(Circulation. 2000;102:III-346.)
© 2000 American Heart Association, Inc.
Myocardial Protection and Vascular Biology |
Myocardial Protection by Preconditioning of Heart With Losartan, an Angiotensin II Type 1–Receptor Blocker
Implication of Bradykinin-Dependent and Bradykinin-Independent Mechanisms
From the Departments of Surgery, University of Connecticut School of Medicine, Farmington (M.S., H.O., N.M., D.K.D), and Baystate Medical Center, Springfield, Mass (R.M.E., J.A.R., J.E.F., D.W.D.).
Correspondence to Richard M. Engelman, MD, Department of Cardiac Surgery, Baystate Medical Center, 759 Chestnut Ave, Springfield, MA 01199.
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Abstract |
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Background—Ischemic preconditioning (PC) represents a state-of-the-art technique for myocardial preservation. Although certain intracellular mediators have been shown to play a role in PC, the exact nature of the trigger for PC is not known. Our previous study demonstrated that intracellular bradykinin released from the heart during ischemia/reperfusion plays a role in myocardial preservation. This study was undertaken to further examine the mechanism of bradykinin-mediated PC.
Methods and Results—Since the bradykinin B2 receptor is likely to provide cardioprotection by blocking angiotensin II formation, we determined the effects of an angiotensin II type 1 (AT1) receptor blocker, losartan, and a bradykinin B2 receptor blocker, HOE 140, on myocardial protection. Isolated rat hearts were perfused initially by the Langendorff mode with Krebs-Henseleit buffer (KHB) for 15 minutes in the absence (control) or presence of losartan (4.5 µmol/L) and/or HOE 140 (10 µmol/L). After conversion to the working mode for 10 minutes (baseline), randomly assigned control and experimental hearts were subjected to 30 minutes of normothermic global ischemia followed by 2 hours of reperfusion. Myocardial function, infarct size, cardiomyocyte apoptosis, and amount of bradykinin/angiotensin released from the hearts were measured at baseline and during reperfusion while in the working mode. Significant postischemic ventricular recovery was demonstrated by improved developed pressure and aortic flow and reduced myocardial infarct size and apoptotic cell death with losartan, whereas the reverse was true for HOE 140. The functional recovery and infarct size–lowering ability of losartan were partially blocked and the antiapoptotic function of losartan was completely blocked by HOE 140.
Conclusions—The results document that losartan reduced whereas HOE 140 increased myocardial ischemia/reperfusion injury by blocking AT1 and bradykinin B2 receptors, respectively, suggesting a role of the bradykinin B2 receptor in PC. Losartan provided cardioprotection through both bradykinin-dependent and bradykinin-independent mechanisms.
Key Words: ischemia • reperfusion • angiotensin • bradykinin • receptors
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Introduction |
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Mammalian hearts have a remarkable ability to adapt themselves to potentially lethal exogenous stresses, which include environmental stresses such as hypoxia, heat shock, and oxidative stress.1 2 3 Increased tolerance to a sustained ischemic insult by hearts previously exposed to cyclic episodes of brief periods of ischemia and reperfusion has been documented in numerous experimental models. This transient adaptive response associated with decreased reperfusion-induced arrhythmias, increased recovery of postischemic contractile function, and reduced infarct size is known as ischemic preconditioning (PC).4
Despite the existence of an astronomic number of papers in the literature demonstrating the cardioprotective abilities of PC, the underlying cytoprotective mechanisms remain elusive. The current hypothesis suggests that the trigger for classic PC is one or more intracellular mediators including catecholamines, adenosine, nitric oxide, bradykinin, and angiotensin.5 6 These intracellular mediators are released into the coronary circulation within minutes of ischemia.7 8 These mediators either alone or in combination potentiate a cascade of signal transduction involving multiple kinases leading to the induction of the expression of genes.9 10
A large number of studies including our own have implicated a role for bradykinin in PC.6 11 We have also demonstrated the cardioprotective abilities of ACE inhibitors.12 The present study reinforces the hypothesis that ACE inhibition potentiates PC through bradykinin B2-receptor activation. Isolated rat hearts were perfused in the absence or presence of losartan (an angiotensin AT1 inhibitor), HOE 140 (a bradykinin B2 blocker) or both losartan and HOE 140 together. The results document that HOE 140 increased myocardial ischemia/reperfusion injury by blocking the bradykinin B2 receptor and increasing angiotensin II formation. This suggests a role of the bradykinin B2 receptor in PC. Losartan reduced ischemia-reperfusion injury by modulating the amount of bradykinin release from the heart. The infarct size–lowering ability of losartan was partially blocked and the antiapoptotic role was completely blocked by HOE 140, documenting both the bradykinin-dependent and bradykinin-independent mechanisms of losartan.
