Therapeutic Activation of Signal Transducer and Activator
`of Transcription 3 by Interleukin-11 Ameliorates Cardiac
`Fibrosis After Myocardial Infarction
`
`Masanori Obana, MS; Makiko Maeda, PhD; Koji Takeda, MD; Akiko Hayama, MS;
`Tomomi Mohri, PhD; Tomomi Yamashita, BS; Yoshikazu Nakaoka, MD, PhD;
`Issei Komuro, MD, PhD; Kiyoshi Takeda, MD, PhD; Goro Matsumiya, MD, PhD;
`Junichi Azuma, MD; Yasushi Fujio, MD, PhD
`
`Background—Glycoprotein 130 is the common receptor subunit for the interleukin (IL)-6 cytokine family. Previously, we
`reported that pretreatment of IL-11, an IL-6 family cytokine, activates the glycoprotein 130 signaling pathway in
`cardiomyocytes and prevents ischemia/reperfusion injury in vivo; however, its long-term effects on cardiac remodeling
`after myocardial infarction (MI) remain to be elucidated.
`Methods and Results—MI was generated by ligating the left coronary artery in C57BL/6 mice. Real-time reverse
`transcription polymerase chain reaction analyses showed that IL-11 mRNA was remarkably upregulated in the hearts
`exposed to MI. Intravenous injection of IL-11 activated signal transducer and activator of transcription 3 (STAT3), a
`downstream signaling molecule of glycoprotein 130, in cardiomyocytes in vivo, suggesting that cardiac myocytes are
`target cells of IL-11 in the hearts. Twenty-four hours after coronary ligation, IL-11 was administered intravenously,
`followed by consecutive administration every 24 hours for 4 days. IL-11 treatment reduced fibrosis area 14 days after
`MI, attenuating cardiac dysfunction. Consistent with a previous report that STAT3 exhibits antiapoptotic and angiogenic
`activity in the heart, IL-11 treatment prevented apoptotic cell death of the bordering myocardium adjacent to the infarct
`zone and increased capillary density at the border zone. Importantly, cardiac-specific ablation of STAT3 abrogated
`IL-11–mediated attenuation of fibrosis and was associated with left ventricular enlargement. Moreover, with the use of
`cardiac-specific transgenic mice expressing constitutively active STAT3, cardiac STAT3 activation was shown to be
`sufficient to prevent adverse cardiac remodeling.
`Conclusions—IL-11 attenuated cardiac fibrosis after MI through STAT3. Activation of the IL-11/glycoprotein 130/STAT3
`axis may be a novel therapeutic strategy against cardiovascular diseases. (Circulation. 2010;121:684-691.)
`
`Key Words: interleukins 䡲 myocardial infarction 䡲 remodeling 䡲 signal transduction
`
`A fter myocardial injury, various kinds of neurohumoral
`
`factors and cytokines modulate cardiac remodeling.
`Among them,
`leukemia inhibitory factor
`(LIF) and
`cardiotrophin-1, which belong to the interleukin (IL)-6 fam-
`ily, play important roles in cardioprotection.1,2 LIF and
`cardiotrophin-1 are secreted from cardiomyocytes in response
`to pathological stress.3–5 These cytokines bind and activate
`LIF receptor in cardiomyocytes.6 Activated LIF receptor
`makes a dimer with glycoprotein 130 (gp130), followed by
`activation of signal transducer and activator of transcription 3
`(STAT3).7 STAT3 activation promotes cardiomyocyte sur-
`vival and vascular formation in the heart.8 –10 Thus, cardiac
`activation of the gp130/STAT3 system may be a potential
`
`therapeutic strategy against cardiovascular diseases; however,
`therapies targeting gp130 have not been proposed.
`
`Clinical Perspective on p 691
`
`The difficulty in therapeutic activation of gp130 is derived
`from its receptor system. Gp130 is expressed ubiquitously as
`the common receptor subunit of IL-6 family cytokines.11 IL-6
`family cytokines bind their specific receptor ␣ subunits,
`followed by activation of a common gp130 receptor. Pleio-
`tropic effects of IL-6 family cytokines are explained by the
`differential expression of receptor ␣ subunits. Most members
`of the IL-6 family, whose receptor ␣ subunits are expressed
`abundantly in inflammatory cells, would evoke severe in-
`
`Downloaded from http://ahajournals.org by on October 29, 2019
`
`Received July 14, 2009; accepted December 4, 2009.
`From the Department of Clinical Pharmacology and Pharmacogenomics, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
`(M.O., A.H., T.M., T.Y., J.A., Y.F.); Department of Clinical Pharmacogenomics, School of Pharmacy, Hyogo University of Health Sciences, Hyogo,
`Japan (M.M., J.A.); and Department of Cardiovascular Surgery (K.T., G.M.), Department of Cardiovascular Medicine (Y.N., I.K.), and Laboratory of
`Immune Regulation, Department of Microbiology and Immunology (K.T.), Graduate School of Medicine, Osaka University, Osaka, Japan.
