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Department of Physiology, New York Medical College, Valhalla, New York 10595
Submitted 3 December 2002 ; accepted in final form 8 May 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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nitric oxide; Western blotting; tempol; mitochondria; SOD1; SOD2; SOD3; p67phox; gp91phox; apocynin
The regulation of myocardial metabolism by NO [specifically endogenous endothelial NOS (eNOS)-derived NO] may be one of its most significant roles. The discovery by Hibbs and colleagues (18) of the biological synthesis of NO by activated macrophages provided support for the concept that NO regulates O2 consumption as part of a host defense mechanism. It has been shown that NO has a physiological role to regulate mitochondrial respiration by binding to cytochrome oxidase (27). For example, Bernstein et al. (5) showed that NO plays a role in the regulation of myocardial O2 consumption during exercise. Shen et al. (23) showed that preventing NO synthesis in vivo led to a significant (10%) rise in whole body O2 use. This increase was due to the loss of the inhibitory effect that NO has on tissue metabolism (23). eNOS, which is the most highly expressed isoform of NOS in vascular tissue under physiological conditions, is responsible for the control of tissue O2 consumption by NO (16).
During heart failure and renal failure, the aging process, and diabetes, NO production decreases or is abolished (27). After the onset of pacing-induced heart failure, endothelium-dependent responses also decrease in peripheral and coronary blood vessels. There is a significantly reduced level of eNOS mRNA, eNOS protein, and cardiac NO production during heart failure (24), a disease of aging. This downregulation of eNOS causes a decrease in NO production (15). The reduction of NO production may contribute to the elevated O2 consumption found in patients with heart failure (33).
We hypothesized that the decreased production of NO contributes to the cardiovascular disease associated with aging. Male Fischer 344 rats of 4, 14, and 23 mo of age, a model of accelerated aging, were used to study the role of NO in the control of myocardial O2 consumption. These age groups represent rats in their juvenile, adult, and senescent stages, and the hemodynamic and morphometric changes at these ages have been previously documented by Anversa et al. (3, 7). A unique focus of our studies was on the ability of NO to control cardiac O2 consumption and the potential role of altered gene expression for eNOS or sources of oxygen-derived free radicals in aging.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Transthoracic two-dimensional Doppler echocardiography studies for cardiac morphology and function. The anatomy and function of rat hearts were assessed with ultrasound techniques. Rats were anesthetized with 65 mg/kg pentobarbital sodium. Transthoracic echocardiography was performed in rats using an Acuson Sequoia 256 equipped with a 15-MHz linear transducer (15L8) in a phased-array format. Generally, the heart was first imaged by the two dimensionally guided M-mode cursor from the parasternal short-axis view. Left ventricular (LV) chamber dimension and wall thickness were measured from these M-mode tracings. LV end-diastolic (LVEDD) and end-systolic chamber dimensions (LVESD) as well as interventricular septum (IVST) and posterior wall thickness (PWT) were measured using American Society of Echocardiography leading-edge techniques. Measurements during three continuous cardiac cycles were averaged. We then imaged the heart by the bidimensional parasternal long-axis view to measure the ejection fraction (EF). EF is calculated as (LVDA – LVSA)/LVDA and expressed as a percent (where LVDA is the LV end-diastolic area, and LVSA is the LV end-systolic area).
Preparation of myocardial tissue and measurement of O2 consumption. The LV free wall was separated; freed of the large coronary arteries, connective tissue, and fat; and cut into pieces weighing 16–60 mg. Fibrous tissues were removed from the hearts of the oldest rats. Tissues were then incubated in Krebs bicarbonate solution containing (in mmol/l) 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4, 1.1 MgSO4, and 5.6 glucose at 37°C, bubbled with 21% O2-5% CO2-74% N2 (pH 7.4) to equilibrate for at least 1.5 h. At the end of the incubation period, two pieces of tissue were placed in a continuously stirred chamber with 3.3 ml of air-saturated Krebs bicarbonate solution containing 10 mmol/l HEPES (pH 7.4). The chambers were sealed using Clark-type platinum O2 electrodes (Yellow Springs Instruments) that were connected to O2 monitors (model YSI 5331) to measure the uptake of O2 by the tissue. Succinate (10–3 mol/l, Sigma; St. Louis, MO), a substrate for complex II, and sodium cyanide (10–3 mol/l, Sigma), an inhibitor of complex IV of the electron transport chain, were added at the completion of the concentration-response curve to each agonist to verify that the changes in O2 uptake were due to mitochondrial respiration. This method has been used previously by our laboratory (16, 17).
