Am J Physiol Heart Circ Physiol 303: H569–H577, 2012.
`First published June 15, 2012; doi:10.1152/ajpheart.00060.2012.
`
`Therapeutic administration of IL-11 exhibits the postconditioning effects
`against ischemia-reperfusion injury via STAT3 in the heart
`
`Masanori Obana,1 Kaori Miyamoto,1 Shiho Murasawa,1 Tomohiko Iwakura,1 Akiko Hayama,1
`Tomomi Yamashita,1 Momoko Shiragaki,1 Shohei Kumagai,1 Akimitsu Miyawaki,1 Kana Takewaki,1
`Goro Matsumiya,2 Makiko Maeda,3 Minoru Yoshiyama,4 Hiroyuki Nakayama,1 and Yasushi Fujio1
`1Laboratory of Clinical Science and Biomedicine, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka,
`Japan; 2Department of Cardiovascular Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan; 3Department
`of Clinical Pharmacogenomics, School of Pharmacy, Hyogo University of Health Sciences; 4Department of Internal Medicine
`and Cardiology, Graduate School of Medicine, Osaka City University, Osaka, Japan
`
`Submitted 26 January 2012; accepted in final form 31 May 2012
`
`Obana M, Miyamoto K, Murasawa S, Iwakura T, Hayama A,
`Yamashita T, Shiragaki M, Kumagai S, Miyawaki A, Takewaki
`K, Matsumiya G, Maeda M, Yoshiyama M, Nakayama H, Fujio
`Y. Therapeutic administration of IL-11 exhibits the postconditioning
`effects against ischemia-reperfusion injury via STAT3 in the heart.
`Am J Physiol Heart Circ Physiol 303: H569 –H577, 2012. First
`published June 15, 2012; doi:10.1152/ajpheart.00060.2012.—Activa-
`tion of cardiac STAT3 by IL-6 cytokine family contributes to cardio-
`protection. Previously, we demonstrated that IL-11, an IL-6 cytokine
`family, has the therapeutic potential to prevent adverse cardiac re-
`modeling after myocardial
`infarction; however,
`it remains to be
`elucidated whether IL-11 exhibits postconditioning effects. To ad-
`dress the possibility that IL-11 treatment improves clinical outcome of
`recanalization therapy against acute myocardial infarction, we exam-
`ined its postconditioning effects on ischemia/reperfusion (I/R) injury.
`C57BL/6 mice were exposed to ischemia (30 min) and reperfusion (24
`h), and IL-11 was intravenously administered at the start of reperfu-
`sion. I/R injury mediated the activation of STAT3, which was en-
`hanced by IL-11 administration. IL-11 treatment reduced I/R injury,
`analyzed by triphenyl tetrazolium chloride staining [PBS, 46.7 ⫾
`14.4%; IL-11 (20 ␮g/kg), 28.6 ⫾ 7.5% in the ratio of infarct to risk
`area]. Moreover, echocardiographic and hemodynamic analyses clar-
`ified that IL-11 treatment preserved cardiac function after I/R. Ter-
`minal deoxynucleotide transferase-mediated dUTP nick-end labeling
`staining revealed that IL-11 reduced the frequency of apoptotic
`cardiomyocytes after I/R. Interestingly, IL-11 reduced superoxide
`production assessed by in situ dihydroethidium fluorescence analysis,
`accompanied by the increased expression of metallothionein 1 and 2,
`reactive oxygen species (ROS) scavengers. Importantly, with the use
`of cardiac-specific STAT3 conditional knockout (STAT3 CKO) mice,
`it was revealed that cardiac-specific ablation of STAT3 abrogated
`IL-11-mediated attenuation of I/R injury. Finally, IL-11 failed to
`suppress the ROS production after I/R in STAT3 CKO mice. IL-11
`administration exhibits the postconditioning effects through cardiac
`STAT3 activation, suggesting that IL-11 has the clinical therapeutic
`potential to prevent I/R injury in heart.
`
`cardiovascular diseases; cytokine; signal transduction
`
`ISCHEMIA-REPERFUSION (I/R) is one of the major causes of myo-
`cardial injury in the clinical setting, especially in the therapeu-
`tic process of acute myocardial infarction. Although various
`kinds of preventive therapies from I/R injury have been pro-
`posed so far, clinical trials revealed that they are insufficient.
`Therefore, it is urgent to develop the therapeutic strategy on a
`
`Address for reprint requests and other correspondence: Y. Fujio, 1-6 Yamada-
`oka, Suita City, 565-0871, Osaka, Japan (e-mail: fujio@phs.osaka-u.ac.jp).
`
`http://www.ajpheart.org
`
`0363-6135/12 Copyright © 2012 the American Physiological Society
`
`Downloaded from www.physiology.org/journal/ajpheart (038.111.035.002) on October 29, 2019.
`
`novel concept to prevent myocardial damage after I/R. Accu-
`mulating evidence has shown that cardiac homeostasis is main-
`tained by a wide range of neurohumoral factors and cytokines,
`suggesting that these factors could be therapeutic targets for
`cardioprotection.
