Statin drugs or 3-hydroxy-3-methyl glutaryl (HMG)-coenzyme A (CoA) reductase inhibitors are widely used as cholesterol-lowering agents based on their ability to block hepatic conversion of HMG-CoA to L-mevalonate in the cholesterol biosynthetic pathway (1). Although the beneficial effects of HMG-CoA reductase inhibitors are primarily attributed to their lipid-lowering effects, statins have actions independent of their lipid effects (2-5). One of the earliest recognizable benefits after therapy with these drugs is normalization of endothelium-dependent relaxation in atherosclerotic coronary arteries (3). Laufs et al. (6,7) showed that the HMG-CoA reductase inhibitors, simvastatin and lovastatin, reversed the downregulation of endothelial nitric oxide synthase (eNOS) by hypoxia and oxidized low-density lipoprotein under cholesterol-clamped conditions. They also showed that prophylactic simvastatin administration protects against ischemic stroke after focal brain ischemia by upregulation of aortic and cerebral eNOS gene expression and NO production in normocholesterolemic mice (8). Like-wise, angiotensin-converting enzyme (ACE) inhibitors have pleiotropic effects beyond their hemodynamic effects that include improved endothelial function, antimitogenic effects, and antithrombotic effects (9). Ramipril, an ACE inhibitor, can reduce cardiomyocyte apoptosis in spontaneously hypertensive rat studies (10). The recently published HOPE trial demonstrated the clinical efficacy of ramipril in reducing morbidity and mortality in patients with cardiovascular risk factors, many of whom were receiving lipid-lowering therapy (11). This has led to an interest in studying whether combined use of statins and ACE inhibitors can act additively or synergistically in various clinical conditions.
ACE inhibitors delay breakdown of bradykinin to cause sustained stimulation of B2 kinin receptors with resultant activation of intracellular eNOS and NO production (12). Amlodipine, a calcium channel antagonist, also has the unique ability to increase endothelial NO production through enhanced kinin availability (13). NO modulates mitochondrial respiration and tissue oxygen consumption through reversible inhibition of cytochrome oxidase, the terminal enzyme complex of the mitochondrial electron-transport chain (14). Previous studies in our laboratory have demonstrated the ability of ACE inhibitors and amlodipine to cause NO-mediated decrease in myocardial oxygen consumption (MVO2) in isolated human and canine myocardium (15,16). Because statins can upregulate NO, the purpose of this study was to determine if short-term statin administration can augment the ability of ACE inhibitors and amlodipine to regulate MVO2 through an NO-dependent mechanism.
Male Wistar rats aged 9-10 weeks were divided into two groups, (a) control group (n = 14) and (b) simvastatin group (n = 10). Simvastatin tablets were crushed and dissolved in 1 ml alcohol each and further diluted with water to form a suspension. The simvastatin group was administered simvastatin suspension (20 mg/kg/day) by oral gavage for 2 weeks. This dose of simvastatin was chosen because simvastatin causes a dose-dependent increase in eNOS with maximal increase seen at 20 mg/kg dose in studies in mice conducted by Endres et al. (8). A dose of ≤160 mg/day has been shown to be safe in humans (17). Up to 55 mg/kg/day of statins failed to cause skeletal myopathy in adult rats, although myopathy was seen in the young rats at doses of ≥15 mg/kg/day (18). Rats were killed with an overdose of pentobarbital. Plasma nitrate and nitrite (NOx) levels and in vitro MVO2 were measured in control and simvastatin group. The protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the "Guiding Principles for the Use and Care of Laboratory Animals" of the National Institutes of Health and the American Physiological Society.
Arterial plasma NOx
Blood samples were drawn from the rat aorta into heparinized syringes. The whole blood was centrifuged at 1,000 g at 4°C, and the plasma was frozen. Plasma NOx was measured using a method developed in our laboratory (19). In brief, the plasma was incubated with Aspergillus nitrate reductase and acidified under an argon atmosphere to convert all the nitrate and nitrite in the plasma sample to NO. The gaseous NO was injected into a chemiluminescence analyzer (Sievers, Inc., Boulder, CO, U.S.A.). In the analyzer, NO combines with ozone to produce photons, the number of which is directly proportional to the amount of NO injected, and thus to the NOx from the plasma sample. Standard curves were generated through the addition of known amounts of potassium nitrite or sodium nitrate (Sigma Chemical, St. Louis, MO, U.S.A.) to plasma pooled from normal rats.