Effect of bradykinin, enalaprilat, and amlodipine on myocardial O2 consumption. The effects of cumulative doses of the B2 kinin receptor agonist bradykinin (10–7–10–4 mol/l, Sigma), the angiotensin-converting enzyme (ACE) inhibitor enalaprilat (10–7–10–4 mol/l), and the calcium channel blocker amlodipine (10–7–10–5 mol/l) on myocardial O2 consumption were examined in tissue from 4-, 14-, and 23-mo-old Fischer 344 rats (n = 15 each). These responses were also examined in the presence of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10–4 mol/l, Sigma) to verify the role of NO production by NOS in the regulation of myocardial O2 consumption.
Effect of S-nitroso-N-acetyl penicillamine on myocardial O2 consumption. Cumulative doses of the NO donor S-nitroso-N-acetyl penicillamine (SNAP; 10–7–10–4 mol/l) on myocardial O2 consumption were examined in tissue from 4-, 14-, and 23-mo-old rats (n = 15 each). These responses were also examined in the presence of L-NAME.
Effect of tempol on myocardial O2 consumption. The effect of the SOD mimetic tempol (10–3 mol/l) on myocardial O2 consumption was examined in tissue from 23-mo-old rats (n = 8) in the presence of cumulative doses of bradykinin, enalaprilat, amlodipine, and SNAP. These responses were also examined in the presence of L-NAME.
Effect of apocynin on myocardial O2 consumption. The effect of apocynin (10–5 M) on myocardial O2 consumption was examined in hearts from 23-mo-old rats (n = 6) in the presence of cumulative doses of bradykinin, enalaprilat, amlodipine, or SNAP. Apocynin has been shown to inhibit NADPH oxidase in endothelial and smooth muscle cells in culture (10, 11) and in blood vessels ex vivo (26).
Immunoblotting. Myocardial tissue was snap frozen in liquid nitrogen and stored at –80°C. For preparation of extracts, tissue was pulverized in liquid nitrogen, followed by homogenization in 5 volumes of lysis buffer [0.05 M Tris · HCl (pH 7.2), 1 mM EDTA, 10 mM dithiothreitol, 1 mg/ml PMSF, 100 µg/ml leupeptin, 100 µg/ml soybean trypsin inhibitor, and 20 µg/ml aprotinin] three times for 15 s at 4°C and sonication for 1 min. Lysates were centrifuged at 10,000 g for 10 min at 4°C and stored at –80°C before use. Protein concentrations of supernatants were measured using a Bio-Rad protein assay (Bio-Rad Laboratories).
Samples of tissue lysate containing 20 µg of protein were loaded onto individual lanes of a 15% polyacrylamide gel containing the detergent SDS prepared using standard techniques. Proteins were separated according to their molecular size. Proteins in the gel were transferred from the gel to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech) using a semidry transfer cell (Bio-Rad Laboratories). Membranes were incubated for 1 h with 5% milk-PBS to block all other free protein binding sites on the membrane. Purified antibodies to eNOS and neuronal NOS (nNOS; 1:1,000 dilution, BD Transduction Laboratories) or inducible NOS (iNOS; 1:5,000 dilution, BD Transduction Laboratories), Cu/Zn SOD (1:1,000 dilution, Santa Cruz Chemicals), Mn SOD (1:1,500 dilution, Santa Cruz Chemicals), extracellular SOD (ecSOD; 1:2,000 dilution, a gift from Lili Fang, University of Colorado, Denver, CO), p67phox, or gp91phox (1:1,000 dilution, Upstate Biotechnologies) in 1% milk-PBS were added, and incubation was continued at 4°C overnight to allow the antibodies to bind to their respective proteins.
The location of binding of these antibodies was detected by incubation with a secondary antibody conjugated to horse-radish peroxidase. The location of antibodies was then visualized by the addition of Super Signal West Pico Chemiluminescent Substrate (Pierce; Rockford, IL), which produces light under the catalytic action of the horseradish peroxidase, and was detected by exposure to X-ray film (Kodak, Rochester, NY).
Quantification of proteins. The relative intensities of the bands in the exposed film were determined by scanning on an Alphaimager 2000 documentation and analysis system (Imgen Technologies; Alexandria, VA), followed by analysis using image software.
