Critical Role of AT₁ Receptor Expression After Ischemia/Reperfusion in Isolated Rat Hearts

Beneficial Effect of Antisense Oligodeoxynucleotides Directed at AT₁ Receptor mRNA

From the Departments of Medicine, Pediatrics, and Physiology, University of Florida, and the VA Medical Center, Gainesville, Fla.

Correspondence to J.L. Mehta, MD, PhD, Department of Medicine, University of Florida College of Medicine, 1600 Archer Rd, PO Box 100277 JHMHC, Gainesville, FL 32610. E-mail mehta{at}medmac.ufl.edu

	Abstract

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Abstract—To examine the relevance of angiotensin II type 1 receptor (AT₁R) expression in the determination of myocardial function after ischemia/reperfusion, Sprague-Dawley rats were treated intravenously with antisense oligodeoxynucleotides (AS-ODNs) directed at AT₁R mRNA (100 µg/rat, n=9) or scrambled antisense oligodeoxynucleotides (Scr-ODNs, 100 µg/rat, n=6). Both AS-ODNs and Scr-ODNs were given along with 300 µg/rat of liposome DOTAP/DOPE, a positive electron carrier (wt:wt=1:1). The hearts from AS-ODN– or Scr-ODN–treated rats were excised 24 hours later, perfused in vitro, and subjected to 25 minutes of global ischemia followed by 30 minutes of reperfusion. Parallel groups of rats were given the specific AT₁R antagonist losartan (10 mg/kg IV, n=6) or saline (n=7) 4 to 6 hours before excising the hearts. Ischemia/reperfusion resulted in a significant increase in myocardial AT₁R expression (autoradiography and binding assay) and myocardial dysfunction, indicated by increases in coronary perfusion pressure and left ventricular end-diastolic pressure and a decrease in developed left ventricular pressure (all P<0.01 versus baseline) in the saline-treated group. AT₁R protein and mRNA levels also increased in ischemic/reperfused myocardial tissues. Administration of AS-ODNs or losartan, but not Scr-ODNs, preserved myocardial function and blocked the increased AT₁R binding after ischemia/reperfusion (both P<0.01). Myocardial AT₁R mRNA levels were not affected by either AS-ODNs or losartan, and the AT₁R protein levels were significantly reduced by AS-ODN, but not losartan, treatment. Plasma angiotensin II levels increased after administration of losartan but not after administration of AS-ODNs. These observations imply a critical role of AT₁R upregulation in determining myocardial function immediately after ischemia/reperfusion. AS-ODNs to AT₁R mRNA may be more beneficial than losartan, because losartan does not affect the plasma angiotensin II level. The sustained increase in AT₁R mRNA, but diminished protein expression, in rat hearts treated with AS-ODNs suggests that AS-ODNs block AT₁R at the translational level.

Key Words: angiotensin II • antisense • AT₁ receptor • ischemia/reperfusion • losartan • mRNA

	Introduction

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The renin-angiotensin system is an important factor in the pathophysiology of ischemic coronary events.^{1 2 3 4 5 6} A deletion polymorphism in the angiotensin-converting enzyme (ACE) gene, which is associated with a high serum level of ACE, has been reported to be related to high risk of myocardial infarction and sudden death.^{1 2 3 4} Plasma renin activity also predicts acute myocardial infarction in patients with hypertension, irrespective of serum cholesterol levels. Angiotensin II (Ang II) contributes to the evolution of ischemic coronary events through its hemodynamic effects.⁷ It also contributes to ischemic events by affecting hemostatic activity.^{8 9} In humans⁸ and in cultured cells,⁹ Ang II has been observed to stimulate the synthesis of the plasminogen activator inhibitor and thereby promote thrombosis. Both ACE inhibitors and Ang II receptor antagonists have been shown to protect myocardium from ischemia/reperfusion injury in experimental animal models.^{10 11} Recent clinical trials^{12 13} have also confirmed the effectiveness of ACE inhibition in preventing recurrent myocardial infarction and the progression of postinfarction left ventricular dysfunction to heart failure.

