Cardiac hypertrophy is a potentially adaptive process

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1 Heart Interferon Regulatory Factor 1 Is Required for Cardiac Remodeling in Response to Pressure Overload Ding-Sheng Jiang, Liangpeng Li, Ling Huang, Jun Gong, Hao Xia, Xiaoxiong Liu, Nian Wan, Xiang Wei, Xuehai Zhu, Yingjie Chen, Xin Chen, Xiao-Dong Zhang, Hongliang Li Downloaded from by guest on November 4, 17 Abstract Interferon regulatory factor 1 (IRF1), a critical member of the IRF family, was previously shown to be associated with the immune system and to be involved in apoptosis and tumor suppression. However, the role of IRF1 in pressure overload induced cardiac remodeling has remained unclear. Using genetic approaches, we established a central role for the IRF1 transcription factor in the regulation of cardiac remodeling both in vivo and in vitro, and we determined the mechanism underlying this process. The expression level of IRF1 was remarkably altered in both failing human hearts and hypertrophic murine hearts. Transgenic mice with cardiac-specific IRF1 overexpression exacerbated aortic banding induced cardiac hypertrophy, ventricular dilation, fibrosis, and dysfunction, whereas IRF1- deficient (knockout) mice exhibited a significant reduction in the hypertrophic response. Similar results were observed in a global IRF1-knockout rat model. Mechanistically, the prohypertrophic effects elicited by IRF1 in response to pathological stimuli were associated with the direct activation of inducible nitric oxide synthase (inos). Furthermore, we identified 1 IRF1-binding site in the promoter region of the inos gene, which was essential for its transcription. To examine the IRF1-iNOS axis in vivo, we generated IRF1-transgenic/iNOS-knockout mice. IRF1 exerted profoundly detrimental effects in these mice; however, these effects were nullified by inos ablation. These data suggest the IRF1 inos axis as a crucial regulator of cardiac remodeling and that IRF1 could be a potent therapeutic target for cardiac remodeling. (Hypertension. 14;64:77-86.) Online Data Supplement Key Words: cardiomegaly fibrosis interferon regulatory factor-1 Cardiac hypertrophy is a potentially adaptive process that occurs in response to pressure or volume overload, including ischemic heart diseases, arterial hypertension, and valvular insufficiency. 1 The hallmarks of cardiomyocyte hypertrophy include enlarged cell size, enhanced protein synthesis, increased assembly and organization of sarcomeres, and re-expression of fetal cardiac gene programs.,3 Under conditions of prolonged overload, the initially compensatory hypertrophic response may become maladaptive, resulting in dilated cardiomyopathy, chronic heart failure, arrhythmias, and sudden death. 4,5 Recently, numerous regulatory pathways have been implicated in the transduction of hypertrophic signaling, including mitogen-activated protein kinase signaling, the phosphatidylinositol 3-kinase/AKT/glycogen synthase kinase-3β pathway, calcium-calmodulin dependent calcineurin phosphorylation, and many others. 6,7 These intracellular signaling pathways modulate transcriptional regulatory proteins, such as GATA4/6, myocyte enhancer factor, nuclear factor κb, and nuclear factor of activated T cells, suggesting that these factors that alter the expression of a diverse set of target genes are redeployed to facilitate hypertrophic growth. 8 1 Thus, identifying novel transcription factors that regulate cardiac hypertrophy is important for the development of new strategies to treat cardiac hypertrophy and heart failure. Interferon regulatory factors (IRFs) constitute a family of transcriptional factor comprising 9 members (IRF1 9) in mammalian cells, which were originally identified as transcriptional regulators of type I interferons. 11 Most recently, we performed intensive studies examining the IRF family members IRF3, IRF4, IRF7, IRF8, and IRF9, revealing the remarkable functional diversity of these transcription factors in the regulation of cardiac hypertrophy both in vitro and in vivo. 3,5,1 14 For example, IRF3, IRF7, IRF8, and IRF9 protected against the pressure overload induced hypertrophic response by inactivating extracellular signal regulated kinases 1 and, inhibitor of Received January 1, 14; first decision February 1, 14; revision accepted March 18, 14. From the Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, China (D.-S.J., L.H., H.X., X.L., N.W., H.L.); Cardiovascular Research Institute (D.-S.J., L.H., H.X., X.L., N.W., H.L.) and College of Life Sciences (J.G., X.-D.Z.), Wuhan University, Wuhan, China; Department of Thoracic and Cardiovascular Surgery, Nanjing Hospital Affiliated to Nanjing Medical University, Nanjing, China (L.L., X.C.); Department of Thoracic and Cardiovascular Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (X.W., X.Z.); and Cardiovascular Division, University of Minnesota, Minneapolis (Y.C.). D.-S. Jiang, L. Li, and L. Huang contributed equally to this work. The online-only Data Supplement is available with this article at /-/DC1. Correspondence to Hongliang Li, Department of Cardiology, Renmin Hospital of Wuhan University, Jiefang Rd 38, Wuhan 436, China. lihl@whu.edu.cn 14 American Heart Association, Inc. Hypertension is available at DOI: /HYPERTENSIONAHA

