Download Adaptive response of the heart to long term anemia induced by iron

Articles in PresS. Am J Physiol Heart Circ Physiol (January 9, 2009). doi:10.1152/ajpheart.00463.2008
Adaptive response of the heart to long term anemia induced by iron deficiency
Yoshiro Naito, Takeshi Tsujino, Mika Matsumoto, Tsuyoshi Sakoda*, Mitsumasa
Ohyanagi*, Tohru Masuyama
Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine,
Nishinomiya, Japan, *Division of Coronary Heart Disease, Department of Internal
Medicine, Hyogo College of Medicine, Nishinomiya, Japan
Running head: adaptive response of the heart to chronic anemia
Word count: 5519,
Word count of abstract: 244,
Total number of figures: 7
There are no financial or other relations that could lead to a conflict of interest.
Corresponding author: Takeshi Tsujino, MD, PhD
Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine,
1-1 Mukogawa-cho, Nishinomiya, 663-8501, Japan
Phone number: +81-798-45-6553, Fax number: +81-798-45-6551
E-mail address: [email protected]
Copyright © 2009 by the American Physiological Society.
Abstract
Anemia is common in patients with chronic heart failure, and an independent
predictor of poor prognosis. Chronic anemia leads to left ventricular (LV) hypertrophy
and heart failure, but its molecular mechanisms remain largely unknown.
We
investigated the mechanisms, including the molecular signaling pathway, of cardiac
remodeling induced by iron deficiency anemia (IDA). Iron deficient diet was given for
20 weeks in weanling Sprague-Dawley rats to induce IDA, and these rats were studied
for the evaluation of molecular mechanisms of cardiac remodeling.
Iron deficient diet
initially induced severe anemia, which resulted in LV hypertrophy and dilation with
preserved systolic function associated with increased serum erythropoietin (Epo)
concentration. Cardiac STAT3 phosphorylation and vascular endothelial growth factor
(VEGF) gene expression increased by 12 weeks of IDA, causing angiogenesis in the
heart. Hereafter, sustained IDA induced upregulation of cardiac hypoxia inducible
factor-1alpha gene expression, maintained upregulation of cardiac VEGF gene
expression and cardiac angiogenesis; however, sustained IDA promoted cardiac fibrosis
and lung congestion with decreased levels of serum Epo concentration and cardiac
STAT3 phosphorylation after 20 weeks of IDA compared to 12 weeks.
Upregulation
of serum Epo concentration and cardiac STAT3 phosphorylation is associated with
2
beneficial adaptive mechanism of anemia induced cardiac hypertrophy, and latter
decreased levels of these molecules may be critical for the transition from adaptive
cardiac hypertrophy to cardiac dysfunction in long term anemia. Understanding the
mechanism of cardiac maladaptation to anemia may lead to a new therapeutic strategy
in chronic heart failure with anemia.
Key words: anemia; cardiac remodeling; erythropoietin; iron; hypertrophy
3
Introduction
Chronic severe anemia is known to cause high-cardiac output heart failure (HF)
(1). Moreover, anemia is common in patients with HF, and many recent observations
have shown that reduced hemoglobin indicates an independent risk of hospitalization
and all-cause mortality in patients with HF (2, 12). Anemia is frequently seen in
patients with not only systolic HF but also diastolic HF (6, 8). Several factors such as
hemodilution, impaired erythropoietin (Epo) secretion, chronic inflammation, and
disturbed iron metabolism are supposed to cause anemia in patients with HF (19);
however, the mechanism of how anemia causes or facilitates HF remains largely
unknown.
Iron deficiency has been reported as a common cause of anemia in patients
with advanced HF (18). A clinical study has also shown that a treatment with iron
supplements in patients with HF is beneficial (20, 28). Thus, iron deficiency anemia
(IDA) is obviously important not only as a primary cause of HF but also as a facilitator
of HF.
Numerous studies have shown that IDA leads to left ventricular (LV)
hypertrophy in developing rats (16, 21, 22, 27), but no data are available for its
molecular mechanisms, including the molecular signaling pathways, of IDA-induced
4
cardiac remodeling.
Recent studies have shown that erythropoietin receptor (EpoR) is expressed in
a variety of cells, including myocardium (30).
Moreover, Epo-EpoR signaling can
stimulate the Jak/STAT, MAPK and PI3K/Akt signaling pathways in hematopoietic and
cardiac cells (23).
Epo treatment improves quality-of-life scores and LV ejection
fraction in patients with HF (14).
Therefore, in the present study, we investigated the molecular mechanism,
including the Epo-EpoR signaling pathway, of the cardiac remodeling in long-term
anemia induced by iron deficiency.
