Heart Rate Reduction by Ivabradine Reduces Oxidative Stress, Improves Endothelial Function, and Prevents Atherosclerosis in Apolipoprotein E–Deficient Mice
Background— Elevated heart rate is associated with increased cardiovascular morbidity. We hypothesized that selective heart rate reduction may influence endothelial function and atherogenesis and tested the effects of the I(f) current inhibitor ivabradine in apolipoprotein E–deficient mice.
Methods and Results— Male apolipoprotein E–deficient mice fed a high-cholesterol diet were treated with ivabradine (10 mg · kg−1 · d−1) or vehicle for 6 weeks (n=10 per group). Ivabradine reduced heart rate by 13.4% (472±9 versus 545±11 bpm; P<0.01) but did not alter blood pressure or lipid levels. Endothelium-dependent relaxation of aortic rings was significantly improved in ivabradine-fed animals (P<0.01). Ivabradine decreased atherosclerotic plaque size in the aortic root by >40% and in the ascending aorta by >70% (P<0.05). Heart rate reduction by ivabradine had no effect on the number of endothelial progenitor cells and did not alter aortic endothelial nitric oxide synthase, phosphorylated Akt, vascular cell adhesion molecule-1, or intercellular adhesion molecule-1 expression but decreased monocyte chemotactic protein-1 mRNA and exerted potent antioxidative effects. Ivabradine reduced vascular NADPH oxidase activity to 48±6% and decreased markers of superoxide production and lipid peroxidation in the aortic wall (P<0.05). The in vivo effects of ivabradine were absent at a dose that did not lower heart rate, in aortic rings treated ex vivo, and in cultured vascular cells. In contrast to ivabradine, treatment with hydralazine (25 mg · kg−1 · d−1 for 6 weeks) reduced blood pressure (−15%) but increased heart rate (37%) and did not improve endothelial function, atherosclerosis, or oxidative stress.
Conclusions— Selective heart rate reduction with ivabradine decreases markers of vascular oxidative stress, improves endothelial function, and reduces atherosclerotic plaque formation in apolipoprotein E–deficient mice.
Received October 16, 2007; accepted March 13, 2008.
Epidemiological studies have shown that elevated heart rate represents a risk factor for cardiovascular morbidity both in primary prevention and in patients with hypertension, coronary artery disease, and myocardial infarction.1–5 Increased heart rate and reduced heart rate variability have been shown to be associated with coronary plaque rupture and subclinical inflammation in healthy middle-aged and elderly subjects.6,7 Experimental data suggest that sustained elevations of heart rate may play a role in the pathogenesis of coronary atherosclerosis. For example, Beere et al8 showed in 1984 that lowering heart rate through ablation of the sinoatrial node in cynomolgus monkeys fed an atherogenic diet decreased coronary atherosclerosis. In later studies, they reported a positive correlation between heart rate and plaque formation at the carotid bifurcation region of monkeys.9 However, the underlying mechanisms and the therapeutic consequences are only partially understood.
Clinical Perspective p 2387
The pacemaker current I(f) plays a central role in determining spontaneous activity of the sinus node. Ivabradine, a selective inhibitor of the I(f) channel, reduces resting and exercise heart rates without affecting cardiac contractility or blood pressure.10,11 Ivabradine exerts antianginal and anti-ischemic effects in patients with stable coronary disease. Clinical trials revealed improved exercise tolerance, increased time to exercise-induced ischemia, and reduced frequency of ambient angina attacks after I(f) channel inhibition.12,13 Ivabradine given orally to mice (10 mg/kg body weight per day) reduces heart rate without influencing left ventricular contractile function and therefore is used as a tool to study the effects of heart rate on vascular biology.14
We hypothesized that selective heart rate reduction by ivabradine may improve vascular function. Therefore, the aim of this study was to determine the effect of ivabradine on endothelial function, atherosclerotic lesion formation, and parameters of vascular oxidative stress in cholesterol-fed apolipoprotein E–deficient (ApoE−/−) mice.
