© 1999 American Heart Association, Inc.
Integrative Physiology |
Activation of Mitochondrial ATP-Sensitive K+ Channel for Cardiac Protection Against Ischemic Injury Is Dependent on Protein Kinase C Activity
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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Abstract—Protein kinase C (PKC) is involved in the second messenger signaling cascade during ischemic and Ca2+ preconditioning. Given that the pharmacological activation of mitochondrial ATP-sensitive K+ (mitoKATP) channels also mimics preconditioning, the mechanisms linking PKC activation and mitoKATP channels remain to be established. We hypothesize that PKC activity is important for the opening of the mitoKATP channel. To examine this, a specific opener of the mitoKATP channel, diazoxide, was used in conjunction with subcellular distribution of PKC in a model of ischemia/reperfusion (I/R). Langendorff-perfused rat hearts were subjected to 40-minute ischemia followed by 30-minute reperfusion. Effects of activation of the mitoKATP channel and other interventions on functional, biochemical, and pathological changes in ischemic hearts were assessed. In hearts treated with diazoxide, left ventricular end-diastolic pressure and coronary flow were significantly improved after I/R; lactate dehydrogenase release was also significantly decreased. The morphology was well preserved in diazoxide-treated hearts compared with nontreated ischemic control hearts. The salutary effects of diazoxide on the ischemic injury were similar to those of Ca2+ preconditioning. Administration of sodium 5-hydroxydecanoate, an effective blocker of the mitoKATP channel, or chelerythrine or calphostin C, an inhibitor of PKC, during diazoxide pretreatment or during continuous presence of diazoxide in the ischemic period, completely abolished the beneficial effects of the diazoxide on the I/R injury. Blockade of Ca2+ entry during diazoxide treatment by inhibiting the L-type Ca2+ channel with verapamil also completely reversed the beneficial effect of diazoxide during I/R. PKC-
was translocated to sarcolemma, whereas PKC-
was
translocated to the mitochondria and intercalated disc, and PKC-
was
translocated to the intercalated disc of the diazoxide-pretreated
hearts. Colocalization studies for mitochondrial distribution with
tetramethylrhodamine ethyl ester (TMRE) and PKC isoforms by
immunoconfocal microscopy revealed that PKC- antibody specifically
stained the mitochondria. ATP was significantly increased in the
diazoxide-treated hearts. Moreover, the data suggest that activation
and translocation of PKC to mitochondria appear to be important for the
protection mediated by mitoKATP channel.
Key Words: mitochondrial KATP channel • preconditioning • ischemia • protein kinase C • ATP
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Introduction |
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Ischemic preconditioning results in a drastic reduction in cellular injury in the ischemic myocardium. Protein kinase C (PKC) is an important component of the signal transduction pathways in the ischemic preconditioning–induced protection. One of the final effectors in this protection has been recently reported to be mitochondrial KATP (mitoKATP) channels.1 Previously, opening of sarcolemmal KATP channels has been shown to shorten the action potential causing depression of contractility.2 3 This was proposed as the mechanism of protection in ischemic myocardium.4 However, recent evidence contradicts this hypothesis, because the degree of action potential shortening can be divorced from the extent of protection and KATP channel openers are protective even in unstimulated cardiac myocytes,5 6 in which action potential abbreviation cannot be a factor.
Cardiac myocytes possess another type of KATP channel in the inner mitochondrial membrane.7 Opening of this channel would tend to dissipate the membrane potential established by the proton pump.8 Such dissipation accelerates electron transfer by the respiratory chain and leads to net oxidation of the mitochondrial matrix. Low concentration of diazoxide (DE) specifically activates mitoKATP channels without any effect on sarcolemmal KATP channels.9 Our recent study shows that stimulation of mitoKATP channels with DE is highly protective against the calcium overload injury and PKC activity is required for the effect of mitoKATP channel.10 11 Because inhibition of PKC can block the effect of mitoKATP channels, we tested the hypothesis that the activation and translocation of PKC are also required for the protection against lethal ischemia by mitoKATP channel. In this study, we investigated the role of the mitoKATP channel in cardiac protection and its dependence on various PKC isoforms. The results of this study show that the effect of mitoKATP channel is blocked by inhibition of PKC, the activation of which is indicated by both Ca2+-dependent and -independent pathways.
