Cholesteryl Ester Transfer Protein (CETP) Expression Protects Against Diet Induced Atherosclerosis in SR-BI Deficient Mice

【摘要】  Objective- To determine whether expression of the human CETP transgene protects against diet-induced atherosclerosis in SR-BI deficient mice.

Methods and Results- SR-BI deficient (-/-) mice were crossed with CETP transgenic (CETPtg) mice to produce a colony of SR-BI -/- x CETPtg mice in a C57Bl/6 background. Age and sex matched groups of genetically modified and wild-type C57Bl/6 mice were fed a high fat, high cholesterol diet for 22 weeks. In both wild-type and SR-BI -/- mice, expression of the CETP transgene reduced the cholesterol content and increased the density of lipoprotein particles in the HDL density range. In SR-BI -/- x CETPtg mice, CETP activity inversely correlated with total plasma cholesterol levels and shifted the buoyant HDL typical of SR-BI deficiency toward a more normal density HDL particle. Atherosclerosis at the level of the aortic arch was evident in both male and female SR-BI deficient mice but occurred to a greater extent in the females. Expression of CETP markedly attenuated the development of atherosclerosis in SR-BI deficient mice fed an atherogenic diet ( P <0.003).

Conclusions- Expression of the human CETP transgene protects SR-BI deficient mice from atherosclerosis, consistent with a role for CETP in remodeling HDL and providing an alternative pathway for the selective uptake of HDL-CE by the liver.

To determine whether expression of the human CETP transgene protects against diet-induced atherosclerosis in SR-BI deficient (-/-) mice, we crossed these with CETP transgenic mice to create SR-BI -/- x CETPtg mice in a C57Bl/6 background. CETP expression reduced HDL cholesterol, increased HDL density, and markedly attenuated the development of atherosclerosis.

【关键词】  CETP SRBI atherosclerosis HDL

Introduction

Cholesteryl ester transfer protein (CETP) is a 74-kDa hydrophobic glycoprotein that plays a central role in human high-density lipoprotein (HDL) metabolism. In humans, CETP mRNA is predominantly expressed in the liver, spleen, and adipose tissue and is secreted to a variable extent from each of these tissues into plasma, where it mediates neutral lipid transport between lipoproteins. 1 Cholesterol effluxed from peripheral tissues is esterified within HDL by lecithin cholesterol acyltransferase (LCAT). This CE can be subsequently transferred by CETP to apolipoprotein B (Apo B)-containing lipoproteins. 2

The overall role of CETP in atherosclerosis is complex. High plasma concentrations of CETP are associated with low HDL cholesterol levels, which are a risk factor for coronary artery disease (CAD). 3 This observation has led to the development of CETP inhibitors as a potential therapy to reduce atherosclerosis. 4,5 On the other hand, under conditions of efficient hepatic Apo B lipoprotein clearance, CETP may promote cholesterol transport from HDL to the liver for eventual secretion into bile.

HDL-CE is also directly returned to the liver by a process known as selective uptake. This involves the reversible incorporation of HDL-derived CE into a plasma membrane pool followed by transfer of the lipid to an inaccessible pool by mechanisms that do not involve coated pit-mediated endocytosis. 6 SR-BI has been shown to be the primary receptor responsible for hepatic HDL-cholesterol clearance in the mouse, 7,8 a species, which intrinsically lacks CETP, 9 whereas the role of the human homologue of SR-BI, CLA-1, in lipoprotein metabolism is less clear. 10 Genetic deficiency of CETP in humans results in a marked increase in plasma concentrations of HDL-CE. 11 This suggests that CETP is required for normal HDL cholesterol clearance in humans. Interestingly, the CE-rich, low-density HDL particles characteristic of SR-BI deficiency in mice are very similar to the HDL particles in CETP deficient patients. These particles contain increased amounts of Apo E and may be cleared by members of the LDL receptor family. 12

CETP and SR-BI each function to reduce HDL size and cholesterol content. CETP also mediates the selective acquisition of CE from HDL by various cell types in a similar fashion to SR-BI. We have previously demonstrated this novel role for CETP in human adipocytes. 13,14 More recently, we have shown that CETP directly mediates selective uptake of HDL-derived CE by hepatocytes, a cell of fundamental importance in HDL metabolism, and have shown that this effect occurs independently of SR-BI or other established lipoprotein receptors 15

Mice deficient in SR-BI have been shown to develop atherosclerosis on a high-fat, high-cholesterol diet. 16 In the present study, we demonstrate that the human CETP transgene protects against diet-induced atherosclerosis in SR-BI deficient mice, thereby providing new and important insight into the possible roles of CETP and SR-BI as alternative routes for HDL-CE clearance by the liver.

