Download Vitamin D signaling pathway plays an important role in the

J Appl Physiol 114: 979–987, 2013.
First published February 21, 2013; doi:10.1152/japplphysiol.01506.2012.
Vitamin D signaling pathway plays an important role in the development
of heart failure after myocardial infarction
Soochan Bae,1 Sylvia S. Singh,1 Hyeon Yu,1,2 Ji Yoo Lee,1 Byung Ryul Cho,1,3 and Peter M. Kang1,2
1
Cardiovascular Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts;
Department of BIN Fusion Technology, Chonbuk National University, Jeonju; 3Division of Cardiology, Kangwon National
University, Chuncheon, South Korea
2
Submitted 17 December 2012; accepted in final form 17 February 2013
heart failure; myocardial infarction; vitamin D; apoptosis; inflammation
CARDIOVASCULAR DISEASE is the leading cause of morbidity and
mortality in developed countries. Coronary heart disease, particularly myocardial infarction (MI) and heart failure, affects
7.9 million and 5.7 million US adults, respectively (39). Occurrence of left ventricular abnormalities related to remodeling
(predominantly progressive chamber dilation, fibrosis, and systolic/diastolic dysfunction) and heart failure often complicates
MI (1). Recent data suggest that the prevalent myocardial
dysfunctions and heart failure are associated with decreased
levels of vitamin D (21, 23, 28). However, the role of vitamin
D signaling in the development of heart failure is not well
characterized.
Vitamin D plays an important physiological role in controlling cardiac functions, and vitamin D-dependent signaling
systems are present in cardiac myocytes and fibroblasts (9).
Vitamin D deficiency has been shown to lead to derangements
in cardiac myocyte contraction, abnormal cardiac relaxation,
Address for reprint requests and other correspondence: P. M. Kang, Cardiovascular Division, Beth Israel Deaconess Medical Center, 3 Blackfan
Circle, CLS 910, Boston, MA 02215 (e-mail: [email protected]).
http://www.jappl.org
proliferation, protein expression, and increased cardiac renin
gene expression (5, 35, 36, 44, 47). Previously, we have
demonstrated that vitamin D therapy prevents the progression
to cardiac hypertrophy (6) and attenuates the development of
heart failure in a salt-sensitive rat model (4). Numerous investigators have also showed that vitamin D therapy is renoprotective in various experimental nephropathy models via its
anti-inflammatory and antifibrotic actions (2, 32, 42, 43).
Because the prevalence of vitamin D deficiency has been
shown to be common and treatable (27), it is imperative to
better understand the mechanism of vitamin D signaling in
heart, which may potentially precede to novel therapeutic
strategies in cardiovascular diseases. However, the cardioprotective mechanisms that are involved are not well understood,
and our understanding of the role of vitamin D in the maintenance of cardiac function remains unclear (15).
In this study, we examine the role of vitamin D signaling in
preventing heart failure after MI with the activation or inhibition of vitamin D signaling using paricalcitol (PC) and vitamin
D receptor (VDR) knockout (KO) mice, respectively. We
hypothesize that vitamin D therapy may attenuate the progression of heart failure after MI through anti-inflammatory, antifibrotic, and antiapoptotic mechanisms.
MATERIALS AND METHODS
Materials. Materials and antibodies were obtained from flowing
sources as follows: PC (provided by Abbott Laboratories, Abbott
Park, IL), VDR antibody (sc-1008) (Santa Cruz Biotechnology, Santa
Cruz, CA), and GAPDH antibody (Cell Signaling Technology, Danvers, MA).
Adult cardiomyocyte culture and apoptotic stimulation. Primary cultures of cardiomyocytes from 6-wk-old Sprague-Dawley rats were
prepared as described (14, 22). Twenty-four hours after PC pretreatment (20 ␮M), cardiomyocytes were exposed to hydrogen peroxide
(H2O2) at 0.1 mM for 24 h to induce apoptosis.
Animal surgery and treatment. Male VDR KO mice (Jackson
Laboratory, Bar Harbor, ME) and their littermates in a C57BL/6
background were used as a model for defective vitamin D signaling.
