Skip to main content
  • American Heart Association
  • Science Volunteer
  • Warning Signs
  • Advanced Search
  • Donate

  • Home
  • About this Journal
    • Editorial Board
    • Meet the Editors
    • Editorial Manifesto
    • Impact Factor
    • Journal History
    • General Statistics
  • All Issues
  • Subjects
    • All Subjects
    • Arrhythmia and Electrophysiology
    • Basic, Translational, and Clinical Research
    • Critical Care and Resuscitation
    • Epidemiology, Lifestyle, and Prevention
    • Genetics
    • Heart Failure and Cardiac Disease
    • Hypertension
    • Imaging and Diagnostic Testing
    • Intervention, Surgery, Transplantation
    • Quality and Outcomes
    • Stroke
    • Vascular Disease
  • Browse Features
    • Circulation Research Profiles
    • Trainees & Young Investigators
    • Research Around the World
    • News & Views
    • The NHLBI Page
    • Viewpoints
    • Compendia
    • Reviews
    • Recent Review Series
    • Profiles in Cardiovascular Science
    • Leaders in Cardiovascular Science
    • Commentaries on Cutting Edge Science
    • AHA/BCVS Scientific Statements
    • Abstract Supplements
    • Circulation Research Classics
    • In This Issue Archive
    • Anthology of Images
  • Resources
    • Online Submission/Peer Review
    • Why Submit to Circulation Research
    • Instructions for Authors
    • → Article Types
    • → Manuscript Preparation
    • → Submission Tips
    • → Journal Policies
    • Circulation Research Awards
    • Image Gallery
    • Council on Basic Cardiovascular Sciences
    • Customer Service & Ordering Info
    • International Users
  • AHA Journals
    • AHA Journals Home
    • Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB)
    • Circulation
    • → Circ: Arrhythmia and Electrophysiology
    • → Circ: Genomic and Precision Medicine
    • → Circ: Cardiovascular Imaging
    • → Circ: Cardiovascular Interventions
    • → Circ: Cardiovascular Quality & Outcomes
    • → Circ: Heart Failure
    • Circulation Research
    • Hypertension
    • Stroke
    • Journal of the American Heart Association
  • Impact Factor 13.965
  • Facebook
  • Twitter

  • My alerts
  • Sign In
  • Join

  • Advanced search

Header Publisher Menu

  • American Heart Association
  • Science Volunteer
  • Warning Signs
  • Advanced Search
  • Donate

Circulation Research

  • My alerts
  • Sign In
  • Join

  • Impact Factor 13.965
  • Facebook
  • Twitter
  • Home
  • About this Journal
    • Editorial Board
    • Meet the Editors
    • Editorial Manifesto
    • Impact Factor
    • Journal History
    • General Statistics
  • All Issues
  • Subjects
    • All Subjects
    • Arrhythmia and Electrophysiology
    • Basic, Translational, and Clinical Research
    • Critical Care and Resuscitation
    • Epidemiology, Lifestyle, and Prevention
    • Genetics
    • Heart Failure and Cardiac Disease
    • Hypertension
    • Imaging and Diagnostic Testing
    • Intervention, Surgery, Transplantation
    • Quality and Outcomes
    • Stroke
    • Vascular Disease
  • Browse Features
    • Circulation Research Profiles
    • Trainees & Young Investigators
    • Research Around the World
    • News & Views
    • The NHLBI Page
    • Viewpoints
    • Compendia
    • Reviews
    • Recent Review Series
    • Profiles in Cardiovascular Science
    • Leaders in Cardiovascular Science
    • Commentaries on Cutting Edge Science
    • AHA/BCVS Scientific Statements
    • Abstract Supplements
    • Circulation Research Classics
    • In This Issue Archive
    • Anthology of Images
  • Resources
    • Online Submission/Peer Review
    • Why Submit to Circulation Research
    • Instructions for Authors
    • → Article Types
    • → Manuscript Preparation
    • → Submission Tips
    • → Journal Policies
    • Circulation Research Awards
    • Image Gallery
    • Council on Basic Cardiovascular Sciences
    • Customer Service & Ordering Info
    • International Users
  • AHA Journals
    • AHA Journals Home
    • Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB)
    • Circulation
    • → Circ: Arrhythmia and Electrophysiology
    • → Circ: Genomic and Precision Medicine
    • → Circ: Cardiovascular Imaging
    • → Circ: Cardiovascular Interventions
    • → Circ: Cardiovascular Quality & Outcomes
    • → Circ: Heart Failure
    • Circulation Research
    • Hypertension
    • Stroke
    • Journal of the American Heart Association
Reviews

Untangling Autophagy Measurements

All Fluxed Up

Roberta A. Gottlieb, Allen M. Andres, Jon Sin, David P.J. Taylor
Download PDF
https://doi.org/10.1161/CIRCRESAHA.116.303787
Circulation Research. 2015;116:504-514
Originally published January 29, 2015
Roberta A. Gottlieb
From the Cedars-Sinai Heart Institute and the Barbra Streisand Women’s Heart Center Cedars-Sinai Medical Center, Los Angeles, CA.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Allen M. Andres
From the Cedars-Sinai Heart Institute and the Barbra Streisand Women’s Heart Center Cedars-Sinai Medical Center, Los Angeles, CA.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jon Sin
From the Cedars-Sinai Heart Institute and the Barbra Streisand Women’s Heart Center Cedars-Sinai Medical Center, Los Angeles, CA.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David P.J. Taylor
From the Cedars-Sinai Heart Institute and the Barbra Streisand Women’s Heart Center Cedars-Sinai Medical Center, Los Angeles, CA.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Tables
  • Info & Metrics

Jump to

  • Article
    • Abstract
    • Overview of Autophagy
    • Examples of Confusing Autophagy Studies and Confounding Variables
    • Methods to Measure Autophagy and Mitophagy
    • Conclusions
    • Sources of Funding
    • Disclosures
    • Footnotes
    • References
  • Figures & Tables
  • Info & Metrics
  • eLetters
Loading

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.

  • autophagy
  • methods
  • microscopy, fluorescence
  • mitochondrial degradation
  • physiology

A photo is static, an instant in time,

Telling nothing that happened next or before;

Yet papers are published and conclusions are drawn

Claiming autophagy’s up when really it’s gone.

Like tea leaves or runes it’s not easy to read

When puzzling out blots of LC3B

When chloroquine’s there or when it’s left out

It’s the increase that matters to tell you what’s what.

Beclin is tricky when it’s gone half away,

Its effects on autophagy go every which way:

Nucleation is up or fusion’s not seen,

So consider with care what it all means.

Like freeways with cars and crowded on-ramps

AVs have cargo and their own traffic jams

Created by leupeptin, CQ or Baf,

LC3 rises at least by a half.

Remember when calculating if flux is intact

It’s the ratio that gives you that crucial fact

LC3 levels of Baf over static

Will yield results that are not so erratic.

To understand the process you must think it through;

Autophagy requires you to use every clue.

Good data help you line up ducks,

Just please be sure to measure flux.

Roberta A. Gottlieb

Overview of Autophagy

Autophagy emerged as a cardiac research topic starting in 2005 with the recognition of its role in hibernating myocardium1 and in the protective response to BNIP32; in the ensuing 8 years, the number of publications on cardiac autophagy have increased 10-fold. Thus, there is a substantial need to have reliable methods to measure the process of autophagy. Autophagy is an essential housekeeping function responsible for eliminating unwanted protein aggregates or organelles.3,4 Autophagy encompasses 3 primary pathways for lysosomal degradation: macroautophagy (the methodological focus of this review), microautophagy, and chaperone-mediated autophagy. Microautophagy is a nonselective form of degradation in which lysosomal membranes directly engulf cytoplasm along the periphery of the organelle. Glycogen and other endosomal components are examples of material that is degraded by microautophagy. Chaperone-mediated autophagy targets cytosolic proteins and translocates them straight across the lysosomal membrane to be degraded. Proteins targeted by chaperone-mediated autophagy must contain a specific targeting motif that is recognized by HSPA8/HSC70, which subsequently directs the cargo for degradation. Ultimately, all 3 processes depend on lysosomal function, failure of which carries pathological consequences. Genetic defects in lysosomal function, such as Danon disease and Pompe disease, are recognized to result in cardiomyopathy because of glycogen accumulation.5–7 Although lysosomal function is essential to macroautophagy—the focus of this review—we do not cover specific methods to assess lysosomal function.