Methods

Animals

The C57Bl6J (wild-type) mouse strain was purchased from Charles River (Wilmington, Mass). Heterozygous SR-BI deficient (strain B6.129S2-Scarb1 tm1Kri) ) mice were purchased from Jackson Laboratory (Bar Harbor, Me) and have been previously characterized. 17 The CETPtg mice (B6,SJL-Tg) were obtained from Taconic Farms (Hudson, NY). SR-BI deficient and CETPtg mice were bred in a C57Bl/6J background to obtain colonies of wild-type mice, SR-BI -/-, CETPtg, and SR-BI -/- x CETPtg. SR-BI -/- x CETPtg mice were selected to have between 2- to 6-fold human CETP activity. All mice were fed a normal chow diet for 6 to 8 weeks and then transferred to an atherosclerosis-promoting, high-fat, high-cholesterol diet (atherogenic diet) for 22 weeks. Animals were anesthetized and exsanguinated by cardiac puncture. Sera were stored at 4°C before discontinuous density gradient ultracentrifugation. Animal experiments were performed in compliance with protocols approved by the University of Ottawa Animal Care Committee.

Diet Composition

The atherogenic diet (product # TD 94059), purchased from Harlan Teklad, consisted of 75% ground 5015 Purina mouse chow, 7.5% casein, 3% dextrose monohydrate, 1.625% sucrose, 1.625% dextrin, 7.5% cocoa butter, 1.25% cholesterol, 1.25% cellulose, 0.875% AIN-76 mineral mix, 0.25% Teklad vitamin mix (40060), and 0.125% choline chloride. The diet did not include sodium cholate, which inhibits bile acid synthesis and may affect reverse cholesterol transport. 18

Discontinuous Density Gradient Ultracentrifugation

Plasma lipoprotein densities were determined by density gradient ultracentrifugation according to the protocols of Terpstra et al 19 with the following modifications. Density solutions of 1.000, 1.006, 1.080, and 1.21 g/mL KBr containing 1 mmol/L EDTA and 0.02% NaN 3 were used to form a step gradient in a 12 mL centrifuge tube. On average, 250 to 500 µL of mouse plasma was mixed with the 1.006 g/mL density solution to make a 3 mL solution. 1 g of KBr and 50 mg of sucrose were then added to the plasma sample. The gradient was made with 3 mL of the 1.000 g/mL solution, under laid with 3 mL of the 1.080 g/mL, 2 mL of the 1.21 g/mL and the approximately 3 mL plasma solution. Plasma was spun at 45 000 g in an SW21 rotor for 18 hours at 8°C. Samples were isolated into 23 fractions and their densities and cholesterol concentrations were determined with a densitometer and a calorimetric total cholesterol kit (Wako Chemical), respectively.

Liver Cholesterol Analysis

Lipids were isolated from approximately 100 mg of snap-frozen liver according to the protocol of Bligh and Dyer. 20 Briefly, lipids were extracted with methanol:chloroform 2:1 at 20 mL/g. After isolation in chloroform, lipids were dried under nitrogen and dissolved in colorimetric reagent. The colorimetric kits for total cholesterol (cholesterol E-test, Wako Chemicals) and free cholesterol (Free cholesterol E-test, Wako Chemicals) were used to quantify the lipids. Because of the large amount of triglycerides in the mice fed a high-fat/high-cholesterol diet, the samples were spun at 50 000 g to remove the triglycerides before measuring the colorimetric products. The CE levels were determined by subtracting the free cholesterol from the total cholesterol levels.

