A method to measure cardiac autophagic flux in vivo

Abstract

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 lysosomotropic 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 lysosomal activity enhanced the rapamycin-induced increase in the number of cells with numerous GFP-LC3-positive autophagosomes. The chloroquine-induced increase of autophagosomes occurred in a dose-dependent manner between 1 microM and 8 microM, 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 A1, an inhibitor of V-ATPase that interferes with fusion of autophagosomes with lysosomes. 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 3 mCherry-LC3 exposed to rapamycin for 4 hr, we observed an increase in mCherry-LC3-labeled autophagosomes 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 1 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.

High-fat diet increases autophagic flux in pancreatic beta cells in vivo and ex vivo in mice

“…Autophagic flux is increased in pancreatic beta cells after high-fat feeding Autophagic flux was assessed in islets of GFP-LC3 mice by quantifying LC3II aggregation on the surface of autophagosomes following in vivo chloroquine injection [7,8]. Basal flux was low, with few fluorescent puncta observed in islets of chow-fed mice even following chloroquine treatment (Fig.…”

“…Eight-week-old male mice were fed ad libitum for 8-10 weeks on either a standard chow (10.88 kJ/g; 8% fat, 21% protein and 71% carbohydrate) or a high-fat diet (19.67 kJ/g; 45% fat, 20% protein and 35% carbohydrate). To estimate autophagic flux in vivo, mice were injected with the lysosomal inhibitor chloroquine (100 mg kg −1 day −1 ; SigmaAldrich, Sydney, NSW, Australia) or saline control daily for 5 days before being killed [7].…”

“…In the current study we have employed in vivo [7] treatments in mice to inhibit lysosomal function, thereby facilitating analysis of true autophagic flux in islets. Autophagic flux was enhanced by high-fat feeding, which we also confirmed using islets treated ex vivo with lysosomal inhibitors.…”

Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

“…1H and I), which is consistent with previous observations 12,13,17 and suggests that 24 h of fasting induces autophagy with efficient flux (with prompt processing of autophagosomes). Indeed, assessment of autophagic flux by concomitant treatment with CQ (chloroquine) to inhibit lysosome acidification and prevent autophagosome processing; 26 and evaluate the ratio of autophagosomes (assessed with punctate GFP-LC3 imaging, Fig. 1J; and autophagosome-bound LC3-II by immunoblotting, Fig.…”

“…26 Ischemia-reperfusion modeling Mice were subjected to reversible left anterior descending artery ligation for 30 min followed by reperfusion, in an openchest procedure, as described. 36 Overall surgical mortality was very low (<3 %).…”