(Circulation. 1997;96:1257-1265.)
© 1997 American Heart Association, Inc.
Articles |
Role of Intracellular Ca2+ in Activation of Protein Kinase C During Ischemic Preconditioning
From the First Department of Medicine (K.N., M.K., H.S., T.M., K.K., M.H.), Osaka University School of Medicine, Osaka, and the Department of Physiology (Y.S., H.M.), Tokai University, Isehara, Japan.
Correspondence to Masafumi Kitakaze, MD, PhD, First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565, Japan. E-mail Kitakaze{at}medone.med.osaka-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background Activation of protein kinase C plays an important role in ischemic preconditioning. Given that protein kinase C is activated by an increase in the intracellular Ca2+ concentration ([Ca2+]i) and that myocardial ischemia and reperfusion increase [Ca2+]i, the effect of transient exposures to Ca2+ on infarct size and the effect of administration of EGTA during ischemic and
1-adrenoceptor–mediated preconditioning on the
limitation of infarct size were investigated in the canine heart.
Methods and Results In open-chest dogs, 5 minutes after the
completion of either three 5-minute infusions of CaCl2 or
four 5-minute infusions of the 1-adrenoceptor agonist
methoxamine into the coronary artery, the
coronary arteries were occluded for 90 minutes; this occlusion
was followed by a 6-hour reperfusion in both the Ca2+
preconditioning and methoxamine groups. Infarct sizes in the
Ca2+ preconditioning (15.8±2.3%) and methoxamine
(10.1±2.2%) groups were significantly (P<.01) smaller
than in the control group (42.5±2.9%), and administration of either
an inhibitor of protein kinase C (GF109203X) or an
inhibitor of ecto-5'-nucleotidase
(,ß-methyleneadenosine 5'-diphosphate) reduced the infarct
size–limiting effect of Ca2+ preconditioning.
Administration of EGTA during ischemic or
1-adrenoceptor–mediated preconditioning inhibited both
the infarct size–limiting effect and the activation of protein kinase
C and ecto-5'-nucleotidase induced by these procedures.
Conclusions [Ca2+]i during
ischemic and 1-adrenoceptor–mediated
preconditioning plays an important role in the infarct
size–limiting effect of these procedures by activating protein
kinase C and ecto-5'-nucleotidase in the canine heart.
Key Words: adenosine • calcium • myocardial infarction • pathology • receptors, adrenergic, alpha
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Ischemic preconditioning, achieved by exposure of the heart to brief periods of ischemia, results in marked tolerance of the myocardium to normally lethal ischemic insults.1 The IS-limiting effect of IP is reduced by exposure to 8-SPT, an antagonist of adenosine receptors.2 Furthermore, we have shown3 that activation of ecto-5'-N, which catalyzes 5'-AMP and the synthesis of adenosine, contributes to IP-induced cardioprotection and that activation of PKC is responsible for the activation of ecto-5'-N. Because myocardial ischemia and reperfusion increases [Ca2+]i in the myocardium,4 5 it is likely that the brief periods of ischemia during IP also increase [Ca2+]i. Furthermore, we have observed3 that activation of PKC in response to
1-adrenoceptor stimulation during IP requires
intracellular Ca2+ and limits IS through activation of
ecto-5'-N, suggesting that an increase in
[Ca2+]i during IP contributes to the
activation of PKC and subsequently, ecto-5'-N, and mediates the
IS-limiting effect of this procedure.
To clarify the role of [Ca2+]i in the IS
limitation, we examined whether transient exposures to
CaCl2 limit IS through activation of PKC, and subsequently,
ecto-5'-N. We also investigated whether administration of the
Ca2+ chelator EGTA during IP or
1-adrenoceptor–mediated preconditioning inhibits the
IS-limiting effect of these procedures.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Instrumentation
Mongrel dogs (body weight, 16 to 24 kg) were anesthetized with sodium pentobarbital (30 mg/kg body wt IV), intubated, and ventilated with room air mixed with oxygen (100% O2 at a flow rate of 1.0 L/min). The chest was opened through the left fifth intercostal space, and the heart was suspended in a pericardial cradle. After administration of heparin (500 U/kg IV), we cannulated the LAD and perfused it with blood from the left carotid artery through an extracorporeal bypass tube. CBF in the perfused area was measured with an electromagnetic flow probe attached to the bypass tube, and CPP was monitored at the tip of the coronary artery cannula. A pair of ultrasonic crystals (5 MHz, 2 mm in diameter; Schuessler) was implanted in the left ventricular anterior wall in the endomyocardial segment in the center of the perfused area to measure segment length. We calculated FS from the equation FS=[(EDL-ESL)/EDL]x100%, where EDL and ESL are end-diastolic and end-systolic segment lengths, respectively.