Materials and Methods
Animals:
Protocol 1: Male 4 weeks old Sprague-Dawley (SD) rats (n=60) were
randomly assigned to the iron deficient and the control diet groups. Regular rat chow
was supplemented with approximately 0.003 % of FeC6H5O7•H2O. Rats of the iron
deficient group (n=30) were given a diet which is not supplemented with
FeC6H5O7•H2O (prepared by Oriental Yeast Inc., Chiba, Japan) and deionized water to
5
reduce the influence of water iron content for 20weeks, while the rats of the control
group (n=30) received the regular rat chow and regular water for 20 weeks. Rats were
maintained on a 12hr light/dark cycle and had free access to food and water.
After 1, 4,
12, 20 weeks of each diet, the rats were evaluated by echocardiography and collected
blood samples and tissues.
Protocol 2: After given iron deficient diet for 12 weeks, twelve rats were
randomized to one of the following treatment groups: Epo analogue, darbepoetin alpha
(Kirin Pharma, Tokyo, Japan), 1.5ug/kg/week (n=6) as previously described (24) and
vehicle (PBS) (n=6). Darbepoetin alpha and vehicle were injected intraperitoneally
once per week. After 8 weeks treatment, the rats were evaluated by echocardiography
and collected blood samples and tissues. All of our experimental procedures were
approved by the Animal Research Committee of Hyogo College of Medicine.
Assessments of Blood Pressure, Hemoglobin, Serum Concentrations of
Iron, Epo, and Tumor Necrosis Factor-alpha, and Renal Function: Systolic blood
pressure (SBP) and heart rate were measured with a non-invasive computerized tail-cuff
system (MK-2000, Muromachi Kikai, Tokyo, Japan). Blood hemoglobin was
measured by the sodium lauryl sulfate-hemoglobin method.
6
Serum concentrations of
iron, BUN, and creatinine were determined by the
2-Nitroso-5-[N-n-propyl-N-(3-sulfopropyl)amino]phenol (Nitroso-PSAP) method, the
urease-glutamate dehydrogenase-UV method, and enzymatic assay (creatinine
amidohydrolase-sarcosine oxidase-peroxidase method), respectively. Serum Epo
concentration was determined by radioimmunoassay as previously described (11).
Serum concentration of tumor necrosis factor-alpha was measured with the Bio-Plex
suspension array system (Bio-Rad, Hercules, CA, USA). 24 hour urine samples were
collected in metabolic cages for measuring urinary volume, protein, and electrolytes
levels.
Echocardiography: Rats were anesthetized with ketamine HCl (50 mg/kg)
and xylazine HCl (10 mg/kg), and were evaluated by transthoracic echocardiographic
studies. We measured LV cavity size and wall thickness, and calculated LV fractional
shortening as previously described (15) with a 12-Mhz phased-array transducer (Aplio,
Toshiba Medical Systems Corp., Odawara, Japan).
LV end-diastolic (LVDd) and
end-systolic (LVDs) dimensions and LV anterior and posterior wall thicknesses were
measured using M-mode tracings. We also recorded pulsed Doppler mitral flow velocity
pattern, and measured peak early diastolic filling velocity (E), peak filling velocity at
atrial contraction (A), their ratio (E/A) and deceleration time as previously described
7
(15).
RNA Extraction and Real-time Quantitative Reverse
Transcription-Polymerase Chain Reaction (RT-PCR): Total RNA was extracted
from the left ventricle using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according
to the manufacturer’s instructions.
Total RNA was DNase-treated and
reverse-transcribed into cDNA using random primers (Applied Biosystems, Foster City,
CA, USA). For detection of atrial natriuretic peptide (Nppa), brain natriuretic peptide
(Nppb), collagen type 3, EpoR, vascular endothelial growth factor (VEGF)-A, hypoxia
inducible factor-1alpha (HIF-1alpha), and ribosomal eukaryotic 18S RNA mRNA levels
in the LV, quantitative RT-PCR was performed with an Applied Biosystems 7900
Real-time PCR System with TaqMan Universal PCR Master Mix and TaqMan Gene
Expression Assays (Applied Biosystems, Foster City, CA, USA) as previously
described (17). The mRNA levels were normalized to the endogenous 18S ribosomal
RNA gene expression.
TaqMan Gene Expression Assays were used as primers and
probes for each gene were as follows: Nppa (assay ID Rn00561661_m1, amplicon size:
58), Nppb (assay ID Rn00580641_m1, amplicon size: 106), collagen type 3 (assay ID
Rn01437683_m1, amplicon size: 130), EpoR (assay ID Rn00566533_m1, amplicon
size: 76), VEGF-A (assay ID Rn00582935_m1, amplicon size: 75), HIF-1alpha (assay
8
ID Rn00577560_m1, amplicon size: 72), and ribosomal eukaryotic 18S RNA (assay ID
Hs99999901_s1, amplicon size:187).
