Heart
Interferon Regulatory Factor 7 Functions as a Novel
Negative Regulator of Pathological Cardiac Hypertrophy
Ding-Sheng Jiang,* Yu Liu,* Heng Zhou,* Yan Zhang, Xiao-Dong Zhang, Xiao-Fei Zhang, Ke Chen,
Lu Gao, Juan Peng, Hui Gong, Yingjie Chen, Qinglin Yang, Peter P. Liu, Guo-Chang Fan,
Yunzeng Zou, Hongliang Li
See Editorial Commentary, pp 663–664
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Abstract—Cardiac hypertrophy is a complex pathological process that involves multiple factors including inflammation
and apoptosis. Interferon regulatory factor 7 (IRF7) is a multifunctional regulator that participates in immune regulation,
cell differentiation, apoptosis, and oncogenesis. However, the role of IRF7 in cardiac hypertrophy remains unclear. We
performed aortic banding in cardiac-specific IRF7 transgenic mice, IRF7 knockout mice, and the wild-type littermates
of these mice. Our results demonstrated that IRF7 was downregulated in aortic banding–induced animal hearts and
cardiomyocytes that had been treated with angiotensin II or phenylephrine for 48 hours. Accordingly, heart-specific
overexpression of IRF7 significantly attenuated pressure overload–induced cardiac hypertrophy, fibrosis, and dysfunction,
whereas loss of IRF7 led to opposite effects. Moreover, IRF7 protected against angiotensin II–induced cardiomyocyte
hypertrophy in vitro. Mechanistically, we identified that IRF7-dependent cardioprotection was mediated through IRF7
binding to inhibitor of κB kinase-β, and subsequent nuclear factor-κB inactivation. In fact, blocking nuclear factor-κB
signaling with cardiac-specific inhibitors of κBαS32A/S36A super-repressor transgene counteracted the adverse effect of
IRF7 deficiency. Conversely, activation of nuclear factor-κB signaling via a cardiac-specific conditional inhibitor of κB
kinase-βS177E/S181E (constitutively active) transgene negated the antihypertrophic effect of IRF7 overexpression. Our data
demonstrate that IRF7 acts as a novel negative regulator of pathological cardiac hypertrophy by inhibiting nuclear f­ actorκB signaling and may constitute a potential therapeutic target for pathological cardiac hypertrophy. (Hypertension.
2014;63:713-722.) Online Data Supplement
•
Key Words: cardiomegaly ◼ fibrosis ◼ interferon regulatory factor 7
C
ardiac hypertrophy occurs in response to increased biomechanical stress (ie, hypertension, valvular disease,
myocardial infarction, or endocrine disorders). It is characterized by enlarged cardiomyocytes, enhanced protein synthesis, accumulation of extracellular matrices, and activation of
the fetal cardiac gene program.1,2 Although exercise-induced
cardiac hypertrophy can enhance heart function, pathological
hypertrophy is an initial compensatory response to increased
cardiac workload, which can ultimately result in heart failure, arrhythmias, and sudden death.3,4 During the past few
decades, several signal transduction pathways have been
suggested to contribute to the development of pathological
cardiac hypertrophy, including the epidermal growth factor receptor tyrosine kinase pathway, the mitogen-activated
protein kinase pathway, phosphatidylinositol 3-kinase/Akt
pathway, the cAMP-response element binding protein pathway, the protein kinase C pathway, and the calcineurin pathway.5–10 Importantly, these signaling pathways converge in the
nucleus to activate various transcriptional regulators, such as
nuclear factor-κB (NFκB), activator protein-1, GATA transcription factors, nuclear factor of activated T cells, and myocyte enhancer factor-2, which drive the expression of fetal
cardiac and stress response genes that promote pathological
cardiac hypertrophy.6–8,11 However, the molecular mechanisms
that mediate the critical transition from compensated hypertrophy to decompensated heart failure have remained elusive.
Therefore, elucidation of these transcriptional reprogramming
processes is essential for identifying potential transcriptional
Received October 17, 2013; first decision November 4, 2013; revision accepted November 13, 2013.
From the Department of Cardiology, Renmin Hospital (D.-S.J., Y.L., H.Z., Y. Zhang, H.L.), Cardiovascular Research Institute (D.-S.J., Y.L., H.Z., Y.
Zhang, H.L.), and College of Life Sciences (X.-D.Z., X.-F.Z., K.C.), Wuhan University, Wuhan, China; Department of Cardiology, Institute of Cardiovascular
Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China (L.G.); Shanghai Institute of
Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, China (J.P., H.G., Y. Zou); Cardiovascular Division, University of Minnesota,
Minneapolis (Y.C.); Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham (Q.Y.); University of Ottawa Heart Institute,
Ottawa, Ontario, Canada (P.P.L.); Department of Pharmacology and Cell Biophysics; University of Cincinnati College of Medicine, Cincinnati, OH (G.-C.F.).
*These are cofirst authors.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.
113.02653/-/DC1.
Correspondence to Hongliang Li, Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute, Wuhan
University, Jiefang Road 238, Wuhan 430060, PR China. E-mail [email protected]
© 2014 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.113.02653
713
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therapies aimed at preventing pathological cardiac hypertrophy and heart failure.
Interferon regulatory factors (IRFs) are a family of transcription factors comprising 9 members (IRF1-9) in mammalian cells. They were initially found to play an essential role
in modulating the expression of type I interferons (IFN-α and
-β),12 but recent studies have indicated that IRFs also play critical roles in antiviral defense, immune response, hematopoietic
development, cell growth/apoptosis, cardiac remodeling, and
oncogenesis.13–15 Among the 9 family members, IRF7 binds to
the DNA consensus sequence GAAWNYGAAANY widely
existing in genes,16 which indicates that IRF7 may modulate a
more diverse range of target genes. In fact, a recent study by
Honda et al17 has described IRF7 as a master transcription factor
in the regulation of type I IFN-dependent immune responses,
suggesting its crucial role in maintaining cellular homeostasis.
Furthermore, Barnes et al18 reported that IRF7 overexpression
in B-lymphoblastoid cells resulted in an altered transcriptional profile of cellular genes. Notably, IRF7 can function as
a transcriptional activator or a repressor, depending on the cell
type and its intrinsic transactivation potential.19,20 Accordingly,
IRF7 has either oncogenic properties or antitumor effects. For
example, IRF7-overexpressing NIH 3T3 (National Institutes of
Health 3T3) cells induce tumor formation in nude mice,21 and
overexpression of IRF7 inhibits the growth of MCF-7 (Michigan
Cancer Foundation-7) breast cancer cells.22 However, the role of
IRF7 in the process of cardiomyocyte remodeling, particularly
in response to chronic pressure overload, has not been investigated. Therefore, we sought to determine the impact of IRF7
deficiency and cardiac-specific IRF7 overexpression on aortic
banding (AB)–induced cardiac hypertrophy.
Here, we report that IRF7 acts as novel negative regulator of
pathological cardiac hypertrophy through suppression of NFκB
signaling. We observed that hypertrophic stress resulted in IRF7
downregulation in both mouse hearts and cardiomyocytes.
Moreover, IRF7-deficient mice revealed an aggravated hypertrophic response on chronic AB, whereas the opposite effect
occurred in IRF7-overexpressing mice. We also discovered that
all regions of IRF7, except for the inhibitory domain (ID: 284467aa), could interact with the kinase domain ­(N/1-300aa) of
inhibitor of κB kinase-β (IKKβ). Furthermore, blocking NFκB
signaling counteracted the effects of IRF7 deficiency, whereas
constitutively active IKKβ negated the antihypertrophic effects
of IRF7. Taken together, our results reveal a previously unrecognized role for IRF7 in the regulation of cardiac hypertrophy.
Methods
All experiments involving animals were approved by the Animal
Care and Use Committee of Renmin Hospital, Wuhan University,
China. An expanded Methods section is available in the online-only
Data Supplement, which includes Reagents, Mice Used in This
Study,17 Aortic Banding,1,8,9,15 Echocardiography and Hemodynamic
Measurements,1,8,9 Histological Analysis, Cultured Neonatal
Rat Cardiac Myocytes and Recombinant Adenoviral Vectors,1
Measurement of the Protein/DNA Ratio, Atrial Natriuretic Peptide
(ANP) Quantification Assays, Immunofluorescence Analysis,
Quantitative Real-Time PCR and Western Blotting,1,8,9 IKKβ Activity
Assays, Luciferase Reporter Assays,23,24 Hydroxyproline Assay,25
Immunoprecipitation, Glutathione S-transferase (GST) Pull-down
Assay, Confocal Microscopy, and Statistical Analysis.
Results
Expression of IRF7 Is Downregulated in
Experimental Hypertrophic Models
IRF7 is expressed constitutively in a variety of tissues, including the heart.26 To determine whether hypertrophic stress can
affect IRF7 expression in cardiomyocytes, we first cultured
neonatal rat cardiac myocytes (NRCMs) with either Ang II
or phenylephrine for 48 hours to induce hypertrophy. Western
blotting results revealed that IRF7 protein levels were markedly
reduced by 52% (Ang II) and 36% (phenylephrine), respectively. At the same time, we observed a significant increase
in 2 hypertrophic markers, β-myosin heavy chain (β-MHC)
and ANP (Figure 1A, n=3 independent experiments, P<0.01
versus PBS), and ANP concentrations in culture medium were
also remarkably increased (Figure S1A in the online-only
Data Supplement, P<0.01 versus PBS). Furthermore, in an
experimental mouse model with AB-induced cardiac hypertrophy (evidenced by elevation of β-MHC and ANP levels,
Figure 1B), IRF7 expression was downregulated by ≈93% in
mouse hearts 4 weeks after AB compared with sham-operated
hearts (Figure 1B, n=3 independent experiments, P<0.01 versus sham). Taken together, these results indicate that hypertrophic stress leads to IRF7 downregulation both in vitro
(cardiomyocytes) and in vivo (intact mouse hearts), suggesting that IRF7 has a role in stress-induced cardiac remodeling.
IRF7 Protects Against Angiotensin II–Induced
Cardiomyocyte Hypertrophy In Vitro
To define the functional contribution of IRF7 to cardiac hypertrophy, we first performed in vitro studies using primary cultured
NRCMs, a well-controlled experimental setting. NRCMs were
Figure 1. Expression of interferon
regulatory factor 7 (IRF7) is downregulated
in experimental hypertrophic models.
A, Representative Western blots of β-myosin
heavy chain (β-MHC), atrial natriuretic
peptide (ANP), and IRF7 in cultured
neonatal rat cardiac myocytes treated
with Ang II (1 μM) and phenylephrine (100
μmol/L) for 48 hours (n=3, *P<0.01 vs PBS).
B, Western blots analysis of β-MHC, ANP,
and IRF7 in a cardiac hypertrophy mouse
model induced by aortic banding at the
indicated time. (n=3, *P<0.01 vs sham). Top,
Representative Western blots. Bottom,
Bar graphs: quantitative results. n indicates
number of independent experiments.