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Methods |
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Isolated Perfused Heart Preparation
Sprague-Dawley rats weighing
300 g were anesthetized
with pentobarbital (80 mg/kg IP). After intravenous
administration of heparin (500 IU/kg), the chests were opened and the
hearts were rapidly excised and mounted on a nonrecirculating
Langendorff perfusion apparatus.1 The
perfusion buffer used in this study consisted of a modified
Krebs-Henseleit bicarbonate buffer (KHB) (in mmol/L: 118 NaCl, 4.7
KCl, 1.2 MgSO4, 1.2
KH2PO4, 25
NaHCO3, 10 glucose, and 1.7
CaCl2, gassed with 95%
O2–5% CO2, filtered
through a 5-µm filter to remove any particulate contaminants, pH 7.4)
that was maintained at a constant temperature of 37°C and was gassed
continuously for the entire duration of the experiment. Left atrial
cannulation was then carried out, and, after allowing for a
stabilization period of 10 minutes in the retrograde perfusion mode,
the circuit was switched to the antegrade working mode, which allows
for the measurement of myocardial contractility as well
as aortic and coronary flows, as described in detail in a
previous report.1 Essentially, it is a left heart
preparation in which the heart is perfused at a constant preload of 17
cm H2O (being maintained by means of a Masterflex
variable speed modular pump, Cole Palmer Instrument Co) against an
afterload of 100 cm H2O.
At the end of 10 minutes, after the attainment of a steady-state
cardiac function, baseline functional parameters were
recorded and coronary effluent samples were collected for
biochemical assays. The circuit was then switched back to the
retrograde mode and hearts were perfused (n=6 per group) with either
KHB alone (control) or KHB supplemented with losartan (4.5
µmol/L), HOE 140 (10 µmol/L), or both for 15 minutes. At the
end of this period, hearts were subjected to global ischemia
for 30 minutes followed by 2 hours of reperfusion. The first 10 minutes
of reperfusion was in the retrograde mode to allow for
postischemic stabilization and thereafter in the antegrade
working heart mode to allow for assessment of functional
parameters. A schematic of the protocol is shown in Figure 1
. Myocardial infarct size and
apoptosis were determined in the heart, whereas creatine
kinase, bradykinin, and angiotensin release were estimated
in the coronary effluent as described below.
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Measurement of Ventricular Function
Aortic pressure was measured with a Gould P23XL pressure
transducer (Gould Instrument Systems Inc) connected to a side arm of
the aortic cannula. The signal was amplified with a Gould 6600 series
signal conditioner and monitored on a CORDAT II real-time data
acquisition and analysis system (Triton Technologies). Heart
rate, developed pressure (defined as the difference of the maximum
systolic and diastolic aortic pressures), and the
first derivative of developed pressure were all derived or calculated
from the continuously obtained pressure signal. Aortic flow was
measured with a calibrated flowmeter (Gilmont Instruments Inc), and
coronary flow was measured by timed collection of the
coronary effluent dripping from the heart.
