DOI: 10.1161/01.res.79.6.1086. An Improved Permeabilization Protocol for the Introduction of Peptides Into Cardiac Myocytes

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

10.1161/01.res.79.6.1086

Crossref journal-article

Ovid Technologies (Wolters Kluwer Health)

Circulation Research (276)

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 of125I-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-fosmRNA (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.

Bibliography

Johnson, J. A., Gray, M. O., Karliner, J. S., Chen, C.-H., & Mochly-Rosen, D. (1996). An Improved Permeabilization Protocol for the Introduction of Peptides Into Cardiac Myocytes. Circulation Research, 79(6), 1086â1099.

Authors 5

John A. Johnson (first)
Mary O. Gray (additional)
Joel S. Karliner (additional)
Che-Hong Chen (additional)
Daria Mochly-Rosen (additional)

References 35 Referenced 71

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10.1161/circ.85.6.1534288
Guthrie TS, Tsuji J, Wells JN. A synthetic pseudosubstrate peptide of protein kinase C inhibits the phorbol-12,13-dibutyrate effect on permeabilized coronary artery smooth muscle. Mol Pharmacol. 1991;39:621-624. (10.1016/S0026-895X(25)11070-5) / Mol Pharmacol (1991)
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Brodde O, Hillemann S, Kunde K, Vogelsang M, Zerkowski H. Receptor systems affecting force of contraction in the human heart and their alterations in chronic heart failure. J Heart Lung Transplant. 1992;11:164-174. / J Heart Lung Transplant (1992)
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10.1074/jbc.270.41.24180
10.1074/jbc.271.40.24962

Dates

Type	When
Created	13 years, 2 months ago (June 11, 2012, 8:47 p.m.)
Deposited	5 months ago (March 30, 2025, 6:19 p.m.)
Indexed	5 months ago (March 31, 2025, 12:18 a.m.)
Issued	28 years, 9 months ago (Dec. 1, 1996)
Published	28 years, 9 months ago (Dec. 1, 1996)
Published Print	28 years, 9 months ago (Dec. 1, 1996)

Funders 0

None

BibTeX

@article{Johnson_1996, title={An Improved Permeabilization Protocol for the Introduction of Peptides Into Cardiac Myocytes: Application to Protein Kinase C Research}, volume={79}, ISSN={1524-4571}, url={http://dx.doi.org/10.1161/01.res.79.6.1086}, DOI={10.1161/01.res.79.6.1086}, number={6}, journal={Circulation Research}, publisher={Ovid Technologies (Wolters Kluwer Health)}, author={Johnson, John A. and Gray, Mary O. and Karliner, Joel S. and Chen, Che-Hong and Mochly-Rosen, Daria}, year={1996}, month=dec, pages={1086–1099} }

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