Download Cardioprotective Effect of Angiotensin

Cardioprotective Effect of Angiotensin-Converting Enzyme
Inhibition Against Hypoxia/Reoxygenation Injury in
Cultured Rat Cardiac Myocytes
Satoaki Matoba, MD; Tetsuya Tatsumi, MD, PhD; Natsuya Keira, MD; Akira Kawahara, MD;
Kazuko Akashi, MD; Miyuki Kobara, MD; Jun Asayama, MD, PhD; Masao Nakagawa, MD, PhD
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Background—Although ACE inhibitors can protect myocardium against ischemia/reperfusion injury, the mechanisms of
this effect have not yet been characterized at the cellular level. The present study was designed to examine whether an
ACE inhibitor, cilazaprilat, directly protects cardiac myocytes against hypoxia/reoxygenation (H/R) injury.
Methods and Results—Neonatal rat cardiac myocytes in primary culture were exposed to hypoxia for 5.5 hours and
subsequently reoxygenated for 1 hour. Myocyte injury was determined by the release of creatine kinase (CK). Both
cilazaprilat and bradykinin significantly inhibited CK release after H/R in a dose-dependent fashion and preserved
myocyte ATP content during H/R, whereas CV-11974, an angiotensin II receptor antagonist, and angiotensin II did not.
The protective effect of cilazaprilat was significantly inhibited by Hoe 140 (a bradykinin B2 receptor antagonist),
NG-monomethyl-L-arginine monoacetate (L-NMMA) (an NO synthase inhibitor), and methylene blue (a soluble
guanylate cyclase inhibitor) but not by staurosporine (a protein kinase C inhibitor), aminoguanidine (an inhibitor of
inducible NO synthase), or indomethacin (a cyclooxygenase inhibitor). Cilazaprilat significantly enhanced bradykinin
production in the culture media of myocytes after 5.5 hours of hypoxia but not in that of nonmyocytes. In addition,
cilazaprilat markedly enhanced the cGMP content in myocytes during hypoxia, and this augmentation in cGMP could
be blunted by L-NMMA and methylene blue but not by aminoguanidine.
Conclusions—The present study demonstrates that cilazaprilat can directly protect myocytes against H/R injury, primarily
as a result of an accumulation of bradykinin and the attendant production of NO induced by constitutive NO synthase
in hypoxic myocytes in an autocrine/paracrine fashion. NO modulates guanylate cyclase and cGMP synthesis in
myocytes, which may contribute to the preservation of energy metabolism and cardioprotection against H/R injury.
(Circulation. 1999;99:817-822.)
Key Words: angiotensin n hypoxia n bradykinin n nitric oxide n myocytes
R
ecent clinical and experimental studies have established the
therapeutic benefits of ACE inhibitors, not only in treating
hypertension and congestive heart failure, but also in reducing
reinfarction, limiting infarct size, and preventing reperfusion
arrhythmias.1–3 These cardioprotective effects of ACE inhibitors
are thought to depend on their ability to attenuate the degradation
of endogenous bradykinin as well as to decrease the synthesis of
angiotensin II from angiotensin I. In addition to altering circulating concentrations of angiotensin II and bradykinin, thereby
effecting hemodynamic changes, ACE inhibitors may participate in cardioprotection through the alteration of localized
angiotensin II or bradykinin concentrations in cardiac tissue.4
Angiotensin II may induce cardiac myocyte necrosis and fibroblast proliferation, thereby exacerbating ischemia-reperfusion
injury.5 In contrast, recent reports have indicated that kinin can
induce an increase in coronary circulation and improve cardiac
function in ischemia-reperfusion injury. Although inhibition of
ACE is also known to cause coronary vasodilation and an
improvement in contractile and metabolic function in ischemiareperfusion injury,6 –9 it is still unknown whether the beneficial
effects of ACE inhibitors are attributable to a direct effect on
cardiac myocytes.
