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ONLINE SUPPLEMENT

Interferon Regulatory Factor 1 is Required for Cardiac Remodeling in Response

to Pressure Overload

Ding-Sheng Jiang1,2*, Liangpeng Li3*, Ling Huang1,2*, Jun Gong4, Hao Xia1,2,

Xiaoxiong Liu1,2, Nian Wan1,2, Xiang Wei5, Xuehai Zhu5, Yingjie Chen6, Xin Chen3,

Xiao-Dong Zhang4, Hongliang Li1,2#

1Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060,

China; 2Cardiovascular Research Institute of Wuhan University, Wuhan 430060,

China; 3Department of Thoracic and Cardiovascular Surgery, Nanjing Hospital

Affiliated to Nanjing Medical University, Nanjing 210006, China; 4College of Life

Sciences, Wuhan University, Wuhan 430072, China; 5Department of Thoracic and

Cardiovascular Surgery, Tongji Hospital, Tongji Medical College, Huazhong

University of Science and Technology，Wuhan 430030. 6Cardiovascular Division,

University of Minnesota, Minneapolis, MN55455, USA

*Ding-Sheng Jiang, Liangpeng Li, and Ling Huang are co-first authors

Running Title: IRF1 modulates cardiac remodeling

#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: lihl@whu.edu.cn

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Materials and Methods Reagents Antibodies against the following proteins were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): IRF1 (sc640, 1:200 dilution), iNOS (sc651, 1:200 dilution), ANP (sc20158, 1:200 dilution), and β-MHC (sc53090, 1:200 dilution). The GAPDH (MB001, 1:10000 dilution) antibody was purchased from Bioworld Technology (Harrogate, UK). The BCA protein assay kit was purchased from Pierce (Rockford, IL, USA). Fetal calf serum (FCS) was ordered from HyClone (Waltham, MA, USA). 1400W (W4262), the cell culture reagents, and other reagents were purchased from Sigma (St. Louis, MO, USA). Human Heart Samples Samples of failing human hearts were collected from the left ventricles of dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) patients undergoing heart transplants.1, 2 Control samples were obtained from the left ventricles of healthy heart donors. This study conformed to the principles outlined in the Declaration of Helsinki. All of the procedures involving human samples were approved by the Human Research Ethics Committee of Renmin Hospital of Wuhan University, and the samples were collected after informed consent. Experimental Mouse Models The animal protocols were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. Mouse IRF1 cDNA was cloned downstream of the cardiac α-myosin heavy chain (α-MHC) promoter. Transgenic mice (C57BL/6J background) were generated by microinjecting the α-MHC-IRF1 construct into fertilized mouse embryos, and they were identified via PCR analysis of tail genomic DNA. The primers used were 5′-ATCTCCCCCATAAGAGTTTGAGTC-3′ and 5′-AAGTCCTGCATGTAGCCTGGAAC-3′. The IRF1-knockout (IRF1-KO) mice (B6.129S2-Irf1tm1mak/J; Stock No. 002762) were purchased from the Jackson Laboratory (Bar Harbor, ME). The IRF1-KO mice were crossed with C57BL/6J mice to produce the F1 generation (IRF1 heterozygotes), which was backcrossed with the C57BL/6J strain to obtain the F2 generation (IRF1 heterozygotes) and then further backcrossed to the C57BL/6J background until the F9 generation (IRF1 heterozygotes). Male and female mice of the F9 generation were crossed with each other to produce IRF1-KO mice in a pure C57BL/6J background. Male α-MHC-IRF1 (TG) mice and their non-transgenic (NTG) littermates as well as IRF1-KO mice and their wild-type (WT) littermates (aged 8 to 10 weeks with body weights of 24-27 g) were used in these experiments. The iNOS-knockout mice (B6.129P2-NOS2tm1Lau/J; Stock No. 002609) were purchased from the Jackson Laboratory; iNOS knockout mice with pure C57BL/6J background were obtained by using the similar method as IRF1 knockout mice (C57BL/6J background). IRF1-TG mice were crossed with iNOS-KO mice to produce IRF1-TG/iNOS-KO mice in a C57BL/6J background. All of the animals were housed in an environment with controlled light cycles (12 hours light/12 hours dark), temperature, and humidity; food and water were available ad