A small-caliber (1 mm), short (70 mm) collecting tube was introduced into a small coronary vein near the center of the perfused area to sample coronary venous blood. Drained venous blood was collected in a reservoir placed at the level of the left atrium and was returned to the jugular vein. The left atrium was catheterized for microsphere injection. The femoral artery was also cannulated for the sampling of reference (control) blood. Hydration was maintained by a slow infusion of saline. Aortic blood pressure was monitored at the tip of the cannula to the femoral artery.
Heart rate averaged 139±2 bpm during control conditions and did not change during the study. The pH and partial pressures of O2 and CO2 in the systemic arterial blood before performing the experimental protocols were 7.42±0.02, 104±4 mm Hg, and 37.5±1.9 mm Hg, respectively.
Experimental Protocols
Protocol 1: Effects of IP and
1-Adrenoceptor–Mediated Preconditioning
After the dogs became hemodynamically stable,
four cycles of coronary occlusion for 5 minutes followed by 5
minutes of reperfusion were performed to precondition the
myocardium before the onset of 90 minutes of
ischemia and a subsequent 6 hours of reperfusion (IP group,
n=8). In a second group of dogs (MTX group, n=6), we administered the
1-adrenoceptor agonist MTX (Sigma Chemical Co) dissolved
in saline (40
µg·kg-1·min-1;
infusion rate, 16.7
µL·kg-1·min-1
IC) for four 5-minute periods separated by 5-minute intervals before
the onset of 90 minutes of coronary occlusion and 6 hours of
reperfusion. In the control group (n=8), 45 minutes after the dogs were
hemodynamically stabilized, they were subjected to 90
minutes of coronary artery occlusion and 6 hours of
reperfusion. Coronary arterial and venous blood
were sampled at 20-minute intervals during 45 minutes of
hemodynamic stabilization for the measurement of plasma
adenosine, lactate, and norepinephrine
concentrations. In addition to the dogs used for the measurement of IS,
other animals were used to measure PKC activity in cytosolic and
membrane fractions of the endocardium as well as cytosolic and
ecto-5'-N activity in the endocardium and epicardium of the LAD area.
Animals were killed immediately after the IP (n=5) or
1-adrenoceptor–stimulated preconditioning (n=5)
procedures; control dogs (n=5) were killed 40 minutes after they
reached hemodynamic stabilization.
Protocol 2: Effects of EGTA During IP and
1-Adrenoceptor–Mediated Preconditioning
Because activation of PKC requires Ca2+,
[Ca2+]i in IP and
1-adrenoceptor–mediated preconditioning may contribute
to the activation of PKC and ecto-5'-N, and subsequently, to the
IS-limiting effect. To test this hypothesis, we examined whether
administration of EGTA (1.7
µmol·kg-1·min-1
IC) beginning 10 minutes before and continuing throughout IP
(IP+EGTA group, n=7) and MTX preconditioning (MTX+EGTA group, n=7)
reduced the IS-limiting effect. The LAD was then occluded for 90
minutes, after which reperfusion was performed for 6 hours. Animals in
the EGTA group (n=7) received EGTA for 45 minutes before
coronary occlusion and reperfusion. In another three groups
(n=5 for each group), corresponding to the IP+EGTA, MTX+EGTA, and EGTA
groups in protocol 1, we measured PKC and ecto-5'-N activities as
described for the earlier protocol.
