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Review
. 2015 Jan 30;116(3):504-14.
doi: 10.1161/CIRCRESAHA.116.303787.

Untangling autophagy measurements: all fluxed up

Roberta A Gottlieb  1 , Allen M Andres  2 , Jon Sin  2 , David P J Taylor  2
Affiliations
Review

Untangling autophagy measurements: all fluxed up

Roberta A Gottlieb et al. Circ Res. .

Abstract

Autophagy is an important physiological process in the heart, and alterations in autophagic activity can exacerbate or mitigate injury during various pathological processes. Methods to assess autophagy have changed rapidly because the field of research has expanded. As with any new field, methods and standards for data analysis and interpretation evolve as investigators acquire experience and insight. The purpose of this review is to summarize current methods to measure autophagy, selective mitochondrial autophagy (mitophagy), and autophagic flux. We will examine several published studies where confusion arose in data interpretation, to illustrate the challenges. Finally, we will discuss methods to assess autophagy in vivo and in patients.

Keywords: autophagy; methods; microscopy, fluorescence; mitochondrial degradation; physiology.

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Conflict of interest statement

Disclosures

RAG is a consultant for Takeda Pharmaceuticals and is a cofounder of TissueNetix, Inc. The other authors have no potential conflicts of interest to disclose.

Figures

Figure 1
Figure 1. Autophagic puncta in hearts of mCherry-LC3 mice on chow or high fat diet (HFD)
A. Representative heart sections of mice from the Mentzer study after 15 wk of chow or Teklad D12492 high fat diet. Cardiac sections (5–6 µm) were made using a cryostat, mounted on glass slides and nuclei stained with Hoechst 33342. Images were acquired with a Nikon TE300 fluorescence microscope equipped with a 60× Plan Apo objective with excitation/emission wavelengths of 560/630 nm. (Unpublished images generously provided by Dr. Bruce Ito and Dr. Robert Mentzer.) B. Representative heart sections of mice from the Abel study after 12 wk of chow or western diet. Images were taken with Zeiss LSM 510 confocal microscope using x63 immersion oil objective with excitation/emission wavelengths of 543/613 nm. (Unpublished images generously provided by Dr. Bharat Jaishy and Dr. E. Dale Abel.)
Figure 2
Figure 2. Optical imaging of autophagy in hearts of mCherry-LC3 mice
A. Mice were imaged at baseline (Pre) and 4 h after rapamycin and chloroquine administration (Post), using a protocol of 3 acquisitions of 15 sec each. Representative images from a single mouse are shown. B. Graph shows the change in fluorescence from baseline to 4 h after rapamycin and chloroquine administration (n=14 mice; each point represents the average of 3 acquisitions). C. Cryosections of typical hearts of mCherry-LC3 mice under fed (left) and fasted (right) conditions, showing the typical increase in total fluorescence as well as the increase in number of fluorescent red puncta (autophagosomes). Nuclei are stained blue with DAPI. (Images from A and B are reprinted with permission from Circulation Research Abstract P066: Imaging Autophagy in Living Mice v109:AP066, 2011. Unpublished images from C provided by Dr. Chengqun Huang and Dr. Roberta A. Gottlieb.)
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