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libitum. All of the animal experiments were performed by an experimenter who was blinded to the animals’ genotypes. Creation of an IRF1-Knockout Rat Model Transcription activator-like effector (TALE) DNA binding domains were designed using an earlier version of targeter designer (https://tale-nt.cac.cornell.edu/node /add/talen-old). TALE repeats were assembled using the “Unit Assembly” method,3 and were then used to construct a modified transcription activator-like effector nuclease (TALEN) expression vector with a T7 promoter for in vitro RNA production. The TALEN expression plasmids were linearized with PmeI (NEB, R0560L) and purified with phenol/chloroform to generate an RNase-free DNA template for in vitro transcription. Mature mRNA was transcribed from the above-mentioned templates using the mMessage mMachine T7 Ultra Kit (Ambion, AM1345), and it was purified using the RNeasy Mini Kit (Qiagen, 74104) following the manufacturer’s instructions. Each TALEN mRNA was diluted to 10 ng/µl with an injection buffer (10 mM Tris-HCl/0.1 mM EDTA, pH 7.4). A 2 pl aliquot of the solution was injected into cytoplasm of one cell stage embryos using the FemtoJet 5247 microinjection system under standard conditions. The surviving embryos were transferred to surrogate mothers.

For F0 offspring genotyping, a 670 bp DNA fragment overlapping the TALEN target site was amplified by PCR with the primers IRF1-FWD (5’-ATAAACAAGC AGCCAACACCTGC-3’) and IRF1-REV (5’-GTCAAGGACCTTTAGTTAGGACC- 3’) using an annealing temperature of 63°C. The completed PCRs were divided into two equal aliquots. The first aliquot was used as a template for sequencing to allow the identification of editing events. The other aliquot was purified and TA cloned following the manufacturer’s instructions (TaKaRa). Eight bacterial colonies per sample were chosen, amplified using M13-47/Rv-M primers, and sequenced. To genotype the F1 and F2 offspring, PCR products from F1 and F2 offspring were genotyped using the DNA restriction enzyme SphI (TaKaRa, 1246A). Aortic Banding Surgery Aortic banding (AB) was performed as described previously.4-6 Briefly, after anesthetization with sodium pentobarbital (50 mg/kg, Sigma) via an intraperitoneal injection, the left chest of each mice was opened to identify the thoracic aorta by blunting dissection at the second intercostal space. We then performed AB by tying a 7-0 silk suture around a specific needle on the thoracic aorta. After ligation, the 27-gauge needle was quickly removed, and adequate constriction of the aorta was confirmed by Doppler analysis. Sham-operated mice underwent the same surgical procedure without constriction of the aorta.

To produce pressure-overloaded cardiac hypertrophy in rats which were 40 days old and weighed 200-250 g, and aortic banding was performed by constricting the abdominal aorta at the suprarenal level as described in published method.7 Briefly, rats were anesthetized, the abdominal aorta was exposed under sterile conditions through a midline abdominal incision. The abdominal aortas were banded against a

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29-gauge needle that was removed before closing the abdominal cavity with a 7-0 silk suture. In addition, Doppler analysis was used to verify adequate constriction of the aortas. Sham-operated rats had an untied ligature placed in the same location. Echocardiography and Hemodynamic Evaluation Echocardiography and hemodynamic measurements was performed essentially as described previously.8, 9 Briefly, echocardiography was performed with a MyLab 30CV ultrasound system (Biosound Esaote Inc.) equipped with a 15-MHz transducer. End-systole and end-diastole were defined as the phase in which the smallest or largest LV area was obtained, respectively. The LV end-systolic diameter (LVESD), LV end-diastolic diameter (LVEDD), and LV wall thickness were measured from the LV M-mode tracing with a sweep speed of 50 mm/s at the mid-papillary muscle level.