2 78 Hypertension July 14 Downloaded from by guest on November 4, 17 κb kinase-β, nuclear factor of activated T cell c1, and myocardin, respectively, whereas IRF4 aggravated pressure overload induced cardiac hypertrophy by activating the transcription of camp response element binding protein in the heart, indicating that the IRFs have more diverse biological functions than previously thought. Like all other IRF members, IRF1 exhibits structural similarities in their DNA-binding domains and have similar binding specificities, which regulates the transcriptional activity of various types of immune-active genes, such as interleukin-1 and inducible nitric oxide synthase (inos), particularly in immune cells. 15 This factor, however, not only functions as a regulator of the interferon system but also is a key transcription factor in the regulation of apoptosis and tumor suppression. 11 For example, Stang et al 16 demonstrated that IRF1 induced ligand-independent caspase-8 mediated apoptosis in breast cancer cells. In addition, hepatocytes from IRF1-deficient mice were resistant to apoptosis induction by ischemia reperfusion injury. 17 However, it has remained unclear whether the transcriptional effectors of IRF1 in the heart play a novel and critical role in the regulation of hypertrophic programming, particularly in response to stress stimuli. Hence, it is important to determine the consequences of deficiency and overexpression of IRF1 in the myocardium after chronic aortic banding (). In the present study, we first demonstrated that IRF1 was significantly altered in heart samples collected from human patients with dilated cardiomyopathy and hypertrophic cardiomyopathy and from mice subjected to. Next, we used both IRF1-overexpressing and IRF1-null mice to determine the role of IRF1 in the murine heart in response to pressure overload. Compared with the controls, a cardiac-specific IRF1 transgene exacerbated cardiac hypertrophy, fibrosis, and dysfunction on stimulation, whereas IRF1 deficiency dramatically suppressed this hypertrophic process. More importantly, for the first time, we generated IRF1-knockout (IRF1 / ) Sprague-Dawley (SD) rats and found that IRF1 played a similar role in response to pressure overload induced cardiac remodeling. Mechanistically, we provided evidence that inos is a crucial target of IRF1 because its transcriptional activity is strongly upregulated in the presence of IRF1. Thus, the IRF1 inos axis could be an important signaling pathway for cardiac remodeling in response to pressure overload. Materials and Methods The animal protocol was approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. 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. A detailed Methods section is available in the online-only Data Supplement, which includes detailed methods on the following: Reagents, Human Heart Samples, 8,14 Animal Models and Procedures, 18 1 Echocardiography and Hemodynamic Evaluation, 5,,3 Histological Analysis, Cardiomyocyte Culture and Infection with Recombinant Adenoviral Vectors, 5,1 Quantitative Real-Time PCR and Western Blotting, 5,14 Luciferase Reporter Assays, Chromatin Immunoprecipitation Assays, and Statistical Analysis. Results IRF1 Expression in Human Failing Hearts and Hypertrophic Mouse Hearts To explore the potential role of IRF1 in the development of cardiac hypertrophy and heart failure, we first analyzed whether IRF1 expression levels were altered in pathological hearts. Western blotting revealed that the IRF1 protein expression levels were significantly downregulated in hearts from patients with both dilated cardiomyopathy and hypertrophic cardiomyopathy compared with hearts from normal donors (Figure 1A and 1B). As shown in Figure 1A and 1B, the decrease in IRF1 was accompanied by increases in the hypertrophic markers β-myosin heavy chain (β-mhc) and atrial natriuretic peptide (ANP). Furthermore, in an experimental mouse model of -induced cardiac hypertrophy (as evidenced by elevated levels of β-mhc and ANP; Figure 1C), the protein expression level of IRF1 was progressively increased in the heart of wildtype (WT) mice subjected to from 3 to 7 days and was then markedly reduced and lower than basal levels at week 4 and week 8 compared with sham-operated hearts (Figure 1C). Collectively, the alterations of IRF1 levels in both dilated cardiomyopathy/hypertrophic cardiomyopathy human hearts and in pressure overload induced hypertrophic mouse hearts indicate that IRF1 may be involved in cardiac hypertrophy. Figure 1. Interferon regulatory factor 1 (IRF1) expression in failing human hearts and experimental mouse model of hypertrophy. A, Western blot analysis of β-myosin heavy chain (β-mhc), atrial natriuretic peptide (ANP), and IRF1 in normal donor hearts (n=6) and hearts collected from human patients with dilated cardiomyopathy (DCM; n=7; P<.5 vs donor hearts). B, Representative Western blots of β-mhc, ANP, and IRF1 in normal donor hearts (n=6) and hearts collected from human patients with hypertrophic cardiomyopathy (HCM; n=4; P<.5 vs donor hearts). C, Representative Western blots of β-mhc, ANP, and IRF1 in an experimental mouse model with aortic banding induced cardiac hypertrophy (n=4; P<.5 vs shams). The data are presented as the mean±sd and are representative of 3 independent experiments.

