Saturated Fatty Acid Evokes Mitochondrial Fragmentation and Dysfunction in an Ex Vivo Model of Insulin-Resistant Myocytes

S170 Journal of Cardiac Failure Vol. 17 No. 9S September 2011 presence of cAMP, Ca2+ spark frequency was much higher in KI than in WT saponin-permeabilized cardiomyocytes, whereas it was largely reversed by co-addition of CaM-Gly-Ser-His. Conclusions: In response to PKA phosphorylation, CaM binding affinity to the CPVT-associated mutant RyR2 was markedly decreased. Correction of the reduced binding affinity of CaM to the mutant RyR2 inhibits aberrant Ca2+ release.

P-079 Enhancement of Calmodulin Binding to Cardiac Ryanodine Receptor Corrects the Defective Channel Gating in Failing Hearts AKIHIRO HINO, MASAFUMI YANO, TAKAYOSHI KATOH, MASAKAZU FUKUDA, TAKESHI SUETOMI, MASAHIRO DOI, SHINICHI OKUDA, SHIGEKI KOBAYASHI, TAKESHI YAMAMOTO, MASUNORI MATSUZAKI Yamaguchi Univ Graduate Sch of Med, Ube, Japan Calmodulin (CaM) modulates the channel function of the cardiac ryanodine receptor (RyR2). Here, we investigated the pathogenic role of RyR2-bound CaM in channel dysfunction in heart failure. Methods and Results: Cardiomyocytes and sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles {normal (N), n54; 4-weeks rapid RV pacing (HF: n54). To assess CaM binding to RyR2, SR was mixed with CaM-SANPAH conjugate, followed by UV photolysis. Then, the RyR2-bound CaM was detected by Western blotting using anti-CaM antibody. The affinity of CaM binding to RyR2 was considerably reduced in failing SR compared to normal SR. To further assess the amount of endogenous CaM bound to RyR2 in cardiomyocytes, we performed the immunofluorescent staining using the anti-CaM antibody. Endogenous CaM was also markedly decreased, indicating the loss of CaM from the RyR2. CaM-(Gly-Ser-His), which was previously reported to show higher binding affinity to the RyR1 than CaM, also showed much higher binding affinity even to the failing SR than (wild-type) CaM. The Ca2+ spark frequency was markedly increased with SR Ca2+ content reduced in failing cardiomyocytes, whereas it was largely reversed by CaM-(Gly-Ser-His). Conclusions: CaM binding affinity to the RyR2 was markedly reduced, associated with aberrant Ca2+ release, in failing hearts. Correction of the reduced CaM binding to the failing RyR2 substantially improved the channel disorder.

P-080 Inhibition of Na+-H+ Exchange as a Mechanism of Rapid Cardioprotection by Resveratrol YASUSHI TESHIMA, LUONG CONG THUC, NAOHIKO TAKAHASHI, SATORU NISHIO, AKIRA FUKUI, OSAMU KUME, KAORI EZAKI, HIROKO MIYAZAKI, MIKIKO NAKAGAWA, TETSUNORI SAIKAWA Department of Laboratory Examination and Diagnostics, Faculty of Medicine, Oita University, Oita, Japan Objective: Resveratrol is a polyphenol abundantly found in grape skins and red wine. In the present study, we investigated whether resveratrol exerts cardioprotective effects against ischemia/reperfusion and also explored its mechanisms. Methods and Results: In isolated perfused rat hearts, left ventricular functional recovery and infarct size improved considerably on treatment with resveratrol (20 mmol/L) 15 min before no-flow global ischemia. In cultured neonatal rat cardiomyocytes exposed to hydrogen peroxide (100 mmol/L), resveratrol significantly attenuated the increase in reactive oxygen species (ROS) and loss of mitochondrial inner membrane potential. Resveratrol also suppressed the increase in intracellular concentrations of Na+ and Ca2+ ([Ca2+]i) because of H2O2 application; however, it did not suppress the ouabain-induced [Ca2+]i increase. We also confirmed that the activation of Na+-H+ exchanger (NHE) was prevented by pre-treatment with resveratrol by measuring the fluctuations in intracellular pH. Furthermore, resveratrol inhibited the H2O2-induced translocation of PKCa from the cytosol to the cell membrane; this translocation is believed to activate NHE. Conclusions: Resveratrol exerts cardioprotection by reducing ROS and preserving mitochondrial function. The PKC-a-dependent inhibition of NHE and subsequent attenuation of [Ca2+]i overload may be a cardioprotective mechanism.

P-081 Saturated Fatty Acid Evokes Mitochondrial Fragmentation and Dysfunction in an Ex Vivo Model of Insulin-Resistant Myocytes TOMOYUKI WATANABE, MASAO SAOTOME, HIDEKI KATO, HIROSHI SATO, HIDEHARU HAYASHI Internal Medicine III, Hamamatsu University School of Medicine, Shizuoka, Japan Objective: The myocardial insulin-resistance is one of deteriorative factors for heart failure. Since mitochondria play pivotal roles in cardiac metabolism, we investigated the mitochondrial morphology and functions in saturated fatty acid (SFA)-induced myocardial insulin-resistance model, which mimics the elevated serum fatty acid level via

compensative hyper-adrenergic state in heart failure. Methods: H9c2 cells were differentiated to cardiac myocytes and cultured with SFA (palmitate, 0.2 mM) for 24hr to induce insulin-resistance. The insulin-mediated glucose uptake and phosphorylation of insulin signaling molecules (IRS-1 and AKT) were evaluated with 2-deoxy-D-glucose and Western blot, respectively. The mitochondrial morphology (by immunocytochemistry), intracellular ATP (with a luciferase assay), and mitochondrial membrane potential (with JC-1) were verified in the SFA-treated myocytes. Results: The SFA-treated myocytes showed reduced insulin-mediated glucose uptake (0.5 6 0.1 fold increase from that without insulin vs. 1.760.1 of control, p!0.01) and suppressed phosphorylation in insulin signaling molecules, indicating insulin-resistance. The SFA-treated cells exhibited fragmented mitochondria (95.760.9%, vs. 4.861.0% of control, p! 0.01), reduced intracellular ATP (7466% decrease from control, p! 0.01), and depolarized mitochondrial membrane potential (565/488nm ratio: 3.060.25, p!0.01 vs. 5.360.22 of control). Conclusion: Our results suggest that the morphological alteration and dysfunction of mitochondria by SFA may correlate with myocardial insulin-resistance

P-082 Development of Post-Infarct Active Cardiac-Targeted Drug Delivery System by Liposomes with Sialyl Lewis X in Rabbits YOSHIHISA YAMADA1, HIROYUKI KOBAYASHI1, MASAMITSU IWASA1, HIROAKI USHIKOSHI1, TAKUMA AOYAMA1, KAZUHIKO NISHIGAKI1, GENZOU TAKEMURA1, TAKAKO FUJIWARA2, HISAYOSHI FUJIWARA3, SHINYA MINATOGUCHI1 1 Department of Cardiology, Gifu University Graduate School of Medicine, Gifu, Japan, 2Kyoto Women’s University, Kyoto, Japan, 3Hyogo Prefectural Amagasaki Hospital, Hyogo, Japan Objective: The purpose of the present study was to develop cardiac-targeted drug delivery system by nano-sized liposomes with Sialyl Lewis X. Methods: We prepared nano-sized EPO-encapsulated liposome with Sialyl Lewis X (SLX). The liposomes were made from the mixture of dipalmitoylphosphatidylcholine, cholesterol, dihexadecylphosphate, ganglioside, dipalmitoylphosphatidylethanolamine and sodium cholate using the improved cholate dialysis method. Rabbits underwent 30 min of coronary occlusion and reperfusion. A liposome in which fluorescent substance Cy5.5 is capsulated (0.5 ml) was intravenously administered immediately after reperfusion. In order to trace the liposome at the subcellular level in vivo, we employed the recently developed SLX liposome encapsulated with colloidal gold (SLX-LipoGold), and 500 ml of SLX-Lipo-Gold were intravenously administered immediately after reperfusion. At 24, 48 hours and 7days after reperfusion, the rabbits were sacrificed with overdose of pentobarbital and the heart was excised. The heart tissues were obtained from ischemic area and non-ischemic area of the heart. These were observed using confocal microscopy and electron microscopy. Results: Confocal microscopy and electron microscopy showed the specific accumulation of liposomes in the infarcted myocardium. Conclusions: Liposome with SLX selectively accumulated in the ischemic area. Nano-size liposome with SLX may be a promising drug delivery system for active targeting treatment of acute myocardial infarction.

P-083 Free Heme Strongly Activates Cardiac Resident Macrophage Through Toll Like Receptor HARUO HANAWA, KAZUHISA HAO, HIROAKI OBATA, MAKOTO TOMITA, TAKESHI KASHIMURA, MASAHIRO ITO, YUKAKO OHNO, MAKOTO KODAMA, YOSHIFUSA AIZAWA Division of Cardiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan Background: Recently, we demonstrated that free heme, which is thought to be massively released from damaged cardiac cells, markedly induced various proinflammatory cytokines and chemokines. On the other hand, Toll-like receptor (TLR) which recognize conserved structures in pathogens, triggers innate immune responses, and recently, has been well known to recognize not only pathogen associated molecular pattern (PAMPS) but also damaged associated molecular pattern (DAMPS). Purpose: We examined the mechanism by which free heme induced proinflammatory cytokines in cells derived from normal hearts. Methods: Gene expressions of proinflammatory cytokines, intracellular reactive oxygen species (ROS) and NF-kB activation were examined in cells derived from normal hearts, macrophage cell line NR8383 and cells derived from TLR4-KO mice. Results: Free heme induced intracellular ROS, and moreover, activated NF-kB and induced proinflammatory cytokines such as IL-1 and TNF-a. Inhibition of NF-kB suppressed expressions of the cytokines but inhibition of ROS production did not suppress them. Expression levels of the cytokines induced by free heme in macrophages derived from TLR4-KO mice were significantly lower than those from wild type mice. Conclusion: These findings suggest that free heme activates