Ang II receptors include at least 2 different subtypes, the AT₁ receptor (AT₁R) and the AT₂ receptor (AT₂R).^{7 14} Both AT₁R and AT₂R (AT₁R>>AT₂R) are expressed in rat heart and distributed in the myocardium.^{15 16 17} Sun and Weber¹⁸ showed that myocardial AT₁R density is significantly increased in association with ACE expression and fibril collagen formation at day 3 through week 8 after myocardial infarction in rats. These investigators suggested that the increased AT₁R binding relates to tissue repair or to fibrogenic response to tissue injury after ischemia. Recent studies from our laboratory showed a marked increase in AT₁R expression in rat myocardium immediately after a brief period of ischemia and reperfusion.¹⁹ To determine the role of increased myocardial AT₁R expression in ischemia/reperfusion injury, we applied both the antisense oligodeoxynucleotides (AS-ODNs) directed at AT₁R mRNA and the AT₁R antagonist losartan in the isolated rat heart model of ischemia/reperfusion.

	Materials and Methods

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ODNs and Vector Liposome
AS-ODNs and scrambled antisense oligodeoxynucleotides (Scr-ODNs) were synthesized as 15-mers targeted to bases +63 to +77 of AT₁R mRNA. The AS-ODNs to AT₁R mRNA and Scr-ODNs were modified by phosphorothioation. Oligodeoxynucleotides (ODNs) were synthesized in the DNA Synthesis Laboratory, University of Florida, Gainesville, as recently described.^{20 21 22 23 24} The antisense sequence targeted to the AT₁R mRNA was 5'-TAACTGTGCCTGCCA-3'. The corresponding sense sequence was 5'-TGGCAGGCACAGTTA-3'. The sequence for scrambled antisense was 5'-CTTACTAGCCTAGGC-3'. This antisense sequence has been shown to effectively inhibit AT₁R expression in rat brain and decrease arterial blood pressure in spontaneously hypertensive rats (SHR), and the effect on blood pressure lasted for 3 to 7 days after a single dose of 50 µg/rat.^{20 21 22 23 24}

Since liposomes have been shown to enhance the uptake of AS-ODNs by tissues,²⁴ DOTAP/DOPE (wt:wt=1:1) liposomes were used in the present studies as the vector for ODNs. DOTAP/DOPE liposomes are positive electron carriers and can combine with negative electron carrier ODNs. The liposome diameter is 142.5±75.5 nmol/L. These cationic liposomes have been shown to be an effective vector for ODNs.²⁵

Animals
Male Sprague-Dawley rats weighing 200 to 225 g (heart weight, 820 to 880 mg each) were injected intravenously with either AS-ODNs (n=14) or Scr-ODNs (n=9) at a dose of 100 µg/rat 24 hours before excising the hearts. DOTAP/DOPE liposomes (300 µg/rat) were given to the rats along with ODNs. Parallel groups of rats were treated with saline (n=15) or 10 mg/kg losartan (n=6) for 4 to 6 hours before the hearts were excised.

Isolated Perfused Heart Model
Twenty-four hours after administration of AS-ODNs or Scr-ODNs or 4 to 6 hours after administration of losartan, rats were anesthetized with sodium pentobarbital (50 mg/kg) intraperitoneally. Blood was collected from the carotid artery into 3.8% sodium citrate (wt:wt=9:1) for measuring the plasma Ang II level. The hearts were excised rapidly and placed in ice-cold Krebs-Henseleit buffer (mmol/L: NaCl 118, KCl 4.7, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 1.25, NaHCO₃ 25, and glucose 11, pH 7.4). Within 1 minute, the hearts were transferred to a perfusion apparatus and perfused via the aorta with oxygen-saturated (95% O₂+5% CO₂) Krebs-Henseleit buffer kept at 37°C with the use of a MasterFlex pump (model 7015-21, Cole-Palmer Instrument Co) according to the modified Langendorff procedure.^{19 26 27} The heart was placed in a semiclosed circulating water–warmed (37°C) air chamber, paced atrially with a Medtronic 5320 pacemaker at a rate of 300 bpm, and perfused at a constant flow (5.5 to 6.0 mL/min). Coronary perfusion pressure (CPP) was measured via a catheter placed just proximal to the aorta and connected to a Gould Statham P23ID pressure transducer. A latex balloon filled with water and connected to a Gould Statham P23ID pressure transducer was inserted into the left ventricle through the left atrium to measure left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), and developed left ventricular pressure (dLVP) (dLVP=LVSP-LVEDP). LVEDP during equilibration was set at 5 to 7 mm Hg. CPP, LVEDP, and LVSP were continuously recorded on a 4-channel recorder (Astro-Med).

Myocardial Ischemia and Reperfusion
In pilot experiments (n=8), hearts perfused with buffer alone were exposed to 40 minutes of ischemia (stop perfusion) followed by 30 minutes of reperfusion. The LVEDP fell to zero during the first few minutes of ischemia, then started to rise at 16 to 18 minutes of ischemia, reached the peak value at 23±1 minutes of ischemia, and then gradually fell again. Therefore, the ischemia time in the present study was kept at 25 minutes.

