INTRACELLULAR CALCIUM, DNASE ACTIVITY AND MYOCYTE APOPTOSIS IN AGING FISCHER-344 RATS

Myocyte apoptosis increases with age in Fischer 344 rats. but the mult iple molecular events implicated in this phenomenon remain to be ident ified. Several defects involving Ca2+ homeostasis, pH, and the express ion of p53 and genes of the Bcl-2 protein family may contribute to the activation of myocyte death. Therefore, changes in intracellular pH, cytosolic Ca2+, DNase I and DNase II were measured in myocytes isolate d by enzymatic digestion from rats of different ages. Moreover, the ex pression of p53, Bcl-2 and Bar in these cells was determined. Measurem ents of intracellular pH by BCECF fluorescence at 3, 12 and 24 months showed that this parameter did not change with age: 3 months, 7.20 +/- 0.05; 12 months. 7.21 +/- 0.07; 24 months, 7.18 +/- 0.09. In contrast , diastolic Ca2+ determined by the Fura 2-AM method increased progress ively from 99.8 +/- 1.9 nM al 3 months to 136.3 +/- 9.6 nM at 24 month s (P<0.001). Concurrently, DNase I activity evaluated by plasmid diges tion assay in myocytes increased 3.2-fold from 3 to 24 months (P<0.02) . Conversely, pH-dependent-DNase II remained essentially constant with age. Western blotting performed on ventricular myocytes did not detec t significant changes in p53, Bar and Bcl-2 proteins with age. Similar ly, immunocytochemically, the fraction of myocytes labeled by p53, Bar and Bcl-2 did not change from 3 to 24 months. In conclusion, myocyte aging is characterized by an increase in diastolic calcium which may a ctivate DNase I triggering apoptosis, independently from the expressio n of p53, Bar and Bcl-2 in the cells. (C) 1998 Academic Press Limited.