Western Blot Analysis: The total protein homogenate (50 µg) from the left
ventricle was separated by SDS-PAGE and transferred onto PVDF membranes. The
expression levels of signaling molecules using antibodies against rabbit
anti-phospho-STAT3 (Tyr705), STAT3, phospho-Akt (Ser473), Akt,
phospho-extracellular signal-regulated kinase (ERK) (Thr202/Tyr204), ERK (Cell
Signaling Technology Beverly, MA, USA; dilution 1:1000), rabbit anti-HIF-1alpha
(Novus Biologicals, CO, USA; dilution 1:1000) and goat anti-actin (Santa Cruz, CA,
USA; dilution 1:1000) were detected by an enhanced chemilumiescence kit (PIERCE,
Rockford, IL, USA). Quantification of the intensity of the bands for phosphorylated
STAT3, Akt, and ERK was normalized with that for native STAT3, Akt, and ERK,
respectively. Expression of HIF-1alpha was standardized on the basis of actin
expression.
Histological Analysis: Midpapillary slices from the hearts were fixed with
buffered 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm-thick sections.
Hematoxylin-eosin, Masson’s trichrome, and Picrosirius red staining were performed
9
using serial sections. Photomicrographs were quantified with the use of NIH Image-J
software to measure the cross-sectional area of cardiomyocytes and to assess the fibrosis
area of myocardium. 100 randomly selected cardiomyocytes in the LV were measured
for cross-sectional area.
Midpapillary slices from the hearts samples were
immunohistochemically stained with von Willebrand Factor antibody (1:1000 dilution,
Dako, Kyoto, Japan).
The number of von Willebrand Factor-positive vessels was
counted to calculate the number of microvessels per cardiomyocytes.
Statistical Analysis: Values are reported as the means ± SEM. Statistical
analysis was performed using one way ANOVA or Student’s t test. Differences among
three groups were assessed by Tukey-Kramer multiple comparison test.
Differences
were considered significant when the probability value was <0.05.
Results
Effects of IDA on Physiological Parameters: Iron deficient diet induced
anemia measured by hemoglobin content (g/dl) in all groups studied (Figure 1A).
There were no significant differences in body weight in each group until 4 weeks;
however, body weight decreased significantly in the IDA group after 12 weeks diet
10
compared to the control group and thereafter (404±13 vs. 457±12 g, p<0.05; Figure 1B),
suggesting growth was inhibited by iron deficiency.
SBP was also comparable
between the control group and the IDA group until 4 weeks after diet and became
progressively decreased in the IDA group compared with the control group after 12
weeks, while heart rate was significantly higher in the IDA group compared with the
control group 4 weeks after diet, but became comparable between the control group and
the IDA group after 12 weeks diet (Figure 1C, D). Chronic IDA lead to reductions of
body weight and blood pressure.
Effects of IDA on Cardiac Function: The heart at 12 and 20 weeks after diet
was larger in IDA relative to controls (Figure 1E). The IDA group displayed a marked
increase in the LV weight to tibia length ratio compared with the control group after 12
weeks diet and thereafter, demonstrating cardiac hypertrophy (Figure 1F). Wet lung
weight to tibia length ratio and wet to dry lung weight ratio, an index of pulmonary
congestion (13), were significantly increased after 20 weeks diet in the IDA group
relative to the control group (Figure 1G, H).
hypertrophy, resulting in cardiac dysfunction.
11
Sustained IDA induced cardiac
Representative M-mode echocardiographic tracings and pulsed Doppler mitral
flow velocity patterns are shown in Figure 2A. LV hypertrophy gradually developed,
reached a peak at 12 weeks in the IDA group. LV dilatation was evident at 12 weeks
and more enhanced at 20 weeks in the IDA group, whereas fractional shortening
slightly increased in the IDA group compared with the control group after 12 weeks diet,
but the differences were not statistically significant (Figure 2B to E). Early diastolic
filling (E) wave was higher in the IDA group compare to the control group at 12 and 20
weeks diet, whereas the ratio of peak early diastolic filling velocity and peak filling
velocity of atrial contraction (E/A ratio) was lower and deceleration time was prolonged
in the IDA group relative to the control group after 12 weeks diet.
Conversely, both E
wave and E/A ratio was increased and deceleration time was shortened in the IDA
group relative to the control group after 20 weeks diet (Figure 2F to H).
Histological analysis showed that the cross-sectional area of cardiomyocytes
was increased in the IDA group after 12 weeks diet compared to the control group and
thereafter (Figure 3A, D). Marked interstitial fibrosis was not detected in the LV of the
IDA group after 12 weeks diet; however, it appeared progressively in the IDA group
compared with the control group after 20 weeks diet (Figure 3B, C, E).
12
Serum Concentrations of Iron and Epo and Cardiac EpoR Expression in
IDA-Induced Cardiac Remodeling: Serum iron concentration was significantly lower
in the IDA group than in the control group even at 1 week diet, while serum Epo
concentrations progressively increased untill 12 weeks but decreased at 20 weeks diet in
the IDA group (Figure 4A, B). Cardiac EpoR gene expression was increased at 12
weeks and thereafter in the IDA group (Figure 4C).