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Materials and Methods |
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DE, chelerythrine (CHE), calphostin C (CN), and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma. Verapamil (VL) hydrochloride was purchased from Research Biochemicals International. Sodium 5-hydroxydecanoate (5-HD) was purchased from ICN Pharmaceuticals, Inc. Tetramethylrhodamine ethyl ester (TMRE) was obtained from Molecular Probes. OCT compound (Tissue-Tek) was from Miles, Inc; rabbit polyclonal isoform-specific anti-PKC antibodies were purchased from Santa Cruz Biotechnology. The following antibodies were raised against isoform-unique peptide sequences within the carboxyl terminus:
, 651 to 672;
ß1, 656 to 671; ß2, 657
to 673; 
, 679 to 697; , 657 to 673; , 722 to 736; 
, 669 to
683; and 
, 573 to 592. Indocarbocyanine-conjugated anti-rabbit IgG
antibody was purchased from Jackson ImmunoResearch Laboratories. DE was
dissolved in DMSO before being added into the perfusion buffer. The
final concentration of DMSO was <0.1%. Other reagents were dissolved
in PBS. All of the agents except DE were directly administered through
the aortic cannula at a rate of 0.2 mL/min by a Harvard infusion pump.
DE was directly perfused through the aorta in a noncirculating
Langendorff apparatus with Krebs-Henseleit (KH) buffer.
Heart Preparation
Hearts from male Sprague-Dawley rats were removed and
retrogradely perfused through the aorta in a noncirculating Langendorff
apparatus with KH buffer, which consisted of (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 the 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) via a pressure transducer (Case).
Hearts were paced at 350 bpm except during ischemia. Pacing was
reinitiated after 3 minutes of reperfusion in all groups. The index of
myocardial function was determined as previously
described.12 All hearts were perfused with the
oxygenated KH buffer for a total of 95 minutes (37°C),
consisting of a 25-minute preischemic period followed by 40
minutes of global ischemia and 30 minutes of reperfusion. The
present study conforms with the Guide for the Care and Use of
Laboratory Animals published by the U.S. National Institutes of
Health (NIH publication No. 85-23, revised 1985).
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 KH buffer as a
normal control for different experimental groups. Hearts were treated
for 11 minutes with vehicle (0.04% DMSO, n=6), after which they were
subjected to ischemia/reperfusion (I/R). The amount of DMSO
used in vehicle control experiments did not affect any
hemodynamic parameters, lactate
dehydrogenase (LDH) release, ATP content, or cell
morphology.1 12
Group 2: I/R
After equilibration, hearts (n=6) were subjected to
ischemia for 40 minutes followed by reperfusion for 30
minutes.
Group 3A: Ca2+ Preconditioning (CPC)+I/R
This group was designed to test whether CPC mimics protection by
opening of mitoKATP channel. Because
Ca2+ plays a significant role in protection of
CPC, DE may increase Ca2+ indirectly (see
Discussion) and activate PKC in addition to activation of
mitoKATP channel. Hearts (n=6) were perfused for
3 cycles of 1 minute each with Ca2+-free KH
buffer followed by 5 minutes with Ca2+-containing
KH buffer, and then the hearts were subjected to I/R as in group 2.
Group 3B: 5-HD+CPC+I/R
The rationale of this experiment is that
Ca2+-mediated activation of PKC is important for
the activation of mitoKATP channels resulting in
attenuation of cellular injury. 5-HD, a blocker of
mitoKATP channels, was used during CPC. Hearts
(n=6) were perfused similarly to group 3A, except that 5-HD (100
µmol/L)1 was infused before CPC.
Group 4: Role of MitoKATP Channels in Cardiac
Protection
A potent activator of the
mitoKATP channel, DE was used before
ischemia. DE opens the sarcolemmal KATP
channel at a higher concentration (K1/2=855
µmoL), whereas it opens the mitoKATP channel at
very low concentration (K1/2=0.4
µmoL).9 This group was designed to test the
hypothesis that an activation of mitoKATP
channels mimics the effect of CPC, which is dependent on stimulation of
PKC. 5-HD, an inhibitor of the
mitoKATP channels, was used to determine the
effect of mitoKATP channels in the prevention of
cell injury. 5-HD blocks both the mitoKATP
channel1 and the action potential
shortening.13
Group 4A: MitoKATP Channel Opener+I/R
Hearts were perfused with KH buffer containing DE for 6 minutes.