`Singapore Exhibit 2003
`The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.893677/DC1.
`Correspondence to Yasushi Fujio, MD, PhD, 1-6 Yamada-oka, Suita City, 565-0871, Osaka, Japan. E-mail fujio@phs.osaka-u.ac.jp
`Lassen v. Singapore et al.
`© 2010 American Heart Association, Inc.
`PGR2019-00053
`DOI: 10.1161/CIRCULATIONAHA.109.893677
`
`Circulation is available at http://circ.ahajournals.org
`
`684
`
`

`

`Therapeutic Activation of Signal Transducer and Activator
`of Transcription 3 by Interleukin-11 Ameliorates Cardiac
`Fibrosis After Myocardial Infarction
`
`Masanori Obana, MS; Makiko Maeda, PhD; Koji Takeda, MD; Akiko Hayama, MS;
`Tomomi Mohri, PhD; Tomomi Yamashita, BS; Yoshikazu Nakaoka, MD, PhD;
`Issei Komuro, MD, PhD; Kiyoshi Takeda, MD, PhD; Goro Matsumiya, MD, PhD;
`Junichi Azuma, MD; Yasushi Fujio, MD, PhD
`
`Background—Glycoprotein 130 is the common receptor subunit for the interleukin (IL)-6 cytokine family. Previously, we
`reported that pretreatment of IL-11, an IL-6 family cytokine, activates the glycoprotein 130 signaling pathway in
`cardiomyocytes and prevents ischemia/reperfusion injury in vivo; however, its long-term effects on cardiac remodeling
`after myocardial infarction (MI) remain to be elucidated.
`Methods and Results—MI was generated by ligating the left coronary artery in C57BL/6 mice. Real-time reverse
`transcription polymerase chain reaction analyses showed that IL-11 mRNA was remarkably upregulated in the hearts
`exposed to MI. Intravenous injection of IL-11 activated signal transducer and activator of transcription 3 (STAT3), a
`downstream signaling molecule of glycoprotein 130, in cardiomyocytes in vivo, suggesting that cardiac myocytes are
`target cells of IL-11 in the hearts. Twenty-four hours after coronary ligation, IL-11 was administered intravenously,
`followed by consecutive administration every 24 hours for 4 days. IL-11 treatment reduced fibrosis area 14 days after
`MI, attenuating cardiac dysfunction. Consistent with a previous report that STAT3 exhibits antiapoptotic and angiogenic
`activity in the heart, IL-11 treatment prevented apoptotic cell death of the bordering myocardium adjacent to the infarct
`zone and increased capillary density at the border zone. Importantly, cardiac-specific ablation of STAT3 abrogated
`IL-11–mediated attenuation of fibrosis and was associated with left ventricular enlargement. Moreover, with the use of
`cardiac-specific transgenic mice expressing constitutively active STAT3, cardiac STAT3 activation was shown to be
`sufficient to prevent adverse cardiac remodeling.
`Conclusions—IL-11 attenuated cardiac fibrosis after MI through STAT3. Activation of the IL-11/glycoprotein 130/STAT3
`axis may be a novel therapeutic strategy against cardiovascular diseases. (Circulation. 2010;121:684-691.)
`
`Key Words: interleukins 䡲 myocardial infarction 䡲 remodeling 䡲 signal transduction
`
`A fter myocardial injury, various kinds of neurohumoral
`
`factors and cytokines modulate cardiac remodeling.
`Among them,
`leukemia inhibitory factor
`(LIF) and
`cardiotrophin-1, which belong to the interleukin (IL)-6 fam-
`ily, play important roles in cardioprotection.1,2 LIF and
`cardiotrophin-1 are secreted from cardiomyocytes in response
`to pathological stress.3–5 These cytokines bind and activate
`LIF receptor in cardiomyocytes.6 Activated LIF receptor
`makes a dimer with glycoprotein 130 (gp130), followed by
`activation of signal transducer and activator of transcription 3
`(STAT3).7 STAT3 activation promotes cardiomyocyte sur-
`vival and vascular formation in the heart.8 –10 Thus, cardiac
`activation of the gp130/STAT3 system may be a potential
`
`therapeutic strategy against cardiovascular diseases; however,
`therapies targeting gp130 have not been proposed.