CETP Transfer Activity Assay

The CETP transfer activity of plasma in the CETPtg mice was determined using a CETP Activity Kit (Roar Biomedical Inc) according to the manufacturer?s recommendations. Briefly, the incubation of a donor and acceptor particle with the mouse plasma containing CETP resulted in transfer of a fluorophore to the acceptor particle. The fluorescence intensity was then measured after exciting it 465 nm. The transfer activity in the unknown mouse samples was compared with the CETP activity of a sample consisting of plasma pooled from 10 normolipidemic human donors. The pooled human plasma was frozen and used as the reference pool for all mouse samples. As the CETP activity in the mice was on average 4-fold greater than human plasma, 2 µL of a 1/10 dilution of the mouse plasma was used to ensure that the fluorescent reading fell within the linear range of the kit.

Quantification of Atherosclerotic Lesions in Tissue Sections

The size of atherosclerotic lesions in the ascending aorta of CETPtg and SR-BI -/- mice was determined from 4 Sudan IV stained serial sections, cut 10 µm thick and collected at 100-µm intervals and captured using a digital CoolSNAP cf camera (Roper Scientific Inc). Lesion analysis began with the first section of tissue that contained the ostia for the coronary arteries, a region that morphologically defines where the aortic sinus becomes the ascending aorta. 21,22 Using the Sudan IV staining as a guide, lesion area defined as intimal tissue within the internal elastic lamina was determined using Image-Pro software (Media Cybernetics).

Statistical Analysis

Results are expressed as the mean±SEM. Where indicated, the statistical significance of the differences between groups was determined using a one-tailed probability value of the unpaired t test. Data were graphed and analyzed in GraphPad Prism v.4.03 software and statistical analyses were performed using GraphPad Instat 3.06 software.

Results

Extent of Atherosclerosis

As previously reported, we observed significant increases in the average lesion area in the aortic root of SR-BI -/- male and female mice compared with their wild-type controls. ( Figure 1A versus 1B and 1E versus 1 F, respectively). 16 We sectioned the aorta in a similar fashion to Van Eck et al, 16 which measures lesion formation in the first 100 um of the tricuspid valve (aortic root), as well as according to the more standard procedure described by Paigen et al, 21,22 which measures lesion formation in the first 300 um of the ascending aorta. The measured areas of these two techniques are greater than 500 um apart and represent 2 different vascular beds with no overlap. We found that there was substantially greater atherosclerosis and greater variability when lesion areas were calculated by the Van Eck procedure. Because lesion formation begins within the aortic root and spreads distally throughout the vascular tree, it is not surprising that others have also demonstrated that lesion size is the greatest around the aortic root. 21,23 These earlier studies also helped explain the considerable variability in lesion area between mice using the Van Eck procedure compared with the Paigen method. According to both protocols, the observed lesion area was much greater in the female mice than the male mice (total lesion area in the females 0.046±0.012 mm 2, 0.015±0.05 mm 2 and in the males 0.036±0.024 mm 2, 0.007±0.003 mm 2 [Van Eck and Paigen, respectively]). Based on the lower variability among mice of the same group, the well-established Paigen method was used for the analyses reported here. Differences in the mean lesion areas in the cross mice versus the SR-BI -/- mice were consistent with a significant atheroprotective effect of CETP transgene expression in SR-BI -/- mice ( Figure 1 IJ red and blue bars). On the other hand, there were no significant differences in lesion areas among the groups of wild-type, CETPtg, and SR-BI -/- x CETPtg mice (Figure IJ red, green and purple bars). Notably, there was no detectable atherosclerosis in wild-type mice or in SR-BI +/+ mice expressing the human CETP transgene.

Figure 1. CETP expression in SR-BI -/- mice protects against diet-induced atherosclerosis. A-H, Representative 10-µm cross sections of ascending aorta of (A, E) wild-type, (B, F) CETPtg, (C, G) SR-BI -/-, and (D, H) SR-BI -/- x CETPtg recipient mice on an atherogenic diet for 22 weeks (A-D, female mice; E-H, male mice). Sections were stained with Sudan IV after sectioning. I, J, Ascending aortic lesions beginning at the ostia were quantified. When analyzed by unpaired one-tailed Student t test, there is significant difference between SR-BI -/- and SR-BI -/- x CETPtg up to 300 µm from the aortic root in the female mice (I) and a significant difference at 100 µm from the aortic route in male mice (J) (* P <0.003, ** P <0.05). There were no significant differences observed between SR-BI -/- and SR-BI -/- x CETPtg in male mice at 200 µm and 300 µm from the ostia or between any of the wild-type, CETPtg, and SR-BI -/- x CETPtg groups ( P 0.05). Data represent mean±SEM (see Table 1 for N).

