Reduced cardiac functional reserve in apolipoprotein E knockout mice

Reduced cardiac functional reserve in apolipoprotein E knockout mice

Reduced cardiac functional reserve in apolipoprotein E knockout mice JON VINCELETTE, BABY MARTIN-MCNULTY, RONALD VERGONA, MARK E. SULLIVAN, and YI-XIN...

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Reduced cardiac functional reserve in apolipoprotein E knockout mice JON VINCELETTE, BABY MARTIN-MCNULTY, RONALD VERGONA, MARK E. SULLIVAN, and YI-XIN WANG RICHMOND, CALIFORNIA

Several years ago, the authors reported that aortic flow velocity under resting conditions was significantly higher in apolipoprotein E knockout (apoE-KO) mice than in age-matched C57Black/6J wildtype (WT) controls. The goal of this study was to examine whether the cardiac functional reserve is impacted in response to a pharmacological stress agent in apoE-KO mice. Cardiac function was measured noninvasively by the Doppler ultrasound method at baseline and at 1 min, 5 min, 10 min, and 20 min after intraperitoneal injection of dobutamine at the doses of 1 ␮g/g, 3 ␮g/g, or 10 ␮g/g in 16-month-old male apoE-KO (n ⴝ 9) and WT (n ⴝ 10) mice under light anesthesia with 1.5% isoflurane via inhalation. The baseline peak and mean aortic flow velocities were 39% to 48% higher, and left ventricular contractility measured by peak acceleration rate of aortic flow velocity was 24% higher in apoE-KO compared with WT mice (P < 0.01). Dobutamine stress dose-dependently increased cardiac function, which, however, was significantly smaller with a right shift of the dose-response curve in apoE-KO mice compared with WT controls. The hypotensive response to dobutamine was not significantly different between the 2 groups. Thus, despite an elevated resting aortic flow velocity and left ventricular contractility, cardiac functional reserve in response to dobutamine stress was significantly reduced in apoE-KO mice, which could be the consequence of coronary atherosclerosis and endothelial dysfunction that limits blood supply to the heart. (Translational Research 2006;148:30 –36) Abbreviations: apoE-KO ⫽ apolipoprotein E knockout; AUC ⫽ area under curve; ECG ⫽ electrocardiogram; MAP ⫽ mean arterial blood pressure; WT ⫽ wildtype

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poE-KO mice are hyperlipidemic and spontaneously develop atherosclerosis consuming a normal diet, and thus, they have been widely used in vascular biology, especially in atherosclerosis research.1 Numerous studies have characterized the morphology and pathohistology of vascular lesions in apoE-KO mice.1-7 Several studies have doc-

From the Department of Pharmacology, Berlex Biosciences, Richmond, California. Submitted for publication December 13, 2005; revision submitted February 22, 2006; accepted for publication March 21, 2006. Reprint requests: Yi-Xin (Jim) Wang, 2600 Hilltop Drive, Richmond, Calif 94549. e-mail: [email protected] 1931-5244/$ – see front matter © 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.lab.2006.03.007

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umented neurologic deficits8-12 and reduced aerobic capacity.13 However, the alterations in other aspects of cardiac function have not been well characterized in this model. Development of atherosclerotic lesions in the coronary arteries can limit cardiac blood supply, which eventually leads to impairment of cardiac function. However, unless it is in the late stage of heart failure, when cardiac function is severely deteriorated, resting cardiac function may not be significantly affected. Indeed, the authors have demonstrated that, in apoE-KO mice with severe atherosclerosis, the resting aortic flow velocity was not reduced; instead, it was even higher than WT mice.14 In laboratory studies and in the clinic, a stress test is commonly used to reveal cardiac dysfunction.15 As controlled exercise is difficult to perform in mice, pharmacological stress is a commonly used

