An Improved Permeabilization Protocol for the Introduction of Peptides Into Cardiac Myocytes: Application to Protein Kinase C Research

• Published in: Circulation research Vol. 79; no. 6; pp. 1086 - 1099
• Main Authors: Johnson, John A, Gray, Mary O, Karliner, Joel S, Chen, Che-Hong, Mochly-Rosen, Daria
• Format: Journal Article
• Language: English
• Published: Hagerstown, MD American Heart Association, Inc 01-12-1996; Lippincott
• Subjects: Animals; Biological and medical sciences; Cell Membrane - drug effects; Cell Membrane - physiology; Fundamental and applied biological sciences. Psychology; Heart; Heart - physiology; Myocardial Contraction - physiology; Peptides - physiology; Protein Kinase C - analysis; Protein Kinase C - metabolism; Rats; Rats, Sprague-Dawley; Saponins; Vertebrates: cardiovascular system; Heart; Performance evaluation; Protein kinase C; Peptides; Rat; Enzyme; Transferases; Rodentia; Enzyme inhibitor; Permeabilizing; Surfactant; In vitro; Uptake; Myocyte; Saponin; Vertebrata; Mammalia; Investigation method; Newborn animal; Myocardium; Circulatory system; Viability
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.RES.79.6.1086

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 I-labeled calmodulin and a 20-kD protein kinase C epsilon fragment into the cells. The intracellular molar concentration of the introduced peptide was approximate equals 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 [sup 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 [sup 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. (Circ Res. 1996;79:1086-1099.)