Cardiovascular Function Dietary Effects on Cardiovascular Toxicology (2004) 04 3746 $25.00 (http://www.cardiotox.

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Characterization of High-Salt and High-Fat Diets on Cardiac and Vascular Function in Mice
Qianli Yu,1,2 Douglas F. Larson,2,* Denise Slayback,1 Tamara F. Lundeen,2 Jeffrey H. Baxter,3 and Ronald R. Watson1
1

College of Public Health, School of Medicine, Health Promotion Science Division, University of Arizona, Tucson, AZ ; 2Sarver Heart Center and Department of Surgery, University of Arizona, Tucson, AZ; and 3Ross Products Division of Abbott Laboratories, Columbus, OH

Abstract
This study compared two established dietary formulations, high salt and high fathigh carbohydrate, separately or in combination on the induction cardiovascular dysfunction. One-month-old C57BL/6J mice were fed one of the following diets for 3 mo: (1) control diet consisting of a high fathigh simple carbohydrate (HFHSC); (2) 8% NaCl diet (HS); or (3) HFHSC diet supplemented with 1% NaCl (HFHS). After 3 mo, the HFHSC mice demonstrated significantly increased end-diastolic volume and end-systolic volume, specifically increases of 35% and 78%, respectively (p < 0.01) and a reduction of ventricular stiffness by 27% (p = 0.015). The HS mice exhibited arterial hypertension with an increase of 33% in maximum end-systolic pressure (p = .024) and a decrease of 44% in arterial elastance (p = 0.020), corroborated by an increase in the heart weight to body weight ratios (p = 0.002) and vascular types I and III collagen (p = 0.03 and p = 0.0008, respectively). The HFHS group revealed a striking response of 230% to the a1-adrenergic challenge (p = 0.00034). These data suggest that the HFHSC diet causes dilated cardiomyopathy, whereas the HS diet produces arterial hypertension and the HFHS diet causes a vascular dysfunctional state that was highly responsive to aadrenergic stimulation. Key Words: Sodium chloride intake; hypertension; vascular function; cardiomyopathy; heart function; conductance catheter; C57BL/6.

*Author to whom all correspondence and reprint requests should be addressed: Douglas F. Larson, College of Medicine, Department of Surgery, Room 4402, University of Arizona, Tucson, AZ 85724. E-mail: dflarson@u.arizona.edu Received: 10/27/03 Revised: 11/10/03 Accepted: 11/10/03 Cardiovascular Toxicology, vol. 4, no. 1, 3746, 2004

Introduction
According to the Centers for Disease Control and Prevention, the principle components of cardiovascular disease are heart disease and stroke. Approximately 61 million Americans, or 25% of the population, live with the effects of stroke or heart disease, making these diseases the first and third causes of deaths each year, respectively. The most common risk factor for cardiovascular and cerebrovascular morbidity and mortality is hypertension. Clinical hypertension is defined as a sys-

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tolic blood pressure above 139 mmHg and a diastolic pressure above 90 mmHg (1). Hypertension is a polygenic trait influenced by both environmental and genetic factors. It has been suggested that the blood pressure of many individuals is sodium-sensitive, rising with total body sodium and the dietary salt load (2). Salt-induced hypertension is commonly studied in rat models, in which several investigators have identified quantitative trait loci (QTLs) linked to blood pressure changes (3). Determination of hypertension has been accomplished in mice by the tail cuff method and telemetry blood pressure measurement; however, attaining valid measurements in mice is difficult (4,5). The study contained herein used male C57BL/6J inbred mice that have six blood pressure QTLs, making the strain genetically susceptible to diet-induced hypertension (46). Five of the loci are concordant with QTLs in rats and four are concordant with QTLs in humans, suggesting that this murine model of salt-induced hypertension adequately parallels human hypertension and is a valuable research tool (6). It has been shown that C57BL/6J mice will develop hypertension when fed 8% NaCl (w/w) in the diet (5, 7) or 1% NaCl (w/v) in the drinking water (6). It has also been reported that C57BL/6J male mice fed a high fat-high simple carbohydrate diet (HFHSC) for 3 mo develop hypertension (4). However, the cardiac and vascular function changes resulting from the high salt or HFHSC have not been defined. Therefore, we investigated the individual or a number of combinations of diets high in sodium and fat to determine which combination will develop the most profound hypertension in C57BL/6J mice. The murine conductance catheter system was used as an all-inclusive method for quantification of the cardiovascular function in mice (811). Unlike other methods of blood pressure analysis, the conductance catheter system affords a thorough and simultaneous analysis of cardiac and vascular function of each mouse. Also, a low-dose phenylephedrine challenge was used to derive a slope of the arterial elastance (Ea) vs time to provide as an additional method to assess vascular compliance. The goal of this study was to compare two established dietary formulations that have been shown to induce arterial hypertension: high salt and high fat high carbohydrate, separately and in combination,
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to define the specific effects of each on the induction of cardiovascular dysfunction.