In the present study, therefore, we focused on the direct molecular actions of an ACE inhibitor on cardiac myocytes. We have
examined the following: (1) whether cilazaprilat, a nonsulfhydryl
ACE inhibitor, directly protects cardiac myocytes against hypoxia/
reoxygenation injury; (2) whether this protective action derives
from an inhibition of angiotensin II synthesis or an accumulation of
bradykinin; (3) whether this protective effect is mediated by prostaglandins, NO, or protein kinase C (PKC); and (4) whether cGMP
is involved in the protective effect. We treated cultured rat neonatal
cardiac myocytes with either modified Tyrode solution, cilazaprilat,
Received July 2, 1998; revision received September 23, 1998; accepted October 5, 1998.
From the Second Department of Medicine, Kyoto Prefectural University of Medicine and Department of Clinical Pharmacology (J.A.), Kyoto
Pharmaceutical University, Kyoto, Japan.
Correspondence to Tetsuya Tatsumi, MD, PhD, Second Department of Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji,
Kamigyo-ku, Kyoto 602-8566, Japan. E-mail [email protected]
© 1999 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
817
818
ACE Inhibitor and Cardiac Myocytes
angiotensin II, bradykinin, or the angiotensin II type 1 receptor
antagonist CV-11974 and subsequently exposed the cells to 5.5
hours of hypoxia followed by 1 hour of reoxygenation. In addition,
we tested whether pretreatment with the kinin receptor antagonist
Hoe 140, the NO synthase inhibitor NG-monomethyl-L-arginine
monoacetate (L-NMMA), the inducible NO synthase inhibitor
aminoguanidine, the cyclooxygenase inhibitor indomethacin, or the
PKC inhibitor staurosporine could block the beneficial effect of
cilazaprilat. Furthermore, we measured the concentration of bradykinin in culture media after treatment with cilazaprilat and monitored the cGMP content in myocytes during hypoxia as well as the
high-energy phosphates in myocytes during hypoxia/reoxygenation.
Methods
Materials
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Cilazaprilat, Hoe 140, and CV-11974 were generous gifts from Nippon
Roch KK, Hoechst Marion Roussel AG, and Takeda Chemical Industries, Ltd, respectively. Angiotensin II and bradykinin were purchased
from Peptide Institute, Inc; L-NMMA from Calbiochem-Novabiochem
International; aminoguanidine from Nacalai tesque Inc; and the remaining reagents from Sigma Chemical Co.
Culture of Neonatal Rat Cardiac Myocytes
Primary cultures of neonatal rat cardiac myocytes were prepared as
previously described with some modifications.10 Briefly, hearts were
removed from 1- to 2-day-old Wistar rats anesthetized by ether under
aseptic conditions and placed in Ca21- and Mg21-free PBS. The
hearts were washed with PBS, and the atria and aorta were discarded.
The ventricles were minced with scissors into 1- to 3-mm3 fragments,
and they were then enzymatically digested 4 times for 10 to 15
minutes each with 7.5 mL of PBS containing 0.2% collagenase
(Sigma type I). The liberated cells were collected by centrifugation
at 300g and incubated in 100-mm culture dishes (Falcon) for 60
minutes at 37°C in a humidified incubator with 5% CO2 air. The
nonadherent cardiac myocytes were harvested and seeded into
60-mm culture dishes (13105 cells/cm2). The myocytes were incubated in Dulbecco’s modified Eagle’s medium (DMEM; Nissui
Pharmaceutical Co) supplemented with 10% FBS (Bioserum Co).
5-Bromo-29-deoxyuridine (BrdU; 100 mmol/L) was added during the
first 48 hours to inhibit proliferation of nonmyocytes.11 Using this
method, we routinely obtained contractile myocyte-rich cultures with
90% to 95% myocytes, as assessed by immunofluorescence staining
with a monoclonal antibody against b-myosin heavy chain. The
myocytes were then incubated in DMEM containing 0.5% FBS
without BrdU, and all experiments were done 36 to 48 hours after
this incubation.
Preparation of Cardiac Nonmyocyte-Rich Culture
Highly enriched cultures of cardiac nonmyocytes (hereafter called
nonmyocyte culture) were prepared by 2 passages of the cells
adherent to the culture dish during the preplating procedure.11 Until
the second passage, cells were maintained in the same culture
medium as above, except that 10% FBS was used and BrdU was not
used. The nature of cells was determined by immunofluorescence
staining with anti-rat factor VIII, anti-desmin, and anti-vimentin for
identification of endothelial cells, smooth muscle cells, and fibroblasts, respectively. After the second passage, only 1% to 2% of cells
were stained positively with anti-desmin or anti-factor VIII. More
than 95% of cells were stained positively with anti-vimentin antibody, indicating that the nonmyocytes consisted of fibroblasts under
our culture conditions.