Animals and Procedures
Animal experiments were conducted in accordance with institutional guidelines and the German animal protection law. Male C57/Bl6 mice (wild-type) and male ApoE−/− mice (C57/Bl6 genetic background; Charles River Laboratories, Sulzfeld, Germany) were used for this study. The animals were maintained in a 22°C room with a 12-hour light/dark cycle and received drinking water ad libitum. All mice were fed a high-fat, cholesterol-rich diet for 6 weeks that contained 21% fat, 19.5% casein, and 1.25% cholesterol (Western-type diet, Ssniff, Soest, Germany) starting at 12 weeks of age. Randomly selected ApoE−/− mice were treated with Western-type diet chow pellets supplemented with commercially available ivabradine (Procoralan, Servier, Neuilly-sur-Seine, France) 10 and 2 mg/kg body weight per day. For indicated mice, NG-nitro-l-arginine methyl ester (L-NAME; Sigma-Aldrich, Taufkirchen, Germany) was added to the drinking water (50 mg/kg body weight per day). An additional group of age-matched ApoE−/− mice was treated with the vasodilator hydralazine 25 mg/kg body weight per day (Sigma) via drinking water.15 Heart rate and blood pressure were measured by a computerized tail-cuff system (BP-2000, Visitech Systems, Apex, NC) in conscious animals. Mice were trained for 3 consecutive days in the prewarmed tail-cuff device to accustom them to the procedure, followed by measurements of heart rate and systolic blood pressure every other day. During each procedure, 20 measurements were obtained and averaged for each individual animal. The mean values of all analyses were used for comparisons. Cholesterol levels were determined by routine chemical methods.
The detailed preparation and the protocol of the tension recording of aortic rings are described in the online Data Supplement and was performed as described.15,16
Polymerase Chain Reactions
The primer sequences and polymerase chain reaction (PCR) protocols are listed in the online Data supplement.16,17
Staining of Atherosclerotic Lesions and Morphometric Analysis
Samples were sectioned on a Leica (Wetzlar, Germany) cryostat (10 μm), starting at the apex and going through the aortic valve area into the ascending aorta. At least 25 consecutive sections per animal were stained with Oil Red O and used for analysis. Lucia Measurement Version 4.6 software (Nikon, Düsseldorf, Germany) was used to measure lipid-staining area and total area of the histological sections. The online Data Supplement gives details.15,16
Western Blot Analysis
Immunoblotting for endothelial nitric oxide synthase (eNOS; BD 610296), phosphorylated eNOS (p-eNOS; BD 612706), phosphorylated Akt (p-Akt; sc 7985-R), and β-actin (sc 1615-R), to control for equal protein loading, with an enhanced chemiluminescence kit (Amersham, Uppsala, Sweden) was performed as described.16,17
Fluorescence-Activated Cell Sorter Analysis and Culture of Spleen-Derived Endothelial Progenitor Cells
Blood and bone marrow were processed as described.17–20 The viable lymphocyte population was analyzed for Sca-1-FITC (E13-161.7, BD PharMingen, San Diego, Calif) and VEGFR-2-PE (Flk-1) (Avas12α1, BD PharMingen). Isotype-identical antibodies (Becton Dickinson, Franklin Lakes, NJ) served as controls in every experiment. Spleen mononuclear cells were isolated and cultured on fibronectin (Sigma) as described.17–20 After 7 days in culture, endothelial progenitor cells (EPCs) were identified by uptake of 1.1′-dioctadecyl-3.3.3′.3′-tetramethylindocarbocyanine–labeled acetylated low-density lipoprotein (DiLDL; 2.4 μg/mL, CellSystems, St Katharinen, Germany) and staining with FITC-labeled Giffonia (bandeiraea) simpliciforia lectin I (lectin; 10 μg/mL) (Vector Laboratories, Burlingame, Calif).
Measurement of Lipid Peroxidation
Aortic tissue was homogenized in PBS (pH 7.4) containing butylated hydroxytoluene (4 mmol/L). Lipid hydroperoxides were determined with the Lipid Peroxidation Assay Kit II (Calbiochem, Gibbstown, NJ) and expressed as micromoles per microgram of protein.16
Measurement of Vascular Superoxide Production and NADPH Oxidase Activity
Superoxide release in intact aortic segments was determined by L-012 chemiluminescence.16,21 To assess vascular superoxide production in situ, dihydroethidium fluorescence of aortic tissue sections was used. Aortas from each treatment group were processed in parallel; images were acquired with identical acquisition parameters and were analyzed and stored digitally (ImageJ 1.37v by W. Rasband, National Institutes of Health, Baltimore, Md).16
NADPH oxidase activity was measured by a lucigenin-enhanced chemiluminescence assay.16,22 Details are given in the Data Supplement.