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method, in which dobutamine is the preferential choice among other pharmacological stress agents, because it dominantly stimulates cardiac function with minimum peripheral hemodynamic effects.16 The current study was designed to expand previous findings from the resting condition to further reveal cardiac reserve dysfunction under dobutamine stress in apoE-KO mice compared with WT controls.14 METHODS Animal preparation and experimental protocol. In this study, 16-month-old male apoE-KO (n ⫽ 9) and age-matched C57BL/6J WT (n ⫽ 10) mice obtained from Jackson Laboratories (Bar Harbor, Maine) were fed normal rodent chow and kept in rooms at controlled temperature (24 °C) and lighting (14:10 h light-dark cycle) with free access to food and water. ApoE-KO mice progressively develop atherosclerotic lesions as they age. Therefore, the authors chose to use relatively old mice with the intention that they have long-term exposure to extensive atherosclerosis that would impact the cardiac function. The number of animals in each group was determined based on the authors’ previous experience. The experimental protocol was approved by the institutional animal care and use committee. Light anesthesia was induced by placing mice in a closed chamber of an anesthesia machine (IMPAC 6; VetEquip, Pleasanton, Calif) ventilated with 1.5% isoflurane for 3-5 min. Cardiac functional measurements were obtained noninvasively using the Doppler ultrasound method (see below). A baseline recording of aortic outflow or blood pressure was made immediately before drug administration (time ⫽ 0 min). Dobutamine (Sigma, St. Louis, Miss) was injected intraperitoneally at doses of 1 ␮g/g, 3 ␮g/g, and 10 ␮g/g at a volume of 3.33 ␮L/g. Aortic flow velocity recordings were made at 1 min, 5 min, 10 min, and 20 min postinjection. In a separate experiment, blood pressure was measured before and 5 min after dobutamine administration. Overall, 1 dose was given per each experiment, with the next dose given on a subsequent day in the same animal. Blood hematocrit was also measured by the standard centrifugation method. Noninvasive cardiac Doppler measurement. Anesthetized mice (9 apoE-KO and 10 WT) were taped supine on a temperature-controlled board for maintaining body temperature at 37°C ⫾ 2°C monitored with a rectal probe (Physitemp, Clifton, NJ). The limbs of the animal were taped to ECG electrodes connected to a high-fidelity ECG amplifier with a 0.1-kHz to 2-kHz bandwidth set to record lead II. Aortic blood flow velocity was measured with a 2-mmdiameter, 20-MHz-pulsed Doppler probe with a 6-10-mm focal distance placed just below the sternum and angled toward the aortic out flow. The sample volume depth and probe position were adjusted to record the maximum velocity when the waveform, direction, and timing were consistent with typical aortic velocity. Valve clicks were often observed in the Doppler spectral signals. The Doppler probe was connected to a modular pulsed Doppler system with a built-in data acquisition system and signal processing software (Indus Instruments Inc., Houston, Tx). The audio Doppler signals

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from the pulsed Doppler module and the amplified ECG signal were sampled and digitized. When the desired signals were obtained, a button was pressed on a remote keypad to save the last 2 s of unprocessed signals to a data file. From the spectral envelopes, the following features were extracted from the average of at least 10 selected cardiac cycles: (1) peak velocity; (2) mean velocity, defined as the flow velocity averaged over the cardiac period (equivalent to stroke distance multiplied by heart rate); (3) stroke distance, the area under the aortic flow velocity curve at each cardiac cycle; (4) peak acceleration, the maximum derivative of the velocity signal; (5) mean acceleration, average acceleration rate; (6) pre-ejection time, the time from the peak of the R-wave of the ECG to the upstroke of velocity; (7) ejection time, the time between the start of the upstroke and when the downstroke returns to the baseline; (8) rise time, the time between the start of the upstroke and when it reaches to the peak; and (9) heart rate. Aortic flow velocity is often used as an index of cardiac output assuming that aortic diameter does not differ between animals of different experimental groups.14,17,18 Noninvasive measurement of blood pressure by the tailcuff method. In a separate experiment (8 apoE-KO and 9

WT), anesthetized mice were placed on a heated platform and the tails inserted through the MC4000 tail-cuff blood pressure monitoring device (Hatteras Instruments, Cary, NC). Blood pressure was measured before and 5 min after dobutamine administration. The data were analyzed using built-in software. Data processing and statistical analysis. Results are presented as mean ⫾ standard error of the mean. To compare total cardiac response with dobutamine stress at each equal dose between the apoE-KO and WT mice, both the duration and the magnitude of the responses were integrated as the AUC over a period of 20 min for each dose. A new doseresponse curve was constituted by the changes in AUC induced by the multiple doses of dobutamine. The statistical analysis was performed by 2-way analysis of variance, followed by the Student–Newman–Keuls test, with 1 factor being WT versus KO and the other doses of dobutamine. Statistical significance was defined by a P value of less than 0.05. RESULTS Cardiac hyperdynamics in apoE-KO mice in the resting condition. As illustrated in the representative wave-

forms in Fig 1, baseline aortic flow velocity was higher in an apoE-KO mouse than in a WT control mouse. Calculated as a group average, cardiac peak and mean aortic velocities were 39% and 48% higher, respectively, in apoE-KO mice compared with WT controls (Table I). The higher cardiac flow velocity in apoE-KO mice was mainly caused by a 57% higher stroke distance because there was no significant difference in the heart rate between the 2 groups. Left ventricular contractility measured by peak, but not mean acceleration rate of aortic flow velocity, was 24% higher in

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Fig 1. Representative original recordings of Doppler spectra from an anesthetized apoE-KO mouse (#6) and a C57 black/6J WT control mouse (#18) before (baseline) and 5 min after intraperitoneal administration of dobutamine at 10 ␮g/kg. The arrows indicate that the response to dobutamine stress was much greater in WT than in apoE-KO mice.

