Prevention of endothelial dysfunction with pure heart rate reduction



By J. Yang and J.-C. Tardif, Canada

Jean-Claude TARDIF, MD, FRCPC, FACC
Ju YANG, BSc
Montreal Heart Institute – University of Montreal – Montreal, CANADA

Endothelial dysfunction plays a major role in the cardiovascular disease continuum, facilitating inflammation, platelet aggregation, and coronary vasoconstriction. Experimental and clinical data clearly suggest that endothelial dysfunction has pro-atherosclerotic and prothrombotic effects and has important predictive value for future cardiovascular events in patients with coronary artery disease. New data demonstrating the effect of increased heart rate on the development of endothelial dysfunction may provide a new understanding about the basis for the association between increased heart rate and cardiovascular outcomes. The protective effect on the endothelium from long-term pure heart rate reduction with ivabradine that we have shown in a dyslipidemic mouse model of endothelial dysfunction could provide an important mechanism for the potential cardioprotective benefits of ivabradine. These data also demonstrate that the β-blocker metoprolol did not provide the same protection despite the same magnitude of heart rate reduction.

Medicographia. 2009;31:420-427 (see French abstract on page 427)

Despite the advances made in the development of efficacious treatments for coronary heart disease (CHD), it remains the first cause of mortality in Western populations. This has led to additional research efforts directed at improving our understanding of the underlying mechanisms responsible for the development of adverse cardiovascular events, in an attempt to find alternative and superior means of treatment. Some of the widely accepted risk factors for CHD that have become targets for therapies include high blood pressure, dyslipidemia, smoking, diabetes, obesity, and physical inactivity.1

An inverse semi-logarithmic relationship has been observed between longevity and resting heart rate in mammals (Figure 1),2,3 although human beings seem to be the exception to this rule, potentially because of the medical advances that prolong human life expectancy. Observation of this relationship has led to the concept that a fixed number of heartbeats is allocated, after which we inevitably expire. An elevated resting heart rate has also been shown in epidemiological studies to be independently associated with mortality from cardiovascular as well as noncardiovascular diseases.4

Heart rate: sinoatrial node and If current

Heart rate in humans is set by the sinoatrial (SA) node. The SA node cells spontaneously depolarize during diastole and initiate the next action potential. It is this characteristic that confers on them their inherent pacemaker ability. The SA node has many intrinsic qualities. Specifically, it has a seemingly fail-safe nature; many currents are responsible for the spontaneous depolarization of the SA node, and the selective blocking of one of these currents does not put one’s life at risk.5 This fact was demonstrated clinically using a drug that specifically and completely inhibits the If current (also referred to as the “funny” current), which is one of the main currents responsible for SA node depolarization. The complete blockade of the If current was shown to reduce the heart rate by approximately 30%.6

Figure 1
Figure 1. Relationship between resting heart rate and life expectancy in mammals, according to size.

After reference 3: Levine HJ. J Am Coll Cardiol. 1997;30:1104-1106. Copyright © 1997, Elsevier.

The If current is an inward flux of sodium and potassium ions and is activated by the hyperpolarization of the cell membrane. The activity of the If current is increased by β-receptor stimulation and inhibited by acetylcholine through intracellular changes in the concentration of cyclic adenosine monophosphate. Thus, drugs with β-blocking capacity such as metoprolol can slow down the heart rate. Ivabradine, a specific If current inhibitor, reduces the heart rate and could be more beneficial because of its higher selectivity in decreasing the heart rate. This issue will be discussed in the following sections.

Drugs available for the treatment of coronary heart disease

In the past decades, β-blockers have been the drug class of choice for the treatment of angina. Their effect is largely attributable to their ability to lower the heart rate. Unfortunately, the use of β-blockers has been linked to side effects such as fatigue, lack of energy, depression, and erectile dysfunction. βBlockers have also been associated with increased symptoms of peripheral arterial occlusive disease and with a potential rebound effect when ceased abruptly,7 worsened symptoms associated with obstructive pulmonary disease,8 and symptomatic conduction block in patients with intrinsic atrioventricular node disease.9 Furthermore, β-blockers have negative inotropic effects10 and can also have negative metabolic effects (on blood glucose11 and lipids12).