`
`Clinical Perspective on p 691
`
`The difficulty in therapeutic activation of gp130 is derived
`from its receptor system. Gp130 is expressed ubiquitously as
`the common receptor subunit of IL-6 family cytokines.11 IL-6
`family cytokines bind their specific receptor ␣ subunits,
`followed by activation of a common gp130 receptor. Pleio-
`tropic effects of IL-6 family cytokines are explained by the
`differential expression of receptor ␣ subunits. Most members
`of the IL-6 family, whose receptor ␣ subunits are expressed
`abundantly in inflammatory cells, would evoke severe in-
`
`Downloaded from http://ahajournals.org by on October 29, 2019
`
`Received July 14, 2009; accepted December 4, 2009.
`From the Department of Clinical Pharmacology and Pharmacogenomics, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
`(M.O., A.H., T.M., T.Y., J.A., Y.F.); Department of Clinical Pharmacogenomics, School of Pharmacy, Hyogo University of Health Sciences, Hyogo,
`Japan (M.M., J.A.); and Department of Cardiovascular Surgery (K.T., G.M.), Department of Cardiovascular Medicine (Y.N., I.K.), and Laboratory of
`Immune Regulation, Department of Microbiology and Immunology (K.T.), Graduate School of Medicine, Osaka University, Osaka, Japan.
`The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.893677/DC1.
`Correspondence to Yasushi Fujio, MD, PhD, 1-6 Yamada-oka, Suita City, 565-0871, Osaka, Japan. E-mail fujio@phs.osaka-u.ac.jp
`© 2010 American Heart Association, Inc.
`
`Circulation is available at http://circ.ahajournals.org
`
`DOI: 10.1161/CIRCULATIONAHA.109.893677
`
`684
`
`

`

`Obana et al
`
`IL-11 as a Novel Cardioprotective Cytokine
`
`685
`
`if administered
`flammation12 as a serious adverse event
`systemically. Therefore, to achieve clinical use of IL-6 family
`cytokines, the cytokine that induces only a tolerable level of
`inflammation should be selected.
`IL-11 is a hematopoietic IL-6 family cytokine with pleiotropic
`effects. IL-11 exhibits thrombopoietic activity, and recombinant
`human IL-11 is used clinically for thrombocytopenia.13 In
`contrast to other IL-6 family members, IL-11 exhibits anti-
`inflammatory activity against chronic inflammatory diseases,
`such as Crohn disease.14 Moreover, recombinant human IL-11
`protects epithelial cells of the intestine from tissue damage,
`suggesting its cytoprotective property.15 Recently, we demon-
`strated that the IL-11 receptor is expressed in cardiomyocytes
`and that pretreatment of IL-11 confers resistance to ischemia/
`reperfusion injury in a murine model as a preconditioning
`effect.16 When the limited level of clinical adverse effects of
`recombinant human IL-11 is considered,13 IL-11 may be a
`candidate to be available clinically as cardiac gp130-targeting
`therapy against heart diseases.
`In this study, we investigated the long-term effects of IL-11
`treatment after myocardial infarction (MI). In addition, we
`report that IL-11 treatment prevents adverse cardiac remod-
`eling through the STAT3 pathway.
`
`Methods
`
`Animal Care
`The care of all animals was in compliance with the Osaka University
`animal care guidelines. The investigation conforms to the Guide for
`the Care and Use of Laboratory Animals published by the US
`National Institutes of Health (National Institutes of Health publica-
`tion No. 85-23, revised 1996).
`
`Coronary Artery Ligation and IL-11 Treatment
`MI was generated by coronary artery ligation according to the
`previous report1 with minor modifications. Briefly, C57BL/6 mice (8
`to 10 weeks old; Japan SLC) were anesthetized and ventilated with
`80% oxygen containing 1.5% isoflurane (Merck). After left-side
`thoracotomy, the left coronary artery was ligated with 7-0 silk
`sutures. Infarction was confirmed by discoloration of the ventricle
`and ST-T changes on ECG. The chest and skin were closed with 5-0
`silk sutures. In preliminary experiments, we confirmed that the
`operation reproducibly generates infarction with the initial area at
`risk 20% to 25% per left ventricular (LV) area, as analyzed by Evans
`blue exclusion assays.16 Sham-operated mice were subjected to
`similar surgery, except that no ligature was placed. Twenty-four
`hours after MI operation, mice were randomly assigned to 2 groups:
`the IL-11 group and control group. In the IL-11 group, recombinant
`human IL-11 (Peprotech) was administered intravenously for 5 days
`consecutively;
`the control group received the same volume of
`phosphate-buffered saline (PBS) during the same period.
`
`Real-Time Reverse Transcription Polymerase
`Chain Reaction
`Real-time reverse transcription polymerase chain reaction (RT-PCR)
`was performed according to the manufacturer’s protocol. Total RNA
`was prepared from hearts at various time points after operation. In
`some experiments, the hearts were cut into 2 pieces: infarct area and
`remote area. The infarct area is the damaged or fibrotic region with
`its surrounding border zone, and the remote area is the portion
`separated from the infarct area by ⬎1 mm.