Evaluation of Myocardial Infarct Size
Hearts to be used for infarct size calculations were taken on
termination of the experiment and immersed in 1% triphenyl tetrazolium
solution in phosphate buffer
(Na2HPO4 88 mmol/L,
NaH2PO4 1.8 mmol/L)
for 10 minutes at 37°C and then stored at -70°C for later
processing. Frozen hearts (including only ventricular
tissue) were sliced transversely in a plane perpendicular to the
apical-basal axis into 0.5-mm-thick sections, blotted dry, placed in
between microscope slides, and scanned on a Hewlett-Packard Scanjet 5p
single-pass flat bed scanner. With the use of NIH 1.61 image processing
software, each digitized image was subjected to equivalent degrees of
background subtraction, brightness, and contrast enhancement for
improved clarity and distinctness. Risk (equivalent to total left
ventricular muscle mass) as well as infarct zones of each
slice were traced, and the respective areas were calculated in terms of
pixels. The weight of each slice was then recorded to facilitate
the expression of total and infarct masses of each slice in grams. The
risk and infarct volumes (in cm3) of each slice
were then calculated on the basis of slice weight to remove the
introduction of any errors caused by nonuniformity of heart slice
thickness. The risk volumes and infarct volumes of each slice were
summed to obtain the risk and infarct volumes for the whole heart.
Infarct size was taken to be the percent infarct volume/risk volume for
any one heart.
Evaluation of Apoptosis
Immunohistochemical detection of apoptotic cells was
carried out with the use of TUNEL, in which residues of
digoxigenin-labeled dUTP are catalytically incorporated into the DNA by
terminal deoxynucleotidyl transferase II, an enzyme
that catalyzes a template-independent addition of
nucleotide triphosphate to the 3'-OH ends of double- or
single-stranded DNA.13 The incorporated
nucleotide was incubated with a sheep polyclonal
antidigoxigenin antibody followed by a FITC-conjugated rabbit
anti-sheep IgG as a secondary antibody as described by the manufacturer
(Apop Tag Plus, Oncor Inc). The sections (n=3) were washed in PBS 3
times, blocked with normal rabbit serum, and incubated with mouse
monoclonal antibody recognizing cardiac myosin heavy chain (Biogenesis
Ltd) followed by staining with TRIRC-conjugated rabbit anti-mouse IgG
(200:1 dilution, Dako Japan). The fluorescence staining was
viewed with a confocal laser microscope (Olympus Co). The number of
apoptotic cells was counted and expressed as a percentage of
total myocyte population.
Measurements of Bradykinin and Angiotensin in
Coronary Effluent
Bradykinin and angiotensin II were assayed by
radioimmunoassay (RIA) with standard RIA kits obtained from Peninsula
Laboratories Inc.
Statistical Analysis
A 2-way analysis of ANOVA followed by Scheffé’s
test was first carried out with the Primer Computer Program
(McGraw-Hill, 1988) to test for differences between groups. If
differences were established, the values were compared by means of a
Student’s t test for paired data. The values were expressed
as mean±SEM. The results were considered significant at a value of
P<0.05.
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Results |
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Effects of Losartan and HOE 140 on Postischemic Ventricular Function
There were no differences in baseline function between the control and treatment groups. As expected, on reperfusion, the absolute values of aortic flow, developed pressure, and dP/dtmax were decreased in all groups as compared with the baseline values, whereas coronary flow did not show a significant change (Figures 2
and 3). Losartan-treated rat hearts
displayed significant recovery of postischemic myocardial
function. This was evidenced by significantly higher pressure and
aortic flow readings throughout the reperfusion period. Significant
differences were observed at all time points. In contrast, HOE
140–treated hearts displayed significantly lower
postischemic recovery in aortic flow, developed pressure,
and dP/dtmax compared with those for the control
group. HOE 140 when combined with losartan significantly
attenuated the losartan-mediated improved
ventricular function. Heart rates varied from 282 to 302
bpm for all groups and did not vary between groups (results not
shown).
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P<0.05
compared with losartan. Black bars indicate control; striped
bars, losartan; dotted bars, HOE 140; and hatched bars,
losartan+HOE 140. R30, R60, and R120 indicate reperfusion at
30, 60, and 120 minutes of reperfusion, respectively.
