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(Circulation. 1999;100:2177.)
© 1999 American Heart Association, Inc.

Basic Science Reports

Cardiovascular Abnormalities in Transgenic Mice With Reduced Brown Fat

An Animal Model of Human Obesity

Presented in part at the 70th Scientific Sessions of the American Heart Association, Orlando, Fla, November 9–12, 1997, and published in abstract form (Circulation. 1997;96[suppl I]:I-465).

Antonio Cittadini, MD; Christos S. Mantzoros, MD; Thomas G. Hampton, PhD; Kerry E. Travers, BS; Sarah E. Katz, BS; James P. Morgan, MD, PhD; Jeffrey S. Flier, MD; Pamela S. Douglas, MD

From the Charles A. Dana Research Institute and the Harvard-Thorndike Laboratory, Cardiovascular and Endocrinology (C.S.M., J.S.F.) Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass.

Correspondence to Antonio Cittadini, MD, Department of Internal Medicine, Federico II Medical School, Via Sergio Pansini, 5 (Edificio 18), 80131 Naples, Italy. E-mail cittadin{at}unina.it

	Abstract

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Background—A new model of murine obesity has recently been developed through transgenic ablation of brown adipose tissue that manifests typical metabolic complications of obesity, including insulin resistance and non–insulin-dependent diabetes mellitus. The cardiovascular phenotype has not been defined.

Methods and Results—Transthoracic echocardiography, aortic catheterization, isolated whole-heart studies, and morphometric histology defined cardiac structure and function in 30 transgenic mice with reduced brown fat and 30 matched wild-type controls. Obesity was indicated by a 77% increase in body weight and was accompanied by elevated systemic pressures (mean aortic blood pressure 85±1 versus 66±2 mm Hg; P<0.01), left ventricular dilation and hypertrophy (mass/body weight 4.0±0.2 versus 2.7±0.3 mg/g; P<0.01), and high cardiac output (cardiac index 3.2±0.4 versus 2.4±0.1 mL · kg^-1 · min^-1; P<0.01). Baseline functional parameters assessed in vitro were not different, but after imposition of zero-flow ischemia, significant relaxation impairment developed in obese mice. Although morphometrically determined myocyte diameters were similar, the percentage of interstitial fibrosis was significantly increased in transgenic mice compared with wild-type controls (7.5±2% versus 4.2±0.2%; P<0.01).

Conclusions—Transgenic ablation of brown adipose tissue is associated not only with obesity but also with systemic hypertension, left ventricular hypertrophy with eccentric remodeling and fibrosis, and high cardiac output, a unique constellation of findings strikingly similar to that seen in human obesity. Mice with reduced brown fat may serve as a new model for the cardiovascular morbid complications associated with obesity in humans.

Key Words: brown fat • hypertrophy • echocardiography

	Introduction

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Obesity is a common disease associated with increased morbidity and mortality, responsible for 7.8% of all healthcare costs.¹ A large portion of the increased risk is related to cardiovascular abnormalities, in particular hypertension and coronary artery disease. Cardiac structural and functional abnormalities, including left ventricular (LV) dilation, hypertrophy, and impaired function, have been reported in numerous studies of human obesity.^{2 3 4} Because ethical considerations limit the use of invasive techniques in humans, experiments with obese animals may offer greater insights into the cardiovascular changes related to obesity and its morbid complications. Nevertheless, such studies are limited.^{5 6 7 8} Furthermore, most rodent models of obesity display features that are atypical of human disease, such as reduced fertility, altered linear growth, elevated levels of corticosteroids, and reduced lean body mass. These peculiarities limit their relevance for studies of cardiovascular pathophysiology. The recent introduction of transthoracic echocardiography in small rodents allows a more accurate assessment of in vivo cardiac physiology, which, combined with classic and innovative in vitro techniques (eg, Langendorff mouse heart), offers powerful tools to examine animal models of human disease.⁹