Other drugs available for the treatment of CHD include calcium channel antagonists and long-acting nitrates. The former have been associated with peripheral edema, constipation, and negative inotropic effects,13 as well as with a higher risk of precipitating or potentiating heart failure or atrioventricular node dysfunction. The use of nitrates has been associated with headaches, lightheadedness, and syncope.14 The continuous use of long-acting nitrates can lead to pharmacological tolerance,15 but intermittent use has been associated with rebound angina.

Ivabradine

Ivabradine, a drug that specifically targets the If current, has generated interest in the medical community because of its higher specificity for decreasing heart rate than the aforementioned drugs. Several studies have been conducted in order to ascertain its safety, efficacy, and noninferiority to other drugs available clinically for the prevention of angina.

When compared with placebo in the absence of any background antianginal therapy, ivabradine has been shown to exert anti-ischemic and antianginal effects with prolonged time to 1-mm ST-segment depression during exercise testing and reduced angina frequency and nitroglycerin use.16 Ivabradine was also evaluated in several noninferiority trials during its drug development program. Ivabradine was compared with the calcium channel antagonist amlodipine,17 and was shown to be noninferior to amlodipine in terms of its antiischemic and antianginal effects. In addition, heart rate both at rest and during exercise decreased significantly more with ivabradine than amlodipine.

Ivabradine was also compared with the β-adrenoceptor antagonist atenolol in the INternatIonal TrIal of the Antianginal effecT of IVabradinE compared with atenolol (INITIATIVE).18 Although atenolol showed greater heart rate reduction, ivabradine was noninferior (and actually even tended to be superior) to the β-blocker in terms of prolonging total exercise duration (primary end point) as well as time to limiting angina and angina onset during exercise tolerance testing. Thus, ivabradine induced a similar or greater improvement in exercise capacity while causing less reduction in rate-pressure-product and heart rate when compared with atenolol. According to these findings, ivabradine possessed greater efficiency in its ability to increase exercise capacity for each beat of heart rate reduction; this phenomenon can likely be linked to its lack of negative inotropic, peripheral vascular, or coronary vasoconstrictor effects.19 Accordingly, this study also concluded that ivabradine was noninferior to atenolol in terms of its antianginal and anti-ischemic effects for all exercise parameters. The long-term beneficial effects of ivabradine have also been established in patientswith stable angina pectoris, as it reduced the frequency of angina over the course of a 1-year study.20

More recently, the results of BEAUTIFUL (morBidity-mortality EvAlUaTion of the If inhibitor ivabradine in patients with coronary disease and left ventricULar dysfunction) have been reported.21 Although the primary end point of the study was not met in the overall population, ivabradine reduced the risks of coronary events by 22% (P=0.023) and fatal and nonfatal myocardial infarction by 36% (P=0.016), and reduced coronary revascularization by 30% (P=0.016) in the subgroup of patients with a baseline heart rate ≥70 bpm.

Ivabradine is generally well tolerated.22 The most common side-effect is phosphenes, which are visual symptoms of a transient nature related to the presence of a channel in the retina that is very similar to the hyperpolarization-activated cyclic nucleotide-gated family of ion channel subunits in the SA node.22,23 These visual symptoms are dose dependent, mild, and spontaneously resolve during treatment or after treatment cessation. Overall, only 1% of patients withdrew from treatment for this reason in clinical trials. Bradycardia has been reported in 2.2% of patients in clinical studies. Ivabradine does not alter the action potential and the corrected QT interval.24 In addition, ivabradine does not have a negative inotropic effect or a rebound effect when therapy is ceased abruptly, and has not been found to interfere with respiratory function.22

The missing link

Over the years, investigators have attempted to identify the missing link between slower heart rate and decreased cardiovascular events. A number of studies point to endothelial dysfunction as this missing link. The hypothesis is that higher heart rate could increase the twisting of large epicardial arteries during systole as well as the number of times per minute that forces are applied to the vascular wall, leading both to fatigue, causing endothelial damage in these vital arteries, and a simultaneous increase in the probability of atherosclerotic plaque rupture in the coronary arteries thereby leading to myocardial infarction. Endothelial dysfunction was already considered an integral part of the events leading to atherosclerosis initiation and progression, and has been shown to be associated with adverse cardiovascular events. We will now review the evidence linking heart rate and endothelial function.