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Effects of Losartan and HOE 140 on Myocardial
Infarction
Thirty minutes of ischemia followed by 2 hours of
reperfusion (control) caused a large myocardial infarct (>30% area of
risk) (Figure 4). Normalized infarct size
in percent (infarct size/area of risk) in the control heart was
34.2±2.0 versus 20.0±1.8 for losartan, 41.3±3.2 for HOE 140,
and 25.4±1.9 for losartan+HOE 140–treated hearts. Thus,
losartan reduced whereas HOE 140 increased infarct size
significantly compared with control. The change in infarct size by
losartan was significantly increased by HOE 140.
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Effects of Losartan and HOE 140 on Cardiomyocyte
Apoptosis
We performed double-antibody staining by using antibody in an Apop
Tag kit and the monoclonal antibody recognizing cardiac myosin heavy
chain to specifically identify cardiomyocyte
apoptosis. A significant number of apoptotic myocytes
as well as nonmyocyte cells were visible in the normal hearts
subjected to 30 minutes of ischemia and 2 hours of reperfusion
(Figure 5A). The number of
apoptotic cells expressed as a percentage of total
cardiomyocyte population (Figure 5B) was higher in
the hearts that were pretreated with HOE 140 compared with control
hearts. In contrast, only a few apoptotic cells and almost no
apoptotic cardiomyocytes were visible in the
losartan-treated hearts. This antiapoptotic property of
losartan was completely abolished by HOE 140 because the number
of apoptotic cells increased significantly (as in the HOE
140–treated group) when hearts were simultaneously treated
with losartan and HOE 140.
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Effects of Losartan and HOE 140 on Release of Bradykinin
and Angiotensin
As expected, an increased amount of bradykinin and
angiotensin II was found in the coronary effluent
from the postischemic control myocardium
(Figure 6). HOE 140 almost completely
blocked the release of bradykinin but dramatically increased the
angiotensin II content of the effluent. Losartan
blocked angiotensin II and dramatically increased
bradykinin generation. Combined treatment of the hearts with HOE 140
and losartan reduced both bradykinin and
angiotensin II content in the perfusate.
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indicates
control; 
, losartan; 
, HOE 140; and 
,
losartan+HOE 140. BL indicates baseline, and 30R, 60R, and 120R
indicate 30, 60, 120 minutes of reperfusion, respectively. |
Discussion |
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The results of our study indicated significant postischemic ventricular recovery with losartan, an AT1 receptor blocker, as demonstrated by improved developed pressure and aortic flow and reduced myocardial infarct size. In contrast, HOE 140 aggravated ischemia-reperfusion injury by reducing the recovery of postischemic contractile function and increasing myocardial infarct size. Additionally, cardiomyocyte apoptosis was reduced with losartan whereas HOE 140 accelerated apoptotic cell death. Ischemia/reperfusion induced the generation of angiotensin II and bradykinin. HOE 140 blocked bradykinin release and augmented the formation of angiotensin II whereas losartan blocked the formation of angiotensin II and augmented bradykinin formation. The results document that losartan reduced whereas HOE 140 increased myocardial ischemia/reperfusion injury by blocking AT1 and bradykinin B2 receptors, respectively, suggesting a role of the bradykinin B2 receptor in PC. Losartan provided cardioprotection in 2 ways: (1) by reducing infarct size and improving ventricular function and (2) by inhibiting cardiomyocyte apoptosis. The antiapoptotic function of losartan was completely blocked and the infarct size–lowering ability was partially blocked by HOE 140, suggesting a bradykinin-dependent and bradykinin-independent function of losartan. This is schematically illustrated in Figure 7
, in which blockage of the
AT1 receptor by losartan prevented
angiotensin II formation, whereas blockage of the
B2 receptor with HOE 140 both limited bradykinin
release and increased angiotensin II formation.
