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Am J Physiol Heart Circ Physiol 282: H1206-H1215, 2002; doi:10.1152/ajpheart.00839.2000
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Vol. 282, Issue 4, H1206-H1215, April 2002

AT1 and AT2 receptor expression and blockade after acute ischemia-reperfusion in isolated working rat hearts

Yi Xu1, Dinender Kumar1,2, Jason R. B. Dyck2, William R. Ford1, Alexander S. Clanachan2, Gary D. Lopaschuk2, and Bodh I. Jugdutt1,2

1 Division of Cardiology, Department of Medicine and 2 Cardiovascular Research Group, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2R7


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We assessed ANG II type 1 (AT1) and type 2 (AT2) receptor (R) expression and functional recovery after ischemia-reperfusion with or without AT1R/AT2R blockade in isolated working rat hearts. Groups of six hearts were subjected to global ischemia (30 min) followed by reperfusion (30 min) and exposed to no drug and no ischemia-reperfusion (control), ischemia-reperfusion and no drug, and ischemia-reperfusion with losartan (an AT1R antagonist; 1 µmol/l), PD-123319 (an AT2R antagonist; 0.3 µmol/l), N6-cyclohexyladenosine (CHA, a cardioprotective adenosine A1 receptor agonist; 0.5 µmol/l as positive control), enalaprilat (an ANG-converting enzyme inhibitor; 1 µmol/l), PD-123319 + losartan, ANG II (1 nmol/l), or ANG II + losartan. Compared with controls, ischemia-reperfusion decreased AT2R protein (Western immunoblots) and mRNA (Northern immunoblots, RT-PCR) and impaired functional recovery. PD-123319 increased AT2R protein and mRNA and improved functional recovery. Losartan increased AT1R mRNA (but not AT1R/AT2R protein) and impaired recovery. Other groups (except CHA) did not improve recovery. The results suggest that, in isolated working hearts, AT2R plays a significant role in ischemia-reperfusion and AT2R blockade induces increased AT2R protein and cardioprotection.

angiotensin II; angiotensin II type 1 receptor; angiotensin II type 2 receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE RENIN-ANGIOTENSIN SYSTEM (RAS) is upregulated during myocardial ischemia, infarction, and ischemia-reperfusion injury (5, 14). ANG II, the major effector molecule of the RAS, binds to two main receptor subtypes (6, 29), the ANG II type 1 (AT1) and the type 2 (AT2) receptor (R) to exert its physiological effects, several of which enhance ischemia-reperfusion injury. One strategy for cardioprotection is therefore to decrease ANG II formation and receptor stimulation with ANG-converting enzyme (ACE) inhibitors, although their efficacy in ischemia-reperfusion is debated (27). A second strategy is to directly reduce ANG II receptor stimulation by using selective AT1R or AT2R antagonists. In the nonworking rat heart, pretreatment (1 wk before ischemia) with the AT1R antagonist TCV-116 reduced reperfusion injury and improved function (43). In the same model, acute treatment (from the onset of ischemia) with the AT1R antagonist losartan attenuated the postischemic mechanical dysfunction (40) and pretreatment (4-6 h before ischemia) with losartan blocked the increase in AT1R binding but did not affect AT1R protein or mRNA after ischemia-reperfusion (41). In the isolated working rat heart (19), we previously showed that the AT2R antagonist PD-123319 enhanced (7), whereas losartan worsened (7, 8), functional recovery after ischemia-reperfusion. We hypothesized that during ischemia-reperfusion in working hearts, AT2R is downregulated and AT2R blockade induces AT2R upregulation and cardioprotection.