_ Vascular endothelium
The endothelium serves many functions in maintaining a state of equilibrium in the cardiovascular tree. Normally, there is a balance between factors promoting and preventing thrombosis, whereby the undamaged endothelium favors anti-thrombosis. The endothelial cells normally host anti-thrombotic molecules on their surface such as heparin sulfate, thrombomodulin, and plasminogen activator, and produce and release antithrombotic molecules such as nitric oxide and prostacyclin in the vessel. On the other hand, once the integrity of the endothelium is compromised, for instance under situations of stress or exposure to traditional cardiovascular risk factors, the endothelium can produce prothrombotic molecules, thus tipping the balance toward prothrombosis in the vessel. Normally functioning endothelium also inhibits smooth muscle cell migration and proliferation. The endothelium is also naturally anti-inflammatory by resisting adhesion of leukocytes under normal conditions, and is impermeable to large molecules. Dysfunctional endothelium, by contrast, promotes inflammation in the vascular wall, contributing to both atherosclerosis progression and acute coronary events. Last but undoubtedly not least, the endothelium can secrete vasodilator molecules, such as nitric oxide and prostacyclin, and vasoconstrictor molecules such as endothelin. Under normal circumstances, the endothelium favors vasodilation over vasoconstriction. This last characteristic promotes the patency of the vessels against progressive narrowing of the lumen in atherosclerosis.25

Endothelial dysfunction is central in the pathogenesis of atherosclerosis. It is the first step leading to the formation of fatty streaks, which is the first in a series of events that can eventually lead to the formation of atherosclerotic plaques and thrombus. Endothelial dysfunction allows lipoproteins to enter in the intima and to then be modified in situ by oxidation and glycation. These events will exacerbate endothelial dysfunction and promote macrophage adhesion to the endothelium and migration into the intima. Subsequently, the generation of extracellular matrix will promote the formation of a fibrofatty lesion, the atherosclerotic plaque. Under conditions of hemodynamic stress and degradation of extracellular matrix, the plaque can rupture and promote the formation of an intraluminal thrombus leading to an acute coronary syndrome (Figure 2).26 As mentioned, normal endothelial function is essential in maintaining the integrity of the cardiovascular system. Therefore, endothelial dysfunction is an early indicator of cardiovascular disease. Thus, one way to demonstrate the efficacy of a cardioprotective drug is to demonstrate its ability to preserve or improve endothelial function.27

Figure 2
Figure 2. Schematic diagram of hypothetical sequence of cellular interactions in atherosclerosis.

Hyperlipidemia and other risk factors are thought to cause endothelial injury, resulting in adhesion of platelets and monocytes and release of growth factors, including platelet-derived growth factor (PDGF), which lead to smooth muscle cell migration and proliferation. Foam cells of atheromatous plaques are derived from both macrophages and smooth muscle cells—from macrophages via the very-low-density lipoprotein (VLDL) receptor and low-density lipoprotein (LDL) modifications recognized by scavenger receptors (eg, oxidized LDL), and from smooth muscle cells by less certain mechanisms. Extracellular lipid is derived from insudation from the vessel lumen, particularly in the presence of hypercholesterolemia, and also from degenerating foam cells. Cholesterol accumulation in the plaque reflects an imbalance between influx and efflux, and high-density lipoprotein (HDL) likely helps clear cholesterol from these accumulations. Smooth muscle cells migrate to the intima, proliferate, and produce extracellular matrix, including collagen and proteoglycans.
After reference 26: Kumar V, Abbas AK, Kausto N, eds. Robbins and Cotran Pathologic Basis of Disease. 7th ed. Copyright © 2005, Elsevier Saunders./em>

Slower heart rate to preserve endothelial function

Several epidemiological studies have previously demonstrated that a lower heart rate is associated with lower rates of adverse cardiovascular events28-30 and death.31 Other studies have suggested that a high heart rate may play a role in the formation and progression of atherosclerotic plaques.32 Strawn et al33 have demonstrated that stress-induced tachycardia is associated with atherosclerosis and dysfunction of coronary artery endothelial cells. A slower heart rate was associated in cynomolgus monkeys with less severe atherosclerotic plaques in coronary and carotid arteries.34,35 In addition, Korshunov and Berk36 have demonstrated the relationship between heart rate in low shear stress conditions and the degree of vascular wall remodeling. Nevertheless, none of these studies evaluated the effects of pure heart rate reduction on endothelial function.