Seven hearts from saline-treated rats were continuously perfused with Krebs-Henseleit buffer for 75 minutes and served as sham controls. Hearts from all other rats, after 20 minutes of equilibration, were subjected to 25 minutes of ischemia followed by 30 minutes of reperfusion. After completion of the experiment, hearts were frozen on dry ice for Ang II receptor analysis (by autoradiography and binding assays), AT₁R protein analysis by Western blot, AT₁R mRNA analysis by reverse transcription (RT)–polymerase chain reaction (PCR), and measurement of the Ang II level.

Determination of Ang II Receptor Expression in Myocardial Sections by Autoradiography
Multiple 20-µm-thick coronal sections of hearts were made at -20°C. The sections were then mounted onto chrome-alum-gelatin–coated slides and incubated with 250 to 300 pmol/L [¹²⁵I-Sar,Ile]Ang II for 2 hours in 10 mmol/L sodium biphosphate buffer (pH 7.2) or buffer containing 10 µmol/L Ang II receptor antagonist [Sar,¹Val,⁵Ala⁸]Ang II, 10 µmol/L AT₁R antagonist losartan, or 10 µmol/L AT₂R antagonist PD123,177. The sections were washed in 10 mmol/L sodium biphosphate buffer (pH 7.2) and dried. Autoradiograms were generated by apposition of slide-mounted tissue sections with x-ray film (Hyperfilm-³H, Amersham) for 3 weeks. Densitometric analysis of the autoradiographs was carried out with Image Systems (MCID M1 software with Tk/M1 Turnkey System with 80486 33-mHz computer, Imaging Research, Inc).^{19 20 21 22 23 24}

Determination of Ang II Receptor Expression in Myocardial Sections by Binding Assay
The ventricular tissues were cut into small pieces and homogenized in ice-cold buffer (50 mmol/L Tris and 150 mmol/L EDTA, pH 4.2). The homogenate was centrifuged at 600g for 10 minutes, and 1 mL of the supernatant was saved for protein assay. The rest of the supernatant was centrifuged at 48 000g for 20 minutes. The pellet was resuspended in Tris-HCl buffer and used for binding assay. Membrane protein (400 µg) was incubated in 500 µL of binding buffer (50 mmol/L Tris, 150 mmol/L EDTA, 1 mg/mL bacitracin, 10 µmol/L 1,14-phenanthroline, and 0.1% BSA) containing 0.05 to 0.45 nmol/L [¹²⁵I-Sar¹,Ile⁸]Ang II at 25°C for 90 minutes. Nonspecific AT₁R and AT₂R binding was determined in the presence of 1 µmol/L Ang II, 1 µmol/L PD123,319 (specific AT₂R antagonist), or 1 µmol/L losartan. Bound radioactivity was separated from free radioactivity on Whatman GF/B filters with a Brandel harvester and counted for 1 minute. All binding assays were performed in triplicate, and the results varied by <10%.

Quantification of AT₁R Protein Expression in Myocardium by Western Analysis
Myocardial tissues were homogenized and lysed in boiling lysis buffer (1% SDS, 0.1% Triton X-100, and 10 mmol/L Tris-HCl, pH 7.4) and centrifuged at 10 000 rpm for 30 minutes at 4°C. The lysate protein from myocardial tissues (15 µg/lane) was separated by 8% SDS-PAGE using a Bio-Rad Mini-Protean cell, transferred to nitrocellulose membrane (Amersham), and then immunoblotted with a primary mouse monoclonal antibody against AT₁R (courtesy of Dr G. Vinson, Queen Mary and Westfield College, London, England) at 1:10 dilution. Anti-mouse horseradish peroxidase–conjugated antibody was used as a second antibody at 1:2500 dilution. The blots were detected by the enhanced chemiluminescence method (ECL Western blot kit, Amersham), as recently described.²⁸