`IL-6 family cytokines contribute to cardioprotection by
`activating various kinds of signaling molecules. In their cyto-
`kine signaling pathways, activation of glycoprotein 130/
`STAT3 axis plays important roles in cytoprotection and angio-
`genesis (6, 21, 22). Thus the activation of STAT3 by IL-6
`family cytokines is considered a potential therapeutic strategy
`for cardiovascular diseases (5). Experimentally, leukemia in-
`hibitory factor shows the antifibrotic effect after myocardial
`infarction (29); however, this cytokine has not been considered
`clinically appropriate because of its proinflammatory proper-
`ties (7). To establish a novel
`therapeutic strategy against
`cardiovascular disease, we focused on IL-11, a member of IL-6
`family cytokines, because its proinflammatory activity is lim-
`ited and because IL-11 exhibits anti-inflammatory activity in
`some cases (3, 23).
`IL-11 exhibits multipotential functions (4). Because IL-11
`has the thrombopoietic activity, recombinant human IL-11 is
`clinically used for thrombocytopenia (9). In addition, IL-11
`also shows nonhematopoietic functions. Previously, we re-
`ported that IL-11 protects cardiomyocytes from H2O2-induced
`cell death through STAT3 activation and has a late precondi-
`tioning effect against I/R injury (16). Recently, we also dem-
`onstrated that the therapeutic treatment of IL-11 reduces ad-
`verse cardiac remodeling after myocardial infarction in murine
`model, concomitant with anti-apoptosis and angiogenesis (20).
`Furthermore, cardiac-specific ablation of STAT3 abrogated
`IL-11-mediated attenuation of adverse cardiac remodeling,
`suggesting that cardiac activation of STAT3 mediates antifi-
`brotic effects.
`In this study, to address the possibility of clinical application
`of IL-11 treatment in the therapeutic process of acute myocar-
`dial infarction as a cardioprotective strategy, we investigated
`its postconditioning effects on I/R injury. In addition, we
`examined whether IL-11 utilizes the cardiac STAT3 signaling
`pathway in its postconditioning effects.
`
`MATERIALS AND METHODS
`
`Animal care. The care of all animals was approved by the Animal
`Singapore Exhibit 2004
`Care and Use Committee of Graduate School of Pharmaceutical
`Sciences, Osaka University. The investigation conforms to the Guide
`Lassen v. Singapore et al.
`for the Care and Use of Laboratory Animals published by the
`PGR2019-00053
`
`H569
`
`

`

`Am J Physiol Heart Circ Physiol 303: H569–H577, 2012.
`First published June 15, 2012; doi:10.1152/ajpheart.00060.2012.
`
`Therapeutic administration of IL-11 exhibits the postconditioning effects
`against ischemia-reperfusion injury via STAT3 in the heart
`
`Masanori Obana,1 Kaori Miyamoto,1 Shiho Murasawa,1 Tomohiko Iwakura,1 Akiko Hayama,1
`Tomomi Yamashita,1 Momoko Shiragaki,1 Shohei Kumagai,1 Akimitsu Miyawaki,1 Kana Takewaki,1
`Goro Matsumiya,2 Makiko Maeda,3 Minoru Yoshiyama,4 Hiroyuki Nakayama,1 and Yasushi Fujio1
`1Laboratory of Clinical Science and Biomedicine, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka,
`Japan; 2Department of Cardiovascular Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan; 3Department
`of Clinical Pharmacogenomics, School of Pharmacy, Hyogo University of Health Sciences; 4Department of Internal Medicine
`and Cardiology, Graduate School of Medicine, Osaka City University, Osaka, Japan
`
`Submitted 26 January 2012; accepted in final form 31 May 2012
`
`Obana M, Miyamoto K, Murasawa S, Iwakura T, Hayama A,
`Yamashita T, Shiragaki M, Kumagai S, Miyawaki A, Takewaki
`K, Matsumiya G, Maeda M, Yoshiyama M, Nakayama H, Fujio
`Y. Therapeutic administration of IL-11 exhibits the postconditioning
`effects against ischemia-reperfusion injury via STAT3 in the heart.
`Am J Physiol Heart Circ Physiol 303: H569 –H577, 2012. First
`published June 15, 2012; doi:10.1152/ajpheart.00060.2012.—Activa-
`tion of cardiac STAT3 by IL-6 cytokine family contributes to cardio-
`protection. Previously, we demonstrated that IL-11, an IL-6 cytokine
`family, has the therapeutic potential to prevent adverse cardiac re-
`modeling after myocardial
`infarction; however,
`it remains to be
`elucidated whether IL-11 exhibits postconditioning effects. To ad-
`dress the possibility that IL-11 treatment improves clinical outcome of
`recanalization therapy against acute myocardial infarction, we exam-
`ined its postconditioning effects on ischemia/reperfusion (I/R) injury.