Myocardial oxygen consumption
Myocardial tissue was isolated from the left ventricular free wall of hearts excised from pentobarbital treated rats. The myocardium was freed of epicardium, endocardium, connective tissue, fat, and large arteries, and was cut into 30- to 50-mg segments. The muscle slices were incubated for 2 h in Krebs bicarbonate buffer (containing the following, in mM: 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.1 MgSO4, 1.2 KH2PO4, and 5.6 glucose) during which 21% O2/5% CO2/74% N2 (room air) was bubbled continuously. MVO2 was measured polarographically in vitro using a Clark-type oxygen electrode (YSI 5331; Yellow Springs Instrument Co. Inc., Yellow Springs, OH, U.S.A.). Oxygen-consumption studies were performed at 37°C in a stirred bath (YSI 5301) containing 3 ml Krebs solution buffered with 10 mM HEPES (pH 7.4). Tissue respiration was calculated as the rate of decrease in oxygen concentration after the addition of muscle slices, assuming an initial oxygen concentration of 224 nmol/ml and was expressed as nanomoles of oxygen consumed per minute per gram of tissue. After measurement of baseline MVO2, cumulative dose-response curves were generated after addition of various pharmacologic agents to separate tissue baths in increasing concentrations. Approximate observation time for each dose of agent was 5-7 min, and new muscle segments were used for each drug tested. Succinate, a substrate for the electron transport chain (1 mM) followed by sodium cyanide, an inhibitor of cytochrome oxidase (1 mM), was added at the end of each drug tested to confirm that the change in MVO2 was reversible and was from mitochondrial sources.
The following agonists that stimulate NO synthesis were added: bradykinin (10−7−10−4M), ramipril (10−7−10−4M), amlodipine (10−7−10−5M), and the NO-releasing drug, S-nitroso N-acetyl penicillamine (SNAP) (10−7−10−4M). To assess the role of endogenous NO production in mediating the effect of these agents on MVO2, these studies were repeated after preincubation of the muscle segments with Nw-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase (10−4M; i.e., eight experiments per rat heart). Each drug was studied using separate pieces of myocardium.
Source of drugs and chemicals
Chemicals (bradykinin, SNAP, L-NAME, nitrite) were purchased from Sigma Chemical, St. Louis, MO, U.S.A. Amlodipine was supplied by Pfizer Pharmaceutical (Groton, CT, U.S.A.). Ramipril was supplied by Hoechst-Roussel Inc (Somerville, NJ, U.S.A.). Simvastatin was obtained from Merck, Sharp and Dohme, Inc. (West Point, PA, U.S.A.).
Statistical analysis Plasma NOx levels and percentage change in MVO2 from baseline were expressed as mean ± SEM. Data were analyzed using an unpaired t test and two-way analysis of variance (ANOVA), with a Student-Newman-Keuls post hoc analysis to identify which means were different (Sigma Stat, Version 1.0; Jandel Scientific, San Rafael, CA, U.S.A.). A value of p < 0.05 was considered statistically significant.
Data from 14 control rats and 10 simvastatin-treated rats are presented. Plasma NOx levels did not change significantly after 2 weeks' simvastatin (19 ± 2.6 μM in control vs. 20 ± 2.5 μM after simvastatin; p = NS). There was no significant difference in baseline MVO2 in isolated left ventricular myocardium from control rats (288 ± 23 nmol/g/min) compared with simvastatin-treated rats (252 ± 11 nmol/g/min; p = 0.09).