Determination of AT₁R mRNA by RT-PCR
RNA was extracted from rat myocardium using the guanidinium thiocyanate–phenol–chloroform method²⁹ and quantified. The quality of isolated RNA was checked by gel electrophoresis. For this purpose, 2 µg of total RNA was denatured in a 50% formamide and 20% formaldehyde mixture for 10 minutes at 65°C. Denatured RNA was fractionated by gel electrophoresis on a 1% agarose gel containing 10% formaldehyde and examined under UV light after staining with ethidium bromide. Two micrograms of the total RNA was digested with DNase I (RNase free) for 10 minutes at 37°C in the presence of 5 U RNase inhibitor. After heat inactivation of DNase, RNA was reverse-transcribed for 50 minutes at 42°C using AMV Reverse Transcriptase (Promega) with oligo(dT) as a primer (Promega) in a 20-µL reaction mixture. The reaction was stopped by heating samples for 15 minutes at 99°C. Ten percent of single-stranded cDNA was used a template for amplification in PCR using Taq polymerase (Promega). Primers were designed on the basis of the AT₁R sequence cloned from the rabbit kidney cortex.³⁰ The sense primer sequence was 5'-TTTGGGAACAGCTTGGCGGT-3', and the reverse primer was 5'-GCCAGCCAGCAGCCAAATAA-3'. PCR reaction was performed at 3 different cycles to ensure that it was performing in the linear range at which there is a fixed relationship between input RNA and densitometric readout. The optimal cycle number for AT₁R mRNA was 30. After 2 minutes at 94°C, amplification was performed at 94°C for 1 minute, 55°C for 2 minutes, and 68°C for 2 minutes for 40 cycles, with a final incubation at 68°C for 7 minutes. Ten microliters of the PCR mixture was separated on a 1% agarose gel stained with ethidium bromide. The DNA bands were photographed and subjected to densitometry. Our PCR negative control was without cDNA (H₂O). The amount of PCR product was determined by comparison of signal density with simultaneously amplified cDNA for GAPDH mRNA (sequence of the primers, 3'-CTCGTGAGCCCAGGATGC and 5'-ACCACCATGGAGAAGGCTGG; the product size was 508 bp).

Determination of Plasma and Myocardial Ang II Levels
Plasma was frozen at -70°C until extraction with methanol on reversed-phase phenylsilylsilica extraction cartridges (Alpco; approximate recovery, 90%). Samples were analyzed by double-antibody Ang II radioimmunoassay (RK-A22, Alpco), as described earlier.²⁴ The assay is sensitive to 0.7 pg/mL (0.7 pmol/L).

Myocardial tissues were boiled in 1 mol/L acetic acid and then homogenized in 1 mol/L acetic acid. The homogenate was centrifuged at 7000 rpm for 20 minutes. The supernatant was then applied to a Sep-Pak C18 cartridge, washed, and eluted with methanol:H₂O:trifluoroacetic acid. The eluent was dried under air at 35°C and dissolved in Tris-HCl buffer (pH 7.4). The assay for myocardial Ang II was similar to that for plasma Ang II (described above).

Data Analysis
CPP, LVEDP, LVSP, and dLVP are expressed in millimeters of mercury. All values in the text are mean±SD. Differences between specific means were examined by ANOVA with the Student-Newman-Keuls test. A value of P<0.05 was considered statistically significant.

	Results

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Cardiac Dynamics During Ischemia and Reperfusion
The basal values of CPP, LVSP, LVEDP, and dLVP were similar in all groups of hearts.

In the control continuously buffer-perfused hearts observed for 75 minutes, there were only minimal (5% to 10%) changes in the indices of cardiac function. In hearts from saline-treated rats, 25 minutes of ischemia followed by 30 minutes of reperfusion resulted in marked cardiac dysfunction, as indicated by several-fold increases in CPP and LVEDP and a 75% decrease in dLVP (all P<0.01 versus preischemia value). A representative example of marked cardiac dysfunction after ischemia/reperfusion is shown in Figure 1, and data from multiple experiments are summarized in Figure 2.

View larger version (41K): [in this window] [in a new window]   Figure 1. Representative experiments showing ischemia/reperfusion-induced cardiac dysfunction and the salutary effects of administration of AS-ODNs directed at AT1R mRNA. Please note that CPP and LVEDP rise and that dLVP falls in the heart from a saline-treated rat after ischemia/reperfusion. Such changes are less marked in the AS-ODN–treated rat heart.

View larger version (30K): [in this window] [in a new window]   Figure 2. Summary of data on ischemia/reperfusion-mediated increase in CPP and LVEDP and decrease in dLVP (LVSP-LVEDP) in hearts from saline-treated rats. Administration of AS-ODNs (n=9), but not Scr-ODNs (n=6), showed a beneficial effect on cardiac function, indicated by smaller increases in CPP and LVEDP and preservation of dLVP. Losartan treatment (n=6) showed only a modest protective effect. Data are mean±SD.

