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

Abstract

In many cell types, DNA fragmentation is a late event of apoptosis which may be lacking. This contrasts with the early translocation of phosphatidylserine (PS) from the internal to the external leaflet of the cell membrane. We examined whether an early PS translocation also occurs during apoptosis induced in adult rat ventricular 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 morphology; (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 as means ± SEM. Prolonged exposure of myocytes to 10 μM staurosporine from day 3 to day 7 of culture resulted in cell shrinkage, typical nuclear alterations, membrane protrusions and fragmentation of the sarcomeric apparatus in the vast majority of myocytes. At this time, 52.4 ± 5.7% of staurosporine-treated myocytes were TUNEL positive (vs 6.1 ± 2.0% in control cultures (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 as early as 35 minutes following staurosporine addition, the percentage of annexin 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 exposure 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), whereas a marked increase in the percentage of TUNEL positive myocytes was observed only from day 5. Finally, myocyte exposure to the membrane-permeant ceramide analog, C2-ceramide (50 μM), resulted in 63.2 ± 3.5% annexin V positive myocytes 4 hours later (vs 17.8 ± 4.4% in CC, p < 0.001), whereas a significant increase in the percentage of TUNEL positive myocytes was detected only the next day (43.7 ± 3.4 vs 9.9 ± 1.3%, p < 0.001). Taken together, these results strongly suggest that the loss of PS asymmetry is an early event of cardiac myocyte apoptosis which precedes DNA fragmentation.