For the hemodynamic analysis, a 1.4-F Millar micro-tip catheter (SPR-839; Millar Instruments, Houston, Texas) was inserted into the right carotid artery and advanced into the left ventricle. After stabilization for 15 minutes, the heart rate, pressure, and volume signals were recorded continuously using a pressure-volume conductance system (MPVS-300 Signal Conditioner, Millar Instruments, Inc.). The results were analyzed with Chart 5.0 software. Histological Analysis Hearts were excised, arrested in diastole with a 10% potassium chloride solution, fixed with 10% formalin, then dehydrated, and embedded in paraffin. Subsequently, the hearts were sectioned transversely close to the apex to visualize the left and right ventricles at 5 µm. The sections were stained with hematoxylin and eosin (HE) for histopathology or with picrosirius red (PSR) to identify collagen deposition and were subsequently visualized by light microscopy. To determine the myocyte cross-sectional area, sections were stained with FITC-conjugated wheat germ agglutinin (WGA, Invitrogen Corp) to visualize the membranes and with DAPI to observe the nuclei. More than 100 LV myocytes were examined in each group. Single myocytes and fibrillar collagen were visualized by microscopy and were measured using a quantitative digital image analysis system (Image-Pro Plus 6.0). Cardiomyocyte Culture and Infection with Recombinant Adenoviral Vectors Primary neonatal rat cardiomyocytes (NRCMs) were prepared from the hearts of 1- to 2-day-old SD rat pups as previously described.8, 10 To overexpress IRF1, the entire coding region of the rat IRF1 gene was subcloned into the pShuttle-cytomegalovirus (CMV) vector using the AdEasy XL Adenoviral Vector System (Stratagene). A similar adenoviral vector encoding the GFP gene was used as a control. To knock down IRF1 expression, we transfected cells with rat shIRF1 constructs (SABiosciences, KR06456G) or the AdshRNA negative control at a multiplicity of infection (MOI) of 100. After adenoviral infection, the cells were grown in DMEM/F12 medium containing 20% FCS, BrdU (0.1 mM, to inhibit fibroblast proliferation), and penicillin/streptomycin without serum for 24 hours, followed by treatment with angiotensin II (Ang II, 1 µmol/L) or PBS for an additional 48 hours. For surface area

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measurements, the cells were stained with a-actinin (Sigma) and were quantitatively analyzed using the Image-Pro Plus software (version 6.0). Quantitative Real-Time PCR and Western Blotting Quantitative real-time PCR and western blotting were performed as previously described.2, 8 Briefly, after total RNA was extracted from ventricles and primary cells using TRIzol reagent (Invitrogen), first strand cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis Kit (Roche, 04896866001). Quantitative real-time PCR was performed using the SYBR Green PCR Master Mix (Roche, 04887352001) to determine the expression levels of our genes of interest, and the results were normalized against GAPDH gene expression. The real-time PCR primers that were used are shown in Supplementary Table S1.

For western blotting, cardiac tissue and cultured cardiac myocytes were lysed in RIPA lysis buffer. Protein extracts (30 µg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and probed with various primary antibodies. After incubation with secondary antibodies for 1 hour at room temperature, the membranes were treated with ECL reagents (170-5061, Bio-Rad) prior to visualization using a FluorChem E imager (ProteinSimple, FluorChem E) according to the manufacturer’s instructions. The specific protein expression levels were normalized to the levels of GAPDH on the same PVDF membrane. Luciferase Reporter Assays The iNOS promoter luciferase (iNOS-luc) plasmid was obtained from Addgene (cat no. 19296), subcloned into the pGL3-basic vector (Promega) and subsequently transferred into by replication-defective adenoviral vectors. Primary neonatal rat cardiomyocytes (NRCMs) cultured in 24-well plates were co-infected with the AdiNOS-luc and AdIRF1 (AdGFP as the control) or AdshIRF1 (AdshRNA as the control) recombinant adenoviruses. After being stimulated with Ang II for 24 hours, the cells were harvested and lysed with 100 µl of passive lysis buffer (PLB, Promega). The cell debris was removed by centrifugation, and the supernatant was used for luciferase assays which were performed using a single-mode SpectraMax® microplate reader according to the manufacturer’s instructions. Chromatin Immunoprecipitation Assays In brief, after being transfected with HA-tagged IRF1 or the pcDNA3.1 control for 48 hours, cultured NIH3T3 cells were fixed with 1% formaldehyde for 10 minutes. The cross-linking reacting was halted by adding 1 ml of ice-cold glycine at room temperature, and the cells were washed, harvested, and sonicated on ice. After preclearing with protein G/A beads, the soluble chromatin was immunoprecipitated using an anti-HA antibody (ab9110, Abcam) or rabbit IgG (2729S, Cell Signaling Technology, Beverly, MA, USA) as a control at 4°C overnight with rotation. The chromatin was then eluted from the beads and reverse cross-linked overnight, and the precipitated DNA was purified using phenol/chloroform extraction. Quantitative real-time PCR was performed for 40 cycles to detect putative IRF1-binding regions

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(P1-P5). The PCR primers for the ChIP assays are listed in Supplementary Table S2. Statistical Analysis The data are presented as the mean ± SD. Differences among groups were assessed using an analysis of variance followed by a post hoc Tukey’s test. Comparisons between two groups were performed using Student's t-test. A value of P<0.05 was considered to indicate a statistically significant difference.