Treatment of rats with AS-ODNs markedly attenuated the ischemia/reperfusion-induced myocardial dysfunction, indicated by preservation of dLVP and minimization of increase in LVEDP and CPP (all P<0.01 versus changes in hearts from saline-treated rats). Treatment of rats with losartan modestly, but significantly (P<0.05), attenuated the ischemia/reperfusion-induced changes in CPP and dLVP. Treatment with Scr-ODNs showed no effect on the ischemia/reperfusion-induced myocardial dysfunction. Data from multiple experiments are summarized in Figure 2.

Myocardial Ang II Receptor Expression After Ischemia and Reperfusion
As determined by autoradiography, sham control (continuously perfused) hearts exhibited some total Ang II receptor and AT₁R binding and some AT₂R binding. Ischemia followed by reperfusion resulted in an immediate and significant increase in total Ang II receptor binding in hearts from saline-treated and Scr-ODN–treated rats (P<0.05 versus sham control hearts). The increase in Ang II receptor binding was primarily due to an increase in AT₁R binding (P<0.05 versus sham control hearts), as AT₂R binding was not affected by ischemia/reperfusion. The ischemia/reperfusion-mediated increase in myocardial Ang II and AT₁R binding was totally abolished (even lower than that in sham control hearts) by AS-ODN or losartan treatment. Results from a representative experiment are shown in Figure 3. Summary of data from several experiments is shown in Figure 4.

View larger version (91K): [in this window] [in a new window]   Figure 3. Representative autoradiographs of Ang II receptor binding in heart from a control rat, a heart from saline-treated rat subjected to ischemia/reperfusion, and a heart from an AS-ODN–treated rat subjected to ischemia/reperfusion. Please note the marked increase in total Ang II and AT1R expression immediately after ischemia/reperfusion; this increase is not present in the heart from the AS-ODN–treated rat. Total Ang II as well as AT1R binding is less in the AS-ODN–treated rat heart than in the control heart.

View larger version (25K): [in this window] [in a new window]   Figure 4. Summary of data on Ang II receptor binding by autoradiography in myocardial sections. Ischemia and reperfusion (Isch-reperf) in hearts from saline-treated rats resulted in a 2- to 3-fold increase in total Ang II binding, mostly AT1 binding, compared with the nonischemic control hearts (n=6). Intravenous injection of losartan (n=4) or AS-ODNs (n=6), but not Scr-ODNs (n=4), blocked Ang II binding on the myocardial sections after Isch-reperf. Data are mean±SD.

As determined by binding assay, sham control (continuously perfused) hearts consistently exhibited AT₁R binding (2.14±0.15 fmol/mg membrane protein, n=3). After ischemia/reperfusion, there was a consistent 50% increase in AT₁R binding (3.25±0.25 fmol/mg membrane protein, n=3) without any change in AT₂R binding. Treatment of rats with AS-ODNs resulted in a decrease in AT₁R binding (2.45±0.15 fmol/mg protein, n=3). AT₂R binding was undetectable in all 3 groups of rat hearts.

Myocardial AT₁R Protein and mRNA Expression
Western analysis of the control continuously perfused hearts using the monoclonal antibody identified a protein band of 41 kDa. A dense pattern of 2 or 3 bands was identified in Western analysis of protein from hearts subjected to ischemia/reperfusion. This pattern of AT₁R expression using the same monoclonal antibody as used by us and its specificity have been recently described by Lu et al.³¹ Pretreatment of rats with AS-ODNs abolished the ischemia/reperfusion-mediated increase in AT₁R protein expression. In contrast, pretreatment of rats with losartan did not affect the ischemia/reperfusion-mediated increase in AT₁R protein expression. A representative experiment and summary of data from 3 separate experiments are shown in Figure 5.

View larger version (57K): [in this window] [in a new window]   Figure 5. AT1R protein expression by Western blot analysis in myocardial tissues from a control rat, 2 vehicle (saline)-treated rats subjected to ischemia/reperfusion, an AS-ODN–treated rat subjected to ischemia/reperfusion, and a losartan-treated rat subjected to ischemia/reperfusion. Please note the marked increase in AT1R protein expression immediately after ischemia/reperfusion in the heart from vehicle-treated rats. Treatment with AS-ODNs decreases the enhanced AT1R protein expression, whereas treatment with losartan had no effect on the enhanced AT1R protein expression. This Western blot analysis is representative of 3 different analyses. See text for details of the methodology.