Protocol 3: Effects of Transient Exposures to CaCl2 on
IS and Activities of PKC and Ecto-5'-N
To test whether transient exposures to CaCl2
(Ca2+ preconditioning) mimic the IS-limiting effect of IP,
we administered CaCl2 into the LAD (8
µmol·min-1·mL-1
of CBF; infusion rate, 16.7
µL·kg-1·min-1)
for three periods of 5 minutes each with 5-minute intervals
(Ca2+ group, n=8). The dogs were then subjected to 90
minutes of coronary occlusion followed by 6 hours of
reperfusion. In five other dogs, we measured PKC and ecto-5'-N
activities after the Ca2+ preconditioning procedure as
described for protocol 1. We also examined the effects on IS and the
activities of PKC and ecto-5'-N of three additional doses of
CaCl2 (4, 6, and 10
µmol·min-1·mL-1
of CBF; infusion rate, 16.7
µL·kg-1·min-1)
administered into the LAD for three periods of 5 minutes each separated
by 5-minute intervals (n=5 for each group). Finally, we also
administered CaCl2 into the LAD (8
µmol·min-1·mL-1
of CBF) for one cycle of 5 minutes with a 5-minute interval (n=5). CBF
is expressed in milliliters per minute.
Protocol 4: Effects of GF109203X Administration During
Ca2+ Preconditioning on IS-Limiting Effect and
Ecto-5'-N Activities
To examine the role of PKC in Ca2+ preconditioning,
we concomitantly infused GF109203X, a selective inhibitor
of PKC. Infusion of GF109203X (40
ng·kg-1·min-1;
infusion rate, 16.7
µL·kg-1·min-1;
Calbiochem) into the LAD was started 5 minutes before and continued
during Ca2+ preconditioning (Ca2++GF109203X
group, n=8). We also determined the effect of GF109203X alone on IS
(GF109203X group, n=7); GF109203X was administered for 45 minutes
before coronary occlusion. We measured ecto-5'-N activity in
the endocardium and epicardium of the LAD area in five animals each
corresponding to the Ca2++GF109203X and GF109203X
groups.
Protocol 5: Effects of AMP-CP and 8-SPT on the IS-Limiting Effect
of Ca2+ Preconditioning
To examine the roles of the activation of ecto-5'-N and
endogenous adenosine in Ca2+
preconditioning, we infused AMP-CP (8
µg·kg-1·min-1;
infusion rate, 16.7 µL · kg-1 ·
min-1 IC; Sigma), a specific and competitive
inhibitor of ecto-5'-N, or 8-SPT (25
µg·kg-1·min-1;
infusion rate, 16.7
µL·kg-1 · min-1
IC; Research Biochemicals) beginning 5 minutes before and continuing
throughout the Ca2+ preconditioning procedure and resuming
for the first 60 minutes of reperfusion (Ca2++AMP-CP group
[n=8] and Ca2++8-SPT group [n=7], respectively). We
also determined the effects of AMP-CP and 8-SPT alone on IS (AMP-CP
group [n=6] and 8-SPT group [n=8]); AMP-CP or 8-SPT was
administered for 45 minutes before coronary occlusion and for
the first 60 minutes of reperfusion. In other dogs, we measured the
ecto-5'-N activity of the endocardium and epicardium in the LAD area
(Ca2++8-SPT group [n=5]; 8-SPT group [n=5]).
Protocol 6: Effects of 8-SPT Only During Ca2+ Exposure
on IS Limitation by Ca2+ Preconditioning
To examine whether increased adenosine
production in response to Ca2+ exposure triggers
the IS-limiting effect of Ca2+ preconditioning, we began
8-SPT infusion 5 minutes before the administration of Ca2+
and continued it until the onset of coronary occlusion
(Ca2++8-SPT [pretreatment] group, n=6). In five other
dogs, we measured the activity of PKC in cytosolic and membrane
fractions of the endocardium as well as the ecto-5'-N activity of the
endocardium and epicardium in the LAD area.