Statistical analysis. Data are presented as means ± SE; n represents the number of animals studied. Comparisons between groups were made using one-way ANOVA, followed by correction for multiple comparisons using Bonferroni's t-test. Statistical significance was accepted at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Baseline O2 consumption in myocardial tissues from 4-, 14-, and 23-mo-old rats. Baseline myocardial O2 consumption was not significantly different among the three age groups [147 ± 5, 147 ± 2, and 137 ± 4 nmol O2 · min–1 · g–1 (n = 7 each) at 4, 14, and 23 mo, respectively, P > 0.05]. These baseline responses were not affected by L-NAME [143 ± 7, 133 ± 8, and 135 ± 9 nmol O2 · min–1 · g–1 (n = 7 each) at 4, 14, and 23 mo, respectively, P > 0.05].
Effect of bradykinin on myocardial O2 consumption. Bradykinin (10–7–10–4 mol/l) caused concentration-dependent decreases in myocardial O2 consumption in the 4-, 14-, and 23-mo-old rats (Fig. 1A; 4 mo: from 9 ± 2% to 36 ± 2%; 14 mo: from 8 ± 1% to 29 ± 1%; 23 mo: from 4 ± 1% to 15 ± 3%). The response to bradykinin in 23-mo-old rats was significantly less than in the 4- and 14-mo-old rats (P < 0.05). The addition of L-NAME significantly inhibited the response to bradykinin at 4 and 14 mo (Fig. 1, A and B). The response to bradykinin with L-NAME was, however, not significantly different in the 23-mo-old rats (Fig. 1, A and B).
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Effect of amlodipine on myocardial O2 consumption. Amlodipine (10–7–10–5 mol/l) caused concentration-dependent decreases in myocardial O2 consumption in the 4-, 14-, and 23-mo-old rats (from 12 ± 2% to 33 ± 2%, from 9 ± 1% to 23 ± 2%, and from 8 ± 2% to 17 ± 3%, respectively). The response to amlodipine in 23-mo-old rats was significantly less than in 4-mo-old rats. The addition of L-NAME significantly inhibited the response to amlodipine at 4, 14, and 23 mo. The response to amlodipine with L-NAME was, however, very small in the 23-mo-old rats.
Effect of enalaprilat on myocardial O2 consumption. Enalaprilat (10–7–10–4 mol/l) caused concentration-dependent decreases in myocardial O2 consumption in the 4-, 14-, and 23-mo-old rats (from 11 ± 1% to 35 ± 3%, from 10 ± 2% to 25 ± 3%, and from 1 ± 1% to 7 ± 2%, respectively). The response to enalaprilat in 23-mo-old rats was significantly less than in hearts from 4- and 14-mo-old rats (Fig. 2). The addition of L-NAME significantly inhibited the response to enalaprilat at 4 and 14 mo but not at 23 mo.
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Effect of SNAP on myocardial O2 consumption. SNAP (10–7–10–4 mol/l) caused concentration-dependent decreases in myocardial O2 consumption in the 4-, 14-, and 23-mo-old rats (from 8 ± 2% to 35 ± 3%, from 6 ± 1% to 35 ± 2%, and from 11 ± 3% to 44 ± 1%, respectively). The response to SNAP in 23-mo-old rats was not significantly different from the 4- and 14-mo-old rats. L-NAME did not significantly alter the response to SNAP at any age.
Effect of tempol on myocardial O2 consumption. The addition of tempol (10–3 mol/l) caused a significant shift in the dose-response curve to all three agonists in tissue from 23-mo-old rats (Fig. 3). Tempol did not have a significant affect on O2 consumption in the presence of SNAP (10–4 mol/l; from 44 ± 1% to –37 ± 2%). eNOS protein during aging. Western blots indicated that eNOS protein did not decrease during aging (Fig. 4). We were unable to detect significant amounts of nNOS or iNOS at any age.
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Levels of SOD in aging. Western blots indicated that levels of Cu/Zn SOD or Mn SOD protein did not vary significantly during aging, whereas ecSOD actually increased significantly (Fig. 5).
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Levels of NADPH subunits in aging. Western blots indicated that levels of p67phox protein did not vary significantly in aging. A slight decrease, however, was observed in tissue from 23-mo-old rats (Fig. 6). In contrast, there was a marked sixfold increase in gp91phox at 23 mo.