Pure heart rate reduction with ivabradine to preserve endothelial function

We performed a study in dyslipidemic mice to document the effects of pure heart reduction on endothelial function.27 Ivabradine was chosen because it reduces heart rate in mice independently of sympathetic activation, and it does not affect blood pressure, myocardial contractility, or intracardial conduction.37,38

Endothelial vasodilator capability was used as the means to show preservation of endothelial function. The experiments were conducted in dyslipidemic mice expressing the human apoprotein-B 100, as they develop changes in endothelialdependent arterial dilation.39-40 Dyslipidemic mice were assigned to 3 months of treatment with ivabradine, metoprolol, or no treatment.

Figure 3
Figure 3. Endothelium-dependent dilation of pressurized renal arteries (100 mm Hg) in response to acetylcholine (control), isolated from (A) 6-month-old wild-type mice (B) dyslipidemic mice (C) dyslipidemic mice treated with ivabradine (10 mg/kg/day; from age 3 to 6 months).

The effects of endothelial nitric oxide synthase inhibition by Nw-nitro-L-arginine (10 mM) and the antioxidant N-acetyl-L-cysteine (1 mM) were tested in separate segments of renal arteries isolated from the same animal. Data are mean ±standard error of the mean. ‡P<0.05 compared with control.
Abbreviations: ACh, acetylcholine; L-NNA, Nw-nitro-L-arginine; NAC, N-acetyl-L-cysteine.
After reference 27: Drouin A, Gendron ME, Thorin E, Gillis MA, Mahlberg-Gaudin F, Tardif JC. Br J Pharmacol. 2008;154:749-757. Copyright © 2008, British Pharmacological Society.

The outcomes in terms of vessel dilation in left and right renal and posterior communicating cerebral arteries were compared between these groups and those of wild-type C57BI/6 mice.

cetylcholine was administered on all preconstricted arteries to measure dilator response. As free radicals impair renal endothelial function in dyslipidemic mice,41 the antioxidant N-acetylcysteine or the inhibitor of endothelial nitric oxide synthase (eNOS) Nw-nitro-L-arginine (L-NNA) was administered before the use of acetylcholine to better understand the mechanisms of drug effects. In cerebral arteries, dilation with acetylcholine was preceded by the inhibition of cyclo-oxygenase (COX) with indomethacin or in the presence of catalase, the latter inactivating the endothelium-derived relaxing factor hydrogen peroxide (H2O2) in these arteries.

Throughout the experiments, it was found that heart rate remained stable in wild-type mice, while it increased in untreated dyslipidemic mice. The use of ivabradine reduced heart rate in dyslipidemic mice by 17% (P<0.05). It was also shown that ivabradine prevented the appearance of left ventricular dysfunction in dyslipidemic mice, as shown by the limited increase in minimal and end-diastolic left ventricular pressure. The maximal left-ventricular systolic pressure and contractility remained unchanged in dyslipidemic mice. Ivabradine did not have any direct vascular effects on the renal and cerebral arteries.

_ Renal arteries
Endothelium-dependent dilation in response to acetylcholine was decreased in untreated dyslipidemic mice compared with those treated with ivabradine, which maintained maximal dilation (Figure 3).27 Therefore the use of ivabradine completely prevented the impaired dilator response to acetylcholine in dyslipidemic mice. The use of the antioxidant N-acetylcysteine fully restored dilation in response to acetylcholine in dyslipidemic mice, whereas it did not affect response to acetylcholine in wild-type mice and in mice treated with ivabradine (Figure 3).40-42 This shows that the endothelial dysfunction in dyslipidemic mice is caused in large part by oxidative stress, which was not increased in wild-type mice and dyslipidemic mice treated with ivabradine. Therefore, ivabradine protected the treated dyslipidemic mice against oxidative stress. Given that ivabradine has no direct antioxidant effects, the protection it afforded might be due to alternative mechanisms such as improvement of the shear stress–dependent stimulation of the endothelium, which favors eNOS and/or prevents nitric oxide or H2O2 degradation, or decreased mechanical fatigue of the arterial wall associated with pure heart reduction. The use of L-NNA, an eNOS inhibitor, did not exacerbate the already altered vasodilator response to acetylcholine in untreated dyslipidemic mice. It did, however, reduce vasodilation in wild-type and dyslipidemic mice treated with ivabradine (Figure 3).27 Since ivabradine has no direct vascular effect, this finding suggests that chronic pure heart reduction preserved the nitric oxide pathway for vasodilation.

