Department of Medicine and Ophthalmology, Boston University School of Medicine, Boston, MA 02118, USA.
Invest Ophthalmol Vis Sci. 2012 Sep 28;53(10):6675-81. doi: 10.1167/iovs.12-9895.
To determine connexin 43 (Cx43) localization in mitochondria and investigate the effects of high glucose (HG) on mitochondrial Cx43 (mtCx43) expression and whether altered mtCx43 channel activity is involved in promoting apoptosis in retinal endothelial cells.
MtCx43 localization was determined using immunostaining, green fluorescent protein (GFP)-tagged Cx43 followed by confocal imaging, and Western blot analysis using protein isolated from mitochondria of rat retinal endothelial cells (RRECs). To assess HG effects on mtCx43 expression, RRECs were grown in normal (5 mM) or HG (30 mM) medium for 7 days, and mtCx43 protein level assessed by Western blot analysis. To determine if mtCx43 channel inhibition affected mitochondrial morphology, RRECs grown sparsely were left untreated or treated with β-glycerrhetinic acid (β-GA), an inhibitor of connexin channels, and imaged using confocal microscopy. Additionally, mitochondria isolated from RRECs were treated with β-GA, and cytochrome c release assessed by Western blot.
Cx43 localization on the mitochondria of RRECs was confirmed with immunofluorescence staining using Cx43 antibody and GFP-tagged Cx43 imaged in live cells. Western blot analysis indicated that Cx43 was located primarily on the inner mitochondrial membrane, and mtCx43 protein level was significantly reduced in RRECs grown in HG condition. Treatment of RRECs with β-GA significantly decreased mtCx43 phosphorylation, induced mitochondrial fragmentation, and isolated mitochondria treated with β-GA showed increased cytochrome c release.
HG-induced downregulation of mtCx43 protein resulting in decreased channel activity may promote mitochondrial morphology changes and cytochrome c release, suggesting a novel mechanism for hyperglycemia-induced apoptosis in diabetic retinopathy.
确定连接蛋白 43（Cx43）在线粒体中的定位，并研究高糖（HG）对线粒体 Cx43（mtCx43）表达的影响，以及改变的 mtCx43 通道活性是否参与促进视网膜内皮细胞凋亡。
使用免疫染色、绿色荧光蛋白（GFP）标记的 Cx43 后进行共聚焦成像以及从大鼠视网膜内皮细胞（RRECs）的线粒体中分离的蛋白质进行 Western blot 分析来确定 mtCx43 的定位。为了评估 HG 对 mtCx43 表达的影响，将 RRECs 在正常（5 mM）或 HG（30 mM）培养基中培养 7 天，并通过 Western blot 分析评估 mtCx43 蛋白水平。为了确定 mtCx43 通道抑制是否影响线粒体形态，将稀疏生长的 RRECs 不进行处理或用β-甘油酸（β-GA）处理，β-GA 是连接蛋白通道的抑制剂，并使用共聚焦显微镜进行成像。此外，用β-GA 处理从 RRECs 分离的线粒体，并通过 Western blot 评估细胞色素 c 的释放。
使用 Cx43 抗体进行免疫荧光染色并在活细胞中成像 GFP 标记的 Cx43 ，证实了 Cx43 在 RRECs 线粒体上的定位。Western blot 分析表明 Cx43 主要位于线粒体内膜上，并且在 HG 条件下生长的 RRECs 中 mtCx43 蛋白水平显著降低。用β-GA 处理 RRECs 可显著降低 mtCx43 磷酸化，诱导线粒体片段化，并且用β-GA 处理的分离线粒体显示出增加的细胞色素 c 释放。
HG 诱导的 mtCx43 蛋白下调导致通道活性降低可能促进线粒体形态变化和细胞色素 c 释放，提示高血糖诱导糖尿病视网膜病变中细胞凋亡的新机制。