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Effect of apocynin on myocardial O2 consumption. Apocynin increased the NO-dependent control of cardiac O2 consumption (Fig. 7) to bradykinin, enalaprilat, or amlodipine but had no effect on the response to SNAP in hearts from 23-mo-old Fischer rats.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Our data indicate that pretreatment with L-NAME had no effect on baseline O2 consumption in hearts from 4-, 14-, and 23-mo-old rats. This suggests that the basal release of NO is small in the absence of an agonist that stimulates NO production in our preparation. Our data suggest that endogenous NO production or biological activity of NO are greatly reduced in tissues from senescent (23 mo old) rats. This reduction in NO bioavailability leads to the decreased ability of bradykinin, for instance, to modulate myocardial mitochondrial O2 consumption. The impairment of NO bioactivity and the resulting elevated O2 consumption rate in aging may contribute to the pathogenesis of heart failure (15, 17, 21, 24). The reduction of NO activity in the 14- and 23-mo-old tissue was not due to decreased eNOS, as evidenced by Western blotting.
One of the most striking findings in this study was that tempol, a SOD mimetic, restored the regulation of cardiac O2 consumption by bradykinin, enalaprilat, and amlodipine in hearts from 23-mo-old Fischer rats to a level that was not different from hearts from 4-mo-old Fischer 344 rats. It has previously been shown that tempol reduces superoxide levels (1). The lack of change in several proteins regulating the production of superoxide in aging (Cu/Zn SOD, Mn SOD, and ecSOD) demonstrates that increased biological activity of superoxide (because we did not directly measure superoxide levels) is not due to loss of SOD in aging. To better understand the mechanism, and because the measurement of the p67phox subunit of NADPH oxidase may not reflect tissue NADPH oxidase levels, we also measured gp91phox. gp91phox was elevated almost sixfold in hearts from 23-mo-old rats, suggesting that NADPH oxidase is a likely source of superoxide. Although we have no explanation for the apparent discrepancy between the measurement of p67phox and gp91phox, we repeated measurements of tissue O2 consumption in hearts from 23-mo-old Fischer rats in the presence of apocynin, a drug that blocks the assembly of NADPH oxidase. Apocynin entirely restored the control of O2 consumption to bradykinin, enalprilat, or amlodipine in hearts from 23-mo-old Fischer rats to that in 4-mo-old Fischer 344 rats. It is therefore likely that NADPH oxidase is responsible for the increase in oxidant stress, not a decrease in the enzyme that produces NO, leading to the impairment of NO bioactivity in aging. Apocynin has been shown to reversibly block the assembly of NADPH oxidase, inhibiting the cytochrome b portion and the generation of superoxide. Apocynin is active in endothelial cells (11), smooth muscle cells (10), and blood vessels (26). Ours is the first study to indicate that apocynin can modulate the control of O2 consumption by the heart. We did not test apocynin or tempol in the youngest Fischer rats; nevertheless, both these agents had substantial effects in hearts from 23-mo-old Fischer 344 rats, the focus of our study.
The mechanism by which enalaprilat, an ACE inhibitor, and amlodipine, a calcium channel agonist, release NO is through a kinin-dependent mechanism that has been described previously (8). Both enalaprilat and amlodipine cause NO release in coronary microvessels, and NO release is blocked by L-NAME. In the 4- and 14-mo-old rats, we observed dose-dependent decreases in myocardial O2 consumption by enalaprilat and amlodipine that were abolished in the presence of L-NAME. ACE inhibitors, such as enalaprilat, have been shown to be effective in the treatment of patients with heart failure (22). Calcium channel blockers, more specifically amlodipine, may reduce the risk of death in patients with certain types of heart failure. Despite the controversy over the use of calcium channel blockers in patients with heart failure, amlodipine may reduce the risk of morbidity and mortality in patients with nonischemic dilated cardiomyopathy (20). Amlodipine has also been shown to prevent cardiac cell death (28, 31). The ability of these agents to release NO and lower mitochondrial respiration may be an important mechanism to maintain the balance between O2 demand and supply. In our study, the decreased suppression of tissue O2 consumption by enalaprilat and amlodipine in the 23-mo-old rats shows that a deficiency in a NO-mediated kinin-dependent mechanism occurs with aging. If these drugs and oxidant radical scavengers can be used together, they may be more effective in the treatment of the cardiovascular diseases associated with aging.
Bradykinin, a B2 kinin receptor agonist, stimulates endogenous NO production through the activation of eNOS. This production is blocked in the presence of L-NAME. Bradykinin caused a significantly smaller reduction in myocardial O2 consumption in the 23-mo-old rats compared with the 4- and 14-mo-old rats. As evidenced by the Western blots, there was no down-regulation of eNOS in the 23-mo-old rats. We also used Western blotting to detect both nNOS and iNOS in the heart. There were no signals for these NOSs in hearts from 23-mo-old Fischer rats. Therefore, the reduced response to bradykinin was not due to a decreased level of eNOS in 23-mo-old Fischer rats.