Gene Expression and Phosphorylation of Signal Pathway in IDA-Induced
Cardiac Remodeling: Iron deficient diet induced the increased expression of fetal-type
cardiac genes, including those for Nppa, in the heart of the IDA group after 4 weeks diet
and thereafter (Figure 5A). Cardiac Nppb gene expression was upregulated in the IDA
group compared to the control group at 12 and 20 weeks after diet (Figure 5B).
Myocardial expression of the collagen type 3 mRNA was decreased during maturational
growth in both groups, in agreement with previous studies (3,9) and upregulated in the
IDA group relative to the control group at 20 weeks after diet (Figure 5C).
In order to clarify the mechanisms of IDA-induced cardiac remodeling, we
studied the molecular signaling pathways in the heart of the IDA group.
Phosphorylation of STAT3 in the myocardial tissue increased in the IDA group relative
13
to the control group at 12 weeks but decreased at 20 weeks in the IDA group. (Figure
5D). In contrast, phosphorylations of Akt and phosphorylation of ERK did not differ
between the control group and the IDA group (Figure 5E, F).
Cardiac VEGF and HIF-1alpha Gene Expression and Angiogenesis in
IDA-Induced Cardiac Remodeling:
Cardiac VEGF gene expression did not differ
between the control group and the IDA group until 4 weeks; however, its expression
was upregulated in the IDA group relative to the control group at 12 and 20 weeks after
diet (Figure 6A). Cardiac HIF-1alpha gene expression decreased with aging in the
control group, which is in agreement with a previous study (29). HIF-1alpha gene
expression was comparable between the control group and the IDA group until 12
weeks, but was upregulated at 20 weeks in the hearts of the IDA group (Figure 6B).
Cardiac HIF-1alpha protein expression was upregulated at 20 weeks in the IDA group
(Figure 6C). The number of microvessels per cardiomyocytes increased at 12 and 20
weeks in the IDA group.
Renal Function and Serum TNF-alpha Levels in Sustained IDA: Since the
cardio-renal syndrome and TNF-alpha play an important role in the anemia observed in
patients with HF (19), we evaluated renal function and serum TNF-alpha levels in both
14
groups (Table1). Serum BUN levels and urinary sodium excretion were increased in
the IDA group compared with the control group, while urinary potassium excretion was
decreased in the IDA group at 12 and 20 weeks after diet. In addition, urinary volume
and serum TNF-alpha levels were increased only at 20 weeks in the IDA group
compared with the control group.
In summary, IDA initially induced in LV hypertrophy with preserved systolic
function, increased serum Epo concentrations, cardiac STAT3 phosphorylation and
VEGF gene expression. Over time, however, the effects of IDA resulted in cardiac
fibrosis and lung congestion with decreased serum Epo levels and cardiac STAT3
phosphorylation.
Effects of Epo on IDA-Induced Cardiac Remodeling: To further explore
whether the preservation of the increased levels of Epo is associated with adaptive
cardiac remodeling, we finally evaluated the cardiac effect of chronic Epo therapy on
the rats after they had been given iron deficient diet for 12 weeks. The administration
of Epo attenuated the downregulation of cardiac STAT3 phosphorylation (Figure 7A)
and prevented cardiac dysfunction in the IDA group (Figure 7B to G). Meanwhile,
blood hemoglobin and SBP were not altered in the IDA group receiving Epo (blood
15
hemoglobin: 3.5±0.3 vs. 4.4±0.3 g/dl, SBP: 94±6 vs. 91±8 mmHg, the IDA group
receiving Epo vs. the untreated IDA group, respectively). The preservation of the
increased levels of Epo prevents the transition from adaptive cardiac hypertrophy to
cardiac dysfunction in sustained IDA.
Discussion
Chronic anemia is common among patients with HF, relating to increased
morbidity and mortality (2, 12). Moreover, chronic severe anemia is known to causes
HF. However, the mechanisms, including the molecular signaling pathway, of HF in
chronic severe anemia are not fully resolved. We tried to reveal the mechanisms
behind the adaptive and maladaptive response of the heart to long term anemia. The
new finding of this study is that the increased serum Epo concentration and cardiac
STAT3 phosphorylation play a compensatory role for the beneficial cardiac remodeling
induced by chronic iron deficiency anemia.
In other words, a decrease in these
molecules may be critical for the transition from adaptive cardiac hypertrophy to cardiac
dysfunction in long-term anemia by iron deficiency.
16
Cardiac Remodeling Induced by Sustained IDA: IDA led to a reduction in
SBP at 12 weeks and induced cardiac hypertrophy, whereas IDA led to cardiac
dysfunction at 20 weeks.