To activate vitamin D signaling, 10 –12-wk-old male mice were
administered with PC or vehicle (Veh) 1 wk before MI and continued
until the end of the experiment. Wild-type (WT) mice underwent
sham or MI operation with either Veh treatment or PC therapy (15
ng/mouse, i.p., 3 times per week). Coronary artery ligation was
performed as described previously (46). After anesthesia with isofluorane (initially 4 –5% in an induction chamber then 2% via intubation
tube) via a precise vaporizer inhaler, each mouse was intubated, and
the heart was exposed and the left anterior descending (LAD) artery
ligated proximally with a 7– 0 silk suture. We confirmed the occlusion
by the pallor of the anterior left ventricular wall. All groups of mice
were age- and sex-matched littermates. For sham operations, the mice
underwent the same procedure except for LAD ligation. Euthanasia
8750-7587/13 Copyright © 2013 the American Physiological Society
979
Downloaded from http://jap.physiology.org/ by 10.220.32.247 on June 15, 2017
Bae S, Singh SS, Yu H, Lee JY, Cho BR, Kang PM. Vitamin D
signaling pathway plays an important role in the development of heart failure
after myocardial infarction. J Appl Physiol 114: 979–987, 2013. First published February 21, 2013; doi:10.1152/japplphysiol.01506.2012.—Accumulating evidence suggests that vitamin D deficiency plays a crucial role
in heart failure. However, whether vitamin D signaling itself plays an
important role in cardioprotection is poorly understood. In this study,
we examined the mechanism of modulating vitamin D signaling on
progression to heart failure after myocardial infarction (MI) in mice.
Vitamin D signaling was activated by administration of paricalcitol
(PC), an activated vitamin D analog. Wild-type (WT) mice underwent
sham or MI surgery and then were treated with either vehicle or PC.
Compared with vehicle group, PC attenuated development of heart
failure after MI associated with decreases in biomarkers, apoptosis,
inflammation, and fibrosis. There was also improvement of cardiac
function with PC treatment after MI. Furthermore, vitamin D receptor
(VDR) mRNA and protein levels were restored by PC treatment.
Next, to explore whether defective vitamin D signaling exhibited
deleterious responses after MI, WT and VDR knockout (KO) mice
underwent sham or MI surgery and were analyzed 4 wk after MI.
VDR KO mice displayed a significant decline in survival rate and
cardiac function compared with WT mice after MI. VDR KO mice
also demonstrated a significant increase in heart failure biomarkers,
apoptosis, inflammation, and fibrosis. Vitamin D signaling promotes
cardioprotection after MI through anti-inflammatory, antifibrotic and
antiapoptotic mechanisms.
Vitamin D Signaling Pathway After Myocardial Infarction
H2O2
*
2.0
†
1.5
1.0
0.5
0.0
Veh
PC
Veh
E
Sham
2.5
*
2.0
1.5
†
1.0
0.5
0.0
PC
Veh
Sham
H2O2
Veh
PC
Veh
Sham
Sham
MI
ANF
BNP
18S
MI
D
Sham
MI
10
8
*
†
6
4
2
*
8
†
6
4
2
0
0
Veh
F
PC
MI
PC
C
Fold of Veh-Sham
2.5
B
PARP-1 Activity
(Fold of Veh-Sham)
Caspase-3 Activity
(Fold of Veh-Sham)
Sham
PC
Veh
Veh-Sham
Veh-MI
2.5
2.0
PC
PC-Sham
PC-MI
*
*
†
†
1.5
1.0
0.5
0.0
ANF
G
Sham
MI
MI
H
Veh
50
Infarct Size (%)
PC
BNP
*
40
Sham
MI
30
*†
20
10
0
Veh
PC
Fig. 1. Effect of paricalcitol (PC) therapy after myocardial infarction (MI) in vitro and in vivo. A–B: effect of PC on Caspase-3 activity (A) and poly(ADP-ribose)
polymerase (PARP)-1 activity (B) after H2O2 (0.1 mM) in adult cardiomyocytes. [N ⫽ 4/group; *P ⬍ 0.05 vs. vehicle (Veh), †P ⬍ 0.05 vs. Veh-H2O2]. C–D: heart
weight over body weight (HW/BW) ratio (C), and lung weight over body weight (LUW/BW) ratio (D) in sham and MI groups with or without PC. (N ⫽ 4 – 6 mice
in each group; *P ⬍ 0.05 vs. sham of each group, †P ⬍ 0.05 vs. Veh-MI). E: representative image of heart failure markers, atrial natriuretic factor (ANF) and brain
natriuretic peptide (BNP) in sham and MI groups with or without PC. F: quantitative analysis of heart failure markers, ANF and BNP. (N ⫽ 4 – 6 mice in each group;
*P ⬍ 0.05 vs. sham of each group, †P ⬍ 0.05 vs. Veh-MI). G: representative photomicrographs from histological sections stained with Masson’s Trichrome. H: infarct
size analysis of sham and MI with and without PC.
