A method to measure cardiac autophagic flux in vivo

摘要: Autophagy, a highly conserved cellular mechanism wherein various components are broken down and recycled through lysosomes, has been implicated in the development of heart failure. However, tools to measure autophagic flux vivo have limited. Here, we tested whether monodansylcadaverine (MDC) lysosomotropic drug chloroquine could be used both vitro model systems. Using HL-1 cardiac-derived myocytes transfected with GFP-tagged LC3 track changes autophagosome formation, autophagy was stimulated by mTOR inhibitor rapamycin. Administration inhibit lysosomal activity enhanced rapamycin-induced increase number cells numerous GFP-LC3-positive autophagosomes. The chloroquine-induced autophagosomes occurred dose-dependent manner between 1 microM 8 microM, reached maximum 2 hour after treatment. Chloroquine also accumulation hydrogen peroxide, while it attenuated that induced Bafilomycin A1, an V-ATPase interferes fusion lysosomes. inhibited 3-methyladenine, which is known early phase process. transgenic mice expressing 3 mCherry-LC3 exposed rapamycin for 4 hr, observed mCherry-LC3-labeled myocardium, further increased concurrent administration chloroquine, thus allowing determination as more precise vivo. MDC injected hr before sacrifice colocalized puncta, validating its use marker This study describes method even non-transgenic animals, using chloroquine.