leptin and leptin receptor long isoform (ob-rb) are upregulated in cardiac myocytes by hypertensive...

S316 Journal of Cardiac Failure Vol. 11 No. 9 Suppl. 2005

P-061Interaction Between ERK and PP2B in Development of Anti-infarctTolerance of the MyocardiumYOSHIHIRO IKEDA, TETSUJI MIURA, TAKAYUKI MIKI, MASAYATANNO, TOSHIYUKI YANO, HIRONORI KOBAYASHI,MASAHIRO NISHIHARA, KAZUYUKI NAITOH, KATSUHIKOOOHORI, KAZUAKI SHIMAMOTOThe Second Department of Internal Medicine, Sapporo medical University,Sapporo, Japan

Purpose: The aim of this study was to examine possible interaction ofextracellular signal-regulated kinase (ERK) and protein phosphates 2B(PP2B) in cardioprotection by delta-opioid receptor activation.Methods: Infarction was induced by 20-min regional ischemia/2-h reperfu-sion in the rat hearts. Infarct size was expressed as % of risk area (%IS/RA). Using separate groups of rats, tissues were sampled from the riskarea for immunoblotting.Results: In controls, phospho-ERK1/2 level was not changed at 5 or 20min after ischemia,but the levelwas elevatedby6fold5minafter reperfusion.Administration of DADLE (1 mg/kg), an delta-opioid receptor agonist,before ischemia increased the phospho-ERK1/2 levels during ischemiawithout effects on their levels after reperfusion and reduced %IS/RA from46.1�3.1% to 23.2�2.5%. PD98059 (2.5 mg/kg) abolished ERK1/2 activa-tion during ischemia and infarct size limitation by DADLE (%IS/RA�43.5�4.3). PP2B inhibitors, cyclosporin-A (5 mg/kg) and FK506 (3.5mg/kg), mimicked the effect of DADLE (%IS/RA�30.5�2.0% and32.1�3.0%, respectively), and the combination of DADLE with thesePP2B inhibitors afforded little further protection (%IS/RA�27.5�2.2%and 21.0�2.1%). The infarct size-limiting effect of FK506 was inhibitedby PD98059 (%IS/RA�43.8�1.7%).Conclusion: These results suggest that activation of ERK1/2 during isch-emia is a part of the mechanism of cardiprotection by PP2B inhibition, andthis mechanism might be involved in delta-opioid-induced cardioprotection.

P-062Mitochondrial KATP Channel Opener Attenuates Ca Overload-inducedMyocyte ApoptosisKAZUYA TAMURA1, MIYUKI KOBARA1, TAKESHI NAKAE1,ERI GOUDA1, TETSUYA TATSUMI2, HIROAKI MATSUBARA2,TETSUO NAKATA1

1Department of Clinical Pharmacology, Kyoto Pharmaceutical University,Kyoto, Japan, 2Department of Cardiovascular Medicine, Kyoto PrefecturalUniversity of Medicine, Kyoto, Japan

Purpose: ATP-sensitive K (KATP) channel openers protect cardiomyocytesagainst ischemia-reperfusion injury. Calcium (Ca) overload plays a crucialrole in ischemia-reperfusion injury. The present study was designed toexamine whether Ca ionophore, ionomycin (0.5-1 µM), induces myocyteapoptosis and KATP channel openers attenuate ionomycin-inducedapoptosis.Methods: Neonatal rat cardiomyocytes were exposed to ionomycin inthe presence or absence of KATP channel openers: diazoxide, a selectivemitochondrial KATP channel opener, or nicorandil, a nonselective KATPchannel opener. Myocyte apoptosis was determined by nuclear stainingwith 4,6-diamidine-2-phenylindole and necrosis was determined by therelease of lactate dehydrogenase (LDH). Activity of caspase-3 was assayedwith colorimetric assay kit. Cytochrome c release was determined byWestern blotting.Results: Ionomycin (0.5 µM) increased the percentage of myocyte apoptosis(11.3�0.1%) without LDH release (75.8�1.2 IU/L), which was associatedwith synchronous cytochrome c release and caspase-3 activation. On theother hand, ionomycin (1 µM) significantly increased LDH release(121.4�4.6 IU/L). Diazoxide and nicorandil significantly decreased boththe percentage of apoptosis (5.7�0.1%, 2.4�0.1%) and necrosis(77.7�2.2 IU/L, 78.7�2.6 IU/L). These agents also suppressed cytochromec release and caspase-3 activation. These protective effects of diazoxidewere abrogated by a mitochondrial KATP channel blocker,5-hydroxydecanoate.Conclusion: Our data demonstrates that Ca ionophore induces myocyteapoptosis as well as necrosis, and that KATP channel openers suppress bothtypes of cell death.

P-063The Gene Expressions of Connective Tissue Growth Factor and BrainNatriuretic Peptide Are Coordinately Regulated in Cardiac MyocytesNORIMICHI KOITABASHI1, MASASHI ARAI MIDORI MORITA1,SHIRO HARA1, KAZUO NIWANO1, ATAI WATANABE1, MASAHIKOKURABAYASHI1, TAKASHI NISHIDA2, SATOSHI KUBOTA2,MASAHARU TAKIGAWA2

1Department of Medicine and Biological Science, Gunma University Gradu-ate School of Medicine, Maebashi, Japan, 2Department of Biochemistry andMolecular Dentistry, Okayama University Graduate School of Medicineand Dentistry, Okayama, Japan

Connective tissue growth factor (CTGF), a profibrotic cytokine, is increasedin human failing heart. We found that CTGF expression levels were closelycorrelated with the mRNA level of BNP, an antifibrotic peptide secretedby cardiac myocytes (CM), in pressure-overloaded rat hearts (N�101,r�0.801, P�0.001). Accordingly, we hypothesized that CM has a commontranscriptional regulatory system on CTGF and BNP genes and competi-tively regulate myocardial collagen production in cardiac fibroblast (CF).In cultured CM, endothelin-1 (ET1) and angiotensin II (AII) induced concor-dant induction of CTGF and BNP mRNA. Serial deletion annalysis ofCTGF gene promoter revealed that binding sites for Nkx2.5 and GATA4,which are also known to be critical in BNP transcription, are responsiblefor ET1 and AII-induced induction of CTGF. In fact, robust expression ofNkx2.5 or GATA4 equally increased CTGF and BNP gene transcription.On the other hands, TGF-beta and aldosterone upregulated CTGF but notBNP. In addition, robust expression of Smad2/4, which is known as targetsof TGF-beta increased CTGF but not BNP. In conclusion, our results suggestthat CM regulates collagen production in CF through both CTGF and BNPsecretion, which have partially common transcriptional control and oppositebiological effects. TGF-beta and/or aldosterone signaling may mediate dis-proportionate increase of CTGF against BNP and may trigger myocardialfibrosis and ventricular dysfunction.

P-064Leptin and Leptin Receptor Long Isoform (ob-Rb) Are Upregulatedin Cardiac Myocytes by Hypertensive StressHIROKI MATSUI1, TOMOYUKI YOKOYAMA2, CHIE TANAKA2,NORIMICHI KOITABASHI1, MASASHI ARAI1, MASAHIKOKURABAYASHI1

1Department of Medicine and Biological Sciences, Graduate School ofGunma University, Gunma, Japan, 2Department of Laboratory Sciences,Gunma University School of Health Sciences, Gunma, Japan

Purpose: Obesity is a major risk factor for the development of cardiovascu-lar desease. Leptin, adipocyte derived hormone, is elevated in patients withobesity. Hyperleptinemia is known to participate in cardiac hypertrophyand hypertension, but these link and mechanism are poorly understood.We therefore examined the expression of leptin (ob) and leptin receptorisoforms (ob-Ra, Rb) in pressure-overloaded rat heart.Method and Result: Pressure overload was produced by ligation of theabdominal aorta of Wistar rats at 1 to 28 days. Using Real-time PCR, ob-Ra mRNAs expression was not changed, while ob and ob-Rb mRNA wassignificantly increased in pressure-overloaded rat heart. Further, ob-Rbwas particularly detected in hypertrophic cardiomyocytes by immunohisto-chemistry. Plasma leptin levels were not different between sham and band-ing groups. To clarify which stress activates ob and ob-Rb expressions, weexamined these mRNA expressions in neonatal rat cardiac myocytes treatedwith angiotensin 2 (A2), endothelin 1 (ET-1) or cyclic mechanical stretch.A2, ET-1, and stretch stimulated ob mRNA expression, but ob-Rb wasactivated only stretch.Conclusion: Our results demonstrated for the first time that leptin andleptin receptor were upregulated in pressure-overloaded rat hearts. Further-more, these activations were confirmed in cardiac myocytes treated withA2, ET-1, and mechanical stretch. Thus, this study has a significant contribu-tion to the elucidation of novel link between obesity, hypertension, andcardiac hypertrophy.