The purpose of this study was to determine whether changes in AT1R/AT2R expression might be related to the recovery of mechanical function after ischemia-reperfusion with or without AT1R/AT2R blockade (with losartan and PD-123319, respectively) in isolated working rat hearts. To gain insight into functional mechanisms, we also determined changes in AT1R/AT2R expression and mechanical function with 1) the adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA), to serve as a positive control that is known to improve functional recovery independent of the RAS or ANG II (7), 2) the ACE inhibitor enalaprilat, to decrease endogenous ANG II formation, 3) the combination of losartan and PD-123319, to block both AT1Rs and AT2Rs and unmask AT1R-AT2R interaction, 4) ANG II as agonist, to enhance AT1R and AT2R stimulation, and 5) the combination of ANG II and losartan, to enhance agonist effects on AT2R.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental animals and isolated working rat heart preparation. All rats were housed and treated according to the guidelines of the Canadian Council on Animal Care and the American Physiological Society. As described previously (7, 8), male Sprague-Dawley rats (250-300 g), which had been acclimatized and fed ad libitum, were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg). Hearts were rapidly excised and placed in ice-cooled Krebs-Henseleit solution (pH 7.4, gassed with 95% O2-5% CO2). Initial Langendorff perfusion of the aorta and coronary arteries was performed with Krebs-Henseleit solution. The pulmonary artery and left atrium were cannulated. After 10 min of Langendorff perfusion, the hearts were switched to working mode by clamping the aortic line and opening the left atrial line. The working hearts were perfused in a closed recirculating system at 37°C in contact with a 95% O2-5% CO2 mixture. Atrial pacing (300 beats/min) was applied during aerobic perfusion. The perfusate (100 ml) was a modified Krebs-Henseleit solution containing 2.5 mmol/l CaCl2, 11 mmol/l glucose, 1.2 mmol/l palmitate prebound to 3% BSA (fraction V), and 100 mU/l insulin. Perfusions were made at constant left atrial preload (11.5 mmHg) and afterload hydrostatic pressure (80 mmHg). Heart rate and aortic systolic and diastolic pressures were recorded (P23 Db; Gould). Cardiac output and aortic flow were measured (Transonic T206). Coronary flow, left ventricular (LV) minute work (J), myocardial oxygen consumption (MVO2, µmol · min-1 · g dry wt-1), myocardial efficiency (%J), and coronary vascular conductance (CVC, ml · min-1 · mmHg-1) were calculated.

Experimental protocol. Hearts were randomly assigned to nine groups of six hearts each: control (no drug, no ischemia-reperfusion), ischemia-reperfusion (no drug), and ischemia-reperfusion with PD-123319 (0.3 µmol/l), CHA (0.5 µmol/l as positive control), losartan (1 µmol/l), enalaprilat (1 µmol/l), PD-123319 (0.3 µmol/l) + losartan (1 µmol/l), ANG II (1 nmol/l), or ANG II (1 nmol/l) + losartan (1 µmol/l). Control hearts were perfused aerobically for 80 min. Hearts in ischemia-reperfusion groups were perfused aerobically in the working mode for 50 min and then subjected to 30 min of global, no-flow ischemia (in the presence or absence of drug) and 30 min of reperfusion. After ischemia, the left atrial inflow was reestablished and pacing (stopped during ischemia) was recommenced after 3 min of reperfusion. The drugs were added to the perfusate 5 min before the onset of ischemia and remained throughout the reperfusion period. To determine whether the drugs modified receptor expression in the absence of ischemia-reperfusion, we studied additional controls with each drug or combination (7 groups) and 80-min aerobic perfusion. At the end of the experiments, LV tissue samples were stored at -70°C and powdered in liquid nitrogen for analysis of AT1R/AT2R protein and mRNA expression (24, 34).

Western blot analysis for AT1R and AT2R proteins. Aliquots of powdered LV tissue (10 mg) were sonicated in homogenization solution (2% SDS, 100 mmol/l dithiothreitol, 60 mmol/l Tris, pH 6.8) at 4°C and boiled at 100°C. The boiled homogenate was subjected to PAGE followed by electrotransfer to nitrocellulose. The nitrocellulose membranes were then blocked with 5% (wt/vol) skimmed milk powder in 1× PBS and 0.05% (vol/vol) of Tween 20 at room temperature. For AT1R protein, the nitrocellulose membranes were incubated with affinity-purified rabbit anti-human AT1R antibody (Santa Cruz Biotechnology) at a dilution of 1:2,000 for 2 h at room temperature. The membranes were washed with PBS-Tween 20 (0.05%; TPBS) three times, followed by incubation with goat anti-rabbit IgG antibody conjugated to peroxidase, and visualized with chemiluminescence detection (ECL Western blot kit, Amersham). Gels with 15 lanes were used. The intensity of bands was quantified by scanning densitometry with standard image analysis software, and images were aligned with the intensity bars for illustrations with the Sigma Gel computer graphics package (SPSS). For AT2R proteins, the same procedure as that for AT1R was used except that incubation with goat anti-human AT2R antibody (Santa Cruz Biotechnology) at a dilution of 1:500 was followed by incubation with donkey anti-goat IgG (BioCan Scientific, Mississauga, ON, Canada).

cDNA probe preparation. cDNAs for AT1aR and AT2R were obtained from the laboratory of Dr. V. Dzau (Harvard Medical School, Boston, MA) and used to prepare probes for analysis of rat AT1R and AT2R mRNA. Human AT2R and mouse AT1aR cDNA were subcloned into the plasmid pcDNA3 (13). The plasmid DNA was then digested using two restriction enzymes that flanked the cDNA for the receptors (AT2R and AT1aR). In both cases, the 1.1-kb cDNA fragments for both receptors were gel purified and 25 ng of each was labeled with [alpha -32P]dCTP (DuPont, Mississauga, ON, Canada) with a random primer method. The labeled probes were separated from unincorporated nucleotides by using Sephadex G-50 spin columns and were used in Northern blot analysis.

