abstracts from the 22nd annual, japanese section of the international society for heart research

doi:10.1016/j.yjmcc.2005.09.014

Journal of Molecular and Cellular Cardiology 39 (2005) 996–1036http://france.elsevier.com/direct/YJMCC/

Abstracts from the 22nd Annual Meeting, Japanese Section of the International Society for Heart Research,

December 15–17, 2005, Osaka, Japan

998 ISHR Japanese Section 22nd Annual Meeting / Journal of Molecular and Cellular Cardiology 39 (2005) 996–1036

Abstracts N° YIA-1Histone acetyltransferase activity of p300 is required for the promotion of left ventricular remodeling following myocardial infarction in adult mice in vivoShoichi Miyamoto a, Teruhisa Kawamura b, Tatsuya Morimoto a, Koh Ono b, Yosuke Kawase c, Toru Kita a, Koji Hasegawa ba Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan. b Division of Translational Research, Kyoto Medical Center, National Hospital Organization, Kyoto, Japan. c Chugai Research Institute for Medical Science, Inc., Pharmacology and Pathology Research Center, Japan

Background. – Left ventricular (LV) remodelingfollowing myocardial infarction (MI) is associated withhypertrophy of surviving myocytes and represents a majorprocess that leads to heart failure. One of the intrinsichistone acetyltransferases, p300, serves as a coactivator ofhypertrophy-responsive transcriptional factors such as acardiac zinc finger protein GATA-4 and is involved in itshypertrophic stimulus-induced acetylation and DNAbinding. However, the role of p300-histone acetyltransferaseactivity in LV remodeling following MI in vivo is unknown.

Methods and results. – To solve this problem, we havegenerated transgenic mice overexpressing intact p300 ormutant p300 in the heart. Due to its two-amino-acidsubstitution in the p300-histone acetyltransferase domain,this mutant lost its histone acetyltransferase activity and wasunable to activate GATA-4-dependent transcription. Thetwo kinds of transgenic mice and the wild-type mice weresubjected to MI or sham operation at the age of 12 weeks.Five weeks later, intact p300 transgenic mice showedsignificantly more progressive LV dilation and diminishedsystolic function following MI than wild-type mice, whilemutant p300 transgenic mice did not. LV levels ofendothelin-1, a downstream target of p300/GATA-4pathway as well as a marker of LV remodeling, were higherin the MI group than the sham-operated group. In intactp300-transgenic mice but not in mutant p300-transgenicmice, this increase was further exaggerated compared withcorresponding wild-type.

Conclusions. – These findings demonstrate that cardiacoverexpression of p300 promotes LV remodeling followingMI in adult mice in vivo and that histone acetyltransferaseactivity of p300 is required for these processes.

Abstracts N° YIA-2Anesthesia for determination of rodent cardiac function by using echocardiographyTakuya Daicho, Yuji Kawahara, Kouichi Tanonaka, Satoshi Takeo

Department of Molecular and Cellular Pharmacology, Tokyo University of Pharmacy and Life Science, Tokyo, Japan

Although combination of ketamine and xylazine is used formeasurement of cardiac function of rodents inechocardiography, this anesthesia induces low heart rate in theanimals. The present study was undertaken to find anappropriate anesthetic condition to determine mouse and ratcardiac function by echocardiography. Echocardiographicmeasurement was performed in male C57BL6 miceanesthetized i.p. with 30 mg/kg pentobarbital (P30) or acombination of 60 mg/kg ketamine and 6 mg/kg xylazine(KX), and in male Wistar rats with an intraperitoneal injectionof 40 pentobarbital (P40) or a combination of 100 mg/kgketamine and 10 mg/kg xylazine (KX). The values for heartrate of conscious mouse and rats were about 700 and 410beats/min, respectively. Heart rate of P30-anesthetized miceor P40-anesthetized rats was approximately 680 or 400 beats/min, whereas that of KX was about 250 or 300 beats/min.Fractional shortening and cardiac output index of the P30-anesthetized mice or the P40-anesthetized rats were greaterthan those of KX-anesthetized animals. Dobutamineincreased heart rate, fractional shortening, and cardiac outputindex of the pentobarbital-anesthetized mice and rats, whereasthe percent responses of KX-anesthetized animals weregreater than pentobarbital-anesthetized ones due to lowerbasal values for the cardiac parameters. Anesthesia with P30for the mouse and P40 for the rat rather than KX may berelevant to the evaluation of cardiac function usingechocardiography.

Abstracts N° YIA-3Presenilin 2 regulates the systolic function of heart by modulating Ca2+ signalingToshihiro Takeda a, Kinya Otsu a, Yoichiro Kusakari b,Makoto Kawai c, Satoshi Kurihara b, Masatsugu Hori aa Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan. b Department of Physiology (II), The Jikei University School of Medicine, Jikei, Japan. c Department of Cardiology, The Jikei University School of Medicine, Jikei, Japan

Genetic studies of families with familial Alzheimer’sdisease have implicated presenilin 2 (PS2) in thepathogenesis of this disease. PS2 is ubiquitously expressedin various tissues including hearts. In this study, weexamined cardiac phenotypes of PS2 knockout (PS2KO)mice in order to elucidate a role of PS2 in hearts. PS2KOmice developed normally with no evidence of cardiachypertrophy and fibrosis. Invasive hemodynamic analysisrevealed that cardiac contractility in PS2KO mice increasedcompared to that in their littermate controls. A study ofisolated papillary muscle showed that peak amplitudes of

ISHR Japanese Section 22nd Annual Meeting

ISHR Japanese Section 22nd Annual Meeting / Journal of Molecular and Cellular Cardiology 39 (2005) 996–1036 999

Ca2+ transients and peak tension were significantly higher inPS2KO mice than those in their littermate controls. PS2KOmouse hearts exhibited no change in expression of calciumregulatory proteins. Since it has been demonstrated that PS2in brain interacts with sorcin, which serves as a modulator ofcardiac ryanodine receptor (RyR2), we tested whether PS2also interacts with RyR2. Immmunoprecipitation analysisshowed that PS2, sorcin and RyR2 interact with each otherin HEK-293 cells overexpressing these proteins or in mousehearts. Immunohistochemistry of heart muscle indicated thatPS2 co-localizes with RyR2 and sorcin at the Z-lines.Elevated Ca2+ attenuated the association of RyR2 with PS2,whereas the association of sorcin with PS2 was enhanced.The enhanced Ca2+ transients and contractility in PS2KOmice were observed at low extracellular [Ca2+], but not athigh levels of [Ca2+]. Taken together, our results suggestthat PS2 plays an important role in cardiac excitation–contraction coupling by interacting with RyR2.

Abstracts N° YIA-4Interleukin-6, oxidized low-density lipoprotein and C-reactive protein in coronary sinus blood samples of subjects with suspected coronary artery diseaseMahmoud R. Mohammed Ramadan, Makoto Kodama, Wataru Mitsuma, Masahiro Ito, Takeshi Kashimura, Taruna Ikrar, Satoru Hirono, Yuji Okura, Yoshifusa AizawaDivision of Cardiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan

Background. – IL-6, oxidized LDL and CRP play a keyrole in atherosclerosis development.

Purpose. – To inspect the association between the levelof these inflammatory markers and CAD severity, theimpact of intervention manipulations, and lipid profile; andto discover the main site of their elaboration.

Methods. – We studied 87 subjects who underwentcoronary intervention for diagnostic, therapeutic or followup purposes. Blood samples were taken during theintervention session from coronary sinus and hepatic veins.High-sensitivity CRP was measured by latex nephelometry,while we measured coronary IL-6 and oxidized LDL usingsandwich ELISA. Subjects were classified according to thecoronary angiographic findings into normal (no evidence ofCAD), mild CAD (1 vessel disease, either treated with PCIor not) and severe CAD group (2 or more vessel disease).

Results. – Mean coronary IL-6 values were elevated insevere than in mild CAD group (3.67 vs. 2.3 pg/ml, P =0.027). In mild CAD patients, mean coronary IL-6 valueswere higher in the PCI subgroup than in those without PCI(2.9 vs. 1.87 pg/ml, P = 0.037) and the same applies for CRP(1.244 vs. 0.498 mg/l, P = 0.032). Coronary oxidized LDLlevels correlated positively with total cholesterol (r = 0.607,P = 0.000), LDL (r = 0.525, P = 0.001), and triglycerides (r= 0.424, P = 0.006) and negatively with HDL (r = –0.423,P = 0.007).

Conclusions. – IL-6 appears to be not only involved inthe pathogenesis of CAD but also in its magnitude. IL-6 andCRP appear to be released from the coronary atheromamostly as a direct response to mechanical squeezing duringPCI in mild CAD cases.

Abstracts N° YIA-5Inhibition of membrane type 1-matrix metalloproteinase (MT1-MMP) prevents Lox-1-mediated RhoA and Rac-1 activation induced by oxidized low-density lipoproteinKoichi Sugimoto a, Toshiyuki Ishibashi a, Hiroshi Ohkawara a, Naotoshi Sugimoto b, Takayuki Sakamoto a, Masashi Kamioka a, Nobuo Sakamoto a, Tamio Teramoto c, Tatsuya Sawamura d, Masahiko Kurabayashi e, Yukio Maruyama aa First Department of Internal Medicine, Fukushima Medical University, Fukushima, Japan. b Department of Physiology, Kanazawa University School of Medicine, Kanazawa, Japan. c Department of Internal Medicine, Teikyo University School of Medicine, Teikyo, Japan. d Department of Bioscience, National Cardiovascular Center Research Institute, Japan. e Department of Cardiovascular Medicine, Gunma University, Gumma, Japan

Background. – This study was conducted to investigatethe possible correlation between membrane type-1 matrixmetalloproteinase (MT1-MMP) and the activation of RhoAand Rac-1 in response to oxidized low-density lipoprotein(ox-LDL) in endothelial cells, as well as the association oflectin-like oxidized low-density lipoprotein receptor-1(LOX-1) with RhoA and Rac-1 activation.

Methods and results. – Ox-LDL (25 µg/ml) activatedRhoA and Rac-1 as determined by pull-down assay as earlyas 5 and 15 minutes in human aortic endothelial cells,respectively. GM6001, an inhibitor of MMPs, preventedthe ox-LDL-induced RhoA and Rac-1 activation as well asRhoA-dependent endothelial nitric oxide synthase (eNOS)downregulation and actin stress fiber reorganization, andRac-1-mediated reactive oxygen species (ROS)generation. Tissue inhibitor of metalloproteinase (TIMP)-2, but not TIMP-1, inhibited ox-LDL-induced RhoA andRac-1 activation, suggesting that MT-MMP(s) may beupstream of ox-LDL-triggered RhoA and Rac-1 activation.Inhibition of MT1-MMP using small-interference RNAprevented ox-LDL-induced RhoA and Rac-1 activation, aswell as the RhoA-dependent eNOS downregulation andRac-1-mediated ROS generation. Finally, inhibition ofLOX-1 by JTX92, an antibody to LOX-1, preventedox-LDL-dependent RhoA and Rac-1 activation as well asthe eNOS downregulation and ROS generation induced byox-LDL.

Conclusions. – The present study provides evidence thatMT1-MMP plays a role in the signaling of LOX-1-mediatedRhoA and Rac-1 activation in ox-LDL stimulation inendothelial cells, suggesting that MT1-MMP may be a goodtarget for treating endothelial dysfunction in coronary arterydisease.


Abstracts N° YIA-6Transmural difference in myocardial crossbridge dynamics and myofilament lattice spacingJuichiro Shimizu a, Naoto Yagi b, Satoshi Mohri c, Takehiro Miyasaka c, Hiroshi Okuyama d, Hiroko Toyota d,Katsuhiko Tsujioka d, Miyako Takaki a, Fumihiko Kajiya c(a Department of Physiology II, Nara Medical University, Nara, Japan. b Japan Synchrotron Radiation Research Institute, Harima, Japan. c Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science, Okayama, Japan. d Department of Physiology, Kawasaki Medical School, Kurashiki, Japan

Purpose. – To reveal the cause of the transmuraldifference in crossbridge (CB) dynamics and the effect ofthe preload on that.

Methods. – We recorded X-ray diffraction images of theisolated and perfused rat left ventricle (LV) at SPring-8 withLV pressure (LVP) and analyzed transmural CB duration(CBD) and myofilament lattice spacing (MLS) usingtransmural variations of epi-myocardial (CBDE and MLSE)and mid-myocardial (CBDM and MLSM) myofilamentorientations at the end-diastolic pressure of 0 and 20 mmHg(EDP0 and EDP20).

Results. – During contraction, normalized LVP andtransmural CBDs temporally harmonized well each other.However, at both EDP0 and EDP20, CBDEs weresignificantly longer than CBDMs (EDP0, CBDE; 195 ± 16msec, CBDM; 172 ± 19 msec, EDP20, CBDE; 226 ± 27 msec,CBDM; 205 ± 23 ms) and also MLSEs were significantlysmaller than MLSMs (EDP0, MLSE; 36.6 ± 1.3 nm, MLSM37.2 ± 1.4 nm, EDP20, MLSE; 35.7 ± 1.4 nm, MLSM; 36.9 ±1.5 nm). MLS is though to be reciprocal proportion tosarcomere length and it decreased with increasing EDP.

Conclusion. – The transmural difference in CB dynamicsmay be partially due to the difference in the amplitude andduration of twitch contraction of regional myocardium atregionally different sarcomere length.

Abstracts N° YIA-7The PI3K-Akt pathway plays a critical role in early cardiomyogenesis by regulating canonical Wnt signalingAtsuhiko T. Naito, Hiroshi Akazawa, Ichiro Shiojima, Issei KomuroDepartment of Cardiovascular Science and Medicine, Chiba University Graduate school of Medicine, Chiba, Japan

We have recently reported that activation ofphosphatidylinositol 3-kinase (PI3K) plays a critical role inthe early stage of cardiomyocyte differentiation of P19CL6cells. We here examined molecular mechanisms of howPI3K is involved in cardiomyocyte differentiation. DNAchip analysis revealed that expression levels of Wnt-3a weremarkedly increased and that the Wnt/β-catenin pathway wasactivated temporally during the early stage of cardiomyocytedifferentiation of P19CL6 cells. Activation of the Wnt/β-

catenin pathway during this period was required andsufficient for cardiomyocyte differentiation of P19CL6cells. Inhibition of the PI3K/Akt pathway suppressed theWnt/β-catenin pathway by activation of GSK-3β anddegradation of β-catenin. Suppression of cardiomyocytedifferentiation by inhibiting the PI3K/Akt pathway wasrescued by forced expression of a non-phosphorylated,constitutively active form of β-catenin. These resultssuggest that the PI3K pathway regulates cardiomyocytedifferentiation through suppressing the GSK-3β activity andmaintaining the Wnt/β-catenin activity.

Abstracts N° YIA-8Overexpression of mitochondrial peroxiredoxin-3 ameliorates left ventricular remodeling and failure after myocardial infarction in miceShouji Matsushima a, Hidenori Matsusaka b, Masaki Ikeuchi b, Tomomi Ide b, Toru Kubota b, Kenji Sunagawa b,Fumiyuki Hattori c, Yasuhiro Hasagawa c, Tatsuya Kurihara c, Shinzo Oikawa c, Shintaro Kinugawa a, Hiroyuki Tsutsui aa Department of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan. b Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. c Biomedical Research Laboratories, Daiichi Suntory Pharma Co., Ltd., Osaka, Japan

Introduction. – Mitochondrial oxidative stress anddamage play a major role in the development andprogression of left ventricular (LV) remodeling and failureafter myocardial infarction (MI). Mitochondrial antioxidantenzyme, peroxiredoxin-3 (Prx-3), may protect the heartagainst this oxidative damage.

Hypothesis. – We thus hypothesized that overexpressionof Prx-3 gene could attenuate LV remodeling after MI inmice.

Methods and results. – We created MI in 12–16-week-old, male Prx-3 transgenic mice (TG + MI; n = 37) andnontransgenic wild-type mice (WT + MI; n = 39) by ligatingthe left coronary artery. Prx-3 protein levels were 1.8-timeshigher in the hearts from TG mice than WT mice with nosignificant changes in other antioxidant enzymes such asglutathione peroxidase, superoxide dismutase, and catalase.At 4 weeks of MI, LV thiobarbituric acid reactivesubstances in the mitochondria were significantly lower inTG + MI than those in WT + MI. LV cavity dilatation anddysfunction were significantly attenuated in TG + MIcompared to WT + MI despite the similar infarct size andaortic pressure. LV end-diastolic pressure and lung weightwere increased in WT + MI, which were also attenuated inTG + MI. Improvement of LV function in TG + MI wasaccompanied by a decrease in myocyte hypertrophy,interstitial fibrosis, and apoptosis in the noninfarcted LV.Mitochondrial DNA copy number and complex enzymeactivities were significantly decreased in WT + MI and thisdecrease was also ameliorated in TG + MI.


Conclusion. – Overexpression of Prx-3 inhibited LVremodeling and failure after MI. Therapies designed tointerfere with mitochondrial oxidative stress by usingantioxidant Prx-3 might be beneficial to prevent cardiacfailure.

Abstracts N° YIA-9Critical roles of muscle regeneration in therapeutic neovascularizationKaoru Tateno, Tohru Minamino, Haruhiro Toko, Hiroshi Akazawa, Issei KomuroDepartment of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chiba, Japan

Understanding the mechanisms of vascular formation hasallowed us to develop a novel strategy for the treatment ofsevere peripheral vascular disease. In particular, thediscovery of bone marrow-derived endothelial progenitorsin the peripheral blood has promoted intensive studies on thepotential of cell therapy for various human diseases.Accumulating evidence has suggested that implantation ofbone marrow mononuclear cells effectively promoteneovascularization in ischemic tissues. It has also beenreported that the implanted cells are not only incorporatedinto the newly formed vessels, but also secrete angiogenicfactors. In the present study, we show that cell therapy usingperipheral mononuclear cells is also very effective for thetreatment of limb ischemia and that implanted cells do notsecret angiogenic factors, but instead stimulate muscle cellsto regenerate and to produce these factors. Reduced abilityof muscle cells to secrete these factors or to regeneratemarkedly impairs neovascularization. Accordingly, wepropose a novel mechanism whereby implanted cellsenhance the regeneration of muscle cells that activelyproduce angiogenic factors, thereby promotingneovascularization in ischemic tissues.

Abstracts N° YIA-10Granulocyte colony-stimulating factor treatment accelerates reendothelialization and decreases neointimal formation after vascular injury in miceToru Yoshioka a, Masafumi Takahashi b, Yuji Shiba b,Chihiro Suzuki b, Hajime Morimoto b, Atsushi Izawa b,Hirohiko Ise b, Uichi Ikeda ca Division of Cardiovascular Medicine, Shinshu University, Matsumoto, Japan. b Division of Cardiovascular Science, Department of Organ Regeneration, Shinshu University Graduate School of Medicine, Matsumoto, Japan. c Division of Cardiovascular Medicine, Shinshu University Hospital/Division of Cardiovascular Science, Department of Organ Regeneration, Shinshu University Graduate School of Medicine, Matsumoto, Japan

Background. – Neointimal formation after percutaneouscoronary intervention (PCI), termed restenosis, limitstherapeutic revascularization. Recent study reported thatgranulocyte colony-stimulating factor (G-CSF)-induced

recruitment of peripheral endothelial progenitor cells(EPCs) enhanced reendothelializaiton of the injured arteryand prevented neointimal formation in splenectomized rats;however, the role of bone marrow-derived EPCs remainsunknown and splenectomy can not be applied to clinicalsettings. We therefore examined the effects of G-CSF onreendothelialization and neointimal formation after vascularinjury in bone marrow transplanted and non-splenectomizedmice.

Methods and results. – Wire-mediated vascular injurywas produced in the femoral artery of C57BL/6 mice. Micewere divided into the following 3 groups: 1) treatment ofsaline (n = 13); 2) treatment of G-CSF (100 mg/kg/day) for10 days after the injury (n = 23); 3) treatment of G-CSF from4 days before the injury through 6 days after (n = 14). G-CSFpretreatment significantly accelerated reendothelialization(74.6% vs. 51.8%, P < 0.05) and decreased neointimalformation after vascular injury (64,316 µm2 vs. 10,1778 µm2,P < 0.05) compared with saline-treated mice, but theseeffects of G-CSF were diminished when G-CSF was startedafter the injury. Flow cytometry analysis showed that G-CSFincreased the number of endothelial progenitor cells (EPCs:CD34+/Flk–/+) in the peripheral circulation, compared withsaline-treated mice (4.32 cell/µL vs. 1.60 cells per µl, P <0.05). Vascular injury was also produced in two types ofmice whose bone marrow was replaced by that of GFP- orTie2/LacZ-transgenic mice. In the reendothelialized arteryof these mice, only few bone marrow-derived EPCs weredetected. Furthermore, G-CSF treatment diminished serumlevels of interleukin (IL)-6 and IL-12p70, known as anti-angiogenic cytokines.

Conclusion. – G-CSF treatment accelerated reendo-thelialization and decreased neointimal formation after thevascular injury even in intact mice, although there was littlecontribution of bone marrow-derived EPCs in thereendothelialized artery. These findings suggest that G-CSFpretreatment has a therapeutic potential for prevention ofrestenosis after PCI.

Abstracts N° O-1-1Apolipoprotein A-I discs-induced nitric oxide production in coronary endothelial cells may suppress reperfusion-induced arrhythmias in ratsSatoshi Imaizumi a, Shin-ichiro Miura a, Yoshinari Uehara a, Yoshihiro Kiya a, Hidenori Urata b, Kerry-Anne Rye c,Kejiro Saku aa Department of Cardiology, Fukuoka University School of Medicine, Fukuoka, Japan. b Department of Internal Medicine, Fukuoka University Chikushi Hospital, Fukuoka, Japan. c Lipid Research Group, The Heart Research Institute, Australia

Although the infusion of reconstituted high-densitylipoprotein (rHDL) produces a rapid regression of coronaryatherosclerosis, the effects of rHDL on ischemia–reperfusion-induced arrhythmias are unknown. In this study,


32 male Wistar rats were divided into two groups: rats thathad been pretreated with or without 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/ApoA-I discs administeredintravenously before left coronary artery occlusion. Theartery was occluded and then subjected to reperfusion. Theduration of ventricular tachycardia and ventricularfibrillation after reperfusion in POPC/ApoA-I-pretreatedrats was much less than that in untreated rats. In addition,plasma nitric oxide (NO) production was significantlyincreased and phospho-extracellular-signal-regulated kinase(p-ERK) activation in the left ventricle was significantlydownregulated in the POPC/ApoA-I-treated group. We alsoperformed an additional experiment using human coronaryartery endothelial cells (ECs) and fetal cardiomyocytes inrats. POPC/ApoA-I activated p-ERK in ECs but not incardiomyocytes. These results suggest that POPC/ApoA-I-induced NO production in ECs may suppress reperfusion-induced arrhythmias and not activate p-ERK incardiomyocytes. These findings represent an exciting newarea in the treatment of ischemia–reperfusion-inducedarrhythmias. HDL-based therapy may hold the promise ofdramatically reducing the incidence of such arrhythmiasafter myocardial infarction.

Abstracts N° O-1-2Essential role of COX-2 in lipopolysaccharide-induced late preconditioning: no-dependent and -independent stages of COX-2-mediated cardioprotectionKen Shinmura, Kayoko TamakiDepartment of Internal Medicine, Keio University School of Medicine, Keio, Japan

A small dose of lipopolysaccharide (LPS) elicits latepreconditioning (PC). Evidence from pharmacologicalinhibition and gene knockout mice demonstrates that iNOSplays an obligatory role in LPS-induced late PC. LPSinduces COX-2 protein in concert with iNOS, but it isunclear whether COX-2 plays a role in LPS-induced late PC.Thus, we investigated the role of COX-2 and the interactionbetween COX-2 and NO using wild-type (WT) and iNOSgene knockout mice (iNOS–/–). Mice were treated with1 mg/kg of LPS 12, 48, 96 and 168 h before. Isolatedperfused hearts were subjected to 25 min of global ischemiafollowed by 40 min of reperfusion. Pretreatment with LPSimproved the recovery of LV function 12, 48, and 96 h, butnot 168 h later in WT. Surprisingly, LPS improved therecovery of LV function 96 h later in iNOS–/–. Westernblotting revealed increased COX-2 protein at all time-pointsin both strains. NS-398 completely abrogatedcardioprotection at all time-points. In WT, iNOS protein wasrapidly upregulated and expression levels returned tobaseline 48 h later. A subsequent increase in nNOSdisappeared 96 h later. PGI synthase were upregulated andassociated with increased myocardial 6-keto-PGF1αcontents 96 h later in both strains. In conclusion, COX-2activity is essential for LPS-induced late PC. During the

early stage, NO derived from iNOS or nNOS is necessary forCOX-2 activity. In contrast, during the final stage, COX-2-mediated cardioprotection is independent of NO, probablybecause upregulation of functionally-coupled PGI synthasecompensates for the decrease in NO.

Abstracts N° O-1-3Intracellular sodium increase and susceptibility to ischemia in hearts from type 2 diabetic db/db miceRyuko Anzawa a, Danielle Feuvray b, Seibu Mochizuki aa Division of Cardiology, Department of Internal Medicine, Jikei University School of Medicine, Jikei, Japan. bUniversite Paris-Sud XI, CNRS UMR 8078, Hopital Marie Lannelongue, France

Background and aim. – An important determinant ofsensitivity to ischemia is altered ion homeostasis, especiallydisturbances in intracellular Na+ (Na+i) handling, which nostudy has so far investigated in type 2 diabetes. Our aim wasto examine susceptibility to ischemia–reperfusion in isolatedhearts from diabetic db/db and control db/+ mice and todetermine whether and to what extent the amount of Na+iincrease during a transient period of ischemia couldcontribute to functional alterations upon reperfusion.

Methods. – Isovolumic hearts were exposed to 30-minglobal ischemia and then reperfused. 23Na nuclear magneticresonance (NMR) spectroscopy was used to monitor Na+iand 31P NMR spectroscopy to monitor intracellular pH(pHi).

Results. – A higher duration of ventricular tachycardia(VT) and the degeneration of VT into fibrillation (VF) wereobserved upon reperfusion in db/db hearts. Recovery of leftventricular developed pressure was reduced. Ischemia-induced Na+i increase was higher in db/db hearts than incontrol hearts, and the rate of pHi recovery was increasedduring reperfusion. Na+/H+ exchange inhibition bycariporide significantly reduced Na+i gain at end ischemia.This was associated with a lower incidence of VT in bothheart groups and with an inhibition of the degeneration ofVT into VF in db/db hearts.

