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O
RIGINAL
A
RTICLE
1,25-Dihydroxyvitamin-D
3
Treatment Reduces CardiacHypertrophy and Left Ventricular Diameter inSpontaneously Hypertensive Heart Failure–Prone (
cp
/+)Rats Independent of Changes in Serum Leptin
Peter Mancuso, PhD,* Ayesha Rahman, PhD,† Stephen D. Hershey, MD,† Loredana Dandu, BS,† Karl A. Nibbelink, MD,† and Robert U. Simpson, PhD†
Abstract:
A number of investigators have observed insufficient 25-hydroxyvitamin D status in patients with congestive heart failure,suggesting a role for vitamin D insufficiency in the pathogenesis of this disorder. We have observed cardiac hypertrophy and collagenaccumulation in rats deficient in vitamin D and in the hearts of vitamin D–receptor knockout mice. Our studies indicate that absenceof vitamin D–mediated signal transduction and genomic activationresults in cardiomyocytes overstimulation including increased con-tractility. These events ultimately lead to cardiomyocyte hypertrophy.In this report, we used spontaneously hypertensive heart failure rats
cp
/+ (hemyzygous for the corpulent gene, a mutant isoform of theleptin receptor) fed a normal and a high-salt diet to assess the potential for activated vitamin D (1,25 dihydroxyvitamin D
3
) to prevent cardiac hypertrophy and increases in cardiac output. After 13weeks, as compared with untreated rats, we observed that 1,25dihydroxyvitamin D
3
treatment in rats fed a high-salt diet resulted inlower heart weight, myocardial collagen levels, left ventricular dia-meter, and cardiac output despite higher serum leptin levels. Thesestudies suggest that 1,25(OH)
2
D
3
treatment may prevent the devel-opment of cardiac hypertrophy, an important contributing factor inthe progression of congestive heart failure.
Key Words:
1,25 dihydroxyvitamin D
3
, cardiac hypertrophy, heart failure, leptin(
J Cardiovasc Pharmacol
TM
2008;0:000–000)
INTRODUCTION
Increased contractility and maladaptive remodeling of the heart resulting in cardiomyocyte hypertrophy and myo-cardial collagen accumulation occurs in vitamin D receptor knockout (VDRKO) mice and vitamin D–deficient rats.
1–3
Indeed, vitamin D deficiency is frequently observed in patientswith congestive heart failure. The possible explanations for insufficient circulating 25 dihydroxyvitamin D
3
(25[OH]
2
D
3
)levels may be a result of inadequate ultraviolet B (UVB) expo-sure and/or inadequate dietary vitamin D intake, a geneticabnormality of the hepatic 25-hydroxylase activity, or increased 25(OH)
2
D
3
catabolism.
4
1,25(OH)
2
D
3
treatment has been shown to reduce plasma renin activity, angiotensin IIlevels, blood pressure, and myocardial hypertrophy.
5,6
How-ever, we recently showed that cardiac hypertrophy associated with vitamin D–receptor (VDR) ablation in VDRKO mice wasnot associated with increased blood pressure or elevated reninor angiotensin II levels.
1
Leptin is an adipocyte-derived pleiotropic hormone that is produced primarily by white adipose tissue and is linearlycorrelated with total body fat mass.
7
Levels of this hormonecan be reduced by fasting and increased by inflammatorystimuli and a high-salt diet.
8–10
In addition, 1,25(OH)
2
D
3
has been shown to inhibit leptin synthesis in human adipocytescultured in vitro.
11
Whereas leptin is recognized for its abilityto regulate energy homeostasis, the long form of the leptinreceptor is expressed in heart and vascular smooth muscle and it can also regulate cardiovascular functions.
12
For example,leptin enhances sympathetic nerve activity, regulates cardiacand vascular contractility, and is an independent risk factor for cardiovascular disease.
13–15
A study by Barouch et al sug-gested that disruption of leptin receptor signaling contributesto murine cardiac hypertrophy.
16
Less is known regarding therole of leptin in the pathogenesis of heart failure, but it appearsthat the levels of this adipokine, corrected for fat mass, areelevated in patients with heart failure with and without cachexia.
