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Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425-2211
Submitted 26 March 2003 ; accepted in final form 5 June 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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superoxide; Akt; Bax; p38 MAPK
Acute myocardial ischemia induced by coronary artery occlusion causes myocardial dysfunction and lethal arrhythmia. Myocardial ischemia-reperfusion (I/R) induces cardiac cell damage and apoptosis (41). Reactive oxygen species (ROS) have been considered culprits because ROS take part in oxygen toxicity and induce cellular damage by oxidation of proteins and lipids through modification of genomic and cellular structures (37). Increased ROS generation has been reported during both the ischemic and reperfusion periods (1). Potential sources of ROS include endothelial enzymes (e.g., xanthine oxidase, NADH/NADPH oxidases, cyclooxygenases, and NOS), myocyte mitochondria, activated neutrophils, and the metabolic products of arachidonic acid and catecholamines. AM produced in cardiac myocytes, fibroblasts, and endothelial and vascular smooth muscle cells may act as an autocrine/paracrine modulator in the process of cardiac remodeling and apoptosis in the heart (31).
We (5, 6, 32, 40) have recently shown that human AM attenuates hypertension and protects against cardiac hypertrophy and fibrosis as well as renal damage in deoxycorticosterone acetate-salt, Dahl salt-sensitive, and two kidney, one-clipped hypertensive rats. Expression of recombinant human AM levels in rat plasma and urine was highest between 5 and 7 days after AM gene delivery (5, 6, 32, 40). In this study, we further investigated the role and mechanisms of action of AM in cardiac protection in acute I/R injury. We show that adenovirus-mediated AM gene transfer significantly attenuated myocardial infarction (MI), ventricular arrhythmia, and apoptosis after I/R via suppression of oxidative stress and activation of the Akt signaling pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Animals. Wistar rats (male, 250–280 g, Harlan Sprague Dawley) were used in this study. The study complied with the standards for care and use of animals as stated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. One week before coronary occlusion, rats were randomly divided into four groups. The first group was injected with saline (control, n = 13), the second group was injected intravenously with Ad.CMV-GFP (n = 12) or Ad.Null (n = 10), and the third group was injected with Ad.CMV-AM (n = 16) at a dose of 2 x 1010 plaque-forming units (pfu)/rat via the tail vein. The fourth group was injected with Ad.CMV-AM (n = 6) 1 wk before infusion of an AM antagonist, human calcitonin gene-related peptide [CGRP(8–37); 20 nmol/kg], through the femoral vein at 15 min before surgery.
Animal surgery and hemodynamic analysis. The surgical procedures were performed as previously described (32). Rats were anesthetized with pentobarbital sodium (50 mg/kg ip), intubated, and then ventilated with room air using a positive-pressure respirator (Harvard Apparatus). Once hemodynamics were stabilized, coronary occlusion was performed by tightening the suture loop for 30 min. Acute myocardial ischemia was deemed successful on the basis of regional cyanosis of the myocardial surface distal to the suture, accompanied by elevation of the ST segment on ECG. The loop was then loosened, and reperfusion was identified on the basis of return of the original color, accompanied by an obvious ST segment change. A microtransducer catheter was inserted into the femoral artery. An ECG was obtained by subcutaneously inserting needle electrodes into the limbs. Mean arterial pressure (MAP) and heart rate (HR) were recorded using a polygraph system (Grass Instruments). MAP and ECG were monitored throughout the experimental period. Ventricular arrhythmia was quantified according to Lambeth conventions. We excluded infarct size analysis of rats in which ventricular fibrillation (VF) persisted for >6 s or in which cardioversion was performed more than four times. The incidences of VF were evaluated as they occurred. Rats with recurrent tachycardia were excluded from further analysis because the ligation occasionally resulted in acute severe MI and malignant ventricular arrhythmia.
Measurement of myocardial infarct size. After a 120-min reperfusion period, the loop around the left anterior descending coronary artery was tightened, and 5% Evans blue was rapidly injected into the left ventricle to distinguish the nonischemic area from the area at risk. The heart was then excised, and the atria, vessels, and right ventricle were dissected. The left ventricle was cut into three to four slices transversely from the base to apex. The slices were incubated at 37°C with 4% triphenyltetrazolium chloride (TTC) for 30 min. Each slice was photographed, and the information was downloaded into a computer. The infarct area (unstained), area at risk (brick red stained), and noninfarct area (blue stained) were measured using NIH Image software. Infarct size expressed as a percentage of the area at risk versus the total area was the average of three to four slices from each heart.