Conclusions. – These findings strongly support thehypothesis of increased Na+i playing a causative role in theenhanced sensitivity to ischemia observed in db/db diabetichearts.

Abstracts N° O-1-4Level of GSK-3β phosphorylation by Akt-dependent and -independent pathways determines the anti-infarct tolerance in rat hearts in situMasahiro NishiharaSecond Department of Internal Medicine, Sapporo Medical University, Sapporo, Japan

We examined the correlations of cardioprotectionafforded by ischemic preconditioning (IPC) anderythropoietin (EPO) with activation of Akt, glycogensynthase kinase-3β (GSK-3β) and STAT3. Myocardial


infarction was induced in rat hearts in situ by 20-mincoronary occlusion and 2-hr reperfusion, and infarct sizewas expressed as % of risk area (%IS/RA). Using separategroups of rats, tissue samples were taken from the risk areaat 5 min after reperfusion for immunoblotting for Akt,GSK-3β and STAT3. IPC with a single cycle of 5-minischemia/5-min reperfusion and EPO (5000 U/kg, i.v.)15 min before ischemia reduced %IS/RA from 56.2 ± 2.4%to 25.2 ± 2.1% and 36.2 ± 2.8%, respectively. Thecombination of IPC and EPO further reduced %IS/RA to8.9 ± 1.9%. Chelerythrine (5 mg/kg), a PKC inhibitor,abolished infarct size limitation by IPC but not that byEPO. Wortmannin (15 µg/kg), a PI3 kinase inhibitor,abolished EPO-induced protection (%IS/RA = 47.9 ±2.8%) but only partly inhibited protection afforded by EPOplus IPC (%IS/RA = 30.2 ± 6.0%). The additive effects ofIPC and EPO on %IS/RA were mirrored by their effects onthe level of phospho-GSK-3β; phospho-GSK-3β (% of thecontrol) was increased to 186 ± 22% by IPC, 163 ± 22% byEPO and 271 ± 22% by EPO plus IPC. Though IPC aloneand EPO alone increased levels of phospho-Akt andphospho-STAT3, the combination of IPC and EPO did notfurther increase the phospho-Akt level or phospho-STAT3level. These results suggest that the level of GSK-3βphosphorylation by Akt-dependent and -independentpathways determines the level of myocardial toleranceagainst infarction.

Abstracts N° O-1-5Fasting serum apolipoprotein B-48 level is a good marker of postprandial hyperlipidemia and coronary heart diseaseShizuya Yamashita a, Taizo Sugimoto a, Ken-ichi Hirano a,Masatsugu Hori a, Yoshiro Masuda b, Atsushi Ikeda b,Takehiko Okuno c, Yoshiaki Uchida c, Kengo Matsumoto d,Toshiharu Kawamoto d, Naohiko Sakai ea Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan. b Japan Tobacco Inc., Japan. c Fujirebio Inc., Japan. d National Hospital Organization Kure Medical Center, Japan. e Itami City Hospital, Japan

Objectives. – Postprandial hyperlipidemia (PH) is one ofthe risk factors for coronary heart disease (CHD) and itsatherogenicity is attributed to the impaired metabolism ofchylomicron remnants. The current study aimed to find auseful marker for PH and CHD risk.

Methods and results. – Serum apo B-48 levels weremeasured by a novel ELISA. Ten male normolipidemicvolunteers were fed a high fat diet and serum TC, TG,HDL-C, LDL-C, apo B-48, apo B-100, RLP-C and RLP-TG levels were measured at 0–8 hour. Fasting serum apoB-48 levels were measured in 137 (88 CHD and 49 non-CHD) patients who underwent CAG. Serum apo B-48,RLP-C and RLP-TG levels were significantly correlatedwith fasting TG. Apo B-48, TG, RLP-C and RLP-TG

levels were increased after fat loading with a peak at 3–4 hour. Incremental area under the curve (iAUC) for TGwas correlated with the TG (r = 0.95, P < 0.0001), RLP-C(r = 0.811, P < 0.01), RLP-TG (r = 0.926, P < 0.001) andapo B-48 (r = 0.775, P < 0.01) levels most significantly at5 hour compared with those at other points. However, atfasting state iAUC was significantly correlated only withserum apo B-48 levels (r = 0.809, P < 0.01), but not withTG, TC, RLP-C or RLP-TG concentrations, suggestingfasting serum apo B-48 levels are a useful marker for PH.Fasting serum apo B-48 levels were significantly higher inCHD patients than in non-CHD patients.

Conclusion. – Fasting serum apo B-48 levels are a simpleand useful marker for PH and can become one of the riskfactors for CHD.

Abstracts N° O-1-6Accelerated intestinal absorption of dietary lipids causes postprandial hypertriglyceridemia in CD36 knockout miceDaisaku Masuda a, Ken-ichi Hirano b, Takahiro Kuwasako c, Chiaki Ikegami a, Masahiro Koseki a, Yumiko Nakagawa-Toyama a, Zhongyan Zhang a, Yoshihiro Tochino a,Masatsugu Hori b, Shizuya Yamashita ba Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka, Japan. b Department of Cardiology, Osaka University Graduate School of Medicine, Osaka, Japan. c Suita Municipal Hospital, Suita, Japan

Background. – Human CD36 deficiency is a monogenicform of metabolic syndrome, we reported that they haddyslipidemia at fastig and postprandial states at the TG 2003.The aim of present study was investigating mechanismsunderlying postprandial hypertriglyceridemia.

Methods and results. – The animals used were CD36knockout and wildtype mice. Oral fat loading test (OFLT)were performed using olive oil, showing that the peak andarea under curves of TG were much greater in CD36knockout mice, which were similar to those observed inhuman CD36 deficiency. And we have performed thefollowing combined experiments. First, we injected TritonWR 1339 at fasting state without OFLT. We found nodifference of serum TG levels between the two groups. Itimplied there was no change in the hepatic TG secretion.To the contrary, when we injected Triton WR 1339 andthen performed OFLT, higher serum TG levels wereobserved in CD36 knockout mice, suggesting that therewas an accelerated intestinal secretion of lipoproteins. Inorder to ascertain this, we analyzed lipids and lipoproteinsof intestinal lymph fluid by puncturing cisterna chili afterOFL. We found higher TG levels in lymph fluid of CD36knockout mice. Finally, in the histological analysis by Oilred O staining of sections of small intestines, oil dropletswere observed much abundant in the earlier phase of OFL.


Conclusions. – These results suggest that the acceleratedfat absorption from small intestine is the cause ofpostprandial hypertriglyceridemia in CD36 deficiency.

Abstracts N° O-2-1Intracoronary ultrasound analyses of coronary plaques containing increased isoprostanes in patients with angina pectorisAkira Nishibe a, Megumu Fukunaga b, Shoichi Senda b,Yuusuke Nakagawa a, Akiko Mastuo a, Osamu Akutagawa a, Megumi Kunishige a, Taku Sakai a, Takeshi Hata a,Yoshiyuki Kijima aa Department of Cardiology, Higashi-osaka City General Hospital, Japan. b Department of Integrated Medicine, Kagawa University Hospital, Kagawa, Japan

Isoprostanes, generated from free radical-catalyzed lipidperoxidation, serve as a sensitive biomarker for oxidantstress in vivo. Enhanced oxidant stress has been proposed asa novel coronary risk factor. We recently reported thatincreased isoprostane content in directionallyatherectomized coronary plaques from patients withunstable versus stable angina. The aim of this study was tocharacterize the relationship between isoprostane content incoronary plaques and their intracoronary ultrasound (ICUS)images in patients with angina pectoris. Twenty-fourpatients with angina pectoris (9 unstable and 15 stableangina) were subjected to directional coronary atherectomy.After solid phase extraction, 8-iso-prostaglandin F2a (8-iso-PGF2a), a representative isoprostane, was measured withenzyme immunoassay. Esterified form of 8-iso-PGF2a wasdefined as subtraction of free form from total 8-iso-PGF2a.Approximately 80% of total 8-iso-PGF2a was a fraction ofesterified form. ICUS visualized morphology of the targetlesions prior to the atherectomy. Membrane-bound form 8-iso-PGF2α content was 9.84 ± 8.76 for UAP and 1.34 ±1.64 ng/g tissue for SEA (P = 0.0014 for UAP versus SEA).The 8-iso-PGF2α content was 12.5 ± 10.74 for presence ofsuperficial calcification and 2.43 ± 3.3 ng/g tissue forabsence (P = 0.01). The 8-iso-PGF2α content was 5.42 ±3.98 for presence of echolucent area and 3.99 ± 8.11 ng/gtissue for absence (P = 0.02). This study clarifiedcharacteristics of vulnerable plaques as: 1) the presence ofsuperficial calcification and/or echolucent area of ICUSimages and 2) their increased isoprostane content.

Abstracts N° O-2-2Apolipoprotein A-I discs containing sphingosine-1-phosphate induced coronary artery endothelial tube formation through Akt/ERK/NO pathwayYoshino Matsuo a, Shin-ichiro Miura a, Yoshihiro Kiya a,Satoshi Imaizumi a, Yoshinari Uehara a, Kerry-Anne Rye b,Keijiro Saku aa Department of Cardiology, Fukuoka University Hospital, Fukuoka, Japan. b Lipid Research Group, The Heart Research Institute, Australia

Reconstituted high density lipoprotein (rHDL) has beenshown to produce the rapid regression of coronaryatherosclerosis in animal models and humans. Sphingosine-1-phosphate (S1P), which is a bioactive lipid in HDL,promotes mitogenesis, endothelial cell motility, and cellsurvival, as well as organization and differentiation into avessel. In this study, we examined the direct role of a newlydeveloped rHDL, apoA-I discs (1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/S1P/ApoA-I) containing S1P,on human coronary artery smooth muscle cell (SMC)proliferation and angiogenesis in human coronary arteryendothelial cells (ECs) as well as cholesterol efflux inmacrophage. The effect of POPC/S1P/ApoA-I oncholesterol efflux in macrophage was similar to that ofPOPC/ApoA-I, which is a traditional disc. In addition,POPC/S1P/ApoA-I induced SMC proliferation through theactivation of phospho-Akt and phospho-extracellular-signal-regulated kinases (p-ERK) 1/2 and EC tubeformation, which was blocked by an inhibitor of endothelialnitric-oxide (NO) synthase activation (L-NAME). POPC/S1P/ApoA-I-induced p-ERK1/2 activation was mediated bya Ras-independent pathway and mainly attributed to S1P-stimutated signaling through endothelial differentiation gene3 (Edg3), Edg5, ATP-binding cassette transporter A1(ABCA1) and ABCG1, but not Edg1. Thus, POPC/S1P/ApoA-I enhances cholesterol efflux and has additionaleffects for cell survival including proliferation andangiogenesis. This new apoA-I disc-based therapy may holdthe promise of dramatically reducing the incidence ofcoronary artery disease and warrants further investigation invivo.

Abstracts N° O-2-3Hyperglycemia induces uncoupling protein 2 expression in the cardiovascular mitochondria in the type 2 diabetic ratsYasuyuki Bito a, Yukiko Minamiyama b,Shigekazu Takemura b, Shigeru Okada ca Department of Cardiovascular Surgery, Graduate School of Medicine, Osaka City University, Osaka, Japan. bDepartments of Hepato-Biliary-Pancreastic Surgery, Graduate School of Medicine, Osaka City University, Osaka, Japan. c Department of Food & Health Science, Graduate School of Medicine and Dentistry, Okayama University, Okayama, Japan

Cardiovascular disease is exceptionally prevalent inpatients with diabetes mellitus. Increasing evidence showsthat the overproduction of reactive oxygen species (ROS),induced by diabetic hyperglycemia, contributes to thedevelopment of several cardiovascular diseases. On theother hand, uncoupling protein 2 (UCP2), which exists onthe inner membrane of the mitochondrial matrix, is reportedto negatively regulate the superoxide production inmitochondria. This study examined the relationship betweenthe pathogenesis of diabetic cardiovascular disease and


UCP2 expression. The Otsuka Long-Evans Tokushima Fatty(OLETF) rat, a model of the spontaneous type II diabetes,develops hyperglycemic obesity with hyperinsulinemia andinsulin resistance after the age of 25 weeks. Blood glucoseand HbA1c levels in OLETF rats were significantly higherthan those of non-diabetic control (LETO) rats at 29 weeksold. These abnormalities in OLETF rats were reverted tonormal levels by 30% calorie restriction from the age of 29to 42 weeks. The mitochondrial ROS productionsignificantly increased in heart and aorta of OLETF rats.UCP2 protein expression in OLETF heart and aorta alsoincreased significantly as compared with those of LETO ratsby Western blot. Calorie restriction significantly reducedboth UCP2 expression and ROS generation in OLETF rats.The data suggest that hyperglycemia induces oxidativedamage to cardiovascular system and a significant increaseof UCP2 protein levels is an adaptation mechanism to inhibitROS production.

Abstracts N° O-2-4Deficiency of a multi-ligand receptor CD36 is associated with insulin resistance and the metabolic syndromeShizuya Yamashita, Daisaku Masuda, Takahiro Kuwasako, Mohamed Janabi, Yumiko Toyama, Ken-ichi Hirano, Naohiko Sakai, Masatsugu HoriDepartment of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan

Oxidized LDL (Ox-LDL) is taken up by macrophages viascavenger receptors and plays an important role in thepathogenesis of atherosclerosis. CD36 is a glycoproteinexpressed on platelets, monocyte-macrophages, adiposetissue, skeletal muscles and heart. We found CD36-deficientpatients and identified several mutations in CD36 gene.CD36-deficient macrophages showed a 50% reduction in thebinding of Ox-LDL, suggesting CD36 is one of the majorreceptors for Ox-LDL. CD36 is expressed on foamedmacrophages in atherosclerotic plaques of human aorta andcoronary arteries. CD36 is also a transporter of long-chainfatty acids (LFCA) and thus CD36 deficiency shows a defectin the uptake of BMIPP, an LFCA analog, by the heart.Furthermore, the secretion of IL1-β and TNF-α frommonocyte-derived macrophages induced by Ox-LDL wasmarkedly reduced and NF-κB activation was attenuated inCD36-deficient subjects, suggesting CD36-mediatedsignaling is impaired in CD36-deficiency. Most of CD36-deficient subjects were accompanied by multiple risk factorssuch as hyperlipidemia (hypertriglyceridemia), increasedremnant lipoproteins and mildly elevated fasting plasmaglucose and blood pressure. Glucose clamp revealed meanwhole body glucose uptake was reduced in CD36-deficientsubjects, indicating the presence of insulin resistance.Frequency of CD36 deficiency was higher in coronary arterydisease (CAD) patients than in controls. Taken together,CD36 deficiency is accompanied by (1) hyperlipidemia withincreased remnant lipoproteins, (2) impaired glucose

metabolism based upon insulin resistance, and (3) mildhypertension, and comprises one of genetic backgrounds ofthe metabolic syndrome, leading to development of CAD.

Abstracts N° O-2-5Probucol enhances the expression of human hepatic scavenger receptor class B type I possibly through species-specific mechanismKen-ichi Hirano a, Chiaki Ikegami b, Ken-ichi Tsujii b,Masahiro Koseki b, Daisaku Masuda b, Yumiko Nakagawa-Toyama b, Yukihiko Ueda c, Iichiro Shimomura b, Shizuya Yamashita d, Masatsugu Hori aa Department of Cardiovascular Medicine, Osaka University, Osaka, Japan. b Department of Metabolic Medicine, Osaka University, Osaka, Japan. c Horizontal Medical Research Organization, Kyoto University, Kyoto, Japan. d Department of Cardiovascular Medicine, Osaka University, Osaka, Japan

Objective. – Scavenger receptor class B type I (SR-BI) isa major receptor for HDL in the liver, which is the terminalof reverse cholesterol transport. The overexpression of SR-BI attenuated experimental atherosclerosis in murinemodels, concomitant with the reduction of plasma HDL-cholesterol levels. Probucol is known to be a potenthypolipidemic drug to regress xanthoma formation andcarotid atherosclerosis, along with a marked reduction ofHDL-cholesterol levels. The aim of the present study was toknow the effect of probucol on the expression of SR-BI andits underlying mechanism.

Methods and results. – We found that probucol increasedthe expression of SR-BI proteins in in vitro human liver cellsand in vivo rabbit model. However, this effect was notobserved in wild type C57Bl6 mice. The decay curve of SR-BI protein was markedly retarded in probucol-treatedHepG2 cells in the presence of cycloheximide, indicatingthat probucol may stabilize human SR-BI protein. In order toknow the underlying mechanism for the observed species-specific effect, we conducted the following host–swapexperiments, in which SR-BI was transfected or expressed inheterologous cells or hosts. Probucol did not increase humanSR-BI protein in the liver of transgenic mice carrying theentire human SR-BI genome. Probucol could stabilize evenmurine SR-BI, when transfected into a human cell line,HepG2, whereas human SR-BI was not stabilized in a mousehepatoma cell line, Hepa 1–6, treated with probucol.

Conclusions. – Probucol increases hepatic SR-BI proteinpossibly through species-specific stabilization of the protein.

Abstracts N° O-2-6The change of lipid compositions by chronic hypoxia increases lipid peroxidation in young rat heart by acute hyperoxic exposureTatsujiro Oka, Toshiyuki ItoiDepartment of Pediatrics Cardiology and Nephrology,


Kyoto Prefectural University of Medicine Graduate School of Medical Science, Japan

The postoperative course of cyanotic patients is generallymore complicated than that of acyanotic patients. It has beenreported that oxygen toxicity with reoxygenation fromcardiopulmonary bypass causes induction of lipidperoxidation. We hypothesized that the change of fatty acidcompositions in the hypoxic young heart caused theacceleration of lipid peroxidation. We also studied whetherl-carnitine (LCAR), suppressing lipid peroxidation, wasassociated with lipid compositions.

Methods. – Four-week-old male Sprague–Dawley ratswere exposed to hypoxic conditions (Hx) for 14 days (FiO2= 0.1), and then to hyperoxic conditions (O2) for 12 hours(FiO2 = 1.0). The fatty acid compositions were analyzed bygas chromatography and mass spectrometry. We measuredmalondialdehyde (MDA) as a lipid peroxidation product,SOD and catalase as antioxidants. All data were expressed asmean ± S.D.

Results. – The concentration of n-6 polyunsaturated fattyacids (PUFA) (C18:2 and C20:4), in the hypoxic heart,exhibited lower than in control heart (C 42.8 ± 2.6%mol, Hx38.0 ± 1.3, P < 0.01). Otherwise, hypoxia resulted inincrease of total n-3 PUFA (C20:5, C22:5 and C22:6) (C:18.4 ± 4.7%mol, Hx: 26.5 ± 2.1, P < 0.001). MDA wassignificantly increased in Hx + O2 (C: 1.9 ± 0.7 mM/mgprotein, Hx + O2: 6.3 ± 2.3, P < 0.001). LCAR reduced theincrease of MDA, but had no effect on n-3 and n-6 PUFAconcentration. SOD was reduced in Hx. Catalase wasunchanged.

Conclusions. – This study suggested that the increase ofn-3 PUFA and the decrease of antioxidant in young rat heartsunder chronic hypoxia augment lipid peroxidation withacute exposure of oxygen.

Abstracts N° O-3-1Upregulated TRPC1 and enhanced store-operated calcium entry in the development of cardiac hypertrophyHiroyuki Watanabe, Hiroshi ItoThe Second Department of Internal Medicine, Akita University School of Medicine, Akita, Japan

Background. – Calcium-calcineurin signaling plays animportant role in the development of cardiac hypertrophy.However, actual Ca2+ source responsible for encoding thehypertrophic responses has been still elusive. Transientreceptor potential (TRP) channels are newly emerging Ca2+

entry channels involved in proliferation and differentiationof various cells, which may serve the function of store-operated Ca2+ entry (SOCE). This study was designed to testthe hypothesis that TRPC1 is an important TRPC isoforminvolved in forming Ca2+-permeable cation channelcomplex that contribute to the development of cardiachypertrophy.

Methods and results. – In neonatal rat cardiac myocytesprimary culture, endothelin-1 (10 nM) increased the

expression of TRPC1 channel, together with the augment ofSOCE, BNP expression and cell surface area (n = 15–25).The 3,5-bis (trifluoromethyl) pyrazole derivative, BTP2,known as a specific TRPC channel blocker, inhibited theincrease in BNP expression and cell size as well as SOCE inendothelin-1 treated cells (n = 5). We also confirmed the up-regulation of TRPC1 in hearts of aorta-banded rat (n = 11).In transient expression analysis using HEK293T, theoverexpression of TRPC1 increased both SOCE and NFATpromoter activity, while co-transfection of dominantnegative forms of TRPC1 suppressed them (n = 25).Immunoprecipitation assays revealed that TRPC1 interactedwith TRPC1 itself, TRPC3, TRPC5, TRPC6 and TRPM4which are expressed in hypertrophic myocardium, implyingTRPC1 forms homo/hetero-multimeric channels with otherTRPs.

Conclusion. – These results suggest that a strongfunctional link between the expression of TRPC‚ P channelsand the development of cardiac hypertrophy. TRPC1 may bepotential therapeutic target to prevent cardiac hypertrophy.

Abstracts N° O-3-2The balance of connective tissue growth factor and brain natriuretic peptide regulates myocardial stiffness in the development of cardiac hypertrophyNorimichi Koitabashi, Masashi Arai, Kazuo Niwano, Atai Watanabe, Masahiko KurabayashiDepartment of Medicine and Biological Science, Gunma University Graduate School of Medicine, Maebashi, Japan

Connective tissue growth factor (CTGF), a cytokine thatpromotes extracellular matrix production, has been shown tobe increased in human failing heart. BNP is also increased infailing heart and has ant-fibrotic effects. Accordingly, wehypothesized that imbalance of profibrotic signal, i.e. CTGFand anti-fibrotic signal, i.e. BNP, is responsible formyocardial fibrosis. In pressure-overloaded rat hearts createdby abdominal aortic banding (AB), both CTGF and BNP wassynergistically upregulated at days 1–7 after AB (r = 0.836,P < 0.0001). This close correlation became weak at laterphase after AB. AB rats at day 28 showed cardiachypertrophy with elevation of passive ventricular stiffnessestimated by pressure–volume relationship. The increase ofventricular stiffness was associated with imbalance betweenCTGF and BNP expression. In cultured cardiac myocytes(CM), G-protein coupled receptor ligands and cyclic stretchincreased both CTGF and BNP mRNA. TGF-β andaldosterone increased CTGF but not BNP in cultured CM.CTGF protein secretion from CM to the culture media wasenhanced by these extrinsic stimuli. Conditioned medium ofCM in which CTGF was overexpressed by these extrinsicstimuli enhanced collagen gene expression in cardiacfibroblasts (CF). This collagen upregulation was blocked byneutralizing antibody against CTGF, suggesting that CTGFproduced by CM plays as a paracrine inducer of collagenproduction in CF. Moreover, the upregulation of collagen


induced by recombinant CTGF protein was blocked byrecombinant BNP in CF. In conclusion, our in vivo and invitro study suggest that CM regulates collagen productionand myocardial stiffness through CTGF secretion due toneurohumoral activation and/or cell stretch under the balanceof antifibrotic action of BNP secreted from same CM.

Abstracts N° O-3-3Regulatory mechanism of cardiac aldosterone system in hypertensive diastolic heart failure ratsTomohito Ohtani a, Toshiaki Mano a, Yasushi Sakata a,Mayu Nishio a, Yasuharu Takeda a, Takeshi Miwa b, Yasuki Nonaka c, Kazuhiro Yamamoto a, Masatsugu Hori aa Department of Cardiovascular Medicine, Osaka University of Graduate School of Medicine, Osaka, Japan. b Osaka University Genome Information Research Center, Osaka, Japan. c College of Nutrition, Koshien University, Koshien, Japan

Background. – We have previously reported thateplerenone, selective mineralocorticoid receptor blocker,effectively prevented the development of diastolic heartfailure (DHF), suggesting crucial roles of cardiacaldosterone system. However, the regulation of cardiacaldosterone system remains unclear.

Methods. – Dahl salt-sensitive rats fed 8% NaCl dietfrom age 7 weeks served as a DHF model. Rats fed 0.3%NaCl diet served as age-matched control. Cardiac steroidswere extracted with silica and C18 cartridges and evaluatedin liquid chromatographic mass spectrometric method (LC–MS). Cardiac aldosterone synthase activity was assessed bythe conversion of 3H-deoxycorticosterone to 3H-aldosterone in left ventricular myocardial homogenate.

Results. – The conversion to 3H-aldosterone could not bedetected in the control and failing hearts. Corticosterone, anintermediate product from deoxycorticosterone toaldosterone, was detected in both control and failing hearthomogenates by LC–MS, however, the production of 3H-corticosterone could not be detected. Cardiac protein level ofmineralocorticoid receptor was significantly higher in theDHF rats than in the control rats.

Conclusions. – Activation of cardiac aldosterone systemin DHF is unlikely explained by enhancement of localsynthesis of aldosterone, but may be partly attributed toupregulation of cardiac mineralocorticoid receptor.

Abstracts N° O-3-4A BMP10 variant found in hypertensive dilated cardiomyopathy decreases binding to Tcap and increases extracellular secretion of BMP10Akinori Kimura, Noritsugu Nakano, Takuro ArimuraDepartment of Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

Expression of bone morphorogenetic protein10 (BMP10)is up-regulated in hearts of Nkx2.5 knock-out mice showing

cardiac hypertrophy with early dilatation of ventriclesresembling to hypertrophic cardiomyopathy (HCM) anddilated cardiomyopathy (DCM). In this study, weinvestigated the possible involvement of BMP10 in humancardiomyopathies. Mutational analysis of BMP10 wasperformed in randomly selected 144 HCM patients and 128DCM patients as well as in 1400 consecutive elderlyautopsied cases including 13 DCM and five HCM patients.A variant, Thr326Ile, was found in four subjects. Two wasdiagnosed as hypertensive DCM and one was diagnosed ashypertension with HCM. A family study in one of thehypertensive DCM patients showed that the variant wasinherited from father showing cardiac hypertrophy andhypertension. In the subjects without cardiac involvement,the variant was found only in one of 617 hypertensivesubjects and none in 766 non-hypertensive subjects,suggesting the association of the variant with hypertensivecardiomyopathy. Transfection of GFP-tagged BMP10showed the localization of BMP10 at the Z-disc in ratneonatal cardiomyocytes. Yeast-two-hybrid assaysdemonstrated the binding of BMP10 to Tcap and that thevariant reduced the binding by 45%. In addition,immunoprecipitation assays using COS7 cells expressingTcap and normal or variant BMP10 demonstrated that thevariant reduced the binding to Tcap and converselyincreased extracellular secretion of BMP10 protein, whichlead to enlargement and sarcomere assembly of neonatal ratcardiomyocytes. These observations strongly suggested theinvolvement of BMP10 in hypertensive cardiomyopathy.