Macroautophagy involves the engulfment of a portion of the cytoplasm by a double-membrane phagophore, which elongates around both selectively and nonselectively targeted cargo. After phagophore closure, the newly formed vesicle, now termed the autophagosome, fuses with the lysosome forming the autolysosome. In the acidic milieu of the lysosome, pH-dependent hydrolases degrade the proteins, lipids, nucleic acids, and carbohydrates of the cargo. The phagophore membrane is thought to arise from multiple membrane sources, including the endoplasmic reticulum. In 1 model, phagophore initiation begins with the distention of the endoplasmic reticulum into a phosphatidylinositol-3-phosphate (PtdIns3P)–rich structure called the omegasome. This process is initiated with the recruitment of the unc-51-like kinase (ULK) complex and the class III PdIns-3-kinase complex, which are targeted to the omegasome via ATG13 and ATG14L1, respectively. From the omegasome emerges the phagophore membrane at which point WIPI2 and ATG16L1 are recruited. ATG12 is conjugated with ATG5 through the action of ubiquitin ligase–like enzymes ATG7 and ATG10; the covalently linked ATG5–ATG12 duo complexes with ATG16L1, enabling the processing of microtubule-associated protein 1A/1B-light chain 3 (LC3). The precursor form of LC3 is truncated by the cysteine protease ATG4 to expose the C-terminal glycine of LC3 to form LC3-I. This in turn is conjugated to the amino group of phosphatidylethanolamine (forming membrane-associated LC3-II) by the action of E1- and E2-like enzymes ATG7 and ATG3. Once the LC3-II–decorated phagophore closes around its target, the ATG12–ATG5–ATG16L complex is released from the membrane. When the autophagosome fuses with the lysosome, LC3-II on the outer face of the autolysosome is released by the action of ATG4; however, LC3 on the inner face is retained and eventually degraded by lysosomal enzymes.

Examples of Confusing Autophagy Studies and Confounding Variables

To highlight the need for appropriate standards for measuring autophagy and interpreting results, we will first present some recent autophagy studies in which the results were contradictory, incomplete, or subject to alternative interpretation, leading to controversial and possibly incorrect conclusions.

Difficulties in Interpreting Autophagic Changes in the Setting of Diet-Induced Obesity

At a recent meeting, contradictory results were reported by 2 groups using mCherry-LC3 transgenic mice, in which the fusion protein is expressed under the control of the αMHC promoter. The group of Abel observed an increase in the number of mCherry-labeled puncta in mice fed a high-fat diet, whereas the group of Mentzer observed a decrease (Figure 1). Diet composition differed as follows: the group of Mentzer used the D12492 60% fat diet, whereas the group of Abel used a Western diet of 40% fat and increased sucrose. Mentzer study enrolled mice at 5 to 6 weeks and maintained for 15 weeks, whereas the Abel study enrolled mice at 8 weeks and maintained them on the diet for 12 weeks. Another important difference was the genetic background of the mice. The Mentzer laboratory used mCherry-LC3 mice in the FVB/N background, whereas the group of Abel backcrossed the line into the C57BL/6 background. Interestingly, both groups arrived at the same conclusion that the obesogenic diet impaired cardiac autophagy. The group of Abel used chloroquine to show that autophagic flux was attenuated. They noted elevated p62/SQSTM1 that did not increase further when flux was inhibited with chloroquine. The group of Mentzer also observed elevated p62/SQSTM1 in the high-fat diet group, and similarly concluded that autophagic flux was impaired. This illustrates the difficulties in determining autophagic activity based on snapshot measurements of LC3 puncta or Western blot and emphasizes the need to assess flux directly (eg, by chloroquine) or indirectly (by p62/SQSTM1).

Figure 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1.

Autophagic puncta in hearts of mCherry-light chain 3 mice on chow or high-fat diet (HFD). A, Representative heart sections of mice from the Mentzer study after 15 weeks of chow or Teklad D12492 HFD. 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 weeks of chow or Western diet. Images were taken with Zeiss LSM 510 confocal microscope using ×63 immersion oil objective with excitation/emission wavelengths of 543/613 nm (unpublished images generously provided by Dr Bharat Jaishy and Dr E. Dale Abel).

Becn1 Haploinsufficient Mice

Deletion of Atg5 results in death in the newborn period unless nutritional support is provided during the first 12 to 24 hours of postnatal life,8 but surviving pups are relatively normal although they develop heart failure and other abnormalities with time. Subsequent studies revealed an alternative autophagic pathway independent of ATG5 and ATG7 but dependent on BECN1, ULK1, and RAB9.9 BECN1, which participates in both pathways, is essential, because the deletion of the corresponding gene is embryonic lethal.10 Subsequently, BECN1 haploinsufficient mice were used as a model of attenuated autophagy in several studies; however, BECN1 also regulates autophagosome–lysosome fusion and when partnered with KIAA0226/Rubicon, it can suppress fusion.11,12 These complexities have led to several contradictory findings: Becn1+/− mice showed less hypertrophy in response to aortic banding, which was used as evidence that autophagy contributed to pathological hypertrophy.13 Becn1+/− mice are observed to have smaller infarcts after ischemia/reperfusion injury, leading the authors to conclude that BECN1-dependent autophagy contributes to reperfusion injury.14 In contrast, chloramphenicol administered to pigs before ischemia or at reperfusion profoundly reduces infarct size and is associated with a substantial upregulation of BECN1.15 Thus, contradictory roles for BECN1 and autophagy emerged from these studies, largely dependent on the interpretation of LC3 autophagic markers. In the study by Zhu et al13 of Becn1+/− mice subjected to aortic banding, they observed fewer autophagosomes in heart tissue and concluded that this indicated decreased autophagy. However, they did not measure autophagic flux; increased autophagic flux would also be consistent with their observation. Subsequent work by Ma et al16 using neonatal rat ventricular myocytes showed that BECN1 knockdown results in accelerated autophagic flux, presumably by reducing the BECN1-KIAA0226 impediment to autophagosome–lysosome fusion.11,12 A criticism of the work of Ma et al16 is that it is limited to cell culture. However, a study by Xu et al17 examined autophagic flux in normal and diabetic mice, comparing wild-type and BECN1 haploinsufficient mice. They measured LC3 levels in the absence and presence of the lysosomal inhibitor bafilomycin A1 (BAF) and reported flux as the difference in LC3-II content between BAF and vehicle, reaching the conclusion that flux was not different between Becn1+/− and wild-type mice. However, when flux is calculated as the fold increase over vehicle control as originally described by Tanida et al,18 they might have reached a different conclusion that would have supported the idea that BECN1 can slow autophagic flux. These controversies over interpretation of the data and the functional effects of BECN1 are reflected in previous work with OVE26 diabetic mice19 (BECN1 suppresses flux) and other studies with the Becn1+/− mice20 (BECN1 deficiency results in insufficient autophagy initiation). Whether diminished autophagy initiation or accelerated flux is the dominant effect may depend on the tissue and the context of pathological stress. Taken together, these studies exemplify the difficulties associated with using BECN1 to modulate autophagy and the challenges associated with interpreting results.

If the dominant effect of BECN1 in the heart is to slow autophagic flux, then it becomes necessary to revisit the conclusions reached by Zhu et al13 and Matsui et al14: BECN1 haploinsufficiency might have accelerated autophagic flux to reduce hypertrophy and infarct size in their models. This illustrates the importance of measuring autophagic flux correctly and emphasizes the profound limitations of static measurement of LC3.