Materials and Methods

Mice
This study was approved by the animal review committee. The procedures in the Guidelines for the Care and Use of Laboratory Animals (DHEW Publication No. [NIH] 85-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205) and Principles of Laboratory Animal Care (published by the National Society for Medical Research) were followed in this study. C57BL/6J, 4wk-old, female mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in the Central Animal Facility at the University of Arizona.

Groups
Four groups of mice were fed the following diets ad libitum for 3 mo. Group I (control) was fed the NIH-31 modified mouse sterilizable diet with 6% fat (w/w) and 0.4% NaCl (w/w) (Harlan Teklad, Madison, WI). Group II (HFHSC) was fed a high-fat diet consisting of 35.5% fat (w/w), 35.4% simple carbohydrates (w/w), and 0.4% NaCl (w/w) (BioServ, Frenchtown, NJ). Group III (high salt [HS]) was fed the AIN-93M Purified Rodent Diet with 8% NaCl (w/w) (Dyets, Inc., Bethlehem, PA). Group IV (high fat, high salt [HFHS]) was fed the HFHSC diet and 1% (w/v) NaCl in the drinking water for 2 mo before termination of the study. Control, HS, and HFHSC diets have similar mineral and vitamin concentrations and are similar in basic composition (Table 2).

Quantification of Left Ventricular and Arterial Mechanics

The Millar Conductance Catheter System was used, as has been previously described by our laboratory (810). All mice were anesthetized with urethane in saline (1000 mg/kg intraperitoneally) and a-chloralose in propylene glycol (50 mg/kg intraperitoneally). The advantage of this anesthetic technique is that it causes minimal cardiac and vascular depression and inhibits sympathetic outflow from the central nervous system that may confound data interpretation (12). The mice were ventilated through a tracheostomy connected to a pressure-controlled
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Table 1 Definition of Hemodynamic Parameters Abbreviation CI CO dP/dtmax dP/dtmin dP/dtmax-Ved dV/dtmin Ea EDPVR EF ESPVR HR Pmax PRSW SV SVI Ved Ves b t weiss Definition Cardiac index (CI = CO/body weight) Cardiac output Maximum derivative of change in systolic pressure over time Maximum derivative of change in diastolic pressure over time Slope describing isovolumic contraction Maximum rate of filling volume change Arterial elastance End diastolic pressurevolume relationship Ejection fraction End systolic pressurevolume relationship Heart rate Maximum arterial pressure Preload recruitable stoke work Stroke volume Stroke volume index (SVI = SV/body weight) End-diastolic volume End-systolic volume Slope of end-diastolic pressurevolume relationship Time constant of isovolumic relaxation

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surevolume relationship; b, the description of cardiac stiffness, was derived from the end-diastolic pressure volume relationship; and CO, SW, PRSW, dP/dtmaxVed, dP/dtmax, dP/dtmin, dV/dtmin cardiac output, stroke work, preload recruitable stroke work, slope describing isovolumic contraction, maximum derivative of change in systolic pressure over time, maximum derivative of change in diastolic pressure over time, maximum rate of filling volume change, and t were measured directly. The Ea and maximum arterial pressure (Pmax) also were measured directly. These computations were performed according to those reported recently by Georgakopoulos (13) and Yang (810) and previously by Glower (14) and Kass (15).

Dynamic Vascular Compliance

To assess dynamic vascular compliance, 50 g/kg (10 L) of phenylephrine was infused through the internal jugular, and the slope of the increased of Ea over time was determined (slope). The first 20 pressurevolume loops after the infusion of the phenylephrine were used to derive the slope (Ea vs time). This a-agonist challenge was used to provide further characterization of the arterial bed compliance.