`C57BL/6 mice were exposed to ischemia (30 min) and reperfusion (24
`h), and IL-11 was intravenously administered at the start of reperfu-
`sion. I/R injury mediated the activation of STAT3, which was en-
`hanced by IL-11 administration. IL-11 treatment reduced I/R injury,
`analyzed by triphenyl tetrazolium chloride staining [PBS, 46.7 ⫾
`14.4%; IL-11 (20 ␮g/kg), 28.6 ⫾ 7.5% in the ratio of infarct to risk
`area]. Moreover, echocardiographic and hemodynamic analyses clar-
`ified that IL-11 treatment preserved cardiac function after I/R. Ter-
`minal deoxynucleotide transferase-mediated dUTP nick-end labeling
`staining revealed that IL-11 reduced the frequency of apoptotic
`cardiomyocytes after I/R. Interestingly, IL-11 reduced superoxide
`production assessed by in situ dihydroethidium fluorescence analysis,
`accompanied by the increased expression of metallothionein 1 and 2,
`reactive oxygen species (ROS) scavengers. Importantly, with the use
`of cardiac-specific STAT3 conditional knockout (STAT3 CKO) mice,
`it was revealed that cardiac-specific ablation of STAT3 abrogated
`IL-11-mediated attenuation of I/R injury. Finally, IL-11 failed to
`suppress the ROS production after I/R in STAT3 CKO mice. IL-11
`administration exhibits the postconditioning effects through cardiac
`STAT3 activation, suggesting that IL-11 has the clinical therapeutic
`potential to prevent I/R injury in heart.
`
`cardiovascular diseases; cytokine; signal transduction
`
`ISCHEMIA-REPERFUSION (I/R) is one of the major causes of myo-
`cardial injury in the clinical setting, especially in the therapeu-
`tic process of acute myocardial infarction. Although various
`kinds of preventive therapies from I/R injury have been pro-
`posed so far, clinical trials revealed that they are insufficient.
`Therefore, it is urgent to develop the therapeutic strategy on a
`
`Address for reprint requests and other correspondence: Y. Fujio, 1-6 Yamada-
`oka, Suita City, 565-0871, Osaka, Japan (e-mail: fujio@phs.osaka-u.ac.jp).
`
`novel concept to prevent myocardial damage after I/R. Accu-
`mulating evidence has shown that cardiac homeostasis is main-
`tained by a wide range of neurohumoral factors and cytokines,
`suggesting that these factors could be therapeutic targets for
`cardioprotection.
`IL-6 family cytokines contribute to cardioprotection by
`activating various kinds of signaling molecules. In their cyto-
`kine signaling pathways, activation of glycoprotein 130/
`STAT3 axis plays important roles in cytoprotection and angio-
`genesis (6, 21, 22). Thus the activation of STAT3 by IL-6
`family cytokines is considered a potential therapeutic strategy
`for cardiovascular diseases (5). Experimentally, leukemia in-
`hibitory factor shows the antifibrotic effect after myocardial
`infarction (29); however, this cytokine has not been considered
`clinically appropriate because of its proinflammatory proper-
`ties (7). To establish a novel
`therapeutic strategy against
`cardiovascular disease, we focused on IL-11, a member of IL-6
`family cytokines, because its proinflammatory activity is lim-
`ited and because IL-11 exhibits anti-inflammatory activity in
`some cases (3, 23).
`IL-11 exhibits multipotential functions (4). Because IL-11
`has the thrombopoietic activity, recombinant human IL-11 is
`clinically used for thrombocytopenia (9). In addition, IL-11
`also shows nonhematopoietic functions. Previously, we re-
`ported that IL-11 protects cardiomyocytes from H2O2-induced
`cell death through STAT3 activation and has a late precondi-
`tioning effect against I/R injury (16). Recently, we also dem-
`onstrated that the therapeutic treatment of IL-11 reduces ad-
`verse cardiac remodeling after myocardial infarction in murine
`model, concomitant with anti-apoptosis and angiogenesis (20).
`Furthermore, cardiac-specific ablation of STAT3 abrogated
`IL-11-mediated attenuation of adverse cardiac remodeling,
`suggesting that cardiac activation of STAT3 mediates antifi-
`brotic effects.
`In this study, to address the possibility of clinical application
`of IL-11 treatment in the therapeutic process of acute myocar-
`dial infarction as a cardioprotective strategy, we investigated
`its postconditioning effects on I/R injury. In addition, we
`examined whether IL-11 utilizes the cardiac STAT3 signaling
`pathway in its postconditioning effects.
`
`MATERIALS AND METHODS
`
`Animal care. The care of all animals was approved by the Animal
`Care and Use Committee of Graduate School of Pharmaceutical
`Sciences, Osaka University. The investigation conforms to the Guide
`for the Care and Use of Laboratory Animals published by the
`
`http://www.ajpheart.org
`
`0363-6135/12 Copyright © 2012 the American Physiological Society
`
`H569
`
`Downloaded from www.physiology.org/journal/ajpheart (038.111.035.002) on October 29, 2019.