Because BECN1 also has the potential to regulate apoptosis through interaction with Bcl-2, its downregulation might benefit the heart through reduction of apoptosis. To examine this, Ma et al16 used chloroquine and found that suppression of autophagic flux was sufficient to exacerbate autophagy after hypoxia/reoxygenation. They found that the cell death was prevented by cyclosporine A but not that pan-caspase inhibitor ZVAD (Z-val-Ala-Asp-fluoromethylketone), suggesting that the mitochondrial permeability transition pore was responsible for triggering necrotic cell death. Other studies have shown the importance of clearing damaged mitochondria to reduce ischemia/reperfusion injury.2,15,21–23 Postconditioning was also shown to restore autophagic flux during reperfusion, whereas inhibition of flux (with chloroquine) abolished the protective effects of sevoflurane postconditioning.24

Histone Deacetylase Inhibitors

Histone deacetylases (HDAC) regulate an expanding number of pathways, and substrate specificity is restricted according to the enzyme subtype.25 In 2011, Cao et al26 reported that trichostatin A, an inhibitor of class I and II HDACs, reversed cardiac hypertrophy because of pressure overload by downregulating autophagy. In their study, autophagy measurements were limited to detection of autophagic puncta and LC3 Western blots. No flux measurements or assessment of p62/SQSTM1 were performed on the hearts although they did assess flux in neonatal rat ventricular myocytes treated with trichostatin A. A careful inspection of their results (their Online Figure I) reveals that autophagic flux was enhanced in trichostatin A–treated cardiomyocytes (lysosomal blockade resulted in a 2.1-fold increase in LC3-II in controls versus a 2.8-fold increase in trichostatin A–treated cells). Confusingly, the authors concluded that because the absolute levels of LC3-II were lower in the trichostatin A group, its effects were because of suppression of autophagy, stating that “suppression of autophagic flux contributes to the salutary effects of HDAC inhibitor therapy.” It should be noted that if autophagic flux was suppressed, LC3-II levels would not increase after lysosomal blockade. The fact that the levels nearly triple after lysosomal blockade suggests that the low level of LC3-II seen in the trichostatin A group (with or without lysosomal blockade) is because of brisk autophagic flux.

In 2013, the same group used a related HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA; vorinostat), to show that HDAC inhibition increased autophagic flux and decreased infarct size in a rabbit model of I/R injury.27 Using mice expressing tandem RFP-GFP-LC3, they observed that SAHA increased autophagic flux. The effect of SAHA on autophagic flux was confirmed in cell culture in which flux was assessed by the gold standard of comparing LC3-II in the presence and absence of lysosomal blockade with bafilomycin. The requirement for autophagy in SAHA’s beneficial effects was further confirmed by downregulating ATG7, which abolished the protective effects of SAHA. These 2 studies again illustrate the challenges in interpreting LC3 results and also support the use of the tandem RFP-GFP-LC3 construct for evaluating autophagic flux. Importantly, they provided further evidence to support the role of autophagy through knockdown of a key factor in the canonical autophagy pathway, ATG7 (we have used dominant negative ATG5 with good effect28). As noted above, BECN1 can have paradoxical effects on autophagy because of its interaction with KIAA0226/Rubicon and suppression of autophagosome–lysosome fusion.

Lessons Learned From Analysis of These Studies

These publications have been widely cited and have influenced our understanding of autophagy. However, the failure to measure flux and the controversy over interpretation of results has resulted in confusion about the role and significance of autophagy. The purpose of our rather detailed dissection of those publications is to point out the challenges associated with analyzing autophagy, to encourage investigators to use proper methodology in assessing autophagy, to call for a thoughtful reevaluation of the conclusions of those studies, and to exhort readers to exercise caution when reading new publications.

Several things emerge from this discussion. Static levels of LC3 or scoring of autophagic puncta is an incomplete assessment of autophagy without the assessment of flux (directly, through lysosomal blockade, or indirectly, inferred from a decrease in p62/SQSTM1). The most definitive way to demonstrate a role for autophagy is to knock down ATG7 or inhibit ATG5. BECN1 has paradoxical effects, and a knockdown of this protein may be confusing when assessing the role of autophagy in a particular organ or disease process. Although not discussed in detail in this review, it should be noted that the transcriptional regulation of autophagy has not been closely correlated with functional autophagic flux: mRNA may be increased when autophagy is impaired, and mRNA levels may be stable even when autophagic flux is active. However, mRNA upregulation is part of the longer term autophagic response. There is a need for additional methods to assess autophagy in tissues and to be able to image autophagy noninvasively.

Additional Variables That May Confound Autophagy Results

Effect of Mouse Strain on Autophagy

In addition to the difference in autophagy noted in the mCherry-LC3 transgenics that may be because of strain differences (FVBN versus C57BL/6), a comparison of cardiac autophagy in C57BL/6 and BALB/c mice conducted by Phyllis Linton’s group revealed substantial differences in autophagy (unpublished data). Although many upstream signals driving autophagy were increased in BALB/c mice when compared with C57BL/6 mice at 3 months, the BALB/c mice had increased aggregate-associated p62/SQSTM1, higher levels of protein carbonylation, and lower levels of LC3-I and -II. Autophagic flux was impaired, evidenced by a failure to increase LC3-II after chloroquine treatment and by the presence of elevated levels of p62/SQSTM1 and ubiquitinated proteins. Strain differences have been noted by others as well.29,30

Circadian Effects

Many investigators have noted a significant diurnal variation in autophagy31–34: we observed that LC3-II levels increased by 40% between 10 am and 2 pm in hearts of FVB/N mice maintained on a 12-hour light/dark schedule (lights off at 6 pm; B. Ito, R.M. Mentzer, R.A. Gottlieb, unpublished data, 2010). The magnitude of this increase is comparable with that achieved by fasting, thus it is important to design experiments to avoid circadian effects.

Effects of Sex on Autophagy

Examination of autophagy (LC3-II levels) in hearts of C57Bl/6 mice revealed that females showed 50% more LC3-I and BECN1 under basal conditions than male animals (M. Gurney and P.-J. Linton, unpublished data, 2012). Sex differences in autophagy in various tissues have been reported by several laboratories.6,35–40 Response to rapamycin also differed according to sex, suggesting sex-related differences in the MTOR signaling pathway.41,42

Methods to Measure Autophagy and Mitophagy

Measurement of Autophagy in Cells and Tissues

Western Blot of LC3 and p62/SQSTM1

Measuring protein expression remains the mainstay method for investigating the process of autophagy. Until recently, the paradigm for measuring autophagy was to examine LC3-II and LC3-I via Western blot. LC3-II is consistently associated with autophagosomes and for that reason is a useful indicator of autophagosome initiation.43–46 Conveniently, lipidation of LC3 alters its conformation such that it has faster mobility in SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Although some groups have used the LC3-II/LC3-I ratio as an indication of autophagic activity, this is unreliable and has been generally discounted.47 Although LC3-I levels can rise in response to an autophagic stimulus, if flux is rapid, levels may remain stable or actually decrease if lipidation outstrips the expression of LC3 or recycling of LC3-II off of autolysosomes by ATG4. Furthermore, LC3-II levels can rise with autophagy, but can rise even more if lysosomal degradation of autophagosomes is impaired. Depending on the particular dynamics of a given cell responding to stress, the ratio can be up, down, or unchanged. LC3-II normalized to a protein loading control is used for flux measurements.

The autophagy receptor protein p62/SQSTM1 can serve as a surrogate marker of increased autophagy when levels are diminished although caveats are appropriate here as well. It is important to understand that p62/SQSTM1 can be present in cells or tissues as a freely soluble form in the cytosol or associated with detergent-insoluble ubiquitinated protein aggregates. The normal response to an autophagic stimulus involves an early increase in p62/SQSTM1 expression in the cytosol, followed by clearance of p62/SQSTM1 associated with aggregates or other cargo. Although not indicated in every instance, assessment of soluble and detergent-insoluble p62/SQSTM1 pools can provide valuable information.48 Like LC3, Western blot analysis of p62/SQSTM1 is only a snapshot of a dynamic process. For that reason, measurement of LC3 and p62/SQSTM1 in the presence or absence of lysosomal blockade with chloroquine or bafilomycin A1 provides essential information about the rate of transit of autophagosome cargo through lysosomal degradation (autophagic flux).49–52 The Table provides the expected changes in LC3 and p62/SQSTM1 under basal conditions, during initiation of autophagy, at equilibrium upregulated autophagy, when autophagic precursors are limited, and in the setting of impaired flux (acute and chronic). Also shown are the expected effects of lysosomal blockade in each scenario. Availability of precursors can be limiting if protein synthesis is suppressed (eg, during ischemia). In that case, brisk autophagic flux is reflected by a substantial depletion of available factors.53 Besides LC3 and p62/SQSTM1, other autophagy-related factors, including ATG16L1, BECN1, and the ATG5–ATG12 complex, commonly rise in parallel with LC3-II and provide supportive evidence, particularly if LC3 blots are of poor quality (see Technical Concerns Specific to LC3 section of this article).