We recently developed a murine model of obesity based on the ablation of brown adipose tissue (BAT) using a transgenic toxigene approach.¹⁰ Mice with reduced BAT (UCP-DTA) develop decreased energy expenditure and hyperphagia leading to obesity.¹¹ More specifically, UCP-DTA mice have decreased body temperature and weight-specific metabolic rate but no differences in locomotor activity compared with normal mice.¹² Moreover, adjustment of food intake in relation to changes in ambient temperature is defective in the UCP-DTA mice.¹³ Importantly, when these mice are raised at thermoneutrality, obesity and hyperphagia are prevented, indicating that BAT deficiency is responsible for the observed hyperphagia and obesity.¹⁴ At 16 days, UCP-DTA mice have a 68% reduction in uncoupling protein content of the interscapular brown fat depot, accompanied by moderate obesity. At 22 to 26 weeks of age, marked obesity develops in association with increased levels of glucose, insulin, and triglycerides and is markedly worsened by a high-fat diet.^{15 16}

Although the metabolic phenotype of these mice resembles human syndrome X, an important cardiovascular risk factor, a systematic investigation of cardiac structure and function has not yet been performed. Such studies would provide insight into the cardiovascular complications of obesity and insulin resistance and simultaneously establish the usefulness of UCP-DTA mice as a new model for studying obesity and insulin resistance and their cardiovascular complications.

The aim of the present studies was to characterize the cardiovascular phenotype of UCP-DTA mice. An integrated approach using transthoracic echocardiography, isolated whole-heart studies, and morphometric histology was used to define cardiac structure and function.

	Methods

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The development of UCP-DTA transgenic mice is described elsewhere.¹⁰ Transgenic FVB mice carrying the toxigene construct were bred as heterozygotes. All the methods are consistent with the Panel on Euthanasia of the American Veterinary Medical Association, conform to the requirements of the American Heart Association, and were approved by the Animal Care Committee of the Beth Israel Deaconess Medical Center. Mice were weaned at the age of 19 days, housed individually, and maintained on a 12:12-hour light-dark cycle under controlled temperature and humidity. Water and food (Purina Chow No. 5008) were available ad libitum. Experiments were performed on male 12-week-old mice.

Echocardiography
Previous reports from our laboratory⁹ have demonstrated the accuracy and reproducibility of transthoracic echocardiography in mice. Briefly, mice were anesthetized with ketamine HCl 100 mg/kg IP (Parke Davis) and xylazine 5 mg/kg IP (Lloyd Laboratories). Echocardiograms were performed with a Hewlett-Packard Sonos 2500 sector scanner equipped with a 7.5-MHz phased-array transducer. Two-dimensionally guided M-mode tracings were recorded with a strip-chart recorder at a paper speed of 100 mm/s. Anterior and posterior wall thickness and LV dimensions were measured in standard fashion,¹⁷ offline (Cardiac Workstation, Freeland Systems), by 1 observer blinded to prior results and were based on the average of 3 consecutive cardiac cycles. LV mass was determined by the cube formula, as well as LV volumes.⁹ Relative wall thickness, stroke volume, and cardiac output were calculated according to standard formulas. When appropriate, structural and functional indexes were normalized to body weight and to fat-free body weight, calculated by multiplying body weight by 0.72 for transgenic and 0.81 for wild-type (total body fat content is 28% and 19% in UCP-DTA and wild-type mice of the same age and sex and on the same diet, respectively¹⁰ ). However, because an ideal frame of reference for expressing cardiac structural and functional data has not been defined in obesity because of the relative underperfusion of fat tissue,¹⁸ we also report absolute values and percent changes from wild-type controls.