Northern blot analysis for AT1R and AT2R mRNA. Total RNA was extracted from rat LV myocardium with the acid guanidinium-thiocyanate-phenol-chloroform extraction method with TRIzol reagent (Life Technologies, Burlington, ON, Canada). Aliquots (20 µg) of total RNA were electrophoretically size-fractionated in a 1% MOPS-3% formaldehyde buffer on a 1% agarose-3% formaldehyde gel. These RNA samples were then transferred to Nytran membrane (Schleicher and Schuell, Keene, NH) and cross-linked under ultraviolet light for 30 s. The membranes were then prehybridized in the solution mixture containing 50% formamide, 5× SSC (sodium chloride-sodium citrate), 5× Denhardt's solution, 0.1% SDS, 0.05 mol/l sodium phosphate buffer (pH 6.8), 0.1% sodium pyrophosphate, and 50 µg/ml sheared herring sperm DNA at 42°C for 3 h. The membranes were then hybridized with a 32P-labeled probe specific for AT1aR and AT2R in the same buffer for 18-24 h at 42°C. Membranes were washed with successively stringent buffers at room temperature in 2× SSC containing 0.1% SDS for 30 min twice, at 1× SSC containing 0.1% SDS for 30 min, and at 55°C in 0.2× SSC containing 0.1% SDS for 45 min. The membranes were exposed to Kodak X-OMAT film (Eastman Kodak, Rochester, NY) with an intensifying screen for 1-2 wk at -80°C. The autoradiograms were quantified by scanning densitometry with image analysis software (Sigma Gel, SPSS). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize the differences in loaded and transferred mRNA.

RT-PCR assay. Northern blot data were verified with semiquantitative RT-PCR. Total RNA from flash-frozen ventricular tissue was isolated with TRIzol reagent as described by the supplier (Life Technologies). The RNA was ethanol precipitated and reconstituted in distilled, deionized water, and the concentration was determined by measurement of the absorbance at 260 nm. All RNA samples were stored frozen at -80°C. Poly A+ RNA was isolated by Oligotex mRNA Kit (Qiagen; Mississauga, ON, Canada). Purity and RNA integrity were assessed by absorbance at 260/280 nm and by agarose gel electrophoresis.

RT-PCR was done with a two-step protocol for RT-PCR method following the manufacturer's instructions. Conditions for RT-PCR included 1 µg of Poly A+ RNA and downstream priming with primers AT1a, AT2, and GAPDH in the presence of Ready-To-Go RT-PCR Beads (Amersham Pharmacia Biotech, Piscataway, NJ) at 42°C for 30 min. After denaturation at 95°C for 5 min, PCR was conducted: 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. The cycle numbers were 36 for AT1a and AT2 and 32 for GAPDH. For all PCR reactions, the number of cycles was found to be in the linear range of product accumulation (not shown). A final PCR extension was performed at 72°C for 5 min. PCR amplifications (10, 11) were done for AT1a, AT2, and GAPDH with the following primers: 1) AT2 receptor, 5'-TTGCTGCCACCAGCAGAAAC-3' (the upstream primer is complementary to nucleotides 3070-3089) and 5'-GTGTGGGCCTCCAAACCATTGCTA-3' (the downstream primer is complementary to nucleotides 4172-4195); the cDNA amplification product is 1125 bp (10); 2) AT1a receptor, 5'-GCACACTGGCAATGTAATGC-3' (the upstream primer is complementary to nucleotides 1370-1389) and 5'-GTTGAACAGAACAAGTGACC-3' (the downstream primer is complementary to nucleotides 1737-1756); the cDNA amplification product is 385 bp (11); and 3) GAPDH, 5'-AATGCATCCTGCACCACCAACTGC-3' (the upstream primer is complementary to nucleotides 524-547) and 5'-GGAGGCCATGTAGGCCATGAG-GTC-3' (the downstream primer is complementary to nucleotides 1055-1078); the cDNA amplification product is 555 bp. PCR products were analyzed by 1.5% agarose gel electrophoresis with a 1-kbp ladder as size marker. All PCR reactions were done at least three times with different RNA preparations (n = 3) and gave identical results. Photographic images were obtained, digitized, and quantified with Sigma Gel (SPSS).