Endothelial cells harvested from bovine aortas and vascular smooth muscle cells isolated from rat thoracic aorta were cultured as described.23,24 Cellular viability was determined by cell count, morphology, and trypan blue exclusion. Cellular protein content was quantified by a modified Lowry assay.
Measurement of Intracellular Reactive Oxygen Species
Intracellular reactive oxygen species production in vascular smooth muscle cells was measured by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence.23,25 Details are given in the Data Supplement.
Results are presented as mean±SEM. Unpaired Student’s t test and ANOVA for multiple comparisons were used when applicable. Post hoc comparisons were performed with the Newman-Keuls test. Values of P<0.05 were considered statistically significant.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Heart Rate, Blood Pressure, and Lipid Levels
Twelve-week-old C57/Bl6 (wild-type) and ApoE−/− mice were fed a Western-type diet and were randomized to oral ivabradine, hydralazine, or vehicle treatment for 6 weeks. The Table shows mean heart rates, systolic and diastolic blood pressures, and serum cholesterol concentrations after 6 weeks. Treatment with 10 mg/kg ivabradine decreased heart rate by −13±2% (−73±2 bpm; P<0.01 versus ApoE−/− vehicle), whereas systolic and diastolic blood pressures were not altered significantly. Heart rate reduction remained consistently stable throughout the whole treatment period. As control for potential effects of ivabradine independently of heart rate, age-matched ApoE−/− mice were fed the highest dose of ivabradine that did not result in a significant reduction in heart rate, namely 2 mg/kg body weight, for 6 weeks. A second control group was treated with ivabradine 10 mg/kg body weight and the NOS inhibitor L-NAME (50 mg/kg body weight per day) for 6 weeks. In a third control group, ApoE−/− mice were treated with the vasodilator hydralazine (25 mg/kg body weight per day). Treatment with hydralazine significantly increased heart rate compared with ivabradine- and vehicle-treated mice. Systolic and diastolic blood pressures were significantly reduced (Figure 1). Serum cholesterol levels were increased in ApoE−/− mice as expected but were not altered by the drug treatments.
Vascular function was assessed in isolated aortic ring preparations. As expected, endothelium-dependent vasodilatation was impaired in ApoE−/− mice.15,16 Treatment with ivabradine (10 mg/kg body weight per day) significantly improved endothelial function in ApoE−/− mice almost to the level of wild-type controls (P<0.05 versus ApoE−/− vehicle). In contrast, the lower dose of ivabradine that did not affect heart rate (2 mg/kg body weight per day) did not alter endothelial function (Figure 2). Cotreatment with ivabradine (10 mg/kg body weight per day) and L-NAME showed a trend toward decreased endothelial function compared with ivabradine alone, suggesting that prevention of eNOS uncoupling does not play a major role. Endothelium-independent vasorelaxation induced by glyceroltrinitrate and vasoconstriction induced by phenylephrine and KCl were similar in all groups.
Atherosclerotic Lesion Formation
The chemokine monocyte chemotactic protein-1 (MCP-1) plays a causal role in the progression from endothelial dysfunction to atherosclerotic lesion development, primarily by inducing leukocyte arrest and transendothelial migration.26 MCP-1 has been shown to be regulated by fluid shear stress.27,28 Reverse-transcription PCR of aortic mRNA showed a marked reduction in MCP-1 expression to 26±7% (Figure 3) after treatment with ivabradine (10 mg/kg body weight per day for 6 weeks; P<0.05). Ivabradine had no effect on the aortic mRNA expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1.
Histomorphometric analysis of atherosclerotic lesions in the aortic sinus and the ascending aorta showed that selective heart rate reduction by ivabradine significantly slows atherogenesis. The atherosclerotic plaque area was reduced by >40% in the aortic root (32±4% versus 18±2%) and by >70% in the ascending aorta (26±4% versus 7±1%) compared with vehicle-treated mice (n=10 per group; P<0.05; Figure 3D through 3I).