The presence of cardiac dysfunction at 20 weeks was
characterized by the increase in LV weight to tibia length ratio and wet-to-dry lung
weight ratio, the increased Nppa, Nppb, collagen type 3 gene expressions, and the
increased myocardial interstitial fibrosis. As far as we know, this is the first report to
evaluate the time course of cardiac function in IDA-induced cardiac remodeling with
echocardiography. In particular, the serial changes in the pulsed Doppler mitral flow
velocity pattern were notable. At 4 weeks, cardiac hypertrophy or LV dilatation could
not be found; however, both were evident at 12 weeks.
What is significant here is that
the fractional shortening was maintained until 20 weeks.
The E/A ratio was low and
the deceleration time was prolonged at 12 weeks; however, the E wave and E/A ratio
were increased with the shortened deceleration time at 20 weeks. Together with our
physiological results, our model of IDA-induced cardiomyopathy may be able to show
diastolic HF at 20 weeks.
Possible Role of Serum Epo and Cardiac STAT3 Phosphorylation in
IDA-Induced Cardiac Remodeling: Anemia causes systemic vasodilatation, decreased
blood viscosity, and sodium and water retention, resulting in high-cardiac output status
17
(31).
IDA induces LV eccentric hypertrophy in developing rats (16, 21, 22, 27);
however, the molecular mechanism responsible for LV remodeling in this setting has
been unknown. Recent studies showed that Epo treatment improves quality-of-life
scores and LV ejection fraction in patients with HF (14), indicating Epo has protective
effects on cardiac diseases.
In addition, EpoR is expressed in a variety of cells not
directly involved in erythropoiesis, including myocardium (30). In the current study,
we found a dynamic variation in serum Epo levels and cardiac EpoR expression in the
process of LV hypertrophy in high-cardiac output status induced by IDA. A recent
study reported that Epo-EpoR system in the nonhematopoietic cells plays an important
protective role against ‘pressure-overload’ cardiac hypertrophy model (4).
Thus,
cardiac Epo-EpoR system seems to contribute as a protective role against not only
concentric hypertrophy induced by ‘pressure overload’ but also eccentric hypertrophy
induced by ‘volume overload’.
IDA induced cardiac hypertrophy and led to an
enhancement in cardiac Epo-EpoR system at 12 weeks. In contrast, serum Epo levels
even decreased and myocardial fibrosis markedly increased at 20 weeks.
Taking these
findings into consideration, Epo is required to maintain cardiac function in the model of
IDA-induced cardiac remodeling.
levels are currently unknown.
The mechanisms of downregulation of serum Epo
At 20 weeks after the change in diet, mild renal
18
insufficiency (slight but significant increase in urinary protein excretion) and increased
serum TNF-alpha concentration were observed.
Thus, these factors may reduce renal
Epo secretion in the IDA group (10).
Moreover, we found marked phosphorylation of STAT3 but not Akt and ERK
in IDA-induced hypertrophied heart.
In addition, the variations in the increased
phosphorylation of STAT3 in the heart correlated with those in the circulating Epo
levels.
Epo-EpoR signaling can stimulate the Jak/STAT, MAPK and PI3K/Akt
signaling pathways in hematopoietic and cardiac cells (23). Although there is no
direct evidence, enhanced phosphorylation of STAT3 via Epo may be involved in
IDA-induced hypertrophied heart and these signalings appeared to play a compensatory
role for IDA-induced cardiac remodeling. In agreement, a recent report showed an
altered phosphorylation of STAT3 but not Akt and ERK signaling in the condition of
pressure overload cardiac hypertrophy in the transgene-rescued Epo receptor-null
mutant mice (4). While serum Epo levels and phosphorylation of STAT3 peaked at 12
weeks and decreased at 20 weeks, cardiac EpoR expression maintained to increase until
20 weeks, suggesting a compensatory mechanism for the decreased Epo-EpoR signaling
in the heart of the IDA group. Epo and hypoxia have been shown to synergistically
induce EpoR expression in the cultured endothelial cells (5). Additionally, TNF-alpha
19
also has been shown to upregulate EpoR expression (7). In the present study, cardiac
HIF-1alpha gene expression and serum TNF-alpha levels further increased at 20 weeks
than 12 weeks in the IDA group. Thus, hypoxia-HIF-1alpha system and TNF-alpha
may have enhanced EpoR expression in the chronic stage for compensating the
declining serum Epo concentration.
Epo has an angiogenic effect (25). However, von Willebrand Factor-positive
cells were observed in the heart of the IDA group even after serum Epo levels decreased
at 20 weeks, suggesting that some signals other than Epo might have been involved in
the neovascularization of IDA heart in the chronic phase.
HIF-1alpha is known to
enhance VEGF expression and finally contribute to neovascularization (26). Thus,
HIF-1alpha may have promoted neovascularization at 20 weeks by regulating VEGF in
the heart of the IDA group.
Conclusions: The upregulation of serum Epo concentration and cardiac STAT3
phosphorylation is associated with beneficial adaptive mechanism of anemia-induced
cardiac hypertrophy. As a result, it can be concluded that the downregulation of these
molecules is critical for the transition from adaptive cardiac hypertrophy to cardiac
remodeling in long-term anemia. Therefore, further understanding of the mechanism
of cardiac maladaptation to anemia should lead to a new therapeutic strategy in chronic
20
heart failure with anemia.