Detection of apoptosis with in situ nuclear DNA fragmentation and DNA laddering. DNA fragmentation was determined using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL; using an in situ cell death detection kit, Roche) (36). TUNEL-positive cardiomyocytes in the ischemic myocardium were carefully distinguished from TUNEL-positive noncardiomyocytes and evaluated under double-blind conditions. The ratio of TUNEL-positive cardiomyocytes to the total number of cardiomyocytes was calculated. DNA laddering was analyzed as described (32, 40). DNA fragments (20 µg each) were separated by 1.2% agarose gel electrophoresis and visualized under ultraviolet light.
Measurement of superoxide production and density. Superoxide production was measured after both ischemic and reperfused periods using a ferricytochrome c reduction assay. Left ventricles were homogenized in lysis buffer [25 mM Tris·HCl (pH 7.4), 1% Triton X-100, 0.1% SDS, and 2 mM EDTA] at 4°C and immediately stored at –80°C. Superoxide production was quantified by a spectrophotometric assay based on rapid reduction of ferricytochrome c to ferrocytochrome c (29). 
-dependent reduction of cytochrome c was corrected for by deducting the activity not inhibited by superoxide dismutase (SOD). After the I/R period, Mn+2/3,3'-diaminobenzidine (DAB) was also used to visualize histochemically superoxide density (2). Cardiomyocytes in the ischemic area of the left ventricle were analyzed in three separate fields for each tissue section. The mean numbers of positively stained capillaries per field were calculated using NIH Image software under blinded conditions.
NADH/NADPH oxidase activity measurements. NADH/NADPH oxidase activity was measured using a lucigenin chemiluminescence assay (22). Heart tissues were excised and then homogenized in lysis buffer. Superoxide production in the cardiac extract in the presence of the substrate NADH (100 µM), NADPH (100 µM), or xanthine (100 nM) in the presence or absence of SOD (34 U/ml) was measured by the lucigenin-derived chemiluminescence. The lucigenin concentration in the final reaction mixture was 75 µM. Light emission levels were expressed as relative light units per minute per milligram of protein.
Western blot analysis for Akt, Bad, Bcl-2, Bax, p38 MAPK, and caspase-3 activation. Heart tissues were homogenized and lysed in extraction buffer containing 10 mmol/l Tris (pH 7.4), 100 mmol/l NaCl, 0.1%SDS, 1% Triton X-100, 5 mmol/l EDTA, 2 mmol/l Na3VO4, and protease inhibitor cocktail (Sigma). Aliquots were resolved by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane, blocked with 5% nonfat milk, and then incubated with primary antibodies that recognize Akt, phospho-Akt, Bad, phospho-Bad (Ser136), p38 MAPK, phospho-p38 MAPK Bax, Bcl-2, and cleaved caspase-3 (Cell Signaling) at 4°C overnight. Bound antibodies were detected by a secondary antibody conjugated to horseradish peroxidase and visualized by ECL-Plus chemiluminescence detection.
Radioimmunoassays for cGMP, cAMP, and human AM levels. The heart was homogenized in 0.1 mol/l HCl at 4°C. Aliquots of the supernatants were used for measuring cardiac cGMP and cAMP levels by radioimmunoassay (RIA) (11). Immunoreactive human AM levels in rat plasma were determined by RIA using rabbit anti-human AM 1–52 anti-serum (Peninsula Laboratories) (6, 32, 40).
Statistical analysis. Data are expressed as means ± SE and were compared between experimental groups with the use of one-way ANOVA, followed by Fisher's protected least-significant-difference test. HR and MAP were compared with the use of two-way ANOVA. Binomially distributed data (VF incidence and sustenance) were compared using the 
2-test and Fisher's exact probability test. A value of P < 0.05 was accepted as significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Figure 2 shows the effect of AM gene transfer on the incidence of ventricular arrhythmias induced by myocardial I/R. Ventricular premature beats were observed from 5 to 15 min in rats with coronary artery occlusion. The incidence of VF after acute I/R injury was similar among the experimental and control groups. AM significantly inhibited the rate of sustained VF compared with control rats (0% vs. 28.6%, n = 11 and 14 respectively, P < 0.05), and the beneficial effect of AM gene transfer was reversed by CGRP(8–37).
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AM gene delivery attenuates cardiomyocyte apoptosis. Figure 3A shows representative apoptotic cardiomyocytes as identified by TUNEL staining after I/R. The ratio of TUNEL-positive cardiomyocytes to total number of cardiomyocytes in the Ad.CMV-AM group was significantly reduced compared with the Ad.CMV-GFP group (25.2 ± 0.1% vs. 42.1 ± 0.9%, n = 7 and 7, P < 0.001; Fig. 3B). The beneficial effect of AM gene delivery on apoptosis in cardiomyocytes was abolished by CGRP(8–37) (25.2 ± 0.1% vs. 38.9 ± 1.6%, n = 7 and 6, P < 0.001). Furthermore, I/R markedly increased DNA laddering compared with that of the sham group, which was reduced after AM gene transfer (Fig. 3C). The protective effect of AM on DNA fragmentation was abolished by CGRP(8–37).
