Introduction

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Acute myocardial ischemia causes significant increase in plasma catecholamine levels, which leads to exacerbation of the ischemic myocardial injury (Waldenstrom et al., 1978; Rona, 1985). The worsening myocardial ischemia is an important factor in cardiac dysfunction. Acute myocardial ischemia also is characterized by increased sensitivity of -adrenoreceptors (-ARs) to catecholamines (Strassere et al., 1990). -ARs form the interface between the sympathetic nervous system and the cardiovascular system (Mukherjee et al., 1979; Maisel et al., 1985; Strassere et al., 1990). Importantly, ₁-AR subtype (₁-AR) is the predominant subtype in the myocardium (Minneman et al., 1995), and its activity and sensitivity are thought to regulate cardiac function via adenylyl cyclase activity (Mukherjee et al., 1979; Thandroyen et al., 1986; Böhm, 1995). Several experimental studies show that density and mRNA of ₁-AR are augmented in the myocardium after acute ischemia (Maisel et al., 1985; Karliner et al., 1989; Ihl-Vahl et al., 1995). Experimental and clinical studies also have demonstrated that therapy with -AR blockers, especially with selective ₁-AR blockers, can protect myocardium against ischemic injury and dysfunction (Ablad et al., 1987; Lu et al., 1990; Schulz et al., 1995), decrease infarct size (Schulz et al., 1995), and reduce the incidence of sudden cardiac death in patients with myocardial infarction (Yusuf et al., 1985).

Although chemical -AR blockers are commonly used in the treatment of ischemic heart disease, these agents often cause central nervous system side effects and the ₂-AR antagonistic activity is associated with an increase in peripheral vascular resistance. Development of antisense-oligodeoxynucleotides (AS-ODNs) against specific receptor mRNA is a novel approach to decrease the synthesis of receptor proteins (Phillips et al., 1994; Phillips, 1997). This approach has potential to be of therapeutic benefit in disease states characterized by up-regulation of these receptors (Phillips et al., 1994; Phillips, 1997; Yang et al., 1998). AS-ODNs directed at ₁-AR mRNA (₁-AS-ODNs) have been reported to reduce blood pressure in spontaneously hypertensive rats (SHR) for prolonged period with a single i.v. injection (Zhang et al., 2000).

We hypothesized that the use of ₁-AS-ODNs may protect myocardium against ischemia-reperfusion-induced dysfunction in the isolated rat heart. This study was undertaken to test this hypothesis. We also examined the effect of ₁-AS-ODNs on lipid peroxidation, the expression of ₁-AR protein, and mRNA in the myocardium after ischemia-reperfusion.

	Materials and Methods

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ODNs and Vector Liposome. ₁-AS-ODNs and inverted-oligodeoxynucleotides (IN-ODNs) control were 15-mers and targeted to 5 to +10 of rat ₁-AR mRNA encompassing the AUG start colon. The sequence of AS-ODNs was 5'-CCGCGCCCATGCCGA-3', and the corresponding IN-ODNs was 5'-AGCCGTACCCGCGCC-3'. These ODNs were modified by backbone phosphorothioation and synthesized in the DNA Synthesis Core Laboratory of the University of Florida.

Animals. Male Sprague-Dawley rats weighing 200 to 250 g were injected i.v. with either AS-ODNs (n = 7) or IN-ODNs (n = 7) at a dose of 200 µg/rat 4 days before excising the hearts. DOTAP/DOPE liposomes (700 µg/rat) were given along with ODNs. Other groups of rats were treated with saline (n = 13), or the selective ₁-AR blocker atenolol (2 mg/kg; n = 7) 6 h before the hearts were excised. These animal studies were approved by the University of Florida Animal Care Committee.

Isolated Perfused Heart Model. All rats were anesthetized with sodium pentobarbital (40 mg/kg i.p). The hearts were excised rapidly and placed in ice-cold Krebs-Henseleit buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 1.25 mM CaCl₂, 25 mM NaHCO₃, and 11 mM glucose, pH 7.4). Within 1 min, the hearts were transferred to an isolated 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, Vernon Hills, IL) according to the modified Langendroff procedure (Neely and Rovetto, 1975; Yang et al., 1998). 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 beats/min, and perfused at a constant flow (5.5-6.0 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 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. All measurements were continuously recorded on a four-channel recorder (Astro-Med, West Warwick, RI).