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References

1. Jiang DS, Zhang XF, Gao L, Zong J, Zhou H, Liu Y, Zhang Y, Bian ZY, Zhu LH, Fan GC, Zhang XD, Li H. Signal Regulatory Protein-Alpha Protects against Cardiac Hypertrophy Via the Disruption of Toll-Like Receptor 4 Signaling. Hypertension. 2014; 63:96-104.

2. Jiang DS, Wei X, Zhang XF, Liu Y, Zhang Y, Chen K, Gao L, Zhou H, Zhu XH, Liu PP, Bond Lau W, Ma X, Zou Y, Zhang XD, Fan GC, Li H. Irf8 Suppresses Pathological Cardiac Remodelling by Inhibiting Calcineurin Signalling. Nat Commun. 2014; 5:3303.

3. Huang P, Xiao A, Zhou M, Zhu Z, Lin S, Zhang B. Heritable Gene Targeting in Zebrafish Using Customized Talens. Nat Biotechnol. 2011;29:699-700.

4. Liu Y, Jiang XL, Liu Y, Jiang DS, Zhang Y, Zhang R, Chen Y, Yang Q, Zhang XD, Fan GC, Li H. Toll-Interacting Protein (Tollip) Negatively Regulates Pressure Overload-Induced Ventricular Hypertrophy in Mice. Cardiovasc Res. 2014; 101:87-96.

5. Zhang Y, Liu Y, Zhu XH, Zhang XD, Jiang DS, Bian ZY, Zhang XF, Chen K, Wei X, Gao L, Zhu LH, Yang Q, Fan GC, Lau WB, Ma X, Li H. Dickkopf-3 Attenuates Pressure Overload-Induced Cardiac Remodelling. Cardiovasc Res. 2014, In Press.

6. Chen K, Gao L, Liu Y, Zhang Y, Jiang DS, Wei X, Zhu XH, Zhang R, Chen Y, Yang Q, Kioka N, Zhang XD, Li H. Vinexin-Beta Protects against Cardiac Hypertrophy by Blocking the Akt-Dependent Signalling Pathway. Basic Res Cardiol. 2013; 108:338.

7. Cantor EJ, Babick AP, Vasanji Z, Dhalla NS, Netticadan T. A Comparative Serial Echocardiographic Analysis of Cardiac Structure and Function in Rats Subjected to Pressure or Volume Overload. J Mol Cell Cardiol. 2005; 38:777-786.

8. Jiang DS, Bian ZY, Zhang Y, Zhang SM, Liu Y, Zhang R, Chen Y, Yang Q, Zhang XD, Fan GC, Li H. Role of Interferon Regulatory Factor 4 in the Regulation of Pathological Cardiac Hypertrophy. Hypertension. 2013; 61:1193-1202.

9. Li H, Tang QZ, Liu C, Moon M, Chen M, Yan L, Bian ZY, Zhang Y, Wang AB, Nghiem MP, Liu PP. Cellular Flice-Inhibitory Protein Protects against Cardiac Remodeling Induced by Angiotensin Ii in Mice. Hypertension. 2010; 56:1109-1117.

10. Jiang DS, Luo YX, Zhang R, Zhang XD, Chen HZ, Zhang Y, Chen K, Zhang SM, Fan GC, Liu PP, Liu DP, Li H. Interferon Regulatory Factor 9 Protects against Cardiac Hypertrophy by Targeting Myocardin. Hypertension. 2014; 63:119-127.

 

 

 

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Supplementary Tables

Supplementary Table S1. The primers for Real-Time PCR.