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Cardiac cGMP, cAMP, and human AM levels. AM gene transfer significantly increased cGMP levels in the ischemic heart compared with the sham group injected with Ad.CMV-GFP (0.14 ± 0.01 vs. 0.07 ± 0.01 pmol/mg protein, n = 6 and 4, P < 0.001; Fig. 4). No significant difference of cardiac cAMP levels was detected between the rats receiving AM gene delivery and control rats (11.5 ± 1.4 vs. 10.8 ± 1.6 pmol/mg protein, n = 6 and 4). Immunoreactive human AM levels in rat plasma at 5 days after gene transfer reached a level of 50.2 ± 3.1 ng/ml (n = 4).
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AM gene transfer reduces cardiac superoxide levels and NAD(P)H oxidase activity. AM gene delivery significantly attenuated cardiac superoxide production during the ischemic period compared with the control group (23.9 ± 5.6 vs. 51.2 ± 7.7 pmol·mg protein–1·min–1, n = 7 and 6, P < 0.001; Fig. 5A). After I/R, AM gene transfer also attenuated superoxide production compared with the control group (21.5 ± 4.1 vs. 49.8 ± 4.2 pmol·mg protein–1·min–1, n = 8 and 7, P < 0.001; Fig. 5B). With the use of the Mn+2/DAB histochemical technique, AM gene transfer also attenuated superoxide density after reperfusion compared with the control group (17.2 ± 3.9 vs. 38.2 ± 8.2 number/mm2, n = 9, P < 0.05; Fig. 5C). AM gene transfer significantly attenuated NADH oxidase activity compared with the control group (2.47 ± 0.16 vs. 4.91 ± 0.95 relative light units·mg protein–1·min–1, n = 6 and 5, P < 0.05; Fig. 5D). Similarly, AM gene transfer also reduced NADPH oxidase activity (0.36 ± 0.06 vs. 0.87 ± 0.09 relative light units·mg protein–1·min–1, n = 6 and 5, P < 0.05). Cardiac xanthine oxidase activities were very low in all the groups.
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Western blot analysis of apoptosis-related proteins. Figure 6A shows representative Western blots of anti-apoptotic and proapoptotic proteins. Quantitative results of phospho-Akt, phospho-Bad, phospho-p38 MAPK, Bcl-2, Bax, and cleaved caspase-3 signals after the I/R period are shown in Fig. 6, B–G. AM gene delivery significantly enhanced phospho-Akt, phospho-Bad (Ser136), and Bcl-2 compared with the control group. However, AM reduced Bax and phospho-p38 MAPK and cleaved caspase-3 activation compared with the control group. Total Akt, Bad, and p38-MAPK levels were not different among these groups.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Expression of human AM mRNA was identified in rat myocardium by RT-PCR Southern blot analysis in rats after intravenous injection of adenovirus containing human AM cDNA (5, 6, 32, 40). However, human AM mRNA was not detected in the heart of rats receiving control virus containing a reporter gene. In addition, we detected secretion of immunoreactive human AM levels in rat serum and urine for 2–3 wk, with the highest level between 5 and 7 days after gene delivery (5, 6, 32, 40). On the basis of these results, we investigated the cardioprotective effect of AM on ischemia and reperfusion injury at 5 days after gene delivery.
Plasma AM levels increase after acute MI, and the levels are correlated with the severity of MI in patients (25). In the rat MI model, cardiac AM synthesis is rapidly induced, suggesting a role of AM in the regulation of the cardiovascular system (26). It seems unlikely that AM-mediated cardioprotection is due to its vasodilatory activity, because the hemodynamic variables such as HR and MAP were not altered throughout the experimental period among experimental and control groups. It is well known that I/R sometimes induces serious ventricular arrhythmia, including VF. Under experimental conditions, we observed short-duration ventricular arrhythmias after I/R, yet there was no significant difference in the incidence of arrhythmia among experimental and control groups (data not shown). On the other hand, we observed serious arrhythmia at the beginning of the ischemic period, and the mortality rate increased up to 39% as a consequence of arrhythmia. However, AM gene transfer significantly attenuated the rate of sustained VF (Fig. 2).
ROS have been shown to induce cellular damage by oxidation of proteins and lipids (37) and therefore might contribute to the genesis of reperfusion arrhythmia due to membrane damage. To clarify the relationship between superoxide radicals and lethal arrhythmia, we measured superoxide levels at the end of the ischemic period. Consistent with a previous report (1), our results showed that superoxide levels were elevated during the ischemic period. These results indicate that AM-mediated suppression of superoxide production may in part contribute to the attenuation of ventricular arrhythmia in the ischemic heart.
