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GATA4 and Suppress Hypertrophic Responses in Cardiomyocytes
S138 Journal of Cardiac Failure Vol. 19 No. 10S October 2013 O-022 ULK1 Plays a Critical Role in Mediating Autophagy in response to Stress Conditions in the Heart YASUHIRO MAEJIMA1, PEIYONG ZHAI2, MONDIRA KUNDU3, JUNICHI SADOSHIMA2 1 Cardiovascular Medicine, Tokyo Medical and Dental University, 2Cell Biology and Molecular Medicine, New Jersey Medical School, 3Pathology, St. Jude Children’s Research Hospital Autophagy plays an important role in mediating protein quality control (QC) in cells. Inhibition of autophagy below physiological levels induces protein aggregation and cellular dysfunction. To elucidate the function of Mammalian UNC-51-like kinase-1 (ULK1), a ortholog of yeast Atg1, in the heart, we used ULK1 knock out mice (ULK1-KO). The autophagic activity of cardiomyocytes (CMs) at baseline was similar in ULK1-KO and wild type mice (WT), and there was no significant difference in cardiac phenotype at 3 months of age. In response to pressure overload (PO) caused by transverse aortic constriction (TAC) for 4 weeks, however, left ventricular weight (LVW)/tibial length (TL) was significantly greater in ULK1-KO than in WT. LV ejection fraction (LVEF) was significantly lower in ULK1-KO than in WT, whereas lung weight/TL was greater in ULK1-KO than in WT. Histological analyses indicated that protein aggregates were significantly accumulated in the peri-nuclear region of CMs in ULK1-KO compared to WT, suggesting that protein aggregates were accumulated in ULK1-KO after TAC. The protein aggregates co-stained with p62 in ULK1-KO, suggesting that autophagic flux is inhibited in ULK1-KO after TAC. In addition, at 18 months of age, ULK1-KO exhibited a greater LVW/TL and lower LVEF than WT even at baseline. These results suggest that ULK1 plays a crucial role in mediating autophagy in the heart therein under stress conditions, thereby mediating protein QC and maintaining cardiac function in the heart.
O-023 Receptor for Activated Protein Kinase C1 Forms a Functional Complex with p300/GATA4 and Suppress Hypertrophic Responses in Cardiomyocytes HIDETOSHI SUZUKI1, YOICHI SUNAGAWA1,2, YASUFUMI KATANASAKA1,2, HIROMICHI WADA2, AKIRA SHIMATSU2, KOJI HASEGAWA2, TATSUYA MORIMOTO1 1 Division of Molecular Medicine, University of Shizuoka, 2Division of Translational Research, Kyoto Medical Center Introduction: A zinc finger protein GATA4 is one of the factors involved in transcriptional regulation during myocardial cell hypertrophy and forms a functional complex with an intrinsic histone acetyltransferase (HAT), p300. HAT activity of p300 is required for acetylation and the transcriptional activation of GATA4 as well as for cardiomyocyte hypertrophy and the development of heart failure in vivo. By tandem affinity purification and mass spectrometric analyses, we identified a receptor for activated protein kinase C1 (RACK1), a multi-functional scaffold protein, as a novel GATA4-binding partner. However, the precise functional relationships among p300, GATA4, and RACK1 remain unknown. Methods and Results: Using glutathione S-transferase pull-down assays, we showed that RACK1 physically interacts with two binding domains in GATA4. Immunoprecipitation-Western blot analysis demonstrated that RACK1 interacted with GATA4 in HEK293T cells. Overexpression of RACK1 reduced the binding between p300 and GATA4 and inhibited p300 induced acetylation of GATA4 and p300/GATA4induced atrial natriuretic factor (ANF) and endothelin-1 (ET-1) promoter activities. Furthermore overexpression of RACK1 in cardiomyocytes significantly inhibited phenylephrine (PE)-induced hypertrophic responses such as myofibrillar organization, an increase in cell size and the activation of the ANF and ET-1 promoters. PE stimulation decreased the association between RACK1 and GATA4 in cardiomyocytes. Conclusions: RACK1 forms a functional complex with p300/GATA4 and plays a repressive role in myocardial cell hypertrophy and gene transcription.
O-024 The Role of mTORC2 and Ribosomal Protein S6 in Cardioprotective Signaling TOSHIYUKI YANO1, MARCELLA FERLITO2, TOSHIYUKI TOBISAWA1, ATSUSHI OGASAWARA1, HIROMICHI MURASE1, ATSUSHI KUNO4, MASAYA TANNO1, TAKAYUKI MIKI1, CHARLES STEENBERGEN3, TETSUJI MIURA1 1 Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University, 2Department of Medicine, Johns Hopkins Medical Institutes, 3 Department of Pathology, Johns Hopkins Medical Institutes, 4Department of Pharmacology, Sapporo Medical University Objectives: The aim of this study is to get new insights into the mechanism by which phosphatidylinositol 3-kinase (PI3K)/Akt signaling is connected to
suppression of cell death. Methods and Results: Using an antibody that detects phosphorylation of serine and/or threonine on Akt consensus sequences (RXRXXS/T), we have found that ischemic preconditioning (IPC) and other Akt activators (insulin, opioids, diazoxide) result in phosphorylation of ribosomal protein S6 (Rps6) at Ser235/236 in mouse hearts and neonatal rat ventricular myocytes. Rps6 interacts with components of mTOR complex 2 (mTORC2), and siRNA-mediated knockdown of Rps6 attenuates insulin- and opioid-induced mTORC2 activation and Akt-Ser473 phosphorylation, indicating that Rps6 activation amplifies mTORC2/Akt signaling. Furthermore, reduction of Rps6 phosphorylation by Rps6 knockdown eliminated the protective effect of opioids on oxidative stressinduced cardiomyocyte necrosis. Finally, the role of mTORC2 on IPC-induced cardioprotection was examined in isolated buffer-perfused mouse hearts. IPC increased mTORC2 activity, leading to phosphorylation of Akt on Ser473. IPCinduced infarct size limitation was lost by pretreatment with dual mTORC inhibitors but not with rapamycin, a mTORC1 inhibitor, which indicates the fundamental role of mTORC2 activation in cardioprotection. Conclusion: Activation of mTORC2 plays a pivotal role in cardioprotection, and Rps6 is a convergence point of cardioprotective signaling, providing positive feedback regulation of mTORC2/ Akt signaling.
O-025 Prognositc Value of Urc acid Levels in Elderly Chronic Heart Failure Patients YASUHIKO MITSUKE1, REIKO NAKAYA2, CHIYO KIRIBA3, HIROSHI TSUTANI3 1 Department of Cardiology, NHO Awara National Hospital, 2Department of Cardiology, NHO Fukui National Hospital, 3Department of Internal Medicine, NHO Awara National Hospital Background: Several circulating neurohormones and inflammatory markers have been shown to have a prognostic significance in chronic heart failure (CHF). Elevated levels of uric acid (UA) have been reported to predict adverse cardiovascular prognosis in CHF patients. However, there were few reports describing clinical value of circulating UA levels in elderly CHF patients. Methods: We studied consecutive 142 Japanese CHF patients older than 75 years of age (85 females, 78.1 +/3.9 years, all serum creatinine levels were !1.0 mg/dl, NYHA II-III, LVEF! 45%). We measured circulating levels of UA, norepinephrine(NE), brain natriuretic peptides (BNP), and interleukin-6 (IL-6). None had evidence of unstable angina, chronic inflammatory disease, collagen disease, or cancer at the time of evaluation. Results: Circulating levels of UA were correlated with circulating levels of IL-6 (r50.56, p!0.03), and BNP (r50.42, p!0.05). Patients were followed up for an average of 38.1months, and 22 patients had death. Patients with events had significantly higher circulating levels of UA, NE, BNP, and IL-6 levels. By multivariate Cox proportional hazard analysis, circulating levels of UA,BNP, and IL-6 were significant predictors in those patients. Conclusion: Our data indicated the possibility that circulating levels of UA were related to the levels of circulating pro-inflammatory cytokines, neurohormones, and prognosis of those elderly CHF patients.