After 5 minutes, washout hearts were subjected to I/R. A dose response
of DE using 1, 20, 40, 80, and 100 µmol/L was studied (n=6
hearts for each subgroup). We found that 80 µmol/L DE had an
optimal effect on the I/R injury. In some hearts (n=6), DE was
present during the entire ischemic period.
Group 4B: Blockade of MitoKATP Channel+I/R
Hearts (n=6) were perfused similarly to those in group 4A,
except 5-HD (100 µmol/L)1 14 was administered alone
for 4 minutes and then with DE together for 6 minutes. After 5-minute
washout, hearts were subjected to I/R.
Group 5: Influence of PKC on MitoKATP Channel
To determine whether DE activates
mitoKATP channels via PKC signaling pathway, PKC
activator, PMA, and 2 PKC inhibitors, CHE
chloride and CN, were used in these studies.
Group 5A: CHE or CN+DE+I/R
Hearts (n=6) were perfused similarly to group 4A, except that
they were pretreated with CHE chloride (6.4 µmol/L) or CN
(0.05 µmol/L) for 4 minutes, followed by combined treatment with
DE for 6 minutes. After 5-minute washout, hearts were subjected to I/R.
The dose of CHE chloride or CN was chosen on the basis of previous
reports.15 16 To determine whether CHE chloride alone
attenuates or increases ischemic injury, hearts were treated
for 10 minutes with CHE chloride (6.4 µmol/L, n=6), and after
5-minute washout, hearts were subjected to I/R.
Group 5B: PMA+I/R
Hearts were perfused with KH buffer containing PMA (100
nmol/L)11 for 6 minutes. After 5-minute washout, hearts
(n=6) were subjected to I/R.
Group 5C: 5-HD+PMA+I/R
To test the hypothesis that a specific
mitoKATP channel inhibitor, 5-HD,
blocks the effect by direct activation of PKC by PMA on the
ischemic injury, hearts were first pretreated with 5-HD
(100 µmol/L) for 4 minutes and then together with PMA (100
nmol/L) for 6 minutes. After 5-minute washout, hearts were subjected to
I/R (n=6).
Group 5D: Activation of MitoKATP Channel After PKC
Blockade
The purpose of these experiments was to determine whether
activation of mitoKATP channel after PKC blockade
could protect the heart against ischemia. Hearts were perfused
similarly to group 4A, except administration of DE was continued during
the ischemic period (n=6).
Group 6: Calcium-PKC Link With MitoKATP Channel
Opening
These experiments tested the hypothesis that activation of
mitoKATP channels is facilitated by the PKC
signaling pathway. The rationale behind these experiments is that DE
may also indirectly influence the L-type Ca2+
channel and increase Ca2+ transient (see
Discussion). The participation of Ca2+ in the
signaling pathway was verified by blocking L-type
Ca2+ channels with VL.
Group 6A: VL+DE+I/R
Hearts were first pretreated with VL (2.5 µmol/L) for 4
minutes and then together with DE for 6 minutes. After 5-minute
washout, hearts were subjected to I/R (n=6).
Group 6B: VL Alone+I/R
To determine whether the concentration of VL used attenuates
ischemic injury, hearts were treated for 10 minutes with VL
(2.5 µmol/L, n=6); after 5-minute washout, hearts were subjected
to I/R. The dose of VL was chosen on the basis of a previous
report.15
Measurement of LDH
LDH, an indicator of myocardial tissue injury, was determined in
the coronary effluent by a coupled-enzyme spectrometric
technique (DU Series 500 spectrophotometer, Beckman Instruments, Inc)
using a Sigma assay kit as described.15 17 LDH was
measured at 3, 5, 10, 20, and 30 minutes of reperfusion. The
accumulated amount was obtained by integrating the area underneath the
individual time-course curve for 30-minute reperfusion.
