Early redistribution of plasma membrane phosphatidylserine during apoptosis of adult rat ventricular myocytes in vitro

In many cell types, DNA fragmentation is a late event of apoptosis which ma y be lacking. This contrasts with the early translocation of phosphatidylse rine (PS) from the internal to the external leaflet of the cell membrane. W e examined whether an early PS translocation also occurs during apoptosis i nduced in adult rat ventriculi myocytes grown in the presence of 10 % fetal calf serum (FCS), by the protein kinase inhibitor staurosporine. Apoptosis was assessed by the observation of: (i) typical alterations in cell morpho logy; (ii) nuclear alterations visualized using the permeant intercalating agent Hoechst 33258; (iii) DNA fragmentation detected by the TUNEL method. PS translocation was detected using annexin V binding. Data are expressed a s means +/- SEM. Prolonged exposure of myocytes to 10 mu M staurosporine fr om day 3 to day 7 of culture resulted in cell shrinkage, typical nuclear al terations, membrane protrusions and fragmentation of the sarcomeric apparat us in the vast majority of myocytes. At this time, 52.4 +/- 5.7 % of stauro sporine-treated myocytes were TUNEL positive (vs 6.1 +/- 2.0 % in control c ultures (CC), p < 0.001) and 69.7 +/- 1.7 % were annexin V positive (vs 21. 1 +/- 1.0 % in CC, p < 0.001). Importantly, PS translocation was detected a s early as 35 minutes following staurosporine addition, the percentage of a nnexin V positive myocytes reaching 10 times the control value (19.2 +/- 2. 7 vs 1.8 +/- 0.8 %, p < 0.001) after 3 hours. A 18-hour staurosporine expos ure of freshly isolated myocytes resulted, at the end of exposure, in 24.3 +/- 1.7 % annexin V positive myocytes (vs 9.6 +/- 0.5 % in CC, p < 0.05), w hereas a marked increase in the percentage of TUNEL positive myocytes was o bserved only from day 5. Finally, myocyte exposure to the membrane-permeant ceramide analog, C2-ceramide (50 mu M). resulted in 63.2 +/- 3.5 % annexin V positive myocytes 4 hours later (vs 17.8 +/- 4.4 % in CC, p < 0.001), wh ereas a significant increase in the percentage of TUNEL positive myocytes w as detected only the next day (43.7 +/- 3.4 vs 9.9 +/- 1.3 %, p < 0.001). T aken together, these results strongly suggest that the loss of PS asymmetry is an early event of cardiac myocyte apoptosis which precedes DNA fragment ation.