J Appl Physiol • doi:10.1152/japplphysiol.01506.2012 • www.jappl.org
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A
Bae S et al.
Terminal deoxynucleotidyl transferase dUTP nick-end labeling
(TUNEL) staining was performed using in situ fluorescein-based Cell
Death Detection Kits (Roche Applied Science, Indianapolis, IN) as
described previously (3). Quantitative analysis of apoptotic nuclei
were performed in border zones of infarcted hearts. To distinguish
cardiomyocyte from noncardiomyocyte nuclei, we triple stained for
nuclei [4=,6-diamidino-2-phenylindole (DAPI) staining], apoptotic
nuclei (TUNEL staining), and cardiomyocytes (␣-actinin staining)
and analyzed the stained sections using confocal microscopy. A
minimum of 200 nuclei/field were counted for each sample.
RT-PCR for mRNA expression. Heart tissues were collected just
below the level of papillary muscles and processed for molecular analysis
4 wk after MI. Semiquantitative RT-PCR was performed as described
previously (4). The mRNA expression levels were analyzed by RT-PCR
by specific primers (see Supplemental Methods; supplemental material
for this article is available online at the Journal of Applied Physiology
website). Ribosomal 18S primers acted as internal controls, and all
RT-PCR signals were normalized to the 18S expression.
HW/BW (mg/g)
was performed by CO2 via a gas cylinder followed by heart extraction.
All procedures were approved by and performed in accordance with
Beth Israel Deaconess Medical Center Institutional Animal Care and
Use Committee guidelines.
Cardiac function analysis. Cardiac function was evaluated by
measuring the left ventricular (LV) pressure-volume (PV) loop to
calculate hemodynamic parameters, including cardiac output (CO),
stroke work (SW), stroke volume (SV), and isovolumetric relaxation
as described previously (3, 13).
Apoptosis assays. The activities of caspase-3 and poly(ADP-ribose)
polymerase (PARP)-1 were determined with colorimetric assay kit
(R&D Systems, Minneapolis, MN) as described previously (3, 13).
Briefly, for caspase-3 activity assay, protein samples were added to
substrates of Acetyl-Asp-Glu-Val-Asp-p-nitroanilide, and enzymecatalyzed release of p-nitroanilide was then measured at 405 nm. For
PARP activity assay, activity was measured at 450 nm by incorporation of biotinylated poly (ADP-ribose) onto histone-coated proteins in
the plate using colorimetric assay kit.
•
LUW/BW (mg/g)
980
Vitamin D Signaling Pathway After Myocardial Infarction
Immunoblot. Immunoblot analyses were also performed as described previously (4). Total protein of VDR from heart tissue was
probed with an anti-VDR rabbit polyclonal antibody. GAPDH was
used as an internal control. Resulting bands were quantified as optical
density (OD) ⫻ band area by NIH ImageJ software (version 1.38⫻;
NIH, Bethesda, MD).
Histology. Hearts were fixed in 10% formalin and paraffin embedded. Sections were stained with Masson-Trichrome at the Histology
Core facility at BIDMC. Infarct size was calculated as described
previously (25). Briefly, the infarct length was calculated by measuring the endo- and epicardial surface length delimiting the infarcted
region. Infarct size percentage was calculated as infarct length divided
by the total LV circumference using ImageJ software.
Statistical analysis. Data were expressed as means ⫾ SE. Comparisons between and within groups were conducted with unpaired
Student’s t-tests and repeated-measures ANOVA using GraphPad
Prism 5.0 (San Diego, CA), respectively. Survival curves after MI
were created by the Kaplan-Meier method and compared by a log rank
test. P values of ⬍0.05 were considered significant.