Hemodynamic Studies
Within 12 hours of the final echocardiogram, mice were anesthetized with ketamine and xylazine at the same doses as used for the echocardiograms. A small cannula (PE-10) was passed via the carotid artery into the aorta under constant pressure monitoring, and the pressure was recorded on a computerized system (Maclab). Peripheral vascular resistance index was calculated as mean arterial blood pressure/cardiac index. Although blood pressure and cardiac index were not measured simultaneously, animals were handled similarly.

Isolated, Isovolumic, Buffer-Perfused Mouse Preparation
Measurement of heart function in the Langendorff preparation in mouse hearts has recently been described in detail.¹⁹ Briefly, the mice were anesthetized with ether and heparinized (500 U/100 g body wt). Each heart was immediately placed in a preweighed beaker containing ice-cold buffer solution. The aorta was slipped over a 20-gauge Luer stub adapter with a stainless steel shaft (Small Parts Inc), through which Krebs-Henseleit solution (see below) was dispensed at a flow rate of 1 mL/min. An incision was made at the root of the pulmonary artery to drain coronary effluent. A constant-flow pump (Masterflex model 7016-20, Cole-Parmer Instrument Co) provided coronary perfusion at a rate of 15 to 20 mL · min^-1 · g heart wt^-1. The pressure was measured via a Statham P23b transducer (Gould) connected to a sidearm. Cardiac temperature was set at 25°C, as measured by a temperature probe. The composition of the perfusate (in mmol/L) was NaCl 118, KCl 4.7, KH₂PO₄ 1.2, CaCl₂ 1.5, MgCl₂ 1.2, NaHCO₃ 23, and dextrose 10.0, saturated with a 95% O₂/5% CO₂ gas mixture to a pH of 7.4±0.2. LV pressure was measured with a tiny fluid-filled balloon inserted into the left ventricle via the mitral valve. After an equilibration period of 15 to 30 minutes at 25°C, the temperature was gradually increased to 30°C, and the hearts were paced at 6 Hz. Hearts of various sizes were compared by a pressure-volume curve obtained in each heart by increasing the balloon volume in steps of 2 to 4 µL, as previously described.²⁰ The volume was increased up to a value of maximal volume (Vol_max) at which the maximal developed pressure occurred. Then, balloon volume was set in each animal at 50% of Vol_max, and LV functional indexes (see below) were obtained. Each LV parameter of interest was subsequently normalized to Vol/Vol_max to achieve comparable loading conditions in hearts of different sizes. Wall thickness, relative wall thickness, and peak systolic, peak diastolic, and developed circumferential wall stresses were derived from LV pressure measurements, balloon volume, and weight of the left ventricle, as previously described by Brooks et al.²¹

The digital LV pressure tracing was analyzed to obtain LV peak systolic pressure, time to peak systolic pressure, peak positive and peak negative dP/dt, end-diastolic pressure, developed pressure, developed wall stress, and time from peak systolic pressure to 90% of relaxation. After the baseline assessment, 15 minutes of zero-flow ischemia was induced by turning off the perfusion pump and simultaneously clamping the perfusion line. Functional parameters were then determined after 20 minutes of reperfusion.

Histology
Specimens for histological examination were obtained from the 5 hearts used for the hemodynamic studies. Each heart was cut into cross sections at 4 levels from apex to base. The tissues were immersion-fixed in 10% buffered formalin. The samples were embedded in paraffin and stained with hematoxylin and eosin for muscle fiber diameter and with Masson’s trichrome for interstitial fibrosis. Quantitative evaluation was carried out by morphometry, according to previously described methods.⁹

Blood Work
Blood was collected from the retro-orbital sinus of animals fasted overnight. Plasma insulin was assayed by radioimmunoassay with rat insulin standards (Linco). Leptin was assayed as reported previously.²²

Statistical Analysis
All values are mean±SEM. Statistical analysis was performed with Statview. After tests for normal distribution, comparisons between the 2 study groups were performed with the unpaired 2-tailed Student’s t test. Linear regression analysis was used as appropriate. A value of P<0.05 was considered significant.