Protein kinase C and p38 mitogen-activated protein kinase. To determine whether protein kinase C (PKC)-epsilon and p38 mitogen-activated protein kinase (MAPK) are activated during AT2R antagonism, we measured these proteins by Western blotting as described by Ping et al. (26) and Zhao et al. (45), respectively. Briefly, the same procedure as for AT1/ AT2R proteins was used, except that incubation with rabbit p38 anti-rat polyclonal or phosphorylated p38 mouse monoclonal antibodies (Santa Cruz Biotechnology) were used for p38 and mouse monoclonal anti-rat nPKC-epsilon antibody (1:100 dilution) followed by anti-mouse IgG HRP conjugate (1:2,000 dilution).

cGMP content. To determine whether cGMP [an index of nitric oxide (NO)] is increased during AT2R antagonism, we measured LV myocardial cGMP content with the commercially available cGMP enzyme immunoassay kit (Amersham Pharmacia Biotechnology, Quebec City, PQ, Canada) and expressed the levels as femtomoles per milligram of wet weight, as described previously (37).

Statistics. Data are shown as means ± SE. Data were analyzed with ANOVA (with repeated measures followed by a Student t-test with the Bonferroni correction for repeated comparisons) and linear regression analysis. Statistical significance was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Recovery of function after global ischemia. For all groups, baseline values of LV work, coronary flow, and cardiac output were essentially similar (Figs. 1 and 2; Table 1). In the ischemia-reperfusion (no drug) group, LV work remained significantly depressed during reperfusion, functional recovery being only 51 ± 10% of the preischemic value (Fig. 1A). Also, coronary flow and cardiac output were both significantly depressed (Table 1). In the PD-123319 and CHA groups, recovery of LV work with reperfusion improved to 82 ± 4 and 81 ± 4%, respectively (P < 0.001 vs. ischemia-reperfusion, no drug; Fig. 1A). Coronary flow and cardiac output were also enhanced in those groups (Table 1). In contrast, the losartan group did not show any recovery of LV work (P < 0.0001 vs. ischemia-reperfusion, no drug), and coronary flow and cardiac output were further impaired compared with the PD-123319 and CHA groups (Table 1). The recovery of LV work with reperfusion in the ANG II or ANG II + losartan (Fig. 1B), enalaprilat (Fig. 2A), or PD-123319 + losartan (Fig. 2B) groups did not differ from that in the ischemia-reperfusion (no drug) group and exceeded that in the losartan group. The effects on coronary flow and cardiac output among the groups were concordant with the effects on LV work (Table 1). Compared with the ischemia-reperfusion group, losartan decreased peak systolic pressure, cardiac output, MVO2, LV work, coronary flow, and myocardial efficiency. In contrast, PD-123319 and CHA improved peak systolic pressure, cardiac output, and LV work. Despite changes in flow, CVC did not change (Table 1).


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  		Fig. 1.   Left ventricular (LV) work during baseline, ischemia and reperfusion. ANG II, 1 nmol/l ANG II; ANG II+Los, 1 nmol/l ANG II + 1 µmol/l losartan; CHA, 0.5 µmol/l N6-cyclohexyladenosine; IR, ischemia-reperfusion alone (no drug); Los, 1 µmol/l losartan; PD, 0.3 µmol/l PD-123319. A: compares IR plus PD, CHA or Los to IR alone. B: compares IR plus ANG II and ANG II + Los to IR alone. Los is shown for reference. * P < 0.05 vs. ischemia-reperfusion (n = 6 per group).



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  		Fig. 2.   LV work during baseline, ischemia, and reperfusion. EN, 1 µmol/l enalaprilat; IR, ischemia-reperfusion alone (no drug); Los, 1 µmol/l losartan; PD, 0.3 µmol/l PD-123319; PD+Los, 0.3 µmol/l PD-123319,+ 1 µmol/l losartan. A: compares IR plus EN and Los to IR alone. B: compares IR plus PD and PD + Los to IR alone; Los is shown for reference. * P < 0.05 vs. ischemia-reperfusion (n = 6 per group).