PC therapy attenuates the development of heart failure after
myocardial infarction. We initially determined whether PC treatment exerts a protective effect against H2O2-induced apoptotic
A
Sham
MI
Sham
MI
COL 1A1
COL 3A1
18S
2
MI
Sham
MI
TNF-α
MCP-1
18S
PC-Sham
PC-MI
*
*
1.5
Fig. 2. Effect of PC on mRNA expression of
biochemical markers of fibrosis, inflammation, and renin-angiotensin system (RAS) after MI. A: representative mRNA expression
of collagenase (COL) 1A1 and 3A1. B: quantitative analysis of mRNA expression of collagenase 1A1 and 3A1. (N ⫽ 4 – 6 mice in
each group; *P ⬍ 0.05 vs. sham of each
group, †P ⬍ 0.05 vs. Veh-MI). C: representative image of inflammatory markers, tumor
necrosis factor-␣ (TNF-␣) and monocyte
chemoattractant protein-1 (MCP-1) in sham
and MI groups with or without PC. D: quantitative analysis of inflammatory markers, TNF-␣
and MCP-1. (N ⫽ 4 – 6 mice in each group;
*P ⬍ 0.05 vs. sham of each group, †P ⬍ 0.05
vs. Veh-MI) E: representative image of RAS
genes [angiotensinogen (ANGTSN), renin, and
renin receptor (ReninR)] in sham and MI
groups with or without PC. F: quantitative
analysis of RAS genes (N ⫽ 4 – 6 mice in each
group; *P ⬍ 0.05 vs. sham of each group,
†P ⬍ 0.05 vs. Veh-MI).
COL3A1
Veh-Sham
Veh-MI
2.0
Fold of Veh-Sham
Sham
PC
†
COL 1A1
D
Veh
*
†
1
0
C
PC-Sham
PC-MI
*
3
PC
Fold of Veh-Sham
Veh
†
†
1.0
0.5
0.0
TNF-α
E
F
ANGTSN
ReninR
Renin
18S
MI
PC
Sham
MCP-1
Veh-Sham
Veh-MI
2.0
MI
Fold of Veh-Sham
Veh
Sham
981
cell death in rat adult cardiomyocytes and whether such protection
is associated with decreased activation of caspase-3 and PARP-1
activities. H2O2 significantly increased activation of caspase-3 and
PARP-1 compared with vehicle control (Fig. 1, A and B). However, PC treatment significantly attenuated activation of caspase-3
and PARP-1. These data suggest that PC protected adult cardiomyocytes against H2O2-induced apoptosis.
Then, to study the effect of activating vitamin D signaling in
cardiac apoptosis in vivo, we examined the mice assigned to
four groups: 1) Veh ⫹ Sham, 2) Veh ⫹ MI, 3) PC ⫹ Sham,
and 4) PC ⫹ MI. Mice underwent sham or MI operation with
either Veh treatment or PC therapy (15 ng/mouse, i.p. 3 times
per week) before MI. We found that PC treatment resulted in
significant decrease in heart weight (HW)/body weight (BW)
ratio (P ⬍ 0.05) and lung weight (LuW)/BW ratio (P ⬍ 0.05)
in PC ⫹ MI group compared with the Veh ⫹ MI group (Fig.
1, C and D). No significant differences were observed in the
sham-operated mice treated with PC or Veh.
We next sought to determine whether the pathophysiological
findings correlated with other biological and molecular measures. The analyses of mRNA expression levels of atrial
natriuretic factor (ANF) and brain natriuretic peptide (BNP) of
Veh-Sham
Veh-MI
B
Bae S et al.
*
PC-Sham
PC-MI
*
*
1.5
†
†
†
1.0
0.5
0.0
ANGTSN
ReninR
Renin
J Appl Physiol • doi:10.1152/japplphysiol.01506.2012 • www.jappl.org
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RESULTS
•
Vitamin D Signaling Pathway After Myocardial Infarction
A
Sham
MI
D
Sham
C
MI
Sham
2.0
MI
Sham
*
1.0
†
0.5
0.0
8
6
*†
4
*
2
0
Veh
PC
Sham
MI
Veh
F
20
900
600
*
300
*†
10
*
5
TAU (ms)
*†
SV (µL)
15
0
0
Veh
PC
PC
Sham
30
MI
*
1500
1200
MI
10
1.5
E
1800
SW (mmHg*µL)
Activation of circulating and tissue RAS leads to ventricular
remodeling after MI, a process that is associated with altering
myocardial apoptosis, myocardial hypertrophy, and increasing
the content of collagen (33). There were significant increases in
mRNA expression of components of the RAS gene 4 wk after
MI (Fig. 2, E and F). The mRNA expressions of angiotensinogen, renin, and renin receptors were significantly reduced by
PC treatment compared with Veh-treated mice after MI.
Effect of PC on apoptosis and cardiac function after MI.
