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ȣ - 500772 348 |
| Increased Glucose Uptake by Amphiphysin SH3 Reduces Hypoxia-induced Apoptosis in Cultured Neonatal Rat Cardiomyocytes. |
| 충북대학교병원 순환기내과¹, 내분비내과 ² |
| 김유경¹, 오태근², 권진숙², 정일하¹, 박노관 ¹, 배장환¹, 황경국¹, 김동운¹, 조명찬¹ |
Backgrounds and Objectives: Amphiphysin was known to play an important role in clathrin-mediated endocytosis. The endocytosis of GLUT4 glucose transporter in adipocytes is thought to occur at least in part via the clathrin-mediated endocytic system. In the cardiomyocytes (CMCs), the role of Amphiphysin was not known. We investigated the effect of glucose-uptake by the Amphiphysin SH3 domain gene expression, defective Amphiphysin, to cultured neonatal rat CMCs under the normoxic or hypoxic conditions. Methods: We ligated Amphiphysin SH3 with lenti-viral vector using multi-cloning sites, and produced lenti-virus for gene transfer using calcium phosphate method at 293 T cell (human embryonic kidney cell). Neonatal rat CMCs (5×105 cells) were infected by lenti-Amphiphysin SH3 virus (1.5×107 IU) for 48 hours. Transfected CMCs by lenti-Amphiphysin SH3 virus (CMCs/Amp-SH3) virus were evaluated by confocal laser microscope. We measured the glucose-uptake of CMCs/Amp-SH3 using glucose transport assay with control at normoxic condition. After hypoxic injury with the hypoxic chamber (1% O2) for 48 hours, we measured the apoptosis of CMCs/Amp-SH3 by microscopic examination and evaluated the survival signals by Western blot analysis. Results: 1) More than 90% of CMCs had GFP expression by confocal laser microscope. Glucose uptake was increased by approximately 5-folds in CMCs/Amp-SH3 than control at normoxic condition.(1.399 vs 0.257 nmol/mg/min) 2) After hypoxic injury for 48 hours, the cell death and apoptosis of CMCs/Amp-SH3 were significantly decreased (approximately 70-80%) by the microscopic examination. The expression of p-akt and survivin was markedly increased at CMCs/Amp-SH3 than control by Western blot analysis. Optical density ratio of p-akt was 2.7 and that of survivin was 5.4. Summary and Conclusion: Amphiphysin SH3 gene increased glucose-uptake in the neonatal rat CMCs at nomoxic condition, and attenuated CMCs’ apoptosis under hypoxic condition via enhanced expressions of akt, survivin. Theses findings suggest the Amphiphysin SH3 is one of the candidates for ischemic protection of myocardium.
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