Nitahara J A, Cheng W, Liu Y, Li B, Leri A, Li P, Mogul D, Gambert S R, Kajstura J, Anversa P
Department of Medicine, New York Medical College, Valhalla, NY, 10595, USA.
J Mol Cell Cardiol. 1998 Mar;30(3):519-35. doi: 10.1006/jmcc.1997.0616.
Myocyte apoptosis increases with age in Fischer 344 rats, but the multiple molecular events implicated in this phenomenon remain to be identified. Several defects involving Ca2+ homeostasis, pH, and the expression 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 isolated by enzymatic digestion from rats of different ages. Moreover, the expression of p53, Bcl-2 and Bax in these cells was determined. Measurements 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 progressively from 99.8+/-1.9 nm at 3 months to 136.3+/-9.6 nm at 24 months (P<0.001). Concurrently, DNase I activity evaluated by plasmid digestion 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 detect significant changes in p53, Bax and Bcl-2 proteins with age. Similarly, immunocytochemically, the fraction of myocytes labeled by p53, Bax 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 activate DNase I triggering apoptosis, independently from the expression of p53, Bax and Bcl-2 in the cells.
在Fischer 344大鼠中，心肌细胞凋亡随年龄增长而增加，但与此现象相关的多个分子事件仍有待确定。涉及钙离子稳态、pH值以及p53和Bcl-2蛋白家族基因表达的若干缺陷可能导致心肌细胞死亡的激活。因此，对不同年龄大鼠经酶消化分离出的心肌细胞内的pH值、胞质钙离子、脱氧核糖核酸酶I（DNase I）和脱氧核糖核酸酶II（DNase II）进行了检测。此外，还测定了这些细胞中p53、Bcl-2和Bax的表达。通过BCECF荧光法在3个月、12个月和24个月时对细胞内pH值进行测量，结果显示该参数并未随年龄变化：3个月时为7.20±0.05；12个月时为7.21±0.07；24个月时为7.18±0.09。相比之下，采用Fura 2-AM法测定的舒张期钙离子浓度从3个月时的99.8±1.9纳米逐渐增加至24个月时的136.3±9.6纳米（P<0.001）。同时，通过质粒消化试验评估的心肌细胞中DNase I活性从3个月到24个月增加了3.2倍（P<0.02）。相反，pH依赖性DNase II随年龄基本保持恒定。对心室肌细胞进行的蛋白质免疫印迹分析未检测到p53、Bax和Bcl-2蛋白随年龄的显著变化。同样，免疫细胞化学分析显示，从3个月到24个月，被p53、Bax和Bcl-2标记的心肌细胞比例没有变化。总之，心肌细胞衰老的特征是舒张期钙离子增加，这可能激活DNase I从而引发细胞凋亡，且与细胞中p53、Bax和Bcl-2的表达无关。