17,18
Although the role of 1,25(OH)
2
D
3
has been studied inVDRKO mice and in various in vitro and ex vivo experiments,less is known about it’s role in relevant animal models of human heart failure.
1,19
The spontaneously hypertensive heart failure (SHHF)–prone rat mimics heart failure progression inhumans.
20,21
SHHF rats possess 1 or 2 copies of the corpulent gene (
cp
), a mutant form of the leptin receptor, which may playa role in the development of heart failure. The SHHF rat develops chronic hypertension and cardiac hypertrophy at anearly age and progresses to congestive heart failure by thesecond year.
22
Increased dietary salt intake has been reported
Received for publication January 10, 2008; accepted March 21, 2008.From the Department of Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan; and
†
Department of Pharmacology,University of Michigan Medical School, Ann Arbor, Michigan.Reprints: Robert U. Simpson, PhD, Department of Pharmacology, 1301MSRB III, Box 632, University of Michigan Medical School, Ann Arbor,Michigan 48109 (e-mail: robsim@umich.edu).Copyright
Ó
2008 by Lippincott Williams & Wilkins
J Cardiovasc Pharmacol
ä
Volume 0, Number 0, Month 2008
1
to induce LV hypertrophy and fibrosis in severe hypertensiverats.
23–25
In this study, we assessed the effects of 1,25(OH)
2
D
3
treatment on cardiac hypertrophy and function in SHHF
(cp/+)
rats fed normal and high-salt diets and determined the influ-ence of 1,25(OH)
2
D
3
treatment on serum leptin levels in bothdietary groups.
MATERIALS AND METHODSAnimals
Male and female SHHF rats (
cp
/+) (Genetic Models,Indianapolis, Indiana), originating from mating of Koletskyand inbred spontaneously hypertensive rats (SHR), were provided either normal rodent chow (10-week-old rats) or acommercially prepared high-salt diet (8% NaCl) (HarlanTeklad diet TD92012, Madison, Wisconsin; 17-week-old rats).All experiments were conducted in compliance with the Ani-mal Care and Use Committee of the University of Michigan.Animals were not exposed to UVand fluorescent light sourcesto preclude de novo 1,25(OH)
2
D
3
synthesis during this time period. All of the rats were provided with access to water and food ad libitum.
1,25(OH)
2
D
3
Treatment
Animals fed the normal or high-salt diet were givensubcutaneous injections of either 15 ng 1,25(OH)
2
D
3
in 1,2- propanediol (Sigma-Aldrich, St. Louis, Missouri);100 g total body weight) or vehicle (ethanol in 1,2-propanediol) for 13weeks.
Echocardiogram Measurements
Two-dimensional and M-mode echocardiographic(ECG) images were recorded on rats anesthetized with iso-flurane using a GE S10-MHz phased-array transducer, con-nected to a General Electric, Vivid 7 Ultrasound System and the AnonyMOUSE/rat ECG Screening System (Mouse Spe-cifics, Boston, Massachusetts). Data were analyzed by pro- prietary software, and all rats’ ECG intervals were analyzed byStudent’s 2-tail
t
-test.
Tissue Harvest and Measurement of Heart/Tibia and Heart/Brain Ratios
After 13 weeks of treatment, rats were weighed,anesthetized with sodium pentobarbital (Nembutal, Abbott Laboratories, North Chicago, Illinois), and heparinized (1500U/kg of body weight) to allow exsanguinations via the inferior vena cava. Hearts were rapidly excised from the chest cavity,washed twice with ice-cold saline, dissected to remove major blood vessels and connective tissue, blotted to remove excessfluid, and weighed. The ventricles were separated and indi-vidually weighed. After removing and weighing the tibias and brains, the heart/tibia and heart/brain weight ratios werecalculated.
Rat Leptin Determination
Rat leptin was assessed in serum samples according tothe manufacturer’s instructions using a commercially availableenzyme immunoassay kit obtained from Cayman Chemical,Inc. (Ann Arbor, Michigan).
