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Heart Rate Reduction by Ivabradine Reduces Oxidative Stress, Improves Endothelial Function and Prevents Atherosclerosis in Apolipoprotein E-Deficient Mice
Florian Custodis, Magnus Baumhäkel, Nils Schlimmer, Franka List, Christoph Gensch, Michael Böhm, and Ulrich Laufs, Klinik für Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin, Universitätsklinikum des Saarlandes, 66421 Homburg/Saar, Germany
Please address correspondence to:
Prof. Dr. med. Ulrich Laufs
Klinik für Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin
Universitätsklinikum des Saarlandes
Tel: 0049-(0)6841-1623000
Fax: 0049-(0)6841-1621331
Email ulrich@laufs.com
Background and Objective
Epidemiological studies have shown that elevated heart rate represents a risk factor for cardiovascular morbidity both in primary prevention as well as 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 [8,9].
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 an effect on cardiac contractility or blood pressure [10,11]. Ivabradine exerts anti-anginal 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 (10mg/kg/d) reduces heart rate without influencing
We hypothesized that selective heart rate reduction by ivabradine may improve vascular function. Therefore, the aim of our study was to determine the effect of ivabradine on endothelial function, atherosclerotic lesion formation, and parameters of vascular oxidative stress in cholesterol fed ApoE-/- mice [15].
Results
Twelve-week-old C57/Bl6 (wild-type) and ApoE-/- mice were fed a Western-type diet and were randomized to oral ivabradine (10 mg/kg/d), hydralazine (25 mg/kg/d), or vehicle treatment for 6 weeks. Ivabradine reduced heart rate by 13.4% (472 ± 9 versus 545 ± 11 bpm) but did not alter blood pressure or lipid levels. Treatment with hydralazine significantly increased heart rate compared to ivabradine- and vehicle-treated mice. Systolic and diastolic blood pressures were significantly reduced.
Vascular function was assessed in isolated aortic ring preparations. As expected, endothelium-dependent vasodilatation was impaired in ApoE-/- mice. Treatment with ivabradine (10 mg/kg/d) improved endothelial function in ApoE-/- mice almost to the level of wildtype controls. 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.
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 trans-endothelial migration [16]. RT-PCR of aortic mRNA showed a marked reduction of MCP-1 expression after treatment with ivabradine. However ivabradine had no effect on the aortic mRNA expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1).
Recent evidence has shown that cardiovascular function and angiogenesis are significantly modulated by circulating endothelial progenitor cells (EPC) from the bone marrow [17,18]. To investigate a possible effect of heart rate reduction by I(f)-channel inhibition on endothelial progenitor cells, DiLDL/lectin positive EPC were expanded from spleen-derived mononuclear cells. As a second method of EPC quantification, Sca-1/VEGFR-2 positive EPC were quantified by FACS analysis. However, treatment with ivabradine had no effect on DiLDL/Lectin positive spleen-derived EPC as well as on EPC in the peripheral blood and the bone marrow.
Endothelial nitric oxide is a key mediator of endothelial function and atherogenesis. In contrast to our initial expectation, 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, phosphorylated eNOS, and phosphorylated protein kinase Akt was not increased.
Heart rate reduction attenuated vascular oxidative stress. Ivabradine reduced vascular NADPH oxidase activity. Vascular release of superoxide radicals was measured by L-012 chemiluminescence assays in aortic segments. ROS release was significantly decreased in ivabradine-treated mice. As a global parameter of oxidative stress, lipid peroxidation of the aortic wall was quantitated. Animals with reduced heart rate displayed downregulation of vascular lipid hydroperoxides compared with vehicle-treated littermates. In addition, in situ-detection of superoxide production was performed by dihydroethidium (DHE) fluorescence microscopy in aortic sections showing a marked reduction of ROS release in ivabradine-treated ApoE-/- mice.
As a control group, mice were treated with hydralazine. Hydralazine acts as a potent arteriolar dilator that stimulates a reflective increase of pulse rate [19]. At the same time, hydralazine lowers blood pressure. As expected, hydralazine treatment lowered blood pressure (99 ± 2 mmHg) and increased heart rate (650 ± 8 bpm). Interestingly, hydralazine treatment did not confer a significant improvement of endothelial function despite robust blood-pressure lowering. Furthermore, in contrast to ivabradine treatment, hydralazine exerted only a minor effect on atherosclerosis that was not statistically significant. Treatment with hydralazine showed no effect on the activity of NADPH oxidase, lipid peroxidation, or superoxide production.
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. Pre-incubation of aortic rings (2 µmol/l, 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. To test if the drug exerts direct cellular effects, cultured bovine aortic endothelial cells (BAEC) were treated (0.2 to 200 µmol/l, 16 hours) and protein expression was examined by Western blot analyses. Expression of p-Akt, eNOS, and p-eNOS was not altered by increasing doses of ivabradine. Similarly, NADPH oxidase activity in BAEC was not altered. To assess a potential direct anti-oxidative action, vascular smooth muscle cells (VSMC) 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 pre-treatment with ivabradine.
Conclusions
Chronic heart rate reduction by ivabradine improves endothelial function and reduces atherosclerotic plaque formation in ApoE-/- mice irrespective of blood pressure and plasma cholesterol levels. Those effects are in part mediated by decreased markers of oxidative stress and downregulation of MCP-1. We propose that the main mechanism by which ivabradine exerts these effects is the reduction of heart rate. 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.
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 is worthwhile to be tested in a prospective clinical investigation.
Funding Sources
This study was supported by the Universität des Saarlandes (HOMFOR, Homburger Forschungsförderungsprogramm). The study was not supported by the manufacturer of Ivabradine. UL and MB are supported by the Deutsche Forschungsgemeinschaft (DFG KFO 196).
References