Culture of Rat Aortic Endothelial Cells
Rat aortic endothelial cells (RAECs) were isolated from male Wistar
rats (weight, 200 to 250 g) by the primary explant technique.12 The
cells were cultured in DMEM supplemented with 10% FBS and 0.15
mg/mL endothelial cell growth supplement. RAECs were grown at
Figure 1. Experimental protocols. Neonatal rat cardiac myocytes
in primary culture were pretreated as described in Methods and
were then exposed to hypoxia for 5.5 hours and reoxygenation for
1 hour. CK activity and bradykinin concentration in culture media
as well as cGMP and high-energy phosphate content in myocytes
were measured as indicated and described in Methods.
37°C in a humidified incubator with 5% CO2 air and serially
passaged with a split ratio of 1:5 using 0.05% trypsin-0.02% EDTA.
Biocoat Cellware (rat-tail collagen type I), 35-mm/60-mm-diameter
tissue culture dishes from Becton Dickinson Labware were used
throughout the RAEC cultures. Experiments were performed on cells
at passage 3 from the primary culture. Characterization as endothelial cells was confirmed by immunofluorescence staining with
antibodies specific for factor VIII.
Experimental Protocols
Figure 1 shows the experimental protocols. Before hypoxic exposure, cell medium was replaced by modified Tyrode solution
(in mmol/L: NaCl 136.9, KCl 2.68, Na2HPO4 z 12H2O 8.1, KH2PO4
1.47, CaCl2 0.9, MgCl2 z 6H2O 0.49; pH 7.4). The cardiac myocytes
were transferred to an environmental chamber at 37°C in a humidified atmosphere flushed with 5% CO2 and ,1% oxygen (F-102,
Iijima Electronics Co) in nitrogen for 5.5 hours and were then
reoxygenated for 1 hour with DMEM.
To examine the role of angiotensin and bradykinin in hypoxia/reoxygenation injury, the myocytes were treated with the following before hypoxia/
reoxygenation: (1) modified Tyrode solution (control); (2) cilazaprilat (1028
to 1025 mol/L); (3) CV-11974 (1028 to 1025 mol/L); (4) angiotensin II (1029
to 1026 mol/L); and (5) bradykinin (1029 to 1026 mol/L). Furthermore, to
examine the mechanism of the cardioprotective effect of cilazaprilat,
myocytes were treated with cilazaprilat (1025 mol/L) before hypoxia/reoxygenation in the presence of (6) Hoe 140 (1026 mol/L), (7) staurosporine
(231029 mol/L), (8) L-NMMA (431024 mol/L), (9) aminoguanidine
(531024 mol/L), (10) indomethacin (1025 mol/L), and (11) methylene blue
(1025 mol/L).
Assay of Creatine Kinase Release
Creatine kinase (CK) activity in culture media was measured before
hypoxia, at the end of 5.5 hours of hypoxia, and after 1 hour of
reoxygenation in all groups (Figure 1). CK activity in culture media
was measured spectrophotometrically at 37°C according to Rosalki’s
procedure. The activity of CK was expressed as IU/L.13
Measurement of Bradykinin Concentration
Bradykinin concentration in the culture media was measured during
hypoxia as shown in Figure 1, according to a previously described
method.14 Briefly, 0.1-mL samples of culture supernatant were acidified
with 5 mL of 0.01 mol/L HCl and extracted twice with 20 mL of diethyl
ether. The aqueous phase was taken to dryness with a rotary evaporator,
and the dried samples were stored at 280°C until assayed. Before assay,
the dried samples were redissolved in 2.5 mL of 0.1 mol/L Tris-HCl
buffer containing 0.2% gelatin, 0.1% neomycin, and 0.01 mol/L EDTA,
Matoba et al
February 16, 1999
819
adjusted to pH 7.4. The incubation mixture for radioimmunoassay
consisted of 0.1 mL of 0.01 mol/L 1,10-phenanthroline HCl, diluent
buffer of 0.5 mL containing the unknown or standard bradykinin, 0.1
mL of antiserum diluted 1:600 with diluent buffer, and 0.1 mL of
(125I-Tyr8)-bradykinin ('8000 cpm) dissolved in normal saline. The
mixture was incubated in a polyethylene tube at 4°C for 24 hours, and
dextran-coated charcoal was used to separate the free labeled antigen
from that bound to antibody. Three replicate tubes containing only
buffer, phenanthroline, and (125I-Tyr8)-bradykinin were incubated and
treated with coated charcoal to determine the amount of labeled antigen
that remained in the supernatant in the absence of antibody. The mean
value of this measurement was subtracted from supernatant radioactivity
after centrifugation of the antibody-containing tubes, and the resultant
value was used to calculate the proportion of label bound to antibody.
Measurement of cGMP in Cardiac Myocytes
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cGMP concentration in myocytes was measured after 1, 3, and 5.5
hours of hypoxia (Figure 1). Cardiac myocytes (2.73106 cells per
dish) were treated with 0.25 mL of ice-cold 6% trichloroacetic acid
and centrifuged at 1000g for 10 minutes. The supernatant was
extracted 3 times with 3 mL of diethyl ether saturated with water,
and the aqueous phase was stored at 280°C. cGMP concentration in
the supernatant was measured by radioimmunoassay.15 Briefly, 0.1
mL of dioxane-triethylamine mixture containing succinic acid anhydride succinylated cGMP was added to the supernatant (0.1 mL).
After a 10-minute incubation, the reaction mixture was added to 0.8
mL of 0.3 mol/L imidazole buffer (pH 6.5). Succinyl cGMP tyrosine
methyl ester (0.1 mL) iodinated with 125I (15 000 to 20 000 cpm in
,10214 mol/L) was added to the assay mixture containing 0.1 mL of
supernatant and 0.1 mL of diluted antisera, and the mixture was
incubated at 4°C for 20 hours. A cold solution of dextran-coated
charcoal (0.5 mL) was added to the mixture in an ice-cold water bath.
The charcoal was spun down, and 0.5 mL of the supernatant was
counted for radioactivity in a gamma spectrometer. The amount of
cGMP was normalized to protein content of cardiac myocytes
assayed by the Lowry method.
Measurement of ATP in Cardiac Myocytes
The ATP content of myocytes was measured before hypoxia, after 3
or 5.5 hours of hypoxia, and after 1 hour of reoxygenation (Figure 1).
Cardiac myocytes (2.73106 cells per dish) were treated with 0.25
mL of 0.6N ice-cold perchloric acid and centrifuged at 1000g for 5
minutes at 4°C. The supernatant was neutralized with KOH to pH 5.0
to 7.0 and, after 10 minutes, was centrifuged at 8000g for 5 minutes
at 4°C to remove the KClO4. The supernatant was used for the
assays. ATP was measured by high-performance liquid chromatography (LC-9A liquid chromatograph, Shimadzu) with a column of
STR ODS-M (Shimadzu).16
Figure 2. Effect of cilazaprilat, CV-11974, angiotensin II, and
bradykinin on hypoxia/reoxygenation-induced CK release. Myocytes were treated with cilazaprilat (1028 to 1025 mol/L),
CV-11974 (1028 to 1025 mol/L), angiotensin II (1029 to 1026
mol/L), or bradykinin (1029 to 1026 mol/L) and were then
exposed to hypoxia for 5.5 hours and reoxygenation for 1 hour.
CK release from myocytes was measured as described in Methods. n56. **P,0.01 vs control.
release. Cilazaprilat reduced CK release in a dose-dependent
fashion to 36% of control at 1025 mol/L. Although CV-11974
is reported to block the effect of angiotensin II and III, it did
not significantly inhibit CK release in the present study
(Figure 2). In addition, there was no significantly additive
increase in CK release after administration of angiotensin II.
In contrast, bradykinin significantly lowered CK release in a
dose-dependent manner, with 1026 mol/L bradykinin reducing CK release to 37% of control.