`Total RNA (1 ␮g) was subjected to first-strand cDNA synthesis
`with oligo(dT) primer. IL-11 was quantified by real-time RT-PCR
`with the use of the ABI-PRISM 7700 sequence detection system
`(Applied Biosystems Inc) with the SYBR green system (Applied
`
`Biosystems). As an internal control, the expression of GAPDH
`mRNA was estimated with the SYBR green system. The primers for
`IL-11 or GAPDH are as follows: IL-11 forward: 5⬘-CTGCCC-
`ACCTTGGCCATGAG-3⬘; IL-11 reverse: 5⬘-CCAGGCGAGACA-
`TCAAGAAAGA-3⬘; GAPDH forward: 5⬘-GCCGGTGCTGAGTAT-
`GTCGT-3⬘; GAPDH reverse: 5⬘-CCCTTTTGGCTCCACCCTT-3⬘.
`
`Immunoblot Analyses
`Immunoblot analyses were performed as described previously.17
`Heart homogenates were prepared in buffer containing 150 mmol/L
`NaCl, 10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, 1% Triton
`X-100, 1% deoxycholic acid, and 1 mmol/L dithiothreitol. Proteins
`were separated by SDS-PAGE and transferred onto the polyvinyli-
`dene difluoride membrane (Millipore). The membrane was immu-
`noblotted with anti-phospho-STAT3 (p-STAT3) (Cell Signaling
`Technology, Danvers, Mass), anti-Bcl-2 (BD Transduction Labora-
`tories), anti-survivin (Santa Cruz Biotechnology, Inc, Santa Cruz,
`Calif), or anti-cleaved caspase 3 (Cell Signaling Technology) anti-
`body. The membrane was reprobed with anti-STAT3 (Santa Cruz
`Biotechnology) or anti-GAPDH (Chemicon, Temecula, Calif) anti-
`body to show equal amount loading.
`
`Histological Estimation of Cardiac Fibrosis
`The frozen sections (5-␮m thick) were prepared from the portion
`⬇300 ␮m distal to the ligation point and stained with Masson’s
`trichrome. Photomicrographs were taken, and fibrotic circumference
`and area were measured with the use of Scion Image (Scion Corpora-
`tion) by a researcher who was blinded to the treatment. Fibrotic
`circumference and area were calculated as a percentage of LV circum-
`ference and area, respectively. Infarct wall thickness was measured
`perpendicular to the infarcted wall at 3 separate regions and averaged.
`
`Analysis of Cardiac Function
`Fourteen days after MI, mice were anesthetized (50 mg/kg pento-
`barbital) and heparinized (50 U) via intraperitoneal injection. The
`hearts were excised rapidly and placed in ice-cold modified Tyrode’s
`solution (140 mmol/L NaCl, 5.4 mmol/L KCl, 1.8 mmol/L CaCl2,
`0.45 mmol/L MgCl2, 0.33 mmol/L NaH2PO4, 5.5 mmol/L glucose,
`5 mmol/L HEPES [pH 7.4]). The aorta was cannulated and retro-
`gradely perfused at a constant pressure of 100 mm Hg with Tyrode’s
`solution bubbled with 80% oxygen at 37°C. The experiments were
`performed at 37°C by immersing the heart in Tyrode’s solution in a
`water-jacketed chamber. The hearts were paced at 420 bpm. The
`fluid-filled balloon was inserted into the LV to monitor cardiac
`function. The balloon was attached to a pressure transducer, which
`was coupled to a 4S PowerLab (AD Instruments). LV developed
`pressure and ⫾dP/dt were measured.
`
`Immunofluorescent Microscopic Analyses
`The hearts were harvested 15 minutes after intravenous injection of
`IL-11, and the frozen sections were prepared. The sections were
`stained with anti-p-STAT3 and anti-sarcomeric ␣-actinin (Sigma)
`antibodies. Alexa Fluor 488 – conjugated goat anti-rabbit IgG (Mo-
`lecular Probes) and Alexa Fluor 546 – conjugated goat anti-mouse
`IgG (Molecular Probes) were used as secondary antibodies. Nuclei
`were also stained with Hoechst 33258.
`Apoptotic cell death was detected by a terminal deoxynucleotide
`transferase–mediated dUTP nick-end labeling (TUNEL) staining
`with an in situ Apoptosis Detection Kit (TaKaRa). The section was
`costained with anti-sarcomeric ␣-actinin antibody to identify the
`cardiac myocytes. For quantitative analyses, apoptotic myocytes
`were counted in number by a researcher who was blinded to the
`assay conditions.