Semiquantification of Collagen mRNA Expression with Reverse Transcriptase Polymerase Chain Reaction
Aortic tissues were stored in a TRIZOL reagent (Gibco BRL). The RNA was extracted and concentrations were determined using a Bio-Rad Biophotometer. The RNA was reverse-transcribed from 5 mg of total RNA in a final volume of 20 L reverse transcriptase (RT) mixture for 2 h at 37C. The RT mixture consisted of 4 L 5X RT buffer 1 M dNTPs, 20 U RNAse inhibitor, 3 g random primers, and Superscript RT enzyme. Polymerase chain reactions (PCRs) were performed in a 50-L reaction volume containing 1 mL cDNA, 5 L 10X PCR buffer, 1 mL 10 mM dNTPs, 2 L 50 mM MgSO4, 1 L 10 mM each primer, and 1 U Taq DNA polymerase. A hot start was applied for 4 min at 95C, and the cycle (denaturation step at 95C for 30 s, annealing step at 60C for 30 s, and an elongation step at 72C) was repeated for the determined number of cycles. Linearity of collagen primer types Ia2 IIIa1 and 18s were determined before assay using cycles 2432. Quantum 18s primers for rRNA internal standards (Ambion, Austin, TX) were used to normalize the
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respirator (RSP 1002, Kent, CT) at a rate of 120 times/ min and with a fraction of inspired oxygen (FiO2) concentration of 1.0. The mice were placed on a surgical table maintained at 37.5C. The external jugular vein was cannulated with a #23 G butterfly, and volume administration was limited to 300 L of hetastarch (6% hetastarch in 0.9% saline, Abbott Laboratories, Columbus, OH). The apical portion of the heart and the inferior vena cava was exposed through a substernal-transverse incision. A Millar conductance catheter 1.4 Fr (Millar Corporation, Houston, TX) was inserted into the apex of the left ventricle with the distal electrode in the aortic root and the proximal electrode in the left ventricular apex. The volume calibration line and parallel conductance were determined, as described previously (10). The parameters of contractility, diastolic function, and left ventricular elastance were expressed as follows (Table 1): Ees was derived from the end-systolic presCardiovascular Toxicology

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Table 2 The Percentage of Major Compositions in Control, High Salt, and High FatHigh Simple Carbohydrate Diets Control Protein Fat Fiber Carbohydrate Mineral mix Vitamin mix NaCl 14 20 0.5 10 0.35 0.1 0.4 High salt 14 20 0.5 10 0.35 0.1 8 High fathigh simple carbohydrate 20 35.5 0.1 35.7 0.4 0.1 0.4

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PCR bands. PCR products were then analyzed by horizontal electrophoresis in a 1% agarose gel. The gels were photographed on an ultraviolet light source and analyzed with a Bio-Rad GS-800 Calibrated Densitometer.

Statistical Analysis
Analysis of variance with multicomparison procedures was used to test the differences among the defined groups. The differences were calculated with a significance level of 0.05 and a power of 0.8. Values obtained from treatment groups were compared with control values using the Students t-test. Comparable nonparametric tests (Kruskal-Wallis and the rank-sum test) were substituted when tests for normality and equal variance failed. All data are reported as mean standard error of the mean.

Results
Major Compositions in Control, HS, and HFHSC Diets
Compared with the control diet, the HS diet has a high percentage of salt ingredients (8%), and the HFHS has a high fat (35.5%)simple carbohydrate (35.7%) composition as well as a high ratio of fat simple carbohydrate to protein of 36.1:1. There is no obvious difference in other basic compositions (Table 2).

Cardiac Hemodynamics
Most striking was the differential response to the two diets, high fathigh simple carbohydrate HFHSC vs HS, related to hemodynamic parameters as illustrated by Fig. 1. Table 3 and Fig. 1 show that the HFHSC
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diet caused a significant increase in dynamic ventricular volumes and a decrease in the ejection fraction (EF), which are characteristic of dilated cardiomyopathy. The end-diastolic and end-systolic volumes were, respectively, increased from 23.5 1.1 and 11.5 0.9 in the control group to 31.8 1.5 and 20.5 1.6 L in the HFHSC group (p < 0.001) These differences are consistent with the decrease in b, which was reduced from 0.15 0.02 to 0.11 0.01 mmHg/ L (p = 0.015). The cardiac index was also decreased by 21% (p = 0.08) in the HFHSC group. These hemodynamic parameters were supported by a decrease in EF from 55.8 3.5% in the control group to 42.3 3.4 % (p = 0.019) in the HFHSC group, which supports the conclusion that this diet may induce a dilated cardiomyopathy producing a cardiac failure state. Conversely to what would be expected, the HFHS groups incidence of a high-fat diet-induced heart failure state was reduced in comparison to the HFHSC group. The HS diet appeared to cause compensatory cardiac functional changes that were consistent with arterial hypertension. The heart weight to body weight ratio was significantly increased (p = 0.002). The minimum rate of filling volume change decreased by 23% (p = 0.023) and coincided with a 33% increase in b. There was significant (p = 0.031) prolongation of t and a decrease of slope describing isovolumic contraction, both of which are parameters characteristic of left ventricular diastolic and systolic dysfunction associated with chronic hypertension.

