Human cardiac sympathetic nerves switch the neurotransmitter from catecholaminergic to cholinergic property in heart failure

S154 Journal of Cardiac Failure Vol. 14 No. 7S September 2008 021 Zac1 is an essential transcription factor for cardiac morphogenesis and cardiac myocyte apoptosis inhibition SHINSUKE YUASA1, TAKESHI ONIZUKA1, KENICHIRO SHIMOJI1, OHNO YOHEI1, KEIICHI FUKUDA2, SATOSHI OGAWA1 1 Cardiology division, Department of Medicine, Keio University school of Medicine, Tokyo, JAPAN, 2Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, Tokyo, JAPAN The transcriptional mechanisms are poorly understood in the heart, despite the identification of several essential cardiac transcription factors. We found that Zac1, which encodes a zinc-finger-type transcription factor and is a maternal imprinting gene, was strongly expressed throughout the murine heart from embryonic stage 8.5. Zac1 was a potent activator of several cardiac genes and bound directly to the atrial natriuretic peptide promoter, upon which Zac1 exerted a strong synergistic transcriptional activity and a physical interaction with Nkx2.5. Nkx2.5 also activated the Zac1 promoter, and Nkx2.5-null hearts showed decreased Zac1 expression. Zac1-mutated mice showed decreased levels of several cardiac genes and severe cardiac malformation. The absence of Zac1 expression resulted in the loss of the apoptosis inhibitor gene Ifi202, thereby increasing the number of apoptotic cells in the heart. These data indicate that Zac1 is a novel and essential cardiac transcription factor that acts both independently and co-operatively with Nkx2.5.

022 Downregulation of ferritin heavy chain increases labile iron pool, oxidative stress and cell death in cardiomyocytes SHIGEMIKI OMIYA, SHUNGO HIKOSO, KINYA OTSU Department of Cardiovascular Medicine Osaka University Graduate School of Medicine Background: Ferritin heavy chain (FHC) regulates the cellular availability of iron, which induces generation of reactive oxygen species. However, the role of FHC in the pathogenesis of heart failue has not been elucidated. Methods and Results: We examined the FHC expression level in murine failing hearts. FHC protein had significantly reduced in failing hearts after both myocardial infarction and transverse aortic constriction compared with corresponding sham-operated hearts (36.6 6 5.3% and 37.6 6 4.5% of sham-operated hearts, respectively, p ! 0.05). Iron deposition and oxidative stress had increased in failing hearts. To clarify the functional significance of FHC downregulation in hearts, we infected rat neonatal cardiomyocytes with adenovirus expressing short hairpin RNA targeted to FHC (Ad-FHCRNAi). Knockdown of FHC by Ad-FHC-RNAi had significantly increased the number of iron deposition positive cells and 4-hydroxy-2-nonenal positive cells (Ad-nonspecific-RNAi infected vs. Ad-FHC-RNAi infected cells, 15.5 6 2.1% vs. 49.8 6 1.2%, 21.4 6 2.9% vs. 48.9 6 4.0%, respectively, p ! 0.05), and resulted in significant reduction in cell viability (45.6 6 8.6% of Ad-nonspecific-RNAi infected cells, p ! 0.05). Reduced cell viability by Ad-FHC-RNAi was significantly attenuated with desferrioxamine treatment, an iron chelator. Conclusions: These findings indicate that increase in free iron mediated through downregulation of FHC induces cardiomyocyte death, and may play an important role in the progression of heart failure.

023 Adult Murine Heart Derived Clonal Expanded Sca-1 Positive Cell Sheet Ameliorates Cardiac Dysfunction after Myocardial Infarction KATSUHISA MATSUURA1, ISSEI KOMURO2, NOBUHISA HAGIWARA1 1 Department of Cardiology, Tokyo Women’s Medical University, Tokyo, Japan, 2 Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chiba, Japan Cardiac progenitor cells (CPC) have been thought to be the promising source of cell therapy. The aim of this study is to clarify the differences between CPC and other somatic stem cells on the contribution when in transplantation for infarct models by using cell sheet technique. We cultured adult murine heart derived clonal expanded Sca-1 positive cells (cSca-1) labeled with RFP or adipose tissue derived mesenchymal cells (ATMC) isolated from GFP mice by using temperature responsive culture dishes and transplanted each of monolayered cell sheet onto the infarcted region just after coronary ligation. At 4 weeks after transplantation, LVDd, LVDs and FS were significantly improved (FS: cSca-1, 0.29 6 0.01; untreated, 0.22 6 0.01; ATMC, 0.24 6 0.01, p ! 0.001) accompanied with increasing the number of capillary density in the border zone in cSca-1 compared with untreated or ATMC. Cytokine array showed several secreting angiogenic factors were abundant in cSca-1 compared to ATMC. Confocal microscopic analysis revealed some RFP(+) cSca-1 cells existed in normal or infarcted areas (normal, 1.1 6 0.4 cells/HPF; infarct, 8.3 6 2.1 cells/HPF) at 4 weeks after transplantation, while none of GFP(+) ATMC

existed. Furthermore about 30% of RFP(+) cells expressed sarcomeric a-actinin or formed vessels in infarcted areas. These findings suggest cardiac progenitor cell sheet transplantation ameliorates cardiac dysfunction via cardiomyogenesis and angiogenesis and might be the most feasible source for cardiac regeneration therapy.

024 Efficacy of Adaptive-Servo Ventilation for a Patient with Chronic Heart Failure who had Cheyne-Stokes Respiration with Central Sleep Apnea YASUHIRO USUI, YOSHIFUMI TAKATA, KIHIRO ASANO, YUKI HASHIMURA, KOTA KATO, HIROKAZU SARUHARA, KAZUKI SIINA, AKIRA YAMASHINA The Second Department of Internal Medicine (Cardiology), Tokyo Medical University, Tokyo, Japan An 82-year-old male patient, who had been diagnosed with chronic heart failure due to dilated cardiomyopathy and combined valvular disease and who atrial fibrillation with complete atrioventricular block, was admitted to our hospital owing to the exacerbation of chronic heart failure. During admission, the patient became aware of drowsiness during daytime hours and was indicated to present periodic apnea during sleep. Polysomonography (PSG) revealed Cheyne-Stokes respiration with severe central sleep apnea as evidenced by an apnea-hypopnea index (AHI) of 93.5 episodes/h. Nocturnal oxygen therapy failed to sufficiently suppress apnea, and arousal reactions occurred frequently. Therefore, we conducted titration by Adaptive Servo-Ventilation (ASV, HEART PAP). Consequently, subjective symptoms and respiratory sleep parameters improved. The patient showed excellent adherence to loading the device at home. PSG at 3 months after implementation of HEART PAP indicated improvement in the AHI to 13.5 episodes/hour, and the patient exhibited marked improvements in breathlessness and awareness of drowsiness during daytime hours. HEART PAP was found to be a useful device for Cheyne-Stokes respiration with central sleep apnea (CSR-CSA) that is associated with chronic heart failure.

025 Molecular and Electrical Remodeling of L- and T- type Ca2 + Channels in Rat Right Atrium with Monocrotaline-Induced Pulmonary Hypertension TAKASHI KOYAMA1, HIROYUKI WATANABE1, TAKAYOSHI OHBA2, TOSHIMITSU KOSAKA1, HITOSHI HASEGAWA1, HIROSHI ITOH1 1 Departments of Cardiology, Akita University School of Medicine, Akita, Japan, 2 Departments of Pharmacology, Akita University School of Medicine, Akita, Japan Background: Atrial arrhythmia is often encountered in cor pulmonale, however, few studies have investigated the ionic mechanism in right atrial cells. Methods and Results: Rats with monocrotaline-induced pulmonary hypertension were used in the present study to examine electrical remodeling of atrial Ca2+ channels. The L-type Ca2+ channel current density was significantly decreased in right atrial cells of monocrotaline-treated rats, accompanied by a significant reduction in mRNA expression of CaV1.2 (a1C) subunit and accessory b2 subunit. Conversely, low voltage-activated Ca2+ current was more marked in the right atrial cells of monocrotaline-treated rats than in those of control rats. The current-voltage relationship and the time course of inactivation resembled closely those of T-type Ca2+ channels, although the current was only slightly inhibited by 10e100 mM Ni2+. No significant differences were observed in the mRNA expression levels of CaV3.1 (a1 G) and CaV3.2 (a1H) or the protein level of the CaV3.1 subunit. In the left atrial cells, neither the current densities of the L- and T-type Ca2+ channels nor the protein levels of CaV1.2 and CaV3.1 were affected by monocrotaline.Conclusions: Pulmonary hypertension causes right atrial hypertrophy, associated with the alteration of electrophysiological molecular properties of Ca2+ channels.

026 Human cardiac sympathetic nerves switch the neurotransmitter from catecholaminergic to cholinergic property in heart failure HIDEAKI KANAZAWA1, MASAKI IEDA1, KENSUKE KIMURA1, TAKAHIDE ARAI1, HARUKO MANABE2, TOKUHIRO KIMURA3, YASUNORI OKADA3, HATSUE UEDA4, SATOSHI OGAWA1, KEIICHI FUKUDA2 1 Department of Cardiology, Keio University School of Medicine, Tokyo, Japan, 2 Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, Tokyo, Japan, 3Department of Pathology, Keio University School of Medicine, Tokyo, Japan, 4Department of Pathology, National Cardiovascular Center, Osaka, Japan Background: Despite upregulation of sympathetic neural tone during congestive heart failure (CHF) there is a paradoxical reduction in norepinephrine (NE) synthesis and reuptake in the cardiac sympathetic nervous system (CSNS). We recent found

The 12th Annual Scientific Meeting that the CSNS switches the neurotransmitter from catecholaminergic to cholinergic property in animal model of CHF. This study is designed to investigate whether these cholinergic transdifferentiation of CSNS may occur in patients with CHF. Methods & Results: We analyzed 8 samples from patients who died of noncardiac causes obtained at autopsy (control group), and 8 samples of patients who died of CHF or hearts of transplant recipients (CHF group). The gross morphology of the CSNS was not different between two groups. HE and Masson trichrome staining showed disorganized cardiomyocytes and interstitial fibrosis in CHF. Immunostaining for tyrosine hydroxylase (TH, sympathetic marker) revealed that the epicardial nerves and stellate ganglia of control group had a predominance of TH+ nerves, while whereas those of CHF group was significantly decreased. Immunostaining for choline transporter (CHT, cholinergic marker) and choline acetyltransferase (ChAT, cholinergic marker) revealed that CHT+ nerves were markedly increased in the epicardial nerves of left ventricle and stellate ganglia had a lot of ChAT+ neurons compared with the control group. Conclusions: These results indicated that human cardiac sympathetic nerves also transdifferentiate into cholinergic property in CHF patients.

027 Restrictive Mitral Annuloplasty for End-stage Heart Failure Due to Idiopathic Dilated Cardiomyopathy SHUNSUKE SAITO1, GORO MATSUMIYA1, TAICHI SAKAGUCHI1, TOMOYUKI FUJITA1, KOJI TAKEDA1, KAZUHIRO TANIGUCHI2, TAKAFUMI MASAI3, YASUSHI SAKATA4, YOSHIKI SAWA1 1 Division of Cardiovascular Surgery, Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan, 2Department of Cardiovascular Surgery, Osaka Rosai Hospital, Osaka, Japan, 3Department of Cardiovascular Surgery, Sakurabashi Watanabe Hospital, Osaka, Japan, 4Division of Cardiology, Department of Internal Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan Objective: Clinical outcomes of restrictive mitral annuloplasty (RMA) for idiopathic cardiomyopathy were studied. Patients and methods: Twenty-three patients with significant left ventricular (LV) dysfunction (LVDdO/ 5 60 mm and LVEF!/ 535%) who underwent RMA for functional mitral regurgitation due to idiopathic cardiomyopathy were studied. Patients’ mean age was 63 6 9 years. MR was grade 3e4 in all patients. Results: There was 1 operative death and 1 late death due to sepsis. NYHA functional status improved from 3.4 6 0.6 to 1.9 6 0.8. MR was grade 2 in 2 patients, and 0e1 in the others. Although mean LVEF remained unchanged after the operation (from 23.3 6 9.9 to 24.4 6 13.9%, p 5 0.221), mean LVDd significantly decreased from 74.6 6 7.8 to 69.0 6 9.0 mm (p 5 0.0069). PA pressure also decreased from 28.5 6 7.9 to 24.4 6 8.9 mmHg (p 5 0.0386), and cardiac index increased from 2.1 6 0.3 to 2.5 6 0.3 L/min/m2 (p 5 0.0021). Histological examination revealed that degree of fibrosis of LV myocardium had significant relation with reduction of LVDd (p 5 0.0369, r 5 -0.69) and PA pressure (p 5 0.0478, r 5 0.63). Conclusions: RMA improved heart failure symptoms and hemodynamics in patients with end-stage heart failure due to idiopathic cardiomyopathy. The degree of fibrosis of LV myocardium was the significant predictor of reverse remodeling and improvement of LV diastolic function after RMA.

028 Enhanced Sensitivity of Ryanodine Receptor to Activation by Luminal Ca2+ may underlie the pathogenic mechanism of lethal arrhythmia HITOSHI UCHINOUMI, MASAFUMI YANO, TAKESHI SUETOMI, MAKOTO OHNO, HIROKI TATEISHI, SHINICHI OKUDA, SHIGEKI KOBAYASHI, TAKESHI YAMAMOTO, YASUHIRO IKEDA, MASUNORI MATSUZAKI Division of Cardiology, Department of Medicine and Clinical Science, Yamaguchi University Graduate School of Medicine Mutations in cardiac ryanodine receptor (RyR2) have been reported to be linked with catecholaminergic polymorphic ventricular tachycardia (CPVT). To study the underlying mechanism of CPVT, we developed knock-in (KI) mice, harboring the Arg-to-Ser (R2474S) mutation :RyR2R2474S/+. The KI mice revealed no structural or histological abnormality in hearts. Echocardiography showed no contractile or relaxation dysfunction at rest. In all KI mice, bidirectional ventricular tachycardia (VT) was observed during or after exercise with treadmill (6/6), but not in wild-type (WT) mice (0/6). In intact cardiomyocytes, the frequency of Ca2+ sparks (SpF; s 1*100 mm 1) was significantly increased in KI mice, but not in WT mice (KI:2.3 6 0.2, WT:0.6 6 0.07, p ! 0.01). In saponin-permeabilized cardiomyocytes with cytoplasmic [Ca2+] ([Ca2+]C) buffered at 100 nM, thapsigargin (SERCA2a inhibitor:1 mM) gradually decreased both SpF and luminal [Ca2+] ([Ca2+]L) due to a decrease in SR Ca2+ content, forming a sigmoid relationship between SpF and [Ca2+]L (SpF -[Ca2+]L). The SpF -[Ca2+]L relationship was



JHFS

S155

markedly shifted to the left in KI cardiomyocytes compared to WT cardiomyocytes, thereby lowering the threshold [Ca2+]L to induce Ca2+ sparks in KI cardiomyocytes. In conclusion, the enhanced sensitivity of the RyR2 channel to activation by [Ca2+]L: i.e. decreased threshold [Ca2+]L to induce spontaneous Ca2+ release, may play a key role in CPVT.

