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p300-mediated Inhibition of Doxorubicin-induced Myocardial Cell Apoptosis Involves Ubiquitin-dependent p53 Degradation
The 9th Annual Scientific Meeting
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JHFS
S289
O-057
O-059
Apoptosis Signal-regulating Kinase 1 is Involved Not Only in Apoptosis But Also in Non-apoptotic Cardiomyocyte Death TETSUYA WATANABE, KINYA OTSU, MASATSUGU HORI Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
The Pathophysiological Role of Endoplasmic Reticulum Stress in the Failing Hearts KEN-ICHIRO OKADA1, TETSUO MINAMINO1, HIDETOSHI OKAZAKI1, OSAMU TSUKAMOTO1, TADASHI ISOMURA2, SEIJI TAKASHIMA1, MASATSUGU HORI1, MASAFUMI KITAKAZE3 1 The Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Osaka, Japan, 2Hayama Heart Center, Kanagawa, Japan, 3National Cardiovascular Center, Osaka, Japan
Purpose: The molecular basis of myocardial cell death in the ischemiareperfused heart still remains to be clarified. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We studied ASK1⫺/⫺ 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 occluded for 30 min then reperfused for 2 h. The presence of apoptosis was detected by DNA ladder formation, terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase-3 activation. Results: In WT hearts, ischemia-reperfusion resulted in significant necrotic injury, as evidenced by a large area of negative triphenyltetrazolium chloride (TTC) staining (32.4⫾11.3% of area at risk). In ASK⫺/⫺ heart, infarct size was substantially smaller at 7.6⫾3.5% (p⬍0.01). The necrotic injury was not accompanied with any evidence of apoptosis such as an increase in TUNEL positive cells, DNA fragmentation or the activation of caspase-3. ASK1⫺/⫺ cardiomyocytes were more resistant to H2O2 - or Ca2⫹-induced apoptotic and non-apoptotic cell death compared with wild-type cells. Conclusions: These data suggest that ASK1 is involved in necrosis as well as apoptosis and that ASK1-dependent necrosis is likely to contribute to myocardial cell death in the ischemia-reperfused heart.
We have recently reported that endoplasmic reticulum (ER) stress, which can initiate apoptotic signaling, was induced in hypertrophic and failing hearts. However, the pathophysiological roles of ER stress in the development of heart failure are not well elucidated. Therefore, we investigated whether the ER-initiated apoptosis can contribute to the pathophysiology of failing hearts. Immunohistochemical analysis revealed that GRP78, a marker of ER stress, and CHOP/GADD153, a death-related transcription factor specifically induced by ER stress, were markedly induced in failing human hearts. Expression of spliced XBP1, another ER stress marker, was also frequently observed in the failing hearts. Quantitative RT-PCR demonstrated that the expression of both GRP78 and CHOP increased in failing human hearts. Furthermore, there was a positive correlation between cardiac expression of BNP and either GRP78 (r⫽0.60, p⬍0.05) or CHOP/GADD153 (r⫽0.53, p⬍0.05), suggesting that ER stress and ERinitiated apoptotic signal were induced as cardiac dysfunction progressed. Pharmacological ER stress inducers caused a significant up-regulation of CHOP/GADD153 and an increase in apoptotic cell death in cultured rat cardiac myocytes. Importantly, the ER stress-induced apoptosis was attenuated by the posttranslational gene silencing of CHOP/GADD153. Thus, we conclude that the ER-initiated apoptosis via CHOP/GADD153-dependent pathways may contribute to the development of heart failure. CHOP/ GADD153 is a logical target for drug development to prevent cardiac apoptosis in the failing hearts.
O-058 O-060
Autophagy is a Protective Mechanism in Glucose-depleted Adult Rat Ventricular Cardiomyocytes RUMI MARUYAMA1, GENZOU TAKEMURA1, SHUSAKU MIYATA1, HIDESHI OKADA1, YIWEN LI1, HIROMITSU KANAMORI1, LONGHU LI1, SHINYA MINATOGUCHI1, TAKAKO FUJIWARA2, HISAYOSHI FUJIWARA1 1 Department of Cardiology, Gifu University Graduate School of Medicine, Gifu, Japan, 2Kyoto Women’s University, Kyoto, Japan
p300-mediated Inhibition of Doxorubicin-induced Myocardial Cell Apoptosis Involves Ubiquitin-dependent p53 Degradation TATSUYA MORIMOTO1, KOH ONO2, TERUHISA KAWAMURA2, TORU KITA1, KOJI HASEGAWA2 1 Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 2Division of Translational Research, Kyoto Medical Center, National Hospital Organization
Although autophagic degenaration/death of cardiomyocytes has been recently reported to be associated with progression of heart failure, autophagy is originally an important physiological process and its pathophysiological significance is largely unknown. We induced autophagy in isolated adult rat ventricular cardiomyocytes (ARVC) by incubation with glucose-depleted/ mannitol-supplemented medium for 96h. The control was ARVC incubated with glucose-containing medium. Intracellular vacuoles were apparent in the glucose-depleted ARVC, which were immunopositive for cathepsin D and LC3. Ultrastructurally, the vacuoles contained degenerated subcellular organelles such as mitochondria, indicating that the vacuoles were autophagic. Serial videomicroscopic recording revealed temporary appearance of protrusion of cell surface, which apperered to reflect the development and disappearance of autophagic vacuoles within ARVC. Autophagic ARVC indeed showed significantly shorter survival intervals as assessed by the dye-exclusion method, compared with controls. We next loaded ARVC with the inhibitors of autophagy: 3-methyladenine, bafilomycin A1, and leupeptin. All of the inhibitors significantly shortened the lifespan of the ARVC in a dose dependent manner. Thus, autophagy seemed proective for the ARVC survival of the present glucose-depletion model. The present study recorded the whole process of autophagy in ARVC and suggested the protective role of autophagy for cell survival. Our findings may imply a possibility that autophagic degeneration of cardiomyocytes in heart failure reflects not a prelude to programmed cell death but represent a resistant mechanism.
