An Improved Permeabilization Protocol for the Introduction of Peptides Into Cardiac Myocytes

Abstract

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 125 I-labeled calmodulin and a 20-kD protein kinase Cε 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 [ 14 C]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 [ 14 C]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.