029 Prognostic value of multiple biomarker method using NT-proBNP, cardiac troponin T and cystatin C on discharge in patients with CHF SHIGERU MATSUI1, JUNICHI ISHII2, MASANORI OKUMURA3, TADASHI NAKANO4, YOSHIHISA MORI6, HIROYUKI NARUSE3, SHINICHIROU MORIMOTO3, MASANORI NOMURA5, HITOSHI HISHIDA3, YUKIO OZAKI3 1 Fujita Health University School of Health Sciences, Toyoake, Japan, 2Department of Joint Research Laboratory of Clinical Medicine, Fujita Health University, Toyoake, Japan, 3Division of Cardiology, Department of Internal Medicine, Fujita Health University School of Medicine, Toyoake, Japan, 4Daido Hospital, Nagoya, Japan, 5 BanBuntane Houtokukai Hospital, Nagoya, Japan, 6Mori Hospital We prospectively evaluated the prognostic value of simultaneous assessment of NTproBNP, troponin T (TnT), and cystatin C on discharge in 225 patients hospitalized for worsening CHF. Results: During a mean follow-up period of 34 months, there were 105 (47%) cardiac events (cardiac deaths or readmission for CHF). On a stepwise Cox regression analysis including 8 clinical and biochemical variables on discharge, elevation (Omedian value) in NT-proBNP (O1308 pg/ml; RR 2.1, P 5 0.001), TnT (O0.01ng/ml; RR 1.6, P 5 0.03), and cystatin C (O1.46 mg/l; RR 1.6, P 5 0.03) were independently associated with cardiac events. The number of elevated biomarkers was correlated with an incremental increase in cardiac mortality and cardiac event rates (Table). Conclusion: Multiple biomarker method using NT-proBNP, TnT and cystatin C on discharge may be highly effective for risk stratification in CHF patients.

Number of elevated biomarkers 0 (n564) 1 (n557) 2 (n557) 3 (n547) Cardiac death n(%) 1 (1/6%) 3 (5.3%) 10 (17.5%) 14 (29.8%) Cardiac event n(%) 14 (21.9%) 23 (40.4%) 34 (59.7%) 34 (72.3%)

030 Acute effects of cardiac resynchronization therapy on endothelial function and hemodynamics KENSHIRO ARAO1, YOSHITAKA SUGAWARA1, YUKO TADA2, CHIKASHI SUGA2, NORIFUMI KUBO2, SHIN-ICHI MOMOMURA2 1 Division of Integrated medicine I, Jichi Medical University Saitama Medical Center, Saitama, Japan, 2Division of Cardiovascular Medicine, Jichi Medical University Saitama Medical Center, Saitama, Japan Aim: The purpose of this study is to explore acute effects of cardiac resynchronization therapy (CRT) on endothelial function as well as cardiac function. Method: We enrolled fifteen patients (male 10 female 5, 60 6 3 years old,ischemia 4 non-ischemia 10) with CHF indicated for CRT. At stable state, electrophysiological study (EPS) was conducted prior to biventricular pacemaker implantation to measure the changes of blood pressure, maximal LV dp/dt and cardiac output. We measured postischemic forearm reactive hyperemia (FBF) with straingauge plethysmography almost one week before and three weeks after biventricular pacemaker implantation. We also examined changes the medical history, body weight and 2D-left ventricular ejection fraction (LVEF) on echocardiogram and blood parameters including blood chemistry of lipid and blood sugar profile, hsCRP, serum creatinine. Result: During the study with average intervals of thirty-five days, patients’ backgrounds of medication, body weight and parameters were not changed. EPS showed increase in LV dp/dt and cardiac output (3.97 6 0.23 vs 4.13 6 0.20 l/min., p ! 0.05) after biventricular pacing. After initiating of CRT, FBF and LVEF were significantly increased (22.7 6 1.3 vs 29.1 6 1.4 ml/min/100 ml tissue, p ! 0.01, 27 6 3 vs 32 6 4%, p ! 0.05). Conclusion: Cronic resynchrnization therapy in patients with CHF improved endothelial function.

031 Thymidylate kinase over-expressing cells showed the mitochondrial myopathy by anti-HIV drug TERUYUKI YANAGISAWA, TAKEYA SATO Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan. Nucleoside reverse transcriptase inhibitors (NRTI) such as azidothymidine (AZT) are currently applied for the highly active anti-retrovirus therapy (HAART) of human immunodeficiency virus (HIV) and reduce rates of morbidity and mortality due to HIV