A METHOD FOR REVERSIBLE PERMEABILIZATION OF ISOLATED RAT VENTRICULAR MYOCYTES

A method is described that enables the cell membrane of isolated rat v entricular myocytes to be permeabilized and resealed while maintaining cell viability. Streptolysin O, a cholesterol-binding cytolysin, was used to form pores in the surface membrane; subsequent incubation with 5 % fetal bovine serum was used to reverse this permeabilization. The efficacy of membrane permeabilization and resealing was ascertained u sing a simultaneous double-staining technique using propidium iodide, a marker for cells with permeabilized membranes, and fluorescein diace tate, a marker for viable cells. This procedure allowed a distinction to be made between dead cells, unpermeabilized cells and viable cells that had been successfully permeabilized and resealed. The accessibili ty of the cell interior during permeabilization was investigated by in cluding fluorescein isothiocyanate (FITC)-labelled dextrans (11, 38 an d 148 kDa) and bovine serum albumin (67 kDa) in the permeabilization b uffer, and localizing the FITC label using confocal microscopy followi ng resealing. The confocal images showed that these molecules entered the cells and were retained after resealing. Following the permeabiliz ation-resealing protocol, cells appeared to have both normal morpholog y and response to electrical stimulation. Thus this appears to be a ch eap, simple and effective method to introduce relatively large molecul es into cardiac myocytes.