Measurement of Tissue ATP
After termination of the experiment, hearts were cut into
transverse slices and were immediately frozen in liquid nitrogen until
freeze-drying. A slice of left ventricle was processed for morphology
and immunocytochemistry. Freeze-dried tissue (50 to 100 mg) was crushed
in a precooled glass tube, and ATP was extracted with 5 mL of cold 6%
trichloroacetic acid. The extract was analyzed for ATP by a
spectrophotometric method.15
Subcellular Localization of PKC Isoforms by
Immunocytochemistry
Subcellular localization of PKC isoforms after various
interventions was performed by immunofluorescence
staining and was compared with control hearts as previously
described.10 15 Control and experimental specimens were
harvested just before I/R. Left ventricular tissue was
blotted and embedded in OCT compound, rapidly frozen in liquid
nitrogen, and then stored at -70°C until use.15
Five-micrometer sections were fixed for 10 minutes in a
70% acetone-30% methanol mixture at –20°C, incubated in 10%
normal goat serum in PBS for 30 minutes to block nonspecific binding,
and incubated with primary antibodies (rabbit polyclonal antibodies
against PKC isoforms PKC-, PKC-ß1,
PKC-ß2, PKC-, PKC-, PKC-, PKC-, and
PKC-). Sections were examined and photographed with an Olympus BH-2
fluorescence microscope equipped with an automatic exposure control
unit. Confocal images were also obtained with a Leitz DMRBE inverted
research fluorescence microscope with a true confocal scanner
(TCS 4D, Leica, Inc). Fluorescence was excited by the 568-nm
line of krypton-argon laser, and the emission at 568 to 580 nm was
recorded. For mitochondrial imaging, additional hearts were
perfused with either 200 nmol/L TMRE alone or together with 80
µmol/L DE for 6 minutes. Specimens for microscopy were prepared in a
manner similar to immunocytochemistry. TMRE is widely used as a
structural marker for mitochondria and preferentially distributes into
negatively charged cellular organelles, such as
mitochondria.18 TMRE was excited with the 488-nm line of a
krypton-argon laser and recorded at >530-nm with an FITC
filter.5 19
Morphological Examination
Heart tissue taken from the left ventricular free
wall at the end of experimental protocols was cut into 1.0-mm pieces
that were fixed with 2.5% buffered glutaraldehyde. A
semiquantitative estimate of cell damage was carried out on
1-µm-thick sections. Approximately 500 cells were analyzed in
each heart by researchers without knowledge of the treatment, and 1 of
3 degrees of cell damage was assigned to each cell. Cell morphology was
assessed according to the classification as previously
described.15 17
Statistical Analysis
All values are expressed as mean±SEM. Group comparisons were
done by 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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Ischemic Control Hearts
A significant decrease in functional recovery was observed in ischemic control hearts. At the end of reperfusion, LVEDP significantly increased, whereas left ventricular developed pressure (LVDP) and coronary flow (CF) were decreased (Table 1
). LDH leakage was significantly
increased on reperfusion (Figure 1), and
ATP levels were reduced to 5.6±0.7 µmol/g dry weight as
compared with the normal control value of 21.5±0.8 µmol/g dry
weight (Figure 2). The pathological
changes were severe, and extensive necrosis was observed (Table 2).
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Role of CPC, MitoKATP Channel, and PKC in Cardiac
Protection
Ca2+-Preconditioned Hearts
A slight increase in LVDP and LVEDP was observed after 3 cycles of
brief Ca2+ depletion and repletion. After I/R, a
significant functional recovery was observed in these hearts (Table 1
).
LDH release was significantly decreased, and ATP contents
were markedly preserved both at the end of ischemia and at
postischemic reperfusion (Figures 1, 2A, and 2B). The cellular structure was extremely well preserved in
hearts after CPC (Figure 3C). A total of
56.2±4.3% cells were normal; 13.8±2.3% and 30.0±3.4% were mildly
and severely damaged, respectively; and the degree of cell damage was
much less than that in the ischemic control hearts (Table 2).
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To clarify the role of mitoKATP channel in CPC, a
specific blocker, 5-HD, was infused before CPC pretreatment. 5-HD
abolished the protective effects of CPC (Table 1; Figures 1, 2A, 2B, and 3D).
MitoKATP Channel and Ischemic Changes
During and after DE treatment, the CF was significantly increased.
