maternal vitamin d deficiency leads to cardiac hypertrophy in rat offspring

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http://rsx.sagepub.com/ Reproductive Sciences http://rsx.sagepub.com/content/17/2/168 The online version of this article can be found at: DOI: 10.1177/1933719109349536 2010 17: 168 originally published online 13 October 2009 Reproductive Sciences Black Oksan Gezmish, Marianne Tare, Helena C. Parkington, Ruth Morley, Enzo R. Porrello, Kristen J. Bubb and Mary Jane Maternal Vitamin D Deficiency Leads to Cardiac Hypertrophy in Rat Offspring Published by: http://www.sagepublications.com On behalf of: Society for Gynecologic Investigation can be found at: Reproductive Sciences Additional services and information for http://rsx.sagepub.com/cgi/alerts Email Alerts: http://rsx.sagepub.com/subscriptions Subscriptions: http://www.sagepub.com/journalsReprints.nav Reprints: http://www.sagepub.com/journalsPermissions.nav Permissions: http://rsx.sagepub.com/content/17/2/168.refs.html Citations: What is This? - Oct 13, 2009 OnlineFirst Version of Record - Feb 3, 2010 Version of Record >> at UVI - Biblioteca Central on April 28, 2014 rsx.sagepub.com Downloaded from at UVI - Biblioteca Central on April 28, 2014 rsx.sagepub.com Downloaded from

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http://rsx.sagepub.com/Reproductive Sciences

http://rsx.sagepub.com/content/17/2/168The online version of this article can be found at:

 DOI: 10.1177/1933719109349536 2010 17: 168 originally published online 13 October 2009Reproductive Sciences

BlackOksan Gezmish, Marianne Tare, Helena C. Parkington, Ruth Morley, Enzo R. Porrello, Kristen J. Bubb and Mary Jane

Maternal Vitamin D Deficiency Leads to Cardiac Hypertrophy in Rat Offspring  

Published by:

http://www.sagepublications.com

On behalf of: 

  Society for Gynecologic Investigation

can be found at:Reproductive SciencesAdditional services and information for    

  http://rsx.sagepub.com/cgi/alertsEmail Alerts:

 

http://rsx.sagepub.com/subscriptionsSubscriptions:  

http://www.sagepub.com/journalsReprints.navReprints:  

http://www.sagepub.com/journalsPermissions.navPermissions:  

http://rsx.sagepub.com/content/17/2/168.refs.htmlCitations:  

What is This? 

- Oct 13, 2009 OnlineFirst Version of Record 

- Feb 3, 2010Version of Record >>

at UVI - Biblioteca Central on April 28, 2014rsx.sagepub.comDownloaded from at UVI - Biblioteca Central on April 28, 2014rsx.sagepub.comDownloaded from

Maternal Vitamin D Deficiency Leads to Cardiac Hypertrophy inRat Offspring

Oksan Gezmish, BSc, Marianne Tare, PhD, Helena C. Parkington, PhD,Ruth Morley, MB, BChir, FRCPCH, Enzo R. Porrello, PhD,

Kristen J. Bubb, BSc, and Mary Jane Black, PhD

The aim of this study was to determine the effect of vitamin D deficiency from conception until 4 weeks of

age on the development of the heart in rat offspring. Sprague-Dawley (SD) rats were fed either a vitamin

D deplete or vitamin D-replete diet for 6 weeks prior to pregnancy, during pregnancy and throughout lac-

tation. Cardiomyocyte number was determined in fixed hearts of offspring at postnatal day 3 and 4 weeks

of age using an optical disector/fractionator stereological technique. In other litters, cardiomyocytes were

isolated from freshly excised hearts to determine the proportion of mononucleated and binucleated cardio-

myocytes. Maternal vitamin D deficiency had no effect on cardiomyocyte number, cardiomyocyte area, or

the proportion of mononucleated/binucleated cardiomyocytes in 3-day-old male and female offspring.

Importantly, however, vitamin D deficiency led to an increase in left ventricle (LV) volume that was

accompanied by an increase in cardiomyocyte number and size, and in the proportion of mononucleated

cardiomyocytes at 4 weeks of age. Our findings suggest that exposure to vitamin D deficiency in utero

and early life leads to delayed maturation and subsequent enhanced growth (proliferation and hypertrophy)

of cardiomyocytes in the LV. This may lead to altered cardiac function later in life.

KEY WORDS: Heart, maternal vitamin D deficiency, rat offspring, cardiomegaly.

INTRODUCTION

Vitamin D is a fat-soluble hormone that is essential for

bone metabolism, cell growth, differentiation, and regu-

lation of minerals in the body. Exposure of the skin to

sunlight is the major source of vitamin D; ultraviolet B

(UVB) radiation induces photolytic conversion of

7-dehydrocholesterol to cholecalciferol (vitamin D3)

that is subsequently converted in the liver to

25-dihydroxyvitamin D3, and further processed in the

kidney to the most abundant and active form 1,

25-dihydroxyvitamin D31-3

Over recent years, there has been a resurgence of

vitamin D deficiency within the community,4,5 and

importantly this has been linked to cardiomegaly in

infants that had been vitamin D deficient in utero.6

The rise in vitamin D deficiency in many populations

throughout the world has been attributed to a variety of

interacting factors such as atmospheric air pollution that

reduces UVB penetration, change to urban lifestyles,

greater time spent indoors at work, reduced sunlight

exposure due to religious practices, deliberate avoidance

of sunlight exposure due to skin cancer risk and increased

awareness of ‘‘sun safe’’ messages, and the migration of

people with increased skin pigmentation to areas away

from the Equator.7-9 In pregnant and breast-feeding

women, the mother is the sole provider of nutrition dur-

ing the critical fetal and suckling periods of development,

From the Departments of Anatomy & Developmental Biology (OG, MJB), and

Physiology (MT, HCP, KJB), Monash University, Clayton, Victoria, Australia;

Department of Paediatrics (RM), The University of Melbourne and Murdoch

Childrens Research Institute, Royal Children’s Hospital, Parkville, Victoria,

Australia; and Department of Physiology (ERP), The University of

Melbourne, Parkville, Victoria, Australia.

Address correspondence to: Mary Jane Black, PhD, Department of Anatomy and

Developmental Biology, Monash University, Victoria 3800, Australia. E-mail:

[email protected].

Reproductive Sciences Vol. 17 No. 2 February 2010 168-176DOI. 10.1177/1933719109349536# 2010 The Author(s)

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and the maternal stores of vitamin D play a crucial role in

the development of the fetus and neonate.10

In recent years, the alarming rise in the number of

women who are vitamin D deficient during pregnancy

and lactation4,11 has raised concern as to the implications

for the fetus12 and particularly for the development of

vital organs such as the heart. Indeed, recent reports sug-

gest that pediatric cardiomyopathy and heart failure may

directly result from maternal vitamin D deficiency.6,13,14

It is well known that vitamin D plays a key regulatory

role in cellular proliferation and differentiation15 and is

linked to maturation of organs during gestation.16,17

Importantly, vitamin D has been found to inhibit prolif-

eration of cardiomyocytes and induces hypertrophy.18,19

It has been suggested that vitamin D regulates cardiomyo-

cyte proliferation by blocking the cells from entering the

S phase of the cell cycle.19 It has been shown in rats that

there is a rise in plasma vitamin D concentrations late in

gestation, and these remain high during lactation.20

Importantly, this is the period in the rat when there is a

switch from proliferation of cardiomyocytes to matura-

tion/terminal differentiation.21 It is therefore likely that,

in the absence of vitamin D, the growth switch from pro-

liferation to terminal differentiation that occurs around

the time of birth will be altered, leading to prolonged car-

diomyocyte proliferation and subsequent cardiomegaly.

Indeed, this may be the cause of the observed cardiome-

galy in the hearts of vitamin D-deficient infants and in

animal models.6,22,23

Because induction of cardiomegaly in early life may

have lifelong adverse consequences on cardiovascular

function, the aim of this study was to gain a better under-

standing of the effect of maternal vitamin D deficiency

during pregnancy and lactation on the growth and

maturation of cardiomyocytes in rats. We analyzed the

hearts of rat offspring at postnatal day 3 when the cardio-

myocytes are actively proliferating21 and at 4 weeks of

age, when cardiomyocyte proliferation has generally

ceased and thus are terminally differentiated.21

METHODS

Animals and Diet Treatment

Four-week-old female Sprague-Dawley (SD) rats were

obtained from Monash Central Animal Services Centre

(Monash University). Female rats were randomly divided

into 2 experimental groups and fed specialized diets

preconception for 6 weeks; during the first 4 weeks,

preconception rats were fed a standard maintenance diet

of either a vitamin D-replete diet containing 1000 IU

of vitamin D3/kg, AIN93M, or a vitamin D-deficient

diet that was of the same composition (including calcium)

as the vitamin D-replete diet, with only vitamin D3

omitted (SF05-064). Rats were then switched to a

growth version of the diets for 2 weeks before pregnancy,

during pregnancy, and throughout lactation, either

AIN93G or SF03-009, respectively. All diets were

commercially available from Glen Forrest Specialty

Feeds, Western Australia. We have previously found that

administration of this vitamin D-deficient diet in rats leads

to markedly reduced 25-(OH)D3 concentrations in com-

parison with control offspring.24 The rats were housed

individually under incandescent lighting (to prevent the

production of endogenous vitamin D3) and maintained

at an ambient temperature of 21�C with a 12-hour

day/night cycle. Rats had access to food and water ad

libitum.

At postnatal day 3, litters were reduced to 8 offspring

by randomly selecting 4 females and 4 males from each

litter to grow out to 4 weeks of age. The remaining

littermates at 3 days of age were weighed, killed by

decapitation, and their hearts excised and immersion

fixed.

At weaning (4 weeks of age), the remaining offspring

were weighed and then either anesthetized and

perfusion-fixed for stereological assessment of cardio-

myocyte number or killed by decapitation for enzymatic

isolation of cardiomyocytes.

The experiments were approved by the Monash

University Biochemistry, Anatomy, and Microbiology

Animal Ethics Committee and the treatment and care

of the animals conformed with the National Health and

Medical Research Council of Australia’s Code of Practice

for the Care and Use of Animals for Scientific Purposes.

Measurement of Serum

25-Hydroxyvitamin D3 (25-(OH)D3) and

Calcium Concentrations

At necropsy of the 4-week-old offspring, serum was

collected and stored at �80�C for later measurement of

25-(OH)D3 and total serum calcium levels. Using liquid

chromatography tandem mass spectrometry (RMIT

Drug Discovery Technologies Pty Ltd [RDDT],

Bundoora, Victoria, Australia), 25-(OH)D3 was deter-

mined. Total calcium was determined on a Konelab

20XTi Random Access Analyser (RDDT).

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Heart Fixation and Processing

Freshly excised hearts from the 3-day-old offspring were

immersion-fixed in 10% buffered formalin for 3 to 4 days.

In the 4-week-old groups, vitamin D-deficient

(n ¼ 8 males, and n ¼ 8 females; each male/female

derived from a different litter) and control offspring

(n ¼ 6 males, and n ¼ 6 females; each male/female

derived from a different litter) were anesthetized and

perfusion-fixed with 2% formaldehyde in 0.1 mol/L

phosphate buffer at a pressure of 80 mm Hg.25 Prior to

fixation, the hearts were arrested in diastole.25 In fixed

hearts, the left ventricle plus septum (LV þ S) and right

ventricle (RV) were separated. Using a razor blade cut-

ting device, the LV þ S was sectioned into 1 mm slices,

and the RV was sectioned into 1.5 mm slices.

The hearts of 3-day-old offspring and ventricular

slices of the 4-week-old offspring were embedded in

glycolmethacrylate. The glycolmethacrylate blocks were

serially sectioned at 20 mm, and every 10th section was

collected and stained with hematoxylin.

Estimation of Heart Wall Volume

Heart wall volume in the 3-day-old hearts was deter-

mined in the glycolmethacrylate-embedded sections.

Every 40th section was projected onto a microfiche mon-

itor and an orthogonal grid superimposed. The number of

grid points overlying the heart tissue was counted, and

volume of the heart wall was determined using the Cava-

lieri principle.26

In the 4-week-old hearts, RV wall volume and LVþS wall volume were determined in the tissue slices. An

orthogonal grid was superimposed over the ventricular

slices; the number of grid points overlying the tissue was

counted and volume determined using the Cavalieri

principle.26

Estimation of Cardiomyocyte Nuclei

Number

To estimate cardiomyocyte number, an optical disector/

fractionator stereological technique was employed,26-28

utilizing a CASTGRID specialized stereological system

(Cast 2002; Olympus, Albertslund, Denmark). Nuclei

were counted in a systematic uniform random sample of

fields. An unbiased counting frame was superimposed

over the heart sections at high magnification (�100

objective lens).

Nuclei were counted in a ‘‘disector’’ of 495.8 mm2

within a depth of 10 mm using the upper and lower

5 mm of the section as a guard area (to account for

inconsistencies in the cut surfaces). Cardiomyocyte nuclei

could be easily identified from other cell types; cardio-

myocyte nuclei were oval-shaped, lightly stained with

visible chromatin, and had prominent nucleoli. An esti-

mate of the total number of cardiomyocyte nuclei per

heart in the 3-day-old hearts and in the ventricles of the

4-week-old hearts was determined by multiplying the

number of nuclei counted using the optical disector by

the inverse of the sampling fractions.26

The total number of cardiomyocytes in the hearts was

determined from the total number of cardiomyocyte

nuclei counts after adjustment for the proportion of

binuclear cells (method described below).

Enzymatic Isolation of Cardiomyocytes

When cardiomyocytes become terminally differentiated

in the rat heart, they are binuclear and can be easily iden-

tified.21 Cardiomyocytes were enzymatically isolated to

examine cardiomyocyte area and the proportion of

mononucleated and binucleated cardiomyocytes.

3-Day-old hearts. Hearts were freshly excised from the

3-day-old pups and the atria removed. Hearts from males

and females were pooled within groups. The pooled

hearts (n ¼ 4-6 hearts per litter; 8 litters per group) were

cut into pieces, and the cardiomyocytes isolated using

collagenase (200 U/mL; Worthington CL-2 281,

SciMar, Australia) and trypsin/DNAase/HBSS (Hanks

balanced salt solution). The enzymatically isolated cardi-

omyocytes within the cell suspension were centrifuged

and the cardiomyocytes resuspended in cardioplegic

relaxing solution (117 mmol/L KCl, 36 mmol/L NaCl,

1 mmol/L MgSO4, 60 mmol/L 4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid [HEPES], 8 mmol/L

ATP-NA, and 50 mmol/L EGTA: pH adjusted to 7.0

with KOH)29 and smeared onto slides, fixed with 2.5%polyethylene glycol in 95% ethanol solution, and stained

with hematoxylin and eosin.

4-Week-old hearts. Hearts were freshly excised from the

4-week-old male and female offspring (n ¼ 7 males and

n ¼ 7 females in the control group; n ¼ 8 males and

n¼ 8 females in the vitamin D-deficient group) and were

analyzed separately. The freshly dissected hearts were

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attached to a Langendorff apparatus via the aortic root and

perfused with calcium-free, bicarbonate-buffered physio-

logical saline solution (120 mmol/L NaCl, 5 mmol/L

KCl, 25 mmol/L NaCO3, 11 mmol/L glucose,

1 mmol/L KH2PO4, 12 mmol/L MgSO4), and oxyge-

nated with carbogen (95% O2; 5% CO2) for 3 minutes

at 35�C to clear any blood remaining in the heart and

then perfused with collagenase type II (Worthington

CL-2, 200 U/mL) to degrade the extracellular matrix,

followed by HEPES-buffered, high Kþ solution (cardio-

plegic relaxing solution) to relax the cardiomyocytes.29

The LV and RV were separated, roughly cut, and put

into separate tubes containing relaxing solution and

gently titrated with a wide bore glass pipette for several

minutes to separate the cardiomyocytes. The isolated car-

diomyocytes were smeared onto slides, fixed with 2.5%polyethylene glycol in 95% ethanol solution, and stained

with hematoxylin and eosin.

Determination of the Proportion of

Binucleated and Mononucleated

Cardiomyocytes

Fixed cardiomyocyte smears, stained with hematoxylin

and eosin, were examined under a light microscope

(Olympus BX50, Tokyo, Japan) using a �40 objective.

Four slides were randomly selected from each litter in the

3-day-old hearts (8 litters per group; n ¼ 8) or each ani-

mal in the 4-week-old hearts with the RV and LV þ S

examined separately (n ¼ 8 per group); a sample of 200

cells29 was examined to determine the proportion of

mononucleated and binucleated cells.

Projected Cardiomyocyte Area

Cardiomyocyte smears stained with hematoxylin and eosin

were also used for analysis of projected cardiomyocyte area.

The cardiomyocytes were examined under a light micro-

scope (�400 magnification). All cardiomyocytes that fell

within each field of view were projected onto a computer

screen, and the outside boundaries of the cells digitally

traced. The projected cardiomyocyte area was measured

using image analysis software (Image Pro Plus, version

4.5). A sample of 50 cells29 from each slide was examined.

Statistical Analysis

Statistical analysis of the data was carried out using Graph-

pad Prism (version 4.03; Graphpad software, San Diego,

California). A 2-way analysis of variance (ANOVA) was

applied to data at 3 days of age and 4 weeks of age to

determine whether there were specific effects due to vita-

min D deficiency (PT) and/or gender (PG) or whether

there was an interaction effect whereby male or female

offspring responded differently to vitamin D deficiency

(PG � T). To determine whether there were significant

differences in projected cardiomyocyte area and the pro-

portion of binuclear cells at 3 days of age, a 2-tailed

unpaired t test was used. Data are expressed as means +standard error of the mean (SEM); significance levels

were set at P < .05.

RESULTS

Serum 25-(OH)D3 and Calcium

Concentrations

In the 4-week-old offspring, the mean serum 25-(OH)D3

concentrations in the vitamin D-deficient group were

markedly reduced (P < .001; males: 5.49 + 0.47 and

females: 7.26 + 0.93 nmol/L) compared with controls

(males: 22.50 + 1.68 and females: 19.3 + 0.96 nmol/

L), whereas serum total calcium levels were in the normal

range in both groups (vitamin D-deficient males 2.75 +0.05 and females 2.77 + 0.12 mmol/L and control males

2.85 + 0.06 and females 3.00 + 0.05 mmol/L).

Body Weights and Heart Volumes

In postnatal day 3 offspring, there was no significant dif-

ference in body weight in either male or female vitamin

D-deficient offspring compared with controls (Table 1).

Compared with males, female offspring had a significantly

increased heart volume (P ¼ .030), and a significant

increase (P¼ .007) in heart volume-to-body weight ratio

in both control and vitamin D-deficient groups at 3 days

of age (Table 1). This gender difference was no longer

evident at 4 weeks of age.

At 4 weeks of age, there was a significant reduction in

body weight (P ¼ .048) in male and female vitamin

D-deficient offspring compared with controls (Table 1).

There was no significant difference in right ventricular

wall volume or right ventricular wall volume to body

weight ratio between vitamin D-deficient and control

offspring (Table 1). However, there was an increase

(P < .0001) in LV þ S wall volume and LV þ S wall

volume to body weight ratio (P < .0001) in vitamin

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D-deficient offspring compared with controls (Table 1

and Figure 1A).

Mononucleated and Binucleated

Cardiomyocytes—Cardiomyocyte

Maturation

In 3-day-old offspring, there was no difference in the pro-

portion of mononucleated or binucleated cardiomyocytes

in control versus vitamin D-deficient offspring. The

majority of the cardiomyocytes were mononucleated in

both groups (control 96.0% + 0.4% and vitamin D defi-

cient 96.8% + 0.8%) with the remainder binucleated

(4.0% + 0.4% and 3.3% + 0.8%, respectively).

By 4 weeks of age, the majority of cardiomyocytes

were binucleated in control and vitamin D-deficient off-

spring. Importantly, there was a significant increase (P ¼.010) in the proportion of mononucleated cardiomyo-

cytes and a significant decrease in binucleated

cardiomyocytes in both right (P ¼ .010) and left (P ¼.0001) ventricles of male and female vitamin

D-deficient offspring when compared with controls

(Table 2), suggesting delayed maturation/differentiation

of cardiomyocytes with vitamin D deficiency.

Projected Cardiomyocyte Area

At postnatal day 3, there was no significant difference in

projected mononucleated cardiomyocyte area in controls

(99.02 + 3.83 mm2) versus the vitamin D-deficient

(98.00 + 4.03 mm2) group.

In 4-week-old offspring, there was a significant

increase (P ¼ .007) in the projected area of cardiomyo-

cytes derived from the LV þ S of vitamin D-deficient

male and female offspring compared with controls

(Figures 1C and 2A). A similar trend of increased pro-

jected cardiomyocyte area (P¼ .065) was observed in the

RV wall of vitamin D-deficient offspring compared with

controls (Figure 2B).

Table 1. Body Weight, Heart Volume, and Heart Volume to Body Weight Ratio in the 3-Day-Old and 4-Week-Old Male and

Female Control and Vitamin D-Deficient Offspringa

Offspring Age Control Males Control Females -Vitamin D Males -Vitamin D Females P Values

3 days n ¼ 8 n ¼ 8 n ¼ 8 n ¼ 8

Body weight (g) 6.03 + 0.47 6.10 + 0.34 5.86 + 0.29 6.02 + 0.38 PG ¼ .700

PT ¼ .691

PT � G¼ .854

Heart volume (mm3) 11.71 + 1.32 14.75 + 0.91 10.02 + 0.78 12.71 + 1.78 PG ¼ .030

PT ¼ .153

PT�G ¼ .897

Heart volume: body

weight (mm3/g)

1.92 + 0.12 2.47 + 0.20 1.72 + 0.08 2.05 + 0.18 PG ¼ .007

PT ¼ .051

PT � G ¼ .481

4 weeks n ¼ 6 n ¼ 6 n ¼ 8 n ¼ 8

Body weight (g) 95.11 + 5.81 89.08 + 4.24 85.32 + 3.32 81.82 + 3.20 PG ¼ .256

PT ¼ .048

PT � G ¼ .993

Left ventricle volume (mm3) 47.56 + 2.12 47.81 + 1.32 61.23 + 1.66 60.13 + 2.01 PG ¼ .822

PT �.0001

PT � G ¼ .721

Right ventricle volume (mm3) 25.08 + 0.43 29.02 + 2.70 26.94 + 1.63 23.35 + 0.77 PG ¼ .457

PT ¼ .734

PT�G ¼ .141

Left ventricle volume: body

weight (mm3/g)

0.51 + 0.04 0.54 + 0.01 0.72 + 0.02 0.74 + 0.03 PG ¼ .467

PT �.0001

PT�G ¼ .935

Right ventricle volume: body

weight (mm3/g)

0.27 + 0.01 0.30 + 0.02 0.31 + 0.01 0.28 + 0.01 PG ¼ .933

PT ¼ .323

PT�G ¼ .151

a Data were analyzed by 2-way analysis of variance (ANOVA) with factors treatment (T: control or vitamin D deficient), gender (G: male or

female) and the interaction (T � G). Values are means + SEM.

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Cardiomyocyte Number

The general cellular morphology of the myocardium did

not appear different between the vitamin D-deficient and

control hearts at 3 days or 4 weeks of age (Figure 1B).

There was no overall difference in cardiomyocyte

number in the 3-day-old control versus vitamin

D-deficient offspring (P ¼ .676). Importantly, in the

4-week-old offspring, there was a significant increase

(P ¼ .019) in cardiomyocyte number in the LV þ S of

vitamin D-deficient offspring compared with controls

(Figure 3A); however, no significant difference (P ¼.265) was observed in the RV (Figure 3B). There was

no effect of gender on left ventricular or right ventricular

cardiomyocyte number.

DISCUSSION

In this study, maternal vitamin D deficiency throughout

pregnancy and lactation had no effect on body weight,

heart size, or cardiomyocyte number in offspring at 3 days

of age, at a time when cardiomyocytes are actively

proliferating. However, by 4 weeks of age, when

cardiomyocyte proliferation had largely ceased, vitamin

D deficiency was associated with left ventricular

hypertrophy, accompanied by a delayed maturation of

cardiomyocytes, and an increase in cardiomyocyte

number and size. These observed cardiac changes were

independent of serum calcium levels. Although differ-

ences in heart growth occurred between males and

females (hearts were bigger in females at postnatal day

3), there was no effect of gender in the growth response

of the hearts to vitamin D deficiency.

Whether the left ventricular hypertrophy in the

vitamin D-deficient offspring was also accompanied with

changes in the interstitial compartment (edema and

extracellular matrix deposition) of the myocardium was

not examined but would be of interest in future

investigations.

It is likely that the differences in response to vitamin

D deficiency at postnatal day 3 and week 4 in the current

study are linked to the timing of cardiomyocyte matura-

tion in the rat. In the rat heart, unlike the human heart,

the majority of cardiomyocytes are still actively proliferat-

ing until 3 to 4 days after birth, thereafter the cardiomyo-

cytes commence maturation, and by 12 days postnatally

all cardiomyocytes are usually terminally differentiated.21

Because vitamin D plays a major role in cellular

differentiation, it is likely to play a key role in this matura-

tion process; indeed, this is well described in other cell

types.15,30,31 Hence, in the hearts of vitamin D-deficient

offspring, the switch to cardiomyocyte differentiation

appears to have been affected, leading to a delay in matura-

tion and an increase in proliferation of cardiomyocytes in

vitamin D-deficient hearts in the critical postnatal period

of days 5 to 6. Future studies examining markers of prolif-

eration during this cardiomyocyte maturational period

will shed light on whether the proliferative potential is

prolonged in the vitamin D-deficient hearts.

Our findings of cardiomegaly in the heart of vitamin

D-deficient offspring are supported by previous studies of

human infants.6,13,14 Importantly, in this regard, there is

recent evidence to suggest that pediatric cardiomyopathy,

which often results in heart failure, may be linked to vita-

min D deficiency during pregnancy, with dark-skinned

infants most at risk. In a recent retrospective study, 16

cases of rickets-associated heart failure were reported in

the southeast of England over a period of 6 years.6 In

addition, a recent autopsy study of an infant suffering

from vitamin D deficiency reported a heart that was twice

20 µm

500 µm

20 µm

Control Vitamin D deficient

50 µm 50 µm

500 µm

A

B

C

Figure 1. Representative cross-sections of the left ventricle plus

septum (LV þ S) stained with hematoxylin and eosin at (A) low

magnification and (B) high magnification and (C) enzymatically

isolated cardiomyocytes taken from 4-week-old female control and

vitamin D-deficient offspring.

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the expected size; the left ventricular wall was hypertro-

phied and exhibited enlarged nuclei, with myofibres that

were stretched and elongated.6 In another study, a

4.5-month-old girl and an 8-month-old boy were

hospitalized due to cardiac failure and, on echocardiogra-

phical examination, both children exhibited an enlarged

LV, and this was accompanied by vitamin D deficiency.32

Table 2. Proportion of Mononucleated and Binucleated Cardiomyocytes in the Left and Right Ventricles of 4-Week-Old

Offspringa

Parameter (%) Control Males (n ¼ 7) Control Females (n ¼ 7) -Vitamin D Males (n ¼ 8) -Vitamin D Females (n ¼ 8)

Left ventricle

Mononucleated 0.50 + 0.26 0.75 + 0.25 3.87 + 0.66b 4.25 + 0.62b

Binucleated 99.50 + 0.26 99.25 + 0.25 96.12 + 0.66b 95.75 + 0.62b

Right ventricle

Mononucleated 0.87 + 0.29 0.25 + 0.16 1.87 + 0.51b 1.12 + 0.29b

Binucleated 99.12 + 0.29 99.75 + 0.16 98.12 + 0.51b 98.87 + 0.29b

a Values are means + SEM.b P � .01 vitamin D-deficient offspring versus control offspring.

Figure 2. Projected cardiomyocyte area in the left ventricle plus

septum (LVþ S; A) and right ventricle (RV; B) in 4-week-old male (&;

n¼ 6 control, n¼ 8 vitamin D deficient) and female (c; n¼ 6 control,

n ¼ 8 vitamin D deficient) offspring. Values are means + SEM.

Figure 3. Cardiomyocyte number in the left ventricle plus septum

(LVþ S; A) and right ventricle (RV; B) of the 4-week-old male (&; n

¼ 6 control, n ¼ 8 vitamin D deficient) and female (c; n ¼ 6 control,

n ¼ 8 vitamin D deficient) offspring. Values are means + SEM.

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Vitamin D deficiency, in addition to leading to an

enlarged heart, is also associated with high blood pres-

sure33,34 and may thus play a role in the etiology of hyper-

tension. In our experimental model of vitamin D

deficiency, we have previously shown that the offspring

exhibit an elevation in blood pressure by 2 months of

age,35 but whether blood pressure is already elevated at

weaning is unknown (blood pressure was not measured

in the current study). Because the cardiomegaly we have

observed is evident by 4 weeks of age (weaning), it is con-

ceivable that this may be a response to an elevation in

blood pressure. Certainly, the increased size in the left

ventricular wall supports an adaptive response to

increased left ventricular load in the vitamin D-deficient

heart, and this would explain why there is hypertrophy

in the LV and not in the RV. Hence, the question arises:

is the exaggerated cardiomyocyte growth response in the

vitamin D-deficient heart a direct effect on the cardio-

myocytes, or indirectly related to left ventricular load

and/or upregulation of the renin-angiotensin system?

Indeed 1,25 dihydroxyvitamin D3 is reported to act as a

negative regulator of the renin-angiotensin system36,37

Further studies are required to elucidate this. In future

studies, it will be important to measure blood pressure

in the offspring at weaning and establish the mechanisms

leading to the exaggerated cardiomyocyte growth

response in the LV.

In conclusion, the findings from this study support

recent clinical findings of cardiomegaly in vitamin D defi-

cient offspring. Whether this leads to adverse effects in

cardiac function later in life is yet to be determined.

ACKNOWLEDGMENTS

This study was supported by the National Health and

Medical Research Council of Australia.

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