Myocardial Ischemia and Reperfusion. Six hearts from saline-treated rats were continuously perfused with Krebs-Henseleit buffer for 80 min and served as sham control. Hearts from other rats, after 20 min of equilibration, were subjected to 30 min of ischemia followed by 30 min of reperfusion. After completion of the experiment, hearts were frozen in liquid nitrogen for ₁-AR density analysis (by radioligand binding assay), ₁-AR protein analysis (by Western blot), ₁-AR mRNA analysis by reverse transcription-polymerase chain reaction (RT-PCR), and measurement of malondialdehyde (MDA).

Quantification of ₁-AR Protein Expression in Myocardium. Myocardial tissues were homogenized and lysed in boiling lysis buffer (1% SDS, 0.1% Triton X-100, and 10 mM Tris-HCl, pH 7.4) and centrifuged at 10,000 rpm for 30 min at 4°C. The lysate protein from myocardial tissues (20 µg/lane) was separated by 8% SDS-polyacrylamide gel electrophoresis with a Bio-Rad Mini-Protean cell, transferred to nitrocellulose membrane (Amersham, Arlington Heights, IL). After incubation in blocking solution (4% nonfat milk; Sigma, St. Louis, MO), membranes were incubated with 1:1000 dilation primary antibody (polycolonal antibody to ₁-AR; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Membranes were washed and incubated with 1:2000 dilution second antibody (Amersham) for 1 h. The membranes were detected with the enhanced chemiluminescence system, and relative intensity of bands of interest was analyzed by NSF-300G Scanner (Microtek, San Clemente, CA), as described previously (Yang et al., 1998; Li et al., 1999).

Determination of ₁-AR mRNA. Total RNA was isolated from rat myocardium with the single step acid-guanidinum thiocyanate-phenol-chloroform method and quantified (Chomczynski and Sacchi, 1987). One microgram of total RNA was reverse transcripted with oligo-dT (Promega, Madison, WI) and M-MLV reverse transcriptase (Promega) at 37°C for 1 h. RT material (1.5 µl) was amplified with Taq DNA polymerase (Promega) with a primer pair specific to ₁-AR (forward primer: 5'-CTCCGAAGCTCGGCATGG-3'; and reverse primer: 5'-GCACGTCTACCGAAGTCCAGA-3'). PCR product was 432 base pairs. For PCR, 35 cycles were used at 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min. The RT-PCR amplified samples were visualized on 1.8% agarose gels with ethidium bromide. A primer pair rat GAPDH was used as control (forward primer: 5'-ATCAAATGGGGTGCTGGTGCT G-3', and reverse primer: 5'-CAGGTTTCTCCAGGCGGCATGTCA-3'). For PCR, 35 cycles were used at 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min. PCR product was 504 base pairs. The amount of PCR product of ₁-AR mRNA was determined by comparison of signal density with simultaneously amplified cDNA for GAPDH mRNA.

Determination of MDA Levels in Myocardium. MDA levels in the myocardium were measured in duplicate by a modification of the method of Ohkawa et al. (1979). Briefly, the ventricular tissues were homogenized. The assay mixture consisted of 0.1 ml of the tissue homogenate, 0.4 ml of 0.9% NaCl, 0.5 ml of 3% SDS, and 3 ml of TBA (thiobarbituric acid reagent, containing equal parts of 0.8% aqueous thiobarbituric acid and acetic acid), and was heated for 75 min at 95°C. Thereafter, 1 ml of cold 0.9% NaCl was added to the mixture, which was cooled and extracted with 5 ml of n-butanol. After centrifugation at 3000 rpm for 15 min, the butanol phase was assayed spectrophotometrically at 532 nm. Tetramethoxypropane (in amounts of 0, 0.1, 0.2, 0.4, 0.8, and 1.0 nmol) served as external standard. MDA levels in myocardium were expressed as micromoles per gram of tissue.

Data Analysis. Data are presented as mean ± S.E. Statistical significance was determined in multiple comparisons among independent groups of data in which ANOVA and the Student-Newman-Keuls test indicated the presence of significant differences. A P value of .05 was considered statistically significant.