Acknowledgements
We thank Noriko Kumon and Utako Kuze for their excellent technical supports, and
Haruyasu Ueda and Haruki Okamura for serum TNF-alpha measurement.
Grants
This study was supported in part by Grant-in-Aid for Young Scientists (B) from the
Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y. Naito).
21
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27
Figure Legends
Figure 1: Effect of Iron Deficient Diet on Physiological Parameters and Cardiac
Function.
Time course of (A) hemoglobin, (B) body weight, (C) systolic blood
pressure, (D) heart rate, (F) LV weight/tibia length ratio, (G) wet lung weight/tibia
length ratio, and (H) wet /dry lung weight ratio in the rats given control (white bar and
circle; n=6) or iron deficient (black bar and circle; n=6) diet.
(E) Representative gross
morphology of the hearts for the control group and the IDA group at 12 weeks and 20
weeks after diet. 1 week, 4 weeks, 12 weeks, and 20 weeks represent 1 week, 4 weeks,
12 weeks, and 20 weeks after diet, respectively. IDA indicates iron deficiency anemia;
LVW, left ventricle weight; TL, tibia length.
*p < 0.05 versus controls at the
corresponding time point, †p<0.05 versus 12 weeks after iron deficient diet.
Figure 2: Echocardiographic Analysis on IDA-Induced Cardiac Remodeling.
(A)
Representative M-mode echocardiogram (top panel) and pulsed Doppler mitral flow
velocity (bottom panel) for the control and IDA rats at 4 weeks, 12 weeks and 20 weeks.
IDA indicates iron deficiency anemia. Time course of (B) diastolic wall thickness of
LV posterior wall (LVPWth), (C) LV end-diastolic dimension (LVDd), (D) LV
28
end-systolic dimension (LVDs), (E) LV fractional shortening (FS), (F) Early diastolic
filling wave (E wave), (G) the ratio of peak early diastolic filling velocity and peak
filling velocity at atrial contraction (E/A ratio), and (H) deceleration time (Dct) in the
rats given control (white circle; n=6) or iron deficient (black circle; n=6) diet.
1 week,
4 weeks, 12 weeks, and 20 weeks represent 1 week, 4 weeks, 12 weeks, and 20 weeks
after diet, respectively. *p < 0.05 versus controls at the corresponding time point.
Figure
3:
Histological
Analysis
on
IDA-Induced
Cardiac
Remodeling.
Representative images of (A) hematoxylin and eosin, (B) Masson’s trichrome, and (C)
Picrosirius red staining of the heart sections. Scale bars: 50 µm.
Quantitative analysis
of (D) cardiac myocyte cross-sectional area and (E) myocardial interstitial fibrosis in the
rats given control (white bar; n=6) or iron deficient (black bar; n=6) diet.
indicates iron deficiency anemia.
IDA
4 weeks, 12 weeks, and 20 weeks represent 4 weeks,
12 weeks, and 20 weeks after diet, respectively. *p < 0.05 versus controls at the
corresponding time point.
Figure 4: Increased serum Epo and cardiac EpoR expression in IDA-Induced
Cardiac Remodeling. Time course of (A) serum iron concentration, (B) serum Epo
29
concentration, and (C) cardiac epor mRNA expression in the rats given control (white
bar; n=6) or iron deficient (black bar; n=6) diet.
Expression of epor gene was
normalized to the endogenous 18S ribosomal RNA gene expression, and the relative
levels of gene expression are plotted in the graph. IDA indicates iron deficiency
anemia. 1 week, 4 weeks, 12 weeks, and 20 weeks represent 1 week, 4 weeks, 12
weeks, and 20 weeks after diet, respectively.
*p < 0.05 versus controls at the
corresponding time point, †p < 0.05 versus 12 weeks after iron deficient diet, #p < 0.05
versus 4 weeks after iron deficient diet.
Figure 5: Increased Gene Expression and Phosphorylation of Signal Pathway in
IDA-Induced Cardiac Remodeling. Time course of (A) atrial natriuretic peptide
(nppa), (B) brain natriuretic peptide (nppb), (C) collagen type 3 gene expression, (D)
expression of phosphorylated (top) and native (bottom) state of STAT3, (E) Akt, and (F)
ERK in the hearts of the rats given control (white bar; n=6) or iron deficient (black bar;
n=6) diet.
Top: Representative Western blot analysis.
Bottom: Densitometric
analysis. Expression of nppa, nppb, and collagen type3 genes was normalized to the
endogenous 18S ribosomal RNA gene expression, and the relative levels of gene
expression are plotted in the graphs. Expression of phosphorylated STAT3, Akt, and
30
ERK was standardized on the basis of native STAT3, Akt, and ERK expression, and the
relative levels of expression are plotted in the graphs.