Cardioprotective Mechanism of Cilazaprilat
Against Hypoxia/Reoxygenation Injury
Reoxygenation-induced CK release in the control and
cilazaprilat-treated groups in the presence of Hoe 140, stau-
Statistical Analysis
Data are expressed as mean6SEM of 6 samples derived from $6
separate experiments. Differences were analyzed by 2-way ANOVA
combined with Scheffé’s test, and a P value of ,0.05 was considered
to be statistically significant.
Results
Effects of Cilazaprilat, CV-11974, Angiotensin II,
and Bradykinin on Hypoxia/Reoxygenation Injury
Treatment of myocytes with 5.5 hours of modified Tyrode
solution followed by 1 hour of DMEM medium under
normoxic conditions or hypoxia alone (5.5 hours) did not
cause a significant release of CK into the myocyte culture
media (data not shown). However, hypoxia followed by
reoxygenation caused a significant increase in CK release, as
shown in the control bar of Figure 2. Figure 2 also shows the
effect of pretreatment with cilazaprilat, CV-11974, angiotensin II, and bradykinin on this reoxygenation-induced CK
Figure 3. Effect of Hoe 140, staurosporine, L-NMMA, aminoguanidine, indomethacin, and methylene blue on cilazaprilatinduced cardioprotection. Myocytes were treated with modified
Tyrode buffer containing cilazaprilat (1025 mol/L) in presence or
absence of Hoe 140 (1026 mol/L), staurosporine (231029 mol/L),
L-NMMA (431024 mol/L), aminoguanidine (531024 mol/L), indomethacin (1025 mol/L), or methylene blue (1025 mol/L) before
hypoxia/reoxygenation. CK release was measured as described
in Methods. n56. *P,0.05, **P,0.01 vs control; ##P,0.01 vs
cilazaprilat.
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ACE Inhibitor and Cardiac Myocytes
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Figure 4. Bradykinin accumulation in culture media during hypoxia and immunofluorescence stainings of myocytes, nonmyocytes, and
RAECs. Myocytes, nonmyocytes, and RAECs, which were pretreated with modified Tyrode buffer (control) or cilazaprilat (1025 mol/L),
respectively, were exposed to 5.5 hours’ hypoxia, and bradykinin concentration in culture media was measured as described in Methods. n56. Myocytes, nonmyocytes, and RAECs were stained with anti-b-myosin heavy chain, anti-vimentin, and anti-factor VIII, respectively. FITC-labeled mouse anti-goat antibody was used as secondary fluorescein. Magnification 3400 and 31000 for upper and lower
panels, respectively. *P,0.05, **P,0.01, †P,0.001 vs control.
rosporine, L-NMMA, aminoguanidine, indomethacin, and
methylene blue is shown in Figure 3. Cilazaprilat (1025
mol/L) significantly reduced CK release compared with
control. However, the protective effect of cilazaprilat was
significantly inhibited by cotreatment with 1026 mol/L Hoe
140 or 431024 mol/L L-NMMA but not 231029 mol/L
staurosporine, 531024 mol/L aminoguanidine, or 1025 mol/L
indomethacin. Cotreatment with 1025 mol/L methylene blue
partially blunted the cardioprotection by cilazaprilat.
cGMP Content in Cardiac Myocytes
During Hypoxia
The time course of cGMP content change in the myocytes is
illustrated in Figure 5. cGMP content in control cells did not
Bradykinin Accumulation in Myocytes and
Nonmyocytes During Hypoxia
Figure 4 show the time course of bradykinin concentration in
the culture media and immunofluorescence stainings of myocytes, nonmyocytes, and RAECs. Hypoxia significantly increased bradykinin levels in the myocytes. Treatment of
myocytes with 1025 mol/L cilazaprilat significantly increased
bradykinin production to 4.5 times that of control after 5.5
hours of hypoxia. In contrast, hypoxia did not enhance
bradykinin production even in the presence of 1025 mol/L
cilazaprilat in nonmyocytes. Hypoxia also significantly increased bradykinin levels in RAECs, and 1025 mol/L cilazaprilat significantly enhanced bradykinin levels to 2 times that
of control after 5.5 hours of hypoxia.