Immunostaining of LC3 Puncta

Detection of autophagosomes by immunostaining of LC3 in fixed cells or tissues is technically challenging because of the high background from LC3-I unassociated with autophagosomes; this can be improved by gentle permeabilization of cells (eg, with digitonin or saponin) to extract free LC3-I without affecting membrane-associated LC3-II.54 This method is technically challenging and has not been widely accepted, however.

Technical Concerns Specific to LC3

LC3 is a fragile low-abundance protein that can be lost from frozen tissue samples with repetitive freeze–thawing, a concern that diminishes for samples stored in SDS-PAGE sample buffer. Heart tissue should be snap-frozen in liquid nitrogen and stored at −80oC until it can be processed. We have compared radioimmunoprecipitation assay buffer (50 mmol/L Tris; 150 mmol/L NaCl; 0.1% SDS; 0.5% sodium deoxycholate; 1% NP-40), a Triton X-100–based detergent extraction buffer (50 mmol/L Tris–HCl; pH, 7.4; 150 mmol/L NaCl; 1 mmol/L EGTA; 1 mmol/L EDTA; 1% Triton-X 100), and radioimmunoprecipitation assay buffer solubilization of tissue ground to a powder at liquid nitrogen temperatures, and have concluded that the most reliable protocol for solubilization of LC3 from frozen heart tissue is with radioimmunoprecipitation assay buffer followed quickly by centrifugation at 1000g to remove nuclei. After removing an aliquot for protein determination, the solubilized sample should be promptly boiled in sample buffer with fresh 2-mercaptoethanol and stored frozen at −80°C until used for gel loading (fresh 2-mercaptoethanol may be added back). LC3 binds more avidly to polyvinylidine difluoride than nitrocellulose (this differential binding is particularly notable for human heart samples) and is readily lost from membranes that are stripped and reprobed. For that reason, LC3 should be the first protein probed for on a membrane. Because of its low molecular weight, transfer time should be kept short (eg, 150 mA for 2 hours). Antibodies to LC3 are available from several sources and vary with respect to isoforms recognized, species specificity, and avidity for LC3-I versus LC3-II. We have found the antibody against LC3A/B (cat 4108; Cell Signaling Technology to be reliable for rodent heart tissue (rat and mouse) and for porcine and human heart samples.

Cell Signaling Markers of Autophagy Induction

Autophagy initiation is controlled by 2 major cell signaling pathways. The AKT/MTOR signaling axis is a well-recognized negative regulator of autophagy, whereas AMPK is known to promote initiation. These opposing signaling pathways converge on ULK1 to dictate the fate of autophagy. ULK1 is important for the nucleation of autophagosomes. Phosphorylation of ULK1 by AMPK on Ser317, 555, and 777 promotes autophagy.55,56 In contrast, phosphorylation of ULK1 at Ser757 by MTOR inhibits autophagy initiation.56 Markers of MTOR activity include MTOR phosphorylation at Ser2448, phosphorylation of ribosomal subunit RPS6 at Ser235/236, and phosphorylation of the translation repressor protein EIF4E-BP1 at Ser65 and Thr70.

mRNA Markers of Autophagy

Measurement of mRNA for autophagy proteins can provide additional insights, but the presence of upregulation at the mRNA level does not necessarily correlate with protein expression or functional autophagy. It can, however, reflect an intact signaling pathway for the induction of autophagy.

Measuring Mitophagy

The specific targeting of mitochondria for autophagic disposal during I/R insult is an important element of cardioprotection.21,22 Because mitochondrial autophagy is a major pathway for turnover of mitochondria, it is important to be able to monitor mitophagy. Examining mitochondrial content via Western blot or mitochondrial DNA:nuclear DNA ratio must be paired with the same measurement in the presence of lysosomal blockade with chloroquine or bafilomycin A1 in order to infer mitophagy. It is important to assess markers in outer mitochondrial membrane (TOMM70 or VDAC) and inner membrane/matrix (COX4I1/COX IV or ACO2/aconitase). This is because outer mitochondrial membrane proteins are also subject to degradation by the ubiquitin proteasome system.57 An observed decrease in mitochondrial mass that is prevented by lysosomal blockade is indicative of mitophagy.

Use of Fluorescent Reporters to Assess Mitophagy

The coral-derived fluorescent protein Keima changes its fluorescence properties over the pH range of 4 to 8; when targeted to the mitochondrial matrix (mitoKeima), it serves to report on delivery of mitochondria to the lysosome.58 MitoKeima holds great promise for monitoring mitochondrial autophagy in cells, and a transgenic reporter mouse would have a considerable use. A somewhat different approach was pioneered by our laboratory: we targeted fluorescent Timer protein to the mitochondrial matrix (MitoTimer). Newly synthesized protein fluoresces green, but the conformation matures >24 to 48 hours to a more stable red fluorescent conformation. This allows detection of mitochondrial biogenesis (green MitoTimer) and mitophagy (decrease in red MitoTimer if new protein is not being made).59,60 Using constitutive expression of MitoTimer electroporated into mouse skeletal muscle, Laker et al61 showed that exercise increases turnover of mitochondria (biogenesis and mitophagy), whereas a high-fat diet slows turnover. The advent of a transgenic MitoTimer mouse will doubtless provide additional insights.

Methods to Assess Autophagy in Intact Animals

Measuring Autophagic Flux In Vivo With Lysosomal Blockade

This method to measure flux introduces a pharmacological blockade that prevents lysosomal/autophagosomal fusion or prevents lysosomal-mediated enzymatic degradation. Several agents are suitable to achieve lysosomal blockade: bafilomycin A1, chloroquine, and ammonium chloride raise the intralysosomal pH, preventing autophagosome fusion with the lysosome,51,62–65 whereas protease inhibitors such as pepstatin A, E-64d, and leupeptin inhibit lysosomal proteases18,47,66 and microtubule inhibitors such as vinblastine prevent trafficking of autophagosomes and lysosomes.67 For in vivo studies, chloroquine (10–50 mg/kg intraperitoneal [IP]),48,68 leupeptin (20–40 mg/kg IP),66 or bafilomycin A1 (2.5 mg/kg IP every 12 hours)69 have been used to block autophagic flux. Mice subjected to lysosomal blockade and untreated comparators are euthanized for tissue harvest at a specified time (usually 2–4 hours) after blockade. Lysosomal inhibition results in the accumulation of autophagosomes that would have progressed through the pathway during that period. Differential accumulation of autophagosomes is assessed by microscopy, or autophagosomal LC3-II is measured by Western blot.18 Intact autophagic flux is indicated by an increase in autophagosome puncta or in LC3-II on Western blot when compared with paired animals without lysosomal blockade and is calculated as a ratio rather than the difference (according to Tanida et al18). Little work has been done to establish whether the magnitude of increase is proportional to the rate of autophagic flux. We have found that the receptor protein p62/SQSTM1 is a fairly reliable surrogate marker of autophagic flux: when flux is intact, p62/SQSTM1 levels should decrease, whereas when flux is absent, p62/SQSTM1 levels will rise. This is helpful when interpreting results of samples obtained before and after an intervention, such as atrial biopsies obtained at the beginning and end of aortic cross-clamp.53

Measuring Autophagic Flux Ex Vivo in Tissue Samples

Kaushik and Cuervo70 have developed a method to measure autophagic flux in small chunks of liver tissue that are minced finely and then divided between 2 wells (with or without lysosomal inhibitors) and then incubated at 37°C for 1 to 2 hours with occasional swirling. The minced tissue is then recovered, homogenized, and processed for Western blotting. We have begun preliminary work to adapt this to heart tissue and are optimistic that this ex vivo flux assay can provide reliable information about the level of autophagy and the presence/absence of autophagic flux in small tissue samples. This approach will eliminate the need for paired animals treated ± lysosomal inhibitors and will also make it possible to monitor flux in freshly obtained biopsies from human tissues.

