New In Vivo Apoptosis Model of Cardiomyocytes and Bcl-2 gene therapy

• Project/Area Number: 13470143
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Circulatory organs internal medicine
• Research Institution: Gifu University
• Principal Investigator: FUJIWARA Hisayoshi Graduate School Medicine Graduate Course, Professor, 大学院・医学研究科, 教授 (80115930)
• Co-Investigator(Kenkyū-buntansha): KOSAI Kenichiro School of Medicine, Associate Professor, 医学部, 助教授 (90258418) ; AKEMURA Genzo T Univ. Hospital, Lecturer, 大学院・医学研究科, 教授 (40283311)
• Project Period (FY): 2001 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥14,000,000 (Direct Cost: ¥14,000,000); Fiscal Year 2002: ¥3,300,000 (Direct Cost: ¥3,300,000); Fiscal Year 2001: ¥10,700,000 (Direct Cost: ¥10,700,000)
• Keywords: apoptosis / Fas-ligand / caspase / sensitivity / clearance rate / ultrastructure / gene therapy / clearance rate / Bcl-2遺伝子治療

Research Abstract

There is no adequate in vivo model of apoptotic cardiomyocyte. The purpose of the present study was to develop a new apoptosis model of heart, and to delineate sensitivity to the apoptotic signal and the clearance rate and ultrastructure of apoptosis in in vivo adult cardiomyocytes and interstitial cells of the heart in comparison with those of hepatocytes and interstitial cells of the liver by use of a direct injection method of soluble Fas ligand (sFL) into adult rat hearts and livers. In addition, it was studied whether Bcl-2 gene therapy can inhibit the Fas-induced apoptosis.
To minimize the systemic influence, soluble Fas ligand was injected directly into in vivo rat hearts and livers (as the control) at concentrations of 0, 0.5, 2, and 5 μg/mL (groups C, F0.5, F2, and F5). Apoptotic cardiomyocytes and apoptotic noncardiomyocytes of the heart were identified with similar incidences only in F5. Their incidents peaked at 12 hours after injecction (2.0±0.09% in cardiomyocytes) and dim inished markedly 24 hours later. Caspase-3 was activated only in F5. Boc-Asp-fmk, a pancaspase inhibitor, inhibited apoptosis, suggesting that the apoptosis sensitivity was regulated upstream of caspase-3. Apoptotic noncardiomyocytes showed typical ultrastructure. In addition to the typical ultrastructure, such as cellular shrinkage, chromatin condensation, and apoptotic bodies, however, apoptotic cardiomyocytes showed unique features : doughnut-like, but not half-moon- or crescent-like, chromatin condensation : frequent plasma membrane rupture even during the early stage ; condensed mitochondria with wrinkled cristae inside ; the appearance of cytoplasmic lipid-like droplets ; and myofibrillar derangement. In the livers, typical apoptosis was induced in hepatocytes and nonhepatocytes of the liver even in the F0.5 group, which were cleared 24 hours later. Bcl-2 gene therapy blocked significantly apoptotic cells.
Compared with liver cells, cardiomyocytes as well as noncardiomyocytes of the heart are more resistant against the apoptotic signal, but the clearance is similarly rapid (within 24 hours). The ultrastructure of apoptotic cardiomyocytes is unique. These findings provide new insights into the dynamics of cell death in the heart.

Research Products

• [Publications] Takemura G et al.: "Characterization of ultrastructure and its relation with DNA fragmentaion in Fas-induced apoptosis of cultcned myocytes"J Pathol. 193(4). 546-556 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Maruyama R et al.: "Dynamic process of apoptosis in adult rat cardiomyocytes analyzed using 48-hour videmomicroseopy and electron microscopy"Am J Pathol. 159(8). 683-691 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Hayakawa et al.: "Sensitivity to apoptosis signal, clearnce rate, and ultrastructure of Fas ligand-induced apoptosis in in vivo adult cardiac cells"Circulation. 105(21). 3039-3045 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] T.Aoyama et al.: "Molecular mechanisms of non-apoptos is by Fas stimulation alone versus apoptosis with an additional actinomysin D in cultured cardiomyocytes"Cardiovasc Res. 55(4). 787-798 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Li Yiwen et al.: "Post-infarct Treatment With an Adenovial Vector Expressing Hepatocyte Growth Factor Relieves Chronic Left Venticular Remodeling and Dysfunction in Mice"Circulation. (in press). (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Hayakawa K et al.: "Inhibition of Granulation Tissue Cell Apoptosis during the Subacute Stage of Myocardial Infarction Improves Cardiac Remodeling and Dysfunction at the Chronic Stage"Circulation. (in press). (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Takemura G, Kato S, Aoyama T, Hayakawa Y, Kanoh M, Maruyama R, Arai M, Nishigaki K. Minatoguchi S, Fukuda K, Fujiwara T, Fujiwara H: "Characterization of ultrastructure and its relation with DNA fragmentaion in Fas-induced apoptosis of cultcned myocytes"J Pathol.. 193 (4). 546-556 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Maruyama R, Takemura G, Aoyama T, Hayakawa K, Koda M, Kawase Y, Que X, Ohno Y, Minatoguchi S, Miyata K, Fijiwara T, Fujiwara H: "Dynamic process of apoptosis in adult rat cardiomyocytes analyzed using 48-hour videmomicroseopy and electron microscopy"Am J Pathol.. 159 (8). 683-691 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Hayakawa K, Takemura G, Koda M, Kawase Y, Maruyama R, Li Yiwen, Minatoguchi S, Fujiwara T, Fujiwara H: "Sensitivity to apoptosis signal, clearnce rate, and ultrastructure of Fas ligand-induced apoptosis in in vivo adult cardiac cells"Circulation. 105(21). 3039-3045 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Aoyama T, Takemuta G, Maruyama R, Kosai K, Takahashi T, Koda M, Hayakawa K, Kawase Y, Minatoguchi S, Fujiwara H: "Molecular mechanisms of non-apoptosis by Fas stimulation alone versus apoptosis with an additional actinomysin D in cultured cardiomyocytes"Cardiovasc Res.. 55(4). 787-798 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Li Y, Takemura G, Kosai K, Yuge K, Nagano S, Esaki M, Goto K, Takahashi T, Hayakawa K, Koda M, Kawase Y, Maruyama R, Okada H, Minatoguchi S, Mizuguchi H, Fujiwara T, Fujiwara H: "Post-infarct Treatment With an Adenovial Vector Expressing Hepatocyte Growth Factor Relieves Chronic Left Venticular Remodeling and Dysfunction in Mice"Circulation. In press. (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Hayakawa K, Takemura G, Kanoh M, Li Y, Koda M, Kawase Y, Maruyama R, Hideshi O, Minatoguchi S, Fujiwara T, Fujiwara H.: "Inhibition of Granulation Tissue Cell Apoptosis during the Subacute Stage of Myocardial Infarction Improves Cardiac Remodeling and Dysfunction at the Chronic Stage"Circulation. In press. (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Hayakawa K et al.: "Inhibition of Granulation Tissue Cell Apoptosis During the Subacute Stage of Myocardial Infarction Improves Cardiac Remodeling and Dysfunction at the Chronic Stage"Circulation. (In press). (2003)
• [Publications] Li Yiwen et al.: "Post-infarction Treatment With an Adenovial Vector Expressing Hepatocyte Growth Factor Relieves Chronic Left Venticular Remodeling and Dysfunction in Mice"Circulation. 107. 2499-2506 (2003)
• [Publications] Matsuo H et al.: "Validation of Collateral Fractional Flow Reserve by Myocardial Perfusion Imaging"Circulation. 105. 1060-1065 (2002)
• [Publications] Sano H et al.: "Blockade of platelet-derived growth factor receptor-beta pathway induces apoptosis of vascular endothelial cells and disrupts glomerular capillary formation in neonatal mice"Am J Pathol. 161(1). 135-143 (2002)
• [Publications] T.Aoyama et al.: "Molecular mechanisms of non-apoptosis by Fas stimulation alone versus apoptosis with an additional actinomysin D in cultured cardiomyocytes"Cardiovasc Res. 55(4). 787-798 (2002)
• [Publications] Hayakawa K et al.: "Sensitivity to apoptosis signal, clearnce rate, and ultrastructure of Fas ligand-induced apoptosis in in vivo adult cardiac cells"Circulation. 105(21). 3039-3045 (2002)
• [Publications] Takemura G et al.: "Characterization of ultrastructure and its relation with DNA fragmentaion in Fas-induced apoptosis of cultcned myocytes"J Pathol. 193(4). 546-556 (2001)
• [Publications] Maruyama R et al.: "Dynamic process of apoptosis in adult rat cardiomyocytes analyzed using 48-hour videmomicroseopy and electron microscopy"Am J Pathol. 159(8). 683-691 (2001)
• [Publications] Wada H et al.: "Tumor necrosis factor-α plays a protective role in active viral myocarditis in mice"Circulation. 103(2). 743-749 (2001)
• [Publications] Matsuo H et al.: "Validation of collateral fractional flow recerve by myocardial perfusion Imaging"Circulation. (In press). (2002)
• [Publications] Hayakawa K et al.: "Sensitivity to apoptosis signal,clearance rate and ultrastructure of Fas Ligand-induced apoptosis in in vivo adult cardiac cells"Circulation. (In press). (2002)