3 Jiang et al IRF1 Modulates Cardiac Remodeling 79 Downloaded from by guest on November 4, 17 IRF1 Aggravates Angiotensin II Induced Cardiomyocyte Hypertrophy After determining that IRF1 levels were altered in pathological hearts, we decided to investigate the functional contribution of IRF1 to cardiac hypertrophy. To address this issue, we first performed gain- and loss-of-function studies in cultured neonatal rat cardiomyocytes (NRCMs), a well-controlled experimental setting. Based on the results of Western blotting, the level of IRF1 was reduced by the infection of cardiomyocytes with adenoviral short hairpin IRF1 (AdshIRF1) and elevated by infection with adenoviral IRF1 (AdIRF1) (Figure S1 in the online-only Data Supplement). Notably, under basal conditions (PBS), neither the reduction nor the elevation of IRF1 had effects on cardiomyocyte morphology and cell size. However, exposure to angiotensin II (Ang II, 1 μmol/l) significantly reduced the cell surface area of AdshIRF1-infected cells by 36% (Figure A and C), whereas Ang II induced cardiomyocyte hypertrophy was enhanced by 49% in AdIRF1- treated NRCMs compared with controls (Figure A and B). Accordingly, the expression of hypertrophy markers (ANP and β-mhc) was profoundly suppressed in the AdshIRF1-infected cardiomyocytes (Figure E) and significantly enhanced in the AdIRF1-infected cells (Figure D) compared with the controls. Taken together, these observations indicate that IRF1 promotes the hypertrophic response in cardiomyocytes. Absence of IRF1 in the Heart Attenuates Pressure Overload Induced Cardiac Hypertrophy To examine the potential function of IRF1 during hypertrophy in vivo, we used a global IRF1 gene knockout (IRF1-KO) mouse model, in which IRF1 protein expression is absent in the heart. The absence of IRF1 in the mouse hearts was confirmed by Western blotting (Figure SA). Notably, at baseline, the IRF1-KO mice did not show any abnormalities in their morphometric or contractile functions (data not shown). However, at 4 weeks after, we observed significant increases in the cardiomyocyte cross-sectional area in wheat germ agglutinin stained left ventricular (LV) tissue sections from the WT mice. However, the increase in cross-sectional area after was blunted in the IRF1-KO mice (Figure 3A and 3B). In addition, the heart weight (HW)/body weight (BW) ratio, the HW/tibia length ratio, and the lung weight/ BW ratio were significantly lower in the IRF1-KO mice than in the WT mice at 4 weeks after (Figure 3C). Moreover, in response to, the IRF1-KO mice exhibited alleviation of cardiac dilation and dysfunction according to echocardiographic and hemodynamic parameters (ie, LV end-diastolic dimension, LV end-systolic dimension, fractional shortening, ejection fraction, dp/dt max, and dp/dt min ) compared with the WT mice (Figure 3D). Consistently, the expression levels of several hypertrophic markers, including ANP, brain natriuretic peptide, and β-mhc, were much lower in the IRF1-KO mice compared with the WT mice after a 4-week course of (Figure SB). To further define the effects of IRF1 deficiency on maladaptive cardiac remodeling, we evaluated fibrosis that is a classical feature of pathological cardiac hypertrophy. Paraffin-embedded slides were stained with picrosirius red to identify fibrotic changes. The extent of fibrosis was quantified based on the collagen volume by visualizing the total Figure. Interferon regulatory factor 1 (IRF1) regulates angiotensin II induced cardiomyocyte hypertrophy in vitro. A, Representative images of cardiomyocytes infected with adenoviral short hairpin IRF1 (AdshIRF1) or adenoviral IRF1 (AdIRF1) and treated with angiotensin II (Ang II; 1 μmol/l) for 48 hours (scale bars, 5 μm). B, Quantification of the cell surface area showing that the overexpression of IRF1 greatly increased Ang II induced hypertrophy compared with expression of adenoviral green fluorescent protein (AdGFP) (n=5+ cells per group, P<.5 vs AdGFP/PBS; #P<.5 vs AdGFP/Ang II). C, Quantification of the cell surface area showing that the knockdown of IRF1 significantly attenuated Ang II induced hypertrophy compared with that observed in adenoviral short hairpin RNA (AdshRNA) cells (n=5+ cells per group, P<.5 vs AdshRNA/PBS; #P<.5 vs AdshRNA/Ang II). D, Real-time polymerase chain reaction (PCR) evaluation of atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-mhc) mrna levels in IRF1-overexpressing cells and control AdGFP cells after treatment with either PBS or Ang II for 48 hours (n=4 independent experiments, P<.5 vs AdGFP/PBS; #P<.5 vs AdGFP/Ang II). E, Real-time PCR analysis of hypertrophy markers (ANP and β-mhc) in AdshIRF1 cells and control AdshRNA cells after treatment with PBS and Ang II for 48 hours (n=4 independent experiments, P<.5 vs AdshRNA/PBS; #P<.5 vs AdshRNA/Ang II).

4 8 Hypertension July 14 Downloaded from by guest on November 4, 17 Figure 3. The absence of interferon regulatory factor 1 (IRF1) attenuates pressure overload induced cardiac hypertrophy. A, Histological analysis of hematoxylin and eosin (HE) stained and fluorescein isothiocyanate conjugated wheat germ agglutinin (WGA) stained wildtype (WT) and IRF1-knockout (KO) hearts at 4 weeks after aortic banding () surgery (n=5 7 mice per experimental group; scale bars, μm). B, Statistical results for the cross-sectional area (n=1+ cells per experimental group). C, Statistical results for the ratios of heart weight (HW)/body weight (BW), lung weight (LW)/BW, and HW/tibia length (TL) in the indicated groups (n=1 18 mice per experimental group). D, Measurements of echocardiographic and hemodynamic parameters in WT and IRF1-KO mice (n=7 1 mice per experimental group). E, Picrosirius red staining of histological sections of the left ventricle (LV) in the indicated groups at 4 weeks after (n=5 7 mice per experimental group, scale bar, 5 μm). F, Quantification of the total collagen volume in WT and IRF1-KO mice after (n=5+ fields per experimental group). P<.5 vs WT/sham; #P<.5 vs WT/. dp/dt max indicates maximum rate of pressure increase; dp/dt min, minimum rate of pressure increase; EF, ejection