Imaging of Autophagic Puncta in Transgenic Mice

Transgenic mice expressing a fluorescent protein fused to LC3 have been used to monitor autophagy in many studies, although as noted above, it is essential to monitor flux either through comparison with mice in which lysosomal function has been inhibited or inferred from changes in p62/SQSTM1. Cryosections are preferable for preserving fluorescence of the fusion proteins. Puncta can be scored in various ways: as the number per unit area, or as the percentage of area occupied by fluorescent puncta (useful when puncta are so numerous that it is difficult to count individual units). Because autophagosomes are typically more numerous in the perinuclear zone, scoring will be more consistent in sections where myocytes are longitudinally arrayed. Normalizing puncta number (or area) to nuclei may be preferable to normalizing to area. It should be noted that transgenics expressing LC3 fusion proteins may also develop protein aggregates, which can easily be mistaken for puncta. It is important to select a line in which expression is relatively low to reduce the occurrence of aggregates. Although the transgene is not under control of a physiological promoter, the abundance of the fusion protein changes rapidly in response to physiological cues, such as exercise, fasting, or ischemic preconditioning,23 suggesting that post-translational regulation dominates. Proteasomal inhibition increased the abundance of mCherry-LC3 (C. Perry, unpublished observations, 2008), consistent with findings reported in astrocytes.71 Several additional caveats should be noted when using LC3 fusion proteins for assessing autophagy. LC3B, which is preferentially detected by one of the commonly used antibodies (rabbit-anti-LC3AB antibody; Cell Signaling [4108S]), and which was used for the fusion proteins, is a member of the ATG8 gene family, which has 7 members in humans: MAP1LC3A, B, B2, C, GABARAP, GABARAPL1, and GABARAPL2/GATE-16. There are tissue-specific differences in the expression of ATG8 family members: GABARAPL2 and GABARAPL1 are expressed predominantly in the central nervous system, whereas GABARAP is more heavily expressed in endocrine glands, and LC3C is predominantly expressed in the lung. Different family members may be responsible for specific roles in protein trafficking and selective target engulfment, and they may be upregulated in response to different cues.72 It has been suggested that GABARAPL1 interacts with starch-binding domain 1 and therefore may play a role in autophagic degradation of glycogen granules (glycophagy). Thus, monitoring LC3B (including LC3B fusion proteins) may be only part of the story. Noncanonical autophagy (independent of LC3B and ATG5) shares some autophagy machinery and often responds to similar cues.73

LC3 fusion proteins can also be monitored by Western blotting: conveniently, the initial degradation of GFP-LC3 in the lysosome gives rise to a fragment that can be readily detected with antibody to GFP.74

Use of Tandem GFP-RFP-LC3 to Assess Flux

This approach uses a tandem fluorescent–tagged LC3 protein, monomeric RFP (or mCherry) coupled to GFP-LC3 (mRFP/mCherry-GFP-LC3), to report autophagy induction and flux.75 A similar approach can be achieved by crossing GFP-LC3 and mCherry-LC3 transgenic mice76 although theoretical disadvantages of the double-transgenic have been noted.20 GFP fused to LC3 loses fluorescence rapidly in the acidic environment of the lysosome, whereas mRFP or mCherry retains fluorescence somewhat longer in the lysosome. Thus, autophagosomes fluoresce in both green and red channels, but after fusion with lysosomes, the green fluorescence is lost, leaving only red fluorescence.47,75 Samples may be fixed; however, it has been suggested that some fixation protocols may artifactually restore green fluorescence by neutralizing the acidic autolysosome.47 The group of Sadoshima embedded heart slices in Tissue-Tek OCT (optimum cutting temperature) compound, and 10 micron cryosections were air-dried and fixed in ethanol before counterstaining with DAPI (4’6-diaminidino-2-phenylindole) and mounting. Because this fails to preserve lysosomal pH, differences in red versus green fluorescence may either be a function of proteolytic degradation of GFP in the lysosome, which must proceed at a faster rate than degradation of mRFP or mCherry or may be lost on neutralization.47 To determine flux, the relative abundance of red-only versus red+green puncta are compared. The presence of red-only puncta indicates intact autophagic flux, although here again, there is little information as to whether the relative ratio of red-only to red+green puncta is a quantitative index of flux. A key advantage of this method is that it allows an estimation of flux in a single animal, rather than having to use paired animals (±chloroquine) for each data point. This was used to study autophagic flux after ischemia/reperfusion.20 The disadvantage is that it is limited to the transgenic mouse line and as such is not available for concurrent use in other mouse lines or other species.

Optical Imaging of Fluorescent Proteins

We hoped to use cardiac-restricted mCherry-LC3 to image autophagy in the heart. mCherry was chosen because its fluorescence properties were better suited to spectral imaging than GFP. We used the Caliper Life Sciences Spectrum In Vivo Imaging System to detect an increase in photon flux from the baseline of 3.19±0.72 to a mean of 3.93±1.10 at 4 hours after administration of rapamycin (2 mg/kg IP) and chloroquine (10 mg/kg IP; P<0.01; n=14; Figure 2). This approach raised hope that it would be possible to image autophagy in vivo. However, to date, there has not been widespread adoption of such an imaging methodology to monitor autophagy. The technique works best in young (small) mice (eg, 6 weeks) and requires thorough depilation to minimize autofluorescence.

Figure 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2.

Optical imaging of autophagy in hearts of mCherry-light chain 3 (LC3) mice. A, Mice were imaged at baseline (Pre) and 4 hours after rapamycin and chloroquine administration (Post), using a protocol of 3 acquisitions of 15 s each. Representative images from a single mouse are shown. B, Graph shows the change in fluorescence from baseline to 4 hours 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 and the increase in number of fluorescent red puncta (autophagosomes). Nuclei are stained blue with DAPI (4'6-diaminidino-2-phenylindole; images from A and B are reprinted from Circulation Research with permission of the publisher [copyright ©2011, Wolters Kluwer Health]. Abstract P066: Imaging Autophagy in Living Mice v109:AP066, 2011. Unpublished images from C provided by Dr Chengqun Huang and Dr Roberta A. Gottlieb, 2010).

Reporting Nanoparticles

Increased activity of the autophagy-associated protease ATG4 can be detected using fluorescent nanoparticles in which a peptide substrate of ATG4 was labeled with fluorescein isothiocyanate and a quenching agent and assembled onto a hydrophobically modified glycol chitosan shell loaded with a red fluorescent lysosomotropic dye (Lysolite Red).77 Activity of ATG4 cleaved the peptide, thereby dequenching the fluorescein isothiocyanate. The purpose of the lysosomal dye was to account for direct (ATG4 independent) lysosomal degradation of nanoparticles (indicated by colocalization of red and green signals). Because the autophagy-independent component is small when compared with the autophagy-dependent pathway, it may be possible to adapt this approach to in vivo imaging. Although this approach was described in 2011, its use for in vivo imaging has not been published.

A related approach was used to monitor the lysosomal compartment, which expands during autophagy.78 A cathepsin-activatable near-infrared fluorochrome (CAF-680) was used to demonstrate an 8-fold increase in fluorescence 2 hours after rapamycin administration followed by ischemia/reperfusion. The signal was localized to lysosomes as documented by colocalization with LAMP2 in immunostained cryosections. Interestingly, Feraheme (Ferumoxytol) nanoparticles that are fluorescently tagged with CyAl5.5 are taken up via endocytosis at a much faster rate when autophagy is stimulated with rapamycin, suggesting that it may be possible to infer autophagic activity from retention of such nanoparticles, which can be readily imaged by MRI.