A significant recovery of LVDP, HR, and CF was observed in DE-treated
hearts as compared with ischemic control hearts (Table 1,
Figure 4). LVEDP was
significantly decreased in the treated hearts, and recovery of LVDP was
amazing (Figure 4). This level of protection is similar to that
described in a recently published study on effects of DE on
ischemia.1 A significant reduction in LDH release
and a dramatic preservation of tissue ATP content both at the end of
ischemia and at reperfusion were observed in contrast to
ischemic control hearts (Figures 1, 2A, and 2B). No significant differences were observed in ATP contents
and LDH release in the DE-pretreated hearts with or without washout
(Figure 5). The cell structure was also
well preserved in DE-pretreated hearts (Table 2, Figure 3E). The blockade of mitoKATP channel with
5-HD completely abolished the recovery of LVDP (Figure 4) and CF
(Table 1).
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PKC-Mediated Signaling Pathways in Cardiac Protection
PKC activation with PMA was itself protective. A significant
functional recovery was observed after I/R (Table 1).
LDH release was significantly decreased and ATP contents were
markedly preserved as compared with ischemic control hearts
(Figures 1, 2A, and 2B). The cell structure was
also well preserved in PMA-pretreated hearts compared with
ischemic control hearts (Table 2). Morphological changes
were similar to those of DE-pretreated hearts (data not shown).
To determine whether stimulation of
mitoKATP channel is mediated via PKC signaling
pathway, hearts were pretreated with CHE or CN before DE treatment. In
these hearts, the contractile function was lost, and CF, LDH release,
tissue ATP levels, and morphological damage were similar to those of
the I/R group (Tables 1 and 2; Figures 1, 2A, 2B, and 3). CHE chloride alone did not have
any effect on the cardiac function (Table 1).
To further clarify the role of PKC in the regulation of
mitoKATP channel, 5-HD was infused before the
treatment with PMA. 5-HD abolished the protective effects induced by
PKC activation (Table 1; Figures 1, 2A, and 2B). There were no significant differences in various
parameters of cell injury between control ischemic
hearts and 5-HD–pretreated hearts after PMA administration (Table 2).
Because DE can indirectly increase cAMP,20 which leads to
elevated [Ca2+]i
transient in myocytes,21 22 we sought to determine whether
a transient increase in
[Ca2+]i by DE treatment
activates PKC and its translocation to mitochondria. The effect
of DE disappeared after pretreatment with VL, and functional recovery
was significantly depressed (Table 1). LDH release was
significantly increased (16.7±1.5 U/g), and ATP content was also
reduced to the level of I/R control hearts (Figures 1, 2A, and 2B). VL alone did not provide any cardiac
protection (Tables 1 and 2; Figures 1, 2A, and 2B). The activation of mitoKATP
channel with DE during blockade of PKC in the ischemic period
did not reduce cell injury.
Immunohistochemical Distribution of PKC Isoforms After Various
Interventions
Representative results from the
immunohistochemical study are shown in Figure 6. In the DE-pretreated hearts,
PKC- was distinctly localized in the cardiac cell sarcolemma, and
PKC- was positively distributed in the mitochondria, which were
confirmed with use of the membrane potential indicator TMRE.
TMRE-treated tissue was also stained with PKC- antibody, which
positively stained mitochondria, intercalated discs, and nuclei.
PKC- was translocated to the intercalated disc in 90% of cells of
DE-pretreated hearts. PKC-ß1 staining was
observed in the nuclear region. PKC-, -, -, and
-ß1 showed diffuse and nonspecific staining in
hearts pretreated with 5-HD, CHE, or VL, similar to results shown in
Figure 6B. PKC-ß2, -, -,
and - staining was less intense and weaker than by PKC-, -,
- and -ß1 isoforms after DE treatment.
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Discussion |
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The major findings of this study are the following: (1) the mitoKATP channel appears to be an end effector of preconditioning against ischemia, and (2) the stimulation of the mitoKATP channel is dependent on PKC activation and translocation to mitochondria. In 1992, Gross et al23 showed that nicorandil, a KATP channel opener, was very effective against infarct size in dogs. Recent studies24 by the same group also showed cardioprotection in dogs treated with bimakalim, a KATP channel opener, which had minimal effect on action potential duration. Such dissociation with action potential shortening has also been shown in several other studies.24 25 These experimental results suggest that sarcolemmal KATP channels may not be the only effector of cardioprotection.