Protocol 7: Effects of Transient Exposures to CaCl2 on
IS and PKC and Ecto-5'-N Activities in Chemically Denervated
Hearts
To clarify whether norepinephrine release
contributes to the IS-limiting effect of Ca2+
preconditioning, we subjected dogs that had undergone chemical
denervation of the heart to the control (protocol 1) and
Ca2+-preconditioning (protocol 3) procedures (denervation
group [n=7] and Ca2++denervation group [n=7],
respectively). Systemic chemical sympathectomy was
performed by an intravenous injection of
6-hydroxydopamine (50 mg/kg) 5 days before the
experiment. Deleterious side effects of
6-hydroxydopamine were prevented by previous injections
of propranolol and phentolamine (1 mg/kg each);
three fractional doses of 6-hydroxydopamine (10, 20,
and 20 mg/kg) were administered over a 24-hour period. Myocardial
tissue from the perfused area of dogs killed immediately after the
experiment was sampled for the measurement of
norepinephrine. Norepinephrine contents of the
myocardium of systemically denervated and
innervated dogs were 11±3 and 366±28 pg/mg tissue
(mean±SEM, n=5, P<.01), respectively. We measured PKC and
ecto-5'-N activities as described for protocol 1 in five animals each,
corresponding to the denervation and Ca2++denervation
groups.
Protocol 8: Effects of IP and Transient Exposures to
CaCl2 on the Microtubular Structure
Although we needed to confirm that infusion of CaCl2
would increase [Ca2+]i or that infusion of
EGTA would attenuate the increase of [Ca2+]i
by IP, we could not measure [Ca2+]i of canine
in vivo heart directly. We have reported6 that the
microtubular structure is sensitive to an increase in
[Ca2+]i. Because intracoronary
administration of CaCl2 disrupts microtubules and EDTA
prevents the disruption of microtubular structures, we evaluated the
change in microtubular structure as a semiquantitative index of
[Ca2+]i. In addition to the dogs used for the
measurement of IS or the activity of PKC and ecto-5'-N, other
animals were used to evaluate the microtubular structure in the
endocardium of the LAD area. Animals were killed immediately after IP
(n=4), IP+EGTA (1.7
µmol·kg-1·min-1
IC, n=3), or CaCl2 exposure
(8µmol · min-1·mL-1
of CBF; infusion rate, 16.7 µL
kg-1·min-1,
n=4) or, for the control group (n=4), 40 minutes after
hemodynamic stabilization was achieved. Samples were processed, and
indirect immunofluorescent staining of microtubules was
observed.6 7 CBF is expressed in milliliters per minute.
Criteria for Exclusion
To ensure that all the animals included for the analysis
of IS data were healthy and exposed to a similar extent of
ischemia, we excluded dogs that fulfilled any of the following
three criteria: subendocardial collateral flow >15 mL·100
g-1·min-1, heart
rate >170 bpm, or more than two consecutive attempts required to
correct ventricular fibrillation with low-energy DC pulses
applied directly to the heart.
Assessment of IS
After 6 hours of reperfusion, the LAD was reoccluded and
perfused with autologous blood, and Evans blue dye was injected into a
systemic vein to determine the anatomic risk area and the
nonischemic area in the heart. The heart was then removed
immediately and sliced into serial transverse sections. The
nonischemic area was identified by blue stain, and the
ischemic region was incubated at 37°C for 20 to 30 minutes in
sodium phosphate buffer (pH 7.4) containing 1% tetraphenyl tetrazolium
chloride (Sigma). IS was expressed as a percentage of the area at
risk.
Measurement of regional myocardial blood flow was determined by using the microsphere technique.3 8
Chemical Analysis
M
O2 was calculated by multiplying
CBF by the coronary arteriovenous blood oxygen difference.
Lactate was measured by using an enzymatic assay, and the LER was
obtained by dividing the coronary arteriovenous difference in
lactate concentration by arterial lactate concentration and
multiplying by 100%. The calcium concentration in coronary
arterial and venous blood was measured by using automated
spectrophotometry (Automatic Analyzer 705, Hitachi) with
o-cresolphthalein complexone in the presence of
8-hydroxyquinoline.9
The methods used for measuring adenosine and norepinephrine concentrations have been described.3 10
Measurements of PKC and Ecto-5'-N Activities
A biopsy specimen (1 to 2 g) of the LAD-perfused
myocardium was obtained before sustained coronary
occlusion in the various treatment groups. Tissue samples were
processed, and the activity of ecto-5'-N was measured by using an
enzymatic assay.3 The activity of PKC was measured by
using an enzyme assay kit (Amersham) that provides a simple and
reliable method of estimating PKC activity without extensive
purification of the enzyme.11 Ecto-5'-N and PKC activities
are expressed as nanomoles per milligram of protein per minute. The
dependence of PKC activity on Ca2+ and phospholipids was
examined by adding 0.5 mmol/L excess EGTA or eliminating
phosphatidylserine from the assay
system.11
Statistical Analysis
Data are expressed as mean±SEM. Statistical significance
was assessed by using an ANOVA and Bonferroni's test. The effect of
endomyocardial collateral blood flow on IS was
analyzed by using an ANCOVA, with regional collateral flow in
the inner half of the left ventricle wall as the covariant.