Delayed enhancement after injection of chelated Gd MRI contrast agents (late gadolinium enhancement [LGE]) has been noted in a variety of conditions and has been attributed to slower clearance from tissue because of fibrosis or the presence of macrophages that take up the agent. However, many of the pathological processes that give rise to fibrosis and LGE are also associated with increased autophagy, including scleroderma,79,80 heart failure,81,82 radiation-induced myocardial injury,83,84 Friedreich ataxia,85,86 and Fabry disease.87,88 It is possible that some instances of LGE are because of upregulation of autophagy rather than fibrosis or macrophage activity, but this will need to be formally tested.

Assessment of Autophagy in Human Subjects

Detection of autophagy in humans is restricted to biochemical or microscopy analysis of biopsies of the tissue of interest or analysis of peripheral blood, thus severely limiting our ability to understand its role in human disease or to develop effective therapies. Kassiotis et al89 reported a decrease in autophagic markers in hearts of patients who received a left ventricular assist device; they inferred that autophagy was an adaptive response in the failing heart, and that mechanical unloading relieved the stress responsible for triggering autophagy. Garcia et al90 studied autophagy markers by western blot and electron microscopy of atrial appendage tissue obtained during coronary artery bypass graft surgery; they noted that postoperative atrial fibrillation was more common in patients with evidence of impaired autophagic flux. Jahania et al53 detected autophagic flux in atrial tissue in patients undergoing cardiopulmonary bypass based on changes in p62/SQSTM1 and other autophagy proteins, and found that the magnitude of p62/SQSTM1 clearance was inversely related to mortality and morbidity risk scores. However, Gedik et al91 examined autophagy markers (by Western blot) in left ventricle of patients who underwent remote ischemic preconditioning before coronary artery bypass graft surgery and saw no evidence of enhanced autophagy compared with nonpreconditioned patients, or did they detect evidence of an autophagic response during early reperfusion in either group. A study conducted by Singh et al92 examined autophagy gene expression by polymerase chain reaction and Western blotting for autophagy proteins and regulators AMPK and MTOR. They identified upregulation of mRNAs for autophagy components, including LC3B, ATG4A, ATG4C, and ATG4D, as well as WIPI1, GABARAP, and GABARAPL2; they also observed an increase in LC3-II from beginning to end of cardiopulmonary bypass and an increase in phospho-AMPK and a drop in protein levels of MTOR. Interestingly, they did not see a drop in p62/SQSTM1 levels. The results differ widely between these various studies, making it difficult to establish specific guidelines to apply to interpreting results, or even to generalize about the findings. Although the studies of atrial tissue noted changes in autophagy, this was not the case for ventricular biopsies, although because no study compared atrial and ventricular samples, it is not possible to know whether the human left ventricle is unresponsive when compared with the right atrium or whether the procedural differences in that study accounted for the lack of change in autophagy markers. More work will be needed to resolve these questions, but the difficulties in obtaining human heart tissue pose substantial obstacles to a comprehensive study.

Systemic conditions that affect autophagy, such as drug exposure, fasting, or inflammation, might be expected to have parallel effects in multiple tissues. That rationale has led some investigators to examine autophagy in peripheral blood leukocytes. Autophagy markers were assessed in CD4 lymphocytes of men subjected to different exercise regimens or sedentary controls after 5 weeks of training; they found that exercise conditioning (especially high-intensity interval training) triggers autophagy and suppresses apoptosis in lymphocytes when the subjects undergo a hypoxic exercise challenge.93 The authors did not go so far as to suggest that the autophagic response in lymphocytes might reliably report on autophagy in other organs, but 2 studies by another group found a significant reduction in autophagy markers (by Western blotting) in leukocytes of patients with coronary artery disease94 or acute myocardial infarction95 when compared with healthy controls. However, none of these studies correlated autophagy markers in lymphocytes with those in heart tissue, so it remains to be seen whether peripheral blood cells represent a reliable reflection of autophagic activity in the heart. Dysregulation of autophagy was also noted by the same group in peripheral leukocytes of patients with sporadic Parkinson disease,96 suggesting that underlying abnormalities that affect autophagy in the brain may be paralleled in leukocytes.

Recent advances in flow cytometry–based approaches to measuring autophagy hold promise for broader use of leukocyte autophagy as an index of organism-wide autophagic competence.97,98 These approaches are based on flow cytometric image analysis of LC3-decorated autophagosomes; however, given the parallel upregulation of lysosomal activity, cathepsin-based fluorogenic reagents or lysosomotropic dyes may provide an adequate readout if appropriate controls and validation studies are included.

Conclusions

Autophagy is increasingly recognized to play an important role in mitigating or exacerbating various cardiovascular diseases; hence, it is important to be able to measure it accurately and to understand the significance of the findings. Given the highly dynamic nature of autophagy, it is essential to be able to account for autophagic flux. Current methods are imperfect, but newer technologies are emerging and there is hope that noninvasive imaging modalities may be developed for in vivo animal and human investigations.

View this table:
  • View inline
  • View popup
Table.

Expected Changes in Common Autophagy Markers in Various Scenarios (Snapshot and After Lysosomal Blockade With CQ)

Sources of Funding

Dr Gottlieb holds the Dorothy and E. Phillip Lyon Chair in Molecular Cardiology in honor of Clarence M. Agress, MD. This work was funded, in part, by National Institutes of Health P01 HL112730 (Dr Gottlieb).

Disclosures

Dr Gottlieb is a consultant for Takeda Pharmaceuticals and is a cofounder of TissueNetix, Inc. The other authors report no conflicts.

Footnotes

  • In December 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.47 days.

  • This Review is in a thematic series on Autophagy in Health and Disease, which includes the following articles:

    Molecular Mechanisms of Autophagy in the Cardiovascular System

    Autophagy in Vascular Disease

    The Role of Autophagy in Vascular Biology

    Therapeutic Targeting of Autophagy: Potential and Concerns in Treating Cardiovascular Disease

    Untangling Autophagy Measurements: All Fluxed Up

    Mitochondria and Autophagy

    Daniel Klionsky, Guest Editor

  • Nonstandard Abbreviations and Acronyms
    HDAC
    histone deacetylase
    LC3
    light chain 3
    LGE
    late gadolinium enhancement
    SAHA
    suberoylanilide hydroxamic acid (vorinostat)
    ULK
    unc-51-like kinase

  • Received October 22, 2014.
  • Revision received January 2, 2015.
  • Accepted January 5, 2015.
  • © 2015 American Heart Association, Inc.

References

  1. 1.↵
    1. Yan L,
    2. Vatner DE,
    3. Kim SJ,
    4. Ge H,
    5. Masurekar M,
    6. Massover WH,
    7. Yang G,
    8. Matsui Y,
    9. Sadoshima J,
    10. Vatner SF
    . Autophagy in chronically ischemic myocardium. Proc Natl Acad Sci U S A. 2005;102:13807–13812. doi: 10.1073/pnas.0506843102.
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    1. Hamacher-Brady A,
    2. Brady NR,
    3. Gottlieb RA,
    4. Gustafsson AB
    . Autophagy as a protective response to Bnip3-mediated apoptotic signaling in the heart. Autophagy. 2006;2:307–309.
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Levine B,
    2. Klionsky DJ
    . Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004;6:463–477.
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Mizushima N,
    2. Levine B,
    3. Cuervo AM,
    4. Klionsky DJ
    . Autophagy fights disease through cellular self-digestion. Nature. 2008;451:1069–1075. doi: 10.1038/nature06639.
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Nishino I,
    2. Fu J,
    3. Tanji K,
    4. et al
    . Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature. 2000;406:906–910. doi: 10.1038/35022604.
    OpenUrlCrossRefPubMed
  6. 6.↵
    1. Reichelt ME,
    2. Mellor KM,
    3. Curl CL,
    4. Stapleton D,
    5. Delbridge LM
    . Myocardial glycophagy - a specific glycogen handling response to metabolic stress is accentuated in the female heart. J Mol Cell Cardiol. 2013;65:67–75. doi: 10.1016/j.yjmcc.2013.09.014.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. Takikita S,
    2. Myerowitz R,
    3. Zaal K,
    4. Raben N,
    5. Plotz PH
    . Murine muscle cell models for Pompe disease and their use in studying therapeutic approaches. Mol Genet Metab. 2009;96:208–217. doi: 10.1016/j.ymgme.2008.12.012.
    OpenUrlCrossRefPubMed
  8. 8.↵
    1. Kuma A,
    2. Hatano M,
    3. Matsui M,
    4. Yamamoto A,
    5. Nakaya H,
    6. Yoshimori T,
    7. Ohsumi Y,
    8. Tokuhisa T,
    9. Mizushima N
    . The role of autophagy during the early neonatal starvation period. Nature. 2004;432:1032–1036. doi: 10.1038/nature03029.
    OpenUrlCrossRefPubMed
  9. 9.↵
    1. Nishida Y,
    2. Arakawa S,
    3. Fujitani K,
    4. Yamaguchi H,
    5. Mizuta T,
    6. Kanaseki T,
    7. Komatsu M,
    8. Otsu K,
    9. Tsujimoto Y,
    10. Shimizu S
    . Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature. 2009;461:654–658. doi: 10.1038/nature08455.
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. Qu X,
    2. Yu J,
    3. Bhagat G,
    4. Furuya N,
    5. Hibshoosh H,
    6. Troxel A,
    7. Rosen J,
    8. Eskelinen EL,
    9. Mizushima N,
    10. Ohsumi Y,
    11. Cattoretti G,
    12. Levine B
    . Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest. 2003;112:1809–1820. doi: 10.1172/JCI20039.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Zhong Y,
    2. Wang QJ,
    3. Yue Z
    . Atg14L and Rubicon: yin and yang of Beclin 1-mediated autophagy control. Autophagy. 2009;5:890–891.
    OpenUrlCrossRefPubMed
  12. 12.↵
    1. Sun Q,
    2. Zhang J,
    3. Fan W,
    4. Wong KN,
    5. Ding X,
    6. Chen S,
    7. Zhong Q
    . The RUN domain of rubicon is important for hVps34 binding, lipid kinase inhibition, and autophagy suppression. J Biol Chem. 2011;286:185–191. doi: 10.1074/jbc.M110.126425.
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    1. Zhu H,
    2. Tannous P,
    3. Johnstone JL,
    4. Kong Y,
    5. Shelton JM,
    6. Richardson JA,
    7. Le V,
    8. Levine B,
    9. Rothermel BA,
    10. Hill JA
    . Cardiac autophagy is a maladaptive response to hemodynamic stress. J Clin Invest. 2007;117:1782–1793. doi: 10.1172/JCI27523.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Matsui Y,
    2. Takagi H,
    3. Qu X,
    4. Abdellatif M,
    5. Sakoda H,
    6. Asano T,
    7. Levine B,
    8. Sadoshima J
    . Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy. Circ Res. 2007;100:914–922. doi: 10.1161/01.RES.0000261924.76669.36.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    1. Sala-Mercado JA,
    2. Wider J,
    3. Undyala VV,
    4. Jahania S,
    5. Yoo W,
    6. Mentzer RM Jr.,
    7. Gottlieb RA,
    8. Przyklenk K
    . Profound cardioprotection with chloramphenicol succinate in the swine model of myocardial ischemia-reperfusion injury. Circulation. 2010;122:S179–S184. doi: 10.1161/CIRCULATIONAHA.109.928242.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    1. Ma X,
    2. Liu H,
    3. Foyil SR,
    4. Godar RJ,
    5. Weinheimer CJ,
    6. Hill JA,
    7. Diwan A
    . Impaired autophagosome clearance contributes to cardiomyocyte death in ischemia-reperfusion injury. Circulation. 2012;125:3170–3181. doi: 10.1161/CIRCULATIONAHA.111.041814.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Xu X,
    2. Kobayashi S,
    3. Chen K,
    4. Timm D,
    5. Volden P,
    6. Huang Y,
    7. Gulick J,
    8. Yue Z,
    9. Robbins J,
    10. Epstein PN,
    11. Liang Q
    . Diminished autophagy limits cardiac injury in mouse models of type 1 diabetes. J Biol Chem. 2013;288:18077–18092. doi: 10.1074/jbc.M113.474650.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Tanida I,
    2. Minematsu-Ikeguchi N,
    3. Ueno T,
    4. Kominami E
    . Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy. 2005;1:84–91.
    OpenUrlCrossRefPubMed
  19. 19.↵
    1. Xie Z,
    2. Lau K,
    3. Eby B,
    4. Lozano P,
    5. He C,
    6. Pennington B,
    7. Li H,
    8. Rathi S,
    9. Dong Y,
    10. Tian R,
    11. Kem D,
    12. Zou MH
    . Improvement of cardiac functions by chronic metformin treatment is associated with enhanced cardiac autophagy in diabetic OVE26 mice. Diabetes. 2011;60:1770–1778. doi: 10.2337/db10-0351.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    1. Hariharan N,
    2. Zhai P,
    3. Sadoshima J
    . Oxidative stress stimulates autophagic flux during ischemia/reperfusion. Antioxid Redox Signal. 2011;14:2179–2190. doi: 10.1089/ars.2010.3488.
    OpenUrlCrossRefPubMed
  21. 21.↵
    1. Andres AM,
    2. Hernandez G,
    3. Lee P,
    4. Huang C,
    5. Ratliff EP,
    6. Sin J,
    7. Thornton CA,
    8. Damasco MV,
    9. Gottlieb RA
    . Mitophagy is required for acute cardioprotection by simvastatin. Antioxid Redox Signal. 2014;21:1960–1973. doi: 10.1089/ars.2013.5416.
    OpenUrlCrossRefPubMed
  22. 22.↵
    1. Huang C,
    2. Andres AM,
    3. Ratliff EP,
    4. Hernandez G,
    5. Lee P,
    6. Gottlieb RA
    . Preconditioning involves selective mitophagy mediated by Parkin and p62/SQSTM1. PLoS One. 2011;6:e20975. doi: 10.1371/journal.pone.0020975.
    OpenUrlCrossRefPubMed
  23. 23.↵
    1. Huang C,
    2. Yitzhaki S,
    3. Perry CN,
    4. Liu W,
    5. Giricz Z,
    6. Mentzer RM Jr.,
    7. Gottlieb RA
    . Autophagy induced by ischemic preconditioning is essential for cardioprotection. J Cardiovasc Transl Res. 2010;3:365–373. doi: 10.1007/s12265-010-9189-3.
    OpenUrlCrossRefPubMed
  24. 24.↵
    1. Zhang YL,
    2. Yao YT,
    3. Fang NX,
    4. Zhou CH,
    5. Gong JS,
    6. Li LH
    . Restoration of autophagic flux in myocardial tissues is required for cardioprotection of sevoflurane postconditioning in rats. Acta Pharmacol Sin. 2014;35:758–769. doi: 10.1038/aps.2014.20.