Table I. Baseline hemodynamic measurements in apoE-KO and WT mice WT (n ⴝ 10)

Peak velocity (cm/s) Mean velocity (cm/s) Stroke distance (cm) Peak acceleration (cm/s2) Mean acceleration (cm/s2) Pre-ejection time (ms) Ejection time (ms) Rise time (ms) Heart rate (Beats/min)

87 ⫾ 2 23 ⫾ 0.5 3 ⫾ 0.1 11098 ⫾ 545 8206 ⫾ 514 15 ⫾ 0.4 49 ⫾ 1 12 ⫾ 0.5 457 ⫾ 9

ApoE-KO (n ⴝ 9)

% Difference

P Value

121 ⫾ 4 34 ⫾ 2 5 ⫾ 0.2 13804 ⫾ 643 8349 ⫾ 432 16 ⫾ 0.5 54 ⫾ 0.7 15 ⫾ 0.7 432 ⫾ 11

39 48 57 24 2 6 11 27 ⫺6

0.000 0.000 0.000 0.005 NS NS 0.001 0.001 NS

Abbreviation: NS, not significant. Notes: The data are calculated from aortic flow velocity wave forms.

apoE-KO mice. Ejection and rise times were 11% and 27% higher, respectively, in apoE-KO mice. No significant difference existed in pre-ejection time and heart rate between the 2 groups. Both the body weight (30 ⫾ 0.3 vs 40 ⫾ 0.9 g) and hematocrit (40 ⫾ 0.5 vs. 43 ⫾ 1.3 %) were significantly lower in apoE-KO than WT mice (P ⬍ 0.02, n ⫽ 10/group). Reduced cardiac functional reserve in apoE-KO mice in response to dobutamine stress. Intraperitoneal injection

of dobutamine increased aortic flow velocity; however, the increase in the peak velocity, as it was directly visible, was greater in WT than in apoE-KO mice (Fig

1). As shown in Fig 2, the dose-dependent increase in acceleration rate of aortic flow velocity quickly reached peak levels and was maintained over the course of the 20-min observation period. To compare the magnitude of the dose-response over the 20-min observation period between apoE-KO and WT mice, the changes in measured parameters after dobutamine administration were integrated as the area under the dose-response curve over a 20-min period (Figs 3-5). The dose-dependent increase in mean acceleration rate of aortic flow velocity induced by dobutamine stress was significantly smaller in apoE-KO mice than in WT controls

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Fig 2. Time course of mean aortic velocity acceleration rate in response to multiple doses of dobutamine in anesthetized C57 black6J WT control (top) and apoE-KO (bottom) mice.

(Fig 3). The increase in peak acceleration rate of aortic flow velocity and heart rate after dobutamine stress was not significantly different between the 2 groups. Dobutamine dose-dependently increased cardiac flow velocity and stroke distance. However, the dose response measured by peak velocity was significantly smaller in apoE-KO mice than in WT controls, and the responses measured by mean aortic flow velocity and stroke distance were completely lost in apoE-KO mice (Fig 4). Dobutamine dose-dependently decreased preejection, ejection, and rise time (Fig 5). Only the response for pre-ejection time was significantly smaller in apoE-KO mice compared with WT controls, with no significant differences in ejection and rise time between the 2 groups. In a separate experiment, blood pressure was measured by the tail-cuff method in anesthetized mice in a condition similar to that for Doppler measurements. Baseline blood pressures were not significantly different between apoE-KO and WT mice (Fig 6). Dobutamine dose-dependently decreased MAP, which was not significantly different between apoE-KO and WT mice.

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Fig 3. Dose-dependent changes of the AUC (over 20 min) of mean (top) and peak (middle) aortic flow velocity acceleration rate, and heart rate (bottom) in response to dobutamine stress in anesthetized C57 black/6J WT control and apoE-KO mice. Statistical significance, p ⬍ 0.05. *The lowest dose point together with the higher dose points that showed significantly different from baseline value; #Significant difference between WT control and apoE-KO mice at the same dose.