	Results

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Body weight was increased by 77% in UCP-DTA mice. Insulin and serum leptin levels were increased by 18 and 16 times, respectively (Table 1). A detailed description of the metabolic consequences of BAT ablation is reported elsewhere.^{10 11 12 13 14 15 16} Maximal, mean, and minimal values of aortic blood pressure were all significantly higher in transgenic mice (Table 1). The average increase of mean blood pressure was 29%.

View this table: [in this window] [in a new window]   Table 1. Animal Characteristics

Echocardiography
Figure 1 depicts representative echocardiographic tracings from a UCP-DTA mouse and a wild-type control. LV mass was increased by 135% in UCP-DTA mice (Table 2) as a result of higher posterior and anterior diastolic wall thickness (+35% and +64%, respectively) and by a concomitant 34% increase of LV cavity diameter. The ratios of LV mass to body weight and to fat-free body weight were significantly increased, indicating LV hypertrophy. The pattern of hypertrophy was eccentric, with unchanged relative wall thickness. Ejection-phase indexes were similar, suggesting normal pump function in vivo in UCP-DTA mice. Cardiac output was increased by 130% in transgenic mice; even after normalization to body weight and fat-free body weight, cardiac output was still significantly higher than in nontransgenic littermates, indicating a high-output syndrome. Peripheral vascular resistance index was slightly but not significantly lower in UCP-DTA mice.

View larger version (38K): [in this window] [in a new window]   Figure 1. Representative echocardiographic monodimensional tracing obtained in a UCP-DTA mouse (left) and a wild-type control (right). Note larger diameters and wall thicknesses of transgenic animals with normal fractional shortening.

View this table: [in this window] [in a new window]   Table 2. Echocardiographic Data

In Vitro LV Function
Systolic and developed pressures were significantly increased in UCP-DTA obese mice compared with control over a wide range of preload, ie, balloon volumes (Figure 2, Table 3), with a trend toward lower developed wall stress. Diastolic pressure–volume curves normalized to Vol_max were superimposable, indicating similar compliance. In both normal and obese mice, wall stress–volume relationships were linear, with high correlation coefficients (r=0.99). Despite similar baseline in vitro function, obese mice had an increased susceptibility to ischemia, with significant prolongation of diastolic relaxation on reperfusion (Table 3).

View larger version (15K): [in this window] [in a new window]   Figure 2. In vitro function in UCP-DTA mice and wild-type controls. Top, Systolic and diastolic pressure-volume curves are shown in wild-type controls () and UCP-DTA mice (•). Note significantly higher values of systolic pressure in transgenic animals for balloon volumes 50% of Volmax. Bottom, Developed wall stress, which was not significantly different between 2 study groups. In both graphs, LV parameter of interest and balloon volumes are normalized to Volmax. See text for experimental conditions.

View this table: [in this window] [in a new window]   Table 3. Comparison of Cardiac Function In Vitro Between UCP-DTA Mice and Wild-Type Controls in Baseline Conditions and After 20 Minutes of Reperfusion (Post-IR)

Morphometric histology showed no differences in myocyte diameter between the groups (6.8±0.4 µm in wild-type versus 7.0±0.9 µm in transgenic, P=NS), whereas percentage of interstitial tissue was significantly higher in obese mice than in controls (4.2±2% versus 7.5±2%, P<0.01).

	Discussion

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The present study demonstrates that mice with BAT ablation display a distinct cardiovascular phenotype in addition to the development of obesity and insulin resistance, manifest by elevation of blood pressure, LV hypertrophy with an eccentric remodeling pattern and increased interstitial tissue, and reduced tolerance to ischemia. Such alterations very closely parallel those observed in human obesity.^{2 4 18 23 24 25 26 27} The UCP-DTA mouse may therefore serve as a new model for studying the cardiac morbid complications of obesity and insulin resistance.