EPCs and eNOS
Recent evidence has shown that cardiovascular function and angiogenesis are significantly modulated by circulating EPCs from the bone marrow.17,29–31 Cardiovascular risk factors impair EPC number and function, whereas vasculoprotection, eg, by statins and physical activity, is associated with increased numbers of EPCs.17,18 To investigate a possible effect of heart rate reduction by I(f) channel inhibition on EPCs, DiLDL/lectin-positive EPCs were expanded from spleen-derived mononuclear cells. As a second method of EPC quantification, Sca-1/VEGFR-2–positive EPCs were quantified by fluorescence-activated cell sorter analysis. However, treatment with ivabradine had no effect on DiLDL/lectin-positive spleen-derived EPCs and on EPCs in the peripheral blood and bone marrow (Figure 4A through 4C).
Endothelial nitric oxide is a key mediator of endothelial function and atherogenesis. However, reduced heart rate was not associated with a significant upregulation of eNOS mRNA in the aorta as determined by real-time PCR. Similarly, protein expression of eNOS, p-eNOS, and phosphorylated protein kinase Akt was not increased (Figure 4D through 4H).
Vascular Oxidative Stress
Reactive oxygen species impair vascular function. The NADPH oxidase is a major source of superoxide radicals in the vascular wall.32 Figure 5 shows that NADPH oxidase activity in the aorta was downregulated to 48±6% in the ivabradine-treated ApoE−/− mice (P<0.05). Vascular release of superoxide radicals was measured by L-012 chemiluminescence assays in intact aortic segments. Reactive oxygen species release was significantly decreased in ivabradine-treated mice (186.5±52.5 versus 44.2±17.2 relative light units · μg−1 · min−1; P<0.05). As a global parameter of oxidative stress, lipid peroxidation of the aortic wall was quantified. Animals with reduced heart rate displayed downregulation of vascular lipid hydroperoxides compared with vehicle-treated littermates (0.94±0.04 versus 0.61±0.08 μmol/μg; P<0.05). In addition, in situ detection of superoxide production was performed by dihydroethidium fluorescence microscopy in aortic sections showing a marked reduction in reactive oxygen species release in ivabradine-treated ApoE−/− mice (124±7 versus 77±5 arbitrary units; P<0.01; Figure 5).
Vascular Effects of Hydralazine
As a control group, mice were treated with hydralazine. As expected, hydralazine treatment lowered blood pressure (99±2 mm Hg) and increased heart rate (650±8 bpm; P<0.05; the Table and Figure 1). Interestingly, hydralazine treatment did not confer a significant improvement in endothelial function despite robust blood pressure lowering (Figure 6A and 6B). Furthermore, in contrast to ivabradine treatment, hydralazine exerted only a minor effect on atherosclerosis that was not statistically significant (Figure 6C and 6D). Treatment with hydralazine showed no effect on the activity of NADPH oxidase, lipid peroxidation, or superoxide production (Figure 6E and 6G).
Test for Potential Direct Effects of Ivabradine Independently of Heart Rate
To investigate potential direct effects of ivabradine on endothelial function, aortic ring preparations from ApoE−/− mice fed the Western-type diet for 6 weeks were treated ex vivo with ivabradine in the organ bath. Increasing concentrations of ivabradine (0.2 to 20 μmol/L) did not induce vasorelaxation. The L-NAME–mediated vasoconstriction was not affected by the presence of ivabradine in the organ bath. Preincubation of aortic rings with ivabradine (2 μmol/L for 5 minutes) did not result in an improved vasorelaxation in the presence of carbachol. The nitroglycerin-mediated vasodilatation was not changed in the presence of ivabradine (Figure 7A through 7C).
To test whether ivabradine exerts direct cellular effects, cultured bovine aortic endothelial cells were treated with ivabradine (0.2 to 200 μmol/L for 16 hours), and protein expression was examined by Western blot analyses. As depicted in Figure 7D, expression of p-Akt, eNOS, and p-eNOS was not altered by increasing doses of ivabradine. Similarly, NADPH oxidase activity in bovine aortic endothelial cells was not altered in the presence of ivabradine (Figure 7E). To assess a potential direct antioxidative action of ivabradine, vascular smooth muscle cells were treated with ivabradine alone and in combination with angiotensin II and the NOS inhibitor L-NAME. Release of reactive oxygen species was detected by H2DCFDA fluorescence. The angiotensin II–induced free radical release was not attenuated by pretreatment with ivabradine (Figure 7F).