                              
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  		Table 1.   Hemodynamic parameters during baseline and reperfusion periods of ischemia-reperfusion protocol

AT1R and AT2R proteins. The Western blot gels for rat hearts displayed a major band for AT1R protein in the 41-kDa molecular mass region [in agreement with Yang et al. (41)] and two bands for AT2R protein in the 44-kDa region [in agreement with Wang et al. (35)]. There were no qualitative or quantitative differences in AT1R protein expression among the nine groups of six hearts (Fig. 3A). In contrast, AT2R protein expression for hearts in the nine groups showed quantitative differences among the groups (Fig. 3B), with significant increase in AT2R protein with ischemia-reperfusion + PD-123319 or ischemia-reperfusion + PD-123319 + losartan compared with control (P < 0.007-0.001) and ischemia-reperfusion (no drug) (P < 0.007-0.001). Also, AT2R protein expression was less (P < 0.02) than control with ischemia-reperfusion (no drug) or ischemia-reperfusion combined with losartan, ANG II, or ANG II + losartan. Compared with ischemia-reperfusion (no drug), enalaprilat increased AT2R protein (P = 0.001). Within the ischemia-reperfusion (no drug) and ischemia-reperfusion + PD-123319 groups, significant positive correlations (linear regression analysis) were found between recovery of LV work (%) and AT2R protein levels (r = 0.94, P = 0.002 and r = 0.99, P = 0.0002, respectively) and negative correlations between recovery of LV work and the narrow range of AT1R protein levels (r = -0.95, P = 0.003 and r = -0.96, P = 0.003, respectively).


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  		Fig. 3.   Changes in ANG II type 1 (AT1R) and type 2 (AT2R) receptor protein expression after ischemia-reperfusion (IR) for the 9 groups. A typical example is shown. A: AT1R protein. B: AT2R protein. Abbreviations as in Figs. 1 and 2. Top, Western blot gels. Bottom, quantitative densitometric analyses. Intensities are expressed as % of control (Con; no IR, no drug). * P < 0.05 vs. control; dagger P < 0.05 vs. IR (n = 6 per group).

AT1R and AT2R mRNAs. RT-PCR (3 blots scanned) confirmed results obtained by Northern blots (6 blots scanned; not shown). Compared with control and ischemia-reperfusion alone, there was a marked increase in AT1R mRNA intensity with ischemia-reperfusion + losartan (P < 0.001) and a lesser increase with ischemia-reperfusion + PD-123319 + losartan (P < 0.001) on RT-PCR (Fig. 4A). Both PD-123319 and ANG II prevented the losartan-induced increase in AT1R mRNA. Compared with control, AT1R mRNA decreased with ischemia-reperfusion alone (P = 0.03) and ischemia-reperfusion combined with ANG II (P = 0.03) or ANG II + losartan (P = 0.03). Compared with control or ischemia-reperfusion alone, AT2R mRNA intensity on RT-PCR (Fig. 4B) increased markedly with ischemia-reperfusion + PD-123319 (P < 0.001) and to a lesser extent with ischemia-reperfusion + PD-123319 + losartan (P < 0.04-0.001). Importantly, losartan prevented the PD-123319-induced increase in AT2R mRNA. Compared with control, AT2R mRNA decreased with ischemia-reperfusion alone (P = 0.01) and ischemia-reperfusion combined with losartan (P = 0.04), ANG II (P = 0.02), or ANG II + losartan (P = 0.03). Compared with ischemia-reperfusion alone, enalaprilat increased AT2R mRNA (P = 0.004).


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  		Fig. 4.   Changes in AT1R and AT2R mRNA expression after IR for the 9 groups by RT-PCR. A typical example is shown. A: AT1R mRNA. B: AT2R mRNA. Abbreviations as in Figs. 1, 2, and 3. Top, immunoblots. Bottom, intensity profiles. bp, number of base pairs. AT1R mRNA is normalized to corresponding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels and shown as % of control. * P < 0.05 vs. control; dagger P < 0.05 vs. IR (n = 3 per group for RT-PCR).

Effect of treatments during aerobic perfusion. The aerobic "controls" did not show significant differences in any of the parameters, including LV work (Fig. 5), AT1R/AT2R protein (Fig. 6), or AT1R/AT2R mRNA (Fig. 7).


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  		Fig. 5.   LV work during aerobic perfusion. ANG II, 1 nmol/l ANG II; ANG II+Los, 1 nmol/l ANG II + 1 µmol/l losartan; CHA, 0.5 µmol/l CHA; IR, ischemia-reperfusion; Los, 1 µmol/l losartan; PD, 0.3 µmol/l PD-123319; PD+Los, 0.3 µmol/l PD-123319 + 1 µmol/l losartan (n = 6 per group). A: compares PD, CHA, and LOS to control. B: compares EN, ANG II, PD + LOS, and ANG II + LOS.



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  		Fig. 6.   AT1R (A) and AT2R (B) protein expression during aerobic perfusion. A typical example is shown. Abbreviations as in Figs. 1-3 P > 0.05 vs. control (n = 6 per group).



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  		Fig. 7.   AT1R (A) and AT2R (B) mRNA expression during aerobic perfusion. A typical example is shown. Abbreviations as in Figs. 1-3 P > 0.05 vs. control (n = 3 per group for RT-PCR).