_ Cerebral arteries
It was first demonstrated in cerebral arteries that smoothmuscle contractile and dilator mechanisms are not affected by either dyslipidemia or ivabradine. Endothelium-dependent dilation induced by acetylcholine was impaired in dyslipidemic mice compared with wild-type mice. The use of ivabradine completely prevented this impairment of vasodilatory capacity in cerebral arteries (Figure 4, page 425).27 H2O2 derived from eNOS is an important relaxing factor in cerebral mouse arteries. In wild-type and ivabradine-treated mice, the administration of catalase (which degrades H2O2) decreased maximal cerebral artery dilation, whereas it had no effect in untreated dyslipidemic mice (Figure 4). COX inhibition using indomethacin produced a similar decrease in cerebral artery maximal dilation in response to acetylcholine in wild-type mice and ivabradine-treated mice, while it did not alter the response to acetylcholine in untreated dyslipidemic mice (Figure 4). These findings show that ivabradine protected cerebral arteries from undesirable changes induced by dyslipidemia.

Figure 4
Figure 4. Endothelium-dependent dilation of pressurized cerebral arteries (60 mm Hg) in response to acetylcholine (control), isolated from (A) 6-month-old wild-type mice (B) dyslipidemic mice (C) dyslipidemic mice treated with ivabradine (10 mg/kg/day; from age 3 to 6 months).

The effects of hydrogen peroxide inactivation by catalase (100 U ml/L) and COX inhibition by indomethacin (10 mM) were tested in separate segments of cerebral arteries isolated from the same animal. Data are mean ±standard error of the mean. ‡P<0.05 compared with control. Abbreviations: ACh, acetylcholine; CAT, catalase; INDO, indomethacin. After reference 27: Drouin A, Gendron ME, Thorin E, Gillis MA, Mahlberg-Gaudin F, Tardif JC. Br J Pharmacol. 2008;154:749-757. Copyright © 2008, British Pharmacological Society.

Figure 5
Figure 5. The effects of chronic treatment with ivabradine and metoprolol from the age of 3 months.

On (A) the percentage increase in heart rate measured under anesthesia in dyslipidemic mice observed at 4.5 and 6 months of age. (B) and (C) Endotheliumdependent dilator responses to acetylcholine of pressurized renal (B) and cerebral (C) arteries preconstricted with phenylephrine, isolated from 6-month-old wildtype mice, dyslipidemic mice, and dyslipidemic mice treated with metoprolol (80 mg/kg/day; from age of 3 to 6 months). Data are mean ±standard error of the mean. ‡P<0.05 compared with wild type; *P<0.05 compared with dyslipidemic mice. Abbreviations: ACh, acetylcholine; DL, dyslipidemic; HR, heart rate; IVA, ivabradine; METO, metoprolol; WT, wild type. After reference 27: Drouin A, Gendron ME, Thorin E, Gillis MA, Mahlberg-Gaudin F, Tardif JC. Br J Pharmacol. 2008;154:749-757. Copyright © 2008, British Pharmacological Society.

_ Superior effects of ivabradine compared with the β-blocker metoprolol
Treating dyslipidemic mice with metoprolol reduced heart rate to the same extent as ivabradine (Figure 5A, page 425).27 In contrast, ivabradine provided superior preservation of endothelial function in renal and cerebral arteries in dyslipidemic mice compared with metoprolol. In renal arteries, metoprolol did not increase renal artery sensitivity to acetylcholine to the same extent as ivabradine (Figure 5B). In cerebral arteries, metoprolol did not prevent the decrease in dilator response to acetylcholine seen in dyslipidemic mice, unlike ivabradine (Figure 5C). The mitigated effects of metoprolol may be related to the coupling between endothelial â-adrenoceptors and eNOS.43

Conclusion

Pure heart rate reduction with ivabradine prevents deterioration of the endothelial function of renal and cerebral arteries in dyslipidemic mice to a greater extent than the β-blocker metoprolol. Changes in endothelial function may represent the link between changes in heart rate and cardiovascular events. These results support the evaluation of the effects of ivabradine on clinical outcomes in patients with coronary artery disease or with cardiovascular risk factors. _

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