective Signal From Cytosol to Mitochondria Circ. Res., August 19, 2005; 97(4): 329 - 336. [Abstract] [Full Text] [PDF]
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L. Willems, K. J. Ashton, and J. P. Headrick Adenosine-mediated cardioprotection in the aging myocardium Cardiovasc Res, May 1, 2005; 66(2): 245 - 255. [Abstract] [Full Text] [PDF]
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M. Mayr, B. Metzler, Y.-L. Chung, E. McGregor, U. Mayr, H. Troy, Y. Hu, M. Leitges, O. Pachinger, J. R. Griffiths, et al. Ischemic preconditioning exaggerates cardiac damage in PKC-{delta} null mice Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H946 - H956. [Abstract] [Full Text] [PDF]
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M. A. Deja, K. S. Golba, M. Kolowca, K. Widenka, J. Biernat, and S. Wos Diazoxide provides protection to human myocardium in vitro that is concentration dependent Ann. Thorac. Surg., January 1, 2004; 77(1): 226 - 232. [Abstract] [Full Text] [PDF]
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L. Argaud, O. Gateau-Roesch, L. Chalabreysse, L. Gomez, J. Loufouat, F. Thivolet-Bejui, D. Robert, and M. Ovize Preconditioning delays Ca2+-induced mitochondrial permeability transition Cardiovasc Res, January 1, 2004; 61(1): 115 - 122. [Abstract] [Full Text] [PDF]
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J. P. Headrick, B. Hack, and K. J. Ashton Acute adenosinergic cardioprotection in ischemic-reperfused hearts Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H1797 - H1818. [Abstract] [Full Text] [PDF]
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J. N. Weiss, P. Korge, H. M. Honda, and P. Ping Role of the Mitochondrial Permeability Transition in Myocardial Disease Circ. Res., August 22, 2003; 93(4): 292 - 301. [Abstract] [Full Text] [PDF]
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S. K. Jain, R. B. Schuessler, and J. E. Saffitz Mechanisms of Delayed Electrical Uncoupling Induced by Ischemic Preconditioning Circ. Res., May 30, 2003; 92(10): 1138 - 1144. [Abstract] [Full Text] [PDF]
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G. Lebuffe, P. T. Schumacker, Z.-H. Shao, T. Anderson, H. Iwase, and T. L. Vanden Hoek ROS and NO trigger early preconditioning: relationship to mitochondrial KATP channel Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H299 - H308. [Abstract] [Full Text] [PDF]
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O. Oldenburg, M. V Cohen, D. M Yellon, and J. M Downey Mitochondrial KATP channels: role in cardioprotection Cardiovasc Res, August 15, 2002; 55(3): 429 - 437. [Abstract] [Full Text] [PDF]
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Z.-Q. Zhao and J. Vinten-Johansen Myocardial apoptosis and ischemic preconditioning Cardiovasc Res, August 15, 2002; 55(3): 438 - 455. [Abstract] [Full Text] [PDF]
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S. Wolfrum, K. Schneider, M. Heidbreder, J. Nienstedt, P. Dominiak, and A. Dendorfer Remote preconditioning protects the heart by activating myocardial PKC{epsilon}-isoform Cardiovasc Res, August 15, 2002; 55(3): 583 - 589. [Abstract] [Full Text] [PDF]
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Y. Ohnuma, T. Miura, T. Miki, M. Tanno, A. Kuno, A. Tsuchida, and K. Shimamoto Opening of mitochondrial KATP channel occurs downstream of PKC-epsilon activation in the mechanism of preconditioning Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H440 - H447. [Abstract] [Full Text] [PDF]
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