O-026 The Relationship between TNF-alpha Levels and Adiponectin Levels in the Patients with Congestive Heart Failure TOMOHIRO NAKAMURA1, YOSHITAKA SUGAWARA2, HIROSHI FUNAYAMA2, CHIKASHI SUGA2, TAKESHI MITSUHASHI2, SHIN-ICHI MOMOMURA2 1 Internal Medicine, Saitama Citizens Medical Center, 2Cardiovascular, Saitama Medical Center, Jichi Medical University Objectives: This study aimed to examine the relationship between circulating tumor necrosis factor-alpha (TNF-alpha) levels and the severity of congestive heart failure (CHF). Background: TNF-alpha, which is one of inflammatory cytokines, appears to be elevated according to the severity of CHF and cardiac performance. In addition, adiponection has roles of cardiovascular protection and antiinflammatory effect. Methods: Ninety-two patients with systolic heart failure (ejection fraction !40%) and twenty control subjects were enrolled. Results: Plasma TNF-alpha levels were significantly increased according to the severity of NYHA class; control: 2.9 6 1.8; NYHA I: 3.6 6 3.3, NYHA II: 6.8 6 5.1, NYHA III: 6.0 6 4.6, NYHA IV: 8.4 6 4.1 pg/ml (p50.0001). Plasma TNF-alpha levels correlated positively with adiponectin (r 5 0.52, p ! 0.0001), BNP (r 5 0.37, p ! 0.0001), norepinephrine (r 5 0.34, p 5 0.0006), arginine vasopressin (r 5 0.36, p 5 0.0005) and creatinine (r 5 0.24, p 5 0.02). In the multiple regression analysis, plasma adiponectin was an independent factor for contributing to plasma TNF-alpha concentration (p 5 0.03). Conclusions: In CHF patients, plasma TNF-alpha levels are increased according to the severity of CHF and have a positive correlation with plasma adiponectin levels. These results may indicate that augmented release of pro- and anti-inflammatory cytokine is involved in the pathogenesis of CHF.
O-023 Receptor for Activated Protein Kinase C1 Forms a Functional Complex with p300/GATA4 and Suppress Hypertrophic Responses in Cardiomyocytes HIDETOSHI SUZUKI1, YOICHI SUNAGAWA1,2, YASUFUMI KATANASAKA1,2, HIROMICHI WADA2, AKIRA SHIMATSU2, KOJI HASEGAWA2, TATSUYA MORIMOTO1 1 Division of Molecular Medicine, University of Shizuoka, 2Division of Translational Research, Kyoto Medical Center Introduction: A zinc finger protein GATA4 is one of the factors involved in transcriptional regulation during myocardial cell hypertrophy and forms a functional complex with an intrinsic histone acetyltransferase (HAT), p300. HAT activity of p300 is required for acetylation and the transcriptional activation of GATA4 as well as for cardiomyocyte hypertrophy and the development of heart failure in vivo. By tandem affinity purification and mass spectrometric analyses, we identified a receptor for activated protein kinase C1 (RACK1), a multi-functional scaffold protein, as a novel GATA4-binding partner. However, the precise functional relationships among p300, GATA4, and RACK1 remain unknown. Methods and Results: Using glutathione S-transferase pull-down assays, we showed that RACK1 physically interacts with two binding domains in GATA4. Immunoprecipitation-Western blot analysis demonstrated that RACK1 interacted with GATA4 in HEK293T cells. Overexpression of RACK1 reduced the binding between p300 and GATA4 and inhibited p300 induced acetylation of GATA4 and p300/GATA4induced atrial natriuretic factor (ANF) and endothelin-1 (ET-1) promoter activities. Furthermore overexpression of RACK1 in cardiomyocytes significantly inhibited phenylephrine (PE)-induced hypertrophic responses such as myofibrillar organization, an increase in cell size and the activation of the ANF and ET-1 promoters. PE stimulation decreased the association between RACK1 and GATA4 in cardiomyocytes. Conclusions: RACK1 forms a functional complex with p300/GATA4 and plays a repressive role in myocardial cell hypertrophy and gene transcription.
O-024 The Role of mTORC2 and Ribosomal Protein S6 in Cardioprotective Signaling TOSHIYUKI YANO1, MARCELLA FERLITO2, TOSHIYUKI TOBISAWA1, ATSUSHI OGASAWARA1, HIROMICHI MURASE1, ATSUSHI KUNO4, MASAYA TANNO1, TAKAYUKI MIKI1, CHARLES STEENBERGEN3, TETSUJI MIURA1 1 Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University, 2Department of Medicine, Johns Hopkins Medical Institutes, 3 Department of Pathology, Johns Hopkins Medical Institutes, 4Department of Pharmacology, Sapporo Medical University Objectives: The aim of this study is to get new insights into the mechanism by which phosphatidylinositol 3-kinase (PI3K)/Akt signaling is connected to
suppression of cell death. Methods and Results: Using an antibody that detects phosphorylation of serine and/or threonine on Akt consensus sequences (RXRXXS/T), we have found that ischemic preconditioning (IPC) and other Akt activators (insulin, opioids, diazoxide) result in phosphorylation of ribosomal protein S6 (Rps6) at Ser235/236 in mouse hearts and neonatal rat ventricular myocytes. Rps6 interacts with components of mTOR complex 2 (mTORC2), and siRNA-mediated knockdown of Rps6 attenuates insulin- and opioid-induced mTORC2 activation and Akt-Ser473 phosphorylation, indicating that Rps6 activation amplifies mTORC2/Akt signaling. Furthermore, reduction of Rps6 phosphorylation by Rps6 knockdown eliminated the protective effect of opioids on oxidative stressinduced cardiomyocyte necrosis. Finally, the role of mTORC2 on IPC-induced cardioprotection was examined in isolated buffer-perfused mouse hearts. IPC increased mTORC2 activity, leading to phosphorylation of Akt on Ser473. IPCinduced infarct size limitation was lost by pretreatment with dual mTORC inhibitors but not with rapamycin, a mTORC1 inhibitor, which indicates the fundamental role of mTORC2 activation in cardioprotection. Conclusion: Activation of mTORC2 plays a pivotal role in cardioprotection, and Rps6 is a convergence point of cardioprotective signaling, providing positive feedback regulation of mTORC2/ Akt signaling.
