Original Article
Role of Interferon Regulatory Factor 4 in the
Regulation of Pathological Cardiac Hypertrophy
Ding-Sheng Jiang,* Zhou-Yan Bian,* Yan Zhang,* Shu-Min Zhang, Yi Liu, Rui Zhang, Yingjie Chen,
Qinglin Yang, Xiao-Dong Zhang, Guo-Chang Fan,# Hongliang Li#
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Abstract—IRF4, a member of the interferon regulatory factor (IRF) family, was previously shown to be restricted in the
immune system and involved in the differentiation of immune cells. However, we interestingly observed that IRF4 was
also highly expressed in both human and animal hearts. Given that several transcription factors have been shown to
regulate the pathological cardiac hypertrophy, we then ask whether IRF4, as a new transcription factor, plays a critical
role in pressure overload–elicited cardiac remodeling. A transgenic mouse model with cardiac-specific overexpression of
IRF4 was generated and subjected to an aortic banding for 4 to 8 weeks. Our results demonstrated that overexpression of
IRF4 aggravated pressure overload–triggered cardiac hypertrophy, fibrosis, and dysfunction. Conversely, IRF4 knockout
mice showed an attenuated hypertrophic response to chronic pressure overload. Mechanistically, we discovered that
the expression and activation of cAMP response element–binding protein (CREB) were significantly increased in
IRF4-overexpressing hearts, while being greatly reduced in IRF4-KO hearts on aortic banding, compared with control
hearts, respectively. Similar results were observed in ex vivo cultured neonatal rat cardiomyocytes on the treatment with
angiotensin II. Inactivation of CREB by dominant-negative mutation (dnCREB) offset the IRF4-mediated hypertrophic
response in angiotensin II–treated myocytes. Furthermore, we identified that the promoter region of CREB contains 3
IRF4 binding sites. Altogether, these data indicate that IRF4 functions as a necessary modulator of hypertrophic response
by activating the transcription of CREB in hearts. Thus, our study suggests that IRF4 might be a novel target for the
treatment of pathological cardiac hypertrophy and failure. (Hypertension. 2013;61:00-00.) Online Data Supplement
•
Key Words: cardiac hypertrophy
■
CREB
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IRF4
C
ardiac hypertrophy, an increase in heart muscle mass, is
the remodeling of the myocardium in response to a variety of extrinsic and intrinsic stimuli.1–3 Although hypertrophic
response is initially compensatory for an increased workload,
long-standing hypertrophy eventually leads to congestive heart
failure, arrhythmia, and sudden death.1–5 Currently, numerous molecules and signaling pathways have been identified to
regulate the process of cardiac hypertrophy and heart failure.
In particular, the provocation of these complex sets of interwoven signaling pathways culminate in the nucleus to activate
a diversified panel of transcription factors, including myocyte
enhancer factor-2 (MEF2), nuclear factor of activated T cells
(NFAT), activator protein-1 (AP-1), and nuclear factor κB
(NF-κB), which induce the activation of the fetal cardiac gene
program leading to pathological hypertrophy.6,7 Nevertheless,
how to coordinate these transcriptional regulations in the
■
pressure overload
■
transcription factor
heart on increased biomechanical stress remains incompletely
understood.
The interferon regulatory factor (IRF) family is a group of
transcription factors, which comprises 9 members (IRF1–9)
in both humans and mice.8–10 IRFs are originally described
as downstream regulators of interferon signaling and play
important roles in the regulation of innate and adaptive
immune response.8–10 Recent studies further demonstrate that
IRFs are involved in multiple biological processes, such as
cell growth, apoptosis, and oncogenesis.10,11 IRF4 (also known
as PU.1 interaction partner [Pip], multiple myeloma oncogene
1 [MUM1], lymphocyte-specific interferon regulatory factor
[LSIRF], NFEM5, and Interferon Consensus Sequence
binding protein for Activated T cells [ICSAT]), as a member of
IRF family, was initially identified in B and T lymphocytes.12
Like all other IRF members, IRF4 contains a highly conserved
Received November 8, 2012; first decision March 18, 2013; revision accepted March 19, 2013.
From the Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, China (D.-S.J., Z.-Y.B., Y.Z., S.-M.Z., H.L.); Cardiovascular
Research Institute, Wuhan University, Wuhan, China (D.-S.J., Z.-Y.B., Y.Z., S.-M.Z., R.Z., H.L.); College of Life Sciences, Wuhan University, Wuhan,
China (Y.L., X.-D.Z., G.-C.F.); Cardiovascular Division, University of Minnesota, Minneapolis, MN (Y.C.); Department of Nutrition Sciences, University
of Alabama, Birmingham, AL (Q.Y.); and Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati,
OH (G.-C.F.).
*D.-S.J., Z.-Y.B., and Y.Z. are joint first authors. #G.-C.F. and H.L. contributed equally to this article.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.
111.00614/-/DC1.
Correspondence to Guo-Chang Fan, Department of Pharmacology and Cell Biophysics, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH
45267-0575. E-mail [email protected]; or Hongliang Li, Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research
Institute, Wuhan University, Jiefang Rd 238, Wuhan 430060, PR China. E-mail [email protected]
© 2013 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.111.00614
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N-terminal DNA-binding domain with a signature tryptophan
pentad and a carboxy-terminal portion of an IRF-association
domain, which regulates the transcriptional activity of IRFs by
mediating protein–protein interactions.12 Although it is well
recognized that IRF4 plays an essential role in the regulation
of lymphoid-, myeloid-, and dendritic cell differentiations,
recent evidence has pointed out that IRF4 to be a tumor
suppressor.11,13,14 Moreover, Eguchi et al8 reported that IRF4
is a critical determinant of the transcriptional response
to nutrient availability in adipocytes. However, whether
IRF4, as a new transcription factor, plays a role in the heart,
especially response to chronic pressure overload, has never
been examined thus far. Hence, it will be very interesting to
determine the consequence of overexpression and ablation of
IRF4 in the myocardium after chronic aortic banding (AB).
In the present study, we first discovered that IRF4 was
downregulated in heart samples collected from both human
patients with dilated cardiomyopathy and animals after AB.
Next, we used IRF4 transgenic (TG) and knockout (KO)
mouse models, which underwent chronic pressure overload
via the AB approach. Our results revealed that either overexpression or deletion of IRF4 did not substantially affect
cardiac phenotype under basal conditions. Remarkably, however, elevation of IRF4 did promote, and absence of IRF4 did
prevent, the development of pressure overload–induced cardiac hypertrophy and heart failure. Mechanistically, we identified that cAMP response element–binding protein (CREB)
is an important target of IRF4 in the regulation of cardiac
hypertrophy.