`
`

`

`H570
`
`POSTCONDITIONING EFFECTS OF IL-11 IN THE HEART
`
`National Institutes of Health (NIH publication No. 85-23, revised
`1996).
`All mice for the experiments were euthanized by inhalation of
`isoflurane in a euthanasia chamber. Death of the animals was con-
`firmed by monitoring the absence of breath after removal of the
`carcass from the euthanasia chamber. A total of 172 mice were used
`in this study.
`I/R model and IL-11 treatment. Murine I/R was generated as
`described previously, with minor modifications (15, 21). Briefly,
`C57BL/6 mice (8- to 12 wk old; Japan SLC) were anesthetized and
`ventilated with 80% oxygen containing 1.5% isoflurane (Merck).
`After left-side thoracotomy, 7-0 silk suture was tied around the left
`coronary artery with a slipknot. Infarction was confirmed by discol-
`oration of the ventricle and ST-T changes in electrocardiogram mon-
`itor. The chest and the skin were closed with 5-0 silk sutures. The
`mice were revived for a 30-min ischemic period, after which the knot
`was released and the heart was allowed to reperfuse for 24 h. By this
`experimental protocol, the mortality was minimized to less than 10%.
`Twenty four hours after reperfusion, the mice were euthanized and the
`slipknot was retied. PBS containing 1.5% Evans blue was injected
`into the left ventricle, and the hearts were removed. Isolated hearts
`were sectioned, and viable myocardium was stained with 2% triphenyl
`tetrazolium chloride (Sigma), as described previously (21). The
`amounts of myocardial area not at risk, area at risk (AAR), and
`infarcted area were quantified with Scion Image (Scion). In the IL-11
`group, basically, 20 ␮g/kg of recombinant human IL-11 (Peprotech)
`was intravenously administered at the start of reperfusion (various
`concentrations in 200 ␮l of PBS/25 g of body wt), whereas the control
`group received the same volume of PBS over the same period. In the
`study concerning the dose-dependent effects of IL-11 on myocardial
`injury, various concentrations (3, 8, 20, 50 ␮g/kg) of IL-11 were used.
`There was no difference in mortality between groups.
`Immunoblot analysis. Immunoblot analyses were performed as
`described previously (18). Briefly, heart homogenates were prepared
`in buffer containing 150 mM NaCl, 10 mM Tris·HCl (pH 7.5), 1 mM
`EDTA, 1% Triton X-100, 1% deoxycholic acid, 1% protease inhibitor
`cocktail, 1 mM dithiothreitol, 1 mM sodium orthovanadate, and 1 mM
`NaF. Proteins were separated by SDS-PAGE and transferred onto the
`polyvinylidene difluoride membrane (Millipore). The membrane was
`immunoblotted with anti-phospho-STAT3 (p-STAT3; Cell Signaling
`
`Technology) or anti-STAT3 (Santa Cruz Biotechnology) antibody.
`The membrane was reprobed with anti-STAT3 or anti-GAPDH (Santa
`Cruz Biotechnology) antibody to show the equal amount loading.
`Electrochemiluminescence system was used for the detection.
`Echocardiographic analysis. Mice were exposed to I/R injury and
`IL-11 (20 ␮g/kg) or PBS, as a control, was administered at start of
`reperfusion. Twenty four hours after reperfusion, two-dimensional
`and motionmode (M-mode) transthoracic echocardiography was per-
`formed using an iE33 model equipped with a 15-MHz transducer
`(Philips Electronics, Andover, MA). Echocardiographic measure-
`ments were taken on M-mode. The investigator was blinded to the
`identity of the mice for analysis. Sham indicates the mice underwent
`thoracotomy without I/R.
`Hemodynamic analysis. Hemodynamics was analyzed according to
`previous report with minor modification (20). Briefly, 24 h after
`reperfusion, mice were anesthetized (50 mg/kg pentobarbital) and
`heparinized (50 units) via intraperitoneal injection. The hearts were
`rapidly excised and placed in ice-cold modified Tyrode’s solution
`containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.45 MgCl2, 0.33
`NaH2PO4, 5.5 glucose, and 5 HEPES (pH 7.4). The aorta was
`cannulated and retrogradely perfused at a constant pressure of 100
`mmH2O with Tyrode’s solution bubbled with 80% oxygen at 37°C.
`Thus 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 beats/min. The fluid-filled balloon was inserted into the
`left ventricle to monitor cardiac function. The balloon was attached to
`a pressure transducer, which was coupled to a 4S PowerLab (AD
`Instruments). Left ventricular developed pressure and maximal and
`minimal change in pressure over time were measured.
