Protein kinase C delta and epsilon mediate positive inotropy in adult ventricular myocytes.
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 Ca(2+)-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.
Kang
Misuk
M
Department of Physiology, Molecular and Cellular Pharmacology Training Program, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA.
Walker
Jeffery W
JW
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D009200:Myocardial Contraction / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics / Q000502:physiology*; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015240:Phorbol 12,13-Dibutyrate; C497259:Prkcd protein, mouse; C497260:Prkcd protein, rat; C497256:Prkce protein, mouse; C497257:Prkce protein, rat; C497258:PRKCD protein, human; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon