maternal vitamin d deficiency leads to cardiac hypertrophy in rat offspring
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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
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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:
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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