A probability value of <.05 was considered statistically significant.
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Results |
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Mortality and Exclusions
We excluded eight dogs from data analysis because the subendocardial collateral flow was >15 mL·100 g-1·min-1. No dogs were excluded because of heart rate >170 bpm. At least one episode of ventricular fibrillation occurred in 36 dogs: ventricular fibrillation that matched the exclusion criterion occurred in 12 of these animals during the 90 minutes of ischemia and in 17 during reperfusion (Table 1
).
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Hemodynamic and Metabolic Parameters
No significant differences in systolic and
diastolic blood pressures or heart rate were detected
immediately before, during, or after 90 minutes of myocardial
ischemia among the various groups of innervated
dogs. Heart rate in the denervated dogs was lower than that in the
innervated dogs. Although EGTA alone had no effect on
baseline systemic and coronary hemodynamic and
metabolic parameters except for FS (from
24.3±0.7% to 16.6±0.5%, P<.05 versus the control
group), an intracoronary administration of EGTA during IP
reduced the coronary arterial and venous difference
in adenosine concentration and the extent of coronary
hyperemic flow measured after the fourth 5-minute exposure to
myocardial ischemia (Table 2). CBF decreased
immediately after administration of the fourth dose of MTX (Table 2)
but returned to the control level after a further 5 minutes (before
ischemia); systolic and diastolic blood
pressures and CPP increased and CBF decreased during MTX administration
due to 1-adrenoceptor–mediated vasoconstriction, which
did not cause myocardial ischemia as revealed by LER (Table 2).
Adenosine concentration in the coronary venous blood
increased during an intracoronary MTX infusion; this increase
was reduced by EGTA, but the adenosine concentration returned
to the control level 10 minutes after the fourth exposure to MTX (Table 2). CBF, FS, MO2, and the
coronary arterial and venous difference in
adenosine concentration increased during administration of
CaCl2 but returned to control values 10 minutes after the
third exposure to Ca2+ (Table 3). CPP, CBF,
MO2, pH in coronary
arterial and venous blood, adenosine and
norepinephrine concentrations in coronary
arterial and venous blood, and LER did not differ
significantly among the various groups of innervated dogs
immediately before the onset of 90 minutes of ischemia.
MO2 was lower and
norepinephrine concentrations in coronary
arterial and venous blood were higher in the denervated
dogs than in the innervated dogs. Intracoronary
infusions of EGTA reduced the Ca2+ concentration in the
perfused blood from 14.8±1.2 to 2.5±0.8 mg/dL; intracoronary
infusion of CaCl2 increased the Ca2+
concentration in the perfused blood from 8.1±0.2 to 19.2±1.2 mg/dL
(P<.01).
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Changes in Microtubular Structures
Microtubular structures in intact nonischemic hearts
appeared throughout the cytoplasm as tortuous, loosely organized
filaments composed mainly of longitudinal and transverse filaments.
Microtubules encircling the nuclei showed dense staining (Fig 1A). In the IP (Fig 1B) and Ca2+ groups (Fig 1C), the microtubular structure was disrupted and observed as dotted
spots; staining around the nuclei had disappeared.
Intracoronary administration of EGTA during the IP procedure
prevented the disruption of microtubules (Fig 1D).