fraction; FS, fractional shortening; LVEDD, LV end-diastolic dimension; and LVESD, LV end-systolic dimension. amount of collagen present in the interstitial and perivascular spaces. Our results showed that both interstitial and perivascular fibrosis were dramatically increased in WT hearts subjected to chronic but were markedly limited in the IRF1-KO hearts (Figure 3E and 3F). Subsequently, these results were confirmed by detecting the mrna levels of fibrotic markers, such as connective tissue growth factor, collagen I, and collagen III, which indicated an attenuated fibrotic response in the IRF1-KO hearts (Figure SC). Together, these results imply that IRF1 deficiency is capable of suppressing pressure overload induced cardiac hypertrophy and fibrosis. Overexpression of IRF1 Aggravates Pressure Overload Induced Cardiac Hypertrophy Given the evidence indicating that IRF1 deficiency has an inhibitory effect on cardiac hypertrophy in the setting of pressure overload, we sought to determine whether overexpression of IRF1 could augment pressure overload induced cardiac remodeling. To this end, we generated transgenic mice overexpressing IRF1 (hereafter referred to as ) targeted to cardiac myocytes using the α-myosin heavy chain promoter upstream of IRF1 (Figure S3A). We established 4 independent lines of transgenic mice (TG1, TG, TG3, and TG4), and Western blotting performed to determine the level of transgene expression revealed a spectrum of IRF1 overexpression, ranging from moderate overexpression in the TG1 mice to high levels in the TG4 mice (Figure S3B and S3C). Under basal conditions, a comparison of the hearts of nontransgenic littermates (hereafter referred to as NTG) and the IRF1-transgenic hearts revealed that both hearts had completely normal morphology and contractile function. We performed these experiments using mice from the highexpressing line (TG4). Morphological and functional cardiovascular analyses of the IRF1-transgenic mice with lower expression levels of IRF1 (line TG1) yielded identical results (data not shown). However, compared with their NTG littermates, the hearts of the mice showed a significant increase in cardiac hypertrophy 4 weeks after being subjected to, as indicated by increased HW/BW, HW/tibia length, and lung weight/bw ratios (Figure 4A). Similarly, the cardiomyocyte cross-sectional area was increased by 8% in the -operated mice compared with the NTGs (Figure 4B and 4C). The mice also displayed pronounced LV dilatation and severe contractile dysfunction in response to hypertrophic stimulation, as indicated by echocardiographic and hemodynamic analyses (Figure S3D). Furthermore, increased the expression of the hypertrophy markers ANP, brain natriuretic peptide, and β-mhc in the hearts of the NTG mice, and this expression was significantly promoted in the mice (Figure S3E). Furthermore, after chronic, the mouse hearts consistently exhibited increased picrosirius red staining in the interstitial and perivascular spaces, as well as increased collagen volumes compared with the control NTGs (Figure 4D and 4E). Subsequent analyses of the expression of fibrosis markers, including connective tissue growth factor, collagen I, and collagen III, also demonstrated an augmented response in the mice compared with the NTGs (Figure S3F). Taken together, these data demonstrate that IRF1 overexpression can promote maladaptive cardiac remodeling in response to pressure overload.

5 Jiang et al IRF1 Modulates Cardiac Remodeling 81 Downloaded from by guest on November 4, 17 Figure 4. The overexpression of interferon regulatory factor 1 (IRF1) aggravates pressure overload induced cardiac hypertrophy. A, The ratios of heart weight (HW)/body weight (BW), lung weight (LW)/BW, and HW/tibia length (TL) determined in NTG and IRF1-transgenic (TG) mice 4 weeks after sham or aortic banding () treatment (n=1 15 mice per experimental group). B, Histological analyses of hematoxylin and eosin (HE) staining and fluorescein isothiocyanate conjugated wheat germ agglutinin (WGA) staining in NTG and mice at 4 weeks after the surgery (n=6 7 mice per experimental group, scale bars, μm). C, Statistical results for the cross-sectional area (n=1+ cells per experimental group). D, Picrosirius red staining to detect fibrosis in NTG and mice subjected to sham or (n=6 7 mice per experimental group, scale bars, 5 μm). E, Statistical results for left ventricle (LV) collagen volume (n=5+ fields per experimental group). P<.5 vs NTG/sham; #P<.5 vs NTG/. IRF1 Mediates Cardiac Hypertrophy Through the Activation of the inos Pathway To determine how IRF1 was linked to the induction of the hypertrophic gene program, we next analyzed the potential molecular involvement of IRF1. Chen et al 4 have demonstrated the detrimental role of inos in cardiac remodeling. Specifically, they found that inos deletion protected against -induced cardiac hypertrophy and heart failure in association with decreased myocardial nitrotyrosine and 4-hydroxy--nonenal. 5 In agreement with these findings, we demonstrated that inos mrna expression was also increased in the myocardium at 4 weeks after (Figure 5A and 5B). Notably, the increase in inos mrna expression was significantly reversed in the IRF1-KO mice compared with the WT controls after pressure overload (Figure 5A), whereas the inos elevation was dramatically enhanced in the cardiacspecific mice (Figure 5B). Western blot analysis further showed that after, IRF1-KO diminished the protein expression of inos (Figure 5C); in sharp contrast, enhanced the expression of inos (Figure 5D). To further confirm the positive correlation between IRF1 and inos expression in the in vitro paradigm, we decreased or increased the expression of IRF1 in vitro by infecting cultured NRCMs with either AdshIRF1 or AdIRF1, followed by treatment with 1 μmol/l Ang II for 48 hours. Similar to the in vivo observations, the level of inos was reduced in the AdshIRF1 cells compared with the control adenoviral short hairpin RNA cells (Figure S4A and S4C), whereas the overexpression of IRF1 enhanced the level of inos compared with that observed in the control cells (Figure