Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria
Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria
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DOI：
10.1007/s00395-009-0007-5
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发表时间：
2009-03-01
影响因子：
9.5
通讯作者：
Schulz, Rainer
中科院分区：
文献类型：
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作者：
Boengler, Kerstin;Stahlhofen, Sabine;Schulz, Rainer
心肌细胞含有肌膜下(SSM)和纤维间(IFM)线粒体，这两种线粒体的呼吸和钙离子滞留能力不同。连接蛋白43(Cx43)位于SSM的内膜，参与缺血预适应(IP)对心肌的保护作用。Cx43形成的通道的功能部分通过Cx43羧基末端残基的磷酸化来调节。本研究的目的是(1)研究Cx43是否也存在于IFM中，以及(2)确定其在线粒体内膜(IMM)中的空间定位。证实了先前的发现，在大鼠脑室，IFM的ADP刺激呼吸大于SSM。在未被肌膜蛋白污染的大鼠和小鼠的标本中，Western印迹分析显示Cx43仅在SSM中表达，而在IFM中不表达(n=6)。用不同浓度的蛋白水解酶K裂解SSM，用Western印迹法检测ATP合成酶α(IMM)、解偶联蛋白3(UCP3，IMM，膜间隙表位)和锰超氧化物歧化酶(MnSOD，基质)的表达。当蛋白酶K浓度为50微克/毫升时，所有被分析蛋白的免疫反应性都完全丧失。5 mU g/ml的蛋白水解酶K使Cx43(19.4+/-A5.8%，n=6)和UCP3(23.0+/-A4%，n=7)的免疫反应活性降低，而ATP合成酶α(49.1+/-A6.4%，n=7)和MnSOD(79.9+/-A17.4%，n=6)的免疫反应活性被较好地保存，提示Cx43的羧基末端指向膜间隙。这一结果在洋地黄素处理的线粒体中得到了证实。综上所述，Cx43仅定位于SSM，其羧基末端指向膜间隙。由于线粒体Cx43的缺失取消了IP的心肌保护作用，SSM和IFM在IP信号转导中的作用明显不同。
Cardiomyocytes contain subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, which differ in their respiratory and calcium retention capacity. Connexin 43 (Cx43) is located at the inner membrane of SSM, and Cx43 is involved in the cardioprotection by ischemic preconditioning (IP). The function of Cx43-formed channels is regulated in part by phosphorylation at residues in the carboxy terminus of Cx43. The aim of the present study was (1) to investigate whether Cx43 is also present in IFM, and (2) to characterize its spatial orientation in the inner mitochondrial membrane (IMM). Confirming previous findings, ADP-stimulated respiration was greater in IFM than in SSM from rat ventricles. In preparations from rats and mice not contaminated with sarcolemmal proteins, Cx43 was exclusively detected in SSM, but not in IFM by Western blot analysis (n = 6). SSM were exposed to different proteinase K concentrations to cleave peptide bonds, and Western blot analysis was performed for ATP synthase alpha (IMM, subunit in the matrix), uncoupling protein 3 (UCP3, IMM, intermembrane space epitope), and manganese superoxide dismutase (MnSOD, matrix). At a proteinase K concentration of 50 mu g/ml, immunoreactivities of all the analyzed proteins were completely lost. The use of 5 mu g/ml proteinase K resulted in similarly reduced immunoreactivities for Cx43 (19.4 +/- A 5.8% of untreated mitochondria, n = 6) and UCP3 (23.0 +/- A 4%, n = 7), whereas the immunoreactivities of ATP synthase alpha (49.1 +/- A 6.4%, n = 7) and MnSOD (79.9 +/- A 17.4%, n = 6) were better preserved, suggesting that the carboxy terminus of Cx43 is directed towards the intermembrane space. The results were confirmed in digitonin-treated mitochondria. Taken together, Cx43 is exclusively localized in SSM, with its carboxy terminus directed towards the intermembrane space. Since loss of mitochondrial Cx43 abolishes IP's cardioprotection, SSM and IFM apparently differ in their function in the signal transduction of IP.