Obesity and the Heart in Humans
LV enlargement in human obesity is well documented,^{2 4} with normotensive individuals displaying eccentric LV hypertrophy (normal or decreased relative wall thickness) as an adaptation to the expanded intravascular volume and low peripheral vascular resistance caused by excess adipose tissue. The presence of LV systolic dysfunction is still debated, with conflicting studies showing either decreased^{3 23 26 27} or normal^{4 24 25} LV systolic performance. High end-diastolic volumes and use of Starling reserve have been postulated as the mechanisms for preservation of function.⁴ Diastolic function by Doppler-derived filling indexes appears to be impaired in obese individuals.²⁸ Circulatory dysfunction is also present,¹⁸ with increases in blood volume and cardiac output necessary to meet the higher metabolic requirements.

The cardiac morphological consequences of hypertension and obesity are the net result of the opposing hemodynamic patterns. Systemic hypertension is associated with contracted intravascular volume, high total peripheral resistance, and normal cardiac output. LV hypertrophy becomes more severe and shows a more concentric pattern when systemic hypertension coexists with obesity.^{4 18 24} Although cardiac function examined at rest may remain normal, the double burden of increased preload and afterload greatly enhance the risk of developing heart failure.

Because an accurate assessment of intrinsic contractility is problematic in humans, rodent models of obesity may offer several advantages in this regard. Nonetheless, few studies are available.

Previous Studies in Animal Models of Obesity
In genetically obese Zucker rats, Segel et al⁸ found that whereas resting isolated heart function at 19 weeks was similar to that of controls, the obese rat showed reduced tolerance to hypoxia. Paradise et al,⁷ studying the same model at 11 to 13 months, found diminished values of unnormalized wall stress, suggesting either reduced intrinsic contractility or dilation, whereas LV chamber compliance was not different from that of controls. LV hypertrophy was suggested by an increased ratio of LV mass to tibial length. It is worth noting that in the Zucker rat, obesity is typically associated with systemic hypertension and hyperleptinemia due to a mutation of the leptin receptor.²⁹ Using JCR:LA obese, insulin-resistant rats, Lopaschuk and Russel⁵ found greater metabolic vulnerability in isolated hearts because they required high levels of insulin and buffer calcium to maintain mechanical function. In vivo analysis of cardiac structure and function was not performed in these earlier rodent investigations.

Hearts in Mice With Reduced Brown Fat
In UCP-DTA mice, high anatomic preload was documented by elevated end-diastolic volumes, while systemic hypertension imposed an afterload excess (30% increase in aortic blood pressure compared with wild-type controls). The consequent 135% increase in LV mass represents a greater extent of hypertrophy than in other rodent obesity models, whereas the increased lean body mass is also at variance with other obese rodents but is a known feature of human obesity. The structural and functional abnormalities displayed by the UCP-DTA mice reflect the dominant impact of obesity rather than hypertension, with eccentric rather than concentric remodeling. UCP-DTA mice also exhibited high cardiac output and stroke volume, and total peripheral resistance index was slightly decreased in UCP-DTA mice, all of which occur in obesity, not hypertension.

Although preload, afterload, and cardiac output were increased, systolic function was within normal limits, whether measured by ejection phase indexes (endocardial and midwall fractional shortening) or isovolumic wall stress, an accurate index of myocardial intrinsic contractility. However, an early impairment of muscle function was suggested by greater susceptibility to ischemia (increase of the time to 90% relaxation and peak negative dP/dt) shown in vitro, perhaps reflecting greater sensitivity of diastolic relaxation to energy depletion,³⁰ and increased fibrosis.

To the best of our knowledge, this is the first report describing wall stress values and the linearity of wall stress–volume relationships in the isolated isovolumic, buffer-perfused beating mouse heart. Use of the wall stress calculation normalized to Vol_max is preferred in comparison of function of hearts of different geometry and size.²⁰ In particular, other findings of decreased unnormalized wall stress may be explained by the larger obese heart⁷ and by the consequent downward and rightward shift of the peak systolic wall stress–volume relationship.²⁰ Our finding of similar normalized wall stress in controls and obese mice supports the hypothesis that even marked obesity per se does not significantly influence baseline intrinsic cardiac contractility.