Vascular Hemodynamics
The HFHSC diet caused no vascular dysfunction. However, the HS and HFHS groups demonstrated
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Fig. 1. Representative pressurevolume loops from the four treatment groups. These representative loops illustrate the dilated cardiomyopathy produced with the high fathigh simple carbohydrate diet and the hypertension caused by the high-salt diet. Control, II. High fathigh simple carbohydrates, III. 8% Salt diet, IV. Combined high fathigh simple carbohydrates with 1% NaCl.

significantly altered vascular function. Table 4 demonstrates that the HS group was hypertensive, as shown in the 33% increase in the Pmax parameter (p = 0.024) and by the 45% increase of the Ea (p = 0.021). Figure 2 shows that the increases of Pmax and Ea correspond with an increased expression of arterial vascular mRNA collagen type Ia2 and IIIa1 (p = 0.03 and p = 0.0008, respectively) that compose the collagen in the adventitial vascular layer. The HFHS group showed a significantly increased Pmax but not Ea. To reveal further vascular dysfunction immediately after a phenylephrine challenge of 50 mg/kg intravenously, the Ea was plotted against time for 20 cardiac cycles. Table 4 and Fig. 3 show the Ea vs time slopes and plots of the vascular compliance changes resulting from this a-adrenergic agonist. There was a 78% (p = 0.035) increase in the slope in the HS group and a 230% (p = 0.0004) increase in the HFHS group. This phenylephrine challenge revealed that the arterial vasculature was highly noncompliant with a HS diet and that the addition of a high fat to the HFHSC diet markedly amplified arterial vascular dysfunction. These data demonstrate that the HFHS diet may
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cause nominal hypertension. However, when combined with a catecholamine release, as may be seen during physiologic conditions of stress, the HFHS diet may cause profound hypertension.

Discussion
The major findings of this cardiac and vascular functional study are as follows: (1) a HFHSC diet induces cardiac dysfunction that is consistent with dilated cardiomyopathy (DCM) without hypertension; (2) the HS diet results in vascular dysfunction and hypertension with compensatory cardiac hypertrophy; and (3) a HFHS diet produces an arterial vasculature that is hyperresponsive to catecholamine stimulation. The dynamic cardiac and arterial vascular dysfunction is related to the remodeling of these tissues in response to the studied diets, whereas the aberrant response to catecholamine stimulation is related either to vascular remodeling or increased adrenergic receptor expression. The mechanism of and further characterization of this cardiac and vascular remodeling in response to these dietary regimens will be the subject of future research.
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Table 3 Cardiac Function: Comparison of HFHS to HS Diets in Induction of Cardiac Dysfunction Parameters n BW HW/BW HR Ved Ves SVI EF CI dP/dtmax PRSW dP/dtmax-Ved b dP/dtmin dV/dtmin t weiss Units Control HFHSC HS 8 28.71.2b 4.80.1b 521.917.5 22.32.7 11.42.0 0.450.03a 58.44.7 23012 9485977 97.13.5a 46941 0.240.03 7019542 52442a 8.81.1a

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HFHS 8 43.92.3a 4.00.2 522.713.8 29.33.3 16.93.1 0.310.03 48.06.1 16620 9303464 117.26.9 62991 0.160.03 6956465 57491 6.80.3a

g mg/g bpm L L L/g % L/min/g mmHg/s mmHg mmHg/L mmHg/L mmHg/s L/s ms

Cardiac function 8 8 37.61.2 47.12.0b 4.10.2 3.80.2 Hemodynamics 563.68.6 544.719.7 23.51.1 31.81.5b 11.50.9 20.51.6b 0.360.03 0.290.02 55.83.5 42.33.4a 20117 15812 Systolic function 9165399 8431657 109.83.4 115.07.4 55234 48088 Diastolic function 0.180.02 0.110.01a 6394273 5797129 67641 67680 5.80.3 6.00.5