The novel finding of the study is the marked reduction in vascular oxidative stress associated with an improvement in endothelial function and inhibition of atherosclerotic plaque formation after selective heart rate reduction with the I(f) channel inhibitor ivabradine in ApoE−/− mice.
ApoE−/− mice fed a Western-type diet represent a well-characterized model of hyperlipidemia-induced endothelial dysfunction.33 Endothelial dysfunction precedes vascular diseases in several vascular beds, including coronary and peripheral artery disease and ischemic stroke.32,34,35 Ivabradine treatment improved endothelium-dependent vasodilatation in the absence of blood pressure or lipid lowering. Improvement in endothelial function was associated with a significant reduction in atherosclerotic plaque volume despite persistent severe hypercholesterolemia. We propose that the main mechanism by which ivabradine exerts these effects is the reduction in heart rate because previous animal studies have shown that a reduction in heart rate by other means, eg, by ablation of the sinoatrial node in cynomolgus monkeys, beneficially affects atherogenesis.8,9
The circadian regulation of heart rate affects cardiovascular morbidity.36 Therefore, it will be important to characterize the modulation of circadian rhythms by ivabradine treatment. Modification of the hemodynamic environment by accelerated heart rate is likely to contribute to enhanced atherosclerosis in certain parts of the vascular tree. In regions of the vasculature where flow reversal and shear stress oscillation dominate flow pattern, high heart rate is associated with expanded periods of low shear stress levels, potentially facilitating atherosclerotic lesion formation.37 In elastic arteries, acute increases in heart rate can be accompanied by reductions in arterial compliance and distensibility.38 Those hemodynamic and tensile effects contribute to the propensity for plaque formation in vessels exposed to high heart rate. Here, we show that improvement in endothelial function and significant slowing of atherogenesis can be achieved by ivabradine, an oral treatment that is well tolerated in mice and humans.13,14
Treating mice with ivabradine at the highest dose that did not reduce heart rate had no effect on endothelial function. Exposure of isolated aortic rings from ApoE−/− mice to ivabradine in the organ bath did not alter vasodilatation. Furthermore, ivabradine showed no effect in cultured endothelial and cultured vascular smooth muscle cells. Taken together, these control experiments show that a direct effect of ivabradine on vascular cells is unlikely and support the reduction of heart rate as the primary mechanism of action.
A pharmacological agent that selectively and permanently increases heart rate is not available. We therefore treated ApoE−/− mice with hydralazine as a control group. Hydralazine acts as a potent arteriolar dilator that stimulates a reflective increase in pulse rate.39 At the same time, hydralazine lowers blood pressure. Decreased systolic and diastolic blood pressures lead to improved endothelial function and reduced atherogenesis in animal models and humans. Interestingly, despite a robust reduction in systolic and diastolic blood pressures in our model, hydralazine did not significantly improve endothelial function or the extent of atherosclerosis. Similarly, hydralazine showed no effect on several parameters of vascular oxidative stress. These data are in agreement with the hypothesis that an increase in heart rate may be associated with negative vascular effects that outweigh the beneficial effects of blood pressure lowering.
Endothelial nitric oxide is a key mediator of endothelial function and is selectively upregulated by steady laminar shear stress.17,32,40 However, in contrast to our expectations, the profound improvement in endothelial function by ivabradine was not associated with upregulation of aortic eNOS mRNA expression. Furthermore, Western blot analysis did not show an upregulation of eNOS or p-eNOS protein nor an increase of p-Akt expression. A potential effect of ivabradine on NOS uncoupling was ruled out by the control experiments with L-NAME in vivo, in isolated aortic rings, and in cell cultures. Therefore, it appears that modulation of eNOS expression is not the primary mechanism. The observed beneficial effects are mediated, at least in part, by the eNOS-independent antioxidative effects of ivabradine.