In our study, the exogenous NO donor SNAP reduced tissue O2 consumption to a degree that was not different in the 4-, 14-, and 23-mo-old rats. These responses were not affected by the addition of L-NAME. The loss of the ability of bradykinin, enalaprilat, and amlodipine, therefore, is not a result of an impairment of the ability of NO to modulate tissue O2 consumption, i.e., altered mitochondrial sensitivity to NO, but rather a reduction in biologically active NO. This is most likely due to superoxide, as indicated by studies using tempol, and due to the activation of NADPH oxidase, as indicated by the present studies using apocynin and measurement of gp91phox.
Western blotting revealed that eNOS protein did not decrease with aging. We hypothesized that the decreased regulation of O2 consumption by NO with aging may be therefore explained by the increased biological activity of superoxide anions in the older rats (9, 12, 30). Tempol, which breaks down superoxide anions, restored the regulation of O2 consumption by all three agonists in the 23-mo-old rat heart to that seen in the 4-mo-old rat heart.
In the present study, we have clearly shown that the stimulation of endogenous NO production failed to reduce tissue O2 consumption in 23-mo-old rats. Therefore, therapeutic use of drugs such as enalaprilat and amlodipine may be less effective in older patients due to increased superoxide. The Fischer 344 rats we used have well-characterized, distinct points in their life cycle. The senescent animals show myocyte loss, scar formation, replacement fibrosis, increased wall stress, and LV weight as well as a decrease in first derivative of pressure development over time, cardiac output, EF, and shortening fraction. Our findings using echocardiography are in accord with those of Anversa et al. (3, 7). Despite the fact that calculated stroke volume increased in the anesthetized 23-mo-old Fischer 344 rats, the EF fell and the heart was dilated. A limitation in our study is the use of nonworking cardiac tissue to study the role of NO in the control of tissue O2 consumption in aging. Therefore, our data reflect the control of a small fraction of the O2 consumed in a working heart. Another study (32) has suggested that a defect in the ability of NO to stimulate guanyl cyclase may be important in aging using anesthetized open chest rabbits. In that study, SNAP had little effect on myocardial O2 consumption in old rabbits or to increase cGMP despite high basal cGMP levels. Weiss et al. (32) concluded that there was a decreased sensitivity to NO, although there was no test of NO production or the role of superoxide anion. We, however, have not found a role for guanyl cyclase in the control of O2 consumption by bradykinin using our methods. A number of other studies (4, 6, 14, 19, 25) have both supported and denied a role for increased oxidant stress in aging. Nevertheless, our results indicate that the biological activity of NO is impaired in old age and that this may have direct consequences on cardiac metabolism.
Several mechanisms exist for production of
radicals (xanthine oxidase, eNOS,
mitochondria, and cyclooxygenase). Levels of
radicals are determined by
production and metabolism (destruction). Levels of Cu/Zn SOD, Mn SOD, and
ecSOD, which break down , and
p67phox, a NAD(P)H oxidase complex subunit, which produces
, were not significantly different in
the young and old animals. Notably, there was a marked upregulation of
gp91phox at 23 mo. Increased
radicals secondary to increased
production (23) are most
likely responsible. Apocynin restored the regulation of cardiac O2
consumption by agonists that stimulate NO production, confirming our
measurments of gp91phox protein and the role of NADPH oxidase in
the generation of superoxide.
We explored the mechanisms that may cause the decrease in NO
bioavailability in 23-mo-old Fischer rats. Levels of eNOS protein were found
to increase in aging, whereas levels of Cu/Zn SOD, Mn SOD, ecSOD, and
p67phox were unchanged. In addition, we demonstrated that there is
an increase bioactivity of radical
in aging and that this was inhibited by apocynin and supported by the
upregulation of gp91phox. This increase may explain the decreased
bioactivity of NO in 23-mo-old Fischer rats. The concept that increased
oxidant stress contributes to decreased NO bioavailability and the possible
onset of cardiac dysfunction with age provides direction for new therapeutic
interventions. Therapeutic use of drugs such as enalaprilat and amlodipine,
which are used to alleviate a wide variety of cardiovascular disorders, may be
less effective in older patients with heart failure due to increased
bioactivity of superoxide anions. Conversely, the combination of these drugs
with antioxidants may restore the control of both cardiac metabolism and
regulation of vascular tone by NO during aging.
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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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-lipoic acid. FASEB J
15: 700–706,
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