`Terminal deoxynucleotide transferase-mediated dUTP nick-end la-
`beling staining. Twenty four hours after reperfusion,
`the frozen
`sections (5 ␮m thick) were prepared from the portion in the middle of
`the infarct zone. Apoptotic cell death was detected by terminal
`deoxynucleotide
`transferase-mediated dUTP nick-end labeling
`(TUNEL) staining with in situ apoptosis detection kit (TaKaRa). The
`sections were costained with anti-sarcomeric ␣-actinin (Sigma) anti-
`body to identify the cardiomyocytes. Nuclei were simultaneously
`stained with Hoechst 33258. For quantitative analyses, apoptotic
`myocytes were counted in number by the researcher who was blinded
`to the assay conditions.
`
`Fig. 1. IL-11 treatment enhanced STAT3
`activation in ischemia/reperfusion (I/R)
`hearts. A: mice were exposed to I/R. At
`indicated time points, mice were euthanized
`and the lysates from hearts were immuno-
`blotted with anti-phospho-specific STAT3
`(p-STAT3) antibody. The blots were re-
`probed with anti-STAT3 antibody or GAPDH
`antibody. Representative data (top) and quan-
`titative analyses of the p-STAT3 (bottom) are
`shown. Data are shown as means ⫾ SD (n ⫽
`3 mice for each condition). *P ⬍ 0.05 vs.
`nonoperation, by 1-way ANOVA followed by
`Bonferroni test. B: mice were exposed to I/R.
`IL-11 (20 ␮g/kg) or PBS, as a control, was
`administered intravenously at start of reperfu-
`sion. Fifteen minutes after treatment, mice
`were euthanized and the lysates from hearts
`were immunoblotted with anti-p-STAT3 anti-
`body. Blots were reprobed with anti-STAT3
`antibody or GAPDH antibody. Representative
`data (top) and quantitative analyses of the p-
`STAT3 (bottom) are shown. Data are shown as
`means ⫾ SD (n ⫽ 4 mice for each condition).
`*P ⬍ 0.05 vs. nonoperation, by 1-way
`ANOVA followed by Bonferroni test.
`
`AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00060.2012 • www.ajpheart.org
`Downloaded from www.physiology.org/journal/ajpheart (038.111.035.002) on October 29, 2019.
`
`

`

`POSTCONDITIONING EFFECTS OF IL-11 IN THE HEART
`
`H571
`
`Fig. 2. Single administration of IL-11 at the time of
`reperfusion attenuated I/R injury. C57BL/6 mice
`were exposed to 30 min ischemia, followed by 24 h
`reperfusion. IL-11 or PBS, as a control, was admin-
`istered intravenously at start of reperfusion. Areas at
`risk were estimated by exclusion of Evans blue. The
`myocardial infarct areas were detected by staining
`with 2% triphenyl tetrazolium chloride (TTC). Rep-
`resentative images are shown (A). Scale bar, 1 mm.
`B and C: risk area size and infarct size were quan-
`titatively estimated. Data are shown as means ⫾ SD
`(n ⫽ 9 mice for PBS; n ⫽ 6 mice for 3 ␮g/kg of
`IL-11; n ⫽ 7 mice for 8 ␮g/kg of IL-11; n ⫽ 9 mice
`for 20 ␮g/kg of IL-11; n ⫽ 6 mice for 50 ␮g/kg of
`IL-11). *P ⬍ 0.05 vs. PBS, by 1-way ANOVA
`followed by Bonferroni test. LV, left ventricular.
`
`Dihydroethidium fluorescence analysis. Dihydroethidium (DHE)
`fluorescence analysis was performed to examine the generation of
`superoxide. The frozen sections (5 ␮m thick) were prepared and
`stained with 10 ␮M DHE in Krebs/HEPES buffer composed of (in
`mmol/l) 99.01 NaCl, 4.69 KCl, 1.87 CaCl2, 1.20 MgSO4, 1.03
`K2HPO4, 25.0 NaHCO3, 20.0 Na-HEPES, and 11.1 glucose (pH 7.4)
`at 37°C for 30 min in a dark. The intensities of fluorescence were
`quantitatively analyzed with Adobe Photoshop Elements 2.0 (Adobe
`Systems) by the researcher who was blinded to the assay conditions.
`Real-time RT-PCR. Real time RT-PCR was performed according to
`the manufacturer’s protocol. Total RNA was prepared from hearts at
`3 h after reperfusion. Total RNA (1 ␮g) was subjected to first-strand
`cDNA synthesis with oligo (dT) primer. The mRNA expression was
`quantified by real-time RT-PCR using the Applied Biosystems
`StepOne Real-Time PCR systems (Applied Biosystems) with SYBR
`green system (Applied Biosystems). As an internal control, the ex-
`pression of GAPDH mRNA was estimated with SYBR green system.