Methods and Materials
The animal protocol was approved by the Animal Care and Use
Committee of Renmin Hospital of Wuhan University. An expanded
Methods section is available in the online-only Data Supplement,
which includes detailed methods on the following: reagents, human
heart samples, experimental animal models and AB surgery, echocardiography and hemodynamics evaluation, histological analysis,
Western blotting and quantitative real-time polymerase chain reaction, CREB-luciferase reporter assays, cardiomyocyte culture and
infection with recombinant adenoviral vectors, immunofluorescence
staining, and statistical analysis.
Results
Expression of IRF4 Is Downregulated in Human
Dilated Cardiomyopathic Hearts and Murine
Hypertrophic Hearts
IRF4 is well known to be expressed in immune cells and adipocytes.8–10 Whether it is detectable in hearts, however, has
never been examined thus far. Hence, our initial experiments
were performed to determine the tissue distribution of IRF4
in mice, using Western blotting. We observed that IRF4 was
highly expressed in brain, heart, liver, lung, kidney, and skeletal muscles, and less expressed in spleen (Figure 1A), which
was confirmed by real-time polymerase chain reaction (Figure
S1A in the online-only Data Supplement). Next, we examined
whether expression levels of IRF4 were altered in diseased
hearts. Human dilated cardiomyopathic samples were collected and revealed that protein levels of IRF4 were reduced
by ≈67%, whereas β–myosin heavy chain (MHC) and atrial
natriuretic factor (2 markers of the hypertrophic heart) were
Figure 1. Interferon regulatory factor 4
(IRF4) is expressed and downregulated
in the heart on hypertrophic stimuli.
A, Western blot analysis of the IRF4
expression in brain, heart, liver, spleen,
lung, kidney, and skeletal muscles
collected from wild-type mice (n=4).
B, Western blot analysis of β–myosin
heavy chain (MHC), atrial natriuretic
peptide (ANP), and IRF4 in normal donor
hearts and dilated cardiomyopathy (DCM)
hearts collected from human patients (n=4,
*P<0.01 vs donor hearts). C, Western blot
analysis of β-MHC, ANP, and IRF4 in an
experimental mouse model with aortic
banding (AB)-induced cardiac hypertrophy
(n=4, *P<0.01 vs shams). D, Western blot
analysis of β-MHC, ANP, and IRF4 in
cardiomyocytes stimulated by angiotensin
(Ang) II (1 μmol/L) and phenylephrine
(PE; 100 μmol/L); n=4 independent
experiments, *P<0.01 vs PBS. Top,
Representative blots. Bottom, Bar graphs
are quantitative results.
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markedly increased, compared with healthy donor hearts
(Figure 1B). Similarly, in an experimental mouse model with
AB-induced cardiac hypertrophy (evidenced by elevation of
β-MHC and atrial natriuretic factor levels, Figure 1C), we
observed that the expression of IRF4 was downregulated by
≈36% in mouse hearts at week 4 after AB, and by ≈65% at
week 8 after AB, compared with sham-operated hearts (Figure
1C; P<0.01 versus shams). The real-time polymerase chain
reaction and immunofluorescence staining results also demonstrated that IRF4 was expressed in the nuclei of cardiomyocytes
under normal conditions and downregulated in hypertrophic
hearts (Figure S1B and S1C). Furthermore, using ex vivo cultured neonatal cardiomyocytes treated with either Ang II (1
μmol/L) or phenylephrine (100 μmol/L) for 48 hours to induce
hypertrophy, we found that the levels of IRF4 were reduced
by ≈56% in these hypertrophic cardiomyocytes (Figure 1D;
P<0.01 versus PBS). Together, these data indicate that IRF4 is
actually expressed in hearts, and its protein levels are significantly decreased in human dilated cardiomyopathy hearts and
pressure overload–induced hypertrophic mouse hearts, as well
as ex vivo Ang II/phenylephrine-treated cardiomyocytes.
Absence of IRF4 in the Hearts Attenuates Pressure
Overload–Induced Hypertrophy
After having determined the alterations of IRF4 levels in
diseased hearts, 1 question would be asked: what is the role
of reduced IRF4 expression in the development of cardiac
hypertrophy and failure? To address this concern, we used
an IRF4-KO mouse model (Jackson Laboratory, Figure 2A)
and subjected them to AB or sham operation for 8 weeks, and
IRF4 KO did not affect blood pressure before or 1 week after
AB (Figure S2). It is important to note here that under basal
conditions, the IRF4-KO mice were viable, fertile, and had
no pathological alterations in cardiac structure and function
at 10 weeks of age (Table S1). However, after 8-week AB,
IRF4-null hearts remarkably exhibited an attenuation of
hypertrophy, as evidenced by the following: (1) shrunk gross
heart (Figure 2B); (2) decreased cardiomyocyte cross-sectional
area (CSA, wheat germ agglutinin [WGA] staining; Figure 2B
and 2C); and (3) reduced ratios of heart weight (HW)/body
weight (BW), lung weight (LW)/BW, and HW/tibia length
(TL) (Figure 2D), compared with AB-treated wild-type hearts
(IRF4+/+). Similar decreases were also observed in the left
ventricular (LV) chamber dimension or wall thickness, as
assessed by echocardiography (left ventricular end-diastolic
dimension: wild-type [WT]/5.30±0.15 versus KO/4.58±0.05;
left ventricular end-systolic dimension: WT/4.15±0.18 versus
KO/3.21±0.07; P<0.01; Figure 2E and Table S2). Accordingly,
LV contraction was significantly improved in IRF4-KO hearts
(fractional shortening [FS]: 30%), compared with WTs (FS:
22%; P<0.01; Figure 2E) on chronic pressure overload.
Moreover, the mRNA expression levels of several hypertrophic
markers, including atrial natriuretic peptide, brain natriuretic
peptide, and β-MHC, were much lower in the IRF4-KO mice
than those of WT mice after 8-week AB (Figure 2F).
To further define the effect of IRF4 deficiency on maladaptive cardiac remodeling, we evaluated fibrosis, a classical feature of pathological cardiac hypertrophy. The extent of fibrosis
was quantified by collagen volume through the visualization
IRF4 Modulates Cardiac Hypertrophy
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of the total amount of collagen present in the interstitial and
perivascular spaces. Our results showed that both interstitial
and perivascular fibrosis were dramatically increased in WT
hearts subjected to chronic AB but markedly limited in KO
hearts (Figure 2G and 2H). Finally, we measured the synthesis
of collagen by analyzing the mRNA levels of fibrotic markers, such as collagen I, collagen III, and connective tissue
growth factor. Our results consistently revealed a decreased
fibrotic response in KO mice (Figure 2I). Collectively, all
these data indicate that deletion of IRF4 blocks pathological
cardiac hypotrophy induced by chronic AB. This suggests that
reduced IRF4 expression observed in human dilated cardiomyopathy hearts may play compensatory role in defending
various stress conditions.
