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Crucumin, a natural p300-specific histone acetyltransferase inhibitor, prevents the development of heart failure in vivo
440
ABSTRACTS / Journal of Molecular and Cellular Cardiology 44 (2008) 435–450
Histologically, the coronary arteries of these two cases were almost the same. Massive eosinophilic infiltration was localized only in adventitia of coronary arteries located in the subepicardial region. Inflammatory infiltration could not be found not only in the intramural coronary arteries, but also in the arteries of other tissues and organs. Immunohistochemically, IL-3 and GM-CSF were positive for the adventitial cells of coronary arteries and for infiltrated eosinophils and macrophages, while these cytokines were negative for the adventitial cells of arteries of other tissues and organs. It is now well documented that eotaxin, IL-3, -5 and GM-CSF are inflammatory cytokines regulating the development and the migration of eosinophils. Our results suggest that the activation of inflammatory cytokines such as eotaxin, IL-3, -5 and GM-CSF in the adventitial cells might induce massive accumulation of eosinophils in the adventitia of the coronary arteries of IECP. Keywords: Coronary periarteritis; Eosinophil; Inflammatory cytokine doi:10.1016/j.yjmcc.2007.07.018
O-3. The effect of voltage- and time-dependent IKur blockade in human atrial fibrillation-induced electrical-remodeling model Kenji Tsujimae1, Shingo Murakami1,2, Yoshihisa Kurachi1,2. 1 Department of Pharmacology, Osaka University Graduate School of Medicine, Japan. 2MEI Center, Osaka University, Suita, Japan Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. The thrombus formation in chronic AF is considered as one of the major causes for stroke in elder patients. Therefore, treatment of AF is of importance. Pharmacological treatment with various anti-arrhythmic drugs to terminate or prevent AF is not satisfactory at present, partly because the drugs act not only on the atrium but also on the ventricle and may cause ventricular arrhythmia, including Torsade de pointes. Recently, the ultra rapid delayed rectifying K+ current (IKur) has become recognized as a potential target of anti-AF drug. Since IKur density is high in the atrium but low or absent in the ventricle, selective IKur blockers may work as anti-AF drugs without ventricular proarrhythmia. New agents which specifically aimed to block IKur have been developed. However, their blockade of IKur exhibited various voltage- and time-dependent kinetics. In the present study, we examined the effects of the drug-induced IKur blockade kinetics on atrial APD prolongation by using normal and remodeled human atrial myocyte models. The IKur-blocker with fast onset effectively prolonged APD with any stimulus frequency, while that with slow recovery prolonged APD in a frequency-dependent manner. We also searched the ideal experimental configuration to find IKur blockers with the fast onset and found that a simple protocol using short steps in voltage clamp experiments is appropriate to differentiate the effectiveness of various drugs in atrial APD prolongation. These results suggest that the voltage and time dependence of the IKur blockade may be the critical criterion in search of effective anti-AF drugs.
Keywords: Atrial fibrillation; IKur; Model study doi:10.1016/j.yjmcc.2007.07.019
O-4. Ethanol modulates expression of T-type Ca2+ channel by regulating transcription factors Csx/Nkx2.5 and GATA4 in neonatal rat myocyte Yan Wang, Masaki Morishima, Toru Shimaoka, Tae-Seong Lee, Mingqi Zheng, Katsushige Ono. Department of Cardiovascular Science, Oita University School of Medicine, Oita, Japan Background and objective: Heavy alcohol abuse is associated with various arrhythmias such as ventricular premature beats, supraventricular tachyarrhythmia, and atrial fibrillation. Although it is reported that chronic ethanol exposure up-regulates the density and function of the L-type Ca2+ channel via protein kinase C (PKC)-dependent mechanism, the action of ethanol on the T-type Ca2+ channel is poorly understood. Purpose of this study was to investigate a long-term effect of ethanol on the T-type Ca2+ channel in cardiomyocytes. Methods: Long-term effects of ethanol on neonatal rat cardiomyocytes were investigated by means of various ethanol concentrations (0.01–1%) and exposure time (0–48 h). Expression of T-type Ca2+ channel α1 subunit isoforms (Cav3.1 and Cav3.2) mRNA was confirmed by real-time PCR. T-type Ca2+ channel current was recorded by patch clamp method. Results: Long-term treatment of cardiomyocytes by ethanol (24 h, 0.1%) significantly increased the expression of T-type Ca2+ channel mRNA (Cav3.1, 24 ± 7%; Cav3.2, 47 ± 18%), and the effect was concentration-dependent (0.01%–0.1%). Peak current of the T-type Ca2+ channel in neonatal rat cardiomyocyte was increased by long-term ethanol exposure (24 h, 49 ± 13%, n = 7), while the activation and the steady-state inactivation curves were unchanged. On the other hand, expression of Csx/Nkx2.5 and GATA4 in neonatal cardiomyocyte was upregulated by ethanol exposure (Csx/Nkx2.5, 64 ± 21%; GATA4, 43 ± 20%). The actions of ethanol on Csx/Nkx2.5 and GATA4 were not affected by a PKC inhibitor (chelerytrine). Summary: It is suggested that ethanol increases the expression of T-type Ca2+ channel in cardiomyocyte depending on transcription factors Csx/Nkx2.5 and GATA4 regardless of PKC activity. Keywords: Ethanol; Csx/Nkx2.5; T-type Ca2+ channel doi:10.1016/j.yjmcc.2007.07.020
O-5. Crucumin, a natural p300-specific histone acetyltransferase inhibitor, prevents the development of heart failure in vivo Tatsuya Morimoto1, Yoichi Sunagawa1, Tomohide Takaya1, Koji Hasegawa1 , Masatoshi Fujita2 , Toru Kita2 . 1Kyoto Medical Center, National Hospital Organization, Japan. 2Kyoto University, Kyoto, Japan
ABSTRACTS / Journal of Molecular and Cellular Cardiology 44 (2008) 435–450
441
Purpose: We have found that curcumin inhibits p300, an intrinsic histone acetyltransferase (HAT)-induced acetylation of histones and GATA-4, and represses phenylephrine-induced hypertrophic responses in primary cardiac myocytes from neonatal rats in culture. To determine whether curcumin can prevent the development of heart failure in vivo, we have utilized a salt-sensitive Dahl (DS) rat model of hypertension and myocardial infarction (MI) rat model. Methods: (1) We randomized 11-week-old DS rats with compensated concentric hypertrophy (n = 56) to oral chronic daily treatment with curcumin (50 mg/kg/day) or vehicle. (2) One week after ligation, 32 rats with moderate-sized MI were randomly subjected to treatment with either curcumin (50 mg/ kg/day) or vehicle. Results: At the age of 17 weeks DS rats, curcumin significantly ameliorated the survival rate (curcumin: 76%, vehicle: 44%, p b 0.001). Curcumin treatment preserved LV fractional shortening both in 17-week-old DS rats (curcumin: 48%, vehicle: 31%, p b 0.05) and in MI rats at 7 weeks after ligation (curcumin: 30%, vehicle: 15%, p b 0.0001). Curcumin can suppress increases in acetylation of GATA4 and myocardial cell diameter in rat heats. Conclusion: A natural compound, curcumin, an inhibitor p300-HAT activity, can prevent the development of heart failure in vivo. Thus, this compound might be applicable to heart failure therapy in humans.
light signal as Ca2+ transient (CaT) and isometric tension induced by electrical field stimulation (0.2 Hz, 30 °C). In KI, the amplitude of CaT was significantly increased, whereas the amplitude of tension was significantly deceased compared to those of WT. The time courses of CaT were prolonged and the time courses of tension were shortened in KI. We plotted the relationship between the peak CaT and the peak tension. This relation was shifted to the right in KI, suggesting a decrease in the Ca2+ sensitivity of the myofilament. These results suggest that the altered CaT, in particular the increased peak, in KI could be partly due to the decrease in the Ca2+ sensitivity of the myofilament and the increased intracellular Ca2+ concentration might underlie the cardiac sudden death in this mouse model of DCM.