Abstracts N° O-3-5Hypoxia inducible factor mediates angiotensin II induced cardiac hypertrophyTomohiro Harada, Koji Maemura, Norihiko Takeda, Yasushi Imai, Tetsuya Saito, Ryozo NagaiDepartment of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

Hypoxia-inducible factor-1α (HIF-1α) and HIF-2α (alsocalled as EPAS1) are transcription factors those play centralroles in cellular response to hypoxia. Recently, it has beenshown that HIFs mediate not only hypoxic response but alsocytokine signaling such as angiotensin II (Ang II) in in vitrostudy. However, precise role of HIFs in vivo remains to beelucidated. We previously generated a dominant negativeform of EPAS1 that inhibits both HIF-1α and HIF-2αfunction. In this study, to elucidate a role of HIFs in cardiacremodeling, we generated transgenic mice overexpressingthe dominant negative EPAS1 (dn-EPAS1) in a heartspecific manner. The transgenic mice were normal at a basalcondition. Then, we infused Ang II to the miceintraperitoneally for 14 days. The blood pressure waselevated to the same level between the dn-EPAS1 and wildtype mice. However, ventricular hypertrophy by AngII wasinhibited in the dn-EPAS1 mice (ventricular weigh to bodyweight; 4.86 ± 0.09 and 5.71 ± 0.17, dn-EPAS and wild-type


mice, respectively, P < 0.01). Hemodynamic study showedthat diastolic dysfunction which was observed in the wildtype was inhibited in the dn-EPAS1 mice. Histological studydemonstrated that both cardiomyocyte hypertrophy andfibrosis were inhibited in the dn-EPAS1 mice. Furthermore,induction of TGFβ, IL-1β, and TNFαmRNAs by Ang IIinfusion was inhibited in the dn-EPAS1 mice. In addition,production of lipid peroxidation, a marker of oxidativestress, in the heart was suppressed in the dn-EPAS1 mice(malondialdehyde + 4-hydroxynonenal diethylacetal level(µmol/mg protein); 0.42 ± 0.01 and 0.59 ± 0.01, P < 0.05).Our results suggest that HIFs play a role in Ang II-inducedcardiac hypertrophy and fibrosis by mediating cytokineinduction and oxidative stress pathways.

Abstracts N° O-3-6Effect of hyperleptinemia on alcohol-induced oxidative stress in Swiss miceBalasubramaniyan Vairappan, Nalini NamasivayamAnnamalai University, Annamalai, India

Our aim was to explore the effect of hyperleptinemia onbody weight, food and water intake, heart rate, oxidativestress and renal sodium handling in alcohol fed mice. Themice were divided into four groups, group 1 were controlanimals, group 2 animals received exogenous mouserecombinant leptin (230 mg/kg body weight i.p.) everyalternate day, group 3 were alcohol fed mice (6.32 g/kg bodyweight) and group 4 alcohol fed mice that received leptin(230 mg/kg body weight i.p.) every alternate day. Theexperiment was terminated after giving leptin injections for15 days. Total body weight, blood pressure, lipidperoxidation, enzymic and non-enzymic antioxidants wereanalyzed. Exogenous leptin administration, significantlydecreased the food and water intake and body weight duringthe experimental period, both in the leptin (group 2) andalcohol treated (group 3) animals. Increased levels of lipidperoxidation (TBARS, CD) and lowered levels of enzymic(SOD, CAT and GPx) antioxidants, and non-enzymic(vitamin E, C and GSH) antioxidants in alcoholic animalswere significantly augmented on leptin administration. Inaddition, leptin treatment significantly increased systolicblood pressure in the control and alcohol treated animals.Ethanol administration resulted in significantly elevated thelevels of plasma creatinine and sodium. The levels weresignificantly lowered on administering leptin (230 mg/kgbody weight) to experimental control (group 2) and alcoholtreated mice (group 3) as compared with untreated normalcontrol (group 1) and alcohol treated animals (group 4),respectively. Administration of recombinant mouse leptin toalcohol fed animals reduced the body weight and increasesthe level of systemic and internal oxidative stress anddecreases renal sodium excretion. These effects maycontribute to the development of leptin-inducedhypertension in alcohol fed experimental models as well asin hyperleptinemic obese individuals.

Abstracts N° O-4-1Delayed propagation of action potentials and altered phosphorylation level of connexin-43 in Ca2+-overloadedguinea pig ventricular musclesNagomi Kurebayashi a, Yuji Nakazato b, Hiroto Nishizawa b, Hiroyuki Daida b, Yasuo Ogawa aa Department of Pharmacology, Juntendo University School of Medicine, Japan. b Department of Cardiology, Juntendo University School of Medicine, Japan

The mechanism of ventricular arrhythmia in Ca2+-overloaded heart remains to be clarified. In this study, weproved the hypothesis that propagation of action potential(AP) is delayed because of inhibition of gap junction channelsin Ca2+ overloaded muscles, resulting in increased probabilityof re-entry. Papillary muscles from guinea pig ventricles wereloaded with fluo-3 and sequential two-dimensional Ca2+

images of surface cells were obtained using a confocalmicroscope. In intact muscles, APs propagatedinstantaneously over a field of view of 0.3 × 0.3 mm. In Ca2+-overloaded muscles made by high frequency stimulationunder anoxic condition, cells themselves already were lessresponsive to electrical stimulation and often showed APalternans. Surprisingly, the delay during cell-to-cellpropagation was often as long as 100 ms. These results meanthat conduction delays in several cells within a small area(~0.5 × 0.5 mm) are enough to allow re-entry of excitation.Because phosphorylation of connexin-43 (Cx43), a primarycomponent of gap junctions in heart, has been reported to beinvolved in propagation of AP, we determined distribution ofphosphorylated and unphosphorylated Cx43 using antiCx43antibodies. In intact muscle, the vast majority of Cx43 wasphosphorylated. In muscles that showed delayed propagation,a fraction of unphosphorylated Cx43 was significantlyelevated. These results suggest that inhibition of gap junctionsby dephosphorylation of Cx43 in addition to inactivation ofNa+ channels may be the cause of the delayed propagation inCa2+ overloaded muscles.

Abstracts N° O-4-2Ca2+ waves and alternans of atrial and ventricular muscles in pigHiroto Nishizawa a, Nagomi Kurebayashi b, Yuji Nakazato a, Hiroyuki Daida aa Department of Cardiology, Juntendo University School of Medicine, Japan. b Department of Pharmacology, Juntendo University School of Medicine, Japan

Ca2+ waves and alternans have been thought to beimportant arrhythmogenic factors, but only a small numberof studies have examined their properties in individual cellsin multicellular preparations. In this study, we comparedcharacteristics of Ca2+ waves and alternans in atrial muscleswith those in ventricular muscles.

Materials and methods. – Atrial trabeculae andventricular papillary muscles were dissected from pig heartsand loaded with fluo-3 or rhod-2. Sequential two-dimensional


Ca2+ images of surface cells were acquired using confocalmicroscope. Experiments were carried out at 25 or 35° C.

Results and discussion. – Healthy atrial muscles showedno waves under resting condition or at low frequencystimulations. At high frequency, muscles often showed Ca2+

waves. Interestingly, onset of Ca2+ transients and initiationof Ca2+ waves were almost simultaneous. The ensembleaverage of Ca2+ signals from ~30 cells which includes Ca2+

transients and waves showed a single peak with a monotonicdecay phase for each excitation. In Ca2+ overloadedventricular muscles, in contrast, Ca2+ waves occurred <=0.3 s after onset of Ca2+ transients. Therefore the ensembleCa2+ signals often showed two peaks, i.e. a larger peakconsisted of Ca2+ transients and a smaller peak Ca2+ waves.The latter corresponds to after-contraction. These resultssuggest that waves in pig atrial muscles used in this studycannot generate DAD. Furthermore, Ca2+ alternans waseasily detected in atrial muscles whereas hardly observed inventricular muscles. In ventricular muscle, instead, actionpotential alternans was observed. These results suggest thatthe mechanisms of generation of arrhythmia may beconsiderably different in atrial and ventricular muscles.

Abstracts N° O-4-3Estimation of Ca2+ content and Ca2+ leakage in the sarcoplasmic reticulum of saponin-treated sarcolipin transgenic mouse myocardiumSatoshi Morimoto a, Makoto Kawai b, Jin O-Uchi a,Yoichiro Kusakari a, Kimiaki Komukai b, Kenichi Hongo b,Takeda Toshihiro c, Kinya Otsu c, Seibu Mochizuki b,Satoshi Kurihara aa Department of Physiology II, The Jikei University School of Medicine, Japan. b Division of Cardiology, The Jikei University School of Medicine, Japan. c Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan

Dysfunction of Ca2+ handling by sarcoplasmic reticulum(SR) has been considered as one of the critical factors in heartfailure. Ca2+ content of SR is determined by the balance ofCa2+ uptake and release of SR and the Ca2+ leakage from SRis also a critical factor in myocardium. We developed a methodto directly estimate functions of SR, which keeps integratedstructure and physiological functions (not vesicular SR). Wetreated thin bundles of ventricular muscle with saponin (50 µg/ml for 30 min) and the preparation was put in a capillary tubewhich was placed on the stage of a fluorescence microscope.Ca2+ was loaded in SR under various conditions and thenreleased from SR by 50 mM caffeine. Ca2+ concentration inthe capillary was measured using fluo-3. We investigated SRfunctions of sarcolipin transgenic mouse myocardium (SLN-TG), in which the activity of Ca2+-ATPase was suppressed. Atshort Ca2+ loading time within 15 s, Ca2+ content of SR inSLN-TG was smaller than that in non-transgenic (NTG). Ca2+

leakage from the SR was estimated by measuring theremaining Ca2+ in SR with various loading time after washing

the preparation with the solution containing EGTA (pCa > 8).The Ca2+ leakage in both SLN-TG and NTG was almostidentical. These results suggest that Ca2+ uptake in SLN-TG isslower without significant changes in Ca2+ releasemechanism. Thus, the method employed in the present study isuseful to estimate altered functions of SR in myocardium fromtransgenic and knockout mouse.

Abstracts N° O-4-4Opposite effects of α1A- and α1B-adrenoceptor stimulation on L-type Ca2+ current through different signaling pathways in rat ventricular myocytesJin O-Uchi a, Kimiaki Komukai b, Yoichiro Kusakari a,Satoshi Morimoto a, Makoto Kawai b, Kenichi Hongo b,Satoshi Kurihara aa Department of Physiology (II), The Jikei University School of Medicine, Japan. b Division of Cardiology, The Jikei University School of Medicine, Japan

Introduction. – Recently, we showed that the effects ofα1-adrenoceptor stimulation (α1ARS) on L-type Ca2+

current (ICa,L) in rat ventricular myocytes can be dividedinto two opposite effects (negative and positive effects) andthe positive effect is protein kinase C (PKC)-dependent (O-Uchi et al., Proc. Natl. Acad. Sci. USA, 2005). It is wellestablished that two α1-adrenoceptor subtypes of α1A andα1B mainly coexist in cardiomyocytes. Therefore, theseresults raise the possibility that the two different effectsα1ARS on ICa,L might be mediated through the two differentreceptor subtypes. In this study, we compared the effects ofα1A- and α1B-adrenoceptor stimulation (α1AARS andα1BARS) on ICa,L and investigated the possible involvementof PKC translocation in the effects.

Methods. – ICa,L was measured using perforated patchclamp method in adult rat ventricular myocytes. Holdingpotential was set at –40 mV and the depolarization pulse to0 mV was applied every 10 s. Western immunoblottinganalysis was employed to check the translocation of PKC.

Results. – We observed biphasic changes in ICa,L (atransient decrease followed by a sustained increase) inducedby non-selective α1-adrenoceptor agonist, phenylephrine.However, α1A-adrenoceptor agonist, A61603, showed onlypositive effect on ICa,L, and the application of phenylephrinewith selective α1A-adrenoceptor blocker, WB4101, causedonly the sustained negative effect. Translocation of novelPKC (δ and ε) from soluble to particle fraction was observedafter α1AARS, but not after α1BARS.

Conclusion. – The opposite changes of Ca,L by α1RS aremediated through two different subtypes of α1A and α1B whichactivate different intracellular signal transduction pathways.

Abstracts N° O-4-5Different Ca2+ dependence of RyR2 activity in ventricular and atrial muscles from swineAkihito Chugun, Nagomi KurebayashiDepartment of Pharmacology, Juntendo University School of Medicine, Japan


It is generally accepted that CICR activity of RyRincluding RyR2 is biphasically regulated by Ca2+ microMCa2+ activates the channel whereas milliM Ca2+ inhibits it.However, we have recently found that [3H]-ryanodinebinding of SR from rat ventricular muscle had polyphasicCa2+ dependence, (J. Pharmacol. Sci. 97, Suppl. 1, 105P), inwhich the binding was activated at 0.1–1 µM Ca2+, reachedplateau level between 3 and 100 µM, again activated at~1 mM Ca2+, and then inhibited by Ca2+ higher than 3 mM.Interestingly, Mg2+ at physiological concentration (~1 mM)increased the activity between 10 and 300 µM Ca2+ to makethe Ca2+ dependence biphasic. In contrast to the case with ratventricle, SR from rabbit ventricular muscle demonstratedtypical biphasic ryanodine binding. To elucidate the cause ofthis difference between rat and rabbit ventricle, we furtherdetermined [3H]-ryanodine binding to SR from otheranimals including swine ventricle and atrium. Ca2+

dependence in swine ventricular SR was similar to that ofrabbit ventricle, whereas swine atrial SR showed rat-typeCa2+ dependence. In the latter, Mg2+ also increased activityof the plateau phase at 3–100 µM Ca2+. These resultssuggest that the difference between rat and rabbit ventriclemay not be attributed to the difference in RyR moleculeitself. Some factor(s) may affect the Ca2+ and Mg2+

regulation of RyR2 activity.

Abstracts N° O-5-1Elevation of serum matrix metalloprotease (MMP)-2 and oxidant stress in patients with acute worsening of chronic heart failureMegumi Kunishige, Yusuke Nakagawa, Taku Sakai, Osamu Akutagawa, Akiko Matsuo, Akira Nishibe, Takeshi Hata, Yoshiyuki KijimaDepartment of Cardiology, Higashi-osaka City General Hospital, Japan

Purpose. – Matrix metalloproteases (MMPs) are a familyof enzymes that catalyze proteolysis of extracellular matrixproteins including collagen fibers. MMP-1 is highly specificfor fibrillar collagen, whereas MMP-2 degrades laminin andfibronectin as well as collagen so that MMP-2 disruptsengagement of cardiomyocytes to extracellular matrix. Wehave recently reported the enhancement of oxidant stressand collagen synthesis at acute worsening of chronic heartfailure (CHF) (J. Am. Coll. Cardiol. 45 Suppl. A, 169A,2005). The aim of this study was to clarify serum levels ofMMPs at acute and chronic phases of CHF.

Methods. – Sixteen patients were enrolled into this studyat their admission due to acute worsening of CHF (mean age65.3 ± 12.5). Etiology of CHF included hypertension,ischemia, valvular disease and idiopathic cardiomyopathy.At their admission (acute phase) and after the successfultreatment using conventional medicines (chronic phase), wemeasured serum levels of MMP-1 and MMP-2.

Results. – After the treatment (12.6 ± 4.1 days later),serum level of brain natriuretic peptide was decreased from

742.8 ± 493.2 to 264.0 ± 291.9 pg/ml. Serum MMP-2 wasenhanced at acute versus chronic phase (1278 ± 275 vs. 856± 217 ng/ml, P < 0.001). MMP-1 did not change (11.1 ± 6.9vs. 11.2 ± 6.0 ng/ml).

Conclusion. – Serum MMP-2, but not MMP-1, wasincreased under the condition of enhanced oxidant stress,suggesting that repetitive worsening of CHF might lead tomyocardial fibrosis and remodeling.

Abstracts N° O-5-2Effect of bepridil on the sub-acute phase of rapid atrial stimulation in the canine model. Possibly a reverse electrical remodeling effectDaisuke Sato, Shinichi Niwan, Masahiko Moriguchi, Sae Sasaki, Masaru Yuge, Ryuta Imaki, Syouji Hirasawa, Takeshi Sasaki, Hiroe Niwano, Tohru IzumiDepartment of Internal Medicine, Kitasato University School of Medicine, Japan

Background. – Several antiarrhythmic agents have beenknown to suppress the progression of atrial electricalremodeling (ER) but their effects are limited especially as adown-stream therapy. In this study, the effect of bepridil onER was evaluated in a sub-acute phase of ER.

Method. – In 10 beagle dogs, the right atrial appendage(RAA) was paced at 400 bpm. The atrial effective refractoryperiod (AERP) and inducibility of atrial fibrillation (AF)were evaluated along the time course. In all dogs, the initialrapid pacing was performed for 2 weeks without anyantiarrhythmic drugs. In five out of 10 dogs, bepridil (15 mg/kg/day) was started at that point and pacing was continuedfor an additional 4 weeks (bepridil group). In the remaining5 dogs, rapid pacing was continued in the same mannerwithout any drug administration (control group).

Result. – The AERPs were shortened during the initial2 weeks in both groups (P < 0.05). In the control group, theAERP exhibited additional shortening in the following 4weeks. In contrast in the bepridil group, the AERP initiallyprolonged in the first week following the bepridiladministration, but the AERP kept gradually prolonging inthe following 3 weeks, then finally recovered to the day 0level. AF inducibility was lower in the bepridil group than inthe control at all time points after the drug administration.

Conclusion. – Bepridil reversed the AERP shortening intwo phases, i.e. a first quick recovery and a followinggradual recovery. The latter was thought possibly to be thedocumentation of the reverse ER effect of bepridil.

Abstracts N° O-5-3Analysis of interval-force relationship of the left ventricle during sustained mechanical alternans in patients with chronic heart failureMakoto Kodama, Mahmoud M. Ramadan, Takeshi Kashimura, Masahiro Ito, Hitoshi Tachikawa, Wataru Mitsuma, Satoru Hirono, Yuji Okura, Kiminori Kato, Haruo Hanawa, Yoshifusa Aizawa


Division of Cardiology, Niigata University Graduate School of Medical and Dental Sciences, Japan

Purpose. – Mechanical alternans is an interesting butpoorly-understood phenomenon of alternating strong andweak beats with a constant beat-to-beat interval. It is apossible linker between failing myocardium and ventricularfibrillation in patients with chronic heart failure. Interval-force relationship is an intrinsic regulatory principle ofmyocardial contractility depending on SR Ca2+ cycling. Inthis study, we analyzed interval-force relationship duringmechanical alternans.

Methods. – During diagnostic cardiac catheterization, wemeasured left ventricular dP/dt using micro-manometer-tipped catheter under programmed stimulation which wasconstructed by 8 beats of basic cycle length stimuli(coupling interval of 600 ms) followed by extra-stimuli ofdecreasing coupling intervals (from 980 to 320 ms). The dP/dt of the extra-beats were plotted and fitted to the mono-exponential equation, and fitted curves were known asmechanical restitution curve. A time constant was calculatedfrom the fitted equation curves for each patient.

Results. – We were able to obtain adequate mechanicalrestitution curves after both strong beats and weak beatsfrom 7 patients. Mechanical restitution curves after weakbeats steeply rose compared with those after strong beats. In5 of 7 cases, the time constant and the expected refractorytime of the curves after weak beats were shorter than thoseafter strong beats. The expected maximum dP/dt did notdiffer after strong and weak beats.

Conclusion. – Mechanical restitution properties weredifferent after strong beats and weak beats duringmechanical alternans. This implies that mechanical alternansoriginates from alternating changes of myocardial Ca2+

cycling property.

Abstracts N° O-5-4Bone marrow stromal cell infusion after myocardil infarction through cardiac vein may improve cardiac function in swineTakatoshi Sato a, Hiroshi Suzuki a, Taro Kusuyama a,Yasutoshi Ohmori a, Teruko Soda a, Fumiyoshi Tsunoda a,Makoto Shoji a, Yoshitaka Iso a, Shinji Koba a, Eiichi Geshi a, Takashi Katagiri a, Keisuke Kawachi b, Kohei Wakabayashi b, Youichi Takeyama ba Third Department of Internal Medicine, Showa University School of Medicine, Japan. b Division of Cardiology, Department of Internal Medicine, Showa University Fujigaoka Hospital, Japan

Purpose. – Recently, bone marrow stromal cell (BMSC)is focused on as the useful source of cell transplantation evenin cardiovascular field. However, there is a few reportsexamined the effects in large animal, and the usefultransplantation method has not been explored. In this study,we investigated the effects of BMSC infusion from coronaryvein in swine myocardial infarction model.

Methods. – To generate myocardial infarction, beadswere placed in left anterior descending coronary artery ofdomestic swine. Bone marrow cells were aspirated, andwere cultivated in BMSC culture medium. Four weeks later,bone marrow stromal cells, labeled with dye were infusedretrogradely from anterior interventricular vein. Mediumwas infused in control group. Left ventriculography wasperformed just before and 4 weeks after cell infusion beforesacrifice. In immunohistochemistry, anti-alpha smoothmuscle actin antibody was used, and vessel number wascalculated in border area. Immunofluorescent staining wasperformed to detect bone marrow stromal cells.

Results. – In left ventriculography, % change of ejectionfraction was significantly improved in BMSC group ascompared with control group (P < 0.05). At 4 weeks, BMSCdetected by red fluorescence were observed in myocardium.In immunohistochemistry, number of alpha smooth muscleactin positive vessels was significantly larger in BMSCgroup in border area (P < 0.05).

Conclusions. – BMSC infusion from coronary vein mayimprove cardiac function after myocardial infarctionthrough inducing angiogenesis.

Abstracts N° O-5-5Immunomodulatory effects of beta-adrenergic stimulation through the Gi-protein signaling pathway on the development of autoimmune myocarditisTakayuki Inomata, Tohru IzumiDepartment of Internal Medicine and Cardiology, Kitasato University School of Medicine, Japan

Background. – 1. Although it is well known thatneurohumoral factors including the sympathetic nervoussystem activated through beta-adrenergic stimulation has amajor role in the development of heart failure, its effect onmyocardial inflammation has not been fully investigated. 2.Daily administration of carvedilol, but not metoprolol,aggravated myosin-induced experimental autoimmunemyocarditis (EAM), while the beta2-selectiveadrenoreceptor agonist formoterol ameliorated myocarditiswhen compared with a vehicle group. These data led to theidea that the difference between beta1- and beta2-adrenoreceptor signals could explain the results. As it is wellknown that different signaling pathways exist concerning G-protein between beta1- and beta2-adrenoreceptorstimulations, inhibitory G (Gi)-protein may become a goodcandidate for the different beta-adrenergic signaling system.

Objective. – We investigated the role of pertussis toxin(PTX), Gi-protein-selective antagonist, for modulating thedisease severity of EAM.

Methods and results. – A single injection of PTX dose-dependently exacerbated active EAM in the induction phasebut ameliorated active EAM in the effector phase.Furthermore, in vivo administration of PTX amelioratedtransfer EAM and in vitro exposure of PTX on


myocarditogenic T cells reduced the disease severity oftransfer EAM in a dose-dependent manner.

Conclusion. – Beta2-adrenergic stimulation variablymodulates the progression of T-cell-induced autoimmunemyocarditis predominantly through Gi signaling pathway.

Abstracts N° O-5-6Pravastatin improved angiotensin II-induced ventricular remodeling through the attenuation of increased MMPs and OPNZhujie Xu, Hiroshi Okamoto, Masatoshi Akino, Hiroyuki TsutsuiDepartment of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Japan

Adult male C57BL/6J mice were randomly divided intofour groups treated with vehicle or angiotensin II (AII; 2 µg/kg m). Pravastatin (10 mg/kg per day) or saline wasadministrated to each group for 4 weeks. AII treatmentsignificantly elevated BP and increased LVW/BW, but notin Pra treatment group. Interestingly, AII-induced increaseof myocyte cross-sectional area and interstitial fibrosisalmost completely were abolished by Pra treatment, but notthe perivascular fibrosis. The expressions of matrixmetalloproteinase (MMP)-2, MMP-3 and osteopontin(OPN) were increased by AII infusion, which wereattenuated in Pra treatment group, whereas transforminggrowth factor-beta (TGFβ1) and collagen I were notdifferent among 4 groups, despite the change in ECMaccumulation by AII. The immunohistochemical staining forOPN and macrophages showed that the OPN expression andthe macrophages accumulation were seen obviously in AII-treated mice. AII treatment increased the concentrations oftotal and LDL cholesterols, but Pra showed no effect. Inconclusion, the hydrophilic statin, pravastatin, improvedangiotensin II-induced left ventricular remodeling bypreventing from cardiac interstitial fibrosis and myocytehypertrophy independent of cholesterol lowering effect, andthese changes may be caused by the attenuation of increasedMMPs and OPN activities.