Primer name Forward Primer Reverse Primer iNOS-Mouse TGCGCCTTTGCTCATGACATCGA ATGGATGCTGCTGAGGGCTCTGTT ANP-Mouse ACCTGCTAGACCACCTGGAG CCTTGGCTGTTATCTTCGGTACCGG BNP-Mouse GAGGTCACTCCTATCCTCTGG GCCATTTCCTCCGACTTTTCTC β-MHC-Mouse CCGAGTCCCAGGTCAACAA CTTCACGGGCACCCTTGGA CTGF-Mouse TGACCCCTGCGACCCACA TACACCGACCCACCGAAGACACAG Collagen I-Mouse AGGCTTCAGTGGTTTGGATG CACCAACAGCACCATCGTTA Collagen III-Mouse CCCAACCCAGAGATCCCATT GAAGCACAGGAGCAGGTGTAGA

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Supplementary Table S2. The PCR primers for chromatin immunoprecipitation (ChIP) assays. Primer name Primer M-iNOS-1F AGATGAGACTCTGGAAGGTTCT -1978 M-iNOS-1R CAGGAGCAACTACACTACGG -1802 M-iNOS-2F GGGTGTTGCCTGGATAAA -1654 M-iNOS-2R GAGAGATGCTGGGTAACCTG -1484 M-iNOS-3F ACCTCAGACAAGGGCAAA -1087 M-iNOS-3R GCCAGGAACACTACACAGAA -828 M-iNOS-4F AGGTAGGACATTATAATCCT -726 M-iNOS-4R ATGGGCTGTGCTGAGTGAAG -539 M-iNOS-5F CCTCCTGATGAATGTGTCCT -284 M-iNOS-5R TCACTCTGTGGTGTATCCTCA -77

M=mouse; F= forward; R=reverse

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Supplementary Table S3. Sequencing results of IRF1 mutant allele in 6 founder rats, revealing different indel mutations in the TALEN target site. The mutation region of founders IRF1 gene was PCR-amplified then sub-cloned into TA vector. SphI site is underlined. N indicates the base. Lines Allele Sequence Indel (bp)

Wild Type AGATCCCATGGAAGCATGCTGCCAAGCACGGTTGGGACATCAACAAGGATG 0

F 1-1 AGATCCCATGGAAGC------------ACGGTTGGGACATCAACAAGGATG △12

F 1-3 AGATCCCATGGAAGC-------CAAGCACGGTTGGGACATCAACAAGGATG △7

F 2-1(allele 1) AGATCCCATGGAAGC--------AAGCACGGTTGGGACATCAACAAGGATG △8

F2-1(allele 2) AGATCCCATGGAAGC-----------CACGGTTGGGACATCAACAAGGATG △12

F 2-2(allele 1) AGATCCCATGGAAGCA-------AAGCACGGTTGGGACATCAACAAGGATG △7

F 2-2(allele 2) AGATCCCATGGAAGC------------ACGGTTGGGACATCAACAAGGATG △12

F 2-3 AGATCCCATGGAAGC------------ACGGTTGGGACATCAACAAGGATG △12

Wild Type AGATCCCATGGAAGC(NNNNNNNNNNN)AAGGTCCTAACTAAAGGTCCTTGA 0

F 1-4 AGATCCCATGGAAGCA-------------CGGTCCTAACTAAAGGTCCTTGA △320(A TO C)

F=founder

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Supplementary Table S4. Germline transmission of the IRF1 knockout founders. Three of the 6 founders were bred to wild type rats to measure germline transmission. The number of F1 pup with wild type and mutant alleles is shown over total number pup for each founder. Specific modifications of the alleles are shown in above (Table 2).

Founders Wild Type

Transmitted Alleles

1 2

1-4 0 0 0

2-1 2/8 1/8 5/8

2-2 4/6 0 2/6

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Supplementary Figures

Supplementary Figure S1. Interferon regulatory factor 1 (IRF1) regulates angiotensin II-induced cardiomyocyte hypertrophy in vitro. IRF1 was knocked down in AdshIRF1-infected cardiomyocytes, whereas IRF1 was increased in AdIRF1-infected myocytes (n=3 independent experiments). Left, representative blots. Right, quantitative results.

 

 

 

IRF1

GAPDH

48kDa

37kDa

*

#

0

1

2

3

IRF1

/GA

PDH

(Fol

d C

hang

e)

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Supplementary Figure S2. The absence of interferon regulatory factor 1 (IRF1) attenuates pressure overload-induced cardiac hypertrophy. A, Representative western blots of IRF1 expression in heart tissues from wild-type (WT) and IRF1-knockout (IRF1-KO) mice (n=4 mice per group). B, 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 mice per experimental group). C, Real-time PCR analyses of fibrotic markers (collagen I, collagen III, and connective tissue growth factor [CTGF]) in the indicated groups (n=4 mice per experimental group). *P<0.05 vs. WT/sham; #P<0.05 vs. WT/AB.