Overexpression of IRF4 Aggravates Pressure
Overload–Induced Cardiac Hypertrophy
Next, we were curious about whether increased IRF4 expression in hearts has any effects on chronic pressure overload. To
this end, we generated a TG mouse model with cardiac-specific overexpression of IRF4. Given that function domains are
highly conserved between human and mouse IRF4 (human
IRF4 protein shares 92% homology with mouse IRF4 protein),8–10 we cloned a human IRF4 cDNA into the downstream
of a cardiac-specific promoter, α-MHC promoter (Figure 3A),
to distinguish mouse endogenous IRF4 from transgene. Four
germ lines of IRF4-TG mice were generated and verified by
Western blotting (Figure 3B). Notably, the expression of mouse
endogenous IRF4 was not affected by the overexpression of
human IRF4 gene (Figure 3B), and transgene IRF4 was specially expressed in the heart (Figure 3C). All IRF4-TG mice
were healthy and showed no apparent cardiac morphological
or pathological abnormalities (Table S1). We then selected the
TG line 8 (Tg8) with the highest level of IRF4 for following experiments. To define the consequence of increased IRF4
expression in hearts on chronic pressure overload, IRF4-TG
mice and their non-transgene (NTG) littermates were subjected to AB for 4 weeks. In contrast to the results observed
in IRF4-KO mice, overexpression of IRF4 aggravated pathological cardiac hypertrophy induced by chronic AB compared
with NTG mice, as determined by examinations of hematoxylin and eosin and WGA–fluorescein isothiocyanate staining
(Figure 3D). The cardiomyocyte CSA and ratios of HW/BW,
HW/TL, and LW/BW were also significantly increased in TG
hearts, compared with NTG hearts on 4-week AB (Figure 3E
and 3F). Accordingly, the cardiac structure and function, as
measured by left ventricular end-diastolic dimension, left ventricular end-systolic dimension, and LVFS, were significantly
aggravated (Figure S3A and Table S3), and the markers of cardiac hypertrophy (atrial natriuretic peptide, brain natriuretic
peptide, and β-MHC) were also dramatically upregulated in
TG mice compared with NTG mice (Figure 3G). We finally
examined the effect of IRF4 overexpression on cardiac fibrosis and found that the collagen present in both interstitial and
perivascular spaces (picrosirius red [PSR] staining), the extent
of cardiac fibrosis quantified by LV collagen volume, and
the expression of fibrosis markers (collagen I, collagen III,
and connective tissue growth factor) consistently increased
in TG mice, compared with NTG mice on chronic AB
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Figure 2. Absence of interferon regulatory factor 4 (IRF4) attenuates pressure overload–induced cardiac hypertrophy. A, Deletion of
IRF4 was confirmed by Western blotting (n=4). IRF4+/+ stands for wild-type (WT) hearts, and IRF4−/− represents knockout (KO) hearts.
B, Histological analyses of the hematoxylin and eosin (HE) staining and the wheat germ agglutinin (WGA) staining of WT and IRF4KO hearts 8 weeks after aortic banding (AB) surgery (n=5–6). C, Statistical results for the cell-sectional area (CSA, n=100+ cells per
group). D, Statistical results for the ratios of heart weight (HW)/body weight (BW), lung weight (LW)/BW, and HW/tibia length (TL) in
the indicated groups (n=12 for WTs and n=14 for KOs). E, Parameters of echocardiographic results for WT and KO mice (n=6–10 per
experimental group). FS indicates fractional shortening; LVEDD, left ventricular end-diastolic dimension; and LVESD, left ventricular
end-systolic dimension. F, Real-time polymerase chain reaction (PCR) analyses of hypertrophic markers (atrial natriuretic peptide [ANP],
brain natriuretic peptide [BNP], and β–myosin heavy chain [MHC]) induced by AB in the indicated mice (n=4 per experimental group).
G, Picrosirius red (PSR) staining on histological sections of the left ventricle (LV) in the indicated groups 8 weeks after AB (n=5–6 per
experimental group, scale bar =50 μm). H, Quantification of the total collagen volume in WT and IRF4-KO mice after AB (n=25+ fields
per experimental group). I, Real-time PCR analyses of the fibrotic markers (collagen I, collagen III, and connective tissue growth factor
[CTGF]) in the indicated groups (n=4). *P<0.05 vs WT/sham; #P<0.05 vs WT/AB.
(Figure S3B–S3D). Together, these gain-of-function data indicate that overexpression of IRF4 promotes cardiac hypertrophy and fibrosis in response to chronic pressure overload.
IRF4 Regulates Angiotensin II–Induced
Cardiomyocyte Hypertrophy Ex Vivo
Considering that chronic overexpression or deletion of IRF4
in vivo may have compensatory effects, which influence the
consequence of IRF4 in pressure overload–induced cardiac
hypertrophy, we then performed ex vivo studies using cultured
neonatal rat cardiomyocytes, a well-controlled experimental
setting. Primary cultured cardiomyocytes were infected with
either AdshIRF4 to knockdown or AdIRF4 to overexpress IRF4
(Figure 4A). Subsequently, these infected cardiomyocytes
were exposed to either angiotensin II (Ang II, 1 μmol/L)
or PBS control for 48 hours, followed by immunostaining
with α-actinin to measure the size of cells. Notably, either
knockdown (AdshIRF4) or overexpression of IRF4 (AdIRF4)
did not alter the size of cultured neonatal cardiomyocytes
under basal PBS treatments, compared with AdshRNA and
AdGFP cells (Figure 4C and 4D). However, in response to Ang
II–induced cell hypertrophy, knockdown of IRF4 significantly
reduced cell size (CSA) by 21.6%, compared with shRNAinfected controls (Figure 4B and 4C). Conversely, Ang II–
induced cell hypertrophy was greatly enhanced by 64.2% in
IRF4-overexpressing neonatal cardiomyocytes, compared
with AdGFP cells (Figure 4B and 4D). Accordingly, the
ratio of protein/DNA and expression of hypertrophy markers
(atrial natriuretic peptide and β-MHC) were dramatically
suppressed in AdshIRF4-infected cardiomyocytes (Figure
4E and Figure S4A), whereas remarkably aggravated in
AdIRF4-infected cardiomyocytes (Figure 4F and Figure
S4B), compared with controls, respectively. These ex vivo
data, consistent with in vivo results (Figures 2 and 3, above),
suggest that downregulation of IRF4 mitigates pathological
cardiac hypertrophy, whereas upregulation of IRF4 promotes
pathological cardiac hypertrophy.
IRF4 Regulates the Expression of CREB in Mouse
Hypertrophic Hearts
Accumulating evidence has indicated that numerous
transcription factors (ie, serum response factor [SRF],
CREB, MEF2C, myocardin, GATA binding protein 4
[GATA4], and NFATc1) are involved in the development
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IRF4 Modulates Cardiac Hypertrophy
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Figure 3. Overexpression of interferon regulatory
factor 4 (IRF4) aggravates pressure overload–
induced cardiac hypertrophy. A, Schematic
diagram of the construction of transgenic (TG)
mice with full-length human IRF4 cDNA under the
control of α–myosin heavy chain (MHC) promoter.