Because adverse LV remodeling after MI has been associated
with increased apoptosis in the myocardium (37), we examined
the effects of PC on cardiomyocyte apoptosis. The number of
TUNEL-positive myocytes in the ischemia border zone were
significantly greater in animals subjected to MI (Veh ⫹ MI)
than in sham-operated animals (Fig. 3, A and B), suggesting
that apoptosis is enhanced during cardiac remodeling. Conversely, the number of TUNEL-positive myocytes in ischemia
border zone was significantly lower in PC ⫹ MI than in Veh ⫹
MI groups (0.39% vs. 1.64%, P ⬍ 0.05). These data confirmed
our notion that PC therapy inhibits apoptosis. Finally, cardiac
function was evaluated by measuring the LV PV loop 4 wk
after MI. PV loop analysis revealed that PC after MI resulted
in a significant improvement of CO, SW, and SV and a
significant reduction in isovolumetric relaxation (␶) compared
with the vehicle group after MI (Fig. 3, C–F).
Effect of PC on VDR expression after MI. Key components
of vitamin D-dependent signaling system are present in the
B
PC
TUNEL + Cardiomyocyte
(%)
Veh
Bae S et al.
*†
20
10
0
Veh
PC
Veh
PC
Fig. 3. Effect of PC on apoptosis and cardiac function after MI. A: representative terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)
staining of cardiomyocytes in sham and MI groups with or without PC. B: quantification of TUNEL-positive cardiomyocytes/total cells. (N ⫽ 4 – 6 mice in each
group; *P ⬍ 0.05 vs. sham of each group, †P ⬍ 0.05 vs. Veh-MI). C–F: hemodynamic measurements by pressure-volume (PV) loop after MI, cardiac output
(CO), stroke work (SW), stroke volume (SV), and tau (␶) in sham and MI groups with or without PC. (N ⫽ 4 – 6 mice in each group; *P ⬍ 0.05 vs. sham of
each group, †P ⬍ 0.05 vs. Veh-MI).
J Appl Physiol • doi:10.1152/japplphysiol.01506.2012 • www.jappl.org
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the LV tissues showed that PC significantly attenuated ANF
(⫺33%) and BNP (⫺29%), which were both increased after
MI (P ⬍ 0.05 for all) (Fig. 1, E and F). We also found that the
infarct size after MI is significantly decreased in PC ⫹ MI
group than in Veh ⫹ MI group (16.8 ⫾ 4.3 vs. 42 ⫾ 6.0) (Fig.
1, G and H). There were no significant differences observed
between the two sham groups.
Effect of PC on fibrosis and inflammation after MI. To
examine the potential vitamin D signaling-induced cardioprotective mechanisms after MI, we examined the effect of PC on
fibrosis, inflammation, and renin-angiotensin system (RAS).
Because studies have shown that vitamin D therapy has an
antifibrotic effect (4, 29, 32, 41), the degree of fibrosis was
quantified by examining the expression of collagen 1A1 and
3A1, the main forms of collagen in the heart. PC effectively
reduced Col1A1 and Col3A1 mRNA levels in the infarct zone
compared with the Veh ⫹ MI group (Fig. 2, A and B),
suggesting that PC treatment attenuates increased fibrosis 4 wk
after MI.
Inflammation is known to play an important role in the
development and progression to heart failure (34). To establish
whether PC suppresses inflammation in the heart, we examined
mRNA level of tissue necrosis factor-␣ (TNF-␣) and monocyte
chemoattractant protein (MCP)-1. TNF-␣ and MCP-1 mRNA
levels were significantly elevated in Veh ⫹ MI than in shamoperated animals (Fig. 2, C and D). These increases were
significantly reduced by PC treatment (P ⬍ 0.05 for both).
•
CO (ml/min)
982
Vitamin D Signaling Pathway After Myocardial Infarction
A
B
Effect of defective vitamin D signaling on apoptosis, cardiac
function, and survival rate after MI. Because our PC study
exhibited elevated VDR expression in cardiomyocytes and
inhibition of apoptosis after MI, we next investigated the
effects of defective vitamin D signaling on apoptosis. TUNELpositive cardiomyocytes exhibited higher levels of apoptosis in
VDR KO mice compared with WT mice 2 days after MI
(4.25% vs. 1.55%, P ⬍ 0.05) (Fig. 6, A and B), thereby
confirming that deletion of VDR does indeed display significant apoptotic effects in the myocardium after MI. PV loop
analysis after MI also demonstrated that cardiac function parameters were significantly worsened in VDR KO ⫹ MI mice
than in WT ⫹ MI group. VDR KO ⫹ MI group displayed a
significant reduction in the CO, SW, and SV and a significant
increase in isovolumetric relaxation (␶) in VDR KO mice
compared with the WT ⫹ MI mice (Fig. 6, C–F).