Abstracts N° O-6-1Apolipoprotein A-I discs may prevent apoptosis through ERK activation in myocytesYoshihiro Kiya a, Shin-ichiro Miura a, Satoshi Imaizumi a,Yoshinari Uehara a, Yoshino Matsuo a, Hedenori Urata b,Kerry-Anne Rye c, Keijiro Saku aa Department of Cardiology, Fukuoka University School of Medicine, Fukuoka, Japan. b Department of Internal Medicine, Fukuoka University Chikushi Hospital, Japan. cLipid Research Group, The Heart Research Institute, Australia

Although the infusion of reconstituted high densitylipoprotein (rHDL) produces a rapid regression of coronaryatherosclerosis in animal models and humans, the effects ofrHDL in pathophysiological remodeling after myocardialinfraction (MI) are unknown. In this study, the effects of

POPC/1-palmitoyl-2-oleoyl-phosphatidylcholine POPC/ApoA-I discs were examined in rats with cardiac dysfunctionafter MI. Fifteen male Wistar rats were divided into threegroups: sham-operated (n = 5), and MI rats that received3 weekly infusions of placebo (MI group, n = 5) or POPC/ApoA-I (containing ApoA-I 6 mg/kg) administeredintravenously (MI + POPC/ApoA-I group, n = 5).Echocardiography was performed in all animals 1 and4 weeks after surgery. The MI + POPC/ApoA-I groupshowed a significant increase in left ventricular (LV)ejection fraction (EF), and a decrease in LV end-systolicdiameter, and a tendency for a decrease in LV end-diastolicdiameter, compared with a progressive deterioration of LVsize and function in the MI group. In addition, the MI +rHDL group showed a significant decrease in the area of MIcompared to that in the MI group. Interestingly, the MI +POPC/ApoA-I group showed a significant activation ofphospho-ERK (extracellular-signal-regulated kinase) butnot p38 mitogen-activated protein kinase or Jun N-terminalkinase compared to the sham-operated and MI groups. Thus,POPC/ApoA-I exerts beneficial morphological effects thatresult in the prevention of LV remodeling and improvedfunction after MI, and may prevent apoptosis through ERKactivation in myocytes. These findings represent an excitingnew area in MI treatment. HDL-based therapy holds thepromise of dramatically reducing the incidence ofpathological remodeling after MI.

Abstracts N° O-6-2Endoplasmic reticulum Ca2+ depletion induces apoptosis independent of caspase-12 signaling pathwayTomoyasu Nakano a, Hiroshi Watanabe b, Hideki Katoh a,Hiroshi Satoh a, Hideharu Hayashi aa Department of Internal Medicine III, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan. b Clinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan

Increased endothelial cell apoptosis has been shown toinitiate atherosclerosis. Despite the observations in manycell types that endoplasmic reticulum (ER) stress causesapoptosis in which caspase-12 plays a crucial role, cytosolicCa2+ signaling responsible for this effect has not beencompletely elucidated in endothelial cells (ECs). Here wehave investigated the interaction of Ca2+ signaling andcaspase-12 cleavage in apoptosis of cultured porcine aorticECs. Cytosolic Ca2+ concentration ([Ca2+]i) was measuredusing fura-2/AM. Apoptosis was assessed by DNA ladderformation, and cleavage of caspase-12 by Western blotting.Thapsigargin (6 µM), an inhibitor of the ER-associatedCa2+-ATPase, increased [Ca2+]i (F340/F380 ratio from 0.74± 0.05 to 5.89 ± 0.52: P < 0.001) and induced persistent ERcalcium depletion, cleavage of caspase-12 and apoptosis.Bradykinin (10 nM) increased [Ca2+]i (F340/F380 ratiofrom 0.77 ± 0.14 to 5.91 ± 0.71: P < 0.001) but induced


neither cleavage of caspase-12 nor apoptosis. However,when ECs were treated with BAPTA/AM (100 µM), BKcaused the Ca2+ depletion of ER and apoptosis without thecleavage of caspase-12. Moreover non-selective caspaseinhibitor, zVAD-fmk (100 µM), inhibited apoptosis andcleavage of caspase-12 in TG-stimulated ECs, and calpaininhibitor, MDL28170 (120 µM), inhibited cleavage ofcaspase-12 but not apoptosis. These results suggested thatthe increase in [Ca2+]i did not play an important role in theinduction of apoptosis in ECs, however, ER Ca2+ depletioninduced apoptosis, which is independent from the caspase-12 linked signaling pathway.

Abstracts N° O-6-3Apoptosis signal-regulating kinase 1 is involved not only in apoptosis but also in non-apoptotic cardiomyocyte deathTetsuya Watanabe, Kinya Otsu, Masatsugu HoriDepartment of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan

Purpose. – The molecular basis of myocardial cell deathin the ischemia-reperfused heart still remains to be clarified.Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays animportant role in stress-induced apoptosis. We studiedASK1–/– mice to examine the role of ASK1 in ischemia–reperfusion injury.

Methods. – The hearts in ASK–/– and wild-type (WT)mice were used, and the left coronary artery was occludedfor 30 min then reperfused for 2 h. The presence of apoptosiswas detected by DNA ladder formation, terminaldeoxyribonucleotidyl transferase-mediated dUTP-biotinnick end labeling (TUNEL) and caspase-3 activation.

Results. – In WT hearts, ischemia–reperfusion resulted insignificant necrotic injury, as evidenced by a large area ofnegative triphenyltetrazolium chloride (TTC) staining (32.4± 11.3% of area at risk). In ASK–/– heart, infarct size wassubstantially smaller at 7.6 ± 3.5% (P < 0.01). The necroticinjury was not accompanied with any evidence of apoptosissuch as an increase in TUNEL positive cells, DNAfragmentation or the activation of caspase-3. ASK1–/–cardiomyocytes were more resistant to H2O2- or Ca2+-induced apoptotic and non-apoptotic cell death comparedwith wild-type cells.

Conclusions. – These data suggest that ASK1 is involvedin necrosis as well as apoptosis and that ASK1-dependentnecrosis is likely to contribute to myocardial cell death in theischemia-reperfused heart.

Abstracts N° O-6-4NF-κB suppresses mitochondrial permeability transition pore opening and apoptosis of ventricular myocytes during hypoxic injuryLorrie A. Kirshenbaum, James Shaw, Delphine Baetz, Kelly M. RegulaUniveristy of Manitoba, Manitoba, Canada

In this report we provide evidence for the operation of thecellular factor NF-κB as a key regulator of the mitochondrialfunction and the cell death of ventricular myocytes duringhypoxia. In contrast to normoxic control cells, ventricularmyocytes subjected to hypoxia displayed a 9.1-fold increase(P < 0.05) in apoptosis as determined by Hoechst 33258nuclear staining and vital dyes. Mitochondrial defectsconsistent with permeability transition pore (PTP) opening,loss of mitochondrial deltaΨm, and Smac release wereobserved in cells subjected to hypoxia. This wasaccompanied by a concomitant increase in the post-mitochondrial caspase 9 and caspase 3 activity in hypoxicmyocytes. Adenovirus mediated delivery of IKKβwtresulted in a significant increase in NF-κB dependent DNAand gene transcription in ventricular myocytes.Interestingly, cells rendered defective for NF-κB activationwith a kinase defective IKKβK-M (IKKβmt) or a non-phosphorylatable form of IκBα were sensitized cells tomitochondrial perturbations and hypoxic injury. Hypoxia-induced, mitochondrial defect and cell death weresuppressed in cells expressing IKKβwt but in not cellsexpressing the kinase defective IKKβ. To our knowledge,the data provide the first direct evidence that IKKβ-mediatedNF-κB activation suppresses hypoxia-induced cell death ofventricular myocytes through a mechanism that impingesupon the mitochondrial death pathway.

Abstracts N° O-6-5AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of ER stressKazuo Trai a, Yoshimune Hiramoto a, Mitsuru Masaki a,Shoko Sugiyama a, Tadashi Kuroda a, Ichiro Kawase b,Masatsugu Hori a, Hisao Hirota aa Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine. b Department of Respiratory Medicine and Rheumatic Diseases, Osaka University Graduate School of Medicine

Oxygen deprivation leads to the accumulation ofmisfolded proteins in endoplasmic reticulum (ER),causing ER stress. Under conditions of ER stress,inhibition of protein synthesis and up-regulation of ERchaperone expression reduce the misfolded proteins inER. AMP-activated protein kinase (AMPK) is a keyregulatory enzyme involved in energy homeostasis duringhypoxia. It has been shown that AMPK activation isassociated with inhibition of protein synthesis viaphosphorylation of elongation factor 2 (eEF2) incardiomyocytes. We therefore examined whether AMPKattenuates hypoxia-induced ER stress in neonatal ratcardiomyocytes. We found that hypoxia induced ERstress, as assessed by the expression of CHOP and BiP andcleavage of caspase12. Knockdown of CHOP or caspase12through siRNA resulted in decreased expression ofcleaved poly-(ADP-ribose) polymerase (PARP) followingexposure to hypoxia. We also found that hypoxia-


induced CHOP expression and cleavage of caspase12were significantly inhibited by pretreatment with5-aminoimidazole-4-carboxyamide-1-b-D-ribofuranoside(AICAR), a pharmacological activator of AMPK. Inparallel, adenovirus expressing dominant-negative AMPKsignificantly attenuated the cardioprotective effects ofAICAR. Knockdown of eEF2 phosphorylation using eEF2kinase-siRNA abolished these cardioprotective effects ofAICAR. Taken together, these findings demonstrate thatactivation of AMPK contributes to protection of the heartagainst hypoxic injury through attenuation of ER stress,and that attenuation of protein synthesis via eEF2inactivation may be the mechanism of cardioprotection byAMP.

Abstracts N° O-6-6A contribution of oxidative stress from mitochondria on remodeling after myocardial infarction: evaluation by in vivo ESRTomomi Ide a, Hiroyuki Tsutsui b, Toru Kubota a, Kenji Sunagawa aa Department of Cardiovascular Medicine, Kyushu University, Japan. b Department of Cardiovascular Medicine, Hokkaido University, Japan

Background. – Mitochondrial electrical respiratorychain is a major source of oxidative stress in failingmyocardium. The copy number of mitochondria DNA(mtDNA) is decreased in failing myocardium in mice aftermyocardial infarction (MI), whereas overexpression ofmitochondrial transcription factor A (Tfam) ameliorate themyocardial remodeling in the same model. Wehypothesized that the protecting effect of Tfam is bydecreasing the oxygen radical production in mitochondria.To test this hypothesis, we have established the methods toestimate oxygen radical formation in living animals byusing in vivo ESR technique and measured the formation ofoxygen radicals.

Methods and results. – Myocardial infarction (MI) byligating left coronary artery was created in transgenic miceof human Tfam (TG) and wild type (WT). The animals wereanesthetized and injected hydroxy-PROXYL, a nitroxidespin probe, intravenously and recorded its signal intensity byESR L-band at the chest level. The semilogarithmic signalclearance was increased in MI compared with Sham-operated mice (0.098 ± 0.015 vs. 0.18 ± 0.021 per min, P <0.01), which was abolished by simultaneous injection ofTiron, or Dimethylthyourea. The increase of the signalclearance was not observed at the head level or at theabdominal level, and not with the injection of the spin proveintra-tracheally. Moreover, the signal decay of the probe intissue homogenate increased in the heart but not in the liver,or the lung. Tfam overexpression could ameliorate theincrease of this signal decay in MI (60 ± 11 vs. 100 ±14 nmol/g).

Conclusions. – Overexpression of Tfam inhibited LVremodeling and failure after MI and it correlates withdecrease of oxygen radical formation.

Abstracts N° P-1-1Lentiviral vector-mediated SERCA2 gene transfer improves the failure heart induced by myocardial infarction in ratKazuo Niwano, Masashi Arai, Norimichi Koitabashi, Atai Watanabe, Masahiko KurabayashiDepartment of Medicine and Biological Science, Gunma University Graduate School of Medicine, Gunma, Japan

Introduction. – Reduced expression of sarcoplasmicreticulum (SR) Ca2+-ATPase (SERCA2) impairs calciumhandling in the cardiac myocytes and contributesimportantly to contractile dysfunction of the heart. Unlikeadenovirus- or adenoassociated vectors, lentivirus can stablyintegrate into host genome of terminally differentiatedcardiac myocytes and thus induces permanent geneexpression. We developed lentivirus-based SERCA2 genetransfer system using hypothermic intracoronary deliverymethod and examined its effect on the cardiac contractilefunction in rat heart failure model.

Results. – Transfection of Lenti-GFP vector (1 × 1011 IU/300 g BW) revealed that the transgene was preferentiallydelivered into heart but far less abundant into liver andspleen. The therapeutic effect of Lenti-SERCA2 vector wascompared with the Lenti-β-Gal control vector in the failureheart induced by myocardial infarction (MI) in rat. Eachvector was introduced into heart 21 days after MI andcontractile performances were monitored byechocardiography. As shown in figure, Lenti-SERCA2group rats showed a significant increase in fractionalshortening by 10% compared with Lenti-β-Gal group ratsfrom 30 to 90 days after gene transfer. Although SERCA2mRNA and protein were elevated after gene transfer, mRNAlevels for other SR genes were not affected. In addition, theexpression of BNP mRNA expression was significantlydecreased in Lenti-SERCA2 group rats.

Conclusion. – Our study showed that the introducedSERCA2 gene was successfully integrated into hearts byusing lentivirus system, and supports the premise that alentivirus-based, SERCA2 gene therapy improves humanheart failure.

Abstracts N° P-1-2β-Blocker ameliorates intracellular Ca2+ handling in heart failure via correction of defective inter-domain interaction within ryanodine receptorMamoru Mochizuki, Masafumi Yano, Hiroki Tateishi, Masunori MatsuzakiDivision of Cardiovascular Medicine, Department of Medical Bioregulation, Yamaguchi University Graduate School of Medicine, Japan


Here, we hypothesized that the interaction between N-terminal (N:1–600) and central (C:2000–2500) domains ofRyR, where many mutations have found in patients withpolymorphic VT (pVT), is defective in heart failure (HF),and that the defectiveness may be corrected by β-blocker,carvedilol (CV).

Methods. – RyR was labeled with the fluorescent probeMCA using DPc10, a peptide, Gly2460-Pro2495 of RyR(contains one pVT mutation), as a site-directing carrier.

Results. – 1) In SR vesicles from untreated paced dogs{CV(–)}, the inter-domain interaction within RyR wasdefective, as confirmed by measuring accessibility of thefluorescent quencher. However, it was not in CV(+) SR. 2)Abnormal Ca2+ leak through RyR was found in CV(–), butnot in CV(+). 3) In the CV(–) SR vesicles, oxidative stressof RyR was augmented, but it normally restored in CV(+).4) Incubation of failing myocytes with CV restored the cellshortening and Ca2+ transient, however, when the myocyteswere incubated together with DPc10 (domain unzipper) theeffect of CV was completely abolished. 5) In contrast,addition of JTV519 (domain stabilizer), normally restoredthe failing myocyte function even in the presence of DPc10.

Conclusions. – Beneficial effect of CV on theintracellular Ca2+ handling and myocyte contractile functionin HF may be caused by correction of defective inter-domaininteraction within RyR by suppressing oxidative stress andhyperadrenergic state.

Abstracts N° P-1-3Transcriptional control of mitochondrial genes determines the contractile function in the failing heart possibly through the transcriptional activation of the serca2 geneAtai Watanabe, Masashi Arai, Norimichi Koitabashi, Kazuo Niwano, Masahiko KurabayashiDepartment of Medicine and Biological Science, Gunma University Graduate School of Medicine, Gunma, Japan

Background. – ATP produced by mitochondrial (Mt)respiratory chain proteins is crucial for the excitation–contraction (E–C) coupling. Transcriptional control of Mtgenes in the failing heart and its significance on the cardiacfunction was examined.

Results. – Myocardial infarction (MI) was created in ratand cardiac function was assessed 7 days after MI. Theamount of complex I and III in the Mt respiratory chain wassignificantly correlated with fractional shortening (I, r =0.62; III, r = 0.47), LVEDP (–0.50, –0.77), +dP/dt (0.48,0.60) and tau (–0.61, –0.80). On the contrary, complex II,which is encoded by nuclear DNA did not change.Messenger RNAs for Mt transcriptional proteins composedof Tfam, a Mt-DNA-binding factor, Tfb2m, a co-factor ofTfam, and Mt-RNA polymerase were correlated withcomplex I and III mRNAs. Interestingly, SERCA2 mRNAlevel was significantly correlated with Tfam (r = 0.53),Tfb2m (r = 0.77) and Mt-RNA polymerase (r = 0.74) and

further with systolic and diastolic function. These resultsraised the possibility that Mt-transcriptional machinery alsoregulates SERCA2 gene transcription. Indeed, robustexpression of Tfam, Tfb2m and Mt-RNA polymeraseactivated SERCA2 gene transcription by 2.43-fold followedby the 1.32 times increase of SERCA2 protein.

Conclusions. – Our study suggested that thetranscriptional activity of the Mt gene determines thesystolic and diastolic function in the failing heart. Moreimportantly, Mt transcription factors may regulate E–Ccoupling not only by producing ATP-generating Mtrespiratory chain proteins but also by directly increasingSERCA2, a protein essential for E–C coupling.

Abstracts N° P-1-4Effects of volatile anesthetics and nitrous oxide on L-type calcium current of rabbit ventricular myocytes and their modulation by phosphorylationLiu Fan a, Seijiro Sonoda a, Hiroto Tsujikawa b, Makino Watanabe b, ChunHong Jin a, Toyoki Kugimiya a, Toyo Miyazaki a, Takao Okada ba Department of Anesthesiology, Juntendo University School of Medicine, Japan. b Department of Physiology, Juntendo University School of Medicine, Japan

We compared the effects of nitrous oxide (N2O) mixed involatile anesthetics and volatile anesthetics alone on L-typecalcium current (ICa,L) of isolated rabbit ventricularmyocytes and examined the influence of phosphorylation(isoproterenol or forskolin) using whole-cell patch clamptechnique. The control solution was bubbled with air. Thechanges of ICa,L were recorded in response to theexperimental gas mixtures containing sevoflurane,halothane with or without N2O; the concentration ofanesthetics was 1 minimum alveolar concentration (MAC).Sevoflurane decreased ICa,L from 4.00 ± 0.34 to 3.50 ± 0.27(n = 16, P < 0.01) and sevoflurane + N2O similarlydecreased it from 4.40 ± 0.29 to 3.90 ± 0.26 pA/pF (n = 18,P < 0.01). Under the presence of ISO (1 µM) sevofluranedecreased ICa,L from 13.43 ± 0.56 to 12.87 ± 0.54 pA/pF (n= 20, P < 0.01). Again N2O did not affect the suppressioneffect of sevoflurane on ICa,L (from 13.26 ± 0.79 to 12.80 ±0.80 pA/pF; n = 18, P < 0.05). However, when Ca2+ channelwas phosphorylated by FSK (10 µM), neither sevofluranenor sevoflurane + N2O decreased ICa,L. While, suppressioneffect of halothane on ICa,L was not affected by FSK, but thesuppression was inhibited when Ca2+ channel wasphosphorylated by ISO. Exposure to halothane + N2O, thesuppression was decreased significantly as Ca2+ channelwas phosphorylated by either ISO or FSK. These resultssuggest that sevoflurane with or without N2O depress ICa,Lsignificantly. However these suppressions are modulateddifferently when Ca2+ channel is phosphorylated. While theeffects of halothane is different from those of sevofluranethat may attenuate Ca2+ influx independently of β-adrenergic stimulation.


Abstracts N° P-1-5Paradoxical effects of endothelin-1 on myofilament Ca2+

sensitivity in twitch and tetanic contraction in mouse ventricular myocytesKazuhide Nishimaru, Masao EndohDepartment of Cardiovascular Pharmacology, Yamagata University School of Medicine, Japan

While ET-1 elicits a negative inotropic effect (NIE) incertain species including mice, alteration of myofilamentCa2+ sensitivity during the induction of NIE has scarcelybeen studied. In the present study, we examined the effect ofET-1 on myofilament Ca2+ sensitivity in single mouseventricular myocytes loaded with the Ca2+ sensitivefluorescent dye indo-1. Twitch or tetanic cell shortening(CS) was recorded with the indo-1 fluorescence signalsimultaneously. In twitch shortening experiments, ET-1elicited rightward shift of the phase-plane diagram of Ca2+

transient (CaT) and CS, suggesting that ET-1 decreasesmyofilament Ca2+ sensitivity. The ET-1-induced shift wasinhibited by wortmannin (1 µM), an inhibitor of PI3-kinaseand MLCK, but not by the PKC inhibitor GF109203X(1 µM). On the contrary, in tetanic contraction experiments,ET-1 produced leftward shift of the instantaneous plot of theCaT versus CS, suggesting that ET-1 increases myofilamentCa2+ sensitivity. The ET-1-induced leftward shift wasabolished by GF109203X but not by wortmannin. Inaddition, it is noteworthy that ET-1 retards the shorteningvelocity with little effects on the rising phase of CaT. Thesuppressive effect of ET-1 on shortening velocity wasinhibited by wortmannin. These findings suggest that ET-1increases the myofilament Ca2+ sensitivity via PKCactivation but suppresses the shortening velocity viawortmannin-sensitive and PKC-insensitive pathway.Apparent decrease in myofilament Ca2+ sensitivity inducedby ET-1 in twitch CS may be explained by the lattermechanism, namely during brief twitch contraction inmouse ventricular myocytes.

Abstracts N° P-1-6Crystal structure of the N-terminal domain of human cardiac troponin C in complex with a calcium-sensitizer; trifluoperazineTomoko Igarashi, Soichi Takeda, Hidezo MoriDepartment of Cardiac Physiology, National Cardiovascular Center Research Institute, Japan

The ability to sensitize cardiac muscle to Ca2+ is an areaof promising therapeutic potential for the treatment forheart failure. Cardiac troponin C (cTnC) is theCa2+-dependent switch for contracting heart muscleand therefore, a potential target for Ca2+-sensitizingcardiotonic drugs. A small hydrophobic compound,trifluoperazine (TFP), has been shown to increase the Ca2+

sensitivity of cardiac muscle preparations and, thus providean excellent model compound for the design of calciumsensitizers. In the present study, we have crystallized the

N-terminal regulatory domain of cTnC (cNTnC) incomplex with TFP. Analysis of eight independently refinedcNTnC structures revealed that they are almost identicaland that two molecules of cNTnC in the crystalline latticesform a tightly associated dimer. The two cNTnCs in thedimer are related by non-crystallographic twofoldrotational symmetry so that their hydrophobic pocketsmerge to form a channel largely occupied by four TFPmolecules. The drug bound complex displays a fully opencNTnC structure, similar to cNTnC bound to theamphiphilic fragment of cardiac troponin I (residues 147–163). The phenothiazine rings of four TFPs are stackedtogether and lie along the hydrophobic cavity of the cNTnCmonomer where the hydrophobic residues in the TnIfragment bind. The current results provide a structuralbasis for the effects of TFP on cTnC and fundamentalinformation for structure based design of new Ca2+-sensitiging compounds.

Abstracts N° P-2-1Modeling of the inactivation process of L-type Ca2+

channelShingo Suzuki a, Chiaki Tsuchikawa b, Ian Findlay c,Yoshihisa Kurachi ba Clinical Genome Informatics Center, Kobe University, Kobe, Japan. b Graduate School of Medicine, Osaka University, Osaka, Japan. c Facultedes Sciences, Universite de Tours, France

Inactivation of the L-type Ca2+ channel regulates theamount of Ca2+ influx into cardiac myocytes. Theinactivation can be driven not only by depolarization(voltage-dependent inactivation, VDI) but Ca2+ influx(Ca2+-dependent inactivation, CDI). Until now, the studyof the modeling for these inactivation processes of the L-type Ca2+ channel has not yet been reported. In this study,we have modeled the VDI and CDI both for the control andthe β-adrenergic stimulation (100 nM isoproterenol). Todevelop the VDI model, we used the experimental timecourse of VDI estimated from the outward-going K+

current through the Ca2+ channel in the absence ofextracellular Ca2+ (I. Findlay, 2002) where the currentshowed biphasic decay. Thus we have determined thegating parameters of VDI for fast inactivation, slowinactivation, and steady-state components. With the VDImodel, the calculated time course of the Ca2+ current wasin the good agreement with the experimental Ba2+ currentwhich possess minimal CDI. After that, we modeled theCDI satisfying the experimental inward Ca2+ current. Themodel CDI was time-dependent where the inactivation ratewas expressed as a function of the Ca2+ current. Thecalculated time course of the inward Ca2+ current was inexcellent agreement with the experimental results underthe control and β-adrenergic stimulation. Therefore wehave successfully established the inactivation model inconsistent with the experimental results.


Abstracts N° P-2-2Na+-independent Mg2+ transport sensitive to 2-aminoethoxydiphenyl borate (2-APB) in pig carotid artery smooth muscleYukihisa Hamaguchi a, Tatsuaki Matsubara b, Toyoaki Murohara aa Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan. b Department of Internal medicine, Aichi-Gakuin University, School of Dentistry, Japan

It is known that Mg2+ is associated with several importantcardiovascular diseases. Recently, transient receptorpotential-melastatin subfamily 7 (TRPM7) has been cloned,and functionally characterized as a passive Mg2+ pathway incultured cells treated with an expression vector. Therefore,the role of this newly identified protein has to be clarified inordinary living cells, for example in the cardiovascularsystem. In the present study, we measured the intracellularfree Mg2+ concentration ([Mg2+]i) using 31P-nuclearmagnetic resonance in pig carotid artery smooth muscle.Removal of extracellular divalent cations (both Ca2+ andMg2+) in the absence of Na+ caused a gradual decrease in[Mg2+]i. 2-APB is a known blocker for TRPM7, a Mg2+-permeable cation channel, and application of this drugattenuated the [Mg2+]i decrease under the divalent cation-free, Na+-free conditions. On the other hand, simultaneousremoval of extracellular Ca2+ and Na+ (K+ substitution) inthe presence of Mg2+ caused a gradual [Mg2+]i rise in anextracellular Mg2+-dependent manner. 2-APB alsoattenuated this [Mg2+]i response, while amiloride, a Na+–/Mg2+ exchange inhibitor, had little effect. Intracellular pH(pHi) gradually decreased, while intracellular ATPconcentration was not significantly altered in Na+-freeexperiments. In conclusion, TRPM7-like Mg2+-permeablechannels exist in vascular smooth muscle, and contribute to[Mg2+]i homeostasis. It has been very recently shown thatknock-down of TRPM7 causes intracellular Mg2+

deficiency. It is speculated that down regulation of TRPM7expression, if occurs in vivo, would decrease [Mg2+]i, andthus constitute a risk factor for cardiovascular diseases.