IDA indicates iron deficiency
anemia. 1 week, 4 weeks, 12 weeks, and 20 weeks represent 1 week, 4 weeks, 12
weeks, and 20 weeks after diet, respectively.
*p < 0.05 versus controls at the
corresponding time point, †p < 0.05 versus 12 weeks after iron deficient diet, #p < 0.05
versus 4 weeks after iron deficient diet, $ p < 0.05 versus 1 week after control diet, ¶p <
0.05 versus 4 weeks after control diet.
Figure 6: Angiogenesis in IDA-Induced Cardiac Remodeling. Time course of (A)
vegf and (B) hif-1alpha mRNA expression in the hearts of the rats given control (white
bar; n=6) or iron deficient (black bar; n=6) diet.
(C) Expression of HIF-1alpha (top)
and actin (bottom) in the hearts of the rats given control (white bar; n=6) or iron
deficient (black bar; n=6) diet. Top: Representative Western blot analysis. Bottom:
Densitometric analysis. Expression of vegf and hif-1alpha genes was normalized to
the endogenous 18S ribosomal RNA gene expression, and the relative levels of gene
expression are plotted in the graphs.
Expression of HIF-1alpha was standardized on
the basis of actin expression, and the relative levels of expression are plotted in the
graphs. (D) Representative images of von Willebrand Factor stained heart sections.
31
Scale bars: 50 µm.
(E) Quantitative analysis of microvessels/myocytes ratio in the
hearts of the rats given control (white bar; n=6) or iron deficient (black bar; n=6) diet.
IDA indicates iron deficiency anemia. 1 week, 4 weeks, 12 weeks, and 20 weeks
represent 1 week, 4 weeks, 12 weeks, and 20 weeks after diet, respectively. *p < 0.05
versus controls at the corresponding time point, #p < 0.05 versus 4 weeks after iron
deficient diet, $ p < 0.05 versus 1 week after control diet, ¶p < 0.05 versus 4 weeks after
control diet.
Figure 7: Effects of Epo administration on IDA-Induced Cardiac Remodeling.
Effects of Epo on (A) cardiac expression of phosphorylated (top) and native (bottom)
state of STAT3, (B) LV weight/tibia length ratio, (C) wet lung weight/tibia length ratio,
(D) LV end-diastolic dimension (LVDd), (E) LV fractional shortening (FS), (F) the ratio
of peak early diastolic filling velocity and peak filling velocity at atrial contraction (E/A
ratio), and (G) deceleration time (Dct) by echocardiography in the rats given control diet
(white bar; n=6), iron deficient diet (black bar; n=6), and iron deficient diet treated with
Epo (gray bar; n=6).
Top: Representative Western blot analysis.
Bottom:
Densitometric analysis. Expression of phosphorylated STAT3 was standardized on the
basis of native STAT3 expression and the relative levels of expression are plotted in the
graphs. IDA indicates iron deficiency anemia; LVW, left ventricle weight; TL, tibia
32
length. 12 weeks and 20 weeks represent 12 weeks and 20 weeks after diet.
* p < 0.05
versus 12 week after control diet, †p < 0.05 versus 20 weeks after iron deficient diet
with Epo treatment.
33
Table 1. Renal function and serum TNF-alpha levels
12w Control
12w IDA
20w Control
20w IDA
Serum BUN (mg/dl)
11.8±1.1
20.3±1.7*
12.7±1.0
17.5±1.2*
Serum creatinine (mg/dl)
0.23±0.01
0.22±0.01
0.24±0.04
0.25±0.01
Urinary volume ( ml/day)
12.2 ±1.5
15.4 ±1.1
12.0 ±1.3
18.1±1.4*
UproV (mg/day)
11.8±1.1
10.7±1.0
12.8±1.3
15.9±2.4＄
UNaV (mEq/day)
1.7±0.1
2.5±0.2*
1.8±0.1
3.3±0.3*
UKV (mEq/day)
3.4±0.2
1.5±0.1*
3.9±0.4
1.9±0.2*
Serum TNF-alpha (pg/ml)
3.0±0.6
4.5±0.5
3.0±0.6
5.6±1.0*
*
p<0.05 vs Control.
＄
p<0.05 vs 12w IDA. IDA indicates iron deficient anemia, 12w
and 20w represent 12 weeks and 20 weeks after diet, UproV; Urinary protein excretion,
UNaV; Urinary sodium excretion, UKV; Urinary potassium excretion.