DISCUSSION

This study is the first to demonstrate that despite an increased peak aortic flow velocity acceleration rate (an index of left ventricular contractility), aortic flow velocity and stoke volume (indexes of cardiac output) in the resting condition, dobutamine-induced increases in the above cardiac functional parameters were significantly smaller, which indicates a reduced cardiac functional reserve in apoE-KO mice compared with WT controls. These observations are consistent with the reports of reduced aerobic capacity in apoE-KO mice,13 compromised cardiac functional reserve in response to pharmacological stress in hyperlipidemic and atherosclerotic minipigs,19,20 and reduced coronary flow reserve in hypercholesterolemic patients without overt coronary stenosis.21,22 Development of atherosclerotic lesions in the coronary arteries can directly limit cardiac blood supply, which could negatively impact cardiac function. However, in the early stages of atherosclerosis and cardio-

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Fig 4. Dose-dependent changes of the AUC (over 20 min) of mean (top) and peak (middle) aortic flow velocity, and stroke distance (bottom) in response to dobutamine stress in anesthetized C57 black/6J WT control and apoE-KO mice. Statistical significance, P ⬍ 0.05. *The lowest dose point together with the higher dose points that significantly different from baseline value. #Significant difference between WT control and apoE-KO mice at the same dose.

vascular diseases when cardiac function is not severely deteriorated, resting cardiac function may not be apparently abnormal. In contrast, the current data confirmed the authors’ previous report that both the peak and the mean aortic flow velocities and stoke volume at resting condition were significantly increased in apoE-KO compared with WT mice.14 Increased resting cardiac function may reflect a higher demand of peripheral blood flow in apoE-KO compared with WT mice. The authors’ previous report that hind limb blood flow significantly increased in high-cholesterol-fed rabbits provides evidence of increased peripheral blood flow or altered vasoregulation in hyperlipidemic and atherosclerotic conditions.23 In this study, the authors used dobutamine to stimulate left ventricular contractility as a means to uncover the potential dysfunction in the cardiac reserve. As dobutamine is a dominant ␤1- and relatively weak ␤2-adrenoceptor agonist, it can maximally stimulate cardiac contractility by activating cardiac ␤1-adrenoceptors with minimum peripheral hemodynamic effects because of its relatively weak vascular

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Fig 5. Dose-dependent changes of the AUC (in 20 min) of preejection time (top), ejection time (middle), and rise time (bottom) in response to dobutamine stress in anesthetized C57 black/6J WT control and apoE-KO mice. Statistical significance, P ⬍ 0.05. *The lowest dose point together with the higher dose points that significantly different from baseline value. #Significant difference between WT control and apoE-KO mice at the same dose.

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␤2-adrenoceptor-mediated vasodilation.15 Thus, comparing with other cardiac stress agents such as isoprotenerol, a nonselective ␤-adrenoceptor agonist, dobutamine has minimum impact on peripheral vascular

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resistance, which can also impact cardiac function via afterload changes. As expected, the authors’ data show that under dobutamine stress, left ventricular contractility and cardiac flow velocity increased dose-dependently, but the response was significantly compromised in apoE-KO mice. The pre-ejection time is another measurement of systolic function. It is usually shortened with an increased volume loading because of the Starling mechanism, or prolonged because of either a reduction in systolic function or a conduction deficit. In this study, dobutamine stress stimulated cardiac systolic function and, thus, dosedependently shortened pre-ejection time. Although the basal pre-ejection time was not significantly different between the 2 groups, the response to dobutamine stress was significantly attenuated, which provides additional evidence of reduced cardiac systolic reserve in apoE-KO mice. The ejection phase of the cardiac index is highly afterload-dependent. The data from this study show that dobutamine had a similar hypotensive effect between apoE-KO and WT mice, which excludes the possibility of afterload contribution to reduced cardiac functional reserve in apoE-KO mice. The authors6 and many others24,25 have reported that endothelium-dependent vasodilation is impaired in atherosclerotic apoE-KO mice. Thus, coronary atherosclerotic lesions or coronary endothelial dysfunction can limit blood supply to the heart when demand has increased in response to dobutamine stimulation. Furthermore, the authors’ data have shown that hematocrit was significantly lower in apoE-KO mice. Thus, a reduced oxygen-carrying capacity of blood in the microcirculation of the heart could impair cardiac functional performance in response to an increased energy demand because of dobutamine stress. Moreover, the apoE-KO mice may also be in compensated heart failure because of the chronically high cardiac outputs. The authors’ previous study has provided morphological evidence that these animals had cardiac hypertrophy, which suggests that remodeling was occurring as early as when the animals were 13 months old.14 Thus, chronic heart failure may also contribute, in part, to the reduced cardiac reserve in apoE-KO mice. One technical limitation of this study is that the Doppler technique can only measure flow velocity, not volume, which is the real unit for the cardiac output. As cardiac output is a function of velocity and crosssectional area of the aorta, which has been shown not significantly different between age-matched apoE-KO and WT mice, the flow velocity can be used as a surrogate index for cardiac output. In summary, this study is the first to demonstrate that despite cardiac hyperdynamics in the resting condition, dobutamine-stimulated increases in cardiac function

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were impaired in apoE-KO mice. Coronary insufficiency because of atherosclerosis and endothelial dysfunction, which impairs blood supply in response to increased demands, may partially contribute to reduced cardiac reserve in apoE-KO mice. Atherosclerosis development in the aorta impacting cardiac afterload could also affect cardiac function.

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