	Results

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Cardiac Dysfunction during Ischemia-Reperfusion. The basal values of CPP, LVEDP, and dLVP were similar in all groups of rat hearts. In the control continuously buffer-perfused hearts observed for 80 min (n = 6), there were only minimal (~5%) changes in the indices of cardiac function. In the hearts from saline-treated rats (n = 7), 30 min of ischemia followed by 30 min of reperfusion resulted in marked cardiac dysfunction, indicating by a significant increase in CPP and LVEDP, and a decrease in dLVP (all P < .01 versus preischemia values).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Summary of data on ischemia-reperfusion-induced changes in CPP, LVEDP, and dLVP in hearts from different group of rats. Note the marked increase in LVEDP and CPP and a decrease in dLVP in the saline-treated rat hearts exposed to a brief period of ischemia-reperfusion. Administration of AS-ODNs, but not IN-ODNs, showed a markedly beneficial effect on cardiac function, indicated by smaller increases in CPP and LVEDP and preservation of dLVP. Treatment with atenolol also showed a modest protective effect. Data from seven rat hearts in each group are expressed as mean ± S.E. (*P < .05 versus saline, P < .05 versus atenolol).

MDA Levels in Myocardium. As shown in Fig. 2, MDA levels in myocardium increased significantly after ischemia-reperfusion (P < .05 versus sham control hearts; n = 6). Pretreatment of rats with AS-ODNs and atenolol attenuated the increase in MDA levels in the myocardium (both P < .05 versus saline pretreatment; n = 7 each group). As expected, IN-ODNs did not affect MDA levels in myocardium.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   MDA levels in the myocardium in each group rats. MDA levels increased markedly after ischemia-reperfusion in the saline-treated rat hearts. Treatment with AS-ODNs and atenolol, but not with IN-ODNs, attenuated the increase in MDA levels. Data from seven rat hearts in each group are shown as mean ± S.E. (*P < .05 versus sham control, P < .05 versus saline). , sham control; , saline + I/R; , AS-ODNs + I/R; , IN-ODNs + I/R; , atenolol + I/R.

Expression of ₁-AR Protein and mRNA in Myocardium. Western analysis of the control continuously perfused hearts showed a distinct ₁-AR protein band of 65 kDa. A similar molecular mass band was observed in hearts from saline-, AS-ODNs-, IN-ODNs-, and atenolol-treated rat hearts. The ₁-AR protein band was very dense in the saline-treated rat hearts, indicating up-regulation of the protein during ischemia-reperfusion. Treatment of rats with AS-ODNs abolished the ischemia-reperfusion-mediated increase in ₁-AR protein expression. Notably, treatment of rats with IN-ODNs or atenolol had no effect on the level of ₁-AR protein. Results of a representative experiment and summary of data from three separate experiments are presented in Fig. 3.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Representative Western blot analysis of 1-AR protein expression in myocardium from each group of rats (top). Please note that 1-AR protein in the saline-treated rat hearts increased (P < .01) after ischemia-reperfusion. Treatment with AS-ODNs decreased (P < .01) the enhanced 1-AR protein in the myocardium after ischemia-reperfusion, whereas treatment with IN-ODNs or atenolol had no effect. Summary of data (mean ± S.E.) from three separate rats in each group (bottom). See text for details of the methodology.

View larger version (54K): [in this window] [in a new window]   Fig. 4.   Expression of 1-AR mRNA in myocardial tissue from each group of rat hearts. GAPDH was amplified as a reference for quantitation of 1-AR mRNA. Please note that 1-AR mRNA expression markedly increased in the myocardium after ischemia-reperfusion in the saline-treated rat hearts. Administration of AS-ODNs inhibited the increased expression of 1-AR mRNA after ischemia-reperfusion. IN-ODNs and atenolol had no effect. The RT-PCR analysis (top) is representative of each group. Summary of data (mean ± S.E.) from three separate rats in each group (bottom). See text for details of the methodology.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