MEASUREMENTS OF SERUM CALCIUM,MAGNESIUM, AND PHOSPHATE
Blood calcium (Arsenazo III), magnesium (FormazanDye), and phosphate (Phosphomolybdate) were measured bythe Special Chemistry Clinical Laboratories, University of Michigan Hospital.
TOTAL COLLAGEN DETERMINATION
Total soluble content was determined using the SircolCollagen Assay Kit (Biocolor, Northern Ireland) accordingto the manufacturer’s instructions. Briefly, hearts were homo-genized in 0.5 M acetic acid containing 1 mg pepsin. Eachsample was incubated for 24 hours at 4
°
C with stirring. After centrifugation, supernatant was assayed. One milliliter of Sircol dye reagent that binds to collagen was added to eachsample and the solutions were mixed for 30 minutes. After centrifugation, the pellet was suspended in 1 mL of the alkalireagent included in the kit and read at 540 nm witha spectrophotometer. Collagen standard solution was used toconstruct a standard curve.
Statistical Analysis
All data are expressed as the mean
6
SEM. Statisticalcomparisons of means were performed using a paired Student’s
t
-test.
RESULTSEffectof1,25(OH)
2
D
3
TreatmentonTotalBodyWeight and Serum Leptin
In rats fed both the normal and high-salt diets, there wasa trend for the 1,25(OH)
2
D
3
treatment to reduce total bodyweight after 13 weeks, but this effect was statisticallysignificant only in the rats fed the high-salt diet (Table 1)
.
As compared with control rats, serum leptin levels were lower in rats fed a normal diet and treated with 1,25(OH)
2
D
3
, but thiseffect did not reach statistical significance (
P
= 0.09; Fig. 1A).In contrast to this result, 1,25(OH)
2
D
3
treatment of rats fed thehigh-salt diet resulted in higher levels of serum leptin (
P
,
0.05; Fig. 1B).
1,25(OH)
2
D
3
Treatment ReducesCardiac Hypertrophy
SHHF (
cp
/+) rats are hypertensive and developsignificantly more hypertrophic hearts than normal animalsthroughout their lifespan. We previously reported that vitaminD
3
deficiency induces increases in heart weight, an indicationof myocardial hypertrophy.
3
To determine if 1,25(OH)
2
D
3
treatment could directly attenuate increases in heart weight inSHHF
(cp/+)
rats, we assessed total heart weight, heart to tibiaweight, and heart to brain weight ratios following 13 weeks of treatment. As shown in Table 1, total heart weights were lower for the 1,25(OH)
2
D
3
-treated fats fed the normal diet. Inaddition, there were significantly lower (
P
= 0.07, 1-tail test)heart to tibia weight ratios in 1,25(OH)
2
D
3
-treated rats fed thenormal diet (
P
= 0.14, 2-tail test) compared with the controls(Table 1). Feeding salt-sensitive rats a high-salt diet is knownto induce hypertension and left ventricular (LV) cardiac
2
q
2008 Lippincott Williams & Wilkins Mancuso et al
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hypertrophy and hasten the development of heart failure.
26
Weused this approach to induce compensatory cardiac hypertro- phy in SHHF (
cp
/+) rats and to determine if 1,25(OH)
2
D
3
treatment could provide a protectiveeffect. In comparison withthe control group, the total heart weight was lower, as was boththe heart to brain and heart to tibia weight ratios (Table 1),which were significantly lower in the SHHF (
cp
/+) ratsreceiving 1,25(OH)
2
D
3
treatment. The results of theseexperiments suggest that 1,25(OH)
2
D
3
attenuates the de-velopment of cardiac hypertrophy in SHHF rats.
Effect of 1,25 (OH)
2
D
3
Treatment on LeftVentricular Myocardial Collagen Content
We assessed total collagen content of the hearts todetermine if 1,25(OH)
2
D
3
influences the extracellular matrixin the myocardial tissue in SHHF (
cp
/+) rats. As compared with vehicle-treated rats, there was slightly less collagen in thehearts of the 1,25(OH)
2
D
3
-treated rats fed the control and high-salt diets but, in both dietary groups, these differenceswere not statistically significant (
P
= 0.44) and (
P
= 0.07;Fig. 2).