Ischemia/reperfusion also resulted in an 2- to 3-fold increase in AT₁R mRNA (adjusted for GAPDH signal) in the myocardium, as determined by RT-PCR. Pretreatment of rats with AS-ODNs did not affect the increase in myocardial AT₁R mRNA level (P<0.05). Pretreatment of rats with losartan appeared to somewhat further increase the myocardial AT₁R mRNA level, but the change was not significant (Figure 6).

View larger version (37K): [in this window] [in a new window]   Figure 6. cDNA of AT1R mRNA in myocardial tissues (RT-PCR) from a control rat, a vehicle (saline)-treated rat subjected to ischemia/reperfusion (Isch-Reperf), an AS-ODN–treated rat subjected to Isch-Reperf, an Scr-ODN–treated rat subjected to Isch-Reperf, and a losartan-treated rat subjected to Isch-Reperf. AT1R mRNA increases after Isch-Reperf, and the increase is similar in AS-ODN– and Scr-ODN–treated rat hearts. AT1R mRNA is highest in the heart from a losartan-treated rat. Densitometric analysis of the AT1R mRNA bands in 3 separate experiments is shown in the graph. See text for details of the methodology.

Plasma and Myocardial Ang II Levels
As shown in Figure 7, plasma Ang II levels in saline-treated, AS-ODN–treated, and Scr-ODN–treated rats were similar. The plasma Ang II level in losartan-treated rats was markedly higher than that in other groups. Ang II levels in myocardium were similar in all groups of rats (Table).

View larger version (20K): [in this window] [in a new window]   Figure 7. Plasma Ang II level after injection of saline (n=14), losartan (n=6), AS-ODNs (n=10), and Scr-ODNs (n=7). Plasma Ang II levels in the AS-ODN– or Scr-ODN–treated rats were similar to those in the control rats (n=14). Treatment of rats with losartan for 4 to 6 hours markedly increased the plasma Ang II level. Data are mean±SD.

	Discussion

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The present study confirms the results of our recent study¹⁹ that ischemia followed by reperfusion results in cardiac dysfunction in isolated perfused rat hearts, and the cardiac dysfunction is associated with an increase in total Ang II receptor expression in the myocardium immediately after ischemia/reperfusion. The marked increase in Ang II receptor expression could be accounted for in its entirety by an increase in AT₁R expression, since AT₂R expression was unchanged. In the present study, we also observed a marked increase in AT₁R protein and mRNA expression in hearts exposed to ischemia/reperfusion. Although the beneficial effects of chemical blockade of AT₁R have been previously shown,^{11 19} this is perhaps the first report on the salutary effect of AS-ODNs directed at AT₁R mRNA on myocardial ischemia/reperfusion injury. AS-ODNs totally abolished the ischemia/reperfusion-induced increase in myocardial AT₁R expression and protected against cardiac dysfunction induced by ischemia/reperfusion in isolated rat hearts without affecting AT₁R mRNA level. Importantly, treatment with AS-ODNs did not affect Ang II levels in plasma.

Ang II is a potent coronary artery constrictor in the rat, which contributes to the ischemic coronary events through its hemodynamic effects.²⁷ All components for the renin-angiotensin system, including mRNA for renin and angiotensinogen, are present in the rat heart.^{6 32 33} At least 2 distinct Ang II receptor subtypes have been defined on the basis of their differential pharmacological and biochemical properties and have been designated as AT₁R and AT₂R.³⁴ To date, evidence from experimental animals indicates that almost all of the known effects of Ang II in adult tissues are attributable to AT₁R and that AT₂R is related to growth and development.³⁴ Since the predominant active subtype of Ang II receptor in myocytes changes from AT₂R to AT₁R in the growing rat,^{14 17} we speculated that the activation of AT₁R must be the primary factor in increased Ang II, resulting in an increase in coronary vascular resistance during ischemia and reperfusion. In accordance with this hypothesis, our study clearly documents an increase in AT₁R expression immediately after ischemia without any change in AT₂R expression.

Feolde et al¹⁷ have demonstrated that Ang II receptors are located on the myocardial cell membrane. Ang II receptor binding by autoradiographic assay used in the present study determines receptors both on the cell membrane and in plasma and does not provide information on the location of AT₁R. Nonetheless, we observed that AT₁R protein expression was markedly increased in the myocardial tissues subjected to ischemia/reperfusion. A dense pattern of 2 or 3 bands (40 to 44 kDa) was identified in Western analysis of protein from hearts subjected to ischemia/reperfusion. This pattern of AT₁R expression using the same monoclonal antibody has been recently described.³¹ These bands are likely to represent immature AT₁Rs that have not been fully glycosylated. Importantly, the AT₁R mRNA level was also concurrently upregulated in these tissues, suggesting that the regulation of Ang II receptors after ischemia/reperfusion is at the mRNA level.