S4B and S4D). These findings indicate that IRF1 may exert its prohypertrophic effects through the regulation of inos. Next, we measured the promoter activity of inos via a luciferase reporter system. NRCMs were infected with either AdIRF1 to overexpress IRF1 or AdshIRF1 to knockdown IRF1. Subsequently, these infected cardiomyocytes were exposed to 1 μmol/l Ang II for 48 hours. Our results showed that compared with the controls, AdshIRF1 increased the inhibition of transcription from the inos promoter in cells treated with Ang II, whereas inos transcription was largely aggravated by the expression of AdIRF1 (Figure 5E). These results indicate that the transcription of inos is promoted by IRF1 expression. Considering that IRF1 primarily acts as a transcription factor, we deduced that IRF1 may modulate cardiac hypertrophy through the direct activation of inos transcription. To investigate this possibility, we performed chromatin immunoprecipitation of hemagglutinin-irf1 in cultured National Institutes of Health 3T3 cells, followed by quantitative real-time polymerase chain reaction of the inos promoter ( to +1 bp around the transcription start site; Figure 5F). We found that hemagglutinin-irf1 chromatin immunoprecipitations were enriched for a conserved inos promoter region (P3) but not for other regions (P1, P, P4, and P5), suggesting that the P3 region contains the primary site for IRF1 binding in vitro (Figure 5G). Thus, IRF1 exerts its prohypertrophic effects through direct binding to the inos promoter to regulate its expression. IRF1-Mediated Detrimental Effect on Cardiac Hypertrophy Is inos Dependent After determining that IRF1 is capable of promoting the transcription of inos, we further investigated whether inos is indispensable for IRF1-dependent cardiac hypertrophy. To address this issue, mice were crossed with inos-ko mice to generate /inos-ko mice (Figure S5A). The generated /inos-ko mice grew normally and were active and fertile. As shown in Figure S5C, the /inos-ko mice displayed significant attenuation of cardiac hypertrophy at 4 weeks after compared with the mice, as evidenced by a reduction in cardiomyocyte size (hematoxylin and eosin staining; Figure S5D) and by the lower ratios of HW/BW, HW/tibia length, and lung weight/bw (Figure S5B). Moreover, the /inos-ko mice showed alleviated cardiac function in response to pressure overload determined by measurements of echocardiographic and hemodynamic parameters (ie, LV end-diastolic dimension, LV end-systolic dimension, fractional shortening, ejection fraction, dp/dt max, and dp/dt min ; Figure S5F), further supporting the theory that inos is the primary target of IRF1

6 8 Hypertension July 14 Downloaded from by guest on November 4, 17 Figure 5. Interferon regulatory factor 1 (IRF1) directly regulates the transcriptional activity of inducible nitric oxide synthase (inos). A and C, Real-time polymerase chain reaction (PCR) assays and representative Western blots of inos in hearts from IRF1-knockout (IRF1-KO) mice 4 weeks after aortic banding (; n=4 mice per experimental group, P<.5 vs wild type [WT]/sham; #P<.5 vs WT/). B and D, Real-time PCR assays and representative Western blots of inos in hearts from IRF1-transgenic () mice at 4 weeks after (n=4 mice per experimental group, P<.5 vs NTG/sham; #P<.5 vs NTG/). E, inos promoter activity was detected via a luciferase assay (n=4 independent experiments; P<.5 vs adenoviral short hairpin RNA (AdshRNA)/PBS or adenoviral green fluorescent protein(adgfp)/ PBS; #P<.5 vs AdshRNA/angiotensin II [Ang II] or AdGFP/Ang II). F, Schematic diagram of 5 pairs of primers (P1 P5) targeted against the 5 putative interferon-stimulated response elements (ISREs) in the inos promoter. G, Chromatin immunoprecipitation experiments were performed to measure the relative enrichment of IRF1 binding at the 5 putative ISREs (n=4 independent experiments). in cardiac hypertrophy. Accordingly, the extent of fibrosis was strongly mitigated in the /inos-ko mice than in the mice (Figure S5C and S5E). In this regard, the /inos-ko mice again did not differ significantly from the inos-ko mice (Figure S5B S5F). In addition, the prohypertrophic effects mediated by IRF1 overexpression were abolished when inos was deleted (Figure S5B S5F), demonstrating that the prohypertrophic effects of IRF1 depend on the presence of inos. To further validate these results, we evaluated whether the detrimental effect of IRF1 could be reversed by blocking inos with a pharmacological inos inhibition (14 W) in vitro. AdIRF1- and adenoviral green fluorescent protein-infected cardiomyocytes were preincubated (1 hour) with 1 μmol/l of 14 W before being treated with Ang II (1 μmol/l). Our results showed that 14 W treatment significantly reversed the detrimental effects of IRF1 overexpression on Ang II induced cardiomyocyte hypertrophy, as evidenced by decreased cell surface area (Figure S6A and S6B) and expression of hypertrophy markers (ANP and β-mhc; Figure S6C), which were consistent with our in vivo results obtained from / inos-ko mice. Collectively, our data demonstrate that inos is required for the prohypertrophic effect of IRF1. Loss of IRF1 in Rat Inhibits the -Induced Hypertrophic Response Given the profound effects of both the absence and overexpression of IRF1 on cardiac hypertrophy in mice, we used the transcription activator like effector nuclease (TALEN) technology for efficient site-specific gene modification to create a novel knockout rat line. Specifically, the IRF1 gene was selected for TALEN-mediated gene inactivation. To generate IRF1-knockout rats, we created a pair of TALEN constructs that were specifically targeted to IRF1 exon 3, which is immediately downstream of the translational start site in the IRF1 cdna sequence (Figure 6A). Mature mrna was transcribed in vitro from linearized TALEN plasmids, and mixtures of mrnas from the TALEN constructs were microinjected into Ntac:SD rat embryos at the single-cell stage. The surviving embryos were then transferred into pseudopregnant females. After screening 9 live births of F rats using DNA sequencing, 6 founders with