期刊
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
卷 38, 期 5, 页码 753-764出版社
ELSEVIER SCI LTD
DOI: 10.1016/j.yjmcc.2005.02.017
关键词
PKC-delta; PKC-epsilon; Golgi; sarcolemma; adenovirus; fluorescent proteins
资金
- NHLBI NIH HHS [P01 HL47053] Funding Source: Medline
To examine cardiac contractile regulation and protein kinase C (PKC) translocation in parallel, the delta and epsilon isoforms of PKC were fused to green fluorescent protein (GFP) and expressed in adult rat ventricular myocytes maintained in short term culture. PKC-delta-GFP and PKC-epsilon-GFP were predominantly cytosolic until phorbol dibutyrate (PDBu) was introduced. PKC-delta-GFP redistributed preferentially to perinuclear structures that co-localized with a Golgi marker, whereas PKC-epsilon-GFP redistributed preferentially to the surface sarcolemma. Myocyte contractile function was assessed by monitoring twitch shortening with field stimulation at 0.5 Hz, 22 degrees C. In myocytes expressing PKC-delta-GFP, PDBu caused a transient negative inotropic response followed by a robust and sustained positive inotropic response that paralleled perinuclear PKC-delta accumulation. In PKC-epsilon-GFP myocytes, PDBu caused a sustained negative inotropic response that paralleled accumulation at the surface sarcolemma, but this response did not differ from myocytes expressing GFP alone. At higher expression levels, PKC-epsilon-GFP myocytes responded more like PKC-delta-GFP myocytes including perinuclear accumulation and a sustained positive inotropic response. Positive inotropic responses were markedly attenuated if PKC translocation was biased toward the surface sarcolemma by use of a more hydrophobic PKC activator, and were completely and selectively blocked by the PKC antagonist bis-indoylmaleimide. In contrast, transient and sustained negative inotropic responses were selectively blocked by the Ca2+-dependent PKC isoform antagonist Go6976. The data indicate that the novel PKC isoforms delta and epsilon have little effect on contractility when accumulating at the cell surface, but produce a strong positive inotropic response upon accumulation at the Golgi or other intracellular sites. (C) 2005 Elsevier Ltd. All rights reserved.
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