B, Representative blots for determination of TG
IRF4 and endogenous IRF4 levels expressed
in the heart tissue from 4 lines of TG and their
control WT mice (n=5). C, Representative Western
blots for evaluation of transgene expression in
different tissues from TG mice as indicated (n=5).
D, Histological analyses of the hematoxylin and
eosin (HE) staining and the wheat germ agglutinin
(WGA) staining of NTG and IRF4-TG mice 4
weeks after the aortic banding (AB) surgery (n=5).
E, Statistical results for the cell-sectional area
(n=100+ cells per group). F, The ratios of heart
weight (HW)/body weight (BW), lung weight (LW)/
BW, and HW/tibia length (TL) in the indicated
groups (n=11–13). G, Real-time polymerase chain
reaction analyses of the hypertrophic markers atrial
natriuretic peptide (ANP), brain natriuretic peptide
(BNP), and β–myosin heavy chain (MHC) induced
by AB in NTG and IRF4-TG mice (n=4). *P<0.05 vs
WT/sham; #P<0.05 vs WT/AB.
of pathological cardiac hypertrophy.4–7 Therefore, it would
be very intriguing to examine whether the expressions/
activities of such transcription factors are interfered by
up- and downregulation of IRF4 in hearts on chronic
pressure overload. Using Western blotting, we determined
the expression levels of SRF, CREB, MEF2C, myocardin,
GATA4, and NFATc1 in AB-induced hypertrophic hearts
collected from both IRF4-overexpressing and -absent
mice. Interestingly, we observed that, with the exception
of CREB (Figure 5A–5D), there were no changes in other
transcription factors (data not shown). Specifically, the
total and phosphorylated protein levels of CREB were
decreased by 45% and 40%, respectively, in AB-induced
hypertrophic hearts of IRF4-null mice, compared with
those of WT controls (IRF4+/+; Figure 5A and 5B). By
contrast, the levels of total and phosphorylated CREB were
more pronounced in AB-treated hearts of IRF4-TG mice
than those hearts of NTG mice (Figure 5C and 5D). To
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Figure 4. Interferon regulatory factor 4 (IRF4) regulates angiotensin II-induced cardiomyocyte hypertrophy ex vivo. A, IRF4 was knocked
down in AdshIRF4-infected cardiomyocytes, whereas IRF4 was increased by 1.7-fold in AdIRF4-infected myocytes (n=3 independent
experiments). Left, Representative blots. Right, Quantitative results. B, Representative images of cardiomyocytes infected with AdshIRF4
or AdIRF4 in response to angiotensin (Ang) II (1 μmol/L). C, Quantitative results of the cell-sectional area showing that knockdown of IRF4
significantly attenuated Ang II–triggered hypertrophy, compared with shRNA-cells (n=50+ cells per group, *P<0.05 vs AdshRNA/PBS;
#P<0.05 vs AdshRNA/Ang II). D, Quantitative results of the cell-sectional area showing that overexpression of IRF4 greatly increased Ang
II–triggered hypertrophy, compared with control green fluorescent protein (GFP) cells (n=50+ cells per group, *P<0.05 vs AdGFP/PBS;
#P<0.05 vs AdGFP/Ang II). E, Real-time polymerase chain reaction (PCR) analysis of hypertrophy markers (atrial natriuretic peptide [ANP]
and β–myosin heavy chain [MHC]) in AdshIRF4 cells and control AdshRNA cells after the treatment with PBS and Ang II for 48 hours (n=4
independent experiments, *P<0.05 vs AdshRNA/PBS; #P<0.05 vs AdshRNA/Ang II). F, Real-time PCR assays for evaluation of mRNA
levels of ANP and β-MHC in IRF4-overexpressing cells and control GFP cells on the treatment with either PBS or Ang II for 48 hours (n=4
independent experiments, *P<0.05 vs AdGFP/PBS; #P<0.05 vs AdGFP/Ang II).
further confirm these findings, we decreased or increased
IRF4 expression ex vivo by infecting cultured neonatal rat
cardiomyocytes with either AdshIRF4 or AdIRF4, followed
by treatment with 1 μmol/L Ang II for 48 hours. Similar to
in vivo observations, the levels of total and phosphorylated
CREB were reduced in AdshIRF4 cells by 39% and 31%,
respectively, compared with control AdshRNA cells (Figure
5E and 5F). Conversely, overexpression of IRF4 enhanced
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Figure 5. Interferon regulatory factor 4 (IRF4) regulates the expression of cAMP response element–binding protein (CREB) in mouse
hypertrophic hearts. A and B, Representative Western blots and quantitative results of the CREB phosphorylation and total levels in the
IRF4 knockout (KO) mice 8 weeks after aortic banding (AB; n=6 hearts, *P<0.05 vs wild-type [WT]/sham; #P<0.05 vs WT/AB). C and D,
Representative Western blots and quantitative results of the CREB phosphorylation and total levels in the IRF4 transgenic (TG) mice
4 weeks after AB (n=6 hearts, *P<0.05 vs NTG/sham; #P<0.05 vs NTG/AB). E and F, Representative Western blots and quantitative
results of the CREB phosphorylation and total levels in AdshIRF4 or AdshRNA adenoviral vector-infected cardiomyocytes on addition
of angiotensin (Ang) II (1 μmol/L) for 48 hours (n=3 independent experiments; *P<0.05 vs AdshRNA/PBS; #P<0.05 vs AdshRNA/Ang
II). G and H, Representative Western blots and quantitative results of the CREB phosphorylation and total levels in AdIRF4 or AdGFP
adenoviral vector-infected cardiomyocytes on addition of Ang II (1 μmol/L) for 48 hours (n=3 independent experiments; *P<0.05 vs
AdGFP/PBS; #P<0.05 vs AdGFP/Ang II).
not only the phosphorylation but also total levels of CREB,
comparable with control cells (Figure 5G and 5H). Together,
both in vivo and ex vivo results consistently indicate
that IRF4-mediated pathological cardiac hypertrophy is
associated with CREB.
Regulation of Cardiac Hypertrophy by IRF4 Is
Largely Dependent on the Activation of CREB
To determine whether IRF4-regulated cardiac hypertrophy is
directly or indirectly associated with CREB, we coinfected
neonatal rat cardiomyocytes with AdshIRF4 plus AdCREB,
or AdIRF4 plus AddnCREB (dominant negative mutation of
CREB to inhibit CREB activity), respectively, followed by
the addition of Ang II for 48 hours. Our results of cell size
analysis show that, among shRNA-control groups (Figure
6A, blue bars), Ang II–induced myocyte hypertrophy was
more pronounced in AdCREB cells than control cells.
These results are consistent with previous reports showing
that activation of CREB promotes cardiac hypertrophy.15,16
Interestingly, we observed that suppression of Ang
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II–induced cell hypertrophy by knockdown of IRF4 (Figure
6A, red bars) was released by increased CREB expression
(AdCREB cells). In the set of gain-of-function experiments,
we observed that Ang II–induced cell hypertrophy was
increased by overexpression of IRF4 (Figure 6B, red bars).