double peak traces were identified (Figure 6B). DNA sequence analysis showed that 8 distinct mutations were identified from 6 individual founders (Table S3). Subsequently, 3 founders (1-4, -1, and -) were backcrossed to the WT Ntac:SD strain to obtain the F1 generation. Three alleles were transmitted through the germline, whereas 1-4 and - (allele 1) failed to be inherited by the F1 offspring (Table S4). Founder -1 (allele 1), which contained an allele with an 8-bp deletion, was selected to establish an IRF1 /+ rat strain. As illustrated in Figure 6C, after digested with SphI, both the 67-bp and 335-bp products were generated from the heterozygous (IRF1 /+ ) samples, and only a 335-bp product was generated from the WT samples (IRF1 +/+ ). Homozygous (IRF1 / ) animals were obtained by sibling matings between heterozygous (IRF1 /+ ) littermates. Polymerase chain reaction amplification and direct sequencing of the homozygous (IRF1 / ) samples further revealed an 8-bp deletion in the genomic DNA when the sequences were compared with those generated from the WT samples (Figure 6D), indicating that

7 Jiang et al IRF1 Modulates Cardiac Remodeling 83 Downloaded from by guest on November 4, 17 Figure 6. Interferon regulatory factor 1 (IRF1) deficiency in rat hearts attenuates aortic banding () induced cardiac hypertrophy. A, Schematic of the Sprague-Dawley (SD) rat IRF1 gene (E3, 1 bp). The translation start site (ATG) is located in E, the transcription activator like effector nuclease (TALEN) target sites are underlined, and the SphI restriction site in the spacer is highlighted in red. B, Representative results from the DNA sequencing of founders. The sequencing chromatogram of heterozygous mutants revealed an indel that resulted in double peak traces. C, Agarose gel photograph illustrating genotyping results obtained via SphI digestion of polymerase chain reaction (PCR) products from wild-type (+/+) and heterozygous (+/ ) rats. D, DNA sequence chromatograms of IRF1- forward+irf1-reverse PCR products illustrating the 8 bp deletion in the homozygous (IRF1 / ) sample. The primer used for sequencing was IRF1-forward. E, The ratios of heart weight to body weight (HW/BW), lung weight (LW)/BW, and HW/tibia length (TL) were determined in IRF1 +/+ and IRF1 / rat 4 weeks after sham or aortic banding () treatment (n=11 rats per experimental group). F, Histological analysis of heart sections from IRF1 +/+ and IRF1 / rats at 4 weeks after (n=6 rats per experimental group). Heart cross-sections were stained with hematoxylin and eosin (HE), indicating hypertrophic growth (top row: scale bars, μm). Wheat germ agglutinin (WGA) staining was performed to identify cell boundaries (second row: scale bars, μm), and fibrosis was detected by picrosirius red staining (third and fourth rows: scale bars, 5 μm). G, Quantification of the cardiomyocyte cross-sectional area in sham- or -induced IRF1 +/+ and IRF1 / rat hearts (n=1+ cells per experimental group). H, Left ventricle (LV) collagen volume in the indicated groups at 4 weeks after surgery (n=5+ fields per experimental group). I, Measurements of echocardiographic parameters in IRF1 +/+ and IRF1 / rats (n=6 rats per experimental group). P<.5 vs IRF1 +/+ /sham; #P<.5 vs IRF1 +/+ /. E indicates exon; FS, fractional shortening; LVEDD, LV enddiastolic dimension; and LVESD, LV end-systolic dimension. the entire IRF1 protein was disrupted because of a frame shift mutation and premature termination. The IRF1-knockout SD rats (hereafter referred to as IRF1 / ) grew normally and were active and fertile. We then determined whether the absence of IRF1 in rat hearts had an effect on cardiac hypertrophy similar to that observed in mice. In this model, at 4 weeks after, the ratios of HW/BW, lung weight/bw, and HW/ tibia length were dramatically lower in the IRF1 / rats compared with the IRF1 +/+ rats (Figure 6E). In addition, compared with the controls, the IRF1 / rats exhibited a blunted hypertrophic response to, as indicated by a smaller ventricular cross-sectional area and decreased cardiac fibrosis (Figure 6F 6H). Accordingly, cardiac structure and function, as measured by LV end-diastolic dimension, LV end-systolic dimension, and fractional shortening, were significantly improved in the IRF1 / rats compared with the IRF1 +/+ rats (Figure 6I). Collectively, the findings from this novel IRF1 / rat model, consistent with findings obtained with traditional mouse IRF1 knockout, further confirm that IRF1 is generally required for cardiac remodeling in response to pressure overload. Discussion Cardiac hypertrophy can be viewed as a disorder of gene regulation, and cardiac transcription factors are key players that regulate inducible gene expression in cardiac myocytes. 6 Thus, the identification of such novel modulators and the elucidation of their regulatory mechanisms are highly warranted. Despite its antiproliferative role in oncogenesis, it remains unclear whether IRF1 orchestrates the hypertrophic transcriptome in cardiomyocytes. In the present study, we used both gain- and loss-offunction approaches to thoroughly decipher the role of IRF1 in chronic pressure overload induced cardiac remodeling. Our data revealed that the absence of IRF1 reduced pressure overload induced hypertrophy, fibrosis, and cardiac dysfunction; conversely, the overexpression of IRF1 rendered the mice more prone to cardiac remodeling. Notably, the blunted hypertrophic response of hearts under pressure overload was also more pronounced in rats lacking IRF1 than IRF1 +/+ rats. Mechanistically, we demonstrated that after, IRF1 binds to the promoter region of inos, thereby initiating its transcriptional activity. Furthermore, in cultured NRCMs, inos was both necessary and sufficient for IRF1-mediated cardiac hypertrophy. Thus, we speculated that in response to pressure overload, IRF1 upregulates inos expression to promote cardiac hypertrophy. More importantly, the detrimental effect of IRF1 was abolished in /inos-ko mice. Therefore, targeting the IRF1 inos axis could be a crucial therapeutic strategy for preventing cardiac hypertrophy and heart failure.