These data represent the mean SEM of cardiac functional parameters after 3 mo of treatment with a HFHSC diet, a HS diet, and a HFHS diet. a = p < 0.05 compared with control. b = p < 0.01 compared with control. HFHSC, high fathigh simple carbohydrate; HS, high salt; HFHS, high fat, high salt; BW, body weight; HW/BW, heart weight/body weight ratio; HR, heart rate; bpm, beats per minute; Ved, end-diastolic volume; Ves, end-systolic volume; SVI, stroke volume index; EF, ejection fraction; CI, cardiac index; dP/dt max, maximum derivative of change in systolic pressure over time; PRSW, preload recruitable stroke work; dP/dtmax-Ved, slope describing isovolumic contraction; dP/dt min, maximum derivative of change in diastolic pressure over time; dV/dt min, maximum rate of filling volume change; t weiss, time constant of isovolumic relaxation.

Table 4 Vascular Function: Comparison of HFHSC to HS Diet Induction of Vascular Dysfunction Parameters Pmax Ea Slope Units mmHg mmHg/L mmHg/L/s Control HFHSC HS 120.712.1a 9.41.5a 4.10.7a HFHS 102.74.5a 8.181.2 7.60.8b

Vascular function 90.92.7 85.72.9 6.50.5 6.00.6 2.30.4 2.90.4

These data represent the mean SEM of arterial vascular functional parameters after 3 mo of treatment with a HFHSC diet, HS diet, and a HFHS diet. a = p < 0.05 compared with control. b = p < 0.01 compared with control. HFHSC, high fathigh simple carbohydrate; HS, high salt; HFHS, high fat, high salt; Pmax, maximum aortic pressure; Ea (arterial elastance), Pmax divided by stroke volume; slope, the time constant of Ea with 50 g/kg of phenylephrine infusion. Cardiovascular Toxicology Humana Press Volume 4, 2004

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Fig. 2. These reverse transcriptase polymerase chain reaction blots of aortic collagen types I and III. These blots illustrate the increased expression of collagen types I and III after 3 mo of a high-salt diet in C57BL/6J male mice.

It has been previously reported that C57BL/6J mice are genetically susceptible to diet-induced hypertension (4,5). To verify these observations, we characterized the cardiac and vascular function in C57BL/
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6J mice fed a control diet supplemented with 8% NaCl (w/w) or a HFHSC diet with and without NaCl supplementation. A number of methods have been used to measure arterial blood pressure in mice, including
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Yu et al.
of diets that are clinically known to cause cardiovascular pathology. The reported technique to challenge the vascular system with a low dose of an a1-adrenergic agonist revealed vascular dysfunction that was not apparent in the static condition of our study. This technique was first described in 1989 by Forster et al. and later by others to characterize the peripheral vascular response in animals and humans in models of congestive heart failure and hypertension (1719). The significantly increased expression of types I and III collagen genes in the salt-treated mice could have accounted for the increased vascular resistance in the presence of phenylephrine-induced peripheral vascular vasoconstriction. Alternatively, the increased response to the phenylephrine challenge also could have been due in part to an increase in a1-adrenoceptor expression. The phenylephrine challenge technique revealed silent hypertension that was not apparent in the nonchallenged mouse. Our study also suggests that there was a significant change in the heart to body weight ratio in the mice in the HS group, which is consistent with a chronic arterial hypertensive state. This cardiac hypertrophy is supported by an increase in the a and decreases in the minimum rate of filling volume change and t. Therefore, these cardiac parameters support the conclusion that the mice in the HS group were chronically hypertensive. The purpose of studying the HFHSC diet was to evaluate this substitute dietary means in terms of the establishment of arterial hypertension. However, this study provided alternate findings that suggest that a HFHSC diet induces DCM without hypertension. Mutation of the fatty acid metabolic pathway resulting in destroyed fatty acid metabolism is one of the mechanisms that account for chronic clinical cardiomyopathy and rhabdomyolysis (20). Animals fed with protein-restricted diets have decreased taurine concentrations, leading to the development DCM secondary to taurine deficiency (21). In our study, high simple carbohydrate and fat intake induces DCM in mice, suggesting that an improper ratio of protein to fat in a simple carbohydrate diet (as in Table 2, wherein the ratio for the control group is 2.1 and the ratio for the HFHSC group is 36.1) can lead to the development of DCM. Moreover, the HFHSC is a ketogenic diet that also has been suggested to be linked to DCM