`
`Immunohistochemical Analyses
`The frozen sections were prepared as described above. Capillary
`density was examined by immunohistochemical staining with the use
`of the Vectastain ABC kit (Vector Laboratories) with anti-CD31
`antibody (BD Biosciences, San Jose, Calif). To estimate capillary
`
`Downloaded from http://ahajournals.org by on October 29, 2019
`
`

`

`Figure 1. The transcript of IL-11 was elevated after MI, and IL-11
`activated STAT3 in cardiomyocytes in murine hearts. A, Expression
`of IL-11 in infarct area or remote area of postinfarct hearts was
`analyzed by real-time RT-PCR. Expression of IL-11 was normalized
`with that of GAPDH. Data are shown as mean⫾SD (n⫽4 mice for
`each condition). *P⬍0.05 vs non-MI (sham) by 1-way ANOVA fol-
`lowed by Bonferroni test. B, IL-11 (8 ␮g/kg) was administered in-
`travenously in mice for the indicated time. The lysates from hearts
`were immunoblotted with anti-p-STAT3 antibody. Blots were
`reprobed with anti-STAT3 antibody. Representative data are
`shown (left). Quantitative analyses of p-STAT3 are shown (right).
`Data are shown as mean⫾SD (n⫽4 mice for each condition).
`*P⬍0.05 vs 0 minutes by 1-way ANOVA followed by Bonferroni
`test. C, Fifteen minutes after injection of IL-11 (8 ␮g/kg) or PBS,
`frozen sections were prepared from the hearts. The sections were
`costained with anti-p-STAT3 and anti-sarcomeric ␣-actinin anti-
`bodies. Hoechst 33258 staining was also performed to identify the
`nuclei. The images are representative of 20 fields obtained from 4
`mice. Bar⫽50 ␮m.
`
`IL-11 Administration Attenuates Cardiac
`Remodeling After MI
`To examine the effects of IL-11 on adverse cardiac remod-
`eling, IL-11 was administered to the mice after MI operation,
`and cardiac fibrosis was histologically estimated at day 14
`after MI (Figure 2). Both fibrotic circumference and fibrotic
`area were reduced by IL-11 in a dose-dependent manner
`(Figures 2B and C). Treatment of IL-11 at 8 ␮g/kg achieved
`a submaximal reduction in fibrotic circumference by 28.9%
`(PBS, 58.6⫾9.6%; IL-11, 41.7⫾10.0%) and fibrotic area by
`33.1% (PBS, 39.8⫾9.3%; IL-11, 26.6⫾7.4%). Interestingly,
`IL-11–treated hearts showed an increase in infarct wall
`thickness compared with PBS-treated hearts (Figure 2D). To
`
`686
`
`Circulation
`
`February 9, 2010
`
`density quantitatively, the number of capillaries was counted by a
`researcher who was blinded to the assay conditions.
`
`Conditional Ablation of the STAT3 Gene in
`Cardiomyocytes of Adult Murine Heart
`The transgenic mice in which Cre recombinase fused to the mutated
`estrogen receptor domains (MerCreMer) were driven by the cardio-
`myocyte-specific ␣-myosin heavy chain (␣-MHC) promoter, desig-
`nated as ␣-MHC-MerCreMer mice, were a gift from Dr Molkentin.18
`We crossed the ␣-MHC-MerCreMer mice with mice that carried
`floxed STAT3 alleles (STAT3flox/flox)19 and produced ␣-MHC-
`MerCreMer/STAT3flox/flox mutant mice. To induce Cre-mediated
`recombination, mice were treated with 20 mg/kg tamoxifen (Sigma,
`St Louis, Mo) by intraperitoneal
`injection once per day for 5
`consecutive days. Five days after the last treatment, the level of
`STAT3 expression decreased dramatically, and the mutant mice
`underwent MI as described above.
`
`Cardiac-Specific Transgenic Mice Expressing
`Constitutively Active STAT3
`Generation of cardiac-specific transgenic mice expressing constitu-
`tively active STAT3 was described previously.9
`
`Statistical Analysis
`Data are presented as mean⫾SD. Comparisons between 2 groups
`were performed with the use of the unpaired t
`test. One-way
`ANOVA with the Bonferroni test was used for multiple compari-
`sons. Differences were considered statistically significant when the
`calculated (2-tailed) P value was ⬍0.05.