Effect of bradykinin
Bradykinin caused a dose-dependent decrease in MVO2 in both control (Fig. 1a) and simvastatin-fed rats (Fig. 1b; p < 0.05). This effect was attenuated by preincubating tissue with L-NAME, NO inhibitor (Figs. 1a and b). Bradykinin caused a significantly larger reduction in MVO2 in simvastatin-fed rats (44 ± 6% at highest dose) compared with control rats (28 ± 5% at highest dose; Fig. 1c).
Effect of ACE inhibitor, ramipril
Ramipril caused a dose-dependent decrease in MVO2 in both control and simvastatin rats, which was attenuated by L-NAME (Figs. 2a and b). Ramipril caused a significantly larger reduction in MVO2 in simvastatin-fed rats (50 ± 8% at highest dose) compared with control rats (35 ± 5% at highest dose; Fig. 2c).
Effect of calcium channel antagonist, amlodipine
Amlodipine caused a dose-dependent decrease in MVO2 in both control and simvastatin-fed rats, which was attenuated by L-NAME (Figs. 3a and b). Amlodipine caused a significantly larger reduction in MVO2 in simvastatin-fed rats (42 ± 3% at highest dose) compared with control rats (32 ± 9% at highest dose; Fig. 3c).
Effect of NO donor, SNAP
SNAP caused a dose-dependent decrease in MVO2 in both control and simvastatin-fed rats, which was not attenuated by L-NAME (Figs. 4a and b). Dose-response curve to SNAP was not significantly different in simvastatin-fed rats (52 ± 7% at highest dose) compared with control rats (55 ± 5% at highest dose), except at the 10−6M dose (Fig. 4c).
The most important finding of this study is that short-term administration of simvastatin enhances NO-mediated inhibition of MVO2. Simvastatin potentiates the ability of ACE inhibitor and amlodipine to decrease MVO2 in isolated rat myocardium.
ACE inhibitors and amlodipine, a calcium channel blocker, have ancillary effects that are independent of their hemodynamic effects. These drugs promote kinin-dependent NO production in coronary microcirculation (12,13). Because NO is a highly diffusible gas, NO produced by coronary endothelium can diffuse into surrounding myocytes to regulate mitochondrial respiration and MVO2(20). Another group of drugs that appear to have a beneficial effect on endothelial function are the statin drugs, which are traditionally used as cholesterol-lowering agents. Statins can upregulate eNOS expression in isolated human endothelial cells independent of cholesterol-lowering effects (7). Our study demonstrates that statin administration can potentiate the ability of ACE inhibitors and amlodipine to regulate MVO2 by increasing NO availability.
Bradykinin, ramipril, and amlodipine caused a NO-dependent decrease in MVO2, which was confirmed by the ability of L-NAME, an inhibitor of NO synthase, to block the effect of these drugs. Bradykinin, ramipril, and amlodipine caused a significantly larger reduction in MVO2 in simvastatin-fed rats compared with controls, an effect that was blocked by inhibiting NO synthesis. The effect of SNAP on MVO2, conversely, was not significantly different between the two groups of rats. SNAP bypasses the step of NO biosynthesis and donates NO directly to the myocardium. Therefore, its effect on MVO2 reflects the functional state of mitochondrial respiration. Thus, our data suggest that baseline mitochondrial function was comparable in both groups and that the enhanced regulation of MVO2 in simvastatin-fed rats was secondary to augmentation of endogenous NO bioavailability.
These findings are important for two reasons. They demonstrate a novel action for statins (i.e., the ability to decrease MVO2 by a NO-dependent mechanism). The importance of this regulation in vivo has been demonstrated in several earlier studies from our laboratory. Bernstein et al. (20) demonstrated that inhibition of NO synthesis in conscious dogs is associated with increased cardiac oxygen consumption during exercise for the same amount of cardiac work, suggesting a decrease in cardiac efficiency in the absence of NO. The importance of this regulation in failing hearts was demonstrated by Recchia et al. (21), who showed that progression to decompensated heart failure in dogs with pacing-induced cardiomyopathy was associated with loss of endogenous NO production and deregulation of MVO2. These studies emphasized the importance of maintaining control over MVO2 in both physiologic and pathologic states. The ability of statins to regulate MVO2, as shown in our study, proposes an additional mechanism of action of statins that may contribute to their cardioprotective effects.
