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Angiotensin II Vaccination Suppresses Cardiac Remodeling after Myocardial Infarction
S198 Journal of Cardiac Failure Vol. 20 No. 10S October 2014 P-054 Prevention of Myocardial Ischemia-Reperfusion Injury in Cardiac-specific SOCS3 Knockout Mice by Enhanced Activation of Cardioprotective Signaling Pathways TAKANOBU NAGATA, HIDEO YASUKAWA, TOYOHARU OBA, KAZUTOSHI MAWATARI, SACHIKO KYOGOKU, HIDEKI OHSHIMA, TOMOKO MINAMI, SHOUICHIROU NOHARA, YUUSUKE SUGI, YOSHIHIRO FUKUMOTO Department of Internal Medicine, Kurume University School of Medicine, Fukuoka, Japan Background: Although JAK-STAT signaling activating cytokines have been shown to prevent myocardial ischemia-reperfusion (IR) injury, little is known about the negative regulatior of the JAK-STAT pathway in the development of IR injury. We previously showed that (SOCS3) is an intrinsic negative regulaton of cytokineinduced STAT3 activation. In this study, we investigated the role of myocardial SOCS3 during IR injury. Methods and Results: Myocardial IR injury was induced by ligation (1-hr) of LAD, followed by different reperfusion time in cardiac-specific SOCS3 knockout mice (SOCS3-CKO) and controls. Evans blue and 1% TTC staining after a 24-hr reperfusion showed a 33% reduction in myocardial infarct size in SOCS3-CKO compared to controls (p!0.05). TUNEL staining revealed that, after 6-hr of reperfusion, apoptotic cells were significantly decreased in SOCS3-CKO. STAT3 phosphorylation was faint at 1-hr after ischemia, marked at 20-min after reperfusion, and suppressed at 3-hr after reperfusion. In contrast, STAT3, AKT, and ERK1/2 phosphorylation after reperfusion was significantly greater in SOCS3CKO than in controls. To investigate the mechanism underlying the prevention of myocardial IR injury in SOCS3-CKO, we examined expression of anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1). Conclusion: These results suggested that myocardial IR injury may have been prevented in SOCS3-CKO by enhanced activation of cardioprotective signaling pathways.
P-055 Evaluation of Mitochondrial ATP Levels In Vivo Identifies G0/G1 Switch Gene 2 as a Therapeutic Target of Ischemic Heart Failure HISAKAZU KATO1, HIDETAKA KIOKA1,2, YOSHIHIRO ASANO1,2, SEIJI TAKASHIMA1,2 1 Department of Medical Biochemistry, Osaka University Graduate School of Medicine, Osaka, Japan, 2Department of Cardiology, Osaka University Graduate School of Medicine, Osaka, Japan Background: Heart tissue consumes more energy than other organs to maintain cell viability and cardiac pump function. Imbalances between energy demand and supply in an ischemic myocardium fall into the mechanical failure of heart. Therefore understanding the mechanism to maintain ATP production in heart would be therapeutic strategy for ischemic heart failure. We recently identified G0/G1 switch gene 2 (G0s2) as an active regulator for ATP-producing enzyme, FoF1-ATP synthase in cultured cardiomyocytes. However, whether G0s2 play roles in regulating ATP production in vivo is still unknown. Hypothesis: In this study, we aimed to clarify whether G0s2 actually affects mitochondrial ATP production using a zebrafish model to accurately measure mitochondrial ATP concentration in vivo. Methods and Results: To assess mitochondrial ATP production in vivo heart, we created transgenic zebrafish model that specifically expresses mitochondria-targeted FRET-based ATP biosensor (Mit-ATeam) in cardiomyocytes. Using Mit-ATeam zebrafish, we observed a decline in mitochondrial ATP concentration in the beating hearts during hypoxia and its recovery by sequential re-oxygenation. Furthermore, we expressed G0s2 in Mit-ATeam zebrafish heart and G0s2-overexpressing cardiomyocytes showed enhanced contractility with increased intra-mitochondrial ATP concentration in hypoxic condition. Conclusion: These results suggest that G0s2 functions as a guardian of hypoxic tissue via enhancing ATP production and could become a therapeutic target for ischemic heart failure.
P-056 Post-infarct Treatment with MicroRNA145 Protects the Heart against Myocardial Ischemia-reperfusion Injury Through Acceleration of Myocyte Autophagy KENSHI HIGASHI1, YOSHIHISA YAMADA1, TAKUMA AOYAMA4, SHINYA BABA1, SHINGO MINATOGUCHI1, KAZUHIKO NISHIGAKI1, GENZO TAKEMURA3, YUKIHIRO AKAO2, SHINYA MINATOGUCHI1 1 Gifu University Graduate School Of Medicine, Gifu, Japan, 2United Graduate School of Drug Discovery and Medical information Science, Gifu University, Gifu, Japan, 3Asahi University, Gifu, Japan, 4Kizawa memorial hospital, Gifu, Japan Background: We previously reported that microRNA145 (miRNA145) significantly reduced the myocardial infarct size and improved the function of left ventricle. It has been reported that autophagy is activated in cardiomyocytes in ischemic heart disease. We investigated whether miRNA145 reduces the myocardial infarct size through acceleration of autophagy. Objective: We aimed to investigate whether administration of miRNA145 would affect myocyte autophagy in a rabbit model of myocardial infarction. Methods: Male Japanese white rabbits underwent 30 min of coronary occlusion followed by 14 days of reperfusion, then received intravenous injection of saline or miRNA145 (0.035 mg/kg) immediately after reperfusion. At 14
days after reperfusion, rabbits were sacrificed and the hearts were removed. Heart tissues were sampled and devided them into three area; remote area, borderline area and infarct area. The morphological changes were investigated by electron microscopy, and the expressions of LC3B were assessed by western blot analysis. Results: Electron microscopy findings showed that cardiomyocyte autophagy was observed in both control and miRNA145 groups. Western blot analysis showed that transition from LC3B-1to LC3B-2 were stronger in miRNA145 group than in the control group. Conclusions: It is suggested that post-infarct treatment with miRNA145 attenuated ischemia-reperfusion injury through acceleration of myocyte autophagy. This was confirmed by western blotting analysis of LC3B.
P-057 Angiotensin II Vaccination Suppresses Cardiac Remodeling after Myocardial Infarction RYO WATANABE, MITSUAKI ISOBE Department of Cardiovascular Medicine, Tokyo Medical and Dental University A growing body of evidence suggests that angiotensin II (AngII) plays a crucial role in the pathogenesis of cardiac remodeling after myocardial infarction (MI), which in turn leads to heart failure. We here examined whether a vaccination against AngII can be effective strategy to prevent cardiac remodeling after MI. Male Sprague-Dawley rats were injected subcutaneously with saline (n57) or AngII vaccine, a conjugate of AngII and keyhole limpet hemocyanin (5mg/rat, n511), on day 0, 14 and 21. On day 28, these rats were subjected to permanent left anterior descending coronary artery ligation. Then, hearts of these rats were harvested on day 56. The anti-AngII antibody titer was increased in the AngII vaccine-injected group [half-maximum binding titer (OD50): AngII vaccine525956788 vs. saline5N.D.]. Although no significant differences of blood pressure or infarct size were observed between two experimental groups, left ventricular (LV) dysfunction and infiltration of macrophages in myocardium were milder in the AngII vaccine injected-group than in the saline-injected group [LV ejection fraction: AngII vaccine541.362.8% vs. saline530.762.6%, p!0.05; infiltration of macrophages: AngII vaccine565618 cells/field vs. saline511969 cells/field, p!0.05]. Furthermore, AngII vaccination did not cause any detrimental changes in cardiac function and myocardium in sham-operated rats. These results suggest that the AngII vaccine may provide a novel promising therapeutic strategy to prevent cardiac remodeling after MI.
P-058 Effects of a Selective Acetylcholine-activated Potassium Channel Blocker on the Regulation of Heart Rate NORIAKI YAMADA1, YOSHIHIRO ASANO1, SATORU YAMAZAKI2, TETSUO MINAMINO1, YASUSHI SAKATA1, MASAFUMI KITAKAZE2, SEIJI TAKASHIMA1 1 Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, 2National Cerebral and Cardiovascular Center Research Institute Sinus bradycardia causes a decreased cardiac output resulting in a variety of clinical symptoms. From a pedigree of familial sinus bradycardia, we previously identified a novel missense mutation in the KCNJ3 gene. KCNJ3 is a member of heterotetrameric acetylcholine-activated potassium channel (KACh channel) in the sinus node, atrioventricular node, and atrial myocardium. The mutation is located in the M1 helix of KCNJ3, which forms the transmembrane pore of the KACh channel. Electrophysiological analyses demonstrated that the KCNJ3 mutation increased the basal current amplitude, and the increased current flow through KACh channels leads to sinus bradycardia. This result suggests that a pharmacological approach to the KACh channel would modulate the heart rate in vivo. We hypothesized that a novel selective KACh channel blocker is a hopeful candidate for the up-regulation of heart rate. We investigated the electrophysiological effects of this drug on the KACh channel activity by two-electrode voltage clamp analysis using Xenopus oocytes. This drug efficiently blocked the potassium current mediated by the KACh channel in the mutant as well as wild type. Furthermore, we generated transgenic zebrafish bearing atrium specific expression of the human KCNJ3 mutant for pharmacological intervention. The transgenic zebrafish showed marked bradycardia. In conclusion, functionally-altered KACh channels cause the pathogenesis of sinus bradycardia, and the KACh channel might be a therapeutic target of heart rate regulation.