AIRF1

GAPDH

48kDa

37kDa

WT IRF1-KO

B

**

*

# # #

0

2

4

6

ANP BNP β-MHC

WT Sham IRF1-KO ShamWT AB IRF1-KO AB

Rel

ativ

e m

RN

A Le

vels

C

** *

#

# #

0

2

4

6

CTGF collagen I collagen III

WT ShamIRF1-KO ShamWT ABIRF1-KO AB

Rel

ativ

e m

RN

A Le

vels

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Supplementary Figure S3. The overexpression of interferon regulatory factor 1 (IRF1) aggravates pressure overload-induced cardiac hypertrophy. A, Schematic diagram of the construction of transgenic (TG) mice with a full-length mouse IRF1 cDNA under the control of the α-myosin heavy chain (MHC) promoter. B, Representative blots for the determination of IRF1 protein expression in heart tissues from 4 lines of TG mice and their non-transgenic (NTG) littermates (n=4 mice per group). C, Quantitation of IRF1 expression in heart tissues from 4 TG and NTG mouse lines. D, Measurements of echocardiographic and hemodynamic parameters (LVEDD, LVESD, FS, EF, dP/dt min, and dP/dt max) in NTG and IRF1-TG mice (n=7-12 mice per experimental group). E, Real-time PCR analyses of the hypertrophic markers ANP, BNP, and β-MHC induced by AB in NTG and IRF1-TG mice (n=4 mice per experimental group). F, Real-time PCR analyses of fibrotic

IRF1

GAPDH

48kDa

37kDa

NTG

A

B

α‐MHC Promoter PolyAIRF1 cDNA

C

2.1 2.7 2.8 3.0

01234

NTG TG1 TG2 TG3 TG4

IRF1

/GAP

DH(F

old

Chan

ge)

*#

020406080

100

EF (%

)

NTGIRF1-TG

ABSham

D

EABSham

* #

0

5

10

15

dPdt

max

(x10

3

mm

Hg/s

ec)

NTGIRF1-TG

ABSham

*#

-15

-10

-5

0

dPdt

min

(x10

3

mm

Hg/s

ec)

NTGIRF1-TG

ABSham

* #

0

2

4

6

LVE

Dd(m

m)

NTGIRF1-TG

F

** *

#

# #

0

5

10

15

CTGF Collagen I Collagen III

NTG ShamIRF1-TG ShamNTG ABIRF1-TG AB

Rel

ativ

e m

RNA

Lev

els

* * *

#

#

#

0

6

12

18

ANP BNP β-MHC

NTG Sham IRF1-TG ShamNTG AB IRF1-TG AB

Rela

tive

mR

NA L

evel

s

ABSham

*#

0

2

4

6

LVES

d(m

m)

NTGIRF1-TG

* #

0

20

40

60

FS (%

)

NTGIRF1-TG

ABSham

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markers (collagen I, collagen III, and CTGF) in the indicated groups (n=4 mice per experimental group). *P<0.05 vs. NTG/sham; #P<0.05 vs. NTG/AB.

Supplementary Figure S4. Interferon regulatory factor 1 (IRF1) directly regulates the transcriptional activity of iNOS. A and C, Real-time PCR assays and representative western blots of iNOS in AdshIRF1 or AdshRNA adenoviral vector-infected cardiomyocytes treated with angiotensin II (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). B and D, Real-time PCR assays and representative western blots of iNOS in AdIRF1 or AdGFP adenoviral vector-infected cardiomyocytes treated with 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).