This study was designed to examine the protective role of AS-ODNs directed at ₁-AR mRNA against cardiac dysfunction after a brief period of ischemia-reperfusion. We also compared the effects of pretreatment with AS-ODNs with a selective ₁-AR blocker atenolol in this process. This study showed that ischemia for 30 min followed by reperfusion for 30 min resulted in significant cardiac dysfunction and lipid peroxidation in the saline-treated rat hearts. Furthermore, ischemia-reperfusion was associated with a marked up-regulation of ₁-AR expression in the hearts. Pretreatment of rats with atenolol decreased cardiac dysfunction after ischemia-reperfusion, but did not affect the expression of ₁-AR protein or mRNA in myocardium. However, pretreatment of rats with ₁-AS-ODNs not only preserved cardiac function and decreased lipid peroxidation after ischemia-reperfusion but also prevented the up-regulation of ₁-AR protein and mRNA expression in the ischemic-reperfused myocardium. The effects of AS-ODNs on cardiac function appeared to be equivalent to those of the commonly used ₁-AR blocker atenolol, but AS-ODNs were superior to atenolol in inhibiting the enhanced expression of ₁-AR induced by ischemia-reperfusion.

There is a generalized stimulation of the sympathetic nervous system during ischemia, perhaps a compensatory response in an attempt to preserve cardiac function. Accordingly, catecholamine levels increase in both plasma and myocardium after myocardial ischemia (Abrahamsson et al., 1983; Rona, 1985; Richard et al., 1994), but the increased catecholamine concentrations have the potential to contribute to increased excitability of myocardium, resulting in arrhythmia (Mukherjee et al., 1979; Maisel et al., 1985, 1987; Ohyanahi et al., 1988; Strassere et al., 1990). There is also an increase in the sensitivity of ₁-ARs during myocardial ischemia (Strassere et al., 1990). This coupled with increased circulating and myocardial catecholamine concentrations can exacerbate cardiac injury and dysfunction.

Rohrer et al. (1996, 1998) found that inhibition of ₁-AR expression, based on studies in ₁-AR knockout mouse, significantly limits the heart rate and blood pressure response during grade treadmill exercise. This observation clearly demonstrates that ₁-AR is important in ₁-AR functional regulation in heart. It is generally accepted that activation of -AR is the first element in the signal transduction chain mediating sympathetic stimulation of the heart (Gudermann et al., 1995). ₁-AR, the dominant -AR subtype in the heart (Ungerer et al., 1993; Minneman et al., 1995), up-regulates cardiac function by mediating adenylyl cyclase activity (Thandroyen et al., 1986; Böhm, 1995). Several experimental studies have shown an up-regulation in ₁-ARs, but not ₂-ARs, during acute ischemia-reperfusion (Maisel et al., 1985; Karliner et al., 1989; Persad et al., 1998). Ihl-Vahl et al. (1995) conclusively demonstrated a rapid up-regulation of -AR mRNA during acute myocardial ischemia; this up-regulation is subtype selective with a specific increase in mRNA level for ₁-ARs, but not for ₂-ARs. They also showed that the increase of ₁-AR mRNA is ischemia time-dependent. Notably, the regulation of ₁-ARs is different in chronic heart failure from that in myocardial ischemia. The number of ₁-ARs is diminished in heart failure and is increased in acute myocardial ischemia.

Pretreatment of animals with -AR blockers does not affect ischemia-reperfusion-induced increase in ₁-AR mRNA level. This pretreatment effect became evident in this study wherein treatment with atenolol did not affect ₁-AR protein (Western analysis) and mRNA (RT-PCR) levels. Other studies also have shown that ₁-AR blockers do not block the expression of ₁-AR expression (Aarons et al., 1980; Aarons and Molinoff, 1982; Heilbrunn et al., 1989). However, our study clearly demonstrates that ₁-AS-ODN blocks the up-regulation of ₁-ARs. We also observed that therapy with a single dose of AS-ODNs prevented the increase in ₁-AR protein. AS-ODNs were given 4 days before excising the heart to permit incorporation of the AS into the ₁-AR mRNA and inhibition of ₁-AR at transcriptional level. Atenolol was given 6 h before excising the heart because of short half-life of this chemical ₁-AR blocker. A previous study in the SHR from our laboratory indeed demonstrated that the ₁-AS-ODNs used in this study decreases ₁-AR mRNA translation in the hearts for up to 20 days (Zhang et al., 2000). The reduction in mRNA could result from inhibition of transcription or induction of RNase H.