`The primers used in this study are as follows: metallothionein (MT) 1,
`forward: CGT AGC TCC AGC TTC ACC AGA TCT C, reverse:
`TGG TGG CAG CGC TGT TCG T; MT-2, forward: GCT TTT GCG
`CTC GAC CCA ATA CTC TC, reverse: GGA GCA GCA GCT TTT
`
`CTT GCA GGA AG; cyclooxygenase (COX)-2, forward: ACT GCC
`CAA CTC CCA TGG GT, reverse: AGT CCA CTC CAT GGC CCA
`GT; MnSOD, forward: AGG AGA GCA GCG GTC GTG TAA ACC
`T, reverse: CGG TGG CGT TGA GGT TGT TCA CGT A; Cu/
`ZnSOD, forward: AGA GCC TGA CAG GTG CAG AGA ACC,
`reverse: ACT TTG GCA TGC GTG TCG CC; redox factor-1, for-
`ward: AGA GAC CAA GAA GAG TAA GGG G, reverse: TGC TTC
`TTC CTT TAC CCA ATC C; peroxiredoxin 5, forward: TCA AGG
`TGG GAG ATG CCA TTC, reverse: AAC CTT GCC TTC TGC CTG
`GT; peroxiredoxin 6, forward: AGA TTC ATG GGG CAT TCT CTT
`TTC C, reverse: TAA GCA TTG ATG TCC TTG CTC CAG;
`isocitrate dehydrogenase, forward: AAG GAG AAG CTC ATC CTG
`CC, reverse: TCA GCT TGA ACT CTT CCA CAC G; glutathione
`reductase, forward: TGA TCA GGC ATG ATA AGG TAC TGA G,
`reverse: CAT CCG TCT GAA TGC CCA CT; glutathione peroxidase
`4, forward: AGG CAG GAG CCA GGA AGT A, reverse: TGA TGG
`CAT TTC CCA GCA TGC; 5-oxoprolinase, forward: TTC CAG
`GGC CAG CTA AAG AAT G, reverse: TCT GTG GAT GTG CCT
`CCC ATG T; nuclear factor-like 1, forward: TGC ACA GTT CCC
`AGC TGA C, reverse: CTT CCA TAG CCT GCA TTT CCA T;
`
`Table 1. Effects of IL-11 on cardiac function at 24 h after reperfusion
`
`Parameter/Group
`
`Ejection fraction, %
`Fractional shortening, %
`Diastolic interventricular septal thickness, cm
`LV, cm
`Diastolic internal diameter
`Diastolic posterior wall thickness
`Systolic internal diameter
`Heart rate-LV, beats/min
`
`Sham
`
`79.3 ⫾ 2.5
`40.8 ⫾ 1.9
`0.085 ⫾ 0.001
`
`0.391 ⫾ 0.033
`0.069 ⫾ 0.003
`0.232 ⫾ 0.026
`494 ⫾ 21
`
`I/R ⫹ PBS
`
`52.2 ⫾ 5.7$
`21.9 ⫾ 3.1$
`0.085 ⫾ 0.012
`
`0.360 ⫾ 0.036
`0.080 ⫾ 0.012
`0.282 ⫾ 0.035
`490 ⫾ 52
`
`I/R ⫹ IL-11
`
`62.9 ⫾ 10.1#*
`28.6 ⫾ 6.2#*
`0.093 ⫾ 0.005
`
`0.378 ⫾ 0.009
`0.077 ⫾ 0.011
`0.270 ⫾ 0.028
`486 ⫾ 45
`
`Values are means ⫾ SD; n ⫽ 3 mice for sham, n ⫽ 6 mice for ischemia-reperfusion (I/R) ⫹ PBS, and n ⫽ 6 mice for I/R ⫹ IL-11. Mice were subjected
`to 30 min of ischemia followed by 24 h reperfusion. IL-11 (20 ␮g/kg) or PBS, as a control, was intravenously administered at the time of reperfusion. $P ⬍
`0.01; #P ⬍ 0.05 vs. Sham; *P ⬍ 0.05 vs. I/R ⫹ PBS, by unpaired t-test. LV, left ventricular.
`
`AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00060.2012 • www.ajpheart.org
`Downloaded from www.physiology.org/journal/ajpheart (038.111.035.002) on October 29, 2019.
`
`

`

`H572
`
`POSTCONDITIONING EFFECTS OF IL-11 IN THE HEART
`
`Table 2. Effects of IL-11 on hemodynamics at 24 h after reperfusion
`
`Parameter/Group
`
`LV developed pressure, mmHg
`⫹dP/dt (mmHg/s)
`⫺dP/dt (mmHg/s)
`
`Sham
`
`79.5 ⫾ 9.9
`2347.8 ⫾ 443.2
`⫺2178.3 ⫾ 387.3
`
`I/R ⫹ PBS
`
`55.6 ⫾ 11.0$
`1532.4 ⫾ 296.1$
`⫺1395.0 ⫾ 326.1$
`
`I/R ⫹ IL-11
`
`69.3 ⫾ 6.7*
`2009.0 ⫾ 321.2*
`⫺1776.7 ⫾ 132.2$*
`
`Values are means ⫾ SD; n ⫽ 4 mice for sham, n ⫽ 5 mice for I/R ⫹ PBS, and n ⫽ 6 mice for I/R ⫹ IL-11. Mice were subjected to 30 min of ischemia followed
`by 24 h reperfusion. IL-11 (20 ␮g/kg) or PBS, as a control, was intravenously administered at the time of reperfusion. $P ⬍ 0.05 vs. Sham; *P ⬍ 0.05 vs. I/R ⫹
`PBS, by unpaired t-test. ⫾dP/dt, maximal and minimal change in pressure over time.