`
`Results
`
`IL-11 Is a Cardiac Cytokine That Activates
`STAT3 in Cardiomyocytes In Vivo
`We analyzed the expression of IL-11 mRNA in hearts at various
`time points after MI. Hearts were separated into infarct area and
`remote area, and the expression of IL-11 mRNA was measured
`by real-time RT-PCR (Figure 1A). The expression of IL-11
`transcript was elevated, with its peak at 1 day after MI, and was
`gradually reduced at both infarct and remote areas. In the infarct
`area, the enhanced expression of IL-11 was sustained for 14
`days. These data indicate that IL-11 is produced in the heart
`during cardiac remodeling after MI.
`Next, we examined whether intravenous administration of
`IL-11 stimulates STAT3 in hearts by immunoblot analysis
`with anti-p-STAT3 antibody (Figure 1B). STAT3 phosphor-
`ylation was induced rapidly and reduced to the basal level
`within 180 minutes after IL-11 injection. IL-11 activated
`STAT3 in the heart in a dose-dependent manner (Figure I in
`the online-only Data Supplement).
`To confirm that STAT3 activation occurred in cardiomyo-
`cytes, we performed immunohistological analyses to detect
`the nuclear localization of activated p-STAT3 (Figure 1C).
`Nuclear staining of p-STAT3 was detected in the IL-11–
`treated hearts but not in untreated hearts. Notably, ⬎90% of
`nuclei of sarcomeric ␣-actinin–positive cells were also posi-
`tively stained with anti-p-STAT3 antibody, indicating that
`IL-11 treatment results in STAT3 activation in cardiomyo-
`cytes in vivo. As is the case with noninfarcted mice,
`p-STAT3 was localized mainly in cardiomyocyte nuclei of
`postinfarct hearts, and IL-11 treatment increased the fre-
`quency of p-STAT3–positive cardiac myocytes (Figure II in
`the online-only Data Supplement).
`
`Downloaded from http://ahajournals.org by on October 29, 2019
`
`

`

`Obana et al
`
`IL-11 as a Novel Cardioprotective Cytokine
`
`687
`
`Figure 2. IL-11 attenuated cardiac fibro-
`sis after MI. Heart sections (3 sections
`from each mouse) were prepared 14
`days after MI and stained with Masson’s
`trichrome. A, Images are representative
`of 24 to 27 obtained from 8 to 9 mice.
`Bar⫽1 mm. B to D, Ratio of fibrotic cir-
`cumference to LV circumference (B),
`ratio of fibrotic area to LV area (C), and
`infarct wall thickness (D) were quantita-
`tively estimated. Data are shown as
`mean⫾SD (n⫽8 mice for PBS; n⫽9 mice
`for 3 ␮g/kg; n⫽9 mice for 8 ␮g/kg; n⫽8
`mice for 20 ␮g/kg). *P⬍0.05 vs PBS by
`1-way ANOVA followed by Bonferroni
`test.
`
`examine the effects of IL-11 on LV hypertrophy, we analyzed
`expression of ␣-skeletal muscle actin mRNA, a well-known
`marker of LV hypertrophy (Figure III in the online-only Data
`Supplement). IL-11 treatment showed a tendency to reduce
`␣-skeletal muscle actin expression, although its reduction was
`not statistically significant.
`To clarify whether IL-11 prevents cardiac dysfunction after
`MI, we measured LV developed pressure and ⫾dP/dt by a
`Langendorff apparatus. As acute myocardial damage, MI rapidly
`impaired LV developed pressure and ⫾dP/dt at day 1 (Table I in
`the online-only Data Supplement) before IL-11 administration
`was started. Because IL-11 treatment attenuated cardiac fibrosis
`that occurred during the following 2 weeks, we examined the
`effects of IL-11 on cardiac function 2 weeks after MI (Table).
`IL-11 treatment ameliorated chronic cardiac dysfunction in a
`dose-dependent manner compared with the PBS-treated group.
`Consistent with the attenuation of fibrosis, treatment of IL-11 at
`a dose of 8 ␮g/kg submaximally prevented cardiac dysfunction.
`Thus, further experiments were performed with the use of IL-11
`at a dose of 8 ␮g/kg.
`We also confirmed the inhibitory effects of IL-11 on
`adverse cardiac remodeling 28 days after MI. IL-11 treatment
`
`Table.
`
`Effects of IL-11 on Cardiac Function at Day 14 After MI
`
`IL-11, ␮g/kg
`
`Parameters
`
`PBS
`
`3
`
`8
`
`20
`
`LVDP, mm Hg
`
`32.4⫾4.4
`
`34.9⫾6.3
`
`45.3⫾6.3*
`
`44.3⫾13.5*
`
`⫹dP/dt,
`mm Hg/s
`
`⫺dP/dt,
`mm Hg/s
`
`884⫾113
`
`958⫾162
`
`1210⫾148*
`
`1243⫾324*
`
`⫺713⫾93 ⫺737⫾176 ⫺1022⫾210* ⫺1043⫾326*
`
`Data are mean⫾SD (n⫽8 mice for PBS; n⫽9 mice for 3 ␮g/kg; n⫽9 mice
`for 8 ␮g/kg; n⫽8 mice for 20 ␮g/kg). LVDP indicates LV developed pressure.