Cardiac myocytes possess KATP channels located in the inner mitochondrial membrane, which also respond to the openers of the sarcolemmal channels.7 26 Activation of any potassium-selective ion channels in the inner mitochondrial membrane would tend to dissipate the membrane potential established by the proton pump.8 DE has been reported to specifically activate mitoKATP channel with little effect on cardiac action potential duration.8 9 Thus, activation of the mitoKATP channel by DE may be necessary for higher ATP production to support increased workload in the heart. Mitochondria are known to play at least 2 roles essential for cell survival, ATP synthesis and maintenance of Ca2+ homeostasis. High-energy production is essential to support the action of ATP-dependent ion pumps, such as Na+/K+ and Ca2+ pumps27 to maintain membrane intactness and calcium ion gradients across various cell membranes, including the sarcolemma, sarcoplasmic reticulum, and mitochondria.28 It is likely that the enhancement of ATP production in the preconditioned heart maintains the Ca2+ homeostasis. We have shown that the effect of DE disappears after inhibition of the L-type Ca2+ channel and PKC, which suggests that DE is a trigger for the activation of both the mitoKATP channel and PKC.
Potential Role of Ca2+ in Modulating
MitoKATP Channels
One of the main theories of the lethal cellular damage induced by
I/R involves massive Ca2+ influx during
reperfusion.28 However, recently it has been reported that
brief ischemia evokes a transient elevation of
[Ca2+]i.29 30
Smith et al31 have demonstrated that
[Ca2+]i was
increased 2- to 4-fold in preconditioning ischemia in isolated
rat heart. We have previously demonstrated that the transient increase
in [Ca2+]i is a trigger
for the induction of CPC, which was strongly protective against
ischemia and Ca2+
paradox.10 11 15 A Ca2+ transient
also exists in the mitochondrial matrix.32 33 34 When rat
myocytes were stimulated with a beta-adrenergic substance and paced at
physiological frequency (4 Hz),
Ca2+ concentration in the mitochondria exceeded
that of cytosolic content, reaching a value of 0.6
µmol/L.35 This Ca2+ increase
stimulates the activity of 3 enzymes (ie, pyruvate dehydrogenase,
isocitrate dehydrogenase, and -ketoglutarate dehydrogenase) and
increases the rate of ATP synthesis.36 37 38 Thus,
[Ca2+]i increase could
act as a second messenger within mitochondria.39 In
addition, [Ca2+]i
transient increase also activates
Ca2+–independent PKC isoforms via phospholipase
C pathway40 and causes their translocation to
mitochondria.
The role of [Ca2+]i in mediating protection against lethal ischemia via PKC signaling pathway has been well established by our previous studies15 and by others.41 42 The mechanism by which DE may increase [Ca2+]i is not determined in this study. However, we have shown that by blocking the L-type Ca2+ channel, the effect of DE dissipates, which suggests the participation of Ca2+ in the signaling pathway leading to protection. In addition, DE is also a known inhibitor of phosphodiesterase,20 43 which degrades cAMP.21 Therefore, an increase in cAMP will influence L-type Ca2+ channel via protein kinases leading to an elevated Ca2+ transient in myocytes.22 This is in agreement with our study44 and work by Asimakis et al45 that isoproterenol elicits preconditioning effect against ischemia. Taken together, this study has shown that blockade of Ca2+ transient by VL in the DE-pretreated hearts prevented cardiac protection, which suggests that activation of the mitoKATP channel by DE may also involve Ca2+. Current studies in progress on isolated myocytes show a significant [Ca2+]i increase on introduction of DE as measured by the use of Fluo-3 (M.A., Y.W., unpublished observation, 1999).
Potential Role of PKC in Modulating MitoKATP
Channel
The role of PKC in preconditioning has been well established by
several investigations.41 46 The present study clearly
demonstrates that [Ca2+]i
plays an important role in activating various second messenger pathways
including PKC. The blockade of
[Ca2+]i transient by VL
inhibited PKC activation and its effect on
mitoKATP channels, which suggests the regulation
of the latter channels by PKC. Similarly, DeWeille et al47
also demonstrated that stimulation of PKC by the phorbol ester phorbol
myristate acetate activated the
KATP channel.
