Janapati V, Wu A, Davis N, Derrico C A, Levengood J, Schummers J, Colvin R A
Department of Biological Sciences, Ohio University, Athens, 45701, USA.
Mech Ageing Dev. 1995 Oct 27;84(3):195-208. doi: 10.1016/0047-6374(95)01656-2.
Altered calcium homeostasis in the senescent heart appears to be the result, at least in part, of decreased Na+/Ca2+ exchange activity. To further investigate the basis of the decrease in Na+/Ca2+ exchange activity, Na+/Ca2+ exchanger gene expression in the heart was compared in 3 and 24 month old male Fischer 344 rats. Sarcolemmal vesicles prepared from left ventricle and septum showed reduced Na(+)-dependent Ca2+ uptake in 24 month old animals when compared to 3 month old animals (0.156 +/- 0.005 and 0.135 +/- 0.008 nmol Ca2+/mg/10 s; mean +/- S.E. for 3 month and 24 month old animals, respectively). Western analysis showed immunodetectable Na+/Ca2+ exchanger protein levels were decreased by 19% in 24 month old animals when compared to 3 month old animals. Poly(A+) RNA was purified from left and right ventricle and left and right atria and subjected to Northern analysis using digoxin labeled cDNA probes for the Na+/Ca2+ exchanger and actin. The Na+/Ca2+ exchanger probe labeled a 7 kb message in both ventricle and atria, while the actin probe labeled both beta-actin (2.2 kb) and alpha-actin (1.4 kb). The steady state level of expression of Na+/Ca2+ exchanger Poly(A+) RNA when normalized to beta-actin, was similar when ventricle and atria were compared. There were no observable differences in Na+/Ca2+ exchanger or alpha-actin Poly(A+) RNA steady state levels when comparing 3 and 24 month old animals. The results suggest that reduced Na+/Ca2+ exchange activity in the left ventricle of 24 month old animals was most likely the result of post-transcriptional modification of the protein that was detectable by Western analysis.
衰老心脏中钙稳态的改变似乎至少部分是由于钠/钙交换活性降低所致。为了进一步研究钠/钙交换活性降低的基础，比较了3月龄和24月龄雄性Fischer 344大鼠心脏中钠/钙交换基因的表达。与3月龄动物相比，从左心室和室间隔制备的肌膜囊泡在24月龄动物中显示出钠依赖性钙摄取减少（分别为0.156±0.005和0.135±0.008 nmol Ca2+/mg/10 s；3月龄和24月龄动物的平均值±标准误）。蛋白质免疫印迹分析显示，与3月龄动物相比，24月龄动物中可免疫检测到的钠/钙交换蛋白水平降低了19%。从左、右心室以及左、右心房中纯化出多聚腺苷酸（Poly(A+)）RNA，并使用地高辛标记的钠/钙交换体和肌动蛋白的cDNA探针进行Northern分析。钠/钙交换体探针在心室和心房中均标记出一条7 kb的信息条带，而肌动蛋白探针标记出β-肌动蛋白（2.2 kb）和α-肌动蛋白（1.4 kb）。当以β-肌动蛋白进行标准化时，心室和心房中钠/钙交换体Poly(A+) RNA的稳态表达水平相似。比较3月龄和24月龄动物时，钠/钙交换体或α-肌动蛋白Poly(A+) RNA的稳态水平没有明显差异。结果表明，24月龄动物左心室中钠/钙交换活性降低很可能是蛋白质转录后修饰的结果，这可通过蛋白质免疫印迹分析检测到。