TABLE 1. Characteristics of Wild-Type and Genetically Modified Mice

CETP Reduces High Plasma Cholesterol Concentrations Associated With SR-BI Deficiency

The expression of CETP in C57Bl/6 mice resulted in a decrease in total cholesterol concentrations ( Table 1 ), a decrease in cholesterol in the HDL density range, and a shift of the HDL particles to a higher density ( Figure 2 A: CETPtg). As previously reported, SR-BI gene deletion in mice increased the amount of cholesterol associated with the HDL-pool and decreased the mean density of the HDL-like particles ( Figure 2 A, wild-type versus SR-BI -/- ). 17 Accordingly, the expression of the CETP transgene led to a substantial decrease in the amount of cholesterol associated with the HDL pool and an increase in the mean density of the HDL particles ( Figure 2 A: wild-type versus CETPtg). In line with this observation, the expression of CETP in the SR-BI deficient mice shifted the HDL peak (1.060 to 1.14 g/mL 24 ) more toward the density range of normal HDL particles ( Figure 2 A). Moreover, this shift in HDL density correlated with the levels of CETP activity in the plasma ( Figure 2 B) and with the total plasma cholesterol levels ( Figure 2 C). At approximately 3.7-fold human CETP activity, the lipoprotein profiles in the HDL density range of the SR-BI -/- x CETPtg mice resembled that of a wild-type mouse ( Figure 2 B blue line). As expected, VLDL cholesterol increased significantly in all groups on the atherogenic diet but CETP expression alone or in the SR-BI -/- background did not lead to significant changes in the concentration of cholesterol in particles of 1.017 g/mL 24 ) density range ( P =0.15 by one way ANOVA, 24.6±2.3 mg/dL SR-BI -/-, 21.3±2.5 mg/dL SR-BI -/- x CETPtg, and 17.9±2.5 mg/dL CETPtg).

Figure 2. Effect of CETP expression on the distribution of lipoprotein cholesterol in mouse plasma. A-C, Blood samples were collected after cardiac puncture. Plasma cholesterol levels and fraction densities were determined by discontinuous density gradient ultracentrifugation and densitometric analysis respectively. A, Cholesterol content of lipoprotein fractions (d 1.000 to 1.180) in mice fed an atherogenic diet for 22 weeks. B, Effects of varying levels of CETP expression on cholesterol content of d 1.000 to 1.180 lipoproteins. Increasing CETP activity in the SR-BI -/- mice leads to progressive normalization of cholesterol content and density of the HDL peak. The dashed lines estimate the cholesterol peak densities for the respective levels of CETP activity. C, Relationship between plasma CETP activity and plasma cholesterol concentration. Increasing CETP activity inversely correlates with total plasma cholesterol concentrations in SR-BI -/- mice. Spearman correlation r =-0.702; two-tailed * P =0.0204.

CETP Expression Alters the Density of "HDL-Like" Particles in the SR-BI -/- Background

In SR-BI -/- and CETPtg mice, the atherogenic diet increased total plasma cholesterol but did not change the overall lipoprotein cholesterol distribution ( Figure 3A and 3 B; Table 2 ). Interestingly, when placed on an atherogenic diet, the HDL peak of the SR-BI -/- x CETPtg mice shifted toward a lower density range.

Figure 3. Effect of CETP expression on diet-induced changes in lipoprotein distributions. A-C, Representative cholesterol distributions in mice fed either a regular chow diet or an atherogenic diet for 22 weeks. Chow fed mice were between 4 to 8 months old and the atherogenic diet fed mice were 8 months old. Blood samples were obtained by cardiac puncture after an overnight fasting period. Symmetrical increases in the cholesterol content of particles in the HDL density range are shown in A and B. An increase in cholesterol content and a shift in peak density of HDL are shown in C. The dotted vertical lines demonstrate the shift to a lower density lipoprotein particle on the atherogenic diet. For corresponding mean peak densities and mean cholesterol concentrations see Table 2.

TABLE 2. CETP Expression Shifts the Mean HDL-Cholesterol Density and Content

CETP Expression Reduces Plasma HDL-Cholesterol Without Increasing Hepatic Cholesterol Accumulation

Given the large decreases in plasma cholesterol levels in CETP expressing mice, we investigated whether this change was accompanied by cholesterol accumulation in the liver. We found that hepatic cholesteryl ester concentrations were significantly increased in SR-BI deficient mice. There were, however, no significant changes in hepatic free or esterified cholesterol concentrations in CETP expressing mice.