However, accelerative effects of IRF4 on Ang II–induced
cell hypertrophy were dramatically dampened down by
overexpression of dominant negative CREB (Figure 6B).
Collectively, these data suggest that detrimental role of IRF4
in pathological cardiac hypertrophy is dependent, at least
partly, on the activation of CREB.
Discussion
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An increasing number of studies have demonstrated that transcription factors are profoundly involved in the development
of cardiac hypertrophy.4,17,18 Accordingly, understanding of
transcriptional regulation mechanisms will provide a new
strategy for the prevention and treatment of cardiac hypertrophy and heart failure. In the present study, we identified
that IRF4, as a transcription factor known in immune cells,
was also highly expressed in human and mouse cardiomyocytes. Intriguingly, the expression of IRF4 was downregulated in human dilated cardiomyopathy hearts and mouse
hypertrophic hearts, suggesting that IRF4 may be involved
in the process of cardiac hypertrophy and failure. Indeed,
using loss-of-function and gain-of-function approaches,
we discovered that IRF4 was a necessary modulator in the
development of pathological cardiac hypertrophy. Absence
of IRF4 in the heart attenuated AB-induced hypertrophic
response, whereas TG mice with cardiac-specific overexpression of IRF4 showed aggravated cardiac hypertrophy,
fibrosis, and dysfunction in response to pressure overload.
Therefore, our study presented here for the first time suggests that blockade of IRF4 transcriptional function might
be a novel remedy for the treatment of pathological cardiac
hypertrophy.
The possible mechanisms by which IRF4 regulates cardiac hypertrophy may be associated with its downstream
targets. IRF4 contains a highly conserved N-terminal DNAbinding domain and the carboxy-terminal portion of an
IRF-association domain, which modulate the transcriptional
activity of IRFs by mediating protein–protein interactions.13
In immune cell system, it has been reported that IRF4 functions as a positive regulator of many downstream transcription factors (ie, NFAT, signal transducer and activator of
transcription 3 [STAT3/6], and forkhead box P3 [FOXP3])
by interacting with its C-terminal transactivation domain.19
However, IRF4-controlled transcription factors involving
in cardiac hypertrophy have never been investigated thus
far. In the present study, we first examined the alterations
of hypertrophy-related transcription factors, including SRF,
CREB, MEF2C, myocardin, GATA4, and NFATc116,20–24 in
IRF4-overexpressing and –KO hearts on chronic pressure
overload. Interestingly, CREB was the only one to be regulated in hypertrophic hearts by IRF4. In fact, using a bioinformatics approach, we identified that there are 3 putative
IRF4 binding sites located in the promoter region of CREB
(Figure S5). The luciferase-reporter assays further showed
that IRF4 increased the transcriptional activity of CREB by
binding with the CREB promoter via the N-terminal DNAbinding domain (Figure S6). Thus, IRF4-mediated regulation
of cardiac hypotrophy may be directly associated with its
downstream target, CREB. Notably, under basal condition,
either overexpression or ablation of IRF4 in mouse hearts did
not alter the total levels of CREB; however, on pressure overload, both total and phosphorylated CREB were increased
in IRF4-TG mice, while being decreased in IRF4-KO mice.
These data suggest that IRF4-mediated regulation of CREB
might be stress-dependent. Indeed, this is supported by our
data showing that blockade of CREB activation offset the
IRF4-elicited hypertrophic response (Figure 6). Currently, it
is well documented that increased CREB expression results
in the activation of fetal gene program (ie, atrial natriuretic
peptide, brain natriuretic peptide, and β-MHC), leading to
cardiac hypertrophy.15 In addition, several previous studies
also suggested that the activation of CREB was essential for
cardiomyocyte hypertrophy25 and Ang II–induced smooth
muscle hypertrophy.26,27 Therefore, we believe that the detrimental role of IRF4 in pathological cardiac hypertrophy is
dependent, at least in part, on the activation of CREB.
Figure 6. Regulation of interferon regulatory factor 4 (IRF4)-mediated cardiac hypertrophy is largely dependent on the activation of
cAMP response element–binding protein (CREB). A, Neonatal rat cardiomyocytes were infected with AdshIRF4 or AdCREB, followed
by the treatment with 1 μmol/L of angiotensin (Ang) II for 48 hours. Cell-sectional areas (CSA) were then measured (see Methods) and
revealed that inhibition of myocyte hypertrophy by knockdown of IRF4 was released by overexpression of CREB. B, IRF4-mediated
promotion of myocyte hypertrophy (middle red bar) was attenuated by inactivation of CREB (AddnCREB, far right red bar). n=50+ cells
per experimental group.
Jiang et al
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
It is important to note here, numerous studies have
shown that IRF4 is closely related to inflammation.28,29 For
example, Mudter et al28 implicated IRF4 as a key regulator of mucosal interleukin-6 production in T cell–dependent
experimental colitis. Lassen et al29 demonstrated that loss
of IRF4 aggravated postischemic renal failure, which is
mediated by tumor necrosis factor-α. Therefore, we further
examined the status of inflammation/inflammatory cell infiltration in AB-treated IRF4-KO and WT hearts. Our results
showed that mRNA levels of interleukin-1β, interleukin-6,
tumor necrosis factor-α, and CD68 were significantly
increased in hearts from both groups (WT and IRF4-KO) on
1-week AB and decreased to basal levels after 4-week AB,
but there were no significant differences between WT and
IRF4-KO hearts on AB surgery (Figure S7). These data suggest that IRF4-KO may not alter the status of inflammation
in AB-treated murine hearts.
In conclusion, the present study demonstrates that upregulation of IRF4 aggravates pressure overload–induced cardiac
hypertrophy, fibrosis, and failure by targeting the CREB transcription factor. Conversely, knockdown/out of IRF4 mitigates the development of pathological cardiac hypertrophy.
These findings suggest that IRF4 might be a novel therapeutic
target for the prevention of cardiac hypertrophy and failure.
Perspectives
The current study provides in vivo and in vitro evidence that
IRF4, a member of IRF transcription factor family, functions
as a positive regulator of the hypertrophic response in the heart
through physically interacting with the CREB promoter, and
thereby activating the myocardial fetal gene program. These
observations suggest that blockade of IRF4 expression/activity
would be beneficial to the heart in prevention/attenuation of
pathological hypertrophy and failure.
Sources of Funding
This work was supported by grants from the National Natural
Science Foundation of China (No. 81100230, No. 81070089, and No.
81200071), the National Science and Technology Support Project
(No. 2011BAI15B02 and No. 2012BAI39B05), and the National
Basic Research Program of China (No. 2011CB503902).
Disclosures
None.
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Hypertension
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Novelty and Significance
What Is New?
•
•
Interferon regulatory factor 4 (IRF4) is identified to be expressed in human and mouse heart tissues, and IRF4 is downregulated in the heart on
hypertrophic stimuli.
