Abstract 17902: Hypoxic Preconditioning Promotes Caveolae-Dependent and Isoform-Selective Translocation of Protein Kinase C in Cardiac Myocytes
Protein kinase C (PKC) has been shown to play an important role in the cardioprotection of ischemic preconditioning. However, the mechanism underlying PKC-mediated cardioprotection is not completely understood. Given that caveolae is critical for cell signaling, we sought to determine whether hypoxic preconditioning (HP) promotes caveolae-dependent translocation of PKC to the plasma membrane. A cellular model of HP from rat heart-derived H9c2 cells or adult rat cardiac myocytes (ARCM) was employed to examine PKC isoforms including PKCα, PKCβ, PKCε, PKCδ and PKCζ. Hypoxia was induced by incubating the cells in an airtight chamber in which O2 was replaced by N2. As previously reported, the protective effect of HP (10 min hypoxia/30 min reoxygenation) against the subsequent hypoxia/reoxygenation-induced injury (H9c2 cells: 120 min hypoxia/160 min reoxygenation; ARCM: 90 min hypoxia/10 min reoxygenation) was observed by cell viability assays. Immunoblot analysis revealed that the immunoreactivity for PKCε or PKCδ (but not PKCα, PKCβ or PKCζ) increased significantly in the membrane fractions from H9c2 cells subjected to HP stimulus when compared with control cells (PKCε: 189.04 ± 27.16%, PKCδ: 230.01 ± 20.72%, n=3, p<0.01 vs. control), whereas the total cellular PKCε or PKCδ level was not altered. This HP-induced translocation of PKCε or PKCδ was blocked by disrupting caveolae using caveolin-3 siRNA (PKCε: 78.69 ± 4.48%, PKCδ: 98.75 ± 12.98%, n=3, p=NS vs. control). Western blot from the purified caveolin-enriched plasma membrane fractions showed that the band intensity for PKCε or PKCδ was enhanced significantly from HP-treated H9c2 cells (PKCε: 186.19 ± 23.56%, PKCδ: 189.73 ± 27.21%, n=3, p<0.01 vs. control) or ARCM (PKCε: 181.29 ± 36.41%, PKCδ: 168.53 ± 27.53%, n=3, p<0.05 vs. control). Furthermore, immunoprecipitation data from the caveolin-enriched membrane fractions of ARCM treated with HP indicated that both PKCε and PKCδ were detected in the anti-caveolin-3 immunoprecipitates. Confocal microscopy further suggested that colocalization of caveolin-3 with PKCε or PKCδ was increased in ARCM subjected to HP. We concluded that HP promotes the selective translocation of PKCε and PKCδ to the caveolin-enriched plasma membrane of cardiac myocytes.
- © 2010 by American Heart Association, Inc.