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PKC and Ecto-5'-N Activities
PKC activity in the membrane fraction of the
myocardium was increased in the IP and Ca2+
groups relative to the control group (Fig 2). In
contrast, PKC activity in the cytosolic fraction did not differ among
these three groups (Fig 2). Total PKC activity was not significantly
changed by IP or Ca2+ exposure. Removal of Ca2+
or phospholipid from the PKC assay reduced PKC activity in the
cytosolic fraction but not in the membrane fraction of the control
group. The increase in PKC activity in the membrane fraction induced by
CaCl2 or IP was also apparent only when the assay was
performed in the presence of both Ca2+ and phospholipid.
Administration of EGTA during IP or
1-adrenoceptor–mediated preconditioning reduced the
increase in PKC activity in the membrane fraction (MTX and MTX+EGTA
groups, 26±2 and 9±1 nmol·mg
protein-1·min-1,
respectively; P<.01). Administration of 8-SPT only
during Ca2+ preconditioning did not reduce PKC activity
in the membrane fraction (26±3 nmol·mg
protein-1·min-1).
Preconditioning with Ca2+ increased PKC activity in the
membrane fraction of chemically denervated hearts (24±2 nmol·mg
protein-1·min-1;
P<.01 versus the control group).
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Activation of ecto-5'-N by IP and
1-adrenoceptor–mediated preconditioning was inhibited
by concomitant administration of EGTA (Fig 3). Exposure
of both innervated and denervated hearts to
CaCl2 increased ecto-5'-N activity in the epicardium (Fig 3A) and endocardium (Fig 3B) to the values similar to those obtained
with IP in innervated hearts. The CaCl2-induced
increase in ecto-5'-N activity was reduced by treatment with GF109203X.
The CaCl2-induced increase in ecto-5'-N activity was not
decreased by treatment with 8-SPT alone during the preconditioning
procedure (Fig 3).
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IS-Limiting Effect of Ca2+ Preconditioning and Effect
of EGTA on IS-Limiting Effect of IP
The area at risk and collateral flow were similar among all
groups. Administration of EGTA reduced the IS-limiting effect of IP and
1-adrenoceptor–mediated preconditioning (Fig 4). Transient Ca2+ exposures mimicked the
IS-limiting effect of IP in both innervated and denervated
hearts. The IS-limiting effect of Ca2+ preconditioning was
prevented by intracoronary administration of AMP-CP or 8-SPT
both before and after ischemia or of GF109203X during exposure
to Ca2+; administration of 8-SPT alone during
CaCl2 exposures did not inhibit the IS-limiting effect of
Ca2+ preconditioning. These results indicate that neither
endogenous adenosine nor norepinephrine
release in response to CaCl2 administration is the trigger
for Ca2+ preconditioning and that activation of PKC
contributes to the IS-limiting effects of IP and
1-adrenoceptor–mediated preconditioning. Similar
results were obtained by plotting IS normalized by risk area against
the collateral blood flow to the inner half of the LAD-dependent
endocardium during the sustained ischemic period (Fig 5).
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) and mean±SEM (
with bars)
are shown. *P<.01, **P<.005 vs control by
Bonferroni's test.
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Effects of Doses of CaCl2 on IS and Activities of PKC
and Ecto-5'-N
Administration of CaCl2 at a dose of 4
µmol ·min-1·mL-1
of CBF did not mimic the IS-limiting effect of IP (IS, 39.4±4.1%) and
did not activate PKC in the membrane fraction of the
endocardium (11.6±2.1 nmol·mg
protein-1·min-1)
or ecto-5'-N in the endocardium (43.8±2.5 nmol·mg
protein-1·min-1).
CaCl2 at a dose of 6
µmol·min-1·mL-1
of CBF did mimic the IS-limiting effect of IP (18.3±2.1%,
P<.01 versus control group) and activated PKC in
the membrane fraction of the endocardium (23.6±1.7 nmol·mg
protein-1 · min-1,
P<.01 versus control group) and ecto-5'-N in the
endocardium (58.2±3.6 nmol·mg
protein-1·min-1,
P<.01 versus control group). CaCl2 at a dose of
10
µmol·min-1·mL-1
of CBF consistently induced more than two episodes of
ventricular fibrillation. One cycle of CaCl2 exposure at
8
µmol·min-1·mL-1
of CBF also reduced IS (18.4±3.5%, P<.01 versus control
group) and activated PKC in the membrane of the endocardium
(25.8±1.3 nmol·mg
protein-1·min-1,
P<.05 versus control group) and ecto-5'-N in the
endocardium (63.6±3.9 nmol·mg
protein-1·min-1,
P<.05 versus control group). Therefore, doses of 6 to
8
µmol·min-1·mL-1
of CBF of CaCl2 appear adequate for cardioprotection.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We have shown that exposures of the heart to CaCl2 induce the translocation of PKC from the cytosolic to the membrane fraction of the endocardium and result in the limitation of IS. We have further demonstrated that activation of ecto-5'-N is important in the PKC-mediated cardioprotection induced by exposure to CaCl2.