It is important to note that despite augmented response to NO agonists, baseline MVO2 was not significantly different between control and simvastatin-fed rats (p < 0.09). These estimations were made in isolated myocardium that is independent of preload and afterload. In the absence of vascular shear stress, a powerful physiologic stimulus for NO production, the differences in basal NO production may have been too small to cause detectable differences in basal oxygen consumption. Not unexpectedly, plasma NOx levels were not significantly different in control and simvastatin-fed rats. We have previously shown that plasma NOx levels alone are not a good indicator of systemic NO production (22) because of the ability of the kidney to excrete NO metabolites efficiently. We also did not measure serum cholesterol levels. However, it is important to recognize that these were normal rats with no heart disease and that simvastatin was administered for a relatively short duration of 2 weeks so that lipid-lowering alone was unlikely to account for the altered regulation of MVO2.
We did not directly measure NOS enzyme expression to determine whether the source of NO was endothelial or inducible NOS. However, the rapid decrease in MVO2 after addition of the agonists suggests stimulation of endothelial rather than inducible NOS, which requires hours rather than minutes for activation. The ability of bradykinin and kinin agonists to stimulate NO production also is specific for endothelial rather than inducible NOS (23).
The second important finding of this study is the demonstration of synergy between a statin drug and an ACE inhibitor or calcium channel blocker. Several studies have now shown that, to a variable degree, statins and ACE inhibitors have similar effects on a number of biologic processes, albeit through different mechanisms (24). However, it is not known whether the effects of these drug classes are additive or synergistic. We previously reported that ACE inhibitors and amlodipine act synergistically to regulate MVO2 in isolated canine and failing human hearts (15). Our study demonstrates that the effect of statins in increasing NO bioavailability is synergistic with ACE inhibitors and amlodipine because this augmentation was seen in the absence of a change in baseline oxygen consumption. The ability of ACE inhibitors and amlodipine to increase local kinin availability and of statins to upregulate eNOS expression may explain the augmentation of NO production by combined use of these drugs. A recent study by Luo et al. (25) reported that simvastatin significantly reduces left ventricular hypertrophy, reduces cardiac tissue ACE activity, and improves left ventricular performance in rats with aortic stenosis. Our study does not exclude the possibility that the synergy between simvastatin and ACE inhibitor was related to reduced cardiac ACE activity. However, the ability of simvastatin to potentiate the effect of amlodipine, which increases NO independent of ACE inhibition, suggests that the synergy among these drugs is not entirely related to ACE inhibition but possibly to other steps in the NO biosynthetic pathway.
This study may have important clinical implications for use of combination of these drugs, especially in conditions of lowered NO availability. Heart failure is associated with reduced endogenous NO production secondary to decreased eNOS expression (26). Restoring this regulation through use of drugs that enhance NO availability, such as ramipril, amlodipine, or statin, may reverse the progression of heart failure. The results of the HOPE trial demonstrate a beneficial effect of ramipril among patients who were already taking other treatments including lipid-lowering agents (11). These benefits included a reduction in all-cause and cardiovascular mortality, myocardial infarction, stroke, heart failure, and other major cardiac events. Only a small part of the benefits were attributable to a reduction in blood pressure, suggesting that these drugs may be exerting additional direct beneficial effects on the heart and/or blood vessels. The ability to regulate myocardial energetics as demonstrated in our study may provide a potential mechanism by which statins may act synergistically with ramipril or amlodipine to improve outcome in the high cardiovascular risk population. Clinical studies to evaluate the combined use of these drugs in high-risk patients is warranted.
Acknowledgment: This work was supported by HL 50142, 53053, 52233, and PO-1HL 43023 from the National Heart, Lung and Blood Institute. A.M. and P.R.F. were medical students and summer research fellows.
K.E.L. was supported by AHA Fellowship 9820046T from New York State Affiliate.
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