[Ca2+]i can also
stimulate Ca2+-independent PKC isoforms via
activation of phospholipase C, which forms inositol triphosphate and
diacylglycerol (DAG). The latter 2 products stimulate
Ca2+-independent isoforms of PKC.40
The role of DAG in the activation of PKC was further supported by our
previous study,12 in which hearts were treated with
1-stearoyl-2-arachidonyl-glycerol, a precursor of DAG, causing
significant protection. The protective effect, as expected, was
abolished with CHE. Similarly, Van Winkle et al48 were
able to block the protection in hearts pretreated with PMA after
administration of 5-HD. It was suggested by their study that protection
occurred with subsequent opening of KATP channels
after activation of PKC. Thus, our study strengthens the idea that
activation of mitoKATP channel after PKC
inhibition is ineffective in reducing ischemic injury.
This study also strongly points out that PKC translocation to mitochondria was essential for the activation of mitoKATP channels. PKC inhibition with CHE and CN totally blocked protection by DE, which suggests a crucial role of PKC in the regulation of mitoKATP channels. The importance of PKC is further emphasized by the findings of Sato et al49 that PKC primes the mitoKATP channel to open earlier and more intensely during prolonged ischemia. On the other hand, Miura et al50 reported that PKC inhibition by CN was not effective in the reduction of infarction in rabbit hearts treated with DE. It is likely that these differences could arise as a result of different animal species and experimental protocols. However, PKC activation by adenosine A1 receptor leads to opening of mitoKATP channels, the effect of which can be blocked by PKC inhibition.50 This and other studies support the fact that various preconditioning stimuli induce myocardial protection against ischemia through mechanisms that appear to be mediated by PKC.
It is well established that DE selectively opens
mitoKATP channels.1 5 The data in
this study point out that DE stimulates both PKC and
mitoKATP channels, thus conferring a greater
protection as suggested by increased ATP preservation and improved
cardiac function. Similar improvement in cardiac function has been
recently reported by Garlid et al.1 Activation of
mitoKATP channel retards calcium uptake and/or
preserves ATP synthetic capability as reviewed by Buja.51
DE-pretreated hearts retained more ATP during ischemia, thus
improving postischemic cardiac function and maintaining
Ca2+ homeostasis as evidenced by less contraction
band formation and Ca2+ accumulation by
mitochondria. The loss of mitoKATP
channel–mediated effects by PKC inhibition further reinforces the
notion that the signal transduction cascades of a large number of
receptors are coupled to PKC activation,42 46 which may
phosphorylate different channels leading to
preconditioning.41 The phosphorylating sites within the
cell need further molecular characterization. Endogenous
PKC exists in various isoforms with specific tissue distribution and
sensitivity. Therefore, it is possible that different isoforms interact
with mitoKATP channel after a specific response
to PKC activation. The simultaneous visualization of
mitochondria and staining with PKC- antibodies expand our existing
knowledge of PKC isoform distribution in cardiac tissue and their
significance in various signaling pathways. We used
immunohistochemistry to determine subcellular localization of PKC
isoforms. Pretreatment with DE clearly caused the translocation of the
PKC- isoform to the mitochondrial sites, intercalated disc, and
nucleus; PKC- into the sarcolemma; PKC- into the intercalated
disc; and PKC-ß1 into the nucleus.
Translocation of PKC- to the sarcolemma was consistent with
our previous study on CPC,52 which suggests that
activation of PKC- isoform by DE is Ca2+
dependent. Translocation of PKC- to the mitochondrial sites,
intercalated disc, and nucleus may regulate intracellular processes not
only by direct activation of mitoKATP channels
but also by phosphorylation of numerous rate-limiting
enzymes or gene transcription.40 41 Translocation of
PKC- to the intercalated disc in DE-treated hearts may facilitate
intercellular communication through cell junctions located in the
intercalated disc of myocytes.
Potential Importance of Our Findings
The present study indicates the potential of
[Ca2+]i and PKC in the
regulation of the mitoKATP channel, which is
important for endogenous cardioprotection against
ischemia. Therefore, the association of PKC with
mitoKATP channel may be an important component of
preconditioning. The pharmacological manipulation of PKC and the
openers of mitoKATP channels could be exploited
in the future for therapeutic purposes.
|
Acknowledgments |
|---|
This work was supported in part by NIH Grants HL 23597 and HL 55678.