Sun and Weber¹⁸ reported that (1) there is relatively low Ang II receptor expression in normal rat myocardium; (2) Ang II receptor expression increases markedly at the site of left ventricular myocardial infarction at day 3 and weeks 1, 2, 4, and 8 after infarction; (3) Ang II receptor expression in the pericardial tissues increases after pericardiotomy; and (4) tissue Ang II receptor expression is displaced by the AT₁R antagonist DuP753 but not by the AT₂R antagonist PD123,177. Sun and Weber¹⁶ also suggested that AT₁R activation plays an important role in mediating the fibrogenic response to tissue injury in the rat heart. Ang II has been shown to possess growth-promoting activity.^{35 36 37 38} Studies by Anversa's group^{39 40} showed that impairment in myocyte contractile function after myocardial infarction in rats is associated with increased Ang II mRNA and an overexpression of c-myc and c-jun. These authors speculated that AT₁R expression in myocytes participates in the reactive growth process of myocytes. Nio et al⁴¹ have also suggested that myocardial infarction in rats leads to increase in AT_1aR gene transcription and protein expression and that therapy with AT₁R, but not AT₂R, antagonists was effective in blocking the increased expression of Ang II receptor subtypes after myocardial infarction. Recently, Harada et al⁴² have reported amelioration of reperfusion arrhythmias in AT_1aR knockout mice. These observations collectively suggest a critical role of AT₁R activation in determination of ischemic injury. The increased AT₁R expression, documented in the present study (by 2 different methodologies: autoradiography and direct receptor binding), may play an important role in determining coronary vascular resistance and perhaps remodeling of the myocardium after ischemia/reperfusion.

Several sequence-specific AS-ODNs directed at AT₁R mRNA have been developed and shown to reduce hypertension in the SHR.^{20 21 22 23 24 43 44 45} A single dose of AS-ODNs to the AT₁R mRNA decreases the number of AT₁R by 25% in the hypothalamic tissue block without affecting AT₂R number and produces a long-lasting decrease in blood pressure.²¹ Antisense delivery in adeno-associated viral vectors results in a prolonged modulation of hypertension.^{43 44} Raizada's group showed that retrovirus-mediated transfer of AS-ODNs to AT₁R mRNA decreases AT₁R number and Ang II action in astroglial and neuronal cells in primary cultures from the brain⁴⁵ and provides long-term control of blood pressure without affecting plasma Ang II levels.⁴⁶ Importantly, our observations of unchanged AT₁R mRNA and plasma Ang II levels in rats treated with AS-ODNs against AT₁R mRNA are in accordance with these early studies.

Since AS-ODNs directed at AT₁R mRNA reduce blood pressure in the SHR^{20 21 22 23 24 43 44 45 46} and since ischemia/reperfusion-induced cardiac dysfunction is associated with increased myocardial AT₁R expression,¹⁹ we hypothesized that suppression of AT₁R expression by AS-ODNs directed at AT₁R mRNA would protect cardiac tissues from ischemia/reperfusion injury. Consistent with this hypothesis, we observed that administration of AS-ODNs to AT₁R mRNA significantly preserved cardiac function after ischemia/reperfusion and abolished the ischemia/reperfusion-mediated increase in total Ang II and AT₁R expression in the myocardium. Such protective effects were not seen in the Scr-ODN–treated group of rats. Chemical blockade of AT₁R with losartan also modestly, but significantly, prevented cardiac dysfunction after ischemia/reperfusion. As expected, losartan blocked AT₁R binding, as measured by autoradiography, without affecting AT₁R protein expression. Since Ang II produces coronary constriction,⁴⁷ the markedly increased AT₁R expression during ischemia/reperfusion must contribute to the increase in coronary vascular resistance and deterioration of cardiac function after ischemia/reperfusion. Notably, the effects of losartan on cardiac dynamics after ischemia/reperfusion were less marked than those of AS-ODN; this finding was probably a reflection of less than total blockade of AT₁R, a feedback increase in Ang II levels, and some undefined effects of losartan.

Ischemia/reperfusion-induced cardiac dysfunction was evaluated in the present study by the measurement of CPP, LVEDP, and dLVP, indices used in several studies in the isolated rat heart model of global ischemia and reperfusion.^{19 47 48 49} All these indices were modified by the use of AS-ODNs directed at AT₁R mRNA. Since the studies were carried out in the isolated heart preparation, it is safe to assume that the peripheral effects of AS-ODNs did not participate in the preservation of cardiac function.