Jiang et al IRF7 Regulates Cardiac Hypertrophy 715
infected with either AdIRF7 (adenoviral IRF7) to overexpress
IRF7 or AdshIRF7 (adenoviral short hairpin IRF7) to knockdown IRF7 (Figure S1B) and subsequently exposed to Ang II
(1 μM) for 48 hours. The cell surface area was then determined
by immunostaining with α-actinin. Notably, under basal conditions neither AdIRF7 nor AdshIRF7 affected the size of cultured
NRCMs compared with control AdGFP (adenoviral green fluorescent protein) and AdshRNA (adenoviral short hairpin RNA)
cells. However, exposure to Ang II significantly reduced the
cell surface area of AdIRF7-infected cells by 29% (Figure 2A
and 2B), whereas Ang II–induced cardiomyocyte hypertrophy
was enhanced by 62% in ­
AdshIRF7-treated NRCMs compared with controls (Figure 2A and 2B). Accordingly, the Ang
II–induced expression of hypertrophic hallmarks (ANP and
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Figure 2. Interferon regulatory factor 7 (IRF7) protects against
angiotensin II–induced cardiomyocyte hypertrophy in vitro. A,
Representative images of cardiomyocytes infected with AdshIRF7
(adenoviral short hairpin IRF7) or AdIRF7 (adenoviral IRF7) in
response to Ang II (1 μM) for 48 hours. B, Quantitative results of
the cell surface area showing that overexpression of IRF7 greatly
attenuated Ang II–triggered hypertrophy compared with control
green fluorescent protein (GFP)-cells (left, bar graph). Conversely,
knockdown of IRF7 significantly increased Ang II–triggered
hypertrophy compared with short hairpin RNA (shRNA)-cells
(right, bar graph; n=100+ cells). C, Real-time polymerase chain
reaction assays for evaluation of the mRNA levels of atrial
natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC) in
­IRF7-overexpressing and knockdown cells under the treatment
with PBS and Ang II for 48 hours (n=3 independent experiments).
D, The ratio of protein/DNA was determined in adenoviral green
fluorescent protein (AdGFP)- and ­AdIRF7-infected neonatal rat
cardiomyocytes, as well as in AdshRNA- and AdshIRF7-myocytes
on the treatment with either PBS or Ang II (1 μM) for 48 hours
(n=3 independent experiments). *P<0.05 vs AdGFP or AdshRNA/
PBS, #P<0.05 vs AdGFP or AdshRNA/Ang II.
β-MHC) and the ratio of protein/DNA were profoundly suppressed in AdIRF7-infected cardiomyocytes (Figure 2C and
2D), whereas significantly enhanced in AdshIRF7-infected
NRCMs (Figure 2C and 2D) compared with controls respectively. These results revealed that IRF7 had an antihypertrophic
effect on cardiomyocytes.
Overexpression of IRF7 Attenuates Pressure
Overload–Induced Cardiac Hypertrophy
We next sought to examine whether increased IRF7 levels in
the heart would attenuate the development of cardiac hypertrophy and failure. Thus, we generated a transgenic (TG) mouse
model with cardiac-specific IRF7 expression, using the α-MHC
promoter (α-MHCp; Figure S2A). Four germ lines of IRF7-TG
mice were created and verified by Western blotting (Figure
S2B). At baseline, the IRF7-TG mice displayed normal cardiac
morphology and contractile function (Table S1). We selected the
­highest-expressing IRF7 line (TG21) to be used in our experiments. IRF7-TG mice, along with their WT littermates (referred
to as NTG), were subjected to AB surgery or a sham operation,
and pressure gradients (mm Hg) were equivalent among all
AB-operated mice (Figure S2C). After 8 weeks of AB, NTG mice
developed massive cardiac hypertrophy, as indicated by increased
heart weight (HW)/body weight (BW), lung weight (LW)/BW,
and HW/tibia length (TL) ratios (Figure 3A–C). These changes
were accompanied by the following features: (1) thickening
of the left ventricular (LV) wall (HE staining, Figure 3D); (2)
increase cardiomyocyte cross-sectional area (WGA [wheat germ
agglutinin] staining, Figure 3D and 3E); and (3) upregulation of
the hypertrophic markers ANP, B-type natriuretic peptide, and
β-MHC (Figure S2E). Consistent with this, NTG mice exhibited
cardiac dilation and dysfunction 8 weeks after AB, as measured
by echocardiographic parameters including LV end-diastolic
dimension (LVEDd), LV ­end-systolic dimension (LVESd), and
LV fractional shortening (LVFS; Figure 3F). Hemodynamic
analysis also showed impaired systolic and diastolic functioning of the LV in NTG mice, as determined by the minimum rate
of pressure (−dp/dt) and the maximum rate of pressure (+dp/
dt) in LV isovolumetric contraction (Table S2). However, this
pathological cardiac growth, which was induced by chronic
pressure overload, was remarkably attenuated in IRF7-TG mice
(Figure 3A–F and Figure S2E), and the cumulative survival
rate of IRF7-TG mice was increased after 8 weeks of AB surgery (Figure S2D).
Fibrosis is a classical feature of pathological cardiac hypertrophy and is characterized by the accumulation of collagen in
the heart.27 To further define the effect of IRF7 overexpression
on maladaptive cardiac remodeling, we examined the effect
of IRF7 on cardiac fibrosis. The extent of fibrosis was quantified by collagen volume through the visualization of the total
amount of collagen present in the interstitial and perivascular spaces. We observed that both interstitial and perivascular
fibrosis were dramatically increased in NTG hearts that were
subjected to AB, but markedly limited in IRF7-TG hearts that
experienced the same treatment (Figure 3G and 3H). Finally,
we measured the synthesis of collagen by analyzing the
mRNA expression levels of fibrotic markers (eg, connective
tissue growth factor, collagen I, and collagen III; Figure S2F).
Our results consistently revealed a decreased fibrotic response
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S3B). In contrast to IRF7-TG mice, IRF7−/− mice aggravated
AB-induced cardiac hypertrophy, as indicated by greater
increases in HW/BW, LW/BW, and HW/TL compared with
AB-treated IRF7+/+ mice (Figure 4A–C). Histological examination of heart sections also revealed an increased cross-sectional area of cardiomyocytes in the IRF7−/− mice (Figure 4D
and 4E). Consistent with these data, hearts from IRF7−/− mice
showed greater hypertrophic marker induction (ANP, B-type
natriuretic peptide, and β-MHC) after 2 weeks of AB compared with controls (Figure S3D). Accordingly, IRF7−/− mice
exhibited deteriorated cardiac dilation and dysfunction, as
observed through echocardiograph and hemodynamic analysis (Figure S3E and Table S3) and decreased cumulative survival rate (Figure S3F). We also assessed the effect of IRF7
deficiency on AB-triggered cardiac fibrosis. Both histological
analysis and fibrotic markers analyses consistently demonstrated an increased fibrotic response in AB-operated IRF7−/−
mice compared with AB-treated IRF7+/+ mice (Figure 4F and
4G and Figure S3G). Collectively, these loss-of-function data
indicate that ablation of IRF7 exaggerates cardiac hypertrophy and fibrosis in response to chronic pressure overload.
IRF7 Suppresses NFκB Signaling
To gain insight into the molecular mechanisms underlying the
negative effects of IRF7 on pathological cardiac hypertrophy,
we next sought to identify IRF7-regulated targets using a Cignal
Figure 3. Overexpression of interferon regulatory factor 7 (IRF7)
attenuates pressure overload–induced cardiac hypertrophy. A–C,
Ratios of heart weight (HW)/body weight (BW), lung weight (LW)/
BW, and HW/tibia length (TL) in the indicated groups (n=12–14).
D, Histological analyses of the hematoxylin-eosin (HE) staining
and the WGA (wheat germ agglutinin) staining of non-transgene
(NTG) and transgenic (TG) mice 8 weeks after the aortic banding
(AB) surgery (n=6). E, Statistical results for the cell sectional area
(n=100+ cells). F, Parameters of the echocardiographic results
for NTG and TG mice (n=6–8). G, PSR (picrosirius red) staining
on histological sections of the LVs from the indicated groups 8
weeks after AB (n=6). H, Statistical results for LV collagen volume
(n=30+ fields). *P<0.05 vs NTG/sham; #P<0.05 vs NTG/AB. n
indicates number of mice per experimental group.
in IRF7-TG hearts (Figure 3G and 3H and Figure S2F). Taken
together, these data imply that IRF7 overexpression suppressed pressure overload–induced cardiac remodeling.
Ablation of IRF7 Exaggerates Pressure
Overload–Induced Hypertrophy
To assess the impact of decreased IRF7 levels on the development of cardiac hypertrophy and failure that are induced by
chronic pressure overload, we next used an IRF7 knockout
mouse model (IRF7−/−), in whom IRF7 expression was absent
in the heart (Figure S3A). At baseline, IRF7−/− mice did not
show any cardiac abnormalities (Table S1). Our preliminary
experiments revealed that almost all IRF7 knockout mice died
after 8 weeks of AB surgery (Figure S3C). Therefore, we subjected IRF7−/− mice and their wild-type littermates (referred to
as IRF7+/+) to AB surgery for 2 weeks, and pressure gradients
(mm Hg) were equivalent among all AB-operated mice (Figure
Figure 4. Ablation of interferon regulatory factor 7 (IRF7)
exaggerates pressure overload–induced hypertrophy. A–C, Ratios
of HW/BW, LW/BW, HW/TL in the indicated groups (n=12–14).
D, Histological analyses of the HE staining and the WGA (wheat
germ agglutinin) staining of WT and IRF7-KO mice 2 weeks after
the aortic banding (AB) surgery (n=6–8). E, Statistical results
for the cell sectional area (n=100+ cells). F, PSR staining on
histological sections of the left ventricles (LVs) from the indicated
groups 2 weeks after AB (n=6–8). G, Statistical results for LV
collagen volume (n=30+ fields). *P<0.05 vs WT/sham; #P<0.05 vs
WT/AB. n indicates number of mice per experimental group.
Jiang et al IRF7 Regulates Cardiac Hypertrophy 717
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45-Pathway Reporter Array kit (SABiosciences: C
­ CA-901 L).
This screening kit provides a comprehensive assay for preliminary monitoring of diverse cell signaling pathways by measuring the activities of downstream transcription factors via a
dual-luciferase reporter system. The results showed that the
activity of NFκB was significantly blocked by IRF7, which
was confirmed by performing an NFκB dual-luciferase reporter
assay in hypertrophic cardiomyocytes (in vitro) and hearts (in
vivo). NRCMs were infected with either AdIRF7 to overexpress IRF7 or AdshIRF7 to knockdown IRF7. Subsequently,
these infected cardiomyocytes were exposed to 1 μM of Ang
II for 48 hours. Our results showed that compared with controls, Ang II–induced NFκB activation was significantly
reduced in the ­AdIRF7-infected NRCMs but greatly enhanced
in the AdshIRF7-infected cardiomyocytes (Figure 5A). IRF7+/+,
IRF7−/−, NTG, and IRF7-TG mice received Ad-NFκB–Luc
injection at ventricular wall immediately after being subjected to AB or sham operation. IRF7 overexpression inhibited
whereas the loss of IRF7 promoted NFκB activation induced
by AB surgery, which was consistent with in vitro experiments
(Figure 5B). Next, we performed an NFκB Signaling Pathway
EpiTect Chip qPCR Array (SABioscience: GM-025A) to determine which genes are regulated by NFκB pathway and further
validated the results by real-time polymerase chain reaction.
The results revealed that genes (eg, Nfkb2, ikbkb [inhibitor
of nuclear factor kappa-B kinase subunit beta], chemokine
[C-C motif] ligand 5 [Ccl5], CSF2 [colony-stimulating factor
2], Smad3 [mothers against decapentaplegic homolog 3], Bax
[B-cell lymphoma 2 associated X protein]) associated with
inflammation, apoptosis, and hypertrophy were significantly
increased (Figure S4A) in the hearts of AB-subjected IRF7−/−
mice. These data indicate that IRF7 may exert its antihypertrophic effects through the inhibition of the NFκB signaling.
It is known that dimeric NFκB (containing p50, p65, and
c-Rel) is inactivated by binding to inhibitors of κB (IκBs),
which sequester NFκB in the cytosol in nonstimulated cells.28
In the classical pathway, the IκB proteins are regulated by
kinases, IKKα, and IKKβ.28 The latter is particularly important because it phosphorylates IκBα at Ser32/36, leading to
ubiquitination/degradation of IκBα by the 26S proteasome
and subsequent NFκB nuclear entry.28 It is also clear that IKKβ
activation involves phosphorylation at 2 key serine residues
(Ser 177/181). Therefore, to elucidate the ways in which IRF7
inhibits NFκB activity, we next examined phosphorylated levels IKKβ and IκBα in hypertrophic cardiomyocytes (in vitro)
and hearts (in vivo). In Ang II–treated NRCMs, IRF7 overexpression significantly limited the kinase activity of IKKβ
(Figure S4B) and activation of IKKβ-IκBα pathway, as evidenced by decreased levels of p-IKKβSer181 and p-IκBαSer32/36
compared with control cells (Figure 5C). In line with this,
IKKβ-mediated proteosomal degradation of IκBα was attenuated in AdIRF7-myocytes, as indicated by increased levels
of total IκBα (Figure 5C). Importantly, similar inhibitory
effects of IRF7 on the IKKβ-IκBα signaling cascades were
also observed in AB-treated IRF7-TG hearts (Figure 5E).
Using loss-of-function strategies, we further demonstrated
that decreased IRF7 levels resulted in pronounced activation
of IKKβ and IκBα in vitro and in vivo experimental hypertrophic models (Figure 5D and 5F and Figure S4C).
Because p65 translocation to the nucleus is important for
NFκB activation,28 we tested the effect of IRF7 on AB-induced
p65 nuclear entry via Western blotting. Lamin B was used as a
loading control for nuclear extracts. We found that AB-induced
phosphorylation of p65 in the nucleus was profoundly attenuated by IRF7 overexpression (Figure 5G) and promoted by
IRF7 depletion (Figure 5H), suggesting that IRF7 suppresses
p65 nuclear translocation. Collectively, these data indicate that
IRF7 negatively regulates the IKKβ–IκBα–NFκB pathway in
cardiomyocytes in response to hypertrophic stimuli.
Additionally, given that NFκB activation has been implicated in the regulation of inflammation and apoptosis in many
cell types, including cardiac myocytes,29 we next measured
the expression profiles of some NFκB-dependent inflammatory factors (tumor necrosis factor α, monocyte chemotactic
protein-1, and interleukin-1β) in animal hearts on chronic
AB. Real-time polymerase chain reaction analysis (Figure
S4D) and Western blotting (Figure S4F) revealed that IRF7
overexpression remarkably blunted AB-induced increases in
tumor necrosis factor α, monocyte chemotactic protein-1, and
interleukin-1β. Conversely, loss of IRF7 elicited the opposite effect (Figure S4E and S4G). Terminal deoxynucleotidyl
transferase dUTP nick-end labeling analysis and Western blotting showed that the percentage of transferase dUTP nick-end
labeling–positive cells, levels of c-Caspase3, and Bax were
increased, whereas full-length Caspase3 and Bcl-2 were downregulated in IRF7 knockout mice (Figure S4I and S4K). On the
contrary, IRF7 overexpression showed the opposite effects on
cardiac apoptosis (Figure S4H and S4J). These results further
support the hypothesis that IRF7 is a novel negative modulator
of the NFκB signaling pathway in the hypertrophic hearts.
IRF7 Directly Interacts With IKKβ
Because stress-induced IκBα degradation (and thus NFκB
activation) is IKKβ dependent,29 our finding of IRF7-mediated
NFκB inhibition prompted us to examine whether IRF7
directly interacted with IKKβ. We first performed coimmunoprecipitation (IP) experiments in HEK293T (human embryonic kidney 293) cells transfected with FLAG-tagged IKKβ
and Myc-tagged IRF7. We found that IRF7 could coprecipitate
along with IKKβ and vice versa (Figure 6A and 5B) which is
consistent with the results of co-IP for endogenous proteins in
NRCMs (Figure S5A and S5B). This interaction was further
confirmed by the GST–pull-down assay (Figure 6C). We next
examined the subcellular localization of IRF7 using immunofluorescent staining. We observed that IRF7 and IKKβ
were finely colocalized in the cytoplasm of HEK293T cells
(Figure 6D). Similar results were observed in H9C2 cells
(Figure S5C). Altogether, these data support that IKKβ can
directly interact with and be regulated by IRF7.
To map which regions of IKKβ and IRF7 mediated the interaction, we next generated a series of deletion mutants of IKKβ and
IRF7 (Figure 6E). Through IP mapping, we found that only the
kinase domain (N/1-300aa), located at the N-terminus of IKKβ,
was required for the interaction (left, Figure 6E and 6F). However,
most regions of IRF7, except for the inhibitory domain (ID: 284467aa), contributed to IKKβ binding (right, Figure 6E and 6F).
Therefore, IRF7 may inhibit IKKβ activity through masking its
kinase domain. Importantly, we measured the kinase activity of
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Figure 5. Interferon regulatory factor 7 (IRF7) suppresses nuclear factor (NF) κB signaling. A, NFκB-luciferase activity in primary
cultured neonatal rat cardiac myocytes (NRCMs) infected with AdIRF7 or AdshIRF7 (n=3, *P<0.05 vs AdGFP/PBS or AdshRNA/PBS,
#P<0.05 vs AdGFP/Ang II or AdshRNA/Ang II). B, Ad-NFκB–Luc was injected into the ventricular wall of IRF7 transgenic (TG) and IRF7
knockout mice, and NFκB-luciferase activity was determined using a Luciferase Assay System kit (n=4).*P<0.05 vs NTG/sham or WT/
sham, #P<0.05 vs NTG/aortic banding (AB) or WT/AB. C, Left, Representative Western blots showing the phosphorylation and total
protein levels of inhibitor of κB kinase-β (IKKβ), IκBα. Right, Quantitative results of the p-IKKβ, p-IκBα, and IκBα in NRCMs infected with
recombinant adenoviruses expression IRF7 and treated with Ang II for 60 min (n=3, *P<0.05 vs AdGFP/PBS, #P<0.05 vs AdGFP/Ang II).
D, Same as shown in C, but cells were instead infected with AdshIRF7 and treated with Ang II for 60 min (n=3, *P<0.05 vs AdshRNA/PBS,
#P<0.05 vs AdshRNA/Ang II). E, Left, Representative Western blots showing that the phosphorylation and total protein levels of IKKβ,
IκBα. Right, Quantitative results of the p-IKKβ, p-IκBα, and IκBα in IRF7-TG mice 2 weeks after AB surgery (n=3, *P<0.05 vs NTG/sham,
#P<0.05 vs NTG/AB). F, Same as shown in (E), but instead data are shown from IRF7-KO mice 2 weeks after AB surgery (n=3, *P<0.05
vs WT/sham, #P<0.05 vs WT/AB). G, Left, Representative Western blots. Right, Quantitative results of nucleus p-P65 and P65 protein
levels in IRF7 TG and NTG mice 2 weeks after AB (n=3, *P<0.05 vs. NTG/AB). H, Left, Representative Western blots. Right, Quantitative
results of the nucleus p-P65 and P65 protein levels in IRF7 knockout and WT mice 2 weeks after AB (n=3, *P<0.05 vs WT/AB). n indicates
number of independent experiments.
Jiang et al IRF7 Regulates Cardiac Hypertrophy 719
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Figure 6. Interferon regulatory factor 7 (IRF7) directly interacts with inhibitor of κB kinase-β (IKKβ). A, Western blot with Flag or Myc
antibody after coimmunoprecipitation (IP) of IRF7 from HEK293T (human embryonic kidney 293) whole-cell lysates using Myc antibody. B,
Western blot with Flag or Myc antibody after co-IP of IKKβ from HEK293T whole-cell lysates using Flag antibody. C, Western analysis of
glutathione S-transferase (GST) pull-down assay using lysates from Rosetta (DE3) E. coli expressing exogenous GST fusion IRF7 protein
(GST-IRF7) incubated with ­Flag-IKKβ–transfected HEK293T cell lysates. D, Representative confocal images showing colocalization of IRF7
and IKKβ in HEK293T cells. E, Schematic representation of the full-length and truncated forms of IKKβ (left) and full length and truncated
versions of IRF7 (right) used for mapping studies. F, Left, Representative Western blot with Flag or Myc antibody after co-IP of full length
and truncated forms of IKKβ from HEK293T whole-cell lysates using Flag antibody. Right, Western blot with Flag or Myc antibody after coIP of full-length IKKβ from HEK293T whole-cell lysates using Flag antibody. All the above results were repeated >3 times.
IKKβ in NRCMs infected with AdIRF7 or AdIRF7-ID (AdGFP
as a control). The results showed that overexpression of wild-type
IRF7 significantly inhibited the kinase activity of IKKβ, however, overexpression of IRF7-ID (IRF7-inhibitory domain) did
not have any effects on its kinase activity (Figure S5D).
Inhibitory Role of IRF7 in Pathological Cardiac
Hypertrophy Is largely Dependent on the NFκB
Signaling Pathway
Next, we examined whether activation or inactivation of the
NFκB signaling cascade would affect the regulatory role of
IRF7 in the development of pathological cardiac hypertrophy.
To address this issue, we generated an inducible mouse model
with the cardiac-specific overexpression of IKKβS177E/S181E, a
constitutively active form of IKKβ (Figure S6A). The inducible IKKβS177E/S181E mice were then mated with IRF7-TG mice
to create IRF7/IKKβS177E/S181E double transgenic mice (Figure
S6B). Cardiac overexpression of IKKβ and IRF7 in these animal
models was validated by Western blotting (Figure S6C). After 4
weeks of AB, the double transgenic mice exhibited a remarkable
pathological cardiac hypertrophy, as evidenced by higher HW/
BW, LW/BW, and HW/TL ratios (Figure S6E–G) compared
with AB-operated IRF7-TG mice. Furthermore, histological
analysis of hearts from double transgenic mice showed a significant increase in myocyte cross-section and collagen contents
compared with AB-operated IRF7-TG mice (Figure S6D and
S6H–J), and cardiac function was also dramatically deteriorated
(Figure S6M). Additionally, pathological cardiac hypertrophy
was remarkably obvious in IKKβS177E/S181E-TG mice after 4
weeks of AB compared with MEM-Cre controls (Figure S6D–
M). Taken together, I­RF7-elicited inhibitory effects on pathological cardiac remodeling seem to be largely IKKβ dependent.
To further verify the role of the NFκB signaling pathway in the negative effects of IRF7 on cardiac hypertrophy,
we overexpressed the nondegradable IκBα super-repressor
(IκBαS32A/S36A) in IRF7−/− hearts by crossing cardiac-specific
IκBαS32A/S36A TG mice with IRF7−/− mice (Figure S7A/B). We
found that IκBαS32A/S36A significantly reversed the detrimental effects of IRF7−/− on AB-induced cardiac hypertrophy,
as determined by histological analysis and ratios of HW/
BW, LW/BW, and HW/TL (Figure S7C–G). Moreover, the
aggravated effects of IRF7−/− on fibrotic response and hypertrophic marker induction were dramatically mitigated by
IκBαS32A/S36A overexpression (Figure S7H–K). Importantly,
cardiac function was significantly improved (Figure S7L).
Collectively, these l­oss-of-function data confirmed that
720 Hypertension April 2014
NFκB pathway inactivation was responsible for IRF7elicited negative action on pathological cardiac hypertrophy.
Discussion
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
In the present study, we used both gain-of-function and loss-offunction approaches to decipher the potential role of IRF7 in
pathological cardiac hypertrophy. For the first time, we observed
that overexpression of IRF7 in the heart limited chronic pressure overload–induced cardiac remodeling, whereas loss of IRF7
resulted in detrimental effects on the development of pathological cardiac hypertrophy. We also found that the expression levels of IRF7 were significantly reduced in hypertrophic hearts
(Figure 1). Thus, IRF7 upregulation may provide a new therapeutic strategy for the treatment of pathological cardiac hypertrophy.
The mechanisms underlying the negative effects of IRF7 on
pathological cardiac remodeling could stem from its direct binding to IKKβ that masks its kinase domain from activation. Except
for the inhibitory domain (residues 284–467), which interferes
with the binding and transactivation function of IRF7,30 other
regions of IRF7 are responsible for the interaction with IKKβ.
One of the regions binding to IKKβ is detected between residues
1 to 246, which contains ­DNA-binding domain and constitutive
activation domain. DNA-binding domain, which specifically
binds to IFN-stimulated response element to initiate the transcription of downstream genes, is shared by each member of IRF
family and has 5 conserved tryptophan repeats.30 Constitutive
activation domain is located between residues 151 and 246
and possesses constitutive activation activity.30 Another IKKβbinding region is found in virus-activated domain and signal
response domain of IRF7. Virus-activated domain is an accessory
inducibility region on viral infection that is required for increasing basal and v­ irus-inducible activity.30,31 Signal response domain
is the site of virus-induced phosphorylation, which regulates the
transactivation function of IRF7.32 Only the kinase domain (residues 1–300) of IKKβ interacts with IRF7. IKKβ kinase domain
contains several serine residues that can be phosphorylated by
upstream kinases, and the phosphorylation is required for activating IKK kinase activity.33,34 In our study, IRF7 directly binds to
the kinase domain of IKKβ, and then suppresses its phosphorylation level and NFκB signaling. The interaction between IRF7 and
IKKβ occurs in the cytoplasm. Hence, besides directly binding to
the DNA sequence to modulate target gene expression, IRF7 also
functions as a regulator by interacting with other proteins. For
instance, it was reported that IRF7 binds to the death domain of
MyD88 (myeloid differentiation primary response gene 88) in the
cytoplasm to regulate the MyD88-dependent IFN gene expression.35,36 Additionally, IRF7 interacts with TRAF6 (tumor necrosis factor receptor associated factor protein 6), a downstream
molecule of MyD88, and activates IFN gene transcription,36 thus
our findings further support the notion that IRF7 functions as a
critical regulator of IKKβ other than a transcription factor.
On binding to IRF7, the downstream IκB would be inactivated, leading to NFκB retained in the cytosol. In support of
this hypothesis, activation of the IKKβ–IκB–NFκB pathway by
aortic banding was remarkably limited in IRF7-overexpressing
hearts but dramatically aggravated in IRF7-null hearts (Figure 5E
and 5F). Furthermore, restoration of IKKβ activity via overexpression of IKKβS177E/S181E offset IRF7-induced inhibitory effects
on pathological hypertrophy (Figure S6). Our findings clearly
indicate that the IKKβ–IκB–NFκB signaling cascade is essential for the development of pathological cardiac hypertrophy. It
is known that NFκB is a key regulator of gene expression programs downstream of diverse signal transduction cascades during a variety of physiological and pathophysiological processes,
such as inflammation, cardiomyocyte growth, and extracellular
matrix remodeling.37–39 NFκB also directly interacts with other
transcription factors that are involved in the development of cardiac hypertrophy. For example, Liu et al40 recently demonstrated
a direct interaction between transcription factors, nuclear factor
of activated T cells and NFκB, which was suggested to integrate
these 2 disparate signaling pathways during cardiac hypertrophy.
Nonetheless, there are some controversial reports on the role of
NFκB signaling in the regulation of cardiac hypertrophy. Some
investigators have shown that NFκB activation is required for the
development of cardiac hypertrophy as well as cardiac dysfunction.40,41 For example, cardiac-specific NFκB inhibition mediated by expression of a stabilized IκBα mutant attenuates Ang
II–induced cardiac hypertrophy.41 Moreover, cardiac-specific
deletion of p65 decreases the hypertrophic response after pressure
overload stimulation, reduces the degree of pathological remodeling, and preserves contractile function.40 In contrast, several
additional studies have disputed the importance of NFκB in the
regulation of cardiac hypertrophy.42,43 For example, Hikoso et al42
reported that c­ ardio-specific knockdown of IKKβ promoted cardiac hypertrophy, dilation, and dysfunction in response to pressure overload. NFκB inhibition by cardiac-specific deletion of
NEMO (NF-kappa B essential modulator), a regulatory subunit
of the IKK complex, augmented cardiac hypertrophy and heart
failure after AB.43 In the present study, we observed that the activation of NFκB was inhibited in the hearts with overexpression
of IRF7, suggesting that IRF7 may attenuate cardiac remodeling
through negative regulation of NFκB signaling. More importantly,
inhibition of the NFκB signaling by overexpression of IκBαS32A/
S36A
combated the deteriorating effects of IRF7 knockout on cardiac dysfunction and dilation in response to chronic AB, whereas
constitutively active IKKβ blocked IRF7-mediated protection
from pathological cardiac remodeling. Therefore, the inhibitory
effects of IRF7 on cardiac hypertrophy may be largely dependent
on inactivation of the NFκB signaling. Our study may add a new
molecule to the increasing list that selectively inhibits the activation of transcription factor NFκB in the hypertrophic hearts. Given
that NFκB is likely to have multiple divergent effects and may
promote or suppress pathogenic processes, future investigation is
needed to determine whether IRF7 has a therapeutic potential for
the treatment of pathological cardiac hypertrophy.
Given that cardiac hypertrophy is a complicated pathological process that involves multiple molecules, our study cannot
rule out other mechanisms that IRF7 is involved. Indeed, as a
transcription factor, IRF7 may directly regulate genes related
to cardiac hypertrophy. This is actually predicted by the online
database. In the present study, we found that IRF7 potentially
binds to the regulatory elements of some prohypertrophic and
profibrotic genes, such as Myh6, connective tissue growth factor, smad3, smad4, Col11a1, and Col4a2. Therefore, it is likely
that IRF7 may have dual roles in regulating cardiac remodeling; however, future work is needed to explore the mechanism
by which IRF7 functions as a transcription factor to modulate
the development of pathological cardiac hypertrophy.
Jiang et al IRF7 Regulates Cardiac Hypertrophy 721
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The limitation of this study is that we used global IRF7
knockout to study the roles of IRF7 in the development of cardiac hypertrophy, and a genome-wide gene expression analysis
has not been performed. Considering that global knockout of
IRF7 may affect other types of cells other than cardiomyocytes
in the heart, we used cultured neonatal rat cardiomyocytes in
which IRF7 was knocked down by shIRF7. Our results showed
that Ang II–caused hypertrophy was enhanced in IRF7knockdown myocytes, which seems consistent with the findings observed in knockout mice on chronic pressure overload.
Additionally, IRF7-TG mice, which overexpressed IRF7 specifically in the heart, showed significant reduction in hypertrophic response. Taken together, both in vivo and in vitro results
consistently reflect the importance of IRF7 in the heart to regulate the development of pathological cardiac hypertrophy.
Notably, interferon regulatory factors (IRFs), consisting of 9
members (IRF1-9) in mammals, represent a group of structurally
similar transcription factors characterized by having a well-conserved N-terminal DNA-binding domain and a C-terminal IRF
association domain. IRFs exert their function either in cooperation or competition with other factors to play an important role
in innate immunity, cell cycle, apoptosis, cancer, metabolic diseases, and cardiovascular diseases.12,13,44,45 Recently, our group
intensively reported the role of IRFs in metabolic diseases and
cardiovascular diseases.8,15,46–49 IRF3 and IRF9 alleviate hepatic
steatosis and insulin resistance by constraining IKKβ/NFκB signaling or activating PPARa (peroxisome proliferator-activated
receptor alpha) target genes, respectively,46,47 but IRF7 promotes
diet-induced obesity and insulin resistance.48 In cardiovascular
system, IRF3 and IRF9 protected against pressure overload–
induced cardiac hypertrophy via suppressing ERK1/2 (extracellular signal-regulated kinases 1/2) or myocardin, respectively,15,49
whereas IRF4 aggravated pressure overload–induced hypertrophic response by activating the transcription of cAMP-response
element binding protein.8 In this study, we demonstrate that
IRF7 protects against pressure overload–induced cardiac hypertrophy by inhibiting IKKβ/NFκB signaling. Therefore, IRFs
have the proper response by regulating an identical yet disparate
set of targeted genes, and further studies are needed to clarify
how these different IRFs interplay each other in the development
of cardiovascular diseases and metabolic diseases.
In conclusion, we have found a novel role for IRF7 in regulating stress-induced cardiac hypertrophy and fibrosis. Importantly,
IRF7 serves a protective function during pathological cardiac hypertrophy by inhibiting the IKKβ–IκB–NFκB signaling pathway. These observations provide new insights into the
mechanisms of cardiac hypertrophy and may have significant
implications for the development of novel strategies for the treatment of pathological cardiac remodeling through targeting IRF7.
Perspectives
By using IRF7 knockout mice and cardiac-specific transgenic
mice, we have provided evidences sufficient to demonstrate
that IRF7 negatively regulates pressure overload–induced cardiac remodeling via suppressing the IKKβ–IκB–NFκB signaling pathway. These observations suggest that increasing of
IRF7 expression could serve as a lead target for the prevention
and treatment of cardiac hypertrophy and failure.
Acknowledgments
We thank Dr Taniguchi Tadatsugu (The University of Tokyo) for generously providing the IRF7 knockout mice. Rui Zhang, Li-Hua Gan,
Ya-Fen Lin, Xue-Yong Zhu, Zhang-Li Li, Miao Yin, and Xin Zhang
are gratefully acknowledged for providing technology help.
Sources of Funding
This work was supported by grants from the National Natural
Science Foundation of China (NO. 81100230, NO. 81070089, NO.
81200071, and NO. 81270306), National Science and Technology
Support Project (NO. 2011BAI15B02, NO. 2012BAI39B05, NO.
2013YQ030923-05, and NO. 2014BAI02B01), the National Basic
Research Program of China (NO. 2011CB503902), and the Key
Project of the National Natural Science Foundation (No. 81330005).
Disclosures
None.
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Novelty and Significance
What Is New?
• Interferon regulatory factor 7 (IRF7) is downregulated in murine hypertrophic hearts and neonatal rat cardiomyocytes treated with Ang II or
phenylephrine.
• IRF7 is a negative regulator of pressure overload–induced cardiac hypertrophy, fibrosis, and cardiac dysfunction.
• IRF7 regulates the development of cardiac hypertrophy via interacting with inhibitor of κB kinase-β and suppressing inhibitor of κB
­kinase-β–nuclear factor-κB signaling pathway.
What Is Relevant?
• Cardiac remodeling induced by pressure overload is close to what occurs
in hypertension.
• The molecular mechanisms of hypertension-induced cardiac remodeling
are not elucidated.
• This finding provides additional information for further understanding the
effect of IRF family proteins on cardiac remodeling and implications for
the development of strategies for the treatment of cardiac remodeling
and heart failure.
Summary
The absence of interferon regulatory factor 7 (IRF7) promotes
pressure overload–induced cardiac remodeling and dysfunction,
whereas cardiac-specific overexpression of IRF7 inhibits the hypertrophic response by interacting with inhibitor of κB kinase-β and
then suppressing the inhibitor of κB kinase-β–nuclear ­factor-κB
signaling. Based on the results of the present study, we propose
that IRF7 could be considered a therapeutic target for the prevention of cardiac hypertrophy.
Interferon Regulatory Factor 7 Functions as a Novel Negative Regulator of Pathological
Cardiac Hypertrophy
Ding-Sheng Jiang, Yu Liu, Heng Zhou, Yan Zhang, Xiao-Dong Zhang, Xiao-Fei Zhang, Ke
Chen, Lu Gao, Juan Peng, Hui Gong, Yingjie Chen, Qinglin Yang, Peter P. Liu, Guo-Chang
Fan, Yunzeng Zou and Hongliang Li
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Hypertension. 2014;63:713-722; originally published online January 6, 2014;
doi: 10.1161/HYPERTENSIONAHA.113.02653
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2014 American Heart Association, Inc. All rights reserved.
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Interferon Regulatory Factor 7 Functions as a Novel Negative Regulator of
Pathological Cardiac Hypertrophy
Ding-Sheng Jiang1,2#, Yu Liu1,2#, Heng Zhou1,2#,Yan Zhang1,2, Xiao-Dong Zhang3, Xiao-Fei Zhang3,
Ke Chen3, Lu Gao4, Juan Peng5, Hui Gong5, Yingjie Chen6, Qinglin Yang7, Peter P. Liu8,
Guo-Chang Fan9, Yunzeng Zou5*, Hongliang Li1,2*
1
Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, China;
Cardiovascular Research Institute of Wuhan University, Wuhan 430060, China; 3 College of Life
Sciences, Wuhan University, Wuhan 430072, China; 4Department of Cardiology, Institute of
Cardiovascular Disease, Union Hospital, Tongji Medical College, Huazhong University of Science
and Technology, Wuhan, PR China; 5Shanghai Institute of Cardiovascular Diseases, Zhongshan
Hospital, Fudan University. Shanghai, China; 6Cardiovascular Division, University of Minnesota,
Minneapolis, MN55455, USA; 7Department of Nutrition Sciences, University of Alabama at
Birmingham, Birmingham, AL35294-3360, USA; 8 University of Ottawa Heart Institute, Ottawa,
Ontario, Canada K1Y 4W7; 9Department of Pharmacology and Cell Biophysics; University of
Cincinnati College of Medicine, Cincinnati, OH 45267;
2
#Ding-Sheng Jiang, Yu Liu, and Heng Zhou are co-first authors
*Yunzeng Zou and Hongliang Li are contributed equally
Running title: IRF7 regulates cardiac hypertrophy
Correspondence to
Hongliang Li, MD, PhD
Department of Cardiology, Renmin Hospital of Wuhan University;
Cardiovascular Research Institute, Wuhan University
Jiefang Road 238, Wuhan 430060, PR China.
Tel/Fax: 86-27-88076990; E-mail: [email protected]
Supplemental Methods
Reagents
Antibodies against the following proteins were purchased from Cell Signaling Technology: IKKβ
(2370, 1:1000 dilution), p-IκBαser32/36 (9246, 1:1000 dilution), IκBα (4814, 1:1000 dilution),
p-p65ser536 (3033, 1:1000 dilution), p65 (4764, 1:1000 dilution), TNFα (3707, 1:1000 dilution),
MCP-1 (2029, 1:1000 dilution), Bcl2 (2870, 1:1000 dilution), Caspase3 (9662, 1:1000 dilution),
C-Caspase3 (9661, 1:1000 dilution) and p-IKKαser180/βser181(2681, 1:1000 dilution). Antibodies
against β-MHC (sc53090, 1:200 dilution), ANP (sc20158, 1:200 dilution), IRF7 (sc9083, 1:200
dilution), and Lamin B (sc6217, 1:200 dilution), were purchased from Santa Cruz Biotechnology. We
also used antibodies against GAPDH (Bioworld Technology; MB001, 1:10000 dilution), Bax
(Bioworld Technology; BS2538, 1:500 dilution); IL-1β (R&D Systems; AF-401-NA, 1:1000
dilution), Flag (Sigma; F3165, 1:1000 dilution), and Myc (Roche; 1:1000 dilution). The BCA protein
assay kit was purchased from Pierce. We used IRDye® 800CW-conjugated secondary antibodies
(LI-COR Biosciences) or Peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch
Laboratories) for visualization. Fetal calf serum (FCS) was purchased from Hyclone, all other
reagents were purchased from Sigma.
Mice Used in This Study
All experiments involving animals were approved by the Animal Care and Use Committee of
Renmin Hospital, Wuhan University. The animal models used for this study are described below.
IRF7 Knockout and Cardiac-Specific IRF7 Overexpressing Mice
Full-length mouse IRF7 cDNA (Origene, MC216147) was cloned downstream of the cardiac
α-myosin heavy chain (α-MHC) promoter. The construct was microinjected into fertilized mouse
embryos to generate cardiac-specific IRF7 transgenic (TG, C57BL/6J background) mice, the
successful generations of these mice were confirmed via PCR analyses of tail genomic DNA using
specific forward (5’-ATCTCCCCCATAAGAGTTTGAGTC-3’) and the reverse (5’-CCAGACTCGG
ATTCCAGTATGTG-3’) primers. IRF7 knockout mice (IRF7-/-, C57BL/6J background, RBRC01420)
were generously provided by Dr Taniguchi Tadatsugu (The University of Tokyo) and shipped by
REKEN1. IRF7-/- mice were identified by PCR analysis of tail genomic DNA using primer 1
(5’-GTGGTACCCAGTCCTGCCCTCTTTATAATCT-3), primer 2 (5’-TCGTGCTTTACGGTATCG
CCGCTCCCGATTC-3), and primer 3 (5’-AGTAGATCCAAGCTCCCGGCTAAGTTCGTAC-3).
The IRF7-/-, IRF7 TG mice and their wild-type littermates (aged 8 to 10 weeks old, with body
weights of 24-27 g) were used in the experiments.
Cardiac-Specific IκBαS32A/S36A-Overexpressing Mice and IκBαS32A/S36A/IRF7-/- Mice
The hemagglutinin-tagged full-length IκBαS32A/S36A super repressor (Addgene, 15264) cDNA was
cloned downstream of the cardiac α-MHC promoter. The construct was microinjected into fertilized
mouse embryos to generate cardiac-specific IκBαS32A/S36A super repressor transgenic mice which
were identified by PCR analysis of tail genomic DNA with forward (5’-ATCTCCCCCAT
AAGAGTTTGAGTC-3’) and reverse (5’-TTCTGGCTGGTTGGTGAT-3’) primers. We crossed
1 IRF7 knock-out with IκBαS32A/S36A transgenic to produce the IκBαS32A/S36A /IRF7+/- mice, which were
then crossed back with IRF7-/- mice to obtain IκBαS32A/S36A/IRF7-/- mice. The IκBαS32A/S36A transgene
and IκBαS32A/S36A/IRF7-/- mice were C57BL/6J background.
Cardiac-Specific Conditional IKKβS177E/S181E-Cre Transgenic and IKKβS177E/S181E-Cre/IRF7
Double Transgenic Mice
To get the IKKβS177E/S181E flox mice, we subcloned Flag-tagged, constitutively active, IKKβS177E/S181E
(Addgene, 11105) into the CAG-CAT-LacZ construct. This construct contains a CMV enhancer and
the chicken β-actin gene (CAG) promoter, which are linked to the chloramphenicol acetyltransferase
(CAT) gene and flanked by loxP sites. In the transgenic mice, IKKβS177E/S181E is blocked by the
presence of the CAT gene if the CAT sequence is excised by Cre recombinase, IKKβS177E/S181E is
expressed. The IKKβS177E/S181E flox mice were crossed with α-MHC–MerCreMer transgene mice
(MEM-Cre-TG) (Myh6-cre/Esr1, Jackson Laboratory, 005650) to obtain the IKKβS177E/S181E-Cre TG
mice. For cardiac specific-overexpression of IKKβS177E/S181E, tamoxifen (Sigma, T-5648) was
injected into 6-week old mice that were positive for the IKKβS177E/S181E gene that contained
MEM-Cre-TG. In particular, 200 mg of tamoxifen was dissolved in 1 ml of ethanol. Approximately
9.0 ml of corn oil (Sigma, C-8267) was added to this solution to attain a final tamoxifen
concentration of 20mg/ml. The emulsion was sonicated prior to its injection into mice, and 80
mg/kg/day of the emulsion was injected intraperitoneally (i.p.) daily for 5 consecutive days.
IRF7-TG mice were crossed with IKKβS177E/S181E-Cre TG mice to produce the IKKβS177E/S181E-Cre
/IRF7 double transgenic (DTG) mice. MEM-Cre-TG mice were used as controls. The MEM-Cre-TG,
IKKβS177E/S181E-Cre TG and DTG mice were C57BL/6J background. All experiments were performed
in male mice with body weights of 24-27g (8 to 10 weeks old).
Aortic Banding
Aortic banding (AB) was performed in accordance with previously described methods2-5. Briefly,
mice were anesthetized with pentobarbital sodium (50 mg/kg, i.p.; Sigma). The left chest was opened
to identify the thoracic aorta by blunting dissection at the second intercostals space after absence of
reflexes was observed. AB was achieved by tying the descending thoracic aorta against a 27G (in
mice with body weights of 24-25g) or 26G (in mice with body weights of 25-27g) needle with a 7-0
silk suture, and then removing the needle. In addition, Doppler analysis was performed to ensure that
adequate constriction of the aorta had been achieved. Pressure gradients (mmHg) were calculated
from the peak blood velocity (Vmax, m/s, PG=4xVmax2) measured by Doppler analyses across the
AB, which was equivalent in all groups of AB-operated mice. A similar sham operation was
performed, but without aortic constriction.
Echocardiography and Hemodynamic Measurements
Mice were anesthetized by using 1.5-2% isoflurane and echocardiography and hemodynamic
measurements were performed at the indicated times in accordance with previously described
methods2-4. Cardiac function and structure were determined by echocardiography using a
Mylab30CV (ESAOTE) machine with a 15-MHz probe. 2-D M-mode measurements of LV internal
diameter were obtained from at least three beats and then averaged. LV end-diastolic dimension
2 (LVEDD) and LV end-systolic dimension (LVESD) were measured at the time of largest and
smallest LV areas, respectively. LV fractional shortening (LVFS) was calculated using the following
formula: LVFS (%) = (LVEDD-LVESD)/LVEDD×100%. For hemodynamic measurements, a
1.4-French Millar catheter-tip micromanometer catheter (SPR-839; Millar Instruments) was inserted
through the right carotid artery into the left ventricle. The pressures and dP/dt were recorded
continuously with an Aria pressure-volume conductance system that was coupled with a
Powerlab/4SP A/D converter and stored, and displayed on a personal computer.
Histological Analysis
Hearts were excised from anesthetized (pentobarbital sodium; 50 mg/kg, i.p.) mice, fixed for > 24
hours in 10% formalin after being arrested with 10% potassium chloride solution, and then
embedded in paraffin. Subsequently, these hearts were sectioned transversely at 5µm. Sections were
stained with either hematoxylin-eosin (HE) for histopathology or picrosirius red (PSR) for the
collagen deposition. The myocyte cross-sectional area (CSA) was measured with a quantitative
digital image analysis system (Image-Pro Plus 6.0) using images that were captured from
FITC-conjugated wheat germ agglutinin (WGA, Invitrogen Corp) stained sections. More than 100
myocytes in the examined sections were outlined for each group of mice.
Cultured Neonatal Rat Cardiac Myocytes and Recombinant Adenoviral Vectors
Primary cultures of neonatal rat cardiomyocytes (NRCMs) were prepared in accordance with
previously described methods2. Briefly, cardiomyocytes from 1- to 2-day-old Sprague-Dawley rats
were isolated in phosphate-buffered saline (PBS) containing 0.03% trypsin and 0.04% type II
collagenase. After the fibroblasts were removed using a differential attachment technique, NRCMs
were seeded at a density of 2×105 cells/well onto six-well culture plates coated with gelatin in a
plating medium that consisted of DMEM/F12 medium supplemented with 20% FCS, BrdU (0.1 mM,
an inhibitor of the proliferation of fibroblasts) and penicillin/streptomycin. After 48 hours, the culture
medium was replaced with serum-free DMEM/F12 for 12 hours prior to stimulation with angiotensin
II (Ang II, 1 µM) or phenylephrine (PE, 100 µM). To over-express IRF7, the entire coding region of
the rat IRF7 gene under the control of the cytomegalovirus promoter was encompassed by
replication-defective adenoviral vectors. A similar adenoviral vector encoding the GFP gene was
used as a control. To knock-down IRF7 expression, three rat shIRF7 constructs were obtained from
SABiosciences (KR54505G), and the construct that produced the most significant decrease in IRF7
levels was selected from three generated AdshIRF7 adenoviruses for further experiments. AdshRNA
was used as the non-targeting control. The NRCMs were infected with adenovirus in diluted media at
a multiplicity of infection (MOI) of 100 for 24 hours.
Measurement of the Protein/DNA Ratio
For determining total cellular protein and DNA content, NRCMs were washed twice with PBS after
stimulated with Ang II or PBS for 48 hours. Then, 0.2N perchloric acid (1 ml) was added to each
well. The samples were centrifuged for 10 min at 10,000 g at 4°C. The precipitates were incubated
for 20 min at 60 °C with 250 µl of 0.3 N KOH. BCA Protein Assay Kit (Pierce, CAT: 23225) was
used to assess protein content, and Hoechst dye 33258 (Invitrogen, CAT: H3569) was used to detect
3 DNA content with salmon sperm DNA (Sigma, CAT: D1626) as a standard.
Atrial Natriuretic Peptide (ANP) Quantification Assays
Enzyme-linked immunosorbent assay (ELISA) was used to measure the production of ANP released
into the cell culture medium by using a Synergy HT reader (Bio-Tek). The cell culture supernatant
was analyzed for ANP using AssayMax Rat ANP ELISA Kit (Assay Pro, Winfield, MO, and Catalog
No. ERA7010-1) following the instructions of the supplier after centrifuging at 2000g for 10 min to
remove any debris. The absorbance was read at 450 nm immediately after the addition of reagents.
Immunofluoresce Analysis
The NRCMs stained with α-actinin antibody to determine cell surface areas by immunofluoresce
staining. In brief, cardiac myocytes were infected with different adenoviruses for 24 hours and
subsequently stimulated with 1 µM Ang II for 48 hours. The cells were then fixed with 3.7%
formaldehyde in PBS for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS
for 40 min, and stained with α-actinin at a dilution of 1:100 dilution using standard
immunofluorescence staining techniques.
Quantitative Real-Time PCR and Western Blotting
Total mRNA was extracted from ventricles and primary cells using TRIzol (Invitrogen), and cDNA
was synthesized with oligo (dT) primers using the Transcriptor First Strand cDNA Synthesis Kit
(Roche). Selected gene differences were confirmed by quantitative real-time PCR using SYBR
Green (Roche) and results were normalized against GAPDH gene expression. Total protein and
nuclear protein were extracted from the ventricles and primary cells in accordance with previously
described approaches2-4. Protein concentrations were determined using Pierce® BCA Protein Assay
Kit (Pierce). A total of 50 µg (for fluorescence) or 15µg (for chemiluminiscence) of protein was used
for SDS-PAGE (Invitrogen) followed by transfer to PVDF membrane (Millipore), which was then
incubated with various primary antibodies overnight at 4°C. After incubation with a secondary
IRDye® 800CW-conjugated antibody (Li-Cor Biosciences, at 1:10000 dilution) or
Peroxidase-conjugated antibody (Jackson ImmunoResearch Laboratories, at 1:10000 dilution),
signals were visualized with an Odyssey Imaging System (Li-Cor Biosciences) or FluorChem E
(Cell Biosciences). The specific protein expression levels were normalized to GAPDH for total
lysate or to Lamin B for nuclear proteins on the same nitrocellulose membrane.
IKKβ Activity Assays
NRCMs infected with AdIRF7 (AdGFP as a control) or AdshIRF7 (AdshRNA as a control) or
AdIRF7-ID grown in 60-mm dishes were lysed in extract buffer (20mM Tris, pH 7.4, 10 mM NaF,
150 mM NaCl, 1 mM Na-orthovanadate, 1% [wt/vol] Triton X-100, 10 µg/ml leupeptin, 10 mM
Na-pyrophosphate, 1 mM PMSF, 1 mM glycerophosphate, and 5 µg/ml aprotinin). The cell lysates
was centrifuged at 10,000g for 30 min at 4°C after incubated for 10 min on a rotating wheel (at 4°C).
250 µl of supernatants were incubated with 1 µg IKKβ antibodies (Cell Signaling Technology,
#8943) and 20 µl Protein A/G-agarose beads (11719394001, 11719386001, Roche) for 2h at 4°C to
immunoprecipitate endogenous IKKβ. The pelleted beads were washed three times with extract
4 buffer and twice with a buffer containing 5 mM MgCl2 and 50 mM Tris (pH 7.4) after centrifugation
on a benchtop centrifuge. 0.5 µg of purified GST-IκBα-1-55 was incubated with immunoprecipitates
containing IKKβ. Reactions were carried out in 5mM MgCl2, 50mM Tris (pH7.4), and 1mM
ATP-Na2 for 30 min at 30°C and ended by the addition of SDS-PAGE sample buffer and then
separated in SDS-PAGE followed by western blotting with indicated antibodies.
Luciferase Reporter Assays
A Cignal 45-Pathway Reporter Array (SABiosciences, CCA-901L) was used to quickly identify
relevant pathways for further analysis. Briefly, 50 µl Opti-MEM® with 200 ng GFP or IRF7 plasmid
was added to each well of the Cignal Finder Array plate, and the reporter assay constructs were
resuspended by gently tapping the side of the plate. Subsequently, 0.6 µl of Attractene Transfection
Reagent (QIAGEN) in 50 µl of Opti-MEM® was incubated at room temperature for 5 min and then
added to each well that contained 50 µl of the diluted nucleic acids. After a 20-minute incubation
form complex formation was completed, 50 µl of a prepared cell suspension (8 × 104 cells in
Opti-MEM® containing 10% of fetal bovine serum) was added to each well that contained
constructs-Attractene complexes. After 16 hours of transfection, the medium was changed to
complete growth medium (DMEM with 10% FBS, 0.1 mM NEAA, 1 mM Sodium pyruvate, 100
U/ml penicillin and 100 µg/ml streptomycin). At 24 hours after co-transfection, the luciferase assay
was performed using the Dual-Luciferase Reporter Assay System (Promega).
To demonstrate the change in NFκB signaling pathway, 40 µl of PBS containing 6x1011 pfu of
Ad-NFκB-Luc (VETOR BIOLABS, Catalog No. 1740) or 6x1011 pfu of Ad-pRL-Luc (VETOR
BIOLABS, Catalog No.1671, served as an internal control) was injected using a 30 G needle at
multiple locations in the ventricular wall of IRF7+/+, IRF7-/-, non-transgenic (NTG), and IRF7 TG
mice according to our published methods6. The luciferase reporter assay in vivo was done as
previously described7. Briefly, after 2 weeks of AB or sham surgery, mice infected Ad-NFκB-Luc
were euthanized with an overdose of pentobarbital solution (200 mg/kg intraperitoneal). The heart
was harvested, homogenized and lysed using tissue lysis buffer from Promega according to the
manufacturer’s protocol. Then, the lysates were centrifuged for 20 min at 10,000 g, and the
supernatant was used to determine both luciferase activity and protein content. Luciferase activity
was measured using a Luciferase Assay System kit (Promega), and a Single-Mode SpectraMax®
Microplate Reader (Molecular Devices). Protein content was determined using the BCA protein
assay kit and was used to normalize luciferase activity per sample.
Hydroxyproline Assay
Hydroxyproline assay was performed to measure total collagen content in ventricular tissue of
indicated group by modified method of Neuman and Logan8. Briefly, followed by vacuum drying of
the samples, the tissue samples were digested with 6N hydrochloric acid overnight at 110 °C. After
resuspending the samples in citric acetate buffer, a colored reaction was done by adding chloramine
T, isopropyl alcohol, and Ehrlich’s reagent. The samples were incubated at 25 °C for 18 h, and
intensity of the red color was measured at 558 nm using Synergy HT (Bio-Tek). Hydroxyproline
content in the unknown samples was calculated with the help of a standard curve. The amount of
collagen was calculated by multiplying hydroxyproline content by a factor of 8.2.
5 Immunoprecipitation
Full-length and deletion mutant human IKKβ expression vectors were constructed by cloning the
human IKKβ cDNA into pcDNA5/FRT/TO-FLAG mammalian expression vector, which was
modified from pcDNA5/FRT/TO (Invitrogen). For immunoprecipitation (IP), HEK293T cells
co-transfected with pcDNA5-Flag-IKKβ and pSicoR-EGFP-Myc-IRF7 for 48 hours were collected,
and lysed in 250 µl IP buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.5%
NP-40) supplemented with protease inhibitor cocktail (Roche). After being centrifuged at 13,000
g for 15 minutes, 250 µl of the sample was incubated with 1 µg specific antibodies and 10 µl
Protein A/G-agarose beads (11719394001, 11719386001, Roche). Subsequently, IP products were
washed with cold IP buffer for 5 to 6 times, and then separated by SDS-PAGE, and western
blotted using the indicated primary antibodies.
Glutathione S-transferase (GST) Pull-down Assay
Purification of GST-IRF7 from Escherichia. Coli lysates was performed and the pull-down assay
was carried out using glutathione-Sepharose 4B beads (GE Healthcare Bio-Sciences AB) according
to the manufacturer’s instructions. Briefly, the GST-IRF7 beads were incubated with
Flag-IKKβ-transfected HEK293T cell lysates (5 µg) in IP buffer (20 mM Tris-HCl, pH 8.0, 150
mM NaCl, 1 mM EDTA, and 0.5% NP-40 supplemented with protease inhibitor cocktail) for 4
hours at 4 °C. After being washed four times with IP lysis buffer (no cocktail), precipitated proteins
were separated in SDS-PAGE followed by western blotting with anti-Flag antibodies (Sigma).
Confocal Microscopy
HEK293T cells were plated on gelatin-coated coverslips in 24-well plates. Upon cotransfection with
pEGFP-myc-IRF7 and pCherry-IKKβ for 48 hours, cells were washed three times in PBS, fixed with
4% paraformaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100 in PBS for 5 minutes,
then incubated in Image-IT™ FX signal enhancer (I36933, Invitrogen) for 30 minutes. After being
washed with TBST for three times and stained with DAPI (1 g/ml, 15 minutes), the coverslides were
mounted with mounting solution (D2522, Sigma) and images were obtained with a confocal
laser-scanning microscope (Fluoview 1000; Olympus).
Statistical analysis
Data are represented in terms of the mean ± SD. A two-tailed Student’s t-test was used to compare
the means of two groups of samples, and one-way ANOVA test with LSD (equal variances assumed)
or Tamhane’s T2 (equal variances not assumed) was applied for multiple groups. A level of P<0.05
was considered statistical significance.
6 1.
2.
3.
4.
5.
6.
7.
8.
References
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GC, Li H. Role of interferon regulatory factor 4 in the regulation of pathological cardiac
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Wang AB, Fassett J, Chen Y, He YW, Yang Q, Liu PP, Li H. Cardiac-specific mindin
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Wilkins BJ, Dai YS, Bueno OF, Parsons SA, Xu J, Plank DM, Jones F, Kimball TR,
Molkentin JD. Calcineurin/nfat coupling participates in pathological, but not physiological,
cardiac hypertrophy. Circ Res. 2004;94:110-118.
Neuman RE, Logan MA. The determination of hydroxyproline. J Biol Chem.
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7 Supplemental Tables
Table S1. Anatomic and Echocardiographic Analysis in 10-12 Week Old Mice
IRF7+/+ mice
IRF7-/- mice
IRF7 NTG mice
IRF7 TG mice
（n=13）
（n=12）
（n=10）
（n=12）
Parameter
BW (g)
27.03±1.36
27.34±2.08
27.87±0.87
27.52±1.57
HW/BW(mg/g)
4.29±0.33
4.20±0.33
4.27±0.32
4.21±0.37
LW/BW(mg/g)
5.29±0.27
5.34±0.43
5.32±0.24
5.11±0.43
HW
6.34±0.36
6.28±0.30
6.46±0.47
6.28±0.46
/TL(mg/mm)
HR (beats/min) 492.20±35.90
576.20±55.54
558.20±48.26
557.00±35.24
LVEDD(mm)
3.60±0.14
3.56±0.21
3.56±0.09
3.58±0.18
LVESD(mm)
1.94±0.19
1.96±0.17
1.96±0.11
1.94±0.15
FS (%)
45.80±4.60
45.20±3.49
45.40±2.30
45.00±4.12
BW=body weight; HW=heart weight; LW=lung weight; TL=tibial length; HR=heart rate;
LVEDD=left ventricular end-diastolic diameter; LVESD=left ventricular end-systolic diameter;
FS=fractional shortening, All values are presented as the mean ± SD.
8 Table S2 Parameters in IRF7 transgenic mice (TG) and wild type littermates (NTG) at 8 weeks after
sham operation or AB
Parameter
NTG- Sham mice
TG -Sham mice
NTG-AB mice
TG -AB mice
（n=12）
（n=14）
（n=12）
（n=14）
BW (g)
28.84±1.20
27.96±2.06
29.08±1.64
28.20±1.88
HR
510.86±41.69
492.29±31.11
477.86±39.85
495.57±22.24
(beats/min)
92.24±5.33
109.53±8.33
128.14±10.07*
157.13±7.45*†
ESP(mmHg)
58.32±7.35
54.97±5.68
24.63±2.38*
36.85±6.92*†
EF (%)
dp/dt max
10298.57±1041.11 10287.75±605.18 6203.33±729.32* 8098.00±755.75*†
(mmHg/sec)
dp/dt min
-8947.00±771.72 -8855.00±1118.43 -4577.67±702.93* -6809.25±1157.63*†
(mmHg/sec)
LVESP=Left ventricular End-systolic Pressure. LVEF=Left ventricular ejection fraction. *P<0.05 vs.
NTG sham operation. †P<0.05 vs. NTG AB after 8 weeks AB.
All values are the mean ± SD.
9 Table S3 Parameters in IRF7-/- and IRF7+/+ mice at 2 weeks after sham operation or AB surgery.
Parameters
BW (g)
HR (beats/min)
ESP(mmHg)
Sham
IRF7+/+ mice
AB
IRF7-/- mice
IRF7+/+ mice
IRF7-/- mice
（n=12）
（n=14）
（n=12）
（n=13）
26.86±1.35
26.74±1.56
26.87±1.18
26.62±1.64
516.29±26.64
503.22±20.32
479.29±27.58
500.00±15.92
93.86±7.25
111.28±8.54
157.72±6.86*
152.35±13.02*
59.52±6.26
54.69±5.57
49.90±5.05*
27.05±4.48*†
EF (%)
dp/dt max
10498.14±755.89
10093.67±749.81 9639.29±1402.39
7294.88±898.34*†
(mmHg/sec)
dp/dt min
-9149.29±667.78
-8998.22±817.50
-8445.71±790.90 -5996.13±1023.53*†
(mmHg/sec)
*P<0.05 vs. IRF7+/+/ sham operation. †P<0.05 vs. IRF7+/+ AB after 2 weeks AB.
All values are presented as the mean ± SD.
10 Supplemental Figures
4
*
*
(B)
3
1.5
AdshRNA
AdshIRF7
AdGFP
AdIRF7
2
IRF7
54kDa
GAPDH
36kDa
1
0
IRF7/GAPDH
ANP in Culture Medium
(Fold Change)
(A)
1.0
P<0.05
P<0.05
0.5
0.0
PBS Ang II PE
Figure S1 (A) The ANP content in culture medium of NRCMs treated with Ang II, PE or PBS (n=3
independent experiments. *P<0.05 vs. PBS. (B) Confirmatory western blot for IRF7 knock-down
and overexpression in AdshIRF7-infected cardiomyocytes and in AdIRF7-infected cardiomyocytes.
Left: Representative blots. Right: Quantitative results.
11 (A)
α-MHC Promoter
Mouse IRF7
PolyA
1374 bp
IRF7/GAPDH
(B)
Tg4 Tg7 Tg12 Tg21 Wt1 Wt7 Wt12 Wt21
IRF7
54kDa
GAPDH
36kDa
(C)
6.6
6
3
2.5
7.6
3.9
1
0
(D)
(F)
50
40
NTG
TG
30
*
20
10
0
*
*#
#
Sham
ANP
AB
Sham
BNP
AB
#
Sham
AB
β-MHC
Relative mRNA Levels
(E)
Relative mRNA Levels
9
8
6
4
NTG
TG
*
*
#
2
0
*
#
#
Sham AB Sham AB Sham AB
CTGF
Collagen I
Collagen III
Figure S2 Overexpression of IRF7 Attenuates Pressure Overload-Induced Cardiac
Hypertrophy. (A) Schematic diagram of the construction of transgenic mice with full-length mouse
IRF7 cDNA under the control of α-myosin heavy chain promoter. (B) Representative blots and
quantitative results for determination of transgenic IRF7 levels expressed in the heart tissue from
four lines of TG and their control WT mice. (C) Pressure gradients (mmHg) were calculated from
the peak blood velocity (Vmax, m/s, PG=4xVmax2) measured by Doppler analyses across the AB,
which were equivalent among NTG and TG AB-operated mice. (D) The cumulative survival rate of
IRF7 NTG and TG mice after 8 weeks of AB (n=12-16 mice per experimental group). (E) Real-time
PCR analyses of the hypertrophic markers ANP, BNP and β-MHC induced by AB in the indicated
mice (n=4 mice per experimental group). (F) Real-time PCR analyses of the fibrotic markers CTGF,
Collagen I, and Collagen III in the indicated mice (n=4 mice per experimental group). *P<0.05 vs.
NTG/sham; #P<0.05 vs. NTG/AB.
12 (A)
IRF7+/+
IRF7-/-
IRF7
54kDa
GAPDH
36kDa
(C)
(D)
Relative mRNA Levels
(B)
40
30
20
0
2
AB
3
0
Sham
*
40
Sham AB
β-MHC
#
20
0
AB
*
IRF7+/+
IRF7-/-
*
1
#
#
Sham AB
BNP
60
#
2
Sham
AB
(G)
Relative mRNA Levels
Sham
IRF7+/+
IRF7-/-
*
Sham AB
ANP
FS (%)
*
LVESd (mm)
LVEDd (mm)
4
#
4
0
(F)
IRF7+/+
IRF7-/-
*
10
(E)
6
IRF7+/+
#
IRF7-/-
20
15
IRF7+/+
IRF7-/-
#
#
#
10
5
0
*
Sham
AB
CTGF
*
Sham
AB
Collagen I
*
Sham
AB
Collagen III
Figure S3 Ablation of IRF7 in Hearts Exaggerates Pressure Overload-induced Hypertrophy.
(A) Deletion of IRF7 was confirmed by western blot. IRF7+/+ stands for wild-type (WT) hearts, and
IRF7-/- represents knockout (KO) hearts. (B) Pressure gradients (mmHg) were equivalent among
IRF7+/+ and IRF7-/- AB-operated mice. (C) The cumulative survival rate of IRF7+/+ and IRF7-/- mice
after 8 weeks of AB (n=13-19 mice). (D) Real-time PCR analyses of the hypertrophic markers ANP,
BNP and β-MHC induced by AB in IRF7 KO and WT mice (n=4). (E) Parameters of
echocardiographic results for IRF7 KO and WT mice (n=7-9). (F) The cumulative survival rate of
IRF7+/+ and IRF7-/- mice after 2 weeks of AB (n=13-17 mice). (G) Real-time PCR analyses of the
fibrotic markers CTGF, collagen I, and collagen III in the indicated mice (n=4). *P<0.05 vs.
WT/sham; #P<0.05 vs. WT/AB. n indicates number of mice per experimental group.
13 Figure S4 IRF7 Suppresses NFκB Signaling. (A) The results of NFκB Signaling Pathway EpiTect
Chip qPCR Array were validated by Real-time PCR analyses (n=4 mice). *P<0.05 vs. WT/AB. (B)
The kinase activity of IKKβ after overexpression IRF7 in NRCMs, Left, Representive Western blot,
Right, Quantitative results; (n=3 independent experiments). (C) The kinase activity of IKKβ after
knockdown IRF7 in NRCMs, Left, Representive Western blot, Right, Quantitative results; (n=3
independent experiments). (D-E) Real-time PCR analyses of TNFα, MCP-1 and IL-1β induced by
14 AB in the (D) IRF7 TG and NTG mice and (E) IRF7 KO and WT mice (n=4 mice, *P<0.05 vs.
NTG or WT/Sham, #P<0.05 vs. NTG or WT/AB). (F) Left, Representative western blots; Right,
Quantitative results of the TNFα, MCP-1 and IL-1β protein levels in IRF7 TG and NTG mice 2
weeks after AB (n=3 independent experiments, *P<0.05 vs. NTG/Sham #P<0.05 vs. NTG/AB). (G)
Left, Representative western blots; Right, Quantitative results of the TNFα, MCP-1 and IL-1β
protein levels in IRF7 KO and WT mice 2 weeks after AB (n=3 independent experiments, *P<0.05
vs. WT/Sham #P<0.05 vs. WT/AB). (H-I) The TUNEL staining of heart tissue of NTG and TG (H);
or IRF7+/+ and IRF7-/- (I) at the indicated time point, TUNEL positive cells: Green; α-actinin: Red;
DAPI: Blue (n=4 mice). *P<0.05 vs. WT/AB or NTG/AB. (J-K) Upper, Representative western
blots showing the protein levels of Caspase3, C-Caspase3; Bax and Bcl2; Bottom, Quantitative
results of the Caspase3, C-Caspase3; Bax and Bcl2 in IRF7 NTG and TG (J) or WT and IRF7 KO
mice (K) after AB or Sham operation (n=3 independent experiments). *P<0.05 vs. NTG/Sham or
WT/Sham, #P<0.05 vs. NTG/AB or WT/AB.
15 (B)
Input
IgG
Input
IP:IRF7
IRF7
IRF7
IKKβ
IKKβ
(C)
(D)
Phospho
GST-IκBα 1-55
IRF7
IKKβ
PBS
DAPI
IP:IKKβ
Merge
Ang II
400
IKKβ Kinase
activity
GST-IκBα 1-55
IB: IKKβ
IgG
IP: anti-IKKβ
% Phospho IκBα
(A)
*
NS
*
300
200
100
0
PBS
Ang II
Figure S5 IRF7 Directly Interacts with IKKβ. (A) Western blot with IRF7 or IKKβ antibody after
co-IP of IRF7 from NRCMs whole cell lysates using IRF7 antibody. (B) Western blot with IRF7 or
IKKβ antibody after co-IP of IKKβ from NRCMs whole cell lysates using IKKβ antibody. (C)
Representative confocal images showing co-localization of IRF7 and IKKβ in H9C2 cells. (D) The
kinase activity of IKKβ after overexpression IRF7 or IRF7-ID in NRCMs, Left, Representive
Western blot, Right, Quantitative results. All the above results repeated at least three times.
16 (A)
(B)
IKKβ flox mice
LoxP
LoxP
CAG promoter
IKKβS177E/S181E-TG
α-MHC-IRF7
IKKβS177E/S181E
CAT
MCM-Cre-MCM
MEM mice
α-MHC promoter
Mer
Cre
DTG(IRF7/IKKβS177E/S181E)
Mer
(C)
Tamoxifen Treatment
LoxP
Recombined allele
IKKβ TG mice
(D)
MEM Cre
IRF7
54kDa
IKKβ
87kDa
GAPDH
36kDa
IKKβS177E/S181E
CAG promoter
AB (4 Weeks)
IKKβS177E/S181E-TG IRF7-TG
DTG
(E)
(F)
10
H&E
P<0.05
15
8
HW/TL(mg/mm)
HW/BW(mg/g)
H&E
P<0.05
6
4
2
6
3
LW/BW(mg/g)
Interstitial
15
P<0.05
10
5
0
12
(M)
8
4
0
LVEDd (mm)
6
4
2
0
P<0.05
P<0.05
P<0.05
400
200
4
3
2
P<0.05
P<0.05
P<0.05
P<0.05
P<0.05
P<0.05
1
0
0
P<0.05
(L)
5
5
P<0.05
ANP
4
40
3
30
2
BNP
1
0
β-MHC
P<0.05
P<0.05
450
300
150
0
6
4
2
0
P<0.05
P<0.05
P<0.05
P<0.05
P<0.05
P<0.05
CTGF
Collagen I
Collagen III
P<0.05
50
P<0.05
FS (%)
P<0.05
16
(K)
600
Relative mRNA Levels
P<0.05
LVESd (mm)
LV collagen volume(%)
20
µg Collagen/100mg Heart
(Hydroxyproline Quantitation)
(J)
(I)
600
Relative mRNA Levels
(H)
P<0.05
Cross Sectional Area(µm 2)
0
(G)
20
P<0.05
9
0
Perivascular
P<0.05
12
P<0.05
MEM Cre
20
IKKβS177E/S181E-TG
10
IRF7-TG
DTG
0
S177E,S181E
Eliminate the Protective Effects of IRF7
Figure S6 Overexpression of IKKβ
Transgenic after Aortic Banding. (A) TG constructs and Cre-mediated recombination event. The
structures of the target gene (CAG-CAT- IKKβS177E,S181E) and their recombined allele are shown. (B)
The breeding strategy for the production of IKKβS177E,S181E/IRF7 DTG mice. (C) Representative
western blots for determination of IRF7 and IKKβ levels in the heart of MEM-Cre,
IKKβS177E,S181E-TG, IRF7-TG, and DTG mice. (D) Histological analyses of HE and PSR staining of
MEM-Cre, IKKβS177E,S181E-TG, IRF7-TG, and DTG mice 4 weeks after the AB surgery (n=6). (E-G)
Results for the ratios of (E) HW/BW; (F) HW/TL; and (G) LW/BW in the indicated groups (n=8-13).
17 (H) Cell sectional area (n=100+ cells); (I) LV collagen volume (n=25+ fields); (J) Hydroxyproline
content per 100mg heart tissue (n=6 mice) in the indicated groups. (K) Real-time PCR analyses of
the hypertrophic markers ANP, BNP and β-MHC induced by AB in the indicated mice (n=4). (L)
Real-time PCR analyses of the fibrotic markers CTGF, collagen I, and collagen III in the indicated
mice (n=4). (M) Parameters of the echocardiographic results for the indicated groups (n=6-10). n
indicates number of mice per experimental group. n indicates number of mice per experimental
group.
18 (A)
(B)
α-MHC-IκBαS32A/S36A
IRF7-/-
IRF7-/-
IRF7-/+ ;
α-MHCIκBαS32A/S36A
IRF7
54kDa
IκBα
39kDa
GAPDH
36kDa
IκBαS32A/S36A/IRF7-/-
(D)
AB (4 Weeks)
(C)
(E)
P<0.05
HW/BW(mg/g)
10
H&E
8
6
4
2
(G)
15
12
P=0.395
9
6
3
0
(I)
P<0.05
6
P<0.05
4
2
P<0.05
P<0.05
0
6
600
P<0.05
400
200
0
P<0.05
LVEDd (mm)
µg Collagen/100mg Heart
(Hydroxyproline Quantitation)
(L)
4
2
0
P<0.05
P<0.05
ANP
BNP
P<0.05
β-MHC
5
P<0.05
4
3
2
Relative mRNA Levels
6
8
P<0.05
400
200
0
5
P<0.05
4
P<0.05
P<0.05
3
2
P<0.05
P<0.05
P<0.05
1
0
CTGF
50
P<0.05
P<0.05
P<0.05
Collagen I
Collagen III
P<0.05
40
FS (%)
P<0.05
(K)
LVESd (mm)
Relative mRNA Levels
LV Collagen Volume(%)
12
P<0.05
600
(J)
P<0.05
18
3
P<0.05
0
(H)
6
Cross Sectional
Area (µm 2)
LW/BW(mg/g)
Interstitial
9
0
18
Perivascular
P<0.05
12
0
(F)
H&E
P<0.05
15
HW/TL(mg/mm)
P<0.05
30
20
1
10
0
0
NTG
IκBαS32A/S36A-TG
IRF7-/IκBαS32A/S36AIRF7-/-
Figure S7 Overexpression of IκBαS32A,S36A Super-repressor Rescues the Phenotype of IRF7
Knock-out after Aortic Banding. (A) Breeding scheme for the production of IκBαS32A,S36A
-TG/IRF7-/- mice. (B) Representative western blots for determination of IRF7 and IκBα levels in the
heart of NTG, IκBαS32A,S36A transgenic, IRF7-/-, and IκBαS32A,S36A -TG/IRF7-/- mice. (C)
Histological analyses of HE and PSR staining of NTG, IκBαS32A,S36A transgenic, IRF7-/-, and
IκBαS32A,S36A -TG/IRF7-/- mice 4 weeks after the AB surgery (n=6). (D-F) Statistical results for the
ratios of (D) HW/BW; (E) HW/TL; and (F) LW/BW in the indicated groups (n=11-14). (G) Cell
sectional area (n=100+ cells); (H) LV collagen volume (n=40+ fields); (I) Hydroxyproline content
19 per 100mg heart tissue (n=6 mice) in the indicated groups. (J) Real-time PCR analyses of the
hypertrophic markers ANP, BNP and β-MHC induced by AB in the indicated mice (n=4). (K)
Real-time PCR analyses of the fibrotic markers CTGF, collagen I, and collagen III in the indicated
mice (n=4). (L) Parameters of the echocardiographic results for the indicated groups (n=6-11). n
indicates number of mice per experimental group.
20