1,25(OH)
2
D
3
Treatment Increases SerumCalcium but Not Magnesium and PhosphateLevels in Rats Fed the High-Salt Diet
Serum calcium, magnesium, and phosphate levels wereassessed in the rats fed both the normal and high-salt diet (datanot shown). Although calcium levelswere higher (
P
,
0.05) in1,25(OH)
2
D
3
-treated rats (normal: 11.6
6
0.7, high salt:11.4
6
0.6 ) relative to the control group (normal: 9.9
6
0.2,high salt: 9.7
6
0.1), these were within the normal calciumlevelfor rats. Serum phosphate and magnesium levelswere not significantly different in 1,25(OH)
2
D
3
-treated SHHF rats fed either diet when compared with the control groups.
1,25(OH)
2
D
3
Reduces Stroke Volume and LeftVentricular Diameter in Rats Fed the High-Saltbut Not the Control Diet
Because we observed that 1,25(OH)
2
D
3
ameliorated cardiac hypertrophy, we assessed the effect of this hormone oncardiac function using echocardiography to estimate strokevolume and LV diameters during diastole and systole. Asshown in Figure 3, 1,25(OH)
2
D
3
did not alter stroke volume or LV diameters in 1,25(OH)
2
D
3
-treated rats fed the control diet.However, the hormone treatment resulted in lower strokevolume and LV diameters (both diastole and systole) in rats fed thehigh-salt diet relativeto the control group. Although we did not observe any differences in heart rate, mean arterial blood pressure, or fractional shortening, we did find that relativeventricular wall thickness was greater in the 1,25(OH)
2
D
3
-treated rats fed the high-salt diet as compared with the controls(Table 2).
FIGURE 1.
Effect of 1,25(OH)
2
D
3
treatment on leptin levels inthe SHHF (
cp
/+) rats fed a normal and a high-salt diet. Serumleptin levels in rats fed a normal diet (A) and a high-salt diet(8% NaCl) (B) treated with or without 1,25(OH)
2
D
3
for 13weeks. Bars represent the mean
6
SEM. n = 5–6 rats/group.
FIGURE2.
Effectof1,25(OH)
2
D
3
treatmentonmyocardialtotalsoluble collagen and left ventricular cardiac myocyte diameter intheSHHF (
cp
/+)rats. Myocardial totalsoluble collagen levelswere assessed in rats fed a normal diet (A) and a high-salt diet(8% NaCl) (B) treated with or without 1,25(OH)
2
D
3
for 13weeks.
P
valuesweredeterminedusingapairedStudent’s
t
-test(mean
6
SEM,
P
,
0.001, n = 40).
TABLE 1.
Morphometric Analysis of SHHF Rats Treated With 1,25(OH)
2
D
3
Normal Diet High-Salt DietControl 1,25(OH)
2
D
3
Control 1,25(OH)
2
D
3
Body wt (g) Initial 217.7
6
46.2 205.6
6
43.6 395.4
6
26.1 388.2
6
23.5After 13 wks 306.7
6
86.8 246.4
6
106.1 435.6
6
14.1 396.4
6
27.0*Heart wt (g) 1.33
6
.04 1.21
6
.03
†
1.50
6
0.04 1.40
6
0.03Heart wt/Tibia wt 1.91
6
0.24 1.46
6
0.44 1.84
6
0.12 1.63
6
0.07*Heart wt/Brain wt 0.63
6
0.09 0.52
6
0.14 0.77
6
0.05 0.71
6
0.02*
Results are presented as the mean
6
SD with n = 5–6 rats.*
P
,
0.05 versus high-salt diet control.
†
P
,
0.05 versus normal diet control.
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2008 Lippincott Williams & Wilkins
3
J Cardiovasc Pharmacol
ä
Volume 0, Number 0, Month 2008
1, 25 (OH)
2
D
3
Reduces Cardiac Hypertrophy
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