Effect on cGMP. Compared with control, LV cGMP content showed a significant decrease with ischemia-reperfusion alone (2.65 ± 0.10 vs. 1.56 ± 0.18 fmol/mg wet wt; P < 0.002) and a significant increase with ischemia-reperfusion + PD-123319 (2.65 ± 0.10 vs. 3.26 ± 0.20 fmol/mg wet wt; P < 0.05) (Fig. 8A).


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  		Fig. 8.   LV cGMP content (A), p-p38 MAPK (B), membrane fraction PKC-epsilon (C), and total PKC-epsilon (D). Abbreviations as in Fig. 2. * P < 0.05 vs. control.

Effect on PKC-epsilon and p38 MAPK. Compared with control, phosphorylated p38 (p-p38; Fig. 8B) and the ratio of p-p38 to p38 (not shown) were significantly increased (P < 0.05) with ischemia-reperfusion alone or combined with PD-123319. Compared with control or ischemia-reperfusion alone (Fig. 8, C and D), significant increases (1.3-fold; P < 0.05) were found in both membrane PKC-epsilon and total cytosolic PKC-epsilon .


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There are two major new findings in this study. First, acute ischemia-reperfusion in isolated working rat hearts induced decreased AT2R protein expression and impaired recovery of mechanical function. Second, acute AT2R blockade produced selective increase in AT2R protein and improved recovery of function. The findings suggest that AT2R plays a significant role in ischemia-reperfusion and that AT2R blockade induces increased AT2R protein and cardioprotection. Although we are aware that expression does not lead to activation unless endogenous agonist is present, we have inferred that increase in AT2R protein expression may lead to increase in AT2R activation. We have provided indirect evidence suggesting that AT2R activation follows AT2R expression. Thus, after ischemia-reperfusion, AT2R was downregulated and correlated with decreased cardiac function, and both these effects on AT2R expression and cardiac function were reversed by the AT2R blocker PD-123319. In addition, our preliminary data suggest that p38, PKC-epsilon , and cGMP were affected by AT2R blockade, providing further evidence that AT2R activation was taking place.

Mechanisms. The molecular and functional changes in response to ischemia-reperfusion and the selected drug treatments in this study provide important insights into AT1R/AT2R functions during cardioprotection. First, the AT2R antagonist may act on myocardium, via AT2R found on cardiomyocytes (35), to enhance recovery of LV function. Our finding that PD-123319 induced selective enhancement of both the AT2R message and its protein in LV myocardium after ischemia-reperfusion, with no effect on AT1R mRNA or protein, provides clear evidence for the effect of the selective blocker on AT2Rs and is indicative of the specificity of action of PD-123319 in eliciting the protective effect. PD-123319 is a known selective AT2R antagonist (with no partial agonist activity) that is widely used to characterize AT2R-mediated mechanisms (5). The responses in this study were not mediated by an indirect effect on the coronary circulation because there was no significant change in CVC with PD-123319 or losartan. In agreement with Yoshiyama et al. (42), exogenous ANG II did not affect flow, CVC, or recovery of function.

Second, AT2R blockade might have improved the balance between AT1R and AT2R stimulation. Although AT1R blockade upregulates the RAS and exposes AT2Rs to increased ANG II levels in vivo (14), the isolated heart in vitro is not exposed to elevated circulating ANG II. However, local ANG II is increased after ischemia-reperfusion (44). In this study, ANG II alone or combined with an AT1R blocker did not improve recovery of function after ischemia-reperfusion, although exogenous ANG II decreased AT1R mRNA and overcame the myocardial depressant effect of the blocker. Exogenous ANG II also decreased AT2R mRNA and protein after ischemia-reperfusion and induced further decrease when combined with the AT1R blocker. Although postischemic recovery of LV function was similar with the combination of AT1R and AT2R blockade compared with ANG II, ANG II + AT1R blockade, ACE inhibition, or ischemia-reperfusion alone, the combination 1) overcame the myocardial depressant effect seen with the AT1R blocker alone and 2) increased AT2R protein (and AT1R/AT2R mRNA) compared with both control and ischemia-reperfusion groups. In support of these findings, AT2R stimulation was recently shown to inhibit responses to AT1R activation (20).

Third, the effects of the AT1R/AT2R blockers during ischemia-reperfusion might involve AT1R and AT2R interaction or cross talk at the mRNA level. Although we did not confirm cross talk in cell culture, AT1R blockade during ischemia-reperfusion caused selective increase in AT1R mRNA and decrease in AT2R mRNA. In combination, the AT1R blocker inhibited the increase in AT2R mRNA and recovery of LV function induced by AT2R blockade, and the AT2R blocker inhibited the increase in AT1R mRNA and deterioration of function induced by AT1R blockade.