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Both bradykinin and angiotensin have been implicated in PC.6 11 In the pig heart, the bradykinin level was found to increase within 3 minutes of PC and was blocked by HOE 140.14 Perfusion of guinea pig hearts with bradykinin for 10 minutes protected the hearts against free radical injury.15 The cardioprotection induced by bradykinin in both the pig and guinea pig was reversed by HOE 140, thus suggesting the role of the bradykinin B2 receptor in such preservation.14 16 In a study with open-chest dogs, the antiarrhythmic effects of PC were abolished by blockade of the bradykinin B2 receptor.17 Similar to bradykinin, ACE inhibitors were found to potentiate a preconditioning effect on the myocardium. For example, enalaprilat, an ACE inhibitor, and an angiotensin II receptor antagonist, EXP 3174, reduced infarct size and augmented the PC effect in the pig heart.18 Another ACE inhibitor, captopril, also potentiated the myocardial infarct size-limiting effect of PC.19
In the present study, losartan was used to block the AT1 receptor. There are at least 2 distinct subtypes of angiotensin II receptors, designated as AT1 and AT2 receptors.20 The best characterized receptor antagonists for AT1 and AT2 are losartan and PD123319 (1-{[4-(dimethylamino)-3-methylphenyl]methyl}-5-(diphenylacetyl)-4,5,6,7tetrahydro-1H-imidazo [4,5-c] pyridine-6-carboxylic acid), respectively.20 Although both the AT1 and AT2 receptors are known to modulate cardiac function, the AT1 receptors particularly affect the contractile and mitogenic action of angiotensin II.21 The activation of the AT1 receptor enhances phospholipase C, resulting in the formation of inositol triphosphate (IP3) and leading to intracellular Ca2+ overloading.22 Thus, blockade of the AT1 receptor makes losartan an excellent antihypertensive drug for treatment of hypertension. In addition, losartan has found its use in the treatment of stroke, malignant nephrosclerosis, and myocardial infarction.
Although ACE antagonism or AT1 receptor blockade has been found to mimic preconditioning, the mechanism(s) of action remains unclear. It has been reported that ACE inhibitors function in part by preserving bradykinin, and captopril, in particular, was found to potentiate the infarct size-limiting effect of PC through the bradykinin B2 receptor in an isolated rabbit heart model.23 Another recent study showed similar results as reported here, namely the bradykinin-dependent cardioprotective effects of losartan against ischemia and reperfusion in rat hearts.24 In the present study, both the postischemic ventricular recovery and infarct size–lowering abilities of losartan were partially inhibited by HOE. Interestingly enough, the losartan-mediated reduction of cardiomyocyte apoptosis was completely abolished by HOE 140.
Our own laboratory has supported the notion that cardiomyocyte apoptosis and necrosis are independent contributors to myocardial infarction, and ischemia and reperfusion lead both to apoptotic cell death and cell necrosis whereas PC results in the decrease in both apoptosis and necrosis.25 The results of this study also demonstrated increased apoptosis in the ischemic reperfused myocardium, with losartan significantly decreasing apoptotic cell death. This was completely reversed by HOE 140, suggesting that the losartan-mediated decrease in cardiomyocyte apoptosis was due to bradykinin B2 receptor activation. These results were further supported by the observation that losartan significantly increased bradykinin formation that was blocked by HOE 140.
In conclusion, this study showed that losartan mimicked the preconditioning effects by its ability to reduce infarct size and to improve postischemic ventricular recovery. These cardioprotective properties of losartan were only partially blocked by HOE 140. In contrast, losartan-mediated decrease in cardiomyocyte apoptosis was completely inhibited by HOE 140, suggesting the apoptotic cell death–lowering ability of losartan was due to the bradykinin B2-receptor activation. This study has documented for the first time that losartan provided cardioprotection by both bradykinin-dependent and bradykinin-independent pathways.
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-34360, HL-22559, HL-33889, and HL-56803 as well as by a Grant-in-Aid from the American Heart Association.
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