Finally, we explored whether mice lacking VDR or defective vitamin D signaling may exhibit significant mortality 4 wk
after MI. We compared survival rates after MI surgery among
WT ⫹ sham, VDR KO ⫹ sham, WT ⫹ MI, and VDR KO ⫹
MI mice. The survival rate was found to be significantly lower
in VDR KO ⫹ MI mice than in WT ⫹ MI mice at 4 wk (77%
vs. 54%, P ⬍ 0.05) (Fig. 6G). All the deaths occurred early
within a week with a 45% decline in VDR-KO mice and 20%
decline in WT-MI mice after MI. These results indicate that
VDR deficiency may cause fulminant and aggressive heart
failure after MI.
DISCUSSION
The key findings of the present study are that 1) the activation of VDR pathway by PC may offer cardioprotection as it
attenuates cardiac dysfunction, cardiac myocyte apoptosis, and
upregulation of proinflammatory cytokines in mouse models
and 2) deficiency of VDR causes fulminant and aggressive
Sham
MI
Sham
MI
VDR
18S
VDR mRNA
(Fold of WT Sham)
Sham
PC
MI
**
1.5
Veh
†
1.0
*
0.5
0.0
Veh
C
D
Sham
VDR
GAPDH
MI
Sham
PC
MI
MI
Sham
2.0
PC
VDR Protein
(Fold of WT Sham)
Veh
983
**
†
1.5
1.0
Fig. 4. Effect of PC on mRNA and protein
expression of vitamin D receptor(VDR) after
MI. A: representative image of tissue mRNA
level of VDR in sham and MI groups with or
without PC. B: quantitative analysis of tissue
mRNA level of VDR. (N ⫽ 4 – 6 mice in each
group; *P ⬍ 0.05 vs. sham of each group,
**P ⬍ 0.05 vs. Veh-sham, †P ⬍ 0.05 vs.
Veh-MI). C: representative image of tissue
protein level of VDR in sham and MI groups
with or without PC. D: quantitative analysis
of tissue protein level of VDR. (N ⫽ 4 – 6
mice in each group; *P ⬍ 0.05 vs. sham of
each group, **P ⬍ 0.05 vs. Veh-Sham, †P ⬍
0.05 vs. Veh-MI). WT, wild-type.
*
0.5
0.0
Veh
PC
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heart (9), and the loss of VDR expression may increase the
likelihood of heart failure progression. Thus we investigated
mRNA and protein levels of VDR after MI. We confirmed that,
4 wk after MI, mRNA and protein expression of VDR were
notably reduced in Veh ⫹ MI group when compared with sham
controls (Fig. 4, A–D). Consistent with other findings in the
kidneys (42), PC amplifies VDR expression in both mRNA and
protein expression in cardiac myocytes after MI. In addition,
the significant reduction of VDR mRNA and protein levels by
MI was abrogated by PC treatment. Together, we conclude that
VDR activation by PC may play a cardioprotective role in the
progression to heart failure caused by MI.
Effect of defective vitamin D signaling on heart failure after
MI. The consequences of VDR disruption on cardiac myocytes
were then examined as we compared the VDR KO mice and
their WT controls. VDR KO mice did not express any VDR
expression in the heart (Fig. 5A). Morphology data showed
that, after MI, the HW/BW ratio and the LuW/BW ratio were
significantly higher in VDR KO mice than in WT group (Fig.
5, B and C). Although there was a modest increase at baseline
in WT sham group, MI caused a much more significant
increase, not only in absolute percentage changes, in HW/BW
ratio and LuW/BW ratio of VDR KO sham mice. Thus these
findings suggest that VDR KO mice developed a more severe
progression to heart failure at 4 wk after MI than WT mice.
We also observed an upregulation in mRNA levels of ANF
and BNP gene expression in VDR KO mice compared with
WT mice after MI (P ⬍ 0.05 for all) (Fig. 5, D and E). In
addition, inflammatory markers TNF-␣ and MCP-1 mRNA
levels were significantly higher in VDR KO-MI groups than in
the WT-MI group (Fig. 5, F and G). VDR KO at baseline did
not show increases in biochemical and inflammatory markers
for heart failure.