Although chemical -AR blockers are effective in the therapy of ischemic heart disease and are widely used in the short- and long-term management of patients with myocardial ischemia (Yusuf et al., 1985), these agents have several undesirable side effects related to their effects on central nervous system, peripheral vascular resistance, and tracheobronchial tree. Even the selective ₁-AR blockers, such as atenolol, lose their cardioselectivity at moderate doses. In addition, these agents need to be taken frequently, at least once daily, due to their short half-life. Furthermore, chemical -AR blockers do not influence -ARs at genomic level. Gene therapy, such as AS-ODNs directed at ₁-AR mRNA, have unique effects. A previous study from our group (Zhang et al., 2000) showed that a single i.v. injection of ₁-AS-ODNs delivered with cationic liposomes markedly decreased blood pressure for 20 days with maximum drop of 38 mm Hg and without significant bradycardia in SHR. However, ₁-AR density in SHR hearts was significantly decreased for 18 days with maximum reduction of 47% on day 4, 33% on day 10, and 29% on day 18, but there was no effect on ₂-AR density. Quantitative autoradiography indicated that the administration of ₁-AS-ODNs decreased ₁-AR density in cardiac ventricles and renal cortex without any effect on distribution of -ARs in brain. The demonstration in this study of preservation of cardiac function and protection of myocardium from lipid peroxidation during ischemia-reperfusion with a single dose of ₁-AS-ODNs with highly selective effects on the expression of ₁-AR in the myocardium complements the observations in SHR. Importantly, IN-ODNs, used as control for ₁-AS-ODNs, did not show any of these effects. These observations in a rat model are consistent with our hypothesis that up-regulation of ₁-AR expression is pathogenetically involved in cardiac dysfunction during ischemia-reperfusion. We suggest that the results of this study set the stage for conduct of similar studies in other models of myocardial ischemia and eventually in humans.

In this study, ischemia-reperfusion-induced cardiac dysfunction was evaluated by the measurement of CPP, LVEDP, and dLVP. These indices of myocardial dysfunction have been used in several studies in the isolated heart model of global ischemia-reperfusion (Yang et al., 1993, 1997, 1998; Kokita et al., 1998; Ozden et al., 1998). Isolated rat, rabbit, or guinea pig heart models provide an inexpensive and reproducible method to evaluate cardiac function and myocardial metabolic alterations during ischemia-reperfusion. We and others have used this model extensively to study regulation of variety of receptors and modulation of cardiac function by agents acting on different receptors (Neely and Rovetto, 1975; Lu et al., 1990; Yang et al., 1993, 1997, 1998; Kokita et al., 1998). In the isolated beating heart, cardiac function can be assessed independent of the influence of circulating blood cells and hormones, which may be considered an important advantage of this model.

MDA, a lipid peroxidation product, has been used as an index of tissue injury after ischemia-reperfusion by several investigators (Kokita et al., 1998; Ozden et al., 1998). Increase in myocardial MDA was attenuated by the use of AS-ODNs in this study. One of the limitations of this study is absence of clear demonstration of AS-ODNs taken up by the heart. Nonetheless, several studies have shown substantial uptake and relatively good stability of phosphorothioate deoxynucleotides in cardiac tissues (Agrawal et al., 1991; Raynaud et al., 1997). Our previous study (Zhang et al., 2000) with the AS-ODNs used in this study showed a dramatic reduction in ₁-AR density in the myocardium. Furthermore, we now show that AS-ODNs decrease the up-regulation of ₁-AR mRNA and protein in the heart during ischemia-reperfusion; thus strongly suggesting that the AS-ODNs were taken up by the myocardium. Because this study was carried out in an isolated heart preparation, the peripheral effects of AS-ODNs were not taken into account. As such, the role of peripheral factors in the preservation of cardiac function cannot be ascertained from this study.

In summary, this study is the first report on the amelioration of cardiac dysfunction and myocardial injury induced by ischemia-reperfusion in the isolated rat heart with a single i.v. injection of AS-ODNs directed at ₁-AR mRNA. This study also provides evidence that the AS-ODNs can block the augmented expression of ₁-AR in the ischemic myocardium. The cardiac dynamic effects of AS-ODNs appear to be equivalent to those of atenolol in the isolated rat heart model of ischemia-reperfusion.