Recent evidence has shown that cardiovascular function and angiogenesis are significantly modulated by circulating EPCs derived from bone marrow. EPCs have been shown to enhance angiogenesis, to promote vascular repair, and to improve endothelial function.17,29–31 Circulating EPCs are regulated, and vascular risk factors have been shown to reduce EPCs.17,18,30 Therefore, we hypothesized that a reduction in heart rate may increase the number of EPCs. However, we did not observe a regulation of EPCs using both EPC culture and fluorescence-activated cell sorter analysis and looking at spleen-, blood-, and bone marrow–derived EPCs. As with any negative result, technical limitations can never be completely excluded; however, other interventions such as physical exercise show clear effects in the same animal model,16,19,20 making a large contribution of EPCs to the observed effects less likely.
The increased release and production of reactive oxygen species is one of the key events in the pathogenesis of endothelial dysfunction and atherosclerosis.32 Treatment with ivabradine potently inhibited vascular oxidative stress, providing an explanation for the observed effects on vascular function. Superoxide derived from the vascular NADPH-oxidase complex has been shown to impair endothelial function and to promote atherogenesis.16,32 Ivabradine-induced reduction in heart rate was associated with marked inhibition of NADPH-oxidase activity and superoxide release, which was confirmed histologically by dihydroethidium staining. In addition, vascular lipid peroxidation as a global marker of oxidative stress was significantly decreased. Taken together, the data of these 4 different assays show a robust antioxidative effect of ivabradine on the vasculature.
Consistent with these findings, ivabradine treatment resulted in a potent downregulation of MCP-1 expression. MCP-1 provides a link between endothelial dysfunction and atherosclerotic lesion formation by inducing leukocyte arrest and transendothelial migration. MCP-1 has been shown to be regulated by fluid shear stress and cyclic strain.26,28,41 Interestingly, cyclic strain–induced MCP-1 expression in both endothelial and vascular smooth muscle cells involves superoxide anion production, which is reduced by ivabradine.41,42 This may explain the selective regulation of MCP-1 compared with intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 that is controlled predominantly by eNOS.43
Chronic heart rate reduction by ivabradine leads to inhibition of vascular oxidative stress, endothelial dysfunction, and atherosclerotic lesion formation in ApoE−/− mice regardless of blood pressure and plasma cholesterol levels. The experiments support the potential of heart rate reduction as an intervention to improve endothelial function and to attenuate progression of atherosclerosis in vascular prevention, in addition to the symptomatic treatment of angina pectoris. Pharmacological inhibition of the I(f) current may represent a novel intervention to prevent endothelial dysfunction that should be tested in a prospective clinical investigation.
We thank Ellen Becker and Simone Jaeger for excellent technical assistance.
Sources of Funding
This study was supported by the Universität des Saarlandes (Homburger Forschungsförderungsprogramm). The study was not supported by the manufacturer of ivabradine. Drs Laufs and Böhm are supported by the Deutsche Forschungsgemeinschaft (DFG KFO 196).
Dr Baumhäkel has received financial support from Servier (France) for basic research that is not related to the present study. Dr Böhm has received honoraria from Servier (France). The remaining authors report no conflicts.
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Epidemiological studies have shown that elevated heart rate is a risk factor for cardiovascular morbidity. Experimental data suggest that sustained elevations of heart rate may play a role in the pathogenesis of coronary atherosclerosis. Ivabradine, a selective inhibitor of the I(f) channel, reduces resting and exercise heart rates without an effect on cardiac contractility. In the present study of apolipoprotein E–deficient mice fed a high-cholesterol diet, ivabradine reduced heart rate without affecting blood pressure or lipid levels. Endothelium-dependent relaxation of aortic rings was significantly improved in ivabradine-fed animals, and ivabradine decreased the development of atherosclerotic lesions. The main underlying molecular observation was a potent antioxidative effect of ivabradine that was mediated by the reduction of heart rate. There was no evidence for a heart rate–independent action of the drug. In summary, long-term heart rate reduction by ivabradine leads to inhibition of vascular oxidative stress, endothelial dysfunction, and atherosclerotic lesion formation in apolipoprotein E–deficient mice, regardless of blood pressure and plasma cholesterol levels. The experiments support the potential of heart rate reduction as an intervention to improve endothelial function and to attenuate progression of atherosclerosis in vascular prevention in addition to the symptomatic treatment of angina pectoris. Pharmacological inhibition of the I(f) current may represent a novel intervention to prevent endothelial dysfunction that should be tested in a prospective clinical investigation.
↵*The first 2 authors contributed equally to this work.
The online Data Supplement, which contains an expanded Methods section, can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.746537/DC1.