A B*

#

0

1

2

3

PBS Ang II

AdshRNAAdshIRF1

Rela

tive

iNO

S m

RNA

Le

vels *

#

0

1

2

3

4

PBS Ang II

AdGFPAdIRF1

Rel

ativ

e iN

OS

mR

NA

Leve

ls

iNOS

GAPDH

135kDa

37kDa

PBS Ang II

AdGFP AdIRF1 AdGFP AdIRF1

iNOS

GAPDH

135kDa

37kDa

PBS Ang II

shRNA shIRF1 shRNA shIRF1

C

DAng IIPBS

*#

0

1

2

3 AdshRNA AdshIRF1

iNO

S/G

APD

H(F

old

Chan

ge)

Ang IIPBS

*

#

0

2

4

6 AdGFP AdIRF1

iNO

S/G

AP

DH(F

old

Chan

ge)

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Supplementary Figure S5. Ablation of iNOS negates the detrimental effects of interferon regulatory factor 1 (IRF1) overexpression on cardiac hypertrophy in IRF1-TG/iNOS-KO mice. A, Schematic representation of the generation of IRF1-TG/iNOS-KO mice. B, The ratios of HW/BW, LW/BW, and HW/TL determined in the indicated groups after sham or AB treatment (n=11-14 mice per experimental group). C, Histological analyses of hematoxylin and eosin (HE) staining and PSR staining in the indicated groups 4 weeks after aortic banding (AB, n=6-8 per experimental group, scale bars=20 µm for HE, scale bars=50 µm for PSR ). D, Statistical results for the ratios of cross-sectional area (CSA, n=100+ cells per experimental group). E, LV collagen volumes in the indicated groups at 4 weeks after AB surgery (n=25+ fields per experimental group). F, Measurements of echocardiographic and hemodynamic parameters (LVEDD, LVESD, FS, EF, dP/dt min, and dP/dt max) in the indicated groups (n=6-12 mice per experimental group). NS: not significant; *P< 0.05 vs IRF1-TG/AB.

A

C

IRF1-TG iNOS-/‐

IRF1-TG/iNOS+/‐

IRF1-TG/iNOS-KO

IRF1-TG/iNOS+/‐

WT IRF1-TG

Sham (4 Weeks)

iNOS-KOIRF1-TG

/iNOS-KO WT IRF1-TG

AB (4 Weeks)

iNOS-KOIRF1-TG

/iNOS-KO

HE

HE

Peri

vasc

ular

Inte

rstit

ial

*

NS

ABSham

NS

ABSham

B

IRF1-TGWT

IRF1-TG /iNOS-KO

iNOS-KO

* *

D

E

NS

Cros

s S

ectio

nal

Are

a (µ

m2 )

ABSham

NS

02468

10

LV C

olla

gen

Vol

ume

(%)

*

*

0

200

400

600

ABSham

0

4

8

12

NS

ABSham

HW

/BW

(mg/

g)

0

6

12

18

LW/B

W(m

g/g)

0

5

10

15

HW/T

L(m

g/m

m)

F

0

20

40

60

80

100

EF

(%)

*NS

ABSham-15

-10

-5

0

dPdt

min

(x10

3

mm

Hg/

sec)

*NS

ABSham

IRF1-TGWT

IRF1-TG /iNOS-KO

iNOS-KO

0

5

10

15

dPdt

max

(x10

3

mm

Hg/s

ec)

*NS

ABSham

ABSham

*NS

0

2

4

6

LVED

d(m

m)

ABSham

*NS

0

1

2

3

4

5

LVES

d(m

m)

ABSham

*NS

0

20

40

60FS

(%)

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Supplementary Figure S6. Blockade of iNOS reverses the detrimental effects of IRF1 overexpression on cardiomyocyte hypertrophy in vitro. A, Representative images of cardiomyocytes stained with α-actinin; B, Quantitative results of cell surface area (n=50+ cells per group); C, Real-time polymerase chain reaction (PCR) evaluation of ANP and β-MHC mRNA levels in AdIRF1- and AdGFP-infected cardiomyocytes treated with Ang II in the presence or absence of the iNOS inhibitor 1400W for 48 hours. (n=4 independent experiments, NS, not significant)

AdGFP+Ang II AdGFP+Ang IIAdIRF1+Ang II AdIRF1+Ang II

1400WPBS

0

2000

4000

6000

8000

10000 PBS1400W

AdGFP+Ang II AdIRF1+Ang II

NS

Cel

l Sur

face

Are

a (µ

m2 )

0

1

2

3

4

5PBS1400W

AdGFP+Ang II

AdIRF1+Ang II

AdGFP+Ang II

AdIRF1+Ang II

Rela

tive

mR

NA

Lev

els

NS

NS

ANP β-MHC

A

B C