© 1996 American Heart Association, Inc.
Articles |
An Improved Permeabilization Protocol for the Introduction of Peptides Into Cardiac Myocytes
Application to Protein Kinase C Research
the Department of Molecular Pharmacology (J.A.J., C.-H.C., D.M.-R.), Stanford (Calif) University School of Medicine; the Cardiology Section (M.O.G., J.S.K.), Veterans Affairs Medical Center, and the Department of Medicine Cardiovascular Research Institute (M.O.G., J.S.K.), University of California, San Francisco (Calif).
Correspondence to John A. Johnson, Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305-5332.
We have developed an improved, less disruptive procedure for the transient permeabilization of neonatal cardiac myocytes using saponin. The method allows delivery of peptides to a high percentage of cells in culture without effects on long-term cell viability. Permeation was confirmed microscopically by cellular uptake of a fluorescently labeled peptide and biochemically by uptake of 125I-labeled calmodulin and a 20-kD protein kinase C
fragment into the cells. The intracellular molar concentration of the introduced peptide was 
10% of that applied outside. We found no significant effects of permeabilization on spontaneous, phorbol ester–modulated, or norepinephrine-modulated contraction rates. Similarly, the expression of c-fos mRNA (measured 30 minutes after permeabilization) and the incorporation of [14C]phenylalanine following agonist stimulation (measured 3 days after permeabilization) were not altered by saponin permeabilization. Finally, permeabilization of cells in the presence of a protein kinase C pseudosubstrate peptide, but not a control peptide, inhibited phorbol ester–induced [14C]phenylalanine incorporation into proteins by 80%. Our results demonstrate a methodology for the introduction of peptides into neonatal cardiac myocytes that allows study of their actions without substantial compromises in cell integrity.
Key Words: neonatal cardiac myocytes • permeabilization method • contraction • hypertrophy • protein kinase C
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