8 84 Hypertension July 14 Downloaded from by guest on November 4, 17 IRF1, as a transcription activator for type I interferons, was initially found to be restricted to immune cells. 15,7 Previous studies have demonstrated the presence of IRF1 in cardiomyocytes, neurons, and other organs and tissues. 8 Intriguingly, Alexander et al 9 reported that IRF1 mrna expression was selectively upregulated in the ischemic brain after middle cerebral artery occlusion in rodents and humans. In addition, IRF1 could be induced after hypoxia or after liver and renal ischemia reperfusion injury. 17 It is important to note here that Yu et al 3 observed that interleukin-18 promotes pressure overload induced cardiac fibrosis and dysfunction by regulating IRF1 expression. However, its relevance with such cardiac remodeling remains largely unknown. In the present study, we found that IRF1 expression level was strikingly varied in failing human hearts and hypertrophic mouse hearts. More importantly, using IRF1-knockout and cardiac-specific IRF1 overexpression mouse model, we found that IRF1 could regulate pressure overload induced cardiac hypertrophy and fibrosis positively. Previous studies showed that IRF1 expression could be regulated by multiple mechanisms. 15 In rodent and human islet cells, interferon-γ could induce IRF1 expression to increase nitric oxide production. 31 Besides, IRF1 can be viewed as a transcription factor that is also regulated by cytokines that participate in Th1 responses. 3 Further studies are needed to clarify the mechanisms involved in IRF1 expression in heart. Initial studies on transcriptional regulation of the interferon-α and interferon-β genes suggested that IRF1 act as transcriptional activators of gene expression. 33 In addition, IRF1 induces the transcription of many genes (eg, PKR, STAT1, and WAF1) during oncogenesis 34 and regulates the expression of inos in macrophages. 35 Notably, Bachmaier et al 36 showed that, in an experimental mouse model of inflammatory heart disease, IRF1 controls inos expression and NO synthesis, which might exert crucially regulative effects in the process of cardiac hypertrophy. Interestingly, in our present study, inos was regulated by IRF1 in hypertrophic hearts. Notably, under basal conditions, neither the overexpression nor the ablation of IRF1 in mouse hearts altered the mrna and protein levels of inos. Inversely, under pressure overload, the inos levels were significantly increased in the mice and reduced in the IRF1-KO mice. Thus, our data indicate that the IRF1-mediated regulation of inos might be stress dependent. Using a bioinformatics approach, we identified a single putative IRF1-binding site in the promoter region of inos. Luciferase reporter assays further showed that IRF1 increased the transcriptional activity of inos by binding to its promoter. Thus, the IRF1-mediated regulation of cardiac hypotrophy may be directly associated with its downstream target, inos. This hypothesis is supported by the findings that the /inos-ko mice still exhibited a reduced hypertrophic response compared with the mice, and a blockade of inos activation offset the IRF1-elicited hypertrophic response. Such findings are consistent with Zhang et al 5 studies showing that pressure overload induced cardiac hypertrophy was attenuated in inos / mice. Moreover, it is well documented that inos protein expression is substantially upregulated in patients with heart failure. 37 Therefore, we speculate that the detrimental role of IRF1 in pathological cardiac hypertrophy is dependent, at least in part, on the activation of inos. The close physiological characteristics between rat and human beings provide highly predictable models for the clinical research on human diseases, especially on complex diseases, including cardiovascular, diabetic, and autoimmune diseases. 38 In cardiovascular research, rats have been used extensively in studies of hypertension, such as spontaneously hypertensive rats. 39 In the past, the production of genetically engineered rats was severely hampered by technical limitations. 4 Recently, advances in gene expression and transgenic systems such as TALEN technology have allowed the direct introduction of genome modifications at targeted genomic loci, enabling efficient gene knockout experiments in rats. 41,4 Here, we first successfully used TALEN technology to knock out IRF1 in an SD rat background, and we obtained results that are consistent with those from our mouse model, evidenced by the finding that the ablation of IRF1 in rats also led to an attenuation of cardiac hypertrophy and fibrosis, and thereby avoiding cardiac dysfunction. To our knowledge, this is the first report describing the use of the TALEN technology to create IRF1 / rats, and our finding emphasize the role of IRF1 as an important regulatory factor in pathological remodeling both in mice and rats. Previous studies have revealed the extensive and crucial functions of 9 members (IRF1-9) of IRFs family in many biological processes, including antiviral response, inflammation regulation, cytokine signaling, cell death, growth, and differentiation. 11,7,43 45 Importantly, our recent findings demonstrated that IRF3, IRF7, IRF8, and IRF9 function as protective factors by inactivating extracellular signal regulated kinases 1 and, inhibitor of κb kinase-β, nuclear factor of activated T cell c1, or myocardin in the process of pressure overload induced cardiac hypertrophy, respectively, 3,1 14 whereas IRF4 plays an opposite role by activating the transcription of camp response element binding protein in the heart. 5 In present study, we demonstrated that IRF1 accelerated pressure overload induced cardiac hypertrophy by the direct activation of inos. An overlapping but distinct set of target genes activated by the IRFs might contribute to shape the appropriate response of various biological processes, including cardiac remodeling. In addition, IRF family members also exhibit versatile functions via cooperating or competing of different transcription factors at the DNA-binding level. 46,47 It is, therefore, possible that cross talk exists among these different IRFs in the development of cardiac hypertrophy and remodeling. Understanding the nature of cross talk between IRF family members will be beneficial to decipher the divergent or opposite effects of different IRFs on cardiac remodeling. In conclusion, our present work demonstrated that IRF1 functions as a novel positive regulator factor of pathological cardiac hypertrophy both in vitro and in vivo. Furthermore, we identified inos as a crucial target of IRF1 and demonstrated that the transcriptional activity of inos is strongly activated by IRF1. Thus, targeting IRF1 might be a useful therapeutic strategy for preventing cardiac hypertrophy and heart failure.