Keywords: Histone acetyltransferase; p300; Heart failure
Objective: The aim of this study was to examine protein kinases responsible for phosphorylation of glycogen synthase kinase-3β (GSK-3β) in response to cardioprotective signalings and the mechanism by which p-GSK-3β inhibits mitochondrial permeability transition pore (mPTP) opening. Methods: First, tissues for immunoblotting were sampled before and after 25-min global ischemia/5-min reperfusion (I/R) in isolated perfused rat hearts. These timings of sampling were based on our previous finding that anti-infarct tolerance afforded by ischemic preconditioning (PC) and erythropoietin (EPO) correlated with p-GSK-3β level at 5 min after reperfusion. Second, rat hearts underwent I/R with or without PC + EPO infusion (5 U/ml) before ischemia. Results: GSK-3β was translocated from the cytosol to mitochondria after reperfusion, and p-GSK-3β level was increased in all fractions. Reperfusion increased GSK-3β bound to the voltage-dependent anion channel (VDAC) and GSK-3β bound to adenine nucleotide translocase (ANT). The level of this GSK-3β–ANT complex was increased by PC + EPO, though GSK-3β–VDAC complex level was unchanged. ANT but not VADAC was co-immunoprecipitated with p-GSK-3β. Chelerythrine alone and wortmannin alone partially suppressed phosphorylation of GSK-3β bound with ANT by PC + EPO. PKC but not Akt was co-immunoprecipitated with GSK-3β. PC + EPO did not change the level of ANT–VDAC complex but suppressed formation of ANT– cyclophilin-D complex after reperfusion. Conclusion: The results suggest that mitochondrial PKC and cytosolic Akt are responsible for phosphorylation of GSK-3β bound with ANT and that p-GSK-3β achieves
doi:10.1016/j.yjmcc.2007.07.021
O-6. Changes in Ca2+ transient and contraction of left ventricular papillary muscles in mouse model of dilated cardiomyopathy Kenichi Hongo1, Satoshi Morimoto1, Makoto Kawai1, Kimiaki Komukai1, Jin O-Uchi1, Sachio Morimoto2, Satoshi Kurihara1. 1 Jikei University, Tokyo, Japan. 2Kyushu University, Fukuoka, Japan Recently, a number of gene mutations of cardiac contractile proteins have been found to cause familiar cardiomyopathy. Among such mutations, mutations of troponin are frequently observed. Deletion mutant of troponin T (ΔK210) is known to develop dilated cardiomyopathy (DCM) and this mutation causes a decrease in the Ca2+ sensitivity of the myofilament measured in vitro (Morimoto et al., Proc. Natl. Acad. Sci. USA. 2002;99:913–8). In this study, we investigated the changes in Ca2+ handling of the cardiac muscles in knock-in mouse model of this troponin T mutation (KI). KI mice exhibited dilatation of heart chambers and frequently suffered with cardiac sudden death. Papillary muscle preparations were dissected from the left ventricle of KI and wild type (WT) mice (8–10 weeks old) hearts and mounted between a fixed hook and a force transducer. To measure intracellular Ca 2+ concentration, aequorin was micro-injected into the superficial cells of the preparation. We simultaneously measured aequorin
Keywords: Ca2+ handling; Troponin T; Dilated cardiomyopathy doi:10.1016/j.yjmcc.2007.07.022
O-7. GSK-3β phosphorylated by PKC and Akt inhibits ANT–cyclophilin-D interaction: A possible mechanism of cardiomyocyte protection Masahiro Nishihara, Tetsuji Miura, Katsuhiko Ohori, Takayuki Miki, Kazuaki Shimamoto. Department of Internal Medicine, Sapporo Medical University, Sapporo, Japan
ABSTRACTS / Journal of Molecular and Cellular Cardiology 44 (2008) 435–450
Histologically, the coronary arteries of these two cases were almost the same. Massive eosinophilic infiltration was localized only in adventitia of coronary arteries located in the subepicardial region. Inflammatory infiltration could not be found not only in the intramural coronary arteries, but also in the arteries of other tissues and organs. Immunohistochemically, IL-3 and GM-CSF were positive for the adventitial cells of coronary arteries and for infiltrated eosinophils and macrophages, while these cytokines were negative for the adventitial cells of arteries of other tissues and organs. It is now well documented that eotaxin, IL-3, -5 and GM-CSF are inflammatory cytokines regulating the development and the migration of eosinophils. Our results suggest that the activation of inflammatory cytokines such as eotaxin, IL-3, -5 and GM-CSF in the adventitial cells might induce massive accumulation of eosinophils in the adventitia of the coronary arteries of IECP. Keywords: Coronary periarteritis; Eosinophil; Inflammatory cytokine doi:10.1016/j.yjmcc.2007.07.018
O-3. The effect of voltage- and time-dependent IKur blockade in human atrial fibrillation-induced electrical-remodeling model Kenji Tsujimae1, Shingo Murakami1,2, Yoshihisa Kurachi1,2. 1 Department of Pharmacology, Osaka University Graduate School of Medicine, Japan. 2MEI Center, Osaka University, Suita, Japan Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. The thrombus formation in chronic AF is considered as one of the major causes for stroke in elder patients. Therefore, treatment of AF is of importance. Pharmacological treatment with various anti-arrhythmic drugs to terminate or prevent AF is not satisfactory at present, partly because the drugs act not only on the atrium but also on the ventricle and may cause ventricular arrhythmia, including Torsade de pointes. Recently, the ultra rapid delayed rectifying K+ current (IKur) has become recognized as a potential target of anti-AF drug. Since IKur density is high in the atrium but low or absent in the ventricle, selective IKur blockers may work as anti-AF drugs without ventricular proarrhythmia. New agents which specifically aimed to block IKur have been developed. However, their blockade of IKur exhibited various voltage- and time-dependent kinetics. In the present study, we examined the effects of the drug-induced IKur blockade kinetics on atrial APD prolongation by using normal and remodeled human atrial myocyte models. The IKur-blocker with fast onset effectively prolonged APD with any stimulus frequency, while that with slow recovery prolonged APD in a frequency-dependent manner. We also searched the ideal experimental configuration to find IKur blockers with the fast onset and found that a simple protocol using short steps in voltage clamp experiments is appropriate to differentiate the effectiveness of various drugs in atrial APD prolongation. These results suggest that the voltage and time dependence of the IKur blockade may be the critical criterion in search of effective anti-AF drugs.
Keywords: Atrial fibrillation; IKur; Model study doi:10.1016/j.yjmcc.2007.07.019
O-4. Ethanol modulates expression of T-type Ca2+ channel by regulating transcription factors Csx/Nkx2.5 and GATA4 in neonatal rat myocyte Yan Wang, Masaki Morishima, Toru Shimaoka, Tae-Seong Lee, Mingqi Zheng, Katsushige Ono. Department of Cardiovascular Science, Oita University School of Medicine, Oita, Japan Background and objective: Heavy alcohol abuse is associated with various arrhythmias such as ventricular premature beats, supraventricular tachyarrhythmia, and atrial fibrillation. Although it is reported that chronic ethanol exposure up-regulates the density and function of the L-type Ca2+ channel via protein kinase C (PKC)-dependent mechanism, the action of ethanol on the T-type Ca2+ channel is poorly understood. Purpose of this study was to investigate a long-term effect of ethanol on the T-type Ca2+ channel in cardiomyocytes. Methods: Long-term effects of ethanol on neonatal rat cardiomyocytes were investigated by means of various ethanol concentrations (0.01–1%) and exposure time (0–48 h). Expression of T-type Ca2+ channel α1 subunit isoforms (Cav3.1 and Cav3.2) mRNA was confirmed by real-time PCR. T-type Ca2+ channel current was recorded by patch clamp method. Results: Long-term treatment of cardiomyocytes by ethanol (24 h, 0.1%) significantly increased the expression of T-type Ca2+ channel mRNA (Cav3.1, 24 ± 7%; Cav3.2, 47 ± 18%), and the effect was concentration-dependent (0.01%–0.1%). Peak current of the T-type Ca2+ channel in neonatal rat cardiomyocyte was increased by long-term ethanol exposure (24 h, 49 ± 13%, n = 7), while the activation and the steady-state inactivation curves were unchanged. On the other hand, expression of Csx/Nkx2.5 and GATA4 in neonatal cardiomyocyte was upregulated by ethanol exposure (Csx/Nkx2.5, 64 ± 21%; GATA4, 43 ± 20%). The actions of ethanol on Csx/Nkx2.5 and GATA4 were not affected by a PKC inhibitor (chelerytrine). Summary: It is suggested that ethanol increases the expression of T-type Ca2+ channel in cardiomyocyte depending on transcription factors Csx/Nkx2.5 and GATA4 regardless of PKC activity. Keywords: Ethanol; Csx/Nkx2.5; T-type Ca2+ channel doi:10.1016/j.yjmcc.2007.07.020
O-5. Crucumin, a natural p300-specific histone acetyltransferase inhibitor, prevents the development of heart failure in vivo Tatsuya Morimoto1, Yoichi Sunagawa1, Tomohide Takaya1, Koji Hasegawa1 , Masatoshi Fujita2 , Toru Kita2 . 1Kyoto Medical Center, National Hospital Organization, Japan. 2Kyoto University, Kyoto, Japan
ABSTRACTS / Journal of Molecular and Cellular Cardiology 44 (2008) 435–450
441
Purpose: We have found that curcumin inhibits p300, an intrinsic histone acetyltransferase (HAT)-induced acetylation of histones and GATA-4, and represses phenylephrine-induced hypertrophic responses in primary cardiac myocytes from neonatal rats in culture. To determine whether curcumin can prevent the development of heart failure in vivo, we have utilized a salt-sensitive Dahl (DS) rat model of hypertension and myocardial infarction (MI) rat model. Methods: (1) We randomized 11-week-old DS rats with compensated concentric hypertrophy (n = 56) to oral chronic daily treatment with curcumin (50 mg/kg/day) or vehicle. (2) One week after ligation, 32 rats with moderate-sized MI were randomly subjected to treatment with either curcumin (50 mg/ kg/day) or vehicle. Results: At the age of 17 weeks DS rats, curcumin significantly ameliorated the survival rate (curcumin: 76%, vehicle: 44%, p b 0.001). Curcumin treatment preserved LV fractional shortening both in 17-week-old DS rats (curcumin: 48%, vehicle: 31%, p b 0.05) and in MI rats at 7 weeks after ligation (curcumin: 30%, vehicle: 15%, p b 0.0001). Curcumin can suppress increases in acetylation of GATA4 and myocardial cell diameter in rat heats. Conclusion: A natural compound, curcumin, an inhibitor p300-HAT activity, can prevent the development of heart failure in vivo. Thus, this compound might be applicable to heart failure therapy in humans.
light signal as Ca2+ transient (CaT) and isometric tension induced by electrical field stimulation (0.2 Hz, 30 °C). In KI, the amplitude of CaT was significantly increased, whereas the amplitude of tension was significantly deceased compared to those of WT. The time courses of CaT were prolonged and the time courses of tension were shortened in KI. We plotted the relationship between the peak CaT and the peak tension. This relation was shifted to the right in KI, suggesting a decrease in the Ca2+ sensitivity of the myofilament. These results suggest that the altered CaT, in particular the increased peak, in KI could be partly due to the decrease in the Ca2+ sensitivity of the myofilament and the increased intracellular Ca2+ concentration might underlie the cardiac sudden death in this mouse model of DCM.
Keywords: Histone acetyltransferase; p300; Heart failure
Objective: The aim of this study was to examine protein kinases responsible for phosphorylation of glycogen synthase kinase-3β (GSK-3β) in response to cardioprotective signalings and the mechanism by which p-GSK-3β inhibits mitochondrial permeability transition pore (mPTP) opening. Methods: First, tissues for immunoblotting were sampled before and after 25-min global ischemia/5-min reperfusion (I/R) in isolated perfused rat hearts. These timings of sampling were based on our previous finding that anti-infarct tolerance afforded by ischemic preconditioning (PC) and erythropoietin (EPO) correlated with p-GSK-3β level at 5 min after reperfusion. Second, rat hearts underwent I/R with or without PC + EPO infusion (5 U/ml) before ischemia. Results: GSK-3β was translocated from the cytosol to mitochondria after reperfusion, and p-GSK-3β level was increased in all fractions. Reperfusion increased GSK-3β bound to the voltage-dependent anion channel (VDAC) and GSK-3β bound to adenine nucleotide translocase (ANT). The level of this GSK-3β–ANT complex was increased by PC + EPO, though GSK-3β–VDAC complex level was unchanged. ANT but not VADAC was co-immunoprecipitated with p-GSK-3β. Chelerythrine alone and wortmannin alone partially suppressed phosphorylation of GSK-3β bound with ANT by PC + EPO. PKC but not Akt was co-immunoprecipitated with GSK-3β. PC + EPO did not change the level of ANT–VDAC complex but suppressed formation of ANT– cyclophilin-D complex after reperfusion. Conclusion: The results suggest that mitochondrial PKC and cytosolic Akt are responsible for phosphorylation of GSK-3β bound with ANT and that p-GSK-3β achieves
doi:10.1016/j.yjmcc.2007.07.021
O-6. Changes in Ca2+ transient and contraction of left ventricular papillary muscles in mouse model of dilated cardiomyopathy Kenichi Hongo1, Satoshi Morimoto1, Makoto Kawai1, Kimiaki Komukai1, Jin O-Uchi1, Sachio Morimoto2, Satoshi Kurihara1. 1 Jikei University, Tokyo, Japan. 2Kyushu University, Fukuoka, Japan Recently, a number of gene mutations of cardiac contractile proteins have been found to cause familiar cardiomyopathy. Among such mutations, mutations of troponin are frequently observed. Deletion mutant of troponin T (ΔK210) is known to develop dilated cardiomyopathy (DCM) and this mutation causes a decrease in the Ca2+ sensitivity of the myofilament measured in vitro (Morimoto et al., Proc. Natl. Acad. Sci. USA. 2002;99:913–8). In this study, we investigated the changes in Ca2+ handling of the cardiac muscles in knock-in mouse model of this troponin T mutation (KI). KI mice exhibited dilatation of heart chambers and frequently suffered with cardiac sudden death. Papillary muscle preparations were dissected from the left ventricle of KI and wild type (WT) mice (8–10 weeks old) hearts and mounted between a fixed hook and a force transducer. To measure intracellular Ca 2+ concentration, aequorin was micro-injected into the superficial cells of the preparation. We simultaneously measured aequorin
Keywords: Ca2+ handling; Troponin T; Dilated cardiomyopathy doi:10.1016/j.yjmcc.2007.07.022
O-7. GSK-3β phosphorylated by PKC and Akt inhibits ANT–cyclophilin-D interaction: A possible mechanism of cardiomyocyte protection Masahiro Nishihara, Tetsuji Miura, Katsuhiko Ohori, Takayuki Miki, Kazuaki Shimamoto. Department of Internal Medicine, Sapporo Medical University, Sapporo, Japan