Fourth, our finding that the adenosine A1 agonist CHA enhances recovery of function without altering AT1R or AT2R mRNA corroborates the view that its beneficial effect does not involve the RAS (7). This finding, together with the association of AT2R upregulation and enhanced function with PD-123319 and increased AT1R mRNA and worsened function with losartan, supports the idea that increased mRNA per se might not be causally related to enhanced functional recovery.

Fifth, our finding of decreased AT1R/AT2R mRNA during ischemia-reperfusion is consistent with increased endogenous ANG II production in working rat hearts. Although no change was seen in AT1R protein during ischemia-reperfusion, AT2R protein decreased by nearly 40% (Fig. 3). This was associated with decrease in mRNA by nearly 50% for AT1R and 60% for AT2R compared with control (Fig. 4). Because these decreases in response to 30 min of ischemia are fairly substantial, they cannot simply be ascribed to cellular damage because 1) they were prevented by the ACE inhibitor enalaprilat (which by itself did not enhance functional recovery) and 2) treatments with the specific AT1R/AT2R blockers had opposing effects on mRNAs and functional recovery. Although enalaprilat (which decreases endogenous ANG II formation) did not enhance functional recovery compared with ischemia-reperfusion alone, recovery with enalaprilat was greater than with AT1R blockade and enalaprilat induced preservation of AT2R mRNA and protein as well as AT1R mRNA during ischemia-reperfusion. Rapid changes in expression of mRNA and/or protein for a variety of proteins involved in cell signaling have recently been reported (1-4, 23, 25, 39), including AT1R (41).

Sixth, the receptor changes after ischemia-reperfusion in our study are consistent with the pharmacological principle that agonists produce receptor downregulation whereas antagonists produce upregulation. The lack of increase in AT1R protein with AT1R blockade and ischemia-reperfusion was probably because the short exposure time was insufficient to increase AT1R protein synthesis (or because protein degradation was enhanced). Similarly, the increase in AT1R mRNA could be due to enhanced synthesis (or increased half-life). Because increased AT2R protein levels followed increased AT2R mRNA expression after similar durations of exposure to AT2R blockade and ischemia-reperfusion, it is possible that the rate of translation might be higher for AT2R than for AT1R. Although AT1Rs are internalized and recycled, this does not seem to be the case with AT2Rs (13).

Potential role of AT2R blockade in cardioprotection. There are very few data on AT2R or its blockade because AT2R was considered to be in low abundance, without significant functional consequences (28). The two distinct subtypes of ANG II receptors were identified on the basis of their inhibition by highly specific and selective nonpeptide ANG II receptor ligands, losartan for AT1R and PD-123319 for AT2R (6, 29). Early studies indicated that AT1Rs are dominant in adult tissues, whereas AT2Rs are abundant in fetal tissues, and most of the physiological actions of ANG II are normally mediated through the AT1R. However, the profile of AT1R and AT2R expression depends on the type of cardiac tissue, the type of preparation, the cell type, and the pathological condition (21). Although AT1Rs and AT2Rs are found in equal proportion in rat myocardium, AT2Rs are dominant in human myocardium, the ratio of AT2R to AT1R being 2:1 (28). AT2Rs, recently reviewed by Horiuchi et al. (14), are reexpressed or upregulated in cardiac hypertrophy, myocardial infarction, and heart failure, whereas AT1Rs are downregulated in heart failure (12). After myocardial infarction in the rat in vivo, AT1R and AT2R mRNA increase and peak at 24 h (46). Thus AT2Rs participate in the pathophysiology of disease in adult hearts. Our findings suggest that AT2Rs play a significant role in acute ischemia-reperfusion in working rat hearts and that the cardioprotective effect of acute AT2R blockade is associated with increased AT2R protein expression.