P-059 Tranilast, Orally Active TRPV2 Antagonist, Ameliorates End-stage Heart Failure in Mice with Dilated Cardiomyopathy KAZUO KOMAMURA, YUKO IWATA Research Institute, National Cerebral and Cardiovascular Center, Suita, Japan Background: Expression of transient receptor potential vanilloid 2 (TRPV2), a calcium-permeable cation channel, increased in the sarcolemma of animal and human myocardium with dilated cardiomyopathy (DCM). We assessed whether a TRPV2 antagonist, tranilast, ameliorated heart failure of DCM mice. Methods and Results: We used 4C30 mice, which has abnormal myocardial calcium handling, as a model of DCM. Sixteen 4C30 mice of 25 weeks old with end-stage heart failure were given no drug (control) or 20 mg/kg/day of carvediol (group C) or 400 mg/kg/day of tranilast
P-055 Evaluation of Mitochondrial ATP Levels In Vivo Identifies G0/G1 Switch Gene 2 as a Therapeutic Target of Ischemic Heart Failure HISAKAZU KATO1, HIDETAKA KIOKA1,2, YOSHIHIRO ASANO1,2, SEIJI TAKASHIMA1,2 1 Department of Medical Biochemistry, Osaka University Graduate School of Medicine, Osaka, Japan, 2Department of Cardiology, Osaka University Graduate School of Medicine, Osaka, Japan Background: Heart tissue consumes more energy than other organs to maintain cell viability and cardiac pump function. Imbalances between energy demand and supply in an ischemic myocardium fall into the mechanical failure of heart. Therefore understanding the mechanism to maintain ATP production in heart would be therapeutic strategy for ischemic heart failure. We recently identified G0/G1 switch gene 2 (G0s2) as an active regulator for ATP-producing enzyme, FoF1-ATP synthase in cultured cardiomyocytes. However, whether G0s2 play roles in regulating ATP production in vivo is still unknown. Hypothesis: In this study, we aimed to clarify whether G0s2 actually affects mitochondrial ATP production using a zebrafish model to accurately measure mitochondrial ATP concentration in vivo. Methods and Results: To assess mitochondrial ATP production in vivo heart, we created transgenic zebrafish model that specifically expresses mitochondria-targeted FRET-based ATP biosensor (Mit-ATeam) in cardiomyocytes. Using Mit-ATeam zebrafish, we observed a decline in mitochondrial ATP concentration in the beating hearts during hypoxia and its recovery by sequential re-oxygenation. Furthermore, we expressed G0s2 in Mit-ATeam zebrafish heart and G0s2-overexpressing cardiomyocytes showed enhanced contractility with increased intra-mitochondrial ATP concentration in hypoxic condition. Conclusion: These results suggest that G0s2 functions as a guardian of hypoxic tissue via enhancing ATP production and could become a therapeutic target for ischemic heart failure.
P-056 Post-infarct Treatment with MicroRNA145 Protects the Heart against Myocardial Ischemia-reperfusion Injury Through Acceleration of Myocyte Autophagy KENSHI HIGASHI1, YOSHIHISA YAMADA1, TAKUMA AOYAMA4, SHINYA BABA1, SHINGO MINATOGUCHI1, KAZUHIKO NISHIGAKI1, GENZO TAKEMURA3, YUKIHIRO AKAO2, SHINYA MINATOGUCHI1 1 Gifu University Graduate School Of Medicine, Gifu, Japan, 2United Graduate School of Drug Discovery and Medical information Science, Gifu University, Gifu, Japan, 3Asahi University, Gifu, Japan, 4Kizawa memorial hospital, Gifu, Japan Background: We previously reported that microRNA145 (miRNA145) significantly reduced the myocardial infarct size and improved the function of left ventricle. It has been reported that autophagy is activated in cardiomyocytes in ischemic heart disease. We investigated whether miRNA145 reduces the myocardial infarct size through acceleration of autophagy. Objective: We aimed to investigate whether administration of miRNA145 would affect myocyte autophagy in a rabbit model of myocardial infarction. Methods: Male Japanese white rabbits underwent 30 min of coronary occlusion followed by 14 days of reperfusion, then received intravenous injection of saline or miRNA145 (0.035 mg/kg) immediately after reperfusion. At 14
days after reperfusion, rabbits were sacrificed and the hearts were removed. Heart tissues were sampled and devided them into three area; remote area, borderline area and infarct area. The morphological changes were investigated by electron microscopy, and the expressions of LC3B were assessed by western blot analysis. Results: Electron microscopy findings showed that cardiomyocyte autophagy was observed in both control and miRNA145 groups. Western blot analysis showed that transition from LC3B-1to LC3B-2 were stronger in miRNA145 group than in the control group. Conclusions: It is suggested that post-infarct treatment with miRNA145 attenuated ischemia-reperfusion injury through acceleration of myocyte autophagy. This was confirmed by western blotting analysis of LC3B.
P-057 Angiotensin II Vaccination Suppresses Cardiac Remodeling after Myocardial Infarction RYO WATANABE, MITSUAKI ISOBE Department of Cardiovascular Medicine, Tokyo Medical and Dental University A growing body of evidence suggests that angiotensin II (AngII) plays a crucial role in the pathogenesis of cardiac remodeling after myocardial infarction (MI), which in turn leads to heart failure. We here examined whether a vaccination against AngII can be effective strategy to prevent cardiac remodeling after MI. Male Sprague-Dawley rats were injected subcutaneously with saline (n57) or AngII vaccine, a conjugate of AngII and keyhole limpet hemocyanin (5mg/rat, n511), on day 0, 14 and 21. On day 28, these rats were subjected to permanent left anterior descending coronary artery ligation. Then, hearts of these rats were harvested on day 56. The anti-AngII antibody titer was increased in the AngII vaccine-injected group [half-maximum binding titer (OD50): AngII vaccine525956788 vs. saline5N.D.]. Although no significant differences of blood pressure or infarct size were observed between two experimental groups, left ventricular (LV) dysfunction and infiltration of macrophages in myocardium were milder in the AngII vaccine injected-group than in the saline-injected group [LV ejection fraction: AngII vaccine541.362.8% vs. saline530.762.6%, p!0.05; infiltration of macrophages: AngII vaccine565618 cells/field vs. saline511969 cells/field, p!0.05]. Furthermore, AngII vaccination did not cause any detrimental changes in cardiac function and myocardium in sham-operated rats. These results suggest that the AngII vaccine may provide a novel promising therapeutic strategy to prevent cardiac remodeling after MI.
P-058 Effects of a Selective Acetylcholine-activated Potassium Channel Blocker on the Regulation of Heart Rate NORIAKI YAMADA1, YOSHIHIRO ASANO1, SATORU YAMAZAKI2, TETSUO MINAMINO1, YASUSHI SAKATA1, MASAFUMI KITAKAZE2, SEIJI TAKASHIMA1 1 Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, 2National Cerebral and Cardiovascular Center Research Institute Sinus bradycardia causes a decreased cardiac output resulting in a variety of clinical symptoms. From a pedigree of familial sinus bradycardia, we previously identified a novel missense mutation in the KCNJ3 gene. KCNJ3 is a member of heterotetrameric acetylcholine-activated potassium channel (KACh channel) in the sinus node, atrioventricular node, and atrial myocardium. The mutation is located in the M1 helix of KCNJ3, which forms the transmembrane pore of the KACh channel. Electrophysiological analyses demonstrated that the KCNJ3 mutation increased the basal current amplitude, and the increased current flow through KACh channels leads to sinus bradycardia. This result suggests that a pharmacological approach to the KACh channel would modulate the heart rate in vivo. We hypothesized that a novel selective KACh channel blocker is a hopeful candidate for the up-regulation of heart rate. We investigated the electrophysiological effects of this drug on the KACh channel activity by two-electrode voltage clamp analysis using Xenopus oocytes. This drug efficiently blocked the potassium current mediated by the KACh channel in the mutant as well as wild type. Furthermore, we generated transgenic zebrafish bearing atrium specific expression of the human KCNJ3 mutant for pharmacological intervention. The transgenic zebrafish showed marked bradycardia. In conclusion, functionally-altered KACh channels cause the pathogenesis of sinus bradycardia, and the KACh channel might be a therapeutic target of heart rate regulation.
P-059 Tranilast, Orally Active TRPV2 Antagonist, Ameliorates End-stage Heart Failure in Mice with Dilated Cardiomyopathy KAZUO KOMAMURA, YUKO IWATA Research Institute, National Cerebral and Cardiovascular Center, Suita, Japan Background: Expression of transient receptor potential vanilloid 2 (TRPV2), a calcium-permeable cation channel, increased in the sarcolemma of animal and human myocardium with dilated cardiomyopathy (DCM). We assessed whether a TRPV2 antagonist, tranilast, ameliorated heart failure of DCM mice. Methods and Results: We used 4C30 mice, which has abnormal myocardial calcium handling, as a model of DCM. Sixteen 4C30 mice of 25 weeks old with end-stage heart failure were given no drug (control) or 20 mg/kg/day of carvediol (group C) or 400 mg/kg/day of tranilast