O-025 Prognositc Value of Urc acid Levels in Elderly Chronic Heart Failure Patients YASUHIKO MITSUKE1, REIKO NAKAYA2, CHIYO KIRIBA3, HIROSHI TSUTANI3 1 Department of Cardiology, NHO Awara National Hospital, 2Department of Cardiology, NHO Fukui National Hospital, 3Department of Internal Medicine, NHO Awara National Hospital Background: Several circulating neurohormones and inflammatory markers have been shown to have a prognostic significance in chronic heart failure (CHF). Elevated levels of uric acid (UA) have been reported to predict adverse cardiovascular prognosis in CHF patients. However, there were few reports describing clinical value of circulating UA levels in elderly CHF patients. Methods: We studied consecutive 142 Japanese CHF patients older than 75 years of age (85 females, 78.1 +/3.9 years, all serum creatinine levels were !1.0 mg/dl, NYHA II-III, LVEF! 45%). We measured circulating levels of UA, norepinephrine(NE), brain natriuretic peptides (BNP), and interleukin-6 (IL-6). None had evidence of unstable angina, chronic inflammatory disease, collagen disease, or cancer at the time of evaluation. Results: Circulating levels of UA were correlated with circulating levels of IL-6 (r50.56, p!0.03), and BNP (r50.42, p!0.05). Patients were followed up for an average of 38.1months, and 22 patients had death. Patients with events had significantly higher circulating levels of UA, NE, BNP, and IL-6 levels. By multivariate Cox proportional hazard analysis, circulating levels of UA,BNP, and IL-6 were significant predictors in those patients. Conclusion: Our data indicated the possibility that circulating levels of UA were related to the levels of circulating pro-inflammatory cytokines, neurohormones, and prognosis of those elderly CHF patients.
O-026 The Relationship between TNF-alpha Levels and Adiponectin Levels in the Patients with Congestive Heart Failure TOMOHIRO NAKAMURA1, YOSHITAKA SUGAWARA2, HIROSHI FUNAYAMA2, CHIKASHI SUGA2, TAKESHI MITSUHASHI2, SHIN-ICHI MOMOMURA2 1 Internal Medicine, Saitama Citizens Medical Center, 2Cardiovascular, Saitama Medical Center, Jichi Medical University Objectives: This study aimed to examine the relationship between circulating tumor necrosis factor-alpha (TNF-alpha) levels and the severity of congestive heart failure (CHF). Background: TNF-alpha, which is one of inflammatory cytokines, appears to be elevated according to the severity of CHF and cardiac performance. In addition, adiponection has roles of cardiovascular protection and antiinflammatory effect. Methods: Ninety-two patients with systolic heart failure (ejection fraction !40%) and twenty control subjects were enrolled. Results: Plasma TNF-alpha levels were significantly increased according to the severity of NYHA class; control: 2.9 6 1.8; NYHA I: 3.6 6 3.3, NYHA II: 6.8 6 5.1, NYHA III: 6.0 6 4.6, NYHA IV: 8.4 6 4.1 pg/ml (p50.0001). Plasma TNF-alpha levels correlated positively with adiponectin (r 5 0.52, p ! 0.0001), BNP (r 5 0.37, p ! 0.0001), norepinephrine (r 5 0.34, p 5 0.0006), arginine vasopressin (r 5 0.36, p 5 0.0005) and creatinine (r 5 0.24, p 5 0.02). In the multiple regression analysis, plasma adiponectin was an independent factor for contributing to plasma TNF-alpha concentration (p 5 0.03). Conclusions: In CHF patients, plasma TNF-alpha levels are increased according to the severity of CHF and have a positive correlation with plasma adiponectin levels. These results may indicate that augmented release of pro- and anti-inflammatory cytokine is involved in the pathogenesis of CHF.