Fig. 3. Slope of arterial elastance (Ea) over time of the four dietary treatment groups. The phenylephrine challenge revealed a significantly increased response to a1adrenergic treatment in the high-salt diet and most striking the vascular response in the HFHSC diet. This technique demonstrates that with nonsignificant static Ea, an a1-adrenergic treatment can elucidate vascular dysfunction. Control, II. High fathigh simple carbohydrates, III. 8% salt diet, IV. Combined high fathigh simple carbohydrates with 1% NaCl.

the tail cuff method (4,7), acute catheterization (16), and telemetry (5). The tail cuff and acute catheterization methods both require restraint, which can elevate blood pressure. Moreover, these methods generate no information related to the integrated analysis of cardiac and vascular function. The Millar conductance catheter system has been shown to provide more than 35 hemodynamic parameters that can provide a detailed characterization of cardiac and vascular function through the analysis of left ventricular pressurevolume loops (810). Moreover, during the ejection phase of the pressure-volume loops, the arterial vascular properties such as Ea can be specifically analyzed through the integration of the stroke volume and systolic blood pressure. Therefore, the conductance catheter system has the advantage of incorporating cardiac and vascular pressures with dynamic volumetric parameters to fully characterize the effects

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(22). Therefore, 3 mo of a HFHSC diet produced DCM through the combined effect of altered fat and carbohydrate substrates in the diet. Most remarkable was the finding that a 2-mo administration of 1% NaCl in the drinking water in the HFHSC diet abrogated the dietary provoked DCM but induced an aberrant vascular response to a1-adrenergic stimulation. Because hypertension is a polygenic, complex, and highly variable phenotype influenced both by genetics and environment, it is difficult to definitively identify the mechanism(s) of hypertension in this murine study. However, it is well-understood that the renin-angiotensin system plays a crucial role in the regulation of blood pressure, electrolyte, and volume homeostasis. Renin secretion is regulated by renal perfusion pressure, renal sympathetic nerve activity, and tubular sodium load (23). The metabolic events of angiotensin II are promotion of increased arterial pressure, sodium retention, mitogenesis of vascular cells, and hypertrophy of cardiac myocytes (7). However, it is unlikely that the renin-angiotensin system is the only mechanism sensitive to salt-load influenced hypertension in this system. Our data suggest that increased salt intake not only influences blood pressure but also induces cardiovascular remodeling and function, suggesting factors in addition to angiotensin-converting enzyme activity are influencing disease pathology. Moreover, we demonstrated that a HFHSC diet does not induce hypertension without supplementation of high salt to the diet. Most striking is the finding that the HFHSC diet induces a DCM that is consistent with a heart failure state. These data underscore the role of diet in the induction of cardiac and vascular dysfunction and demonstrate the advantage of an integrated analytical system to define cardiac and vascular function for the characterization of cardiovascular disease processes.

