A method to measure cardiac autophagic flux in vivo
A method to measure cardiac autophagic flux in vivo
复制标题
DOI：
10.4161/auto.5603
复制
发表时间：
2008-04-01
期刊：
影响因子：
13.3
通讯作者：
Gottlieb, Roberta A.
中科院分区：
文献类型：
--
作者：
Iwai-Kanai, Eri;Yuan, Hua;Gottlieb, Roberta A.
自噬是一种高度保守的细胞机制，其中各种细胞成分通过溶酶体进行分解和再循环，它与心力衰竭的发展有关。然而，体内测量自噬通量的工具一直有限。在此，我们测试了单丹磺酰尸胺（MDC）和溶酶体促渗药物氯喹是否可用于在体外和体内模型系统中测量自噬通量。利用转染了绿色荧光蛋白标记的微管相关蛋白1轻链3（GFP - LC3）的HL - 1心脏来源的心肌细胞来追踪自噬体形成的变化，雷帕霉素（一种哺乳动物雷帕霉素靶蛋白抑制剂）刺激自噬。给予氯喹抑制溶酶体活性增强了雷帕霉素诱导的具有大量GFP - LC3阳性自噬体的细胞数量的增加。氯喹诱导的自噬体增加在1μM到8μM之间呈剂量依赖性，并在处理后2小时达到最大值。氯喹还增强了过氧化氢刺激的细胞中自噬体的积累，而减弱了由巴弗洛霉素A1（一种干扰自噬体与溶酶体融合的V - ATP酶抑制剂）诱导的自噬体积累。自噬体的积累被3 - 甲基腺嘌呤抑制，已知3 - 甲基腺嘌呤可抑制自噬过程的早期阶段。利用暴露于雷帕霉素4小时的表达红色荧光蛋白 - LC3（mCherry - LC3）的转基因小鼠，我们观察到心肌中mCherry - LC3标记的自噬体增加，同时给予氯喹进一步增加了自噬体数量，从而能够将通量确定为体内自噬活性的更精确测量指标。在处死前1小时注射的MDC与mCherry - LC3斑点共定位，验证了其作为自噬体标记物的用途。本研究描述了一种使用MDC和氯喹在体内（甚至在非转基因动物中）测量自噬通量的方法。
Autophagy, a highly conserved cellular mechanism wherein various cellular components are broken down and recycled through lysosomes, has been implicated in the development of heart failure. However, tools to measure autophagic flux in vivo have been limited. Here, we tested whether monodansylcadaverine (MDC) and the lysosornotropic drug chloroquine could be used to measure autophagic flux in both in vitro and in vivo model systems. Using HL-1 cardiac-derived myocytes transfected with GFP-tagged LC3 to track changes in autophagosome formation, autophagy was stimulated by mTOR inhibitor rapamycin. Administration of chloroquine to inhibit lysosornal activity enhanced the rapamycin-induced. increase in the number of cells with numerous GFP-LC3-positive autophagosornes. The chloroquine-induced increase of autophagosomes occurred in a dose-dependent manner between 1 mu M and 8 mu M, and reached a maximum 2 hour after treatment. Chloroquine also enhanced the accumulation of autophagosomes in cells stimulated with hydrogen peroxide, while it attenuated that induced by Bafilomycin A(1), an inhibitor of V-ATPase that interferes with fusion of autophagosomes with lysosornes. The accumulation of autophagosomes was inhibited by 3-methyladenine, which is known to inhibit the early phase of the autophagic process. Using transgenic mice expressing mCherry-LC3 exposed to rapamycin for 4 hr, we observed an increase in mCherry-LC3-labeled autophagosornes in myocardium, which was further increased by concurrent administration of chloroquine, thus allowing determination of flux as a more precise measure of autophagic activity in vivo. MDC injected I hr before sacrifice colocalized with mCherry-LC3 puncta, validating its use as a marker of autophagosomes. This study describes a method to measure autophagic flux in vivo even in non-transgenic animals, using MDC and chloroquine.