Novel methods for measuring cardiac autophagy in vivo.
Novel methods for measuring cardiac autophagy in vivo.
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DOI：
10.1016/s0076-6879(08)04016-0
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发表时间：
2009
影响因子：
--
通讯作者：
Gottlieb, Roberta A.
中科院分区：
文献类型：
--
作者：
Perry, Cynthia N.;Kyoi, Shiori;Hariharan, Nirmala;Takagi, Hiromitsu;Sadoshima, Junichi;Gottlieb, Roberta A.
自噬是一种高度保守的细胞机制，其中各种细胞组分被分解并通过溶酶体再循环，在心脏中组成性发生，并且在某些情况下可以用作心脏保护机制。它与心力衰竭的发展有关，并在缺血-再灌注损伤后上调。自噬通量（autophagic flux）是自噬囊泡形成和清除的量度，是评价该途径功效的重要量度，然而，在体内测量通量的工具受到限制。在这里，我们描述了使用单丹酰尸胺（MDC）和亲溶酶体药物氯喹来测量体内模型系统中的自噬通量，特别关注其在心肌中的使用。该方法允许测定通量作为体内自噬活性的更精确测量，其方式与巴弗洛霉素A1用于测量细胞培养物中的通量的方式大致相同。在处死前1小时注射MDC，与mCherry-LC 3斑点共定位，验证其作为自噬体标志物的用途。本章提供了一种方法来测量在转基因和非转基因动物体内的自噬通量，使用MDC和氯喹，此外还介绍了mCherry-LC 3小鼠和这种动物模型在心脏自噬研究中的优势。此外，我们还综述了在心肌梗死、缺血/再灌注、压力超负荷和营养饥饿等病理条件下诱导心肌自噬的几种方法。
Autophagy, a highly conserved cellular mechanism wherein various cellular components are broken down and recycled through lysosomes, occurs constitutively in the heart and may serve as a cardioprotective mechanism in some situations. It has been implicated in the development of heart failure and is up-regulated following ischemia-reperfusion injury. Autophagic flux, a measure of autophagic vesicle formation and clearance, is an important measurement in evaluating the efficacy of the pathway, however, tools to measure flux in vivo have been limited. Here, we describe the use of monodansylcadaverine (MDC) and the lysosomotropic drug chloroquine to measure autophagic flux in in vivo model systems, specifically focusing on its use in the myocardium. This method allows determination of flux as a more precise measure of autophagic activity in vivo much in the same way that Bafilomycin A1 is used to measure flux in cell culture. MDC injected 1 h before sacrifice, colocalizes with mCherry-LC3 puncta, validating its use as a marker of autophagosomes. This chapter provides a method to measure autophagic flux in vivo in both transgenic and nontransgenic animals, using MDC and chloroquine, and in addition describes the mCherry-LC3 mouse and the advantages of this animal model in the study of cardiac autophagy. Additionally, we review several methods for inducing autophagy in the myocardium under pathological conditions such as myocardial infarction, ischemia/ reperfusion, pressure overloading, and nutrient starvation.