Bae S et al.
•
984
Vitamin D Signaling Pathway After Myocardial Infarction
B
Sham
HW/BW (mg/g)
WT
VDR KO
VDR
GAPDH
C
MI
10
*
6
4
2
0
MI
Sham
MI
BNP
18s
*
4
2
2.0
WT
*†
*
*
Sham
VDR KO
MI
Sham
MI
Fold of WT Sham
G
WT
18S
WT-MI
VDRKO-MI
1.0
0.5
0.0
F
MCP-1
WT-Sham
VDRKO-Sham
1.5
ANF
TNF-α
VDR KO
3
2
BNP
*†
*†
*
*
WT-Sham
WT-MI
VDRKO-Sham
VDRKO-MI
1
0
TNF-α
MCP-1
Fig. 5. Effect of defective vitamin D signaling on heart failure after MI. A: VDR protein expression in WT and VDR knockout (KO) mice heart. B–C: HW/BW
ratio (B) and LUW/BW ratio (C) in sham and MI groups. (N ⫽ 4 – 6 mice in each group; *P ⬍ 0.05 vs. sham of each group, †P ⬍ 0.05 vs. WT-MI).
D: representative image of tissue mRNA level of ANF and BNP in sham and MI groups. E: quantitative analysis of tissue mRNA level of ANF and BNP. (N ⫽
4 – 6 mice in each group; *P ⬍ 0.05 vs. sham of each group, †P ⬍ 0.05 vs. WT-MI). F: representative image of tissue mRNA level of TNF-␣ and MCP-1 in
sham and MI groups. G: quantitative analysis of tissue mRNA level of TNF-␣ and MCP-1. (N ⫽ 4 – 6 mice in each group; *P ⬍ 0.05 vs. sham of each group,
†P ⬍ 0.05 vs. WT-MI).
progression of heart failure after MI because they lack cardioprotective signaling through the VDR pathway. These findings
demonstrate that vitamin D therapy could be an effective,
preventive, and therapeutic candidate against progression of
heart failure after MI.
Vitamin D and its biological activities are mediated by a
specific high-affinity receptor, the VDR, a member of the
superfamily of nuclear receptors for steroid hormones (16).
Analogous to other members of the steroid family, VDR acts as
a ligand-activated transcription factor, and, upon activation by
its ligand, VDR forms a heterodimeric complex with its obligatory partner, retinoid X receptor, and binds to the vitamin D
response element. Hence, loss of VDR could lead to an
eradication of vitamin D signaling, even without any reduction
in levels of active vitamin D (42). Several cardioprotective
mechanisms of vitamin D signaling could be postulated from
our study. Earlier studies have stated that there may be a direct
connection between vitamin D and apoptosis (31, 42) although
this connection had not been shown in cardiomyocytes. A
study using an obstructive nephropathy rat model, consistent
with our findings, demonstrated that vitamin D treatment
upregulated VDR levels in vehicle rats and significantly decreased the number of TUNEL-positive cells in obstructed
animals (20). Additionally, pretreatment with 1,25(OH)2D3
(calcitriol) increases VDR gene expression in cardiomyocyte
but has no effect in cardiac fibroblasts (10). Collectively, these
results suggest that upregulation/activation of liganded VDR
signaling may protect cardiomyocyte against MI-induced apoptosis. Therefore, beneficial effects of PC may be linked to attenuation of cardiac dysfunction after MI. The results from our study,
to our knowledge, are the first to suggest that vitamin D therapy
could be a possible approach to the reduction of apoptosis in
cardiomyocytes in MI-induced heart failure.
We showed that vitamin D therapy significantly decreased
cardiac fibrosis and extracellular matrix remodeling, a pathological feature of heart failure, after MI. Tan et al. (42)
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ANF
Fold of WT Sham
Sham
†
6
*†
2.5
VDR KO
*
8
VDR KO
E
WT
MI
0
WT
D
Sham
10
†
*
8
Bae S et al.
LUW/BW (mg/g)
A
•
Vitamin D Signaling Pathway After Myocardial Infarction
A
WT
•
985
Bae S et al.
B
VDR KO
TUNEL + Cardiomyocyte
(%)
Sham
Sham
MI
MI
5
*
4
3
*
2
1
0
WT
*†
2
1500
*
1000
†
*
500
WT
*
†
0
VDR KO
WT
F
*
10
5
0
0
MI
15
SV (µL)
SW (mmHg*µL)
*
4
20
MI
8
6
Sham
Sham
2000
MI
CO (ml/min)
E
D
Sham
10
VDR KO
G
VDR KO
WT
100
VDR KO
WT-Sham
Sham
80
WT-MI
Survival (%)
*
20
TAU (ms)
VDR KO-Sham
MI
25
*
15
10
5
VDR KO-MI
60
*
†
40
20
0
WT
VDR KO
0
0
4
8
12
16
20
24
28
Time after MI (d)
Fig. 6. Effect of defective vitamin D signaling on apoptosis, cardiac function, and survival rates after MI. A: representative TUNEL staining of cardiomyocytes
in sham and MI groups. B: quantification of TUNEL-positive cardiomyocytes/total cells. (N ⫽ 4 – 6 mice in each group; *P ⬍ 0.05 vs. sham of each group, †P ⬍
0.05 vs. WT MI). C–F: hemodynamic measurements by PV loop in sham and MI groups. (N ⫽ 4 – 6 mice in each group; *P ⬍ 0.05 vs. sham of each group,
†P ⬍ 0.05 vs. WT MI). G: survival rates after MI in WT and VDR KO mice. Survival curves up to 4 wk after MI were created by Kaplan-Meier method and
compared by a log-rank test. (N ⫽ 10 –15 mice in each group; *P ⬍ 0.05 vs. sham of each group, †P ⬍ 0.05 vs. WT-MI).
observed that dramatic downregulation of VDR in the fibrotic
kidney was completely restored by administration of vitamin
D, resulting in an increase in VDR expression and further
suggesting that deficiency in vitamin D signaling in the diseased kidney is far greater than it was anticipated earlier.
Mizobuchi et al. (32) also showed that vitamin D therapy
suppresses the progression of perivascular fibrosis and myocardial arterial vessel thickness and inhibits collagen synthesis
after MI in vivo, presumably due to the upregulation/activation
of VDR signaling. These findings coincide with our data where
vitamin D demonstrates a significant increase in VDR expression after MI. Hence, these data along with the findings from
this study suggest that cardiac fibrosis after MI may be regulated by the degree of vitamin D signaling in the heart.
Activation of circulating and tissue RAS plays a key role in
the pathophysiology of heart failure and ventricular remodel-
J Appl Physiol • doi:10.1152/japplphysiol.01506.2012 • www.jappl.org
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C
†
986
Vitamin D Signaling Pathway After Myocardial Infarction
GRANTS
This work was supported in part by NIH Grants R01 HL65742 (P. Kang),
the World Class University program (R31-20029) from Ministry of Education,
Science and Technology, South Korea (P. Kang), and research support from
Abbott Laboratories (P. Kang).
DISCLOSURES
The study was supported in part by Abbott Laboratories.
AUTHOR CONTRIBUTIONS
Author contributions: S.B., S.S.S., B.R.C., and P.M.K. conception and
design of research; S.B., S.S.S., H.Y., J.Y.L., and B.R.C. performed experiments; S.B., S.S.S., H.Y., J.Y.L., B.R.C., and P.M.K. analyzed data; S.B.,
S.S.S., H.Y., J.Y.L., and P.M.K. interpreted results of experiments; S.B.,
S.S.S., H.Y., J.Y.L., and P.M.K. prepared figures; S.B., S.S.S., and P.M.K.
drafted manuscript; S.B., S.S.S., and P.M.K. edited and revised manuscript;
S.B., S.S.S., H.Y., J.Y.L., B.R.C., and P.M.K. approved final version of
manuscript.
Bae S et al.
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additional beneficial effects of vitamin D after MI. Several
experimental studies have exhibited that vitamin D therapy
effectively reduces renin transcript levels and plasma renin
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In patients with end-stage heart failure, significantly low
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with high selectivity and efficacy to treat the heart failure
phenotype and high-renin-associated dysfunctions (40).
In summary, we have shown that activation of vitamin D
signaling attenuates the progression to heart failure after MI,
but defective VDR signaling exacerbates this deleterious effect. Given our evolving understanding of vitamin D and
cardiovascular diseases, future studies are needed to determine
whether vitamin D therapy could be an effective therapeutic
agent for the treatment of heart failure.
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•