Rapid turnover of connexin43 in the adult rat heart
Rapid turnover of connexin43 in the adult rat heart
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DOI：
10.1161/01.res.83.6.629
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发表时间：
1998-09-21
影响因子：
20.1
通讯作者：
Saffitz, JE
中科院分区：
文献类型：
--
作者：
Beardslee, MA;Laing, JG;Saffitz, JE
缝隙连接分布的重构是心肌梗死愈合后心律失常的重要解剖学基础。这一过程的基本机制知之甚少，但可能涉及间隙连接蛋白（连接蛋白）合成，组装成通道和降解的变化。在新生儿心肌细胞的原代培养物中，主要的心脏间隙连接蛋白连接蛋白43（Cx43）的半衰期仅为1.5至2小时，但尚不清楚Cx43的快速周转是否发生在成人心脏中，或者是在体外积极重建连接的解聚的新生儿心肌细胞所特有的。为了表征成年心脏中连接蛋白的周转动力学，并阐明其在间隙连接重塑中的潜在作用，我们测量了Cx43周转动力学，并表征了离体灌注成年大鼠心脏中Cx43降解所涉及的蛋白水解途径。用含[S-35]蛋氨酸的Krebs-Henseleit缓冲液标记心脏40分钟，然后用非放射性缓冲液进行0、60、120和240分钟的追踪灌注。在每个时间点，4个心脏中Cx43放射性的定量免疫沉淀测定产生单指数衰减曲线，表明Cx43半衰期为1.3小时。通过用溶酶体或蛋白酶体蛋白水解的特异性抑制剂灌注离体大鼠心脏4小时来阐明负责Cx43降解的蛋白水解途径。免疫印迹分析表明用溶酶体或蛋白酶体途径抑制剂灌注的心脏中Cx43含量显著增加（约30%）。大部分的Cx43在心脏灌注溶酶体抑制剂由磷酸化的亚型，而非磷酸化的Cx43选择性地积累在心脏灌注一个特定的蛋白酶体抑制剂。这些结果表明，Cx43在成人心脏中迅速翻转，并通过多种蛋白水解途径降解。Cx43降解的调节可能在心肌损伤后缝隙连接重构中起重要作用。
Remodeling of the distribution of gap junctions is an important feature of anatomic substrates of arrhythmias in patients with healed myocardial infarcts. Mechanisms underlying this process are poorly understood but probably involve changes in gap junction protein (connexin) synthesis, assembly into channels, and degradation. The half-life of the principal cardiac gap junction protein, connexin43 (Cx43), is only 1.5 to 2 hours in primary cultures of neonatal myocytes, but it is unknown whether rapid turnover of Cx43 occurs in the adult heart or is unique to disaggregated neonatal myocytes that are actively reestablishing connections in vitro. To characterize connexin turnover dynamics in the adult heart and to elucidate its potential role in remodeling of gap junctions, we measured Cx43 turnover kinetics and characterized the proteolytic pathways involved in Cx43 degradation in isolated perfused adult rat hearts. Hearts were labeled for 40 minutes with Krebs-Henseleit buffer containing [S-35]methionine, and then chase perfusions were performed with nonradioactive buffer for 0, 60, 120, and 240 minutes. Quantitative immunoprecipitation assays of Cx43 radioactivity in 4 hearts at each time point yielded a monoexponential decay curve indicating a Cx43 half-life of 1.3 hours. Proteolytic pathways responsible for Cx43 degradation were elucidated by perfusing isolated rat hearts for 4 hours with specific inhibitors of either lysosomal or proteasomal proteolysis, Immunoblot analysis demonstrated significant increases (approximate to 30%) in Cx43 content in hearts perfused with either lysosomal or proteasomal pathway inhibitors. Most of the Cx43 in hearts perfused with lysosomal inhibitors consisted of phosphorylated isoforms, whereas nonphosphorylated Cx43 accumulated selectively in hearts perfused with a specific proteasomal inhibitor. These results indicate that Cx43 turns over rapidly in the adult heart and is degraded by multiple proteolytic pathways. Regulation of Cx43 degradation could play an important role in gap junction remodeling in response to cardiac injury.