Permeability of rat heart myocytes to cytochrome c

Rat heart myocytes undergoing progressive damage demonstrate morphological changes of shortening and swelling followed by the formation of intracellul ar vacuoles and plasma membrane blebbing. The damaged myocytes displayed im paired N,N'-tetramethyl-p-phenyldiamine (TMPD) ascorbate-stimulated respira tory activity which was restored by the addition of reduced cytochrome c to the cell culture medium. To clarify the role played by cytochrome c in the impairment of cell respiration, polarographic, spectrophotometric and fluo rescence as well as electron microscopy imaging experiments were performed. TMPD/ascorbate-stimulated respiratory activity returned to control levels, at approximately 20 mu M cytochrome c, establishing the threshold below wh ich the turnover rate by cytochrome c oxidase in the cell depends on cytoch rome concentration. Mildly damaged cardiac myocytes, as indicated by cell s hortening, retention of visible striations and free-fluorescein exclusion, together with the absence of lactate dehydrogenase leakage and exclusion of trypan blue, were able to oxidize exogenous cytochrome c and were permeabl e to fluorescein-conjugated cytochrome c. The results, while consistent wit h an early cytochrome c release observed at the beginning of cell death, el ucidate the role played by cytochrome c in the kinetic control of mitochond rial electron transfer under pathological conditions, particularly those in volving the terminal part of the respiratory chain. These data are the firs t to demonstrate that the sarcolemma of cardiac myocytes, damaged but still viable, is permeable to cytochrome c.