`*P⬍0.05 vs PBS by 1-way ANOVA followed by Bonferroni test.
`
`prevented cardiac fibrosis and preserved cardiac function
`(Figure IV in the online-only Data Supplement).
`To examine whether IL-11 reduces infarct size, infarct size
`was measured by Evans blue exclusion 2 days after MI. There
`was no significant difference in infarct size (PBS, 23.0⫾7.1%;
`IL-11 [8 ␮g/kg], 22.0⫾4.3%; n⫽4 mice for each group).
`
`IL-11 Treatment Exhibits Antiapoptotic and
`Proangiogenic Activity in the Heart
`Granulocyte colony-stimulating factor (G-CSF) was reported
`to prevent cardiac remodeling after MI, accompanied by
`antiapoptotic and proangiogenic effects through STAT3.20
`Because IL-11 activated STAT3 in cardiomyocytes (Figure
`1), we examined the effects of IL-11 on cardiomyocyte
`survival and vascular formation in postinfarct myocardium.
`To evaluate the antiapoptotic effects of IL-11, TUNEL
`staining was performed (Figure 3A). TUNEL-positive cardio-
`myocytes were detected mainly at the border zone, adjacent
`to the ischemic area, at both day 2 and day 4 after MI.
`Importantly, IL-11 treatment significantly reduced the num-
`ber of apoptotic cardiomyocytes compared with the PBS
`group. To further confirm the cytoprotective effects,
`the
`cleavage of caspase 3 was examined by immunoblot analyses
`with cleaved caspase 3–specific antibody (Figure 3B). Com-
`pared with PBS treatment, IL-11 significantly reduced the
`band intensity for cleaved caspase 3. To address the antiapo-
`ptotic pathways downstream of the IL-11 signal, we exam-
`ined the expression of cell survival proteins (Figure 3C).
`Immunoblot analysis revealed that Bcl-2 and survivin pro-
`teins, both of which have been characterized as downstream
`targets of STAT3,21–23 were increased in the IL-11 group
`(Bcl-2, 1.9-fold; survivin, 1.5-fold).
`Because activation of STAT3 mediated vascular formation
`during cardiac remodeling,20,24 capillary density was immu-
`
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`688
`
`Circulation
`
`February 9, 2010
`
`Figure 3. IL-11 treatment suppressed car-
`diomyocyte apoptosis after MI. A, The fre-
`quency of apoptotic cardiomyocytes was
`estimated by TUNEL staining 2 days and 4
`days after MI. The sections were costained
`with anti-sarcomeric ␣-actinin antibody and
`Hoechst 33258 dye. The images are repre-
`sentative of 60 to 100 obtained from 4 to 5
`mice (15 to 20 fields from each mouse) (left).
`Arrowheads show TUNEL-positive, apoptot-
`ic cardiomyocytes. Bar⫽50 ␮m. Quantifica-
`tion of the apoptotic cardiomyocytes is
`shown (right). Visual fields (15 to 20) were
`randomly selected. Data are shown as
`mean⫾SD (n⫽5 mice for PBS [2 days]; n⫽4
`mice for IL-11 [2 days]; n⫽5mice for PBS [4
`days]; n⫽5 mice for IL-11 [4 days]). *P⬍0.05
`vs PBS by unpaired t test. B, The lysates
`from hearts at day 2 after MI were immuno-
`blotted with anti-cleaved caspase 3 and
`anti-GAPDH antibodies. Arrows indicate the
`cleaved caspase 3 fragments with molecular
`weight 17 and 19 kDa (left). Quantitative
`analysis of cleaved caspase 3 fragments is
`shown (right). Data are shown as mean⫾SD
`(n⫽6 mice for PBS; n⫽5 mice for IL-11).
`*P⬍0.05 vs PBS by unpaired t test. C, Rep-
`resentative results of immunoblotting with
`anti-Bcl-2 and anti-survivin antibodies. Blots
`were reprobed with anti-GAPDH antibody to
`show equal amount loading. Experiments
`were repeated 5 times with similar results.