The biological activity of AM has been shown to be mediated by second messengers and cAMP- and NO-cGMP-dependent pathways (16, 30). In this study, we showed that AM gene transfer increased cardiac cGMP levels in the myocardium after I/R. Consistent with this result, our previous study (5) also showed that AM gene transfer resulted in increased cardiac cGMP levels in streptozotocin-induced diabetic rats. cGMP is an indicator of NO production, and NO has been shown to inhibit NAD(P)H oxidase activity and thus superoxide formation in vascular cells (12). Therefore, it is likely that AM may protect against myocardial injury by functioning as an antioxidant through a NO-cGMP signaling pathway.
Myocardial I/R injury has been shown to induce myocardial apoptosis and necrosis (18, 41). We showed that AM gene transfer significantly attenuated myocardial apotposis after I/R injury as assessed by the TUNEL assay, DNA laddering, and caspase-3 activation (Figs. 3 and 6). Several signaling pathways evoke cardiomyocyte apoptosis (8). A redox-regulated system, which is activated by oxygen radicals, is one of the possible proapoptotic signaling pathways in cardiomyocytes. Potential sources of NAD(P)H oxidases could originate from endothelial cells, vascular smooth muscle cells, fibroblasts, and neutrophils (24, 38). AM is synthesized in cardiomyocytes, endothelium, and vascular smooth cells; therefore, the beneficial effect of AM may be attributed to autocrine/paracrine regulation. Neutrophils produce high levels of superoxide and prefer NADPH to NADH as a substrate for oxidation (28). We showed that NADH oxidase activity was markedly higher than NADPH oxidase activity and cardiac superoxide levels were lower than the estimated "burst" level derived from neutrophils. The results indicate that superoxide production in the heart might not originate from neutrophils. However, we do not have the data to support the speculation that increased superoxide formation in the I/R group originates from endothelial cells, vascular smooth muscle cells, or fibroblasts. In addition, AM gene delivery significantly attenuated superoxide levels, NAD(P)H oxidase activity, Bax, and p38 MAPK activation after the I/R period (Fig. 6). ROS induce activation of p38 MAPK family members in cardiac cells (34) and stimulate the expression of Bax, a proapoptotic factor, in bovine luteal cells, resulting in apoptosis (27). The effects of AM have been shown to be mediated by pathways linked to NO (12), which suppresses oxidative stress via inhibition of NADPH oxidase in neutrophils (4). These results indicate that binding of AM to its receptor in the heart activates the second messenger NO/cGMP, which suppresses ROS, leading to inhibition of Bax expression and p38 MAPK activation.
Akt-mediated signaling pathway is known to promote cell survival. Activation of Akt has been shown to enhance Ca2+-independent activation of eNOS and thus increase NO-cGMP formation (9). We showed that AM gene transfer increased phosphorylation of Akt, which leads to increased phospho-Bad (Ser136) and Bcl-2, but reduced Bad and caspase-3 activation. Bad has been shown to be a proapoptotic member of the Bcl-2 family that binds to Bcl-2 and Bcl-XL, resulting in cell death through the release of cytochrome c from mitochondria and thus the activation of caspases. Phospho-Akt may inhibit apoptosis by phosphorylating the Bad component of the Bad/Bcl-XL complex to enable cell survival (21, 41). Phosphorylation of Bad prevents the binding of Bad to Bcl-XL and Bcl-2, thus reducing Bad and increasing the survival factors Bcl-2 and Bcl-XL. Increased NO/cGMP levels could inhibit ROS production, leading to suppression of Bax expression and p38 MAPK activation. Taken together, these results indicate that AM protects against apoptosis after I/R injury via Akt-Bad and ROS signaling pathways.
Perspectives. Occlusion of the coronary artery causes cardimyocyte dysfunction and eventual tissue necrosis. Reperfusion relieves ischemia and prevents extensive damage by providing cells with oxygen. However, reperfusion initiates a series of events that results in ventricular infarction and arrhythmia and accelerates myocardial apoptosis and necrosis. AM is a potent vasodilator and natriuretic peptide that plays an important role in cardiovascular and renal function. Elevated AM production could be a biological attempt to compensate for cardiac and renal damage. Using an adenovirus-mediated gene transfer approach, we demonstrated that AM exhibits cardiac protection against I/R-induced MI and apoptosis. One advantage of this technique is that one injection of the AM gene construct could provide a continuous supply of AM with a long-lasting effect (for weeks or months) without apparent side effects. Understanding the role of AM and signaling mechanisms in I/R-induced myocardial apoptosis could provide the impetus for developing AM-based therapeutics to prevent myocyte loss and cardiac dysfunction. Therefore, successful application of this technology may have a protective effect in coronary artery diseases.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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