Mechanism of Activation of PKC Induced by Exposure to
CaCl2
An underlying assumption of our study is that exposure of the
myocardium to CaCl2 increases
[Ca2+]i. The results of several studies
support this hypothesis.12 13 De Tombe et
al14 have shown that increases in extracellular
Ca2+ increase myocardial contractility as a
result of increased [Ca2+]i, and Marban et
al12 report that increases in extracellular
Ca2+ increase the average
[Ca2+]i. We also observed that microtubular
structures are sensitive to an increase in
[Ca2+]i and that intracoronary
administration of CaCl2 disrupts microtubular structures,
as shown by immunohistochemical staining.6 It is thus
likely that an increase in the extracellular Ca2+
concentration results in Ca2+ influx via Ca2+
channels and Na+/Ca2+ exchanges and a
consequent increase in [Ca2+]i in myocardial
cells.13
The increases in PKC activity in response to exposure to
CaCl2 could be achieved by at least four means. Exposure to
CaCl2 increases
MO2,15 which may increase
adenosine release in the heart.11
Adenosine induces translocation of PKC from the cytosolic to
the membrane fraction in a Gi protein–dependent
manner,16 and adenosine mimics the IS-limiting
effect of IP in the rabbit heart.17 Indeed, we have also
shown that transient exposures to high doses of adenosine can
activate ecto-5'-N and limit IS in the canine
heart.18 However, in the present study, although
MO2 and adenosine release were
slightly increased during exposure to CaCl2, the
concomitant administration of 8-SPT during Ca2+
preconditioning did not inhibit the IS-limiting effect or activation of
PKC and ecto-5'-N. Given that we confirmed that the dose of 8-SPT used
in the present study is sufficient to inhibit the cardiovascular
effects of endogenous adenosine during myocardial
ischemia, the present results suggest that the amount of
adenosine released during exposure to CaCl2 is not
sufficient to induce cardioprotection via activation of PKC.
Second, transient exposures to Ca2+ may induce myocardial subendocardial ischemia due to an imbalance between myocardial oxygen supply and demand. We did not detect either lactate release or a decrease in the pH of coronary venous blood during Ca2+ exposure, suggesting that myocardial ischemia did not occur during Ca2+ preconditioning.
Third, exposure to Ca2+ may increase the release of
norepinephrine,19 which would then stimulate
1-adrenoceptors and activate PKC as a result of
diacylglycerol formation.10 However, the concentration of
norepinephrine did not change during and after the
administration of CaCl2 in innervated hearts.
Furthermore, transient Ca2+ exposures limited IS even in
denervated hearts. Thus, endogenous
norepinephrine does not appear to mediate the effects of
Ca2+ preconditioning.
The most likely possibility is that exposure to Ca2+ increases PKC activity directly by increasing [Ca2+]i. Increases in [Ca2+]i activate phospholipase C,20 the enzyme that catalyzes polyphosphoinositide hydrolysis and thereby generates diacylglycerol.21
In this experiment, we administered drugs, including EGTA and
CaCl2, into a coronary bypass tube. Since we
perfused the LAD with blood from the left carotid artery through a
bypass tube and the left circumflex artery is dominant in the canine
heart, systemic hemodynamic and metabolic
parameters including systemic blood pressure, dP/dt, and
heart rate did not change by infusion of either CaCl2 or
EGTA via the LAD. On the other hand, the FS of the LAD-perfused area
increased during infusion of CaCl2 but returned to baseline
5 minutes after the third transient infusion of CaCl2
(Table 3
).