`
`GAPDH, forward: GCC GGT GCT GAG TAT GTC GT, reverse:
`CCC TTT TGG CTC CAC CCT T.
`Cell culture and reagents. Cardiomyocytes were cultured as de-
`scribed previously (16). Briefly, cardiac ventricles of 1-day-old Wistar
`rats were minced and cells were isolated with 0.1% trypsin (Difco
`Laboratories) and 0.1% collagenase type IV (Sigma). To eliminate the
`nonmyocyte population, isolated cells were plated and incubated for 1
`h at 37°C. Nonattached cells were collected as cardiomyocytes and
`cultured in DMEM/Ham’s F-12 (DMEM/F-12) containing 5% neo-
`natal calf serum. More than 90% cells were identified as cardiomyo-
`cytes, assessed by immunostaining with anti-sarcomeric specific ␣-ac-
`tinin antibody.
`STAT3 Stealth RNAi, MT Stealth RNAi, and control Stealth RNAi
`were purchased from Invitrogen. Cardiomyocytes were transfected
`with these small interfering RNA (siRNA) using Lipofectamine RNAi
`MAX (Invitrogen) in DMEM/F-12 containing 5% neonatal calf se-
`rum. Cardiomyocytes were cultured in serum-free DMEM/F-12 con-
`taining IL-11 and/or H2O2 at the indicated concentrations. Apoptotic
`cells were detected by Annexin V staining, as described in a previous
`report (16).
`Conditional ablation of STAT3 gene in cardiomyocytes of adult
`murine hearts. Cardiac STAT3 conditional knockout mice were gen-
`erated as described previously with minor modifications (20). In brief,
`the cardiac-specific transgenic mice overexpressing Cre recombinase
`fusion protein to the mutated estrogen receptor domains (MerCreMer)
`under the control of ␣-myosin heavy chain (␣-MHC) promoter were
`crossed with STAT3 flox mice (STAT3flox/flox) to produce ␣-MHC-
`MerCreMer/STAT3flox/flox mice. To activate Cre-recombinase activ-
`ity, ␣-MHC-MerCreMer/STAT3flox/flox
`or ␣-MHC-MerCreMer/
`STAT3wild/wild mice, as control mice, were intraperitoneally injected
`with 8 mg/kg of tamoxifen (Sigma) dissolved in corn oil (Sigma) once
`a day for 14 consecutive days. After tamoxifen treatment, the mutant
`mice underwent I/R as described above.
`
`Statistical analysis. Data were presented as means ⫾ SD. The
`comparison between two groups was performed using an unpaired
`t-test. One-way ANOVA with Bonferroni test was used for compar-
`isons of multiple groups. Differences were considered statistically
`significant when the calculated P value was less than 0.05.
`
`RESULTS
`
`IL-11 treatment enhanced STAT3 activity in I/R hearts.
`Because it is known that STAT3 is activated during I/R, we
`first confirmed that STAT3 is endogenously activated in hearts
`at various time points after I/R in our system (Fig. 1A).
`Immunoblot analyses with anti-p-STAT3 antibody revealed
`that STAT3 phosphorylation was slightly induced at a 30-min
`ischemia period. It is important that STAT3 was dramatically
`activated at 1 h after reperfusion. These data indicated that
`STAT3 signals are endogenously activated during I/R.
`Next, we examined whether IL-11 administration further
`enhanced STAT3 activity during I/R (Fig. 1B). Mice were
`exposed to I/R injury with intravenous injection of IL-11 at
`a dose of 20 ␮g/kg or PBS at start of reperfusion. We
`analyzed the activation of STAT3 at 15 min after reperfu-
`sion by immunoblot analysis, based on the previous findings
`that
`the intravenous administration of
`IL-11 activated
`STAT3 with its peak at 15 min in nonoperated hearts (20).
`Fifteen minutes after
`reperfusion,
`I/R stimuli
`induced
`STAT3 activation, which was enhanced by IL-11 treatment,
`relative to control.
`The therapeutic treatment of IL-11 exhibits the postcondi-
`tioning effects against I/R injury. Because STAT3 activity was
`reinforced by IL-11 injection at start of reperfusion in I/R
`
`Fig. 3. IL-11 treatment suppressed cardiomyocytes apoptosis after I/R. A: frequency of apoptotic cardiomyocytes was estimated by terminal deoxynucleotide
`transferase-mediated dUTP nick-end labeling (TUNEL) staining 24 h after myocardial infarction. The sections were costained with antisarcomeric ␣-actinin
`antibody and Hoechst 33258 dye. The images shown are representative of 75 to 120 images obtained from 5 to 6 mice (15 to 20 fields from each mouse).