While excess of an intrinsic histone acetyltransferase p300 results in overgrowth of cardiomyocytes, p300 depletion by doxorubicin results in apoptosis of these cells. Our previous study demonstrates that overexpression of p300 in the heart rescues cardiac myocytes from doxorubicininduced apoptosis. However, precise mechanisms regarding p300-mediated inhibition of apoptosis are unclear. p300 protein possesses ubiquitin ligase activity for p53 tumor suppressor gene product. Thus, p300 is able to inhibit p53-mediated apoptosis. The present study investigated regulation of ubiquitin-dependent p53 degradation in cardiac myocytes. A proteasome inhibitor MG132 increased both p300 and p53 protein levels in these cells, suggesting that ubiquitin-dependent degradation is involved in homeostasis of these proteins. Notably, treatment of myocytes with doxorubicin decreased protein levels of p300 but markedly increased those of p53. We show here that treatment with doxorubicin decreases poly-ubiquitinated p53 in cardiac myocytes. In addition, we have found that doxorubicin reduces the binding between p53 and p300 in cardiomyocytes. Thus, doxorubicin may suppress ubiquitin-dependent p53 degradation, in part, by dissociating p53 from p300 as well as by decreasing p300 protein levels. Finally, overexpression of p300 in cardiomyocytes suppressed doxorubicinmediated increase in p53 levels as well as inhibited doxorubicin-induced apoptosis. These findings suggest that p300, by its ligase activity, promotes ubiquitin-dependent degradation of p53, providing a novel mechanism of p300-mediated inhibition of doxorubicin-induced apoptosis in cardiac myocytes.
•
JHFS
S289
O-057
O-059
Apoptosis Signal-regulating Kinase 1 is Involved Not Only in Apoptosis But Also in Non-apoptotic Cardiomyocyte Death TETSUYA WATANABE, KINYA OTSU, MASATSUGU HORI Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
The Pathophysiological Role of Endoplasmic Reticulum Stress in the Failing Hearts KEN-ICHIRO OKADA1, TETSUO MINAMINO1, HIDETOSHI OKAZAKI1, OSAMU TSUKAMOTO1, TADASHI ISOMURA2, SEIJI TAKASHIMA1, MASATSUGU HORI1, MASAFUMI KITAKAZE3 1 The Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Osaka, Japan, 2Hayama Heart Center, Kanagawa, Japan, 3National Cardiovascular Center, Osaka, Japan
Purpose: The molecular basis of myocardial cell death in the ischemiareperfused heart still remains to be clarified. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We studied ASK1⫺/⫺ 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 occluded for 30 min then reperfused for 2 h. The presence of apoptosis was detected by DNA ladder formation, terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase-3 activation. Results: In WT hearts, ischemia-reperfusion resulted in significant necrotic injury, as evidenced by a large area of negative triphenyltetrazolium chloride (TTC) staining (32.4⫾11.3% of area at risk). In ASK⫺/⫺ heart, infarct size was substantially smaller at 7.6⫾3.5% (p⬍0.01). The necrotic injury was not accompanied with any evidence of apoptosis such as an increase in TUNEL positive cells, DNA fragmentation or the activation of caspase-3. ASK1⫺/⫺ cardiomyocytes were more resistant to H2O2 - or Ca2⫹-induced apoptotic and non-apoptotic cell death compared with wild-type cells. Conclusions: These data suggest that ASK1 is involved in necrosis as well as apoptosis and that ASK1-dependent necrosis is likely to contribute to myocardial cell death in the ischemia-reperfused heart.
We have recently reported that endoplasmic reticulum (ER) stress, which can initiate apoptotic signaling, was induced in hypertrophic and failing hearts. However, the pathophysiological roles of ER stress in the development of heart failure are not well elucidated. Therefore, we investigated whether the ER-initiated apoptosis can contribute to the pathophysiology of failing hearts. Immunohistochemical analysis revealed that GRP78, a marker of ER stress, and CHOP/GADD153, a death-related transcription factor specifically induced by ER stress, were markedly induced in failing human hearts. Expression of spliced XBP1, another ER stress marker, was also frequently observed in the failing hearts. Quantitative RT-PCR demonstrated that the expression of both GRP78 and CHOP increased in failing human hearts. Furthermore, there was a positive correlation between cardiac expression of BNP and either GRP78 (r⫽0.60, p⬍0.05) or CHOP/GADD153 (r⫽0.53, p⬍0.05), suggesting that ER stress and ERinitiated apoptotic signal were induced as cardiac dysfunction progressed. Pharmacological ER stress inducers caused a significant up-regulation of CHOP/GADD153 and an increase in apoptotic cell death in cultured rat cardiac myocytes. Importantly, the ER stress-induced apoptosis was attenuated by the posttranslational gene silencing of CHOP/GADD153. Thus, we conclude that the ER-initiated apoptosis via CHOP/GADD153-dependent pathways may contribute to the development of heart failure. CHOP/ GADD153 is a logical target for drug development to prevent cardiac apoptosis in the failing hearts.
O-058 O-060
Autophagy is a Protective Mechanism in Glucose-depleted Adult Rat Ventricular Cardiomyocytes RUMI MARUYAMA1, GENZOU TAKEMURA1, SHUSAKU MIYATA1, HIDESHI OKADA1, YIWEN LI1, HIROMITSU KANAMORI1, LONGHU LI1, SHINYA MINATOGUCHI1, TAKAKO FUJIWARA2, HISAYOSHI FUJIWARA1 1 Department of Cardiology, Gifu University Graduate School of Medicine, Gifu, Japan, 2Kyoto Women’s University, Kyoto, Japan
p300-mediated Inhibition of Doxorubicin-induced Myocardial Cell Apoptosis Involves Ubiquitin-dependent p53 Degradation TATSUYA MORIMOTO1, KOH ONO2, TERUHISA KAWAMURA2, TORU KITA1, KOJI HASEGAWA2 1 Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 2Division of Translational Research, Kyoto Medical Center, National Hospital Organization
Although autophagic degenaration/death of cardiomyocytes has been recently reported to be associated with progression of heart failure, autophagy is originally an important physiological process and its pathophysiological significance is largely unknown. We induced autophagy in isolated adult rat ventricular cardiomyocytes (ARVC) by incubation with glucose-depleted/ mannitol-supplemented medium for 96h. The control was ARVC incubated with glucose-containing medium. Intracellular vacuoles were apparent in the glucose-depleted ARVC, which were immunopositive for cathepsin D and LC3. Ultrastructurally, the vacuoles contained degenerated subcellular organelles such as mitochondria, indicating that the vacuoles were autophagic. Serial videomicroscopic recording revealed temporary appearance of protrusion of cell surface, which apperered to reflect the development and disappearance of autophagic vacuoles within ARVC. Autophagic ARVC indeed showed significantly shorter survival intervals as assessed by the dye-exclusion method, compared with controls. We next loaded ARVC with the inhibitors of autophagy: 3-methyladenine, bafilomycin A1, and leupeptin. All of the inhibitors significantly shortened the lifespan of the ARVC in a dose dependent manner. Thus, autophagy seemed proective for the ARVC survival of the present glucose-depletion model. The present study recorded the whole process of autophagy in ARVC and suggested the protective role of autophagy for cell survival. Our findings may imply a possibility that autophagic degeneration of cardiomyocytes in heart failure reflects not a prelude to programmed cell death but represent a resistant mechanism.
While excess of an intrinsic histone acetyltransferase p300 results in overgrowth of cardiomyocytes, p300 depletion by doxorubicin results in apoptosis of these cells. Our previous study demonstrates that overexpression of p300 in the heart rescues cardiac myocytes from doxorubicininduced apoptosis. However, precise mechanisms regarding p300-mediated inhibition of apoptosis are unclear. p300 protein possesses ubiquitin ligase activity for p53 tumor suppressor gene product. Thus, p300 is able to inhibit p53-mediated apoptosis. The present study investigated regulation of ubiquitin-dependent p53 degradation in cardiac myocytes. A proteasome inhibitor MG132 increased both p300 and p53 protein levels in these cells, suggesting that ubiquitin-dependent degradation is involved in homeostasis of these proteins. Notably, treatment of myocytes with doxorubicin decreased protein levels of p300 but markedly increased those of p53. We show here that treatment with doxorubicin decreases poly-ubiquitinated p53 in cardiac myocytes. In addition, we have found that doxorubicin reduces the binding between p53 and p300 in cardiomyocytes. Thus, doxorubicin may suppress ubiquitin-dependent p53 degradation, in part, by dissociating p53 from p300 as well as by decreasing p300 protein levels. Finally, overexpression of p300 in cardiomyocytes suppressed doxorubicinmediated increase in p53 levels as well as inhibited doxorubicin-induced apoptosis. These findings suggest that p300, by its ligase activity, promotes ubiquitin-dependent degradation of p53, providing a novel mechanism of p300-mediated inhibition of doxorubicin-induced apoptosis in cardiac myocytes.