Cytosolic PKC activity tended to decrease but did not reach statistical significance. Accordingly, total PKC activity tended to increase but showed no significant changes, while PKC in the membrane fraction was markedly activated. There may be two possible reasons for this. First, when a small part of cytosolic PKC is translocated to the plasma membrane, the assay system used in the present study may not detect decreases in cytosolic PKC. In this case, however, we could detect significant changes in PKC activity in the membrane fraction. Another possibility is the contamination of inhibitors in the cytosolic fraction of the total PKC activity. Although it is difficult to address which possibility is more likely, we can conclude that PKC activity of the membrane fraction is increased due to exposures to CaCl2.
Role of Increases in [Ca2+]i and
1-Adrenoceptor Activation in IP-Induced
Cardioprotection
Myocardial ischemia and reperfusion are characterized by
an increase in [Ca2+]i5 and the
release of norepinephrine, both of which appear to mediate
the IS-limiting effect of IP by activating PKC, because activation of
PKC plays an important role in IP.22 23 24 25 We3
and others26 27 have shown that
1-adrenoceptor stimulation plays a key role in mediating
the IS-limiting effect of IP. Furthermore, we have shown that the PKC
activation induced by 1-adrenoceptor stimulation is
important for cardioprotection because it activates
ecto-5'-N,3 and we have suggested that the
activated PKC is Ca2+ dependent in the canine
heart.28 Therefore, an increase in
[Ca2+]i may be required for activation of PKC
induced by 1-adrenoceptor stimulation and may decrease
the activation threshold of the enzyme.29 Both pathways,
an increase in [Ca2+]i and
1-adrenoceptor activation, may mediate IP-induced
cardioprotection in the canine heart independently or they may be
interdependent. Indeed, exposures of cardiac tissue to
1-adrenoceptor agonists stimulate
phosphoinositide hydrolysis, resulting in an increase
in [Ca2+]i triggered by inositol
1,4,5-trisphosphate,30 and 1-adrenoceptor
activation enhances intracellular alkalization through
Na+/H+ exchange and increases Ca2+
influx as a result of the subsequent activation of
Na+/Ca2+ exchange in adult rat
cardiomyocytes.31 In turn, PKC activity
typically depends on
[Ca2+]i.20 21 Thus, both
increases in [Ca2+]i and
1-adrenoceptor activation are linked to each other and
to activation of PKC and ecto-5'-N. The relative importance of these
two pathways may depend on the severity, duration, and number of
episodes of transient ischemia during IP.
Pathophysiological Role of Transient
Ca2+ Overload in Ischemia and Reperfusion
Injury
Ca2+ overload during ischemia and reperfusion
results in reversible or irreversible cellular injury,32
and the administration of EGTA during sustained ischemia and
reperfusion protects against such injury.33 Indeed, an
intracoronary infusion of EDTA during the initial 10 minutes of
reperfusion attenuates the severity of myocardial stunning in canine
hearts.6
On the other hand, transient exposures to CaCl2 before sustained ischemia limited IS, and the administration of EGTA during IP reduced the IS-limiting effect of this procedure. These observations suggest that Ca2+ overload during sustained ischemia and reperfusion is deleterious but that transient Ca2+ overload before sustained ischemia may induce cardioprotection.
Transient Ca2+ overload induced by brief periods of exposure to 10 mmol/L (Ca2+)o causes contractile dysfunction and ATP depletion in perfused ferret hearts.34 In the present study intracoronary infusions of CaCl2 increased the Ca2+ concentration in the perfused blood from 0.07 to 0.17 mmol/L, but transient CaCl2 infusion at a low dose did not cause contractile dysfunction.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We thank Noriko Tamai, Kayoko Yoshida, Yukiyo Nomura, Kiyomi Okuto, Shinya Suzuki, and Makoto Hasegawa for technical assistance.
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Footnotes |
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Presented in part at the 67th Annual Scientific Sessions of the American Heart Association, Dallas, Tex, November 11-14, 1994, and the 17th Congress of the European Society of Cardiology, Amsterdam, Netherlands, August 20-24, 1995. Published in abstract form (Circulation. 1994;90[suppl I]:I-209).
Received October 31, 1996; revision received February 24, 1997; accepted February 28, 1997.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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