`
`nohistochemically estimated with anti-CD31 antibody (Fig-
`ure 4). In the border zone, capillary density was increased by
`IL-11 in a dose-dependent manner. Because the frequency of
`apoptosis was higher at day 2 than at day 4 (30.9⫾7.1 versus
`5.7⫾2.8 TUNEL-positive cardiomyocytes/mm2,
`respec-
`tively; P⬍0.01), cardiomyocyte loss occurs mainly at days 1
`to 3. On the basis of this finding, to clarify the relation
`between antiapoptosis and angiogenesis mediated by IL-
`11, we compared the effectiveness between cytokine treat-
`ment at days 1 to 3 and that at days 3 to 5 (Table II in the
`online-only Data Supplement). IL-11 treatment at days 1 to
`3 attenuated cardiac fibrosis, whereas that at days 3 to 5
`did not. Interestingly, treatment at days 1 to 3 increased
`capillary density, whereas that at days 3 to 5 did not.
`Therefore, IL-11–mediated increase in capillary density is
`likely to be closely associated with improved myocyte
`viability.
`
`Activation of STAT3 Is Necessary and Sufficient
`for IL-11–Mediated Prevention of
`Cardiac Remodeling
`To assess the importance of cardiac STAT3 activation in
`IL-11–mediated attenuation of adverse remodeling, we gen-
`erated cardiac-specific conditional STAT3-deficient mice
`(STAT3 CKO mice) by establishing ␣-MHC-MerCreMer
`mice on STAT3flox/flox background (Figure 5A). The ablation
`of the STAT3 gene did not
`induce notable histological
`alterations at day 14 after sham operation. IL-11 treatment
`ameliorated postinfarct fibrosis in ␣-MHC-MerCreMer mice
`on STAT3wild/wild background after MI (Figure V in the
`online-only Data Supplement), as in the case with nontrans-
`
`genic mice (Figure 2). Importantly, IL-11–mediated preven-
`tion against adverse cardiac remodeling was abrogated in
`STAT3 CKO mice (Figure 5C and 5D). Interestingly, en-
`largement of LVs was observed in STAT3 CKO mice
`compared with wild-type mice exposed to MI without IL-11
`treatment (18.02⫾3.20 mm [n⫽6] versus 15.08⫾1.81 mm
`[n⫽8]; P⬍0.05), probably because STAT3 is endogenously
`activated after MI, even without IL-11 treatment (Figures II
`and VI in the online-only Data Supplement), and contributes
`to the prevention of adverse cardiac remodeling. Consis-
`tently, we also confirmed that IL-11–mediated attenuation of
`cardiac dysfunction was canceled in STAT3 CKO mice
`(Figure VII in the online-only Data Supplement). Moreover,
`the increase in capillary density, which was observed in
`response to IL-11 in the mice with STAT3wild/wild back-
`ground, was abrogated by STAT3 gene ablation (Figure VIII
`in the online-only Data Supplement). These data indicate that
`STAT3 is required for IL-11–mediated amelioration of ad-
`verse cardiac remodeling after MI.
`To reinforce the importance of STAT3 in cardioprotection,
`transgenic hearts expressing constitutively active STAT3
`(caSTAT3) were exposed to MI, and cardiac fibrosis was
`estimated. In caSTAT3 transgenic hearts, cardiac fibrosis was
`reduced by 47% compared with nontransgenic littermates
`(wild-type) (Figure 6). Moreover, similar to IL-11–treated
`hearts, caSTAT3 transgenic hearts showed an increase in
`fibrotic wall thickness. Importantly, cardiac dysfunction was
`ameliorated in caSTAT3 transgenic mice compared with
`wild-type mice (Figure IX in the online-only Data Supple-
`ment). Therefore, activation of STAT3 in cardiomyocytes
`was sufficient to suppress cardiac remodeling after MI.
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`

`Obana et al
`
`IL-11 as a Novel Cardioprotective Cytokine
`
`689
`
`Figure 5. STAT3 was required for IL-11–mediated amelioration
`of adverse remodeling after MI. A, Generation of STAT3 condi-
`tional knockout mice. B, After tamoxifen treatment, sham or MI
`operation was performed in ␣-MHC-MerCreMer/STAT3flox/flox
`(flox/flox; n⫽5 mice for sham; n⫽6 mice for MI) or ␣-MHC-Mer-
`CreMer/STAT3wild/wild (wild/wild; n⫽4 mice for sham; n⫽6 mice
`for MI) mice, followed by IL-11 administration. Heart sections (3
`sections from each mouse) were prepared 14 days after MI and
`stained with Masson’s trichrome. The images are representative
`of 12 to 18 obtained from 4 to 6 mice. Bar⫽1 mm. C, LV cir-
`cumference was quantitatively estimated. D, Ratio of fibrotic
`circumference to LV circumference was quantitatively estimated.
`Data are shown as mean⫾SD. *P⬍0.05, #P⬍0.01 vs STAT3wild/wild
`by unpaired t test.
`
`IL-11 belongs to the IL-6 cytokine family, which uses gp130
`as its common receptor. Interestingly, LIF, a member of the IL-6
`cytokine family, has been r