| 김유경, 황경국¹, 오태근², 권진숙¹, 정일하¹, 한아름¹, 심태진¹, 박노관¹, 이원익¹, 배장환¹, 김동운¹, 조명찬¹ |
Objective: Amphiphysin was known to play an important role in clathrin-mediated endocytosis. In the cardiomyocytes(CMCs), the role of Amphiphysin was not known. We investigated the effect of the mutated Amphiphysin SH3 domain gene expression, defective Amphiphysin, versus Amphiphysin full-length in cultured neonatal rat CMCs under the normoxic or hypoxic conditions. Design and Methods: We ligated Amphiphysin SH3 and full-length with lenti-GFP viral vector using multi-cloning sites, and produced lenti-virus for gene transfer using calcium phosphate method at 293T cell(human embryonic kidney cell). Neonatal rat CMCs (5×105cells) were infected by lenti-Amphiphysin SH3 virus or Amphiphysin full-length(1.5×107IU) for 48 hours. Transfected CMCs by lenti-Amphiphysin SH3 virus(CMCs/Amp-SH3) or lenti-Amphiphysin full-length(CMCs/Amp-F) 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 or CMCs/Amp-F by microscopic examination and evaluated the survival signals by Western blot analysis. Results:1) More than 80% of CMCs had GFP expression by confocal microscope. Glucose uptake was increased approximately 2-folds in CMCs/Amp-SH3 than control in normoxic condition.(457 vs 206 pmol/mg/min) 2) After hypoxic injury for 48 hours, the cell death and apoptosis of CMCs/Amp-SH3 were decreased by FACS analysis(8.97 vs 6.18 %). The expressions of p-Akt, p-GSK-3ß, p-Erk, Survivin and GLUT4 were enhanced at CMCs/Amp-SH3 by Western blot (OD ratios of CMCs/Amp-SH3 compared to CMCs/Lenti-GFP: p-Akt:1.6, p-GSK-3ß:2.98, p-Erk:1.92, Survivin; 2.26, and GLUT4:3.51). The expressions of p-Akt, p-Erk, Survivin were reduced at CMCs/Amp-F. (OD ratios of CMCs/Amp-F: p-Akt:0.49, p-Erk;0.63, Survivin;0.75). Conclusion: Compared to Amphiphysin full-length, Amphiphysin SH3 gene attenuated CMCs’ apoptosis under hypoxic condition via enhanced expressions of p-Akt,p-GSK-3ß,p-Erk, survivin and GLUT4. Theses findings suggest the Amphiphysin SH3 is one of the candidates for ischemic protection of myocardium.
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