IRF4 is a necessary modulator responsible for the development of pressure overload–induced cardiac hypertrophy, fibrosis, and cardiac dysfunction in vivo and Ang II–induced cardiomyocyte hypertrophy in vitro
through regulating the transcription and phosphorylation of cAMP response element–binding protein.
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
What Is Relevant?
•
•
Several transcription factors have been elucidated to contribute to cardiac hypertrophy.
Effects of IRF4, as an immune transcription factor, on the development
of cardiac hypertrophy and its underlying mechanisms are not known.
•
This study advances our understanding the role of transcription factors
in the process of cardiac hypertrophy and provides a basis for searching novel therapeutic targets to prevent cardiac hypertrophy and heart
failure.
Summary
Our findings demonstrate that absence of IRF4 protects hearts
against chronic pressure overload–induced cardiac hypertrophy,
fibrosis, and cardiac dysfunction, whereas overexpression of myocardial IRF4 aggravates pathological hypertrophic response by
regulating the transcription and phosphorylation of cAMP response
element–binding protein. These observations suggest that IRF4
might be a novel target for the treatment of cardiac hypertrophy
and heart failure.
Role of Interferon Regulatory Factor 4 in the Regulation of Pathological Cardiac
Hypertrophy
Ding-Sheng Jiang, Zhou-Yan Bian, Yan Zhang, Shu-Min Zhang, Yi Liu, Rui Zhang, Yingjie
Chen, Qinglin Yang, Xiao-Dong Zhang, Guo-Chang Fan and Hongliang Li
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
Hypertension. published online April 15, 2013;
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2013 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
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ONLINE SUPPLEMENT
Role of Interferon Regulatory Factor 4
In the Regulation of Pathological Cardiac Hypertrophy
Ding-Sheng Jiang1, 2 #, Zhou-Yan Bian1, 2 #, Yan Zhang1, 2#, Shu-Min Zhang1, 2,
Yi Liu3, Rui Zhang 2, Yingjie Chen4, Qinglin Yang5, Xiao-Dong Zhang3,
Guo-Chang Fan3, 6*, Hongliang Li1, 2 *
1
Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060,
China;
2
Cardiovascular Research Institute of Wuhan University, Wuhan 430060, China;
3
College of Life Sciences, Wuhan University, Wuhan 430072, China;
4
Cardiovascular Division, University of Minnesota, Minneapolis, MN 55455, USA;
5
Department of Nutrition Sciences, University of Alabama at Birmingham,
Birmingham, AL 35294-3360, USA;
6
Department of Pharmacology and Cell Biophysics; University of Cincinnati College
of Medicine, Cincinnati, OH 45267
Running Title: IRF4 modulates cardiac hypertrophy
# Ding-Sheng Jiang, Zhou-Yan Bian and Yan Zhang are co-first authors
* Correspondence to
Or Correspondence to
Guo-Chang Fan, PhD
Department of Pharmacology and Cell
Biophysics
University of Cincinnati
231 Albert Sabin Way
Cincinnati, OH 45267-0575
Phone: (513) 558-2340
Fax: (513) 558- 2269
Email: [email protected]
Hongliang Li, MD, PhD
Department of Cardiology,
Renmin Hospital of Wuhan University
Cardiovascular Research Institute,
Wuhan University;
Address: Jiefang Road 238,
Wuhan 430060, PR China.
Tel/Fax: 86-27-88076990;
E-mail: [email protected]
1
Methods and Materials
Reagents
Antibodies against the following proteins were purchased from Cell Signaling
Technology: murine IRF4 (#4948), phospho-CREB (#9198) and total-CREB (#9198).
The antibody against human IRF4 (#sc56713) was purchased from Santa Cruz Inc.
The GAPDH (MB001) antibody was purchased from Bioworld Technology. The BCA
protein assay kit was purchased from Pierce. We used IRDye® 800CW-conjugated
secondary antibodies (LI-COR Biosciences) for visualization. Platinum Taq DNA
polymerase High Fidelity (#11304-029) was purchased from Invitrogen. The pGL3
basic vector (E1751) and the Dual-Luciferase® Reporter (#E1960) Luciferase kit were
purchased from Promega. The FUGENE® HD Transfection reagent (#04709713001)
was purchased from Roche. Fetal calf serum (FCS) was purchased from Hyclone. Cell
culture reagents and all other reagents were purchased from Sigma.
Human heart samples
Left ventricle (LV) samples were collected from human patients with dilated
cardiomyopathy (DCM) who were under treatment following heart transplantation.
Control samples were obtained from normal heart donors. This study complied with
the protocol approved by the Renmin Hospital of Wuhan University Human Research
Ethics Committee, and samples were collected after informed consent.
Experimental animal models and aortic banding surgery
The animal protocol was approved by the Animal Care and Use Committee of
Renmin Hospital of Wuhan University. Human IRF4 cDNA was cloned downstream
of the cardiac α-myosin heavy chain (α-MHC) promoter. Transgenic mice were
generated by microinjecting the α-MHC-IRF4 construct into fertilized mouse embryos
and identified by PCR analysis of the tail genomic DNA. IRF4-knockout mice were
purchased from the Jackson Laboratory (Stock Number: 009380). Male α-MHC-IRF4
(TG) and non-transgenic (NTG) littermates, IRF4-knockout mice and their wild-type
(WT) littermates, ages 8 to 10 weeks (with body weights of 24-27 g), were used in
these experiments. Aortic banding (AB) was conducted as described previously1-5.
Doppler analysis was performed to ensure that adequate constriction of the aorta, and
similar pressure overload was achieved. Pressure gradients (mmHg) were calculated
from the peak blood velocity (Vmax, m/s, PG=4xVmax2) measured by Doppler
analyses across the AB. Control animals underwent the same procedure without aortic
banding (sham groups).
Echocardiography and hemodynamics evaluation
Echocardiography was performed using a MyLab 30CV ultrasound (Biosound Esaote
Inc.) with a 10-MHz linear array ultrasound transducer. The left ventricle (LV) was
assessed in both the parasternal long-axis and short-axis views at a frame rate of 120
Hz. LV end-systolic diameter (LVESD) and LV end-diastolic diameter (LVEDD) were
measured from the M-mode tracing with a sweep speed of 50 mm/s at the
mid-papillary muscle level.
For hemodynamic measurements, a 1.4-F Millar microtip catheter (SPR-839,
Millar Instruments, Houston, Texas) was inserted into the left ventricle through the
right carotid artery. After stabilization for 15 minutes, the pressure, volume signals
and heart rate were recorded continuously using a pressure-volume conductance
system (MPVS-300 Signal Conditioner, Millar Instruments, Inc., USA). The results
were analyzed with Chart 5.0 software.