Other studies. There are no other reports comparing effects of acute AT1R/AT2R blockade, ACE inhibition, and ANG II on AT2R mRNA or protein after ischemia-reperfusion in adult rat hearts in vitro. In contrast, there are many reports involving AT1R blockade. In nonworking rat hearts, LV function improved gradually and modestly after ischemia-reperfusion with both acute losartan (40) and chronic losartan pretreatment (41), and losartan did not affect AT1R mRNA or protein after ischemia-reperfusion but decreased AT1R binding (41). Others found that pretreatment with 1) ANG II augments reperfusion injury independent of a vasocontrictive effect (42, 43), 2) losartan improves recovery of LV function after ischemia-reperfusion (36), and 3) TCV-116 is cardioprotective to ischemia-reperfusion and decreased ANG II (43). In a pig model of in vivo ischemia-reperfusion, 30-min pretreatment with the AT1R blocker candesartan decreased infarct size, whereas PD-123319 produced a slight nonsignificant decrease (17% vs. 22%) compared with placebo (16). The reason for the discrepancy is not clear, but it may be related to the greater hemodynamic load and metabolic demand in isolated working vs. nonworking hearts (19). In addition, the time window during which treatment is applied might be important. Thus pretreatment could modify receptor status and responses to ischemia-reperfusion. Because chronic pretreatment with an AT1R antagonist increases plasma and myocardial ANG II (36), withdrawal before an acute ischemia-reperfusion experiment produces heightened stimulation of AT1Rs and AT2Rs. In fact, AT1R stimulation was shown to be cardioprotective in rabbit hearts (18). Intrinsic AT1R activation may also contribute to functional recovery after AT2R blockade (8), possibly via an AT1R-mediated positive inotropism.

Although our findings with AT2R blockade during acute ischemia-reperfusion in this study and others (7, 8) suggest that acute increase in AT2R expression and potential AT2R activation might be harmful, this does not necessarily conflict with the postulated beneficial role of the mild-to-modest AT2R stimulation during chronic AT1R blockade. This concept (16, 17, 33) assumes that during chronic AT1R blockade, shunting of ANG II to AT2Rs induces AT2R activation and unopposed AT2R effects involving bradykinin, PGs, and NO, but data on AT2R protein or mRNA or downstream signaling are needed. Under chronic conditions, AT2Rs in coronary endothelial cells exert antigrowth and antiproliferative effects that are offset by the growth-promoting effects of AT1R stimulation (28). AT2Rs also mediate apoptosis (38), so that AT2R blockade could inhibit apoptosis. However, in rats with chronic heart failure 2 mo after infarction, a beneficial effect of AT1R blockade on LV remodeling was attenuated by chronic AT2R blockade with PD-123319 (17). Several studies have implicated NO and free radicals in the expression of ANG II receptors (15, 31).

In this study, we found increases in PKC-epsilon and cGMP in the combined ischemia-reperfusion and PD-123319 hearts, suggesting that the protective effect of AT2R antagonism might involve signaling through these molecules. Signaling through PKC-epsilon and cGMP has been implicated in ischemic preconditioning (26). Although the p38 MAPK pathway has been implicated in adenosine-induced ischemic preconditioning, we did not find an increase in p38 in the combined ischemia-reperfusion and CHA group, but p38 increased significantly in the PD-123319 group. We did not find a consistent correlation between the activity of p38, PKC-epsilon , or cGMP and functional recovery or expression of AT1R/AT2Rs in the other ischemia-reperfusion groups.

As reviewed by Jalowy et al. (16), AT1R blockade is not universally beneficial in all models of ischemia-reperfusion in all species (7, 8) or in other models (22, 30, 32), as is often assumed (9, 16, 17, 40, 41). Although the beneficial effects of long-term AT1R blockade after infarction (9) support the role of AT1R during cardiac remodeling after infarction, AT2Rs are likely involved (12, 20, 38). Whether AT2R blockade might be beneficial in vivo during more prolonged acute ischemia-reperfusion or recurrent ischemic episodes after infarction requires further study. Further studies are also needed to define the pathways involved.

In conclusion, in the isolated working rat heart, 1) decreased AT2R protein is associated with impaired recovery of mechanical function after acute ischemia-reperfusion and 2) increased AT2R protein after acute AT2R blockade is associated with enhanced recovery, suggesting a potential link between increased AT2R protein expression and cardioprotection.


    ACKNOWLEDGEMENTS

We are grateful to Drs. Jukka Lehtonen, Masatsugu Horiuchi, and Victor J. Dzau (Harvard Medical School) for the generous supply of cDNAs for AT1R and AT2R. We thank Catherine Graham for assistance with typing.


    FOOTNOTES

10.1152/ajpheart.00839.2000

This study was supported in part by Grant MT-13403 from the Canadian Institutes of Health Research.

Present address of W. R. Ford: Department of Pharmacology, Cambridge University, Cambridge, UK CB2 1QJ.

Address for reprint requests and other correspondence: B. I. Jugdutt, 2C2.43 Walter Mackenzie Health Sciences Ctr., Div. of Cardiology, Dept. of Medicine, Univ. of Alberta, Edmonton, AB, Canada T6G 2R7 (E-mail: bjugdutt{at}ualberta.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 August 2000; accepted in final form 7 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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