Abstracts N° P-2-3Effects of aldosterone on the expression of gap junction protein connexin43 in cultured rat ventricular myocytesShinsuke Suzuki a, Tomoko Ohkusa a, Takashi Satoh a,Tomo Matsumoto a, Yuji Hisamatsu a, Masafumi Yano a,Kenji Yasui b, Itsuo Kodama c, Masunori Matsuzaki aa Division of Cardiovascular Medicine, Department of Medical Bioregulation, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan. b Department of Bioinformation Analysis, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan. c Department of Circulation, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan

The renin–angiotensin–aldosterone system (RAAS)affects cellular structure and electrophysiology. Modulationof intercellular coupling through gap junction (GPJ) couldlead to an alteration in conduction velocity and conductionblock. Previously, we have reported that tachyarrhythmiacauses a prominent increase of connexin 43 (Cx43), which ismediated by angiotensin II-MAPKs pathways, and it isaccompanied by alterations of conduction properties. Here,we hypothesized that aldosterone (Ald) might modulate theexpression of Cx43 that initiates GPJ remodeling and alterselectrophysiological properties of cardiomyocytes.

Methods and results. – We examined the effects of Aldon Cx43 expression in cultured neonatal rat ventricularmyocytes. Culture was exposed to Ald for 24 hours, andCx43 expression was characterized by Western blotting andreal-time RT-PCR. Exposure to 10–6–10–4 M Ald decreasethe expression of Cx43 (protein). Interestingly, treatmentwith 10–8 M Ald induced ~1.4 increase of Cx43 expression.Aldosterone is known to bind to two types of binding sites:mineralocorticoid (MR) and glucocorticoid (GR) receptors.To clarify the effects of dose dependency of Ald on Cx43expression, Ald was applied to cardiomyocytes pretreatedwith GR antagonist, mifepristone or MR antagonist,eplerenone. Mifepristone inhibited the decrease of Cx43 ofcardiomyocytes exposed to 10–4 M Ald, however did notaffect the increase of Cx43 of cardiomyocytes induced by10–8 M Ald. The increase of Cx43 of cardiomyocytesexposed to 10–8 M Ald was only inhibited by eplerenone. Aneffective prevention of RAAS activation duringdevelopment of GPJ remodeling might be a new therapeuticstrategy for the treatment of arrhythmias.

Abstracts N° P-2-4Effects of dronedarone on the currents of Xenopus oocytes co-expressing HERG and KvLQt1/mink channelsKuniaki Ishii, Masao EndohDepartment of Cardiovascular Pharmacology, Yamagata University School of Medicine, Yamagata, Japan

Dronedarone, a noniodinated derivative of amiodarone,inhibits both IKr and IKs, although IKr is blocked much morepotently than IKs. We have previously examined the effectsof E-4031 (an IKr blocker) and chromanol 293B (an IKsblocker) on the currents of oocytes co-expressing HERG andKvLQT1/minK channels (hereafter the IK). Inhibition of theIK by E-4031 was reverse frequency-dependent, and that bychromanol 293B was frequency-dependent. In this study, weinvestigated the effects of dronedarone (an IKr + IKs blocker)on the IK and compared its effects with those of amiodarone.Membrane potential of oocytes co-expressing HERG andKvLQT1/minK was stepped to +20 mV from a holdingpotential of –80 mV for 400 ms followed by 100-ms rampvoltage to –80 mV. Effects of the drugs were evaluated bycalculating the integral of the recorded currents.Dronedarone at 10 µM blocked the IK by 54.6 ± 7.3% at afrequency of 0.1 Hz (n = 6) and by 33.7 ± 6.9% at 1.67 Hz


(n = 7). Higher concentration of dronedarone (30 µM)blocked the IK by 74.8 ± 4.8% at 0.1 Hz and by 53.7 ± 5.8%at 1.67 Hz. Amiodarone at 30 µM blocked the IK by 49.5 ±4.4% at 0.1 Hz (n = 4) and by 13.7 ± 8.5% at 1.67 Hz (n =3). Thus, dronedarone has greater effects on the IK thanamiodarone and its effects seem to be less frequency-dependent, which is probably ascribed to inhibition ofKvLQT1/minK channels.

Abstracts N° P-2-5Hydrogen peroxide induced increase of L-type Ca2+

current is mediated through PKC pathway in rabbit ventricular myocytesChunhong Jin a, Hiroto Tsujikawa b, Makino Watanabe b,Takao Okada ba Department of Anesthesiology, School of Medicine, Juntendo University, Japan. b Department of Physiology, School of Medicine, Juntendo University, Japan

One of the reactive oxygen species, Hydrogen peroxide(H2O2), is produced during post ischemic reperfusion. It isreported that H2O2 increases L-type Ca2+ current ICa,L,while catalase, a scavenger of H2O2, can alter response to βsimulation of ICa,L. Although it is reported that PKClocalization is altered by H2O2, the mechanism of H2O2-induced modulation of Ca2+ channel is not yet clear. Toinvestigate the involvement of cAMP-dependentmechanisms in the H2O2-induced increase in ICa,L, the effectof modulation of PKA and PKC activation were examinedby using whole-cell patch clamp technique on isolated rabbitventricular myocytes. H2O2 alone induced 1.20 ± 0.04-folds(n = 20) increase in ICa,L. H2O2-induced increase in ICa,Lwas not affected by 300 nM forskolin (1.24 ± 0.10-folds, n =7) and 1 µM forskolin (1.15 ± 0.14-folds, n = 6), a PKAactivator. In the presence of H-89 (100 µM), a PKAinhibitor, the ICa,L increased 1.12 ± 0.05-folds (n = 7) byH2O2. H2O2-induced ICa,L increase was suppressed bybisindolylmaleimide (1 µM) (0.80 ± 0.06-folds, n = 6), aPKC inhibitor. These results suggest that H2O2 increaseICa,L in the isolated rabbit ventricular myocytes, and thisincrease of ICa,L is mediated through PKC but not PKApathway.

Abstracts N° P-2-6Investigation of the block and facilitation effects by nifekalant in a HERG channelYukio Hosaka a, Mitsuhiko Yamada b, Yoshihisa Kurachi aa Division of Molecular and Cellular Pharmacology, Department of Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan. b Department of Molecular Pharmacology, Shinshu University School of Medicine, Japan

Background. – Nifekalant, a blocker of cardiac delayedrectifier K+ current (IKr) channels encoded by a humanether-a-go-go related gene (HERG), is reported as the drugclinically effective for lethal ventricular tachyarrhythmias.

But the kinetic properties of nifekalant on HERG channelsare not studied sufficiently. So we attempted to analyze indetail the properties and find the characteristics.

Methods and results. – By using the standard two-microelectrode voltage clamp technique on HERG channelsexpressed in Xenopus oocytes, nifekalant blocked HERGchannels in a use-dependent and non frequency-dependentmanner, and caused a negative shift of the voltage-dependence of activation. Nifekalant increased HERGchannel current at low voltages only in the presence of aprevious strong depolarizing pulse, and so this pulse couldseparate the facilitation effect from the block effect. Withoutthe facilitation effect, nifekalant blocked HERG channelcurrent in a weak voltage-dependent manner. On the otherhand, with the facilitation effect, nifekalant caused asignificant negative shift in the voltage-dependence ofactivation. In the case of E-4031, a methanesulfonanilideanti-arrhythmic drug, the facilitation effect was not found.

Conclusions. – The strong depolarizing prepulse allowsnifekalant to increase the HERG channel currents, and thefacilitation effect might be the unique characteristic ofnifekalant.

Abstracts N° P-3-1NAD(P)H oxidase-derived superoxide anions are not involved in endothelium-dependent hyperpolarization mediated by hydrogen peroxide in miceAya Takaki a, Keiko Morikawa a, Ender Tekes a, Yoshinori Murayama a, Kenji Sunagawa a, Hiroaki Shimokawa ba Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan. b Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan

Background. – We have identified that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor (EDHF) in animals andhumans and that endothelial NO synthase (eNOS)-derivedsuperoxide anions are an important source for EDHF/H2O2in mice, where endothelial Cu,Zn-superoxide dismutaseplays a pivotal role to produce EDHF/H2O2 by dismutatingsuperoxide anions to H2O2. In endothelial cells, NAD(P)Hoxidase is one of major superoxide-generating enzymes.However, it remains to be examined whether NAD(P)Hoxidase-derived superoxide anions also are coupled with theEDHF/H2O2 responses. In this study, we addressed thispoint in angiotensin II-infused mice.

Methods and results. – An osmotic mini-pump withangiotensin II or vehicle was implanted subcutaneously toC57BL/6N male mice (10–16 weeks old, 2 µg ofangiotensin II/kg/min for 1 week). Isometric tensions andmembrane potentials of small mesenteric microvessels wererecorded. The angiotensin II infusion significantly increasedarterial blood pressure. In those mice, both EDHF-mediatedrelaxations and hyperpolarizations in response toacetylcholine were significantly reduced and the NO-


mediated relaxations were enhanced. Endothelium-independent relaxations to sodium nitroprusside andNS1619 (a direct opener of KCa channels) were comparablebetween the two groups. In angiotensin II-infused mice,antihypertensive treatment with hydralazine (10 mg/kg/dayin drinking water for 1 week) normalized blood pressure butfailed to improve EDHF-mediated responses. Catalase, aspecific scavenger of H2O2, inhibited EDHF-mediatedrelaxations, and apocynin, a specific NAD(P)H oxidaseinhibitor, did not acutely ameliorate EDHF-mediatedresponses in angiotensin II-infused mice.

Conclusions. – These results indicate that NAD(P)Hoxidase-derived superoxide anions do not contribute to theEDHF/H2O2 responses in mice.

Abstracts N° P-3-2Fenofibrate increases phosphorylation of AMP-activated protein kinase in human umbilical vein endothelial cells and C2C12 cellsHisashi Murakami, Ryuichiro Murakami, Toshihisa Hirai, Toru Asai, Ryotaro Takahashi, Hideo Matsui, Kenji Okumura, Toyoaki MuroharaDepartment of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan

Fibrates are peroxisome proliferator-activated receptoralpha (PPARα) ligands that have been known to improveendothelial function and glucose tolerance due to lipid loweringeffect. AMP-activated protein kinase (AMPK) has beenreported to stimulate NO production of endothelial cells viaphosphorylation of endothelial NO synthase (eNOS) andglucose uptake of skeletal muscle cells via translocation ofGLUT4 to cell membranes. We hypothesized that fibratesimprove endothelial function and glucose tolerance via not onlylipid metabolism but also the AMPK cascade. To test thishypothesis, we assessed the effect of fibrates onphosphorylation of AMPKα subunit at the activation site(threonine 172) using human umbilical vein endothelial cells(HUVEC) and C2C12 cells. Phosphorylated-AMPK wasincreased when HUVEC and C2C12 cells were treated withfenofibrate. In parallel with its increase in phosphorylated-AMPK, phosphorylation of eNOS at serine 1177 in HUVECwas increased, which was not inhibited by either PI3K inhibitor(wortmannin) or PKA inhibitor (H-89). Fenofibrate alsoincreased 2-deoxy-[3H] D-glucose uptake of C2C12 cells.Neither bezafibrate nor WY14643 increased phosphorylated-AMPK. Furthermore neither RNA polymerase inhibitor(actinomycin D) nor eukaryotic translation inhibitor(cycloheximide) blocked the rise of phosphorylated-AMPK byfenofibrate. Fenofibrate had no evident effect on theintracellular AMP/ATP ratio, which is generally thought toregulate AMPK activity. We conclude that fenofibrate activatesAMPK via the pathway distinct from both the PPARα-genomicaction and the inhibition of ATP generation. Fenofibratepossibly improves endothelial function and glucose tolerance atleast in part via the activation of AMPK cascade.

Abstracts N° P-3-3Vascular dysfunction in the newly developed heritable post-prandial hypertriglyceridemic rabbitsAkira Ishihata a, Yumi Katano ba Department of Physiology, Yamagata University School of Medicine, Yamagata, Japan. b Department of Theoretical Nursing and Pathophysiology, Yamagata University School of Medicine, Yamagata, Japan

We have recently segregated a new line of rabbit namedPHT, which showed post-prandial hypertriglyceridemia.The objective of the present study was to investigate thechanges in vascular function and the progression ofatherosclerosis in PHT rabbits. Thoracic Aorta was isolatedfrom PHT rabbits (3 or 10 months old) and Japanese whiterabbits (JW), and the developed tension of the ringpreparation was measured. Histological examination wascarried out with hematoxylin–eosin and elastica-Massontrichrome staining to detect atherosclerotic lesions. JWrabbits had no atherosclerotic lesions, while 10 months oldPHT rabbits had significant atherosclerotic lesions in theaortic luminal surface. The vascular relaxing responses toacetylcholine in the endothelium-intact aortic preparationswere not altered in the 3 months old PHT rabbits. However,the relaxing responses were significantly attenuated in the10 months old PHT rabbits. Sodium nitroprusside relaxedthe phenylephrine-precontracted vessels denuded ofendothelium equivalently in each group of rabbits. Incontrast, isoproterenol-induced vascular relaxation wassignificantly decreased in the endothelium-denuded vesselsfrom 10 months old PHT rabbits. This study showed that therapid progression of atherosclerosis and the vasculardysfunction in the endothelial cells as well as vascularsmooth muscle cells in PHT rabbits. These results suggestthat post-prandial hypertriglyceridemia may be an importantrisk factor for promoting atherosclerosis. This newlydeveloped PHT rabbits line may become a useful animalmodel for studies on the role of hypertriglyceridemia inatherosclerosis and atherosclerosis-related diseases.

Abstracts N° P-3-4Individual change of the autonomic nervous function accompanied with the posture change from supine to upright position in healthy young subjectsMakie Higuchi, Naoko KouzumaDivision of Pharmacology, Kyushu University of Nursing and Social Welfare, Japan

In our previous studies, we showed the deleterious effectsof a high sympathetic activity during underperfusion on thecardiac function, particularly in diabetic hearts. A typicalsympathetic acceleration was caused by the posture changefrom supine to standing position. Because of the individualvariation, in the present study, we examined the relationshipbetween the sympathetic and parasympathetic nervous


functions during the posture change, and/or the heart rate,blood pressure, lifestyle, subjective symptom.

Methods. – Autonomic nervous activities were assessedby power spectral analysis of heart rate variability during 25-min supine and 25-min upright positions in 27 healthy youngvolunteers.

Results. – Parasympathetic nervous function waspredominant in supine, while sympathetic nervous functionwas predominant in upright position. Depending on thesympathetic nervous reactivity, subjects were divided into3 groups: high (19%), middle (56%) and low level (26%).In the high level group with greater diastolic bloodpressure and heart rate, the parasympathetic nervousfunction was significantly low both in supine and uprightpositions, and the subjects tended to have worse subjectivesymptom.

Conclusion. – The results indicate that an assessment ofthe autonomic nervous activity, particularly parasympatheticnervous function of objects in supine position is significantfor the individual care.

Abstracts N° P-3-5EDHF-mediated responses are absent in triply NOSs-knockout miceEnder Tekes a, Keiko Morikawa a, Aya Takaki a, Yoshinori Murayama a, Masoto Tsutsui b, Nobuyuki Yanagihara b,Kenji Sunagawa a, Hiroaki Shimokawa aa Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Japan. bDepartment of Pharmacology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807-8555, Japan

Background. – The nature of endothelium-derivedhyperpolarizing factor (EDHF) still remains to beelucidated. We have demonstrated that endothelium-derivedhydrogen peroxide (H2O2) is an EDHF and that endothelialnitric oxide synthase (eNOS) is a major source of H2O2/EDHF, where Cu,Zn-superoxide dismutase (SOD) plays animportant role as an EDHF synthase by dismutating eNOS-derived superoxide to H2O2/EDHF. We aimed to investigatethe effects of the genetic disruption of all NOS isoforms onEDHF-mediated responses.

Methods and results. – We have successfully developedtriply NOSs–/– mice. Isolated small mesenteric arteries andthe aorta of 10–16-week-old male wild-type and triplyNOSs–/– mice were used for isometric tension andmembrane potential recordings. In wild-type mice,endothelium-dependent relaxations of the aorta weremarkedly inhibited by Nω-nitro-L-arginine (L-NNA, 10–4

M), whereas those of mesenteric artery were resistant to thecombination of L-NNA and carboxy-PTIO, but were highlysensitive to the combination of charybdotoxin (10–7 M)and apamin (10–6 M) (n = 7 each), indicating a primary roleof EDHF in mesenteric arteries. Importantly, EDHF-mediated responses (in the presence of 10–5 M

indomethacin and 10–4 M L-NNA) were totally absent inmesenteric arteries of triply NOS–/– mice (n = 7), whereasendothelium-independent relaxations to NS1619 wereunaltered and those to sodium nitroprusside were ratherenhanced (n = 9 each). Furthermore, endothelium-dependent hyperpolarizations of mesenteric arteries weremarkedly decreased in triply NOS–/– mice (n = 4).Antihypertensive treatment with hydralazine normalizedblood pressure but failed to improve EDHF-mediatedresponses in those mice.

Conclusions. – These results indicate that EDHF-mediated responses are closely coupled to endothelial NOSssystem.

Abstracts N° P-3-6Increased chylomicron remnants (CR) in diabetic patients in relation to coronary heart disease (CHD)—analysis by a novel ELISA for apo B-48Shizuya Yamashita a, Taizo Sugimoto a, Ken-ichi Tsujii b,Masahiro Koseki a, Daisaku Masuda a, Yumiko Toyama a,Ken-ichi Hirano a, Yoshiaki Uchida c, Naohiko Sakai ba Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan. b Itami City Hospital, Japan. c Fujirebio Co., Japan

Objectives. – We have developed a novel ELISA for apoB48 which can precisely determine serum CR levels. Thepresent study aimed to evaluate CR metabolism in diabeticsubjects in relation to CHD.

Subjects and methods. – Fasting serum apo B-48 levelswere measured by novel ELISA in 363 healthy [TL]volunteers (197 normolipidemic [NL] and 166hyperlipidemic) and assessed correlations to fasting plasmaglucose (FPG), IRI, HbA1c, HOMA-IR, and adiponectinlevels.

Results. – In 363 subjects, TG, HbA1c, IRI, HOMA-IRwere significantly higher, while HDL-C and adiponectinwere significantly lower in apo B-48 ≥ 6.5 µg/ml (85percentile, n = 56) group than in apo B-48 < 6.5 group (n =307). Among 197 NL subjects, TG, FPG and HbA1c levelswere significantly higher and HDL-C levels weresignificantly lower in apo B-48 ≥ 3.9 (85 percentile) groupthan in apo B-48 < 3.9 group. When 363 TL and 197 NLsubjects were divided into IGT (FPG ≥ 110 mg/dl and/orHbA1c ≥ 5.8%) and NGT group, IGT subjects showedsignificantly higher fasting apo B-48 levels in both TL (5.2± 4.2 vs. 3.9 ± 3.8, P < 0.05) and NL (3.5 ± 1.6 vs. 2.5 ±1.7, P < 0.01) subjects, indicating impaired CR metabolismin IGT subjects. Fasting apo B-48 levels were significantlyhigher in CHD patients (n = 88) than in non-CHD patients(n = 49) (5.8 ± 3.3 vs. 4.1 ± 2.5 µg/ml, P < 0.01) inassociation with higher TG and HbA1c and lower HDL-Clevels.

Conclusion. – CR metabolism is impaired in diabeticpatients despite normolipidemia, and serum apo B-48


levels may become a good marker for CR metabolism andCHD risk.

Abstracts N° P-4-1Oxytocin and trichostatin a induce differentiation of cardiac side population cells into beating cardiomyocytesTomomi Oyama, Toshio Nagai, Katsuhisa Matsuura, Hiroshi Wada, Koji Iwanaga, Issei KomuroDepartment of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chiba, Japan

Although there have been many reports indicating thatside population (SP) cells exist in a variety of organs and areone of the candidates for the somatic stem cells, there is littleinformation about the characters of cardiac SP cells. Here wefirst report that both oxytocin and trichostatin A inducedifferentiation of cardiac SP cells into beatingcardiomyocytes. Cardiac SP cells were isolated fromneonatal rat hearts by flow cytometer combined withHoechst 33342 differential staining. SP cells wereconfirmed by disappearance of their fraction by treatmentwith verapamil, and expression of Bcrp1 mRNA andproteins. Immunohistochemical staining using anti-Bcrp1antibody revealed that Bcrp1 positive cells exist in theinterstitial space and blood vessel wall. When cardiac SPcells were cultured and treated with oxytocin or trichostatinA, some of cardiac SP cells differentiated into spontaneouslybeating cells in which sarcomere formation was observed.RT-PCR revealed that the differentiated cells expressedcardiac genes including Nkx-2.5, GATA4 and MEF2C,which were not expressed before the treatment.Immunocytochemical staining showed expression of cardiactranscription factors and contractile proteins such as GATA4and MLC2v in differentiated cardiac SP cells. Cardiac SPcells differentiated into adipocytes and osteocytes under theappropriate conditions. These results suggest that cardiac SPcells are population of cardiac stem cells/progenitor cellsand further understanding of the molecular mechanisms ofdifferentiation, expansion and migration of cardiac stemcells may enable us to establish the new therapeuticstrategies.

Abstracts N° P-4-2In vitro evidence of cardiomyocyte like cells derived from dedifferentiated fat cellsShin-Ichiro Yokoyama a, Medet Jumabay a, Noboru Fukuda a, Taro Matsumoto a, Koichi Kano b, Hideo Mugishima c,Satoshi Saito aa Department of Internal Medicine, Nihon University School of Medicine, Japan. b Department of Animal Sciences, College of Bioresource Sciences, Nihon University, Japan. cDivision of Cell Regeneration and Transplantation, Nihon University School of Medicine, Japan

Background. – Recent insights of stem cell biologyindicate possibility of regenerative medicine for myocardial

infarction by the cell transplantation, which led to numerousinvestigations to identify a putative source of transplantedcells. Current study was undertaken to evaluatededifferentiated fat (DFAT) cells as source for thecardiomyocyte regeneration.

Methods and results. – Mature adipocytes wereseparated from subcutaneous fatty tissue of mice and thefloating population of mature adipocytes were isolated.They were dedifferentiated into fibroblast-like cells. DFATcells have significant expansion capability as similarmesenchymal stem cells. In in vitro, DFAT cells wereintroduced to differentiate into cardiomyocyte-like cells bymorphological observation. To identify the phenotype ofthe cardiomyocyte-like cells at molecular level, the geneexpression of clones was compared with control cells frommyocardial cells. RT-PCR analysis revealed that thecardiomyocyte-like cells expressed cardiac-specificmRNAs such as GATA4, Nkx 2.5, ANP, MLC-2v, andMLC-2. Pharmacological studies were performed toexamine functions of the cardiomyocyte-like cells, inwhich isoproterenol induced a dose-dependent increases inthe spontaneous contraction rate measured as beat perminute.

Conclusions. – Present study indicate that the matureadipose cells can be dedifferentiated into fibroblast-likecells, it could represent a useful source of cardiomyocyte,and that the adult mature adipose tissue will be an abundant,expendable source of autologous implantation cell asregenerative medicine for severe heart diseases.

Abstracts N° P-4-3Interaction of angiogenic behavior of endothelial cells with peptide svvyglr in vitroNaomasa Kawaguchi a, Yoshinosuke Hamada a, Shunzo Onishi b, Nariaki Matsuura aa Department of Molecular Pathology, Osaka University Graduate School of Medicine and Health Sciences, Japan. bTane General Hospital, Japan

Objectives. – In this report we found that angiogenicgrowth peptide, which is a synthetic peptide containing thefragment SVVYGLR placed on Osteopontin (OPN) as asingle unit peptide, had useful vascularization (tubeformation with cell differentiation) functions. Its effect wasequivalent to VEGF, which is known to have an importantangiogenesis function. This report showed for the first timethat SVVYGLR played a pivotal role in vascularization byendothelial cells.

Methods. – Synthesis and verification of peptide;Peptide was synthesized with a high efficiency solid-phasemethod by using an automatic peptide synthesizer. Cellculture; Transformed rat lung endothelial cells (TRLECcells) were used. Evaluation of cells ability ofproliferation; Cells were assessed by the WST-1 assay.Evaluation of cells ability of adhesion; Cells were assessedby the Cell adhesion assay. Evaluation of cells ability of


migration; Cells were assessed by the Wound assay.Evaluation of tube formation ability (Three-dimensionalassay).

Results. – 1. In an adhesion assay, we found that the cellsability of adhesion increased significantly compared withthat of the control group, but there was no difference in theability of adhesion between the various peptideconcentrations (P < 0.005). 2. In a migration assay, the cellsability of migration increased significantly compared with200 or 2000 ng/ml SVVYGLR and the control group(P < 0.005).

Conclusions. – Thus, we found that SVVYGLR peptidedid not influence the cells ability of proliferation but couldinfluence the abilities of adhesion and migration. This studyshowed that SVVYGLR plays the role of ideally assistingangiogenesis by vascular endothelial cells.

Abstracts N° P-4-4Cardioprotective effects of granulocyte colony-stimulating factorHiroshi Hasegawa, Hiroyuki Takano, Koji Iwanaga, Masashi Ohtsuka, Yingjie Qin, Yuriko Niitsuma, Kazutaka Ueda, Tomohiko Toyoda, Hiroyuki Tadokoro, Issei KomuroDepartment of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Japan

Objectives. – The aim of this study was to investigate theeffect of granulocyte colony-stimulating factor (G-CSF) onchronic myocardial ischemia in swine.

Background. – We have recently reported that G-CSFprevents cardiac remodeling and dysfunction after acutemyocardial infarction in mice and swine. It remains unclearwhether G-CSF has beneficial effects on chronic myocardialischemia.

Methods. – An ameroid constrictor was placed on leftcircumflex coronary artery (LCx) of swine. The presence ofmyocardial ischemia was verified at 4 weeks after theoperation and the animals were randomized into thefollowing two groups: 1) administration of vehicle (controlgroup, n = 10); 2) administration of G-CSF (10 µg/kg/day)for 7 days (G-CSF group, n = 10).

Results. – Echocardiographic examination revealed thatthe G-CSF treatment prevented left ventricular dilation anddysfunction at 8 weeks after the operation. Stressechocardiography revealed that G-CSF ameliorates theregional contractility of chronic myocardial ischemia.Morphological analysis revealed that the extent ofmyocardial fibrosis of the ischemic region was less in the G-CSF group than in control group. There were more vesselsand less apoptotic cells at the ischemic region of the heart ofthe G-CSF group than control group. Moreover, Akt1 wasmore strongly activated in the heart of the G-CSF group thancontrol group.

Conclusions. – These findings suggest that G-CSFimproves cardiac function of chronic myocardial ischemia

through decreases in fibrosis and apoptotic death, and anincrease in vascular density in the ischemic region.

Abstracts N° P-4-5Highly regular honeycomb-patterned biodegradable scaffold promotes topographical control of myoblast proliferation and differentiation: a novel scaffold for myocardial regenerative therapyAtsuhiro Saito a, Satoshi Taketani b, Keiko Arai c, Masaru Tanaka d, Masatsugu Shimomura e, Yoshiki Sawa ba Tissue Engineering and Cell Therapy group, Foundation for Biomedical Research and Innovatio. b Department of Cardiovascular Surgery, Osaka University Graduated School of Medicine. c Dissipative-Hierarchy Structures Laboratory, RIKEN Frontier Research System. d Creative Research Initiative “Sousei”, Hokkaido University. eResearch Institute for Electronic Science, Hokkaido University

Background. – Cell therapy for myocardial repair aftermyocardial infarction is a new and promising strategy.Skeletal myoblast transplantation has now entered theclinical stage. However, cell therapy generally needs toculture the cell for a long period in order to accrue asufficient number of cells. To solve this problem, it needs touse a suitable scaffold for cell attachment, proliferation,differentiation and several functions. In this study, wehypothesized that the optimum surface of the scaffold forskeletal myoblast may accelerate the cell attachment,proliferation, differentiation and morphological changes.We evaluated the effect of surface topography in the skeletalmyoblast behavior using the highly regular patternedbiodegradable films by simple casting technique.

Methods. – A chloroform solution of poly-ε-caprolactone(PCL) and an amphiphili colymer was used. Honeycomb-patterned PCL films were prepared by casting polymersolution under moist airflow. Unpatterned films werefabricated by the polymer solution under dry atmosphere.Myoblasts were isolated from 2-week-old male SD rats andseeded onto polymer substrates with several pore sizes. Themyoblast on each polymer substrate was observed bydesmin staining.

Results. – The myoblast proliferation was significantlyaccelerated on the laminin-coated honeycomb-patternedfilm with 6 µm pores as compared to the films with otherpore size and without laminin. On the stretched honeycomb-patterned films, the myoblast morphology was correctlyaligned to the stretch direction and not aligned on the normalfilms. Further, the length of myoblast on the stretched filmswas significantly longer and better differentiation than thaton the non-stretched films.

Conclusion. – The highly regular patternedbiodegradable films with controlled pore size and surfacetopography have a strong effect on the skeletal myoblastproliferation and differentiation. The honeycomb-patterned


films may be a novel candidate of the scaffold for celltherapy using skeletal myoblast.

Abstracts N° P-5-1Changes in Ca2+ handling predicted from mechanically unloaded O2 consumption of left ventricular wall slices of isoproterenol-induced hypertrophied rat heartsMiyako Takaki, Daisuke YamashitaDepartment of Physiology II, Nara Medical University School of Medicine

Aim. – The aim of the present study was to evaluate thecomposition of O2 consumption of left ventricular wallslices at mechanically unloaded conditions fromisoproterenol (Iso)-induced hypertrophied rat heartsassociated with increased collagen production.

Methods. – Osmotic mini-pump containing Iso wassubcutaneously implanted for 3 days to induce hypertrophy.O2 consumption per minute (mVO2) was measured withoutand with 1-Hz field stimulation by oximetric system. DeltamVO2 corresponds to O2 consumption for Ca2+ handling inexcitation–contraction (E–C) coupling.

Results. – Basal metabolic mVO2 was significantlysmaller than that of the normal heart slices. A sarcoplasmicreticulum (SR) Ca2+ pump inhibitor, cyclopiazonic acid(CPA; 30 µM) significantly decreased delta mVO2, but thedecrease in delta mVO2 was significantly smaller inhypertrophied hearts (43.6 ± 14.7% of first control) than innormal hearts (74.1 ± 18.3% of first control). Furthermore,the decrease in E–C coupling mVO2 by an NCX inhibitor,KB-R7943 was significantly larger in hypertrophied hearts(36.9% of first control) than in normal hearts (25.9% of firstcontrol).

Discussion. – Results indicated that O2 consumption bySR Ca2+ pump was largely decreased in hypertrophiedhearts. It might be predictable that the recirculation fraction(RF) of [Ca2+]i into SR was decreased to 60.7% [43.6 × 2/(43.6 × 2 + (100 – 43.6))] in hypertrophied hearts from85.2% [74.1 × 2/(74.1 × 2 + (100 – 74.1))] in normal hearts.The contribution of forward mode NCX for extrusion ofCa2+ (1-RF) would be more prominent in hypertrophiedhearts (100 – 60.7 = 39.3%) than in normal hearts (100 –85.2 = 14.8%).

Abstracts N° P-5-2Angiotensin II receptor blockade ameliorates myocardial remodeling and dysfunction in diabetes by inhibiting connective tissue growth factorShouji Matsushima a, Hidenori Matsusaka b, Masaki Ikeuchi b, Tomomi Ide b, Toru Kubota b, Kenji Sunagawa b,Hiroyuki Tsutsui aa Department of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan. bDepartment of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

Purpose. – Angiotensin II (AII) regulates extracellularmatrix remodeling by activating transforming growthfactor-β (TGF-β)/connective tissue growth factor (CTGF)and matrix metalloproteinases (MMPs). However, little isknown about the role of AII and its down stream mediatorsin cardiac remodeling in diabetes. We thus hypothesizedthat AII receptor antagonist (ARB) could attenuatediabetes-induced cardiac remodeling via these profibroticfactors.

Methods. – After injection of streptozotocin (200 mg/kg,ip), mice were randomized to ARB (candesartan 1 mg/kg/day, po; n = 7) or vehicle (n = 7) for 8 weeks.

Results. – ARB treatment ameliorated left ventricular(LV) diastolic dysfunction as assessed by the time constantof relaxation (tau; 10.9 ± 0.6 vs. 9.1 ± 0.6 ms, P < 0.05)without affecting LV systolic function in diabetic mice.ARB also decreased myocyte hypertrophy and interstitialfibrosis. These beneficial effects of ARB were associatedwith the decrease in myocardial CTGF protein levels, but notin TGF-β and MMP-2. To further determine the effects ofARB on the expression of CTGF in vitro, CTGF proteinlevels were assessed in cultured neonatal rat cardiacfibroblasts. AII directly increased CTGF protein levels,which was inhibited by AT1 receptor antagonist(candesartan 10–7 mol/l), but not AT2 receptor antagonist(PD123319 10–7 mol/l).

Conclusion. – AII receptors play an important role inmyocardial remodeling and dysfunction in diabetes, at leastin part, via activating the expression of CTGF.

Abstracts N° P-5-3Cardiac Na+/Ca2+ exchanger is highly phosphorylated and inhibited by PKCα in phenylephrin-treated hypertrophic cardiomyocytesYuki Katanosaka, Yuko Iwata, Munekazu Shigekawa, Shigeo WakabayashiNational Cardiovascular Center Research Institute

The Na+/Ca2+ exchanger (NCX) is the predominantCa2+ extrusion system in beating cardiomyocytes. Ourprevious work showed that phenylephrine-treatedhypertrophic cardiomyocytes exhibited the low NCX1activity that was markedly inhibited by calcineurin up-regulated in such cells. We now report the role of anothersignaling molecule protein kinase C (PKC) in function ofNCX1. We found that NCX1 is highly phosphorylated byPKC only when calcineurin is activated in cardiomyocytes.This phosphorylation was blocked with PKC inhibitorcalphostin C, but not with protein kinase A inhibitor H89.Expression experiment in CCL39 fibroblastic cellsrevealed that PKC-induced phosphorylation occurred atSer249, Ser250, and Ser357 of dog NCX1 but they were notdephosphorylated by calcineurin, indicating thatcalcineurin does not directly modulate the phosphorylationlevel of NCX1, but is required for PKC-inducedphosphorylation and depression of the exchange activity.


Knockdown experiment using siRNA revealed that PKCƒαisoform is involved in phosphorylation of NCX1. Our datasuggest that PKC-induced phosphorylation depresses theNCX activity in hypertrophic cardiomyocytes and that itmay play an important role in pathogenesis of heartmuscles in which both calcineurin and PKCƒα arechronically activated.

Abstracts N° P-5-4Voglibose is beneficial for heart failure in mice with LV pressure overloadYulin Liao a, Seiji Takashima a, Hui Zhao a, Yoshihiro Asano a, Yasunori Shintani a, Tetsuo Minamino a, Jiyoong Kim b, Masashi Fujita a, Masafumi Kitakaze b, Masatsugu Hori aa Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Japan. b Cardiovascular Division of Medicine, National Cardiovascular Center, Japan

Background. – Diabetes is a well-known risk factor forthe development of cardiovascular disease, especially forheart failure. Intriguingly, emerging evidence shows areciprocal relation exists between heart failure and glucoseabnormalities. Clinical investigations by others and usdemonstrated that the alpha-glycosidase inhibitors, acarboseand voglibose were beneficial in the treatment of heartfailure. Thus, we hypothesized that abnormal glucosemetabolism might be induced by cardiac remodeling andcontrol of blood glucose levels might delay or prevent thedevelopment of heart failure.

Methods and results. – This study was to determinewhether decreasing postprandial glucose levels byvogliboseis associated with prevention or amelioration ofheart failure in mice, and if so, try to explore the potentialmechanism. Transverse aortic constriction (TAC) wasemployed to C57BL/6 male mice to generate cardiachypertrophy and heart failure. Four weeks after TAC,plasma glucose levels were markedly decreased invoglibose-treated mice. The heart-to-body weight ratio andthe lung-to-body weight ratio were lower; and LV fractionalshortening was higher in the voglibose-treated groups thanin the TAC group. Invasive hemodynamic examinationshowed an improvement of dP/dTmax/LVSP in voglibosetreated mice. Western blot analysis demonstrated thatvoglibose treatment significantly decreased myocardialexpression of NAPDH oxidase subunit p47phox. In neonatalrat cardiac myocyte culture, high glucose culturesignificantly increased the protein synthesis of myocytes andexpression of protein p47phox. The NADPH oxidaseinhibitor apocynin abrogated the enhanced protein synthesisof myocytes induced by high glucose.

Conclusions. – These findings indicated that vogliboseattenuates cardiac remodeling mediated by a decrease ofmyocardial oxidant stress.

Abstracts N° P-5-5Left ventricular functions under dobutamine-stress in hypertrophic cardiomyopathy patients: an assessment with radionuclide ventriculographyCheng-Yi ChengDepartment of Nuclear Medicine, Tri-Service General Hospital, Taipei, Taiwan, Japan

Hypertrophic cardiomyopathy (HCM) is one of the non-coronary diseases that can deteriorate the left ventricular(LV) function. To study the left ventricular systolic function(ejection fraction, EF; peak ejection rate, PEF) and diastolicfunction (peak filling rate, PFR) in patients with HCM, restradionuclide ventriculography (RNV) and low-dosedobutamine stress (5, 10, 15, and 20 mg/kg per min) RNVwere performed for nine normal subjects (N) and 10 HCMpatients with normal rest LV function demonstrated byechocardiography. In normal subject group and HCM group,dobutamine stress could significantly increase the LVEF (N:59 + 7% to 78 + 8%, P < 0.05; HCM: 56 + 5% to 67 + 8%,P < 0.05) and LVPER (N: 3.3 + 0.6 end-diastolic volumes/sto 5.1 + 0.7 end-diastolic volumes/s, P < 0.05; HCM: 3.1 +0.6 end-diastolic volumes/s to 4.6 + 0.4 end-diastolicvolumes/s, P < 0.05). PFR in N revealed significantlyincreased, from 3.5 + 0.8 to 4.6 + 0.7 end-diastolic volumes/s, P < 0.05. In contrast to N, HCM demonstrated similar PFR(3.3 + 0.8 to 3.0 + 0.6 EDV/s, NS) during dobutamineinfusion. It is therefore concluded that HCM may deterioratethe LV diastolic function with preserved LV systolicfunction during low-dose dobutamine infusion. Our datamay suggest 1) Dobutamine-stress RNV is a reliable andsafe non-invasive method in the evaluation of leftventricular functional status in clinically asymptomaticHCM patients, and 2) the HCM patients may result indeteriorated diastolic function with preserved systolicfunction during increased myocardial oxygen demand.

Abstracts N° P-5-6Thyroid hormone induced cardiac hypertrophy in a phosphoinositide 3-kinase pathway dependent mannerRie Kosugi a, Tetsuo Shioi b, Kayo Maeda Watanabe a, Yoji Machida a, Tohru Izumi aa Kitasato University School of Medicine, Sagamihara, Japan. b Kyouto University School of Medicine, Kyouto, Japan

Background. – Thyroid hormone (T4) induces cardiachypertrophy. Activation of phosphoinositide 3-kinase(PI3K) is shown to be necessary and sufficient to promoteheart growth in mice. We assessed the hypothesis that PI3Kis involved in T4 induced cardiac hypertrophy.

Methods and results. – To examine if T4 activates theeffectors of PI3K, L-thyroxine (T4, 10 µg/g) or saline wasintraperitoneally administered to 10-week-old male mice.The mice were sacrificed 1, 4, 8, or 24 hours after theinjection. Akt, p70 ribosomal S6kinase1 (S6K1), ribosomalS6 Protein (S6) are downstream effectors of PI3K. The


amount of phosphorylated Akt, S6K1, or S6 in the hearttissue was analyzed by Western blotting. To examine ifPI3K is necessary for T4 induced cardiac hypertrophy, T4(10 µg/g per day) was daily administered to transgenic miceexpressing dominant-negative form of PI3K in the heart(dnPI3K mice) or non-transgenic mice (NTg mice) for14 days. The amount of phosphorylated Akt was increasedat 1 hour (1.6-fold vs. saline injected NTg), 4 hours (1.6-fold), and 8 hours (2.2-fold) after T4 injection. The S6K1phosphorylation increased at 4 hours (6.5-fold) and 8 hours(13.7-fold). The S6 phosphorylation increased at 8 hours(2.7-fold). Heart weight/body weight of T4 treated NTgmice was increase by 27% compared with the saline treatedNTg mice. In contrast, heart weight/body weight of T4treated dnPI3K mice was increased by 9% compared withsaline treated dnPI3K mice.

Conclusion. – Thyroid hormone induced cardiachypertrophy in a PI3K pathway dependent manner.

Abstracts N° P-6-1Diacylglycelol kinaseζ attenuated left ventricular remodeling and improved survival after myocardial infarctionTakeshi Niizeki, Yasuchika Takeishi, Takanori Arimoto, Takahashi Hiroki, Yo Koyama, Isao KubotaDepartment of Cardiology, Pulmonology, and Nephrology, Yamagata University School of Medicine, Yamagata, Japan

Background. – Left ventricular (LV) remodelingcontributes to cardiac dysfunction and mortality aftermyocardial infarction (MI). Although precise cellularsignaling mechanisms of LV remodeling are not fullyelucidated, Gq-coupled receptor signaling pathwayincluding diacylglycerol (DAG) and protein kinase C (PKC)are involved in this process. DAG kinase (DGK)phosphorylates DAG and controls cellular DAG levels, thusacting as a negative regulator of PKC. We previouslyreported DGK inhibited angiotensin II-induced activation ofthe DAG-PKC signaling and subsequent cardiachypertrophy. The purpose of this study was to examinewhether DGK modified LV remodeling after MI.

Methods and results. – Left anterior descending coronaryartery was ligated in transgenic (TG) mice with cardiac-specific overexpression of DGKζ and wild-type (WT) mice.We detected translocation of PKC-alpha and epsilon, andupregulation of fetal genes 4 weeks after MI in WT mousehearts. However in DGKζ-TG mouse hearts, neithertranslocation of PKC nor fetal gene induction was observed.LV remodeling, determined as the development of LVdilation and increases in LV weight, was attenuated inDGKζ-TG mice compared to WT mice (P < 0.05 and P <0.01, respectively). Furthermore, the increase of cross-sectional cardiomyocyte area and myocardial fibrosis in thenon-infarcted area in DGKζ-TG mice were restrainedcompared to WT mice (P < 0.01 and P < 0.01, respectively).

The survival rate after MI was higher in DGKζ-TG micethan in WT mice (P < 0.05).

Conclusion. – These results demonstrate the firstevidence that DGKζ suppresses LV remodeling andimproves survival after MI.

Abstracts N° P-6-2Gap junction remodeling is an important arrhythmogenic substrate during the development of heart failure in cardiomyopathic hamsterTakashi Sato a, Ohkusa Tomoko a, Haruo Honjo b, Shinsuke Suzuki a, Masafumi Yano a, Itsuo Kodama c, Masunori Matsuzaki aa Division of Cardiovascular Medicine, Department of Medical Bioregulation, Yamaguchi University Graduate School of Medicine, Japan. b Department of Hormonal Regulation, Research Institute of Environmental Medicine, Nagoya University, Japan. c Department of Circulation, Research Institute of Environmental Medicine, Nagoya University, Japan

In the clinical setting, lethal ventricular arrhythmias oftenoccur during the development of heart failure. Differences inthe distribution of gap junction are considered to be animportant factor in the origin of reentrant arrhythmias. Weinvestigated the alterations in connexin (Cx)43 during thedevelopment of heart failure in UM-X7.1 cardiomyopathichamster (CM) hearts. We analyzed Cx43 expressions(protein and mRNA) of the left ventricular (LV)myocardium by western blotting and RT-PCR.Echocardiographic and electrophysiological data wereobtained. In CM, LV hypertrophy had developed at 10 w,and LV global hypokinesis at 20 w. In CM of 20 w, randomreentry was easily induced by programmed pacing, andconduction velocity was remarkably decreased in CM. Theinterstitial fibrosis was significantly increased in CMcompared with G at 20 w. The relative expression level ofCx43 protein and mRNA were significantly lower in CM at20 w than in G (0.52 ± 0.29 vs. 1.20 ± 0.12, 0.58 ± 0.14 vs.0.87 ± 0.12, P < 0.05, respectively). The relative expressionlevel of serine 255-phosphorylated Cx43, which initiates thedown-regulation of gap junctional intercellularcommunication, was markedly increased in CM (1.1 ± 0.4)compared with G (0.4 ± 0.4, P < 0.01) at 20 w.

Conclusions. – In cardiomyopathic hamster hearts, inaddition to an increase of interstitial fibrosis, downregulationand abnormal serine-phosphorylation of Cx43 may result inabnormal cell-to-cell communication and alter theelectrophysiologic properties of the ventricle, leading to theinitiation and perpetuation of ventricular arrhythmias.

Abstracts N° P-6-3Defective inter-domain interaction within the ryanodine receptor plays a key role in the pathogenesis of heart failureHiroki Tateishi, Masafumi Yano, Mamoru Mochizuki, Masunori Matsuzaki


Division of Cardiovascular Medicine, Department of Medical Bioregulation, Yamaguchi University Graduate School of Medicine, Japan

Background. – Two domains within the ryanodinereceptor (RyR) of sarcoplasmic reticulum (SR) {N-terminal(0–600) and central (2000–2500) domains}, where manymutations have been found in patients with arrhythmogenicright ventricular cardiomyopathy (ARVC) and polymorphicVT, have been recently shown to interact with each other asa regulatory switch for channel gating. Here, we investigatedwhether the destabilization of RyR in heart failure (HF) isproduced by defective inter-domain interaction.

Methods and results. – RyR was fluorescently labeledwith methylcoumarin acetate (MCA) using DP176, asynthetic peptide corresponding to His163-Ser195 of RyR(contains one ARVC mutation), as a site-directing carrier.The DP176 mediated a specific MCA labeling of the RyR.Addition of DP176 induced an unziping of the interactingdomains, which was confirmed by measuring theaccessibility of the fluorescence quencher. The DP176-induced domain unzipping resulted in Ca2+ leak throughRyR. In the SR from failing dogs, the domain unzipping hasalready taken place together with Ca2+ leak. Abenzochiazepine derivative JTV519 (domain stabilizer)normalized DP176-induced domain unzipping and Ca2+

leak in normal SR, and restored the normal domain zipping,and the spontaneous Ca2+ leak in failing SR. In the DP176-introduced myocytes both cell shortening and Ca2+ transientwere deteriorated, but normally restored with JTV519.

Conclusions. – Defectiveness of the inter-domaininteraction within RyR plays a key role in the pathogenesisof HF and thus correction of the defective inter-domaininteraction may be a new therapeutic strategy against HF.

Abstracts N° P-6-4Alteration in cardioprotective signaling provoked by erythropoietin in post-infarct remodeled myocardiumTakayuki Miki, Tetsuji Miura, Jun Sakamoto, Hironori Kobayashi, Masahiro Nishihara, Katsuhiko Ohori, Akari Takahashi, Kazuaki ShimamotoSecond Department of Internal Medicine, Sapporo Medical University School of Medicine, Japan

Objective. – We examined whether myocardial responseto activation of the erythropoietin (EPO) receptor ismodified by post-infarct remodeling.

Methods and results. – Four weeks after a shamoperation (Sham) or coronary artery ligation (CL), isolatedrat hearts were subjected to 25-min global ischemia/2-hreperfusion. Infarct size was expressed as % of the leftventricle (%I/LV), from which scarred infarct by CL wasexcluded. The heart weight was 25% larger in CL, but therewas no inter-group difference in plasma EPO levels (14.4 ±0.5 vs. 14.5 ± 1.1 mU/ml) or EPO receptor protein levels inthe myocardium. EPO infusion (5 U/ml) before ischemiasignificantly reduced %I/LV from 57.8 ± 4.1 to 36.2 ± 4.2 in

Sham and from 58.1 ± 5.0 to 35.2 ± 4.0 in CL. This EPO-induced protection in Sham was abolished by LY-294002(5 µM), a PI3K inhibitor, but both LY-294002 andwortmannin (100 nM) failed to abolish EPO-inducedprotection in CL. A guanylyl cyclase inhibitor, ODQ(2 µÊM), and a blocker of the mitoKATP channel, 5-HD(100 µM), inhibited EPO-induced protection in both Shamand CL. EPO infusion significantly increased phospho-Aktlevel by 45% in Sham but not in CL. Suppressor of cytokinesignaling (SOCS)-1 protein level was higher by 50% in CLthan in Sham, though SOCS-3 levels were similar.

Conclusion. – Post-infarct remodeling disrupts cellularsignaling from the EPO receptor to PI3K presumably byincreasing SOCS-1 protein. However, in the remodeledmyocardium, lack of PI3K/Akt activation by the EPOreceptor is compensated by a mechanism upstream of theguanylyl cyclase/mitoKATP channel pathway to achieveEPO-induced protection.

Abstracts N° P-6-5Overexpression of glutathione peroxidase attenuates cardiac remodeling and diastolic dysfunction in diabetic miceShouji Matsushima a, Tomomi Ide b, Hidenori Matsusaka b,Masaki Ikeuchi b, Toru Kubota b, Kenji Sunagawa b,Shintaro Kinugawa a, Hiroyuki Tsutsui aa Department of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan. bDepartment of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

Purpose. – Oxidative stress plays an important role in thestructural and functional abnormalities of diabetic heart. Wedetermined whether the overexpression of glutathioneperoxidase (GSHPx) gene could attenuate left ventricular(LV) remodeling and dysfunction in diabetes mellitus (DM).

Methods. – We induced DM by injection ofstreptozotocin (160 mg/kg, ip) in male GSHPx transgenicmice (TG + DM) and non-transgenic wild-type littermates(WT + DM).

Results. – GSHPx activity was higher in the hearts of TGmice compared to WT mice (68 ± 5 vs. 45 ± 3 nmol/min mgprotein, P < 0.05), with no significant changes in otherantioxidant enzymes. LV thiobarbituric acid-reactivesubstances measured in TG + DM at 8 weeks weresignificantly lower than those in WT + DM (58 ± 3 vs. 71 ±5 nmol/g, P < 0.05). Systolic function was preserved normalin both WT + DM and TG + DM mice. In contrast, diastolicfunction was impaired in WT + DM and was improved inTG + DM as assessed by the time needed for relaxation of50% maximal LV pressure to baseline value (tau; 13.5 ± 1.2vs. 8.9 ± 0.7 ms, P < 0.01). Improvement of LV diastolicfunction was accompanied by a decrease in interstitialfibrosis and myocyte hypertrophy. Myocardial matrixmetalloproteinase-2 zymographic activity and TGF-β


expression was increased in WT + DM but was attenuated inTG + DM.

Conclusion. – Overexpression of GSHPx gene inhibitedLV remodeling and diastolic dysfunction in DM. Therapiesdesigned to interfere with oxidative stress might bebeneficial to prevent diabetic cardiac abnormalities.

Abstracts N° P-7-1Senescence-like changes in cardiac myocytes by oxidative stress is novel mechanism of cardiac dysfunctionYasuhiro Maejima a, Susumu Adachi a, Hiroshi Ito b,Mitsuaki Isobe aa Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Japan. b Second Department of Internal Medicine, Akita University, Japan

Senescence is an important phenomenon in cellphysiology. Senescence-like phenotypes can be induced byoxidative stress in proliferating cells. In the present study,we examined whether senescence-like change also occurs incardiac myocytes by oxidative stress and plays as one ofmajor mechanisms of myocardial dysfunction. Cardiacmyocytes taken from aged rats showed that the positivestaining ratio of senescence-associated β-galactosidase(SAβ-gal) significantly increased, and the protein level ofcyclin-dependent kinase inhibitors (cdk-I) accumulatedcompared with those of young rats. Deterioration oftroponin-I phosphorylation and decrease of telomeraseactivity were seen in the aged cardiac myocytes. Treatmentwith low-dose doxorubicin (DOX, 10–7 mol/l) in culturedneonatal rat ventricular myocytes (NRVM) did not induceapoptosis, but caused oxidative stress which was confirmedby 2′,7′-dichlorofluorescin diacetate staining. In the DOX-treated NRVM, the positive staining ratio of SAβ-gal, cdk-Iexpression, deterioration of troponin-I phosphorylation anddecrease of telomerase activity were observed as similarchanges as aged cardiac myocytes. The changes of mRNAexpressions in the aged cells were seen in the DOX-treatedNRVM (downregulation: ƒα-MHC, GATA4, Nkx2.5,upregulation: ANP, Angiotensin II). Thus, low-dose DOX-treated NRVM indicated characteristic changes that weresimilar to that of aged cardiac myocytes and suggested thatoxidative stress induces senescence-like alterations inNRVM. These findings may indicate novel mechanism ofcardiac dysfunction induced by oxidative stress.

Abstracts N° P-7-2G 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 MAPK by angiotensin receptor stimulation in rat neonatal cardiomyocytesHitoshi KuroseKyushu University

We examined signal transduction mechanism of reactiveoxygen species (ROS) production and the role of ROS in

angiotensin II-induced activation of mitogen activatedprotein kinases (MAPKs) in rat neonatal cardiomyocytes.ROS production was necessary for activation of c-Jun NH2-terminal kinase (JNK) and p38 MAPK, as an NADPHoxidase inhibitor, diphenyleneiodonium, inhibited theactivation. The angiotensin II-induced activation of JNK andp38 MAPK was also inhibited by the expression of Gα12/13-specific regulator of G protein signaling (RGS) domain, aspecific inhibitor of Gα12/13, but not by a RGS domainspecific for Gαq. Angiotensin II receptor stimulation rapidlyactivated Gα13, which was completely inhibited by Gα12/13-specific RGS domain. Furthermore, Gα12/13- but not Gαq-specific RGS domain inhibited angiotensin II-induced ROSproduction. Dominant negative Rac inhibited JNK and p38MAPK activation, but did not affect ERK activation.Dominant negative Rac also inhibited angiotensin II-stimulated ROS production. Rac activation was mediated byRho and Rho-kinase, as Rac activation was inhibited by C3toxin and a Rho-associated kinase inhibitor Y27632. Theseresults suggest that Gα12/13-mediated ROS productionthrough Rho and Rho-kinase, and Rac is essential for JNKand p38 MAPK activation.

Abstracts N° P-7-3NHE inhibitor cariporide attenuates mitochondrial death pathwayHideyuki Ishida a, Takako Toda b, Toshie Kadono b, Minako Hoshiai b, Yu Eguchi a, Shinpei Nakazawa b, Hiroe Nakazawa aa Department of Physiology, Tokai University School of Medicine, Japan. b Department of Pediatrics, Yamanashi University School of Medicine, Japan

Recent reports suggest that NHE inhibitor cariporideinteracts with mitochondrial death pathway. However, effectsof NHE inhibitor on mitochondrial death pathway remainelusive. We therefore examined whether cariporideattenuates the [Ca2+]m overload and inhibits themitochondrial permeability transition pore (PTP) opening.With the use of a Nipkow confocal system, the mitochondrialCa2+ concentration ([Ca2+]m) and the mitochondrialmembrane potential (ψm) in rat ventricular myocytes weremeasured by loading cells with rhod-2 and JC-1 respectively.The exposing cells to ouabain (1 mM) evoked [Ca2+]moverload and increased the intensity of rhod-2 fluorescence to149 ± 6% of baseline (P < 0.001). Co-administration ofcariporide (1 mM) or diazoxide (0.1 mM), significantlyattenuated the ouabain-induced [Ca2+]m overload (120 ± 5%and 113 ± 5% of baseline; P < 0.05 vs. ouabain). Althoughdiazoxide significantly reduced the intensity of JC-1fluorescence (P < 0.05 vs. ouabain), cariporide did not affectthe JC-1 fluorescence. These results indicate that cariporideattenuates the [Ca2+]m overload without accompanyingdepolarization of ψm. Cariporide and 0.4 mM cyclosporineA(CsA), a inhibitor of PTP opening, attenuated phenylarsineoxide (PAO), a potent opener of PTP, induced hyper-


contracture (from 59 ± 3% to 50 ± 4% and to 50 ± 4%,P < 0.05 vs. PAO). In contrast, diazoxide could not inhibitthe PAO-induced hypercontracture. These results indicatedthat cariporide attenuates [Ca2+]m overload and therebypreventing the following PTP opening. Moreover, cariporidecan directly affect the PTP. Such effects might potentially beattributed to the mechanisms of cardioprotection afforded byNEH inhibitor.

Abstracts N° P-7-4A novel protective factor against ischemia/reoxygenation, adenylate kinase 2, suppresses hypoxia/reoxygenation-induced cell deathEmi Maeno, Kaori Matsushita, Asuka Shiga, Yoshiki SawaDepartment of Surgery, Osaka University Graduate School of Medicine, Japan

Even the current myocardial protections for infarctmyocardium have still limitation because of simultaneouslyexecuted multiple acts of death signal transduction. For thepurpose of search for the novel resistant genes againstischemia/reperfusion (I/R) injury, we have performed cDNAlibrary screening and analysis of isolated genes. Morerecently, we found adenylate kinase 2 (AK2), and it ispresent in the mitochondrial intermembrane space, inapoptotic cells only is translocated into the cytosolconcomitantly with cytochrome c. We hypothesized thatAK2 may play a pivotal role in suppression of H/R inducedcell death. Cultured cardiomyocytes were exposed tohypoxic condition using anaerobic culture incubator (> 1%O2) in glucose- and serum-free medium just like starvationand anoxia in infarct area in vivo, followed by reoxygenationin glucose- and serum-containing medium in 20% O2.Almost all dying cardiomyocytes indicated involvement ofnecrosis in vitro model of H/R. We selected about 50% cellviable condition for screening of resistant gene against H/R.We isolated some genes from survival transfectants keepingmouse embryo and human fetal heart cDNA libraries.Because we reported that acute myocardial infarction in vivoincreased fetal isoforms and decrease adult isoforms. Weanalyzed cell viability after H/R stress of transfectantsoverexpressed selected genes in rat neonatal cardiomyocytesand human embryo kidney (HEK) 293 cells. Cell viabilityafter H/R stress in AK2 transfectants was more than that inheat shock protein (HSP) 70 transfectants known H/Rresistant gene. Overexpression of AK2 isolated fromsurvival cells treated with H/R stress led to suppress celldeath by H/R stress in two cell types. These data suggest thatAK2 is effective in gene therapy of H/R injured tissue.

Abstracts N° P-7-5Early apoptotic event promotes dedifferentiation in cardiac myocyte: a speculated mechanism of autologous preservationEmi Maeno a, Satoru Kitagawa-Sakakida b, Asuka Shiga a,Yoshiki Sawa a

a Department of Surgery, Osaka University Graduate School of Medicine, Japan. b Department of Molecular Pharmacology, Osaka University Graduate School of Medicine, Japan

Adult cardiac myocytes show dedifferentiationcharacterized by morphological appearance, cellproliferation and fetal gene expression in culture eventhough cardiac myocytes are terminally differentiated cellsand lose fetal characters. On the other hand, cardiac myocyteapoptosis occurs in the infarct zones, where fetal geneexpression can also be observed. We therefore hypothesizedthat cardiac myocytes exposed to apoptotic stimuli mayshow dedifferentiated and induce myocardial preservation.The first, we made a comparison of gene expression betweeninfarct zones and dedifferentiated cardiac myocytes. In vitrostudy, terminally differentiated rod-shaped cardiacmyocytes derived from adult rat heart became rounded andproliferated in culture. In vivo study, we examined somecharacters at early time (2–24 h) after ligation of proximalleft anterior descending coronary artery of adult rat hearts.Analysis of α-myosine heavy chain (MHC), β-MHC,GATA4, Nkx2.5, desmin, smooth muscle α-actin, orsarcomeric α actinin expression by quantitative RT-PCRand immunohistochemical staining showed that de-differentiated cardiac myocytes resembled infarct tissue intheir expression patterns. The second, apoptotic volumedecrease (AVD) is known to early apoptotic event, and itsinhibitor (DIDS) was added to the isolation buffer andculture medium. Cardiac myocytes isolated by enzymaticdigestion of minced ventricular tissue almost were toundergo apoptotic cell death or dedifferentiated. DIDS wascytoprotective during the isolation and preventeddedifferentiation during cultivation of cardiac myocytes, asjudged by the morphological appearance (lack of blebformation and cytoskeleton defects) and the fetal/adult geneexpression. Adult cardiac myocyte showed dedifferentiationin culture, and expressed marker genes as same as infarctmyocardium. Inhibit of early apoptotic events prevented celldeath and dedifferentiation. These data suggest that cardiacmyocytes exposed to apoptotic stimuli is dedifferentiatedand induce autologous preservation mechanism.

Abstracts N° P-7-6Dual role of myocardin on hypertrophy and apoptosis in cardiac myocytesSeimi Satomi-Kobayashi, Tomomi Ueyama, Ryuji Toh, Tomoya Masano, Mitsuhiro Yokoyama, Seinosuke KawashimaDivision of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan

Background. – Most cases of heart failure are precededby cardiac hypertrophy and, therefore, it has been suggestedthat hypertrophy makes cardiac myocytes more sensitive toapoptosis. Myocardin is a transcriptional cofactor for serum


response factor, and its expression begins in the cardiaccrescent and continues through adulthood. However, itsfunction in the postnatal heart is not determined.

Methods and results. – First, we examined mRNAexpression of myocardin in mice hearts. In hypertrophiedhearts induced by aortic banding, myocardin was markedlydecreased compared to sham hearts, whereas it did notchange in swimming-induced hypertrophied hearts. Basedon these findings, we examined whether myocardin wasinvolved in the pathogenesis of hypertrophy and apoptosis.We used recombinant adenoviruses expressing wild-type ofmyocardin (wtMC) and the dominant negative mutant ofmyocardin (dnMC). In rat cultured cardiac myocytes,wtMC induced hypertrophic responses, whereas dnMCinhibited α-adrenergic agonist phenylephrine (PE)-inducedhypertrophic responses. TUNEL staining analysis showedthat PE stimulation with dnMC markedly increased theexpression of apoptotic cells (26.3 ± 2.2%, P < 0.01),although PE stimulation alone had no effects (15.9 ± 2.1%vs. 17.8 ± 3.3% control). PE stimulation with wtMCdecreased apoptotic cells (13.0 ± 2.1%). Regarding themechanism of anti-apoptotic action of myocardin, wtMCincreased Bcl-2 expression via modifying its transcriptionalactivity, and Bcl-2 inhibitor blocked the anti-apoptoticeffect of wtMC.

Conclusion. – Myocardin induces hypertrophic response,and also has an anti-apoptotic effect on cardiac myocytes byenhancing Bcl-2 expression. Myocardin may play animportant role in the mechanism of heart failure bymodulating both hypertrophy and death of cardiacmyocytes.

Abstracts N° P-8-1Mechanisms for ischemic-reperfusion injury related to reverse-mode NCX and calpainYoshiro Yoshikawa a, Hiroji Hagiwara a, Yoshimi Ohga b,Chikako Takenaka Nakajima b, Tsuyoshi Tsuji a, Yamato Tamura a, Shigeki Taniguchi a, Miyako Takaki ba Department of Thoracic and Cardiovascular Surgery, Nara Medical University School of Medicine, Japan. bDepartment of Physiology II, Nara Medical University School of Medicine, Japan

We hypothesized that left ventricular (LV) contractilefailure similar to that after high Ca2+ infusion occurs afterischemic-reperfusion. To test this hypothesis, weinvestigated LV mechanical work and energetics in thecross-circulated rat hearts that underwent 15-min globalischemia and 60-min reperfusion (I/R). Mean systolicpressure–volume area (PVA; total mechanical energy perbeat) at midrange LV volume (PVAmLVV) wassignificantly decreased. Mean VO2 (myocardial oxygenconsumption per beat) intercept (mainly VO2 for the totalCa2+ handling in E–C coupling) of VO2–PVA linearrelation was significantly decreased without change in itsslope. After reperfusion with a Na+/Ca2+ exchanger (NCX)

inhibitor, KB-R7943 (KBR, 10 µM) or calpain inhibitor-1(CI, 10 µM), each decrease in mean PVAmLVV and VO2intercept was reduced. Mean VO2 for the total Ca2+

handling was significantly higher than control between 10and 15 min after I/R. This energy-wasting process wascompletely blocked by KBR, but not blocked by CI. Therewere no significant differences in O2 costs of LVcontractility among normal, post I/R and post I/R withKBR or with CI hearts. KBR and CI almost completelyprotected proteolysis of α-fodrin. This results revealed thatan NCX inhibitor, KBR and CI antagonized the I/R injuryby inhibiting the impairment of the total Ca2+ handling inE–C coupling due to the proteolysis of α-fodrin caused byactivation of reverse-mode NCX or calpain.

Abstracts N° P-8-2Michigrinid, a novel oral hypoglycemic agent, does not abolish the cardioprotective effect of ischemic preconditioning on ischemia/reperfusion injuryKazuhiko Ogawa, Ikuo Taniguchi, Hisashi Takatsuka, Chikara Mori, Hideki Sasaki, Mitsuyuki Shimizu, Satoshi Takeda, Seibu MochizukiDivision of Cardiology, Internal Medicine, The Jikei University School of Medicine

Background and purpose. – Glibenclamide (Glib), non-selective SU receptor (SUR) blocker of sulfonylurea,abolishes the cardioprotective effect of ischemicpreconditioning (IP), presumably by inhibiting the openingof mitochondrial KATP channel in myocytes. Mitiglinide(Miti), a novel oral hypoglycemic agent, selectively blocksSUR1, which is thought to have no influence on theprotective action of IP. To clarify whether Michi attenuatesthe cardioprotective effect of IP, we evaluated reperfusionventricular tachyarrhythmias (rVT) in isolated perfused ratheart.

Methods. – Isolated rat hearts were conducted to perfusein working heart system, and subjected to 10 min of diastolicischemia followed by 20 min of reperfusion. Hearts wereassigned to one of the following groups after 5 min of initialaerobic perfusion: (1) nonIP performed (IP–); (2) IPperformed (IP+); (3) IP+ with Glib (2× 10–7 mol/l); (4) IP+with Miti (10–6 mol/l). IP protocol is 3 cycles of 2 min ofdiastolic ischemia followed by 5 min of reperfusion beforelong (10 min) ischemia.

Results. – IP+ had shorter duration of rVT than IP– (6.7± 5.2 min vs. 15.5 ± 4.4 min; P < 0.05). This protectiveaction of IP was not abolished by Michi [6.7 ± 5.2 min (IP+)vs. 3.9 ± 2.3 min (IP+ with Michi)]. However, Glibabolished the inhibiting effect of rVT (6.7 ± 5.2 min (IP+)vs. 14.0 ± 2.9 min (IP+ with Glib); P < 0.05).

Conclusions. – Glib attenuated the cardioprotectiveeffect of IP, however Miti preserve the beneficial effects ofIP, suggesting that Miti did not block SUR2 and inhibit theopening of mitochondrial KATP channel in myocyte.


Abstracts N° P-8-3Myocardial susceptibility to ischemia/reperfusion injury in spontaneously type 2 diabetic rats: comparison with streptozotocin induced diabetic ratsChikara Mori, Ikuo Taniguchi, Ogawa Kazuhiko, Takatsuka Hisashi, Sasaki Hideki, Shimizu Mitsuyuki, Seibu MochizukiDivision of Cardiology, Internal Medicine, The Jikei University School of Medicine

Background and purpose. – Although diabetics havesignificantly greater incidence and severity of ischemic heartdisease compared to non-diabetic population, experimentalresults have reported both increased and decreasedsusceptibility to ischemic injury under differentexperimental conditions. We investigated the myocardialsusceptibility to ischemia/reperfusion injury in type 1 andtype 2 diabetic model rats.

Method. – To clarify the differences between type 1 andtype 2 diabetes in their myocardial susceptibility toischemia/reperfusion injury, we evaluated reperfusionventricular tachyarrhythmias (R-VT) using isolated heartperfusion system. We used drug (streptozotocin) induceddiabetic rats as models for type 1 diabetes and OLETF ratsas models for spontaneous type 2 diabetes. After 20 min ofinitial aerobic perfusion, rats were subjected to 10 min ofischemia followed by 20 min of reperfusion using theworking heart method. The duration of R-VT was recordedas a marker of susceptibility to ischemia/reperfusion injury.

Results. – Type 1 diabetic rats had a significantly shorterR-VT duration than control rats (6.7 ± 5.2 min vs. 15.5 ±4.4 min, P < 0.05). There was no significant difference,however, in the R-VT duration between type 2 diabetic ratsand control rats (16.5 ± 8.1 min vs. 17.7 ± 2.0 min).

Conclusion. – These results show that type 1 diabetic ratshave less myocardial susceptibility to ischemia/reperfusioninjury than control rats, and that type 2 diabetic rats havesusceptibility similar to control rats. This suggests that type1 diabetes is less vulnerable to ischemia/reperfusion injurythan type 2 diabetes.

Abstracts N° P-8-4A selective inhibitor of Na+/Ca2+exchanger, SEA0400, preserves cardiac function and high-energy phosphates against ischemia/reperfusion injuryChun Feng Niu, Hiroshi Satoh, Tsuyoshi Urushida, Hideki Katoh, Hideharu HayashiInternal Medicine III, Hamamatsu University School of Medicine, Japan

The Ca2+ overload by Ca2+ influx via Na+/Ca2+

exchanger (NCX) is one of the major mechanisms inmyocardial ischemia/reperfusion (I/R) injury. Weinvestigated the protective effects of a novel selectiveinhibitor of NCX, SEA0400, on cardiac function and energymetabolism during I/R. Langendorff-perfused rat heartswere exposed to 35 min in global ischemia and 40 min

reperfusion. Using 31P nuclear magnetic resonancespectroscopy, cardiac phosphocreatine (PCr), ATP levelsand pHi were monitored. (1) SEA0400 at 0.1 and 1 µM didnot change the basic cardiac function. (2) SEA0400 at0.1 µM and 1 µM throughout I/R improved the recovery ofleft ventricular developed pressure (LVDP) after reperfusion(27.6 ± 4.9 mmHg in control, 101.2 ± 19.3 mmHg in 0.1 µMand 115.5 ± 13.3 mmHg in at 1 µM SEA0400, means ± S.E.,n = 6, P < 0.05). SEA0400 also reduced LV end-diastolicpressure and increased coronary flow after reperfusion. (3)SEA0400 improved the recoveries of PCr and ATP afterreperfusion, but did not alter pHi during I/R. (4) There weresignificant linear correlations between LVDP and PCr (r =0.73, P < 0.05), and LVDP and ATP (r = 0.75, P < 0.05). (5)SEA0400 did not reduce but rather increased both theincidence and the duration of reperfusion ventriculararrhythmias. (6) SEA0400 at 1 µM only during reperfusionalso improved significantly both the contractile function andenergy metabolism. In conclusion, the selective inhibition ofNCX may be effective to preserve high-energy phosphatesand to improve cardiac function after reperfusion, but maynot be able to prevent fatal arrhythmias. The preservation ofhigh-energy phosphates contributes, at least partly, to theSEA0400-induced improvement of contractile function afterreperfusion.

Abstracts N° P-8-5Cardioprotection without cardiosuppression against ischemia-reperfusion injury by PAK-200, a dihydropyridine analogue with inhibitory effect on Cl–but not Ca2+

Iyuki Namekata, Miku Tamura, Jyunya Kase, Hikaru Tanaka, Koki ShigenobuDepartment of Pharmacology, Toho University School of Pharmaceutical Sciences, Japan

We examined the effects of a dihydropyridine analogue,PAK-200, on guinea pig myocardium during no-flowischemia and reperfusion. PAK-200 (1 µM) had no effectson contractile force, heart rate and action potential undernormal condition. In isolated ventricular cardiomyocytes,PAK-200 had no effects on basal peak inward and steadystate currents, but inhibited the isoprenaline-induced timeindependent Cl– current. In an ischemia-reperfusion modelwith coronary-perfused right ventricular tissue, contractileforce was decreased during 30 min no-flow ischemia, whilethe resting tension was increased. Upon reperfusion,transient arrhythmias were observed and contractile forcereturned to less than 50% of preischemic values. PAK-200had no effect on the decline in contractile force during theno-flow ischemia, but reduced the rise in resting tension.PAK-200 significantly improved the recovery of contractileforce after reperfusion to about 80% of the preischemicvalue. PAK-200 was also shown to attenuate the decrease intissue ATP and the depolarization of mitochondrialmembrane potential during ischemia. Thus, PAK-200


inhibits Cl- channels in cardiac muscle and may haveprotective effects against ischemia-reperfusion injurythrough novel mechanisms.

Abstracts N° P-8-6Adrenomedullin potentiates the opening of mitochondrial Ca2+-activated K+ channels and confers cardioprotection in rabbit heartsHirofumi Nishida, Toshiaki Sato, Haruaki NakayaDepartment of Pharmacology, Chiba University Graduate School of Medicine

Mitochondrial Ca2+-activated K+ (mitoKCa) channels areactivated by protein kinase A (PKA) and protect themyocardium against ischemic injury. Adrenomedullin(ADM) increases cyclic AMP levels in cardiac myocytesand reduces myocardial infarct size. To look for possiblemechanistic links between mitoKCa channel and ADM, wemeasured mitochondrial flavoprotein fluorescence in rabbitventricular myocytes and infarct size after no-flow ischemia(30 min) and reperfusion (120 min) in Langendorff-perfusedrabbit hearts. The mitoKCa channel opener NS1619 (30 µM)reversibly oxidized flavoprotein, an index of mitoKCachannel activity, to 21 ± 3% of the maximum value inducedby 2,4-dinitrophenol. This effect of NS1619 was completelyantagonized by the mitoKCa channel blocker paxilline(2 µM). ADM (10 nM) alone had no effects on flavoproteinfluorescence (4 ± 3%), but ADM significantly increased theNS1619-induced flavoprotein oxidation to 41 ± 5% (P <0.05 vs. NS1619 alone). KT5720 (200 nM), a potent PKAinhibitor, prevented the effect of ADM (31 ± 7%, P = NS vs.NS1619 alone). 8-Bromo-cAMP (0.5 mM) mimicked theeffect of ADM and significantly increased the NS1619-induced flavoprotein oxidation to 43 ± 2% (P < 0.05 vs.NS1619 alone). Treatment with ADM (10 nM) for 10 minbefore ischemia significantly reduced infarct size from 57 ±7% in controls to 33 ± 6% (P < 0.05). The infarct size-limiting effect of ADM was attenuated by paxilline (59 ±2%, P < 0.05 vs. ADM). These results indicate that ADMpotentiates the opening of mitoKCa channels in a PKA-dependent manner and further suggest that infarct size-limiting effect of ADM is mediated by activation of mitoKCachannel.

Abstracts N° P-9-1Hypoxia by high concentration of Mg perfusate protects hearts from hypoxia-reoxygenation injuryShanshuang Li, Jinrong Wu, Jun Takeda, Mariko Nakatani, Makino Watanabe, Takao OkadaDepartment of Physiology, Juntendo University School of Medicine, Tokyo, Japan

Objective. – We have reported that high Mg appliedthrough out the period of hypoxia can protect rat hearts fromhypoxia/reoxygenation injury. The purpose of the present

study was to examine when and how long high Mg appliedduring hypoxia is effective.

Methods. – Langendorff-perfused rat hearts weresubjected to 1) control (CT) group: 30 min hypoxia and30 min reoxygenation. 2) Hypoxic perfusate containing12 mM Mg was given at the first 5 min, 3) 10 min or 4)15 min and then perfused with normal hypoxic solution fortotal 30 min hypoxia and then 30 min reoxygenation. 5)Hypoxic solution containing 12 mM Mg was given at thesecond 5 min or 6) the last 15 min during hypoxia and theothers same as 2 group.

Results. – Recoveries of pressure-rate product (PRP) in 2,3, 4 groups were not different from those perfused with highMg throughout hypoxia and were all higher than those of CTat the end of 30 min reoxygenation. Recoveries of PRP in 5,6 groups were similar as those of in 2, 3, 4 groups at the first5–10 min of reoxygenation period, but the recovery wassuppressed and there was no difference from CT at the endof 30 min reoxygenation.

Conclusions. – High concentration of Mg applied at thebeginning of hypoxia was important and necessary forprotecting rat hearts from hypoxia/reoxygenation-inducedinjury.

Abstracts N° P-9-2The functional and histological changes of mitochondria by cardiac hypoxia-reoxygenation injury and protective effect of ischemic post-conditioningJinrong Wu, Makino Watanabe, Shanshuang Li, Chunzi Li, Takao OkadaDepartment of Physiology, Juntendo University School of Medicine, Japan

Ischemic post-conditioning shows the protective effecton the cardiac ischemic-reperfusion injury. The changes ofmitochondrial function and morphology by cardiac hypoxia-reoxygenation injury and protective effect of ischemic post-conditioning were examined. Male SD rat hearts wereexcised and perfused with Langendorff manner. Post-conditioning was 3 cycles of 10 sec ischemia and 10 secreperfusion before reoxygenation. The Left ventriculardeveloped pressure, heart rate and coronary perfusionpressure were monitored throughout experiments.Mitochondria were isolated after perfusion and itscytochrome C content was quantitated. Moreover,mitochondrial ultrastructure and parameters of morphologywere observed by using the electron microscope. In post-conditioning group, the recovery of pressure rate product(PRP) by reoxygenation was 61.58 ± 3.93% and significantlyhigher than that of control group (47.26 ± 3.62%). Thecytochrome C content of mitochondria was decreased byreoxygenation, but the extent of decrease was significantlyinhibited by post-conditioning. Mitochondria were swollenby hypoxia and incompletely recovered by reoxygenation.The recovery of mitochondria was improved by post-conditioning. These results suggest that ischemic post-


conditioning protects mitochondria from cardiac hypoxia-reoxygenation injury, which facilitate the functionalrecovery of the hearts.

Abstracts N° P-9-3Direct protective effects of G-CSF on myocardium during ischemia-reperfusion injuryKazutaka Ueda, Hiroyuki Takano, Hiroshi Hasegawa, Yuriko Niitsuma, Yingjie Qin, Masashi Ohtsuka, Issei KomuroDepartment of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine

Background. – G-CSF (granulocyte colony-stimulatingfactor) has been recently reported to prevent cardiacremodeling and dysfunction in acute myocardial infarctionand myocardial ischemia-reperfusion (IR) models. In thisstudy, we examined whether G-CSF has direct protectiveeffects on the myocardium exposed to IR injury.

Methods. – Rat hearts were perfused according to theLangendorff method and subjected to a global 35 minischemia and 120 min reperfusion with or without G-CSF(300 g/ml, each group; n = 5). G-CSF administration wasstarted at the onset of reperfusion. Infarct size was assessedby triphenyltetrazolium chloride (TTC) staining. Toexamine G-CSF-induced signaling pathways, perfused rathearts were subjected to 35 min ischemia and 7 minreperfusion (n = 5, per group). To identify the role ofsignaling pathways that are activated by G-CSF, hearts werepreperfused with inhibitors of PI3K (LY294002, 5 µM),Jak2 (AG490, 5 µM) or ERK (PD98059, 10 µM).

Results. – G-CSF significantly reduced the infarct sizemeasured by TTC staining (G-CSF group; 32 ± 13%, controlgroup; 58 ± 12%, P < 0.01). G-CSF significantly increasedthe phosphorylation of Akt, Jak2, STAT3 and ERK in thehearts subjected to ischemia followed by 7 min reperfusion.The reduction of infarct size afforded by G-CSFadministration was abolished in the presence of LY294002or AG490, but not PD98059. LY294002 or AG490 did notinfluence the infarct size in control group. In vitro study, G-CSF directly induced activation of Akt, Jak2 and STAT3 inrat cardiac myocytes.

Conclusions. – These results suggest that G-CSF actsdirectly on the myocardium during IR injury and hascardioprotective effects even if G-CSF administration isstarted after reperfusion. G-CSF-induced activation of Aktand Jak2-STAT3 may be important for its cardioprotectiveeffects.

Abstracts N° P-9-4Protein kinase C (PKC) activation reduces sarcoplasmic reticulum calcium content and induces cardioprotectionKen Yamamura a, Charles Steenbergen b, Elizabeth Murphy aa Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, NIH. b Department of Pathology, Duke University Medical Center

Activation of protein kinase C (PKC) is cardioprotective,but the mechanism(s) by which PKC mediates protection isnot fully understood. As PKC has also been welldocumented to modulate sarcoplasmic reticulum (SR) Ca2+,and since altered SR Ca2+ handling during ischemia isinvolved in cardioprotection, we examined the role PKCmediated alterations in SR Ca2+ in cardioprotection. Usingisolated adult rat ventricular myocytes we found thataddition of 1,2-dioctanoyl-sn-glycerol (DOG) to activatePKC under conditions that reduced myocyte death followingsimulated ischemia and reperfusion also reduced SR Ca2+.We examined the effect of DOG on SR Ca2+ using fura-2fluorescence to monitor calcium transients and caffeine-releasable SR Ca2+. Caffeine-releasable SR Ca2+ wassignificantly reduced (by ~65%) after 10 min of DOGtreatment compared to untreated myocytes (P < 0.05). Weexamined the mechanism by which PKC alters SR Ca2+ andpresent the novel finding that DOG treatment reduced thephosphorylation of phospholamban (PLB) at Ser16. Thiseffect is mediated by PKC-epsilon, since a PKC-epsilonselective inhibitory peptide blocked the DOG mediateddecrease in phosphorylation of PLB. Using immuno-precipitation, we further demonstrated that DOG increasedthe association between protein phosphatase-1 (PP-1) andPLB. These data suggest that activated PKC-epsilon reducesSR Ca2+ content through PLB dephosphorylation, and thatreduced SR Ca2+ may be important in cardioprotection.

Abstracts N° P-9-5Utility of 16-slice multislice spiral computed tomography for follow-up study of coronary stent implantationMakoto Watanabe a, Shiro Uemura a, Hajime Iwama a,Satoshi Okayama a, Yukiji Takeda a, Hiroyuki Kawata a,Manabu Horii a, Tamio Nakajima a, Sinji Hirohashi b,Kimihiko Kichikawa b, Akira Ookura c, Yoshihiko Saito aa Nara Medical University. b Department of Radiology, Nara Medical University. c Department of Radiology, Kokuho Central Hospital

Aims. – Although multislice spiral computed tomography(MSCT) is a promising technique for non-invasive coronaryangiography, its usefulness remains unclear. Our aim was tocompare the utility of MSCT with that of invasive coronaryangiography for evaluating coronary stent patency.

Methods and results. – Thirty-one patients were enrolledafter coronary stent implantation. Just before follow-upcoronary angiography, 16-slice MSCT was performed. Afterassigning an Image Score based on luminal visibility (1 =poor, 2 = fair and 3 = good), factors influencing imagequality were analyzed. Among 42 implanted stents, 33(78%) were assigned an Image Score of 3, two (5%) a scoreof 2, and seven (17%) a score of 1. Image Scores amongstents with diameters ≥ 3.5 mm were significantly (P < 0.05)higher than among smaller stents (≤ 3.0 mm). Stent strutthickness did not affect image quality, but coronarycalcification significantly (P < 0.01) reduced luminal image


quality. After excluding seven stents with Image Scores of 1,MSCT’s sensitivity, specificity, and positive and negativepredictive values for detection of stent patency were 83%,90%, 63% and 96%, respectively.

Conclusion. – MSCT can provide useful and valuableclinical information for assessing stent patency during thechronic phase when patients are treated with relatively largediameter coronary stents.

Abstracts N° P-9-6A novel cellular phenotype of macrophages with deficiency of atp-binding cassette transporter-A1 (ABCA1)Masahiro Koseki a, Ken-ichi Hirano b, Daisaku Masuda a,Zhongyan Zhang a, Chiaki Ikegami a, Yumiko Nakagawa-Toyama a, Yukiko Shimada c, Yoshiko Ohno-Iwashita c,Toshihide Kobayashi d, Satoshi Sato e, Iichirou Shimomura a, Masatsugu Hori b, Shizuya Yamashita ba Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Japan. b Department of Cardiology, Osaka University Graduate School of Medicine, Japan. c Department of Protein Biochemistry, Tokyo Metropolitan Institute of Gerontology. d The RIKEN, Wako,

Saitama. e Department of Biophysics, Graduate School of Science, Kyoto University

Background. – It is well known that macrophages withABCA1 deficiency present a defective lipid efflux and asubsequent accumulation of intracellular lipid droplets,called foam cell formation. However, phenotypes of thesecells are still largely unknown.

Objective. – We investigated whether ABCA1 deficiencyinfluences the cholesterol-rich microdomains of outerplasma membrane with two newly developed probes bothselectively binding to these domains; a protease-nicked andbiotinylated derivative of perfringolysin O and a fluoresceinester of polyethylene glycol-derivatized cholesterol.

Results. – Western blot analyses and the confocal laserscanning microscopy revealed that these two probes recognizeda greater volume of cholesterol-rich microdomains in ABCA1-deficient macrophages and fibroblasts obtained from patientswith Tangier disease and ABCA1 knockout mice. Thephenotype was corrected by the gene introduction of ABCA1.

Conclusion. – The ABCA1-deficient cells exhibited anovel phenotype, the alteration of cholesterol-richmicrodomains. These results suggest that ABCA1 mayregulate the formation and function of the importantstructure on the plasma membrane.


Author Index (Referring to Abstract Numbers)

Adachi, S., P-7-1Aizawa, Y., YIA-4, O-5-3Akazawa, H., YIA-9, YIA-7Akino, M., O-5-6Akutagawa, O., O-5-1, O-2-1Anzawa, R., O-1-3Arai, K., P-4-5Arai, M., O-3-2, P-1-3, P-1-1Arimoto, T., P-6-1Arimura, T., O-3-4Asai, T., P-3-2Asano, Y., P-5-4Baetz, D., O-6-4Bito, Y., O-2-3Cheng, C.-Y., P-5-5Chugun, A., O-4-5Daicho, T., YIA-2Daida, H., O-4-1, O-4-2Endoh, M., P-1-5, P-2-4Fan, L., P-1-4Feuvray, D., O-1-3Findlay, I., P-2-1Fujita, M., P-5-4Fukuda, N., P-4-2Fukunaga, M., O-2-1Geshi, E., O-5-4Hagiwara, H., P-8-1Hamada, Y., P-4-3Hamaguchi, Y., P-2-2Hanawa, H., O-5-3Harada, T., O-3-5Hasagawa, Y., YIA-8Hasegawa, H., P-4-4, P-9-3Hasegawa, K., YIA-1Hata, T., O-5-1, O-2-1Hattori, F., YIA-8Hayashi, H., O-6-2, P-8-4Higuchi, M., P-3-4Hirai, T., P-3-2Hiramoto, Y., O-6-5Hirano, K., O-2-5, O-1-6,

P-9-6, O-1-5, O-2-4, P-3-6Hirasawa, S., O-5-2Hiroe, N., P-7-3Hirohashi, S., P-9-5Hirono, S., O-5-3, YIA-4Hirota, H., O-6-5Hisamatsu, Y., P-2-3Hisashi, T., P-8-3Hongo, K., O-4-3, O-4-4Honjo, H., P-6-2Hori, M., O-6-3, O-1-5,

YIA-3, O-2-4, O-1-6,O-3-3, O-2-5, O-6-5, P-5-4

Horii, M., P-9-5Hosaka, Y., P-2-6Hoshiai, M., P-7-3

Ide, T., O-6-6, P-6-5,YIA-8, P-5-2

Igarashi, T., P-1-6Ikeda, A., O-1-5Ikeda, U., YIA-10Ikegami, C., O-2-5, O-1-6,

P-9-6Ikeuchi, M., YIA-8, P-5-2,

P-6-5Ikrar, T., YIA-4Imai, Y., O-3-5Imaizumi, S., O-1-1, O-6-1,

O-2-2Imaki, R., O-5-2Inomata, T., O-5-5Ise, H., YIA-10Ishibashi, T., YIA-5Ishida, H., P-7-3Ishihata, A., P-3-3Ishii, K., P-2-4Iso, Y., O-5-4Isobe, M., P-7-1Ito, H., O-3-1, P-7-1Ito, M., O-5-3, YIA-4Itoi, T., O-2-6Iwama, H., P-9-5Iwanaga, K., P-4-4, P-4-1Iwata, Y., P-5-3Izawa, A., YIA-10Izumi, T., O-5-5, P-5-6, O-5-2Janabi, M., O-2-4Jin, C., P-2-5, P-1-4Jumabay, M., P-4-2Kadono, T., P-7-3Kajiya, F., YIA-6Kamioka, M., YIA-5Kano, K., P-4-2Kase, J., P-8-5Kashimura, T., O-5-3, YIA-4Katagiri, T., O-5-4Katano, Y., P-3-3Katanosaka, Y., P-5-3Kato, K., O-5-3Katoh, H., O-6-2, P-8-4Kawachi, K., O-5-4Kawaguchi, N., P-4-3Kawahara, Y., YIA-2Kawai, M., O-4-3, YIA-3,

O-4-4Kawamoto, T., O-1-5Kawamura, T., YIA-1Kawase, I., O-6-5Kawase, Y., YIA-1Kawashima, S., P-7-6Kawata, H., P-9-5Kazuhiko, O., P-8-3Kichikawa, K., P-9-5Kijima, Y., O-5-1, O-2-1

Kim, J., P-5-4Kimura, A., O-3-4Kinugawa, S., P-6-5, YIA-8Kirshebaum, L.A., O-6-4Kita, T., YIA-1Kitagawa-Sakakida, S., P-7-5Kitakaze, M., P-5-4Kiya, Y., O-6-1, O-2-2, O-1-1Koba, S., O-5-4Kobayashi, H., P-6-4Kodama, I., P-2-3Kodama, M., O-5-3, YIA-4Koitabashi, N., O-3-2, P-1-1,

P-1-3Komukai, K., O-4-4, O-4-3Komuro, I., YIA-7, YIA-9,

P-4-1, P-9-3, P-4-4Koseki, M., P-9-6, O-2-5,

O-1-6, P-3-6Kosugi, R., P-5-6Kouzuma, N., P-3-4Koyama, Y., P-6-1Kubota, I., P-6-1Kubota, T., O-6-6, YIA-8,

P-5-2, P-6-5Kugimiya, T., P-1-4Kunishige, M., O-5-1, O-2-1Kurabayashi, M., O-3-2,

P-1-1, P-1-3, YIA-5Kurachi, Y., P-2-6, P-2-1Kurebayahi, N., O-4-2, O-4-1,

O-4-5Kurihara, S., YIA-3, O-4-4,

O-4-3Kurihara, T., YIA-8Kuroda, T., O-6-5Kurose, H., P-7-2Kusakari, Y., YIA-3, O-4-4,

O-4-3Kusuyama, T., O-5-4Kuwasako, T., O-1-6, O-2-4Li, C., P-9-2Li, S., P-9-1, P-9-2Liao, Y., P-5-4Machida, Y., P-5-6Maeda-Watanabe, K., P-5-6Maejima, Y., P-7-1Maemura, K., O-3-5Maeno, E., P-7-4, P-7-5Mano, T., O-3-3Maruyama, Y., YIA-5Masaki, M., O-6-5Masano, T., P-7-6Masuda, D., O-1-6, O-2-4,

P-9-6, O-2-5, P-3-6Masuda, Y., O-1-5Matsubara, T., P-2-2Matsui, H., P-3-2

Matsumoto, K., O-1-5Matsumoto, T., P-2-3Matsumoto, T., P-4-2Matsuo, A., O-2-1, O-5-1Matsuo, Y., O-2-2, O-6-1Matsusaka, H., YIA-8,

P-5-2, P-6-5Matsushima, S., YIA-8,

P-5-2, P-6-5Matsushita, K., P-7-4Matsuura, K., P-4-1Matsuura, N., P-4-3Matsuzaki, M., P-1-2, P-6-3,

P-6-2, P-2-3Miki, T., P-6-4Minamino, T., P-5-4Minamino, T., YIA-9Minamiyama, Y., O-2-3Mitsuma, W., YIA-4, O-5-3Mitsuyuki, S., P-8-3Miura, T., P-6-4Miura, S., O-1-1, O-2-2,

O-6-1Miwa, T., O-3-3Miyamoto, S., YIA-1Miyasaka, T., YIA-6Miyazaki, T., P-1-4Mochizuki, M., P-1-2, P-6-3Mochizuki, S., O-1-3, P-8-3,

P-8-2, O-4-3Mohammed Ramadan, M.R.,

YIA-4, O-5-3Mohri, S., YIA-6Mori, C., P-8-3, P-8-2Mori, H., P-1-6Moriguchi, M., O-5-2Morikawa, K., P-3-1, P-3-5Morimoto, H., YIA-10Morimoto, S., O-4-3Morimoto, S., O-4-4Morimoto, T., YIA-1Mugishima, H., P-4-2Murakami, H., P-3-2Murakami, R., P-3-2Murayama, Y., P-3-1, P-3-5Murohara, T., P-2-2, P-3-2Murphy, E., P-9-4Nagai, R., O-3-5Nagai, T., P-4-1Naito, A.T., YIA-7Nakagawa-Toyama, Y.,

O-1-6, O-2-5, P-9-6Nakagawa, Y., O-5-1, O-2-1Nakajima, T., P-9-5Nakano, N., O-3-4Nakano, T., O-6-2Nakatani, M., P-9-1Nakaya, H., P-8-6


Nakazato, Y., O-4-1, O-4-2Nakazawa, H., P-7-3Nakazawa, S., P-7-3Namasivayam, N., O-3-6Namekata, I., P-8-5Niitsuma, Y., P-9-3, P-4-4Niizeki, T., P-6-1Nishibe, A., O-2-1, O-5-1Nishida, H., P-8-6Nishihara, M., O-1-4, P-6-4Nishimaru, K., P-1-5Nishio, M., O-3-3Nishizawa, H., O-4-2, O-4-1Niu, C.F., P-8-4Niwan, S., O-5-2Niwano, H., O-5-2Niwano, K., P-1-1, O-3-2,

P-1-3Nonaka, Y., O-3-3Ogawa, K., P-8-2Ogawa, Y., O-4-1Ohga, Y., P-8-1Ohkawara, H., YIA-5Ohkusa, T., P-2-3, P-6-2Ohmori, Y., O-5-4Ohno-Iwashita, Y., P-9-6Ohori, K., P-6-4Ohtani, T., O-3-3Ohtsuka, M., P-4-4, P-9-3Oikawa, S., YIA-8Oka, T., O-2-6Okada, S., O-2-3Okada, T., P-2-5, P-9-2,

P-9-1, P-1-4Okamoto, H., O-5-6Okayama, S., P-9-5Okumura, K., P-3-2Okuno, T., O-1-5Okura, Y., YIA-4, O-5-3Okuyama, H., YIA-6Onishi, S., P-4-3Ono, K., YIA-1Ookura, A., P-9-5Otsu, K., YIA-3, O-4-3,

O-6-3O-Uchi, J., O-4-4, O-4-3Oyama, T., P-4-1Qin, Y., P-4-4, P-9-3Regula, K.M., O-6-4Rye, K.-A., O-1-1, O-2-2,

O-6-1

Saito, T., O-3-5Saito, A., P-4-5Saito, S., P-4-2Saito, Y., P-9-5Sakai, N., O-2-4, P-3-6, O-1-5Sakai, T., O-5-1, O-2-1Sakamoto, J., P-6-4Sakamoto, N., YIA-5Sakamoto, T., YIA-5Sakata, Y., O-3-3Saku, K., O-2-2, O-6-1, O-1-1Sasaki, H., P-8-2, P-8-3Sasaki, S., O-5-2Sasaki, T., O-5-2Sato, D., O-5-2Sato, S., P-9-6Sato, T., O-5-4Satoh, T., P-6-2, P-2-3Sato, T., P-8-6Satoh, H., P-8-4, O-6-2Satomi-Kobayashi, S., P-7-6Sawa, Y., P-7-4, P-7-5, P-4-5Sawamura, T., YIA-5Senda, S., O-2-1Shaw, J., O-6-4Shiba, Y., YIA-10Shiga, A., P-7-4, P-7-5Shigekawa, M., P-5-3Shigenobu, K., P-8-5Shimada, Y., P-9-6Shimamoto, K., P-6-4Shimizu, J., YIA-6Shimizu, M., P-8-2Shimokawa, H., P-3-1, P-3-5Shimomura, I., O-2-5, P-9-6Shimomura, M., P-4-5Shinmura, K., O-1-2Shinpei, N., P-7-3Shintani, Y., P-5-4Shioi, T., P-5-6Shiojima, I., YIA-7Shoji, M., O-5-4Soda, T., O-5-4Sonoda, S., P-1-4Steenbergen, C., P-9-4Sugimoto, K., YIA-5Sugimoto, N., YIA-5Sugimoto, T., O-1-5, P-3-6Sugiyama, S., O-6-5Sunagawa, K., O-6-6, P-3-1,

YIA-8, P-5-2, P-6-5, P-3-5

Suzuki, C., YIA-10Suzuki, H., O-5-4 Suzuki, S.,

P-2-1Suzuki, S., P-2-3, P-6-2Tachikawa, H., O-5-3Tadokoro, H., P-4-4Takahashi, A., P-6-4Takahashi, H., P-6-1Takahashi, M., YIA-10Takahashi, R., P-3-2Takaki, A., P-3-1, P-3-5Takaki, M., P-5-1, YIA-6,

P-8-1Takako, T., P-7-3Takano, H., P-9-3, P-4-4Takashima, S., P-5-4Takatsuka, H., P-8-2Takeda, J., P-9-1Takeda, N., O-3-5Takeda, S., P-1-6Takeda, S., P-8-2Takeda, T., YIA-3, O-4-3Takeda, Y., O-3-3Takeda, Y., P-9-5Takeishi, Y., P-6-1Takemura, S., O-2-3Takenaka-Nakajima, C.,

P-8-1Takeo, S., YIA-2Taketani, S., P-4-5Takeyama, Y., O-5-4Tamaki, K., O-1-2Tamura, M., P-8-5Tamura, Y., P-8-1Tanaka, H., P-8-5Tanaka, M., P-4-5Taniguchi, I., P-8-2, P-8-3Taniguchi, S., P-8-1Tanonaka, K., YIA-2Tateishi, H., P-6-3, P-1-2Tateno, K., YIA-9Tekes, E., P-3-5, P-3-1Teramoto, T., YIA-5Tochino, Y., O-1-6Toda, T., P-7-3Toh, R., P-7-6Toko, H., YIA-9Toyama, Y., O-2-4, P-3-6Toyoda, T., P-4-4Toyota, H., YIA-6Trai, K., O-6-5

Tsuchikawa, C., P-2-1Tsuji, T., P-8-1Tsujii, K., O-2-5, P-3-6Tsujikawa, H., P-2-5, P-1-4Tsujioka, K., YIA-6Tsunoda, F., O-5-4Tsutsui, H., O-6-6, O-5-6,

P-5-2, P-6-5, YIA-8Tsutsui, M., P-3-5Uchida, Y., O-1-5, P-3-6Ueda, K., P-9-3, P-4-4Ueda, Y., O-2-5Uehara, Y., O-1-1, O-6-1,

O-2-2Uemura, S., P-9-5Ueyama, T., P-7-6Urata, H., O-1-1, O-6-1Urushida, T., P-8-4Vairappan, B., O-3-6Wada, H., P-4-1Wakabayashi, K., O-5-4Wakabayashi, S., P-5-3Watanabe, A., P-1-3, O-3-2,

P-1-1Watanabe, H., O-3-1Watanabe, H., O-6-2Watanabe, M., P-9-2, P-2-5,

P-1-4, P-9-1Watanabe, M., P-9-5Watanabe, T., O-6-3Wu, J., P-9-2, P-9-1Xu, Z., O-5-6Yagi, N., YIA-6Yamada, M., P-2-6Yamamoto, K., O-3-3Yamamura, K., P-9-4Yamashita, D., P-5-1Yamashita, S., O-1-5, O-2-4,

P-3-6, O-2-5, O-1-6, P-9-6Yanagihara, N., P-3-5Yano, M., P-1-2, P-6-3, P-6-2,

P-2-3Yasui, K., P-2-3Yokoyama, M., P-7-6Yokoyama, S., P-4-2Yoshikawa, Y., P-8-1Yoshioka, T., YIA-10Yu, E., P-7-3Yuge, M., O-5-2Zhang, Z., P-9-6, O-1-6Zhao, H., P-5-4


Subject Index

Angiogenesis, YIA-9, O-2-2, P-4-3, P-9-6

Animal models of humandisease, O-2-3, O-2-6,P-3-3, P-8-2, P-8-3

Apoptosis, O-6-2, O-6-4,P-7-6

Arrythmias, O-4-1, O-4-2,O-5-2, O-5-4, P-2-3, P-6-2,P-8-3

Atherosclerosis, YIA-4,YIA-10, O-1-5, O-2-1,O-2-4, O-2-5, P-3-3, P-3-6, P-9-5, P-9-6

Autonomic, reflex, and neurohumoral control of circulation, P-3-4

Calcium cycling/excitation-contraction coupling,YIA-3, O-3-1, O-4-1,O-4-2, O-4-3, O-4-4,O-4-5, O-5-3, P-1-2,P-1-5, P-5-1, P-6-3, P-8-1

Cardiovascular pharmacology, YIA-2, O-1-1, O-6-1,P-1-5, P-3-2, P-3-4,P-5-4, P-8-4, P-9-3

Catecholamine, O-5-5, YIA-2Cell Death, O-6-3, O-6-4,

P-7-3, P-7-4, P-7-5Cell signaling/signal

transduction, YIA-1, YIA-5, O-1-1, O-1-4, O-2-2, O-6-2,

O-6-3, O-6-5, P-2-5, P-5-6, P-6-4, P-7-1, P-7-2, P-9-3,P-9-4

Coronary circulation, O-1-5

Developmental biology,YIA-7

Diabetes, O-1-3, O-2-3,P-3-6, P-5-2, P-6-5,P-8-2, P-8-3

Diastolic heart failure, O-3-2,O-3-3

Echocardiography, YIA-2Electrophysiology, O-5-2,

P-1-4, P-2-1, P-2-3, P-6-2Energy metabolism, P-8-1

Fibrosis, O-5-6, O-6-1

Gene expression/regulation,YIA-7, O-2-5, O-6-4,P-1-1, P-1-3, P-7-5

Gene therapy, P-1-1, P-7-4Genetically altered mice,

YIA-1, O-1-2, P-3-5, P-6-1Genetics of cardiovascular

disease, O-3-4Growth factors/cytokines,

O-3-2

Heart failure, YIA-1, YIA-8, O-3-4, O-5-1, O-5-3, O-6-6, P-1-1, P-1-2, P-1-3, P-1-6,

P-5-4, P-5-6, P-6-2, P-6-3, P-7-5, P-7-6, P-9-2

Hypertrophy, O-3-1, O-3-2, O-5-6, P-5-1, P-5-3, P-5-5, P-5-6, P-6-1, P-7-6

Imaging, YIA-6, O-2-1, O-4-1, O-4-2, P-9-5

Inflammation, O-5-5Ion channels/membrane

transport, O-1-3, O-3-1, O-4-4, P-1-4, P-2-1, P-2-2, P-2-4, P-2-5, P-2-6, P-5-3, P-7-3, P-8-4, P-8-5, P-8-6

Ischemic biology, YIA-4,O-6-5, P-4-4, P-7-3, P-7-4, P-8-1, P-8-4, P-8-5, P-8-6, P-9-1, P-9-2, P-9-3, P-9-4

Lipid and lipoproteinmetabolism, O-1-1, O-1-5,O-1-6, O-2-2, O-2-4, O-2-5,O-2-6, O-6-1, P-3-6, P-9-6

Myocardial biology, YIA-6,O-2-6, O-4-5, P-1-5, P-4-4,P-7-1, P-8-5, P-9-2

Myocardial cardiomyopathy disease, O-3-4, O-5-5, P-5-5

Myogenesis, YIA-9, P-4-2,P-4-5

Nanotechnology, P-4-5Necrosis, O-6-3, P-6-4

Nuclear cardiology, P-5-5Nitric oxide, O-1-2, P-3-2,

P-3-3, P-3-5

Obesity, O-3-6Oxidant stress, YIA-8, O-2-1,

O-2-3, O-3-5, O-3-6, O-5-1, O-6-6, P-2-5, P-5-4, P-6-5, P-7-1

Preconditioning, O-1-2, O-1-4

Receptor pharmacology,O-2-4, O-4-4

Regenerative medicine, YIA-7, O-5-4, P-4-2, P-4-3, P-4-5

Remodeling, YIA-8, O-3-5,O-5-1, O-5-6, O-6-6, P-4-4, P-5-2, P-6-1, P-6-4, P-6-5

Renin-angiotensin-aldosterone,O-3-3, O-3-5, P-2-3, P-5-2

Restenosis, YIA-10, P-9-5

SarcoEndoplasmic reticulum, YIA-3, O-4-3, O-4-5, P-1-2, P-1-3, P-5-1, P-6-3, P-9-4

Smooth muscle, P-2-2Stress adaptation, P-3-4Stem cell, P-4-1, P-4-2Structural biology, YIA-6,

P-1-6

Vascular medicine, YIA-5,P-3-1, P-3-2, P-4-3

abstracts from the 22nd annual, japanese section of the international society for heart research

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