Figure 5. cGMP content in hypoxic myocytes. Myocytes were
treated with modified Tyrode buffer (control), bradykinin (1026
mol/L), or cilazaprilat (1025 mol/L) in presence or absence of
L-NMMA (431024 mol/L), aminoguanidine (531024 mol/L), or
methylene blue (1025 mol/L) before exposure to 5.5 hours of
hypoxia. cGMP content in myocytes was determined as
described in Methods. n56. **P,0.01, †P,0.001 vs control.
Matoba et al
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Figure 6. ATP content in myocytes. Myocytes were pretreated
with modified Tyrode buffer (control), bradykinin (1026 mol/L), or
cilazaprilat (1025 mol/L) in presence or absence of L-NMMA
(431024 mol/L) and were then exposed to 5.5 hours of hypoxia
and/or 1 hour of reoxygenation; ATP content in myocytes was
determined as described in Methods. n56. *P,0.05, **P,0.01
vs control. H/R indicates hypoxia/reoxygenation.
change appreciably during the 5.5 hours of hypoxia. However, cilazaprilat 1025 mol/L markedly increased cGMP
content with increased time of hypoxia. This augmentation of
cGMP production by cilazaprilat was blunted by cotreatment
with 431024 mol/L L-NMMA or 1025 mol/L methylene blue.
In contrast, 531024 mol/L aminoguanidine did not block the
cilazaprilat-induced increase in cGMP content. Bradykinin
(1026 mol/L) significantly promoted cGMP production
throughout the hypoxic period.
ATP Content in Cardiac Myocytes During
Hypoxia/Reoxygenation
The time course of ATP content change in myocytes is shown in
Figure 6. Exposure to 6.5 hours of normoxic culture conditions
alone did not affect ATP content in the myocytes. In the control,
hypoxia significantly lowered ATP content in a time-dependent
manner, and reoxygenation induced a further decrease in ATP. Both
1025 mol/L cilazaprilat and 1026 mol/L bradykinin significantly
inhibited this hypoxia/reoxygenation-induced decline in ATP. The
ATP content in the myocytes treated with cilazaprilat was 1.31
times that of control after 5.5 hours’ hypoxia and 4.84 times that of
control after 1 hour of reoxygenation. L-NMMA 431024 mol/L
totally inhibited this cilazaprilat-induced preservation of ATP.
Discussion
In the present study, we have demonstrated that an ACE
inhibitor, cilazaprilat, shows remarkable protection against hypoxia/reoxygenation injury in a dose-dependent fashion. Recent
reports demonstrated the existence of a local renin-angiotensin
system within the myocardium4 and indicated that angiotensin II
type 1 receptor antagonist improves ischemia-reperfusion injury.17 In the present study, however, pretreatment with the
angiotensin II receptor antagonist CV-11974 or angiotensin II
did not worsen CK release (Figure 2), which strongly suggests
that cilazaprilat reduced myocardial hypoxia/reoxygenation injury independently of angiotensin II synthesis.
February 16, 1999
821
Our results also show that the cardioprotective effect of
cilazaprilat is blocked by Hoe 140, which suggests that its
effect is mediated by the bradykinin B2 receptor (Figure 3).
Furthermore, we demonstrated that bradykinin production
during hypoxia was significantly increased by cilazaprilat
(Figure 4). It has been reported that a local kinin-kallikrein
system exists in the heart18 and that myocardial bradykinin
levels are further enhanced by ischemia or ACE inhibitors
through their inhibitory effect on the degradation of kinins.19
It is still unclear which cells produce the bradykinin in the
heart. One recent report20 suggested that the coronary vascular endothelium is the main source of the release of kinins.