Figure 1
*
*
*
400
200
0
0
1week 4weeks 12weeks 20weeks
1week 4weeks 12weeks 20weeks
E
F
Control IDA
Control IDA
12weeks
20weeks
*
120
800
†
*
80
40
*
*
20
0
1week 4weeks 12weeks 20weeks
*
600
400
200
0
0
1week 4weeks 12weeks 20weeks
1week 4weeks 12weeks 20weeks
G
40
5mm
160
Heart Rate (bpm
m)
*
*
600
D
60
H
*
40
20
0
1week 4weeks 12weeks 20weeks
Wet/Dry Lung Weight (g/g)
*
800
Wet Lung Weigght/TL (mg/mm)
10
Body Weight (gg)
20
C
Sysstolic Blood Pressure (mmHg)
B
LVW/TL (mg/mm)
Hemoglobin (g/ddL)
A
5
*
4
3
2
1
0
1week 4weeks 12weeks 20weeks
Figure 2
A
Control
IDA
Control
IDA
Control
IDA
5mm
12weeks
10
*
*
2
9
8
7
6
*
120
*
60
5
4
0
1week 4weeks 12weeks 20weeks
0
1week 4weeks 12weeks 20weeks
0
1week 4weeks 12weeks 20weeks
H
60
3
2
*
1
0
1week 4weeks 12weeks 20weeks
Dct ((msec)
E/A
A ratio
*
40
40
3
G
*
80
200msec
E
6
*
0
1week 4weeks 12weeks 20weeks
0
1week 4weeks 12weeks 20weeks
F
D
FS (%)
3
20weeks
LV
VDs (mm)
C
LV
VDd (mm)
B
E wave (cm/sec)
LVP
PWth (mm)
4weeks
*
*
30
0
1week 4weeks 12weeks 20weeks
Figure 3
4weeks
A
12weeks
20weeks
D
Control
Cross-sectional area (µm2)
600
IDA
4weeks
B
12weeks
20weeks
Control
*
*
400
200
0
4weeks
IDA
12weeks
E
*
4weeks
Control
IDA
12weeks
20weeks
Fibrosis aarea (%)
3
C
20weeks
2
1
0
4weeks
12weeks
20weeks
Figure 4
B
300
150
*
*
*
*
0
1week
4weeks
12weeks
20weeks
C
Relative epor eexpression
3
*
*
2
1
0
1week
4weeks
12weeks
20weeks
Serum
m Epo concentrationn (mU/mL)
Serum
m iron concentrationn (µg/dL)
A
#
*
1200
†
*
600
*
*
0
1week
4weeks
12weeks
20weeks
Figure 5
#
*
Relative nppa eexpression
25
20
*
15
10
*
5
C
#
†
*
3
#
*
2
1
0
0
1week 4weeks 12weeks 20weeks
D
4weeks
1week 4weeks 12weeks 20weeks
Relative collagen ttype3 expression
B
Relative nppb eexpression
A
2
1
$
¶
0
1week 4weeks 12weeks 20weeks
E
F
12weeks 20weeks
4weeks
12weeks 20weeks
4weeks
p-STAT3
p-Akt
p-ERK
STAT3
Akt
ERK
Control IDA Control IDA Control IDA
Control IDA Control IDA Control IDA
12weeks 20weeks
Control IDA Control IDA Control IDA
5
4
3
†
2
p-ERK
K/ERK
#
*
p-Akkt/Akt
p-STAT
T3/STAT3
*
1
1
1
0
0
4weeks
12weeks
20weeks
0
4weeks
12weeks
20weeks
4weeks
12weeks
20weeks
Figure 6
A
B
C
Hif-1alpha
p
2
*
1
0
2
Relattive HIF-1alpha exppression
#
*
Relattive hif-1alpha exprression
*
1
$
¶
0
1week
4weeks 12weeks 20weeks
Control
IDA
2
*
1
0
1week
4weeks 12weeks 20weeks
D
Control
IDA
E
4weeks
12weeks
20weeks
Control
IDA
Miicrovessels/Myocytes ratio
Relative vegf expresssion
R
actin
2
*
*
1
0
4weeks
12weeks
20weeks
Figure 7
A
12weeks
B
20weeks
C
p-STAT3
STAT3
*
*
p-ST
TAT3/STAT3
*
2
†
1
†
0
20
†
Control IDA
12weeks
20weeks
*
*
40
20
Control IDA
Control IDA
IDA
+ IDA
Epo
12weeks
20weeks
12weeks
20weeks
F
G
†
*
3
60
2
30
20
2
†
*
*
1
Dct (msec)
4
E/A ratio
6
*
†
40
FS (%)
8
†
*
0
50
*
60
IDA
+ IDA
Epo
E
†
LVDd (mm)
*
0
IDA
+ IDA
Epo
D
10
Wet Luung Weight/TL (mgg/mm)
40
Control IDA IDA IDA
+
Epo
p
LV
VW/TL (mg/mm)
3
†
*
*
†
*
30
10
0
0
0
Control IDA
IDA
+ IDA
Epo
12weeks
20weeks
Control IDA
IDA
+ IDA
Epo
12weeks
20weeks
0
Control IDA
IDA
+ IDA
Epo
Control IDA
IDA
+ IDA
Epo
12weeks
20weeks
12weeks
20weeks