| 심태진, 황경국, 박노관, 최은주, 박건재, 이원익, 배장환, 김동운, 조명찬 |
Objectives; The 3'-phosphoinositide-dependent kinase-1 (PDK1) has been shown that automatic gain control kinase-signaling pathway regulates diverse cellular processes, including cell survival, proliferation, and metabolic responses. However, the involvement of PDK1 in cardiomyocytes (CMCs) has yet to be elucidated. This study investigated the effects by overexpression of PDK1 gene to cultured neonatal rat CMCs under the hypoxia/reoxygenation (H/R) conditions. Methods; The effects of lentiviral-PDK1 transfection on H/R were examined using neonatal rat CMCs. We ligated PDK1 with lenti-viral vector using multi-cloning site, and produced by lenti-virus for gene transfer using calcium phosphate method at 293T cell. CMCs (5×105 cells) were infected by lenti-PDK1 (1.5×107 IU) (CMCs/PDK1) and then hypoxia was created by incubating cells for 24 hours using GasPak. By replacing the medium saturated with 95% air and 5% CO2 for 6 hours, the cells were exposed to normoxic atmosphere (reoxygenation). The extents of apoptosis were evaluated by flow cytometry (FACS) with Annexin-Ⅴ-phycoerythrin staining. The expression of proteins related with cell survival pathway and apoptosis (p-Akt/Akt, p-GSK-3β/GSK-3β, Bax-α, Bcl-2 and Caspase-3) measured by Western blot. Result; More than 90% of CMCs had GFP expression by confocal laser microscope. Compared to lenti-GFP as a control, overexpression of PDK1 was confirmed at CMCs/PDK1. In CMCs/PDK1, apoptosis was significantly decreased by 39.6 % compared with control (38.3 vs 79.9 %, p<0.01). The expressions of p-Akt/Akt, p-GSK-3β/GSK-3β, Bcl-2/Bax-α were enhanced at CMCs/PDK1 by Western blot (optical density (OD) ratios: p-Akt /Akt; 1.8, p-GSK-3β/GSK-3β; 1.7, Bcl-2/Bax-α; 1.3). Caspase-3, the final executor of apoptosis signal, was significantly decreased (OD ratio: 0.6). Conclusion; Although the mechanism of PDK1 is not fully elucidated at this study, PDK1 may up-regulates the survival-related proteins in CMCs under H/R injury. These findings suggest that lenti-PDK1 gene therapy has considering potential for myocardial ischemic protection.
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