However, it is likely that myocytes also play a significant role
because bradykinin levels were significantly enhanced by
cilazaprilat after hypoxia in myocytes but not in nonmyocytes
under our culture conditions (Figure 4). In addition, we have
confirmed that nonmyocytes consist of 95% fibroblasts and
1% to 2% endothelial cells. Furthermore, the enhancement in
bradykinin production by cilazaprilat in myocytes was greater
than that in RAECs. Although these results were obtained
from in vitro studies using neonatal cardiac myocytes, the
present data strongly suggest that myocytes may be an
important source of bradykinin and that ACE inhibitors may
act directly on myocytes through a local kinin-kallikrein
system, thereby contributing to cardioprotection in an autocrine/paracrine fashion. Indeed, recent data indicate that
kallikrein activity can be detected in primary cultures of
neonatal rat cardiocytes and in heart slices.18
Previous observations have also demonstrated the existence of functional bradykinin B2 receptors on cardiomyocytes, which are coupled to the activation of phospholipase C,
the subsequent generation of inositol 1,4,5-triphosphate or
diacylglycerol, an increase in cytosolic Ca21 levels, and the
activation of PKC.21–23 Elevated cytosolic Ca21 can stimulate
phospholipase A2 and cyclooxygenase, as indicated by the
production of prostaglandins.24 Furthermore, the increase in
cytosolic Ca21 through the B2 receptor may also stimulate
myocyte constitutive NO synthase (cNOS).25,26 In the present
study, the cardioprotective effect of cilazaprilat was significantly blocked by L-NMMA but not by staurosporine, aminoguanidine, or indomethacin (Figure 3), therefore suggesting that the effect of cilazaprilat is not mediated by PKC,
inducible NOS, or prostaglandins but rather by a cNOSassociated, bradykinin B2 receptor–mediated pathway.
The present study also indicates that the protective effect of
cilazaprilat is mediated by cGMP, because methylene blue
significantly blocked this effect (Figure 3). In addition, cGMP
content was significantly increased in hypoxic myocytes after
treatment with cilazaprilat, and this augmentation of cGMP was
blunted by cotreatment with L-NMMA and methylene blue but
not aminoguanidine (Figure 5). The data therefore suggest that
cGMP production in myocytes treated with cilazaprilat is mediated by a cNOS-NO–guanylate cyclase signaling pathway. It
has been reported previously that cGMP can improve the energy
state in the ischemic heart.27 Indeed, in the present study,
cilazaprilat as well as bradykinin significantly preserved the
ATP content of myocytes (Figure 6).
Although the role of cGMP in regulating myocardial
contraction remains controversial, recent reports suggest that
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ACE Inhibitor and Cardiac Myocytes
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cGMP can regulate sarcolemmal Ca21 influx through L-type
Ca21 channels (ICa) by activation of a cGMP-stimulated
phosphodiesterase28,29 or by cGMP-dependent protein kinase
(PKG)30,31 and can reduce the myofilament response to Ca21
via activation of an endogenous cGMP-dependent protein
kinase (cGMP-PK).32 Furthermore, cGMP has been recently
reported to mediate the negative inotropic effect of NO.33,34 It
is therefore tempting to speculate that NO production induced
by cilazaprilat modulated myocyte contractility and contributed to the energy-sparing effect, although we cannot exclude
the possibility that other effects of NO, such as radical
scavenge action, also contribute to cardioprotection.35
In conclusion, the present study demonstrates that cilazaprilat can protect isolated myocytes against hypoxia/reoxygenation injury, possibly as a result of bradykinin accumulation and the resultant production of NO by cNOS in hypoxic
myocytes in an autocrine/paracrine fashion. NO may increase
cGMP synthesis in myocytes, which may consequently modulate their contractility and may contribute to energy preservation and cardioprotection.
Acknowledgments
The authors are grateful to Dr Henry Fliss, Department of Cellular
and Molecular Medicine, University of Ottawa, Canada, for the
generous academic advice, and to Dr Shinji Fushiki, Department of
Dynamic Pathology, Kyoto Prefectural University of Medicine, for
the technical advice of immunohistochemical examination.
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Cardioprotective Effect of Angiotensin-Converting Enzyme Inhibition Against
Hypoxia/Reoxygenation Injury in Cultured Rat Cardiac Myocytes
Satoaki Matoba, Tetsuya Tatsumi, Natsuya Keira, Akira Kawahara, Kazuko Akashi, Miyuki
Kobara, Jun Asayama and Masao Nakagawa
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Circulation. 1999;99:817-822
doi: 10.1161/01.CIR.99.6.817
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