9 Jiang et al IRF1 Modulates Cardiac Remodeling 85 Downloaded from by guest on November 4, 17 Perspectives The current study provides in vivo and in vitro evidences that IRF1, a member of the IRF transcription factor family, functions as a positive regulator of the hypertrophic response in the heart by binding to the inos promoter to activate inos expression, thereby activating the cardiac fetal gene program. These observations suggest that an intervention of IRF1 expression could be useful for the prevention and treatment of cardiac hypertrophy and heart failure. Sources of Funding This work was supported by grants from the National Natural Science Foundation of China (No. 8113, No , No. 8171, No , No , and No ), National Science and Technology Support Project (No. 11BAI15B, No. 1BAI39B5, No. 13YQ393-5, and No. 14BAIB1), the Key Project of the National Natural Science Foundation (No ), and the National Basic Research Program of China (No. 11CB539). None. Disclosures References 1. 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10 86 Hypertension July Umar S, van der Laarse A. Nitric oxide and nitric oxide synthase isoforms in the normal, hypertrophic, and failing heart. Mol Cell Biochem. 1;333: Aitman TJ, Critser JK, Cuppen E, et al. Progress and prospects in rat genetics: a community view. Nat Genet. 8;4: Rapp JP. Genetic analysis of inherited hypertension in the rat. Physiol Rev. ;8: Lazar J, Moreno C, Jacob HJ, Kwitek AE. Impact of genomics on research in the rat. Genome Res. 5;15: Joung JK, Sander JD. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol. 13;14: Tesson L, Usal C, Ménoret S, Leung E, Niles BJ, Remy S, Santiago Y, Vincent AI, Meng X, Zhang L, Gregory PD, Anegon I, Cost GJ. Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol. 11;9: Guo S, Li ZZ, Jiang DS, Lu YY, Liu Y, Gao L, Zhang SM, Lei H, Zhu LH, Zhang XD, Liu DP, Li H. Irf4 is a novel mediator for neuronal survival in ischaemic stroke. Cell Death Differ. 14, In Press. 44. Zhang SM, Gao L, Zhang XF, Zhang R, Zhu LH, Wang PX, Tian S, Yang D, Chen K, Huang L, Zhang XD, Li H. Interferon regulatory factor 8 modulates phenotypic switching of smooth muscle cells by regulating the activity of myocardin. Mol Cell Biol. 14;34: Wang XA, Zhang R, Jiang D, et al. Interferon regulatory factor 9 protects against hepatic insulin resistance and steatosis in male mice. Hepatology. 13;58: Taniguchi T, Ogasawara K, Takaoka A, Tanaka N. IRF family of transcription factors as regulators of host defense. Annu Rev Immunol. 1;19: Ikushima H, Negishi H, Taniguchi T. The IRF family transcription factors at theinterface of innate and adaptive immune responses. Cold Spring Harb Symp Quant Biol. 13; In Press. Novelty and Significance Downloaded from by guest on November 4, 17 What Is New? Interferon regulatory factor 1 (IRF1) is significantly altered in failing human hearts and hypertrophic murine hearts. IRF1 is a positive regulator of aortic banding induced cardiac hypertrophy, fibrosis, and cardiac dysfunction in vivo and of angiotensin II induced cardiomyocyte hypertrophy in vitro. The detrimental role of IRF1 in cardiac remodeling was further established by using the IRF1 deficiency rats model. IRF1 binds to inducible nitric oxide synthase promoter region and regulates its transcriptional activity, thus regulating the development of pathological cardiac hypertrophy. What Is Relevant? Several transcription factors have been demonstrated to contribute to cardiac hypertrophy. The role of IRF1, an immune transcription factor, in the development of cardiac hypertrophy and its underlying mechanisms have not been elucidated. The results of this study expand our understanding of the effects of IRF family proteins on cardiac remodeling and have implications for the development of strategies for treating cardiac hypertrophy and heart failure. Summary This study demonstrates that the absence of IRF1 protects the heart against chronic pressure overload induced cardiac hypertrophy, fibrosis, and cardiac dysfunction, whereas the overexpression of IRF1 aggravates the pathological hypertrophic response by regulating the transcriptional activity of inducible nitric oxide synthase. These observations will provide a new target to prevent/treat pathological cardiac hypertrophy and heart failure.

11 Interferon Regulatory Factor 1 Is Required for Cardiac Remodeling in Response to Pressure Overload Ding-Sheng Jiang, Liangpeng Li, Ling Huang, Jun Gong, Hao Xia, Xiaoxiong Liu, Nian Wan, Xiang Wei, Xuehai Zhu, Yingjie Chen, Xin Chen, Xiao-Dong Zhang and Hongliang Li Downloaded from by guest on November 4, 17 Hypertension. 14;64:77-86; originally published online April 14, 14; doi: /HYPERTENSIONAHA Hypertension is published by the American Heart Association, 77 Greenville Avenue, Dallas, TX 7531 Copyright 14 American Heart Association, Inc. All rights reserved. Print ISSN: X. Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Data Supplement (unedited) at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Hypertension is online at: