title: Hypoxic preconditioning promotes the translocation of protein kinase C ε binding with caveolin-3 at cell membrane not mitochondrial in rat heart.
abstract: Protein kinase C has been shown to play a central role in the cardioprotection of ischemic preconditioning. However, the mechanism underlying PKC-mediated cardioprotection is not completely understood. Given that caveolae are critical for PKC signaling, we sought to determine whether hypoxic preconditioning promotes translocation and association of PKC isoforms with caveolin-3. A cellular model of hypoxic preconditioning from adult rat cardiac myocytes (ARCM) or H9c2 cells was employed to examine PKC isoforms by molecular, biochemical and cellular imaging analysis. Hypoxia was induced by incubating the cells in an airtight chamber in which O2 was replaced by N2 with glucose-free Tyrode's solution. Cells were subjected to hypoxic preconditioning with 10 minutes of hypoxia followed by 30 minutes of reoxygenation. Western blot data indicated that the band intensity for PKCε, PKCδ or PKCα, but not PKCβ and PKCζ was enhanced significantly by hypoxic preconditioning from the caveolin-enriched plasma membrane interactions. Immunoprecipitation experiments from the caveolin-enriched membrane fractions of ARCM showed that the level of PKCε, PKCδ and PKCα in the anti-caveolin-3 immunoprecipitates was also increased by hypoxic preconditioning. Further, our FRET analysis in H9c2 cells suggested that there is a minimum FRET signal for caveolin-3 and PKCε along cell peripherals, but hypoxic preconditioning enhanced the FRET signal, indicating a potential interaction between caveolin-3 and PKCε. And also treatment of the cells with hypoxic preconditioning led to a smaller amount of translocation of PKCε to the mitochondria than that to the membrane. We demonstrate that hypoxic preconditioning promotes rapid association of PKCε, PKCδ and PKCα with the caveolin-enriched plasma membrane microdomain of cardiac myocytes, and PKCε via direct molecular interaction with caveolin-3. This regulatory mechanism may play an important role in cardioprotection.
authors: a Department of Molecular Biology ; College of Basic Medical Sciences; Jilin University ; Changchun; Jilin , China.|b China-Japan Union Hospital; Jilin University ; Changchun; Jilin , China.
authors: Hongmei
authors: H
authors: Yu
authors: c NSF Nanoscale Science and Engineering Center (NSEC); The Ohio State University ; Columbus , OH USA.
authors: Zhaogang
authors: Z
authors: Yang
authors: b China-Japan Union Hospital; Jilin University ; Changchun; Jilin , China.
authors: Su
authors: S
authors: Pan
authors: b China-Japan Union Hospital; Jilin University ; Changchun; Jilin , China.
authors: Yudan
authors: Y
authors: Yang
authors: a Department of Molecular Biology ; College of Basic Medical Sciences; Jilin University ; Changchun; Jilin , China.
authors: Jiayi
authors: J
authors: Tian
authors: a Department of Molecular Biology ; College of Basic Medical Sciences; Jilin University ; Changchun; Jilin , China.
authors: Luowei
authors: L
authors: Wang
authors: a Department of Molecular Biology ; College of Basic Medical Sciences; Jilin University ; Changchun; Jilin , China.
authors: Wei
authors: W
authors: Sun
mesh_terms: D000818:Animals; D051244:Caveolin 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D005786:Gene Expression Regulation; D000860:Hypoxia / Q000235:genetics* / Q000378:metabolism / Q000517:prevention & control; D019157:Ischemic Preconditioning, Myocardial; D008929:Mitochondria, Heart / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D061251:Primary Cell Culture; D011485:Protein Binding; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D064546:Protein Kinase C beta / Q000235:genetics / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D000099221:Protein Kinase C zeta
chemical_list: D051244:Caveolin 3; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D034741:RNA, Small Interfering; D000099221:Protein Kinase C zeta; C495549:Cav3 protein, rat
keywords: FRET; PKC; caveolin; mitochondria; preconditioning