`Arrowheads show TUNEL-positive, apoptotic cardiomyocytes. Scale bar, 50 ␮m. B: quantification of the apoptotic cardiomyocytes is shown. Data are shown
`as means ⫾ SD [n ⫽ 5 mice for PBS; n ⫽ 6 mice for IL-11 (20 ␮g/kg)]. *P ⬍ 0.05 by unpaired t-test.
`
`AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00060.2012 • www.ajpheart.org
`Downloaded from www.physiology.org/journal/ajpheart (038.111.035.002) on October 29, 2019.
`
`

`

`POSTCONDITIONING EFFECTS OF IL-11 IN THE HEART
`
`H573
`
`Fig. 4. IL-11 treatment attenuated reactive
`oxygen species (ROS) production after I/R.
`Twenty four hours after reperfusion,
`the
`heart sections were stained with dihydro-
`ethidium (DHE). A: representative fluores-
`cent images are shown. Scale bar, 100 ␮m.
`B: fluorescence intensity at risk area was
`estimated. Data are shown as means ⫾ SD
`[n ⫽ 4 mice for PBS; n ⫽ 5 mice for IL-11
`(20 ␮g/kg)]. *P ⬍ 0.05 by unpaired t-test.
`C: three hours after reperfusion, total RNA
`was prepared from I/R hearts and real time
`RT-PCR was performed for metallothionein
`(MT) 1 and 2. The expression of MT1 and
`MT2 were normalized with that of GAPDH.
`Data are shown as means ⫾ SD [n ⫽ 6 mice
`for PBS; n ⫽ 7 mice for IL-11 (20 ␮g/kg)].
`*P ⬍ 0.05 by unpaired t-test.
`
`model, we examined the postconditioning effects of IL-11 on
`I/R injury (Fig. 2). Mice were subjected to 30 min of left
`coronary artery ligation followed by 24 h reperfusion. IL-11 or
`PBS, as a control, was administered intravenously at start of
`reperfusion. Although there was no significant difference in
`AAR between IL-11 treatment and control group, the infarct
`size relative to AAR was decreased by single treatment of
`IL-11 in a dose-dependent manner (Fig. 2C). Treatment of
`IL-11 at 20 and 50 ␮g/kg significantly reduced the infarct size
`by 38.8 and 39.2%, respectively (PBS, 46.7 ⫾ 14.4%; 20
`␮g/kg of IL-11, 28.6 ⫾ 7.5%; 50 ␮g/kg of IL-11, 28.4 ⫾
`13.7%). Because it is confirmed that IL-11 treatment at a dose
`of 20 ␮g/kg achieved the maximal effect, further studies were
`performed with the use of IL-11 at a dose of 20 ␮g/kg. Next,
`we investigated the cardiac function after I/R by echocardio-
`graphic and hemodynamic analysis. Echocardiographic analy-
`
`sis revealed that ejection fraction and fraction shortening were
`dramatically reduced 24 h after reperfusion (Table 1). Further-
`more, hemodynamic analysis elucidated that left ventricular
`developed pressure and maximal and minimal change in pres-
`sure over time were reduced by I/R (Table 2). It is intriguing,
`however, that IL-11 treatment at a dose of 20 ␮g/kg signifi-
`cantly preserved cardiac function.
`We also examined the effects of the timing of IL-11 treat-
`ment on cardioprotection. Mice were exposed to I/R injury, and
`IL-11 was treated at 3 h after reperfusion. As a result, IL-11
`was less effective in cardioprotection when administered at 3 h
`than immediately after reperfusion (data not shown). There-
`fore, the therapeutic window of IL-11 is likely to be the early
`time point after myocardial infarction.
`These findings suggest that the administration of IL-11 at
`reperfusion has the therapeutic potential to prevent I/R injury.
`
`Table 3. The expressions of cytoprotective genes
`
`Gene
`
`Non–I/R ⫹ IL-11
`
`Metallothionein 1
`Metallothionein 2
`Cyclooxygenase-2
`MnSOD
`Cu/ZnSOD
`Redox factor-1
`Peroxiredoxin 5
`Peroxiredoxin 6
`Isocitrate dehydrogenase
`Glutathione reductase
`Glutathione peroxidase 4
`5-Oxoprolinase
`Nuclear factor-like 1
`
`6.73 ⫾ 1.57*
`4.07 ⫾ 0.82*
`0.41 ⫾ 0.20
`1.09 ⫾ 0.08
`1.09 ⫾ 0.12
`2.77 ⫾ 3.07
`1.02 ⫾ 0.12
`1.06 ⫾ 0.11
`1.08 ⫾ 0.04
`0.68 ⫾ 0.29
`1.03 ⫾ 0.12
`1.52 ⫾ 0.45
`1.28 ⫾ 0.04
`
`I/R ⫹