Discussion

SR-BI has been shown to be the primary receptor responsible for hepatic HDL-cholesterol clearance in the mouse, a species that intrinsically lacks CETP. Despite a marked increase in HDL-cholesterol concentrations, SR-BI deficient mice develop increased atherosclerosis as compared with wild-type mice when fed a high-fat, high-cholesterol diet. We previously reported that, when expressed in mouse liver, CETP functions analogously to SR-BI to mediate selective uptake from HDL. 15 Importantly, we now demonstrate that expression of the human CETP transgene protects against diet-induced atherosclerosis in the SR-BI -/- background.

The effect of CETP expression on atherosclerosis susceptibility in animal models has been extensively studied and appears highly contingent on the level of CETP expression and on the genetic and metabolic context (reviewed by Parini and Rudel 25 ). Unlike mice, rabbits naturally express CETP. Plasma CETP concentrations in cholesterol-fed rabbits are 10-fold that of humans and inhibition of CETP expression or function using anti-sense oligonucleotides, 26 a CETP vaccine 27 or a CETP inhibitor 28 reduces atherosclerosis in this model. Similarly, very high levels of simian CETP transgene expression were shown to promote atherosclerosis in male C57Bl/6 mice. 29 However, as shown here, more moderate CETP expression that elicits CE transfer activity at 2.6- to 5.8-fold that of normal human plasma, does not promote atherosclerosis in C57Bl/6 mice fed an atherogenic diet. In other studies, both ApoE and LDL receptor knockout mice developed more extensive atherosclerosis when crossed with CETP transgenic mice. 30 Westerterp et al recently demonstrated that in mice bearing the apoE Leiden gene, resulting in defective apoB lipoprotein clearance, CETP expression increases atherosclerosis. 31 Thus, by promoting cholesterol enrichment of ApoB-containing lipoproteins, high plasma CETP activity has proatherogenic effects if the clearance of these particles is impaired. In other genetic backgrounds, including mice made hypertriglyceridemic by overexpression of the ApoCIII gene, CETP expression was consistently atheroprotective in both male and females. 32 Introduction of the CETP transgene corrected the dysfunctional HDL and also reduced aortic atherosclerosis in mice deficient in LCAT. 33

Accordingly, our studies add additional insight into the overall function of CETP in lipoprotein remodeling and reverse cholesterol transport. In wild-type mice fed an atherogenic diet, CETP expression shifts the HDL toward a denser, lipid poor fraction. At the level of CETP expression achieved in this study, no effect on atherosclerosis was noted. In contrast to the CETP expressing mice, the plasma HDL peak was dramatically increased in SR-BI knockout mice and consisted of cholesterol rich, low-density particles. However, as previously noted, these large HDL appear "dysfunctional" in terms of atherosclerosis protection 16 and are associated with impaired fertility in females. 34 Thus, in the context of SR-BI -/- deficiency, we demonstrate that CETP expression partially "normalizes" the density and concentration of circulating HDL ( Figure 2 A) and protects against atherosclerosis ( Figure 1 IJ).

In mice deficient in SR-BI, CETP may protect against atherosclerosis through a number of mechanisms. As shown here ( Figure 2 ), expression of CETP in SR-BI -/- mice normalizes HDL composition, which may make these HDL better acceptors of cellular cholesterol. Macrophages also synthesize and secrete CETP 35 and CETP may directly increase cholesterol efflux. 36,37 Impairment of the final pathway for the direct return of HDL cholesterol to the liver by selective uptake is causative of the abnormal HDL composition in SR-BI deficiency and may contribute to atherosclerosis susceptibility. Thus, in this metabolic context, CETP may be atheroprotective by increasing the flux of HDL-derived cholesterol to the liver indirectly via transfer to apoB lipoproteins or by directly mediating hepatocyte HDL cholesteryl ester selective uptake. 15 Quantification of liver CE levels indicated that CETP in the SR-BI -/- background mediates this transfer without increased deposition of hepatic cholesterol.

Our group has previously reported that CETP mediates the transfer of CE from HDL to hepatocytes by a process that does not require the presence of SR-BI or other known lipoprotein receptors. 15 Using adenovirus-mediated CETP (ad-CETP) expression in primary mouse hepatocytes from either wild-type, LDL receptor -/-, or SR-BI -/- mice, we demonstrated that CETP enhanced the selective accumulation of HDL-derived 3 H-CE independently of known lipoprotein receptors. When mice were treated with torcetrapib to prevent CETP-mediated transfer of CE to apoB lipoproteins and infected with ad-CETP, we noted a prompt 37% decrease in HDL-C versus ad-luciferase infected controls.

CETP also has a well established role in mediating the transfer of CE from HDL to apoB-containing lipoproteins, which can be a final vehicle for reverse cholesterol transport. Despite the large decreases in cholesterol in particles in the HDL density range, we did not observe significant increases in VLDL cholesterol levels in mice expressing CETP, consistent with effective hepatic clearance of apoB/E lipoproteins. Thus, in the context of SR-BI deficiency, CETP may promote the clearance of HDL-derived cholesterol indirectly via transfer to apoB lipoproteins or directly by hepatocyte selective uptake in an analogous fashion to SR-BI. These results have recently been supported by work of Zhou et al 38 that further suggested CETP promotes CE clearance to the liver independent of LDL receptor family-mediated endocytosis. The relevance of these findings to humans is likely to depend on the adequacy of the SR-BI (CLA-1) pathway for hepatocyte selective uptake of HDL-CE. As discussed above, CETP has both pro- and antiatherogenic effects and the optimal levels of CETP protein and activity for cardiovascular protection are not clear. Interestingly, probucol increases plasma CETP concentrations, 39 and despite a marked decrease in HDL cholesterol, results in regression of tendon xanthomata in patients with familial hypercholesterolemia 40 and reduces progression of carotid atherosclerosis. 41 Isolated case reports suggest that this agent may confer protection against CAD in patients with partial CETP deficiency and elevated HDL-C concentrations. 42

Conclusions

As we have shown here, CETP expression protects against diet-induced atherosclerosis in SR-BI deficient mice. This work provides insight into the multiple functions of CETP and suggests that the composition of HDL particles and the dynamics of cholesterol flux through the HDL pool are central to its atheroprotective function. Analogous to murine SR-BI -/- deficiency, genetic deficiency of CETP in humans results in a marked increase in plasma concentrations of HDL-CE, 11 suggesting a limited role for SR-BI (CLA-1) or a requirement for CETP in selective uptake in humans. Inhibition of CETP activity results in a marked increase in plasma HDL concentrations. Although a recent clinical study of torcetrapib did not demonstrate a decrease in coronary events, other compounds, which inhibit CETP, are under investigation. On the other hand, therapies which increase CETP 39 might be of benefit in a subset of patients with low plasma CETP and CE-rich HDL.

Figure 4. Effect of CETP expression on hepatic free cholesterol and cholesteryl ester levels in mice fed an atherogenic diet. No significant differences were noted in hepatic free cholesterol levels (light gray bars) among the different groups (* P =0.32 by nonparametric two-way ANOVA). Hepatic CE levels (black bars) inversely correlated with the presence of the SR-BI gene ( P <0.002, columns 1 and 2 vs columns 3 and 4 by unpaired t test with Welch correlation), and the expression of CETP in the SR-BI deficient background did not significantly increase the accumulation of CE ( P =0.09). There were also no significant differences in hepatic free cholesterol or cholesteryl ester concentrations between the CETPtg and wild-type mice ( P <0.23, P <0.15). Data represent mean±SEM. n=6 (3 males, 3 females).

Acknowledgments

The authors thank Ross Milne and Yves Marcel for critical review of the manuscript.

Sources of Funding

This work was supported by grants from Canadian Institutes of Health Research (CIHR) # 44360 (to R.M.) and by an Ontario Graduate Scholarship in Science and Technology and a Heart and Stroke Foundation of Canada Doctoral award (to C.H.). S.C.W is the recipient of a Great-West Life & London Life New Investigator Award from the Heart and Stroke Foundation of Canada.

Disclosures

None.

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作者单位：Lipoprotein and Atherosclerosis Research Group & Division of Cardiology, University of Ottawa Heart Institute, Ottawa, Canada