AS-ODNs and Scr-ODNs were synthesized as 15-mers targeted to bases +63 to +77 of AT₁R mRNA. As indicated earlier, the AS-ODNs used in the present study target AT₁AR mRNA and inhibit mRNA translation without any effect on mRNA transcription.²³ Consistent with this concept, AT₁R protein expression was decreased, and AT₁R mRNA was unaffected by AS-ODN treatment.

The present study showed a marked increase in plasma Ang II level in losartan-treated rats, but not in AS-ODN– or Scr-ODN–treated rats, whereas the myocardial Ang II levels were similar among all the groups of rats. The increase in plasma Ang II level in the losartan-treated group is probably secondary to loss of the negative-feedback effect of Ang II on the renin-producing cells in the kidney and is consistent with the concept of upregulation of the renin-angiotensin system after AT₁R blockade. The absence of an increase in plasma Ang II levels in AS-ODN–treated rats is an interesting observation. Although the precise basis for the absence of increase in Ang II levels in plasma after AS-ODN administration is not known, a similar observation has also been made by Iyer et al⁴⁶ in SHR treated with AS-ODNs to AT₁R mRNA. This phenomenon may in part relate to the fact that administration of AS-ODNs decreased AT₁R protein expression, whereas treatment with losartan had no effect on AT₁R protein expression. Chemical blockers of AT₁R-like losartan may also have effects besides blocking AT₁R activity. This explanation, however, remains unsubstantiated and needs direct evidence. With long-term use of AS-ODNs, it is possible that the endogenous Ang II levels may rise. Nonetheless, increase in Ang II level is detrimental in the context of myocardial ischemia/reperfusion injury.⁴⁷

Although the overwhelming body of evidence suggests that almost all of the known hemodynamic effects of Ang II are mediated by AT₁R activation, AT₂R activation under certain conditions may be important. Ford et al⁵⁰ reported that AT₂R blockade markedly decreased ischemia/reperfusion injury in the isolated rat heart, whereas AT₁R inhibition was detrimental. However, these investigators did not examine Ang II receptor status in their preparation, which makes it difficult to discern the effects of AT₁R and AT₂R blockade. Yamada et al⁵¹ have suggested that it is the AT₂R activation that mediates apoptotic cell death in PC12W cells (rat pheochromocytoma cell line) and R3T3 cells (mouse fibroblast cell line). Both these cell lines express abundant AT₂R but not AT₁R. Cigola et al⁵² have recently demonstrated that Ang II induces apoptosis in rat cardiac myocytes and that the Ang II–induced DNA damage is inhibited by the AT₁R antagonist losartan but not by the AT₂ R blocker PD123,319. In recent studies from our laboratory in human coronary artery endothelial cells, we found that Ang II–mediated apoptosis is primarily caused by AT₁R activation.⁵³ Importantly, rat cardiac myocytes and human coronary artery endothelial cells express primarily AT₁R subtypes. These observations also imply that cells that exhibit primarily AT₂R subtypes develop apoptosis by AT₂R activation, whereas cells that exhibit AT₁R subtypes develop apoptosis by AT₁R activation.

In this first report of the use of AS-ODNs directed at AT₁R mRNA in the modulation of ischemia/reperfusion injury, we describe almost total abolition of the increase in myocardial AT₁R receptor binding and protection against cardiac dysfunction. Use of AS-ODNs decreases the elevated AT₁R protein expression, but it does not affect the increased AT₁R mRNA. The increase in plasma Ang II level in the losartan-treated group is consistent with the upregulation of the renin-angiotensin system after AT₁R blockade. The absence of an increase in plasma Ang II levels in AS-ODN–treated rats is an intriguing observation. Although the precise basis for the absence of an increase in Ang II levels in plasma after AS-ODN administration is not known, the lack of an increase in plasma Ang II, if confirmed in subsequent studies, may make the use of AS-ODNs an attractive approach in the treatment of ischemia/reperfusion injury.

	Acknowledgments

 
This study was supported by a Grant-in-Aid from the American Heart Association, Florida Affiliate, Inc (St Petersburg, Fla), a Merit Review Award from the VA Central Office, and an NIH (MERIT) Award. The authors thank Reed Pedlow for superb assistance in preparing the illustrations and Christina Jimenez for secretarial assistance.

Received December 1, 1997; accepted June 1, 1998.

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