Leptin, the product of the ob gene, is an adipocyte-secreted protein that signals the brain regarding the amount of energy stored in the adipose tissue.³¹ Leptin may have sympathetic and cardiorenal actions,^{32 33} because it increases norepinephrine turnover and sympathetic nerve activity to both thermogenic BAT and other organs, including the kidneys, the hindlimbs, and the adrenals.^{32 33} In addition, hyperleptinemia and leptin resistance are associated with hyperinsulinemia and insulin resistance. Furthermore, because leptin is a potassium-sparing natriuretic factor,^{32 34} leptin resistance may be related to sodium and volume retention. Therefore, leptin may also play a relevant cardioregulatory role, in addition to the control of body fat.

The obese hyperinsulinemic UCP-DTA mouse has increased leptin levels and is resistant to exogenous leptin administration even before developing obesity.³⁵ Although UCP-DTA mice are resistant to the weight- and food intake–reducing effect of leptin,³⁶ they appear to be sensitive to other actions of leptin, including the regulation of hypothalamic NPY expression and the activity of the CRH-ACTH-adrenal axis.³⁶ Therefore, hyperleptinemia may provide a novel mechanism by which hypertension develops, in addition to the well-known link between hyperinsulinemia and insulin resistance and hypertension.³⁷ Specifically, it is possible that the hypertension and volume overload displayed by UCP-DTA mice may be secondary to the renal long-term chronic sympathetic activation with subsequent sodium retention induced by hyperleptinemia, because kidneys appear to be leptin sensitive. Whether hyperleptinemia with resistance to its neural effects is the primary cause of obesity because of the decrease of energy expenditure or is a compensatory mechanism for other unknown pathogenetic factors remains an open issue.³⁷

Special Considerations and Study Limitations
The absence of systolic dysfunction in UCP-DTA mice at 12 weeks does not exclude the possibility that overt cardiac dysfunction would appear later in life. In fact, the enlarged and fibrotic hearts of young UCP-DTA mice work at a distinct metabolic and mechanical disadvantage, as shown by the impaired recovery after global ischemia, which aging and/or additional disease states can only exacerbate. Our data are cross-sectional, however, and future longitudinal research is needed.

In the present study, the assessment of systemic blood pressure under anesthesia was not ideal, because it lowers blood pressure 20% to 30%. However, both groups were handled similarly, and results most likely reflect actual intergroup differences.

The possibility of "leaky" DTA expression in cardiac or surrounding tissues theoretically exists in this transgene. However, considering that UCP, which was used to drive the expression of DTA, is BAT-specific³⁸ and that the clinical phenotype of UCP-DTA mice is very different from the one observed after DTA exposure, this possibility appears very unlikely.

Clinical Implications
As characterized, UCP-DTA mice represent a novel and faithful model of human obesity on the basis of on their phenotypic characteristics. The physiological/clinical characteristics of this strain, ie, hyperinsulinemia, hyperleptinemia, obesity, diabetes, hyperlipidemia,^{10 11 12 13 14 15 16 36} and now hypertension and cardiac abnormalities, make this strain relevant for the study of the development of cardiac abnormalities in humans. Future longitudinal assessment of the molecular basis for cardiac changes in this model is likely to provide additional insight into the critical links between obesity and cardiovascular disease.

	Acknowledgments

 
This work was supported in part by NIH grants HL-31117 and HL-511307-01 (Dr Morgan), DK-46930 (Dr Flier), and RR-01032-22S2 (Dr Mantzoros).

Received January 25, 1999; revision received June 24, 1999; accepted July 2, 1999.

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