Histological analysis
Hearts were excised and arrested in diastole with 10% potassium chloride solution,
fixed by 10% paraformaldehyde and embedded in paraffin. Subsequently, these hearts
were sectioned transversely close to the apex to visualize the left and right ventricles
at 5 µm. Sections of each heart at the mid-papillary muscle level were stained with
hematoxylin-eosin (HE) for histopathology or with picrosirius red (PSR) for the
collagen deposition. To determine the myocyte cross-sectional area, sections were
stained with FITC-conjugated Wheat germ agglutinin (WGA, Invitrogen Corp) for
membranes and with DAPI for nuclei. More than 100 myocytes in sections were
outlined in each group. Single myocyte and fibrillar collagen were visualized by
microscopy and measured with a quantitative digital image analysis system
(Image-Pro Plus 6.0).
Western blotting and quantitative real-time PCR
Cardiac tissue and cultured cardiac myocytes were lysed using a RIPA buffer, and the
protein concentration was determined with a BCA protein assay kit. Protein extracts
(50 µg) were separated by SDS-PAGE and were transferred to nitrocellulose
membranes and probed with various primary antibodies. After incubation with a
secondary IRDye® 800CW-conjugated antibody, signals were visualized with an
Odyssey Imaging System. The specific protein expression levels were normalized to
GAPDH on the same nitrocellulose membrane.
For real time-PCR, total RNA was extracted from the frozen mouse tissue using
a TRIzol (Invitrogen) and reverse-transcribed into cDNA using oligo (dT) primers
with a Transcriptor First Strand cDNA Synthesis Kit. PCR amplifications were
quantified using the SYBR Green PCR Master Mix (Applied Biosystems) and
normalized to GAPDH gene expression.
CREB-luciferase reporter assays
Nucleotides -1310 to +51 of the human CREB gene were amplified from the genome
of 293T cells by PCR with Platinum Taq DNA polymerase High Fidelity and inserted
into the pGL3 basic vector as a CREB-F1 vector. H9C2 cells (2 × 106 per well) were
seeded on 24-well plates the day before transfection. Using a FUGENE® HD
Transfection reagent, cells at 50-80% confluence were transfected with 1 µg of
pGL3-CREB. A 50 ng of the expression plasmid (pRL-Thymidine Kinase, pRL-TK)
was co-transfected as a transfection efficiency control, and luciferease activities were
normalized based on TK activities. After 24 hours, cells were harvested, washed three
times with phosphate-buffered saline (PBS) and lysed in 100 µl of the passive lysis
buffer (PLB) according to the manufacturer’s instructions (Promega). Cell debris was
removed by centrifugation, and the supernatant was used for the luciferase assay
using Single-Mode Microplate Readers SpectraMax® (Molecular Devices).
Cardiomyocyte culture and infection with recombinant adenoviral vectors
The replication-defective adenoviral vectors expressing the full-length IRF4 gene and
a control adenoviral vector expressing the GFP protein were used in this study. The
shIRF4 that led to the greatest decrease in IRF4 levels was selected for further
experiments and the shRNA virus was used as a control. We infected cardiac
myocytes with Ad-shRNA, Ad-shIRF4, Ad-GFP and Ad-IRF4. The cultures of
cardiac myocytes were prepared as described in our previous studies with a minor
revision2, 3. Cardiomyocytes isolated from hearts of 1- to 2-day-old Sprague-Dawley
rats were seeded at a density of 3 × 105/well onto six-well culture plates in MEM
supplemented with 10% FCS and penicillin/streptomycin. Subsequently, these
myocytes were infected with AdIRF4, Ad-shIRF4 and/or AdCREB, Addn-CREB for
12 hours with 10 MOIs. The culture medium was then replaced with serum-free
medium for 12 hours, followed by stimulation with 1 µM Ang II for 48 hours.
AdCREB was purchased from Applied Biological Materials Inc. (Cat. # 000407A).
Addn-CREB was constructed in our lab with the CREB Dominant-Negative Vector
which was ordered from Clontech (Cat. # 631925).
Measurement of the Protein/DNA Ratio
For the quantitative analysis of total cellular protein and DNA content, neonatal rat
cardiomyocytes were washed twice with PBS (phosphate-buffered saline) after
stimulated with Ang II or PBS for 48 hours. Then, 0.2N perchloric acid (1 ml) was
added to cells. The samples were centrifuged for 10 min at 10,000 ×g. The
precipitates were incubated for 20 min at 60 °C with 250 µl of 0.3 N KOH. Protein
content was analyzed by the BCA method using BCA Protein Assay Kit (Pierce, CAT:
23225). DNA content was detected using Hoechst dye 33258 (Invitrogen, CAT: H3569)
with salmon sperm DNA (Sigma, CAT: D1626) as a standard.
Immunofluorescence Staining
Neonatal rat cardiomyocytes (NRCM) were cultured on cover slips, infected with
Ad-IRF4 or Ad-shIRF4, and treated with Ang II (1 µM) for 48 hours. Cardiomyocytes
were fixed with 3.7% formaldehyde in PBS for 15 minutes at room temperature;
washed three times; permeabilized with 0.1% Triton X-100 in PBS for 40 minutes;
and stained with α-actinin (1:100 dilution), using standard immunofluorescence
staining techniques. For the tissue section staining, the procedure was the same as that
used for the NRCM staining after de-parafinisation step (as described above).
Statistical analysis
Data are shown as means ± SEM. Differences among groups were assessed by
ANOVA followed by Tukey’s post hoc test. Comparisons between two groups were
performed by Student's t-test. A value of P<0.05 was considered to be a statistically
significant difference.
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against cardiac hypertrophy and fibrosis during biomechanical stress of pressure
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Supplemental Tables
Table S1 Anatomic and Echocardiographic Analysis in 10-Week Old Mice.
IRF4+/+ mice
IRF4-/- mice
NTG mice
TG mice
（n=12）
（n=13）
（n=10）
（n=12）
BW(g)
27.26±0.68
27.32±0.33
26.93±0.44
27.56±0.42
HW/BW(mg/g)
4.38±0.10
4.22±0.11
4.28±0.10
4.17±0.10
5.35±0.12
5.17±0.10
5.24±0.13
5.15±0.10
6.49±0.09
6.26±0.12
6.42±0.12
6.22±0.12
549±15
525±9
558±22
512±17
LVEDD(mm)
3.84±0.09
3.76±0.06
3.64±0.07
3.63±0.08
LVESD(mm)
2.10±0.08
2.12±0.05
1.96±0.04
2.08±0.10
FS (%)
45.2±1.5
43.4±0.9
46.0±1.1
43.0±1.8
Parameter
LW/BW(mg/mg)
HW/TL(mg/mm)
HR (beats/min)
BW=body weight; HW=heart weight; LW=lung weight; TL=tibia length; HR=heart rate;
LVEDD=left ventricular end-diastolic diameter; LVESD=left ventricular end-systolic diameter;
FS=fractional shortening, All values are presented as means ± SEM.
Table S2 Parameters in IRF4-/- and IRF4+/+ mice at 8 weeks after sham operation or
AB
Parameters
BW(g)
HW/BW(mg/g)
LW/BW(mg/g)
HW/TL(mg/mm)
Sham
IRF4+/+ mice
IRF4-/- mice
AB
IRF4+/+ mice
IRF4-/- mice
（n=15）
（n=11）
（n=12）
（n=14）
27.43±0.40
27.62±0.55
28.72±0.48
28.95±0.25
4.14±0.07
4.08±0.08
8.25±0.21*
6.31±0.13*†
4.89±0.01
4.81±0.11
11.47±1.03*
6.92±0.17*†
6.35±0.10
6.24±0.13
12.93±0.31*
9.99±0.19*†
HR(beats/min)
547±12
LVEDD(mm)
3.67±0.10
3.55±0.04
5.30±0.15*
4.58±0.05*†
LVESD(mm)
2.02±0.11
2.00±0.05
4.15±0.18*
3.21±0.07*†
FS (%)
45.3±1.7
LVESP (mmHg)
LVEF (%)
111.76±3.46
536±18
43.7±1.2
110.02±4.37
496±19*
22.0±1.5*
126.90±8.07
498±8
29.9±1.3*†
158.66±4.41
55.1±2.2
57.4±1.4
29.0±1.5*
45.2±3.8*†
dp/dt max (mmHg/sec)
9780.5±318.4
10216.5±446.8
6150.2±226.8*
8267.0±311.9*†
dp/dt min (mmHg/sec)
-8339.7±320.5
-8864.8±311.2
-4835.2±223.2*
6907.3±439.3*†
LVESP=Left ventricular End-systolic Pressure. LVEF=Left ventricular ejection fraction.
*P<0.05 versus IRF4+/+/ sham operation. †P<0.05 versus IRF4+/+ AB after 8 weeks AB.
All values are presented as means ± SEM.
Table S3 Parameters in IRF4 transgenic mice (TG) and wild type littermates (NTG) at
4 weeks after sham operation or AB.
NTG- Sham mice
TG -Sham mice
NTG-AB mice
TG -AB mice
（n=13）
（n=13）
（n=13）
（n=11）
BW (g)
27.62±0.33
27.80±0.39
28.03±0.28
26.18±0.70
HW/BW(mg/g)
4.00±0.13
4.19±0.09
6.48±0.11*
8.40±0.35*†
LW/BW(mg/mg)
4.95±0.14
5.05±0.13
7.13±0.10*
10.87±1.30*†
HW/TL(mg/mm)
6.12±0.15
6.46±0.13
10.03±0.19*
11.94±0.37*†
522±36
500±13
LVEDD(mm)
3.57±0.06
3.62±0.06
4.61±0.04*
5.00±0.10*†
LVESD(mm)
1.99±0.06
2.07±0.02
3.14±0.02*
3.61±0.08*†
FS (%)
44.3±0.8
42.8±1.1
31.9±0.5*
27.6±1.2*†
107.02±1.94
100.97±1.49
61.3±0.9
57.3±2.0
37.1±1.5*
26.7±1.4*†
dp/dt max (mmHg/sec)
10370.6±427.0
10698.4±328.0
7825.7±352.5*
5959.4±227.5*†
dp/dt min (mmHg/sec)
-8330.9±273.5
-8956.1±411.7
-6949.7±282.0*
-4534.2±275.6*†
Parameter
HR (beats/min)
LVESP (mmHg)
LVEF (%)
491±8
171.92±3.85*
480±10
122.83±2.51†
*P<0.05 versus NTG mice after 4 weeks sham operation. †P<0.05 versus NTG mice after 4
weeks AB operation. All values are means ± SEM.
Supplemental Figures
Figure S1 IRF4 is expressed and downregulated in murine hearts upon
hypertrophic stimuli. (A) The levels of IRF4 mRNA in different tissues of
C57BL/6 mice under normal conditions. (B) The levels of IRF4 mRNA in hearts of
C57BL/6 mice after AB or Sham operation. (C) The immunofluorescence results
showed that IRF4 was expressed in the nuclei of cardiomyocytes and downregulated
in hypertrophic murine hearts (red: IRF4; blue: nucleus; green: α-actinin). *P<0.05 vs.
sham
Figure S2 The left ventricular end-systolic pressure (LVESP) in IRF4+/+ and IRF4-/mice 1 week after AB, and there is no significant difference between these two group
neither upon Sham nor AB surgery. *P<0.05 vs. sham
Figure S3 Overexpression of IRF4 aggravates pressure overload-induced cardiac
hypertrophy. A, Parameters of echocardiographic results for NTG and IRF4-TG
mice upon 4-week aortic banding (AB) or sham operations (n=6-9 mice per
experimental group, *P<0.05 vs. NTG/sham; #P<0.05 vs. NTG/AB). LVEDD=left
ventricular end-diastolic diameter; LVESD=left ventricular end-systolic diameter;
FS=fractional shortening. B, PSR staining of LV sections in the indicated groups upon
4-week AB (scale bar=50µm). C, Quantification of the total collagen volume in
AB-treated NTG and IRF4-TG mice (n=25+ fields per experimental group, *P<0.05
vs. NTG/sham; #P<0.05 vs. NTG/AB). D, Real-time PCR analyses of the fibrotic
markers (collagen I, collagen III and CTGF) in the indicated mice. n=4 mice per
experimental group, *P<0.05 vs. NTG/sham; #P<0.05 vs. NTG/AB.
Figure S4 IRF4 regulates angiotensin II-induced cardiomyocyte hypertrophy ex
vivo. The ratio of protein/DNA was determined in AdshRNA- and AdshIRF4-infected
neonatal rat cardiomyocytes (A), as well as in AdGFP- and AdIRF4-myocytes (B)
upon the treatment with either PBS or Ang II (1µM) for 48 h.
Figure S5 Phylogenetic foot-printing identified three conserved IRF4-binding sites
(sites 1, 2 and 3) in the promoter region of human CREB and mouse CREB orthologs.
Figure S6 Schematic constructs of various deletions in the human CREB promoter.
Putative IRF4 binding sites are indicated and intercepted in order (left). H9c2 cells
were co-transfected with each deletion mutant of the CREB promoter and
pcDNA3.1-GFP or pcDNA3.1-IRF4. After 24 h, cells were harvested for a luciferase
assay. Values of the corresponding luciferase activity are shown for four deletion
mutants of the CREB promoter (right). *P<0.05 vs. controls. Similar results were
observed in 3 additional independent experiments.
Figure S7 The status of inflammation/ inflammatory cell infiltration was examined in
WT vs. IRF4 KO hearts upon 1-week and 4-week AB, as determined by the mRNA
levels of IL-1β, IL-6, TNFα, and CD68. The results showed that mRNA levels of
IL-1β, IL-6, TNFα, and CD68 were significantly increased at 1 week after AB and
decreased to basal levels at 4 weeks after AB, but the status of inflammation/
inflammatory cell infiltration has no significance difference between WT and IRF4
KO hearts upon AB.