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2. Geleijnse, J.M., Kok, F.J., and Grobbee, D.E. (2003). 2 Blood pressure response to changes in sodium and potassium intake: a metaregression analysis of randomised trials. J. Hum. Hypertens. 17:471480. 3. Garrett, M.R., Dene, H., Walder, R., Zhang, Q.Y., Cicila, 3 G.T., Assadnia, S., et al. (1998). Genome scan and congenic strains for blood pressure QTL using Dahl salt-sensitive rats. Genome Res. 8:711723. 4. Mills, E., Kuhn, C.M., Feinglos, M.N., and Surwit, R. 4 (1993). Hypertension in CB57BL/6J mouse model of noninsulin-dependent diabetes mellitus. Am. J. Physiol. 264: R73R78. 5. Gros, R., Van Wert, R., You, X., Thorin, E., and 5 Husain, M. (2002). Effects of age, gender, and blood pressure on myogenic responses of mesenteric arteries from C57BL/6 mice. Am. J. Physiol. Heart Circ. Physiol. 282: H380H388. 6. Sugiyama, F., Yagami, K., and Paigen, B. (2001). Mouse 6 models of blood pressure regulation and hypertension. Curr. Hypertens. Rep. 3:4148. 7. Carlson, S.H., Oparil, S., Chen, Y.F., and Wyss, J.M. 7 (2002). Blood pressure and NaCl-sensitive hypertension are influenced by angiotensin-converting enzyme gene expression in transgenic mice. Hypertension 39:214218. 8. Yang, B., Larson, D.F., and Watson, R.R. (1999). Age8 related left ventricular function in the mouse: analysis based on in vivo pressure-volume relationships. Am. J. Physiol. 277:H1906H1913. 9. Yang, B., Larson, D.F., Kelley, R.R., Beischel, J., and 9 Watson, R.R. (2000). Conductivity: an issue for the application of conductance catheter system in mice. Cardiovasc. Eng. 5:5760. 10. 10 Yang, B., Larson, D.F., Beischel, J., Kelley, R., Shi, J., and Watson, R.R. (2001). Validation of conductance catheter system for quantification of murine pressure-volume loops. J. Invest. Surg. 14:341355. 11. 11 Yu, Q., Montes, S., Larson, D.F., and Watson, R.R. (2002). Effects of chronic methamphetamine exposure on heart function in uninfected and retrovirus-infected mice. Life Sci. 71:953965. 12. 12 Dalkara, T., Irikura, K., Huang, Z., Panahian, N., and Moskowitz, M.A. (1995). Cerebrovascular responses under controlled and monitored physiological conditions in the anesthetized mouse. J. Cereb. Blood Flow Metab. 15: 631638. 13. 13 Georgakopoulos, D., Mitzner, W.A., Chen, C.H., Byrne, B.J., Millar, H.D., Hare, J.M., et al. (1998). In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry. Am. J. Physiol. 274:H1416H1422. 14. 14 Glower, D.D., Spratt, J.A., Snow, N.D., Kabas, J.S., Davis, J.W., Olsen, C.O., et al. (1985). Linearity of the FrankStarling relationship in the intact heart: the concept of preload recruitable stroke work. Circulation 71:9941009. 15. 15 Kass, D.A., Yamazaki, T., Burkhoff, D., Maughan, W.L., and Sagawa, K. (1986). Determination of left ventricular end-systolic pressure-volume relationships by the conductance (volume) catheter technique. Circulation 73: 586595.

Acknowledgments
This study was supported by grants from Ross Products Division, NIH HL 63667, and the Steinbron Foundation for Heart Failure Research.

References
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16. 16 Li, Y.C., Kong, J., Wei, M., Chen, Z.F., Liu, S.Q., and Cao, L.P. (2002). 1,25-Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system. J. Clin. Invest. 110:229238. 17. Forster, C., Carter, S.L., and Armstrong, P.W. (1989). Alpha 1-adrenoceptor activity in arterial smooth muscle following congestive heart failure. Can. J. Physiol. Pharmacol. 67:110115. 18. Creager, M.A., Hirsch, A.T., Dzau, V.J., Nabel, E.G., 18 Cutler, S.S., and Colucci, W.S. (1990). Baroreflex regulation of regional blood flow in congestive heart failure. Am. J. Physiol. 258:H1409H1414. 19. White, M., Fourney, A., Mikes, E., and Leenen, F.H. (1999). Effects of age and hypertension on cardiac responses to the alpha1-agonist phenylephrine in humans. Am. J. Hypertens. 12:151158.

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20. 20 Roe, C.R., Sweetman, L., Roe, D.S., David, F., and Brunengraber, H. (2002). Treatment of cardiomyopathy and rhabdomyolysis in long-chain fat oxidation disorders using an anaplerotic odd-chain triglyceride. J. Clin. Invest. 110:259269. 21. 21 Sanderson, S.L., Gross, K.L., Ogburn, P.N., Calvert, C., Jacobs, G., Lowry, S.R., et al. (2001). Effects of dietary fat and L-carnitine on plasma and whole blood taurine concentrations and cardiac function in healthy dogs fed protein-restricted diets. Am. J. Vet. Res. 62:16161623. 22. 22 Best, T.H., Franz, D.N., Gilbert, D.L., Nelson, D.P., and Epstein, M.R. (2000). Cardiac complications in pediatric patients on the ketogenic diet. Neurology 54:23282330. 23. Hackenthal, E., Paul, M., Ganten, D., and Taugner, R. (1990). Morphology, physiology, and molecular biology of renin secretion. Physiol. Rev. 70:10671116.

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