Received February 16, 1999; accepted July 26, 1999.
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References |
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K. Mubagwa and W. Flameng Adenosine, adenosine receptors and myocardial protection: An updated overview Cardiovasc Res, October 1, 2001; 52(1): 25 - 39. [Abstract] [Full Text] [PDF]
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M. Xu, Y. Wang, A. Ayub, and M. Ashraf Mitochondrial KATP channel activation reduces anoxic injury by restoring mitochondrial membrane potential Am J Physiol Heart Circ Physiol, September 1, 2001; 281(3): H1295 - H1303. [Abstract] [Full Text] [PDF]
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Y. Wang, E. Takashi, M. Xu, A. Ayub, and M. Ashraf Downregulation of Protein Kinase C Inhibits Activation of Mitochondrial KATP Channels by Diazoxide Circulation, July 3, 2001; 104(1): 85 - 90. [Abstract] [Full Text] [PDF]
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R. A. Ockaili, P. Bhargava, and R. C. Kukreja Chemical preconditioning with 3-nitropropionic acid in hearts: role of mitochondrial KATP channel Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H2406 - H2411. [Abstract] [Full Text] [PDF]
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S. Ghosh, N. B. Standen, and M. Galinanes Failure to precondition pathological human myocardium J. Am. Coll. Cardiol., March 1, 2001; 37(3): 711 - 718. [Abstract] [Full Text] [PDF]
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R. M. Fryer, Y. Wang, A. K. Hsu, and G. J. Gross Essential activation of PKC-{delta} in opioid-initiated cardioprotection Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1346 - H1353. [Abstract] [Full Text] [PDF]
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 C. Ozcan, E. L. Holmuhamedov, A. Jahangir, and A. Terzic Diazoxide protects mitochondria from anoxic injury: Implications for myopreservation J. Thorac. Cardiovasc. Surg., February 1, 2001; 121(2): 0298 - 306. [Abstract] [Full Text] [PDF]
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M. Xu, Y. Wang, K. Hirai, A. Ayub, and M. Ashraf Calcium preconditioning inhibits mitochondrial permeability transition and apoptosis Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H899 - H908. [Abstract] [Full Text] [PDF]
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S. Wang, J. Cone, and Y. Liu Dual roles of mitochondrial KATP channels in diazoxide-mediated protection in isolated rabbit hearts Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H246 - H255. [Abstract] [Full Text] [PDF]
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J. Huh, G. J. Gross, H. Nagase, and B. T. Liang Protection of cardiac myocytes via {delta}1-opioid receptors, protein kinase C, and mitochondrial KATP channels Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H377 - H383. [Abstract] [Full Text] [PDF]
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B. O'Rourke Myocardial KATP Channels in Preconditioning Circ. Res., November 10, 2000; 87(10): 845 - 855. [Abstract] [Full Text] [PDF]
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T. Pain, X.-M. Yang, S. D. Critz, Y. Yue, A. Nakano, G. S. Liu, G. Heusch, M. V. Cohen, and J. M. Downey Opening of Mitochondrial KATP Channels Triggers the Preconditioned State by Generating Free Radicals Circ. Res., September 15, 2000; 87(6): 460 - 466. [Abstract] [Full Text] [PDF]
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E. Takashi, Y. Wang, and M. Ashraf Activation of Mitochondrial KATP Channel Elicits Late Preconditioning Against Myocardial Infarction via Protein Kinase C Signaling Pathway Circ. Res., December 3, 1999; 85(12): 1146 - 1153. [Abstract] [Full Text] [PDF]
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S. Ovide-Bordeaux, R. Ventura-Clapier, and V. Veksler Do Modulators of the Mitochondrial KATP Channel Change the Function of Mitochondria in Situ? J. Biol. Chem., November 17, 2000; 275(47): 37291 - 37295. [Abstract] [Full Text] [PDF]
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 P. E. Light, C. Bladen, R. J. Winkfein, M. P. Walsh, and R. J. French Molecular basis of protein kinase C-induced activation of ATP-sensitive potassium channels PNAS, August 1, 2000; 97(16): 9058 - 9063. [Abstract] [Full Text] [PDF]
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