    OpenUrlCrossRefPubMed
  25. 25.↵
    1. Olsen CA,
    2. Ghadiri MR
    . Discovery of potent and selective histone deacetylase inhibitors via focused combinatorial libraries of cyclic alpha3beta-tetrapeptides. J Med Chem. 2009;52:7836–7846. doi: 10.1021/jm900850t.
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Cao DJ,
    2. Wang ZV,
    3. Battiprolu PK,
    4. Jiang N,
    5. Morales CR,
    6. Kong Y,
    7. Rothermel BA,
    8. Gillette TG,
    9. Hill JA
    . Histone deacetylase (HDAC) inhibitors attenuate cardiac hypertrophy by suppressing autophagy. Proc Natl Acad Sci U S A. 2011;108:4123–4128. doi: 10.1073/pnas.1015081108.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Xie M,
    2. Kong Y,
    3. Tan W,
    4. et al
    . Histone deacetylase inhibition blunts ischemia/reperfusion injury by inducing cardiomyocyte autophagy. Circulation. 2014;129:1139–1151. doi: 10.1161/CIRCULATIONAHA.113.002416.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Yitzhaki S,
    2. Huang C,
    3. Liu W,
    4. Lee Y,
    5. Gustafsson AB,
    6. Mentzer RM Jr.,
    7. Gottlieb RA
    . Autophagy is required for preconditioning by the adenosine A1 receptor-selective agonist CCPA. Basic Res Cardiol. 2009;104:157–167. doi: 10.1007/s00395-009-0006-6.
    OpenUrlCrossRefPubMed
  29. 29.↵
    1. Martyniszyn L,
    2. Szulc-Dabrowska L,
    3. Boratyńska-Jasińska A,
    4. Badowska-Kozakiewicz AM,
    5. Niemiałtowski MG
    . In vivo induction of autophagy in splenocytes of C57BL/6 and BALB/c mice infected with ectromelia orthopoxvirus. Pol J Vet Sci. 2013;16:25–32.
    OpenUrlPubMed
  30. 30.↵
    1. Pinheiro RO,
    2. Nunes MP,
    3. Pinheiro CS,
    4. D’Avila H,
    5. Bozza PT,
    6. Takiya CM,
    7. Côrte-Real S,
    8. Freire-de-Lima CG,
    9. DosReis GA
    . Induction of autophagy correlates with increased parasite load of Leishmania amazonensis in BALB/c but not C57BL/6 macrophages. Microbes Infect. 2009;11:181–190. doi: 10.1016/j.micinf.2008.11.006.
    OpenUrlCrossRefPubMed
  31. 31.↵
    1. Bray MS,
    2. Young ME
    . Diurnal variations in myocardial metabolism. Cardiovasc Res. 2008;79:228–237. doi: 10.1093/cvr/cvn054.
    OpenUrlAbstract/FREE Full Text
  32. 32.↵
    1. Ma D,
    2. Li S,
    3. Molusky MM,
    4. Lin JD
    . Circadian autophagy rhythm: a link between clock and metabolism? Trends Endocrinol Metab. 2012;23:319–325. doi: 10.1016/j.tem.2012.03.004.
    OpenUrlCrossRefPubMed
  33. 33.↵
    1. Pfeifer U,
    2. Scheller H
    . A morphometric study of cellular autophagy including diurnal variations in kidney tubules of normal rats. J Cell Biol. 1975;64:608–621.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    1. Young ME,
    2. Razeghi P,
    3. Cedars AM,
    4. Guthrie PH,
    5. Taegtmeyer H
    . Intrinsic diurnal variations in cardiac metabolism and contractile function. Circ Res. 2001;89:1199–1208.
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    1. Campesi I,
    2. Straface E,
    3. Occhioni S,
    4. Montella A,
    5. Franconi F
    . Protein oxidation seems to be linked to constitutive autophagy: a sex study. Life Sci. 2013;93:145–152. doi: 10.1016/j.lfs.2013.06.001.
    OpenUrlCrossRefPubMed
  36. 36.↵
    1. Oliván S,
    2. Calvo AC,
    3. Manzano R,
    4. Zaragoza P,
    5. Osta R
    . Sex differences in constitutive autophagy. Biomed Res Int. 2014;2014:652817. doi: 10.1155/2014/652817.
    OpenUrlPubMed
  37. 37.↵
    1. Chen C,
    2. Hu LX,
    3. Dong T,
    4. Wang GQ,
    5. Wang LH,
    6. Zhou XP,
    7. Jiang Y,
    8. Murao K,
    9. Lu SQ,
    10. Chen JW,
    11. Zhang GX
    . Apoptosis and autophagy contribute to gender difference in cardiac ischemia-reperfusion induced injury in rats. Life Sci. 2013;93:265–270. doi: 10.1016/j.lfs.2013.06.019.
    OpenUrlCrossRefPubMed
  38. 38.↵
    1. Cosper PF,
    2. Leinwand LA
    . Cancer causes cardiac atrophy and autophagy in a sexually dimorphic manner. Cancer Res. 2011;71:1710–1720. doi: 10.1158/0008-5472.CAN-10-3145.
    OpenUrlAbstract/FREE Full Text
  39. 39.↵
    1. Le TY,
    2. Ashton AW,
    3. Mardini M,
    4. Stanton PG,
    5. Funder JW,
    6. Handelsman DJ,
    7. Mihailidou AS
    . Role of androgens in sex differences in cardiac damage during myocardial infarction. Endocrinology. 2014;155:568–575. doi: 10.1210/en.2013-1755.
    OpenUrlCrossRefPubMed
  40. 40.↵
    1. Lista P,
    2. Straface E,
    3. Brunelleschi S,
    4. Franconi F,
    5. Malorni W
    . On the role of autophagy in human diseases: a gender perspective. J Cell Mol Med. 2011;15:1443–1457. doi: 10.1111/j.1582-4934.2011.01293.x.
    OpenUrlCrossRefPubMed
  41. 41.↵
    1. Fok WC,
    2. Chen Y,
    3. Bokov A,
    4. Zhang Y,
    5. Salmon AB,
    6. Diaz V,
    7. Javors M,
    8. Wood WH III.,
    9. Zhang Y,
    10. Becker KG,
    11. Pérez VI,
    12. Richardson A
    . Mice fed rapamycin have an increase in lifespan associated with major changes in the liver transcriptome. PLoS One. 2014;9:e83988. doi: 10.1371/journal.pone.0083988.
    OpenUrlCrossRefPubMed
  42. 42.↵
    1. Miller RA,
    2. Harrison DE,
    3. Astle CM,
    4. et al
    . Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. Aging Cell. 2014;13:468–477. doi: 10.1111/acel.12194.
    OpenUrlCrossRefPubMed
  43. 43.↵
    1. Barth S,
    2. Glick D,
    3. Macleod KF
    . Autophagy: assays and artifacts. J Pathol. 2010;221:117–124. doi: 10.1002/path.2694.
    OpenUrlCrossRefPubMed
  44. 44.↵
    1. Tanida I,
    2. Waguri S
    . Measurement of autophagy in cells and tissues. Methods Mol Biol. 2010;648:193–214. doi: 10.1007/978-1-60761-756-3_13.
    OpenUrlCrossRefPubMed
  45. 45.↵
    1. Mizushima N,
    2. Yoshimori T,
    3. Levine B
    . Methods in mammalian autophagy research. Cell. 2010;140:313–326. doi: 10.1016/j.cell.2010.01.028.
    OpenUrlCrossRefPubMed
  46. 46.↵
    1. Menzies FM,
    2. Moreau K,
    3. Puri C,
    4. Renna M,
    5. Rubinsztein DC
    . Measurement of autophagic activity in mammalian cells. Curr Protoc Cell Biol. 2012;Chapter 15:Unit 15.16. doi: 10.1002/0471143030.cb1516s54.
    OpenUrl
  47. 47.↵
    1. Klionsky DJ,
    2. Abdalla FC,
    3. Abeliovich H,
    4. et al
    . Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy. 2012;8:445–544.
    OpenUrlCrossRefPubMed
  48. 48.↵
    1. Gurney MA,
    2. Huang C,
    3. Ramil JM,
    4. Ravindran N,
    5. Andres AM,
    6. Sin J,
    7. Linton PJ,
    8. Gottlieb RA
    . Measuring cardiac autophagic flux in vitro and in vivo. Methods Mol Biol. 2015;1219:187–197. doi: 10.1007/978-1-4939-1661-0_14.
    OpenUrlCrossRefPubMed
  49. 49.↵
    1. Verschooten L,
    2. Barrette K,
    3. Van Kelst S,
    4. Rubio Romero N,
    5. Proby C,
    6. De Vos R,
    7. Agostinis P,
    8. Garmyn M
    . Autophagy inhibitor chloroquine enhanced the cell death inducing effect of the flavonoid luteolin in metastatic squamous cell carcinoma cells. PLoS One. 2012;7:e48264. doi: 10.1371/journal.pone.0048264.
    OpenUrlCrossRefPubMed
  50. 50.↵
    1. Egger ME,
    2. Huang JS,
    3. Yin W,
    4. McMasters KM,
    5. McNally LR
    . Inhibition of autophagy with chloroquine is effective in melanoma. J Surg Res. 2013;184:274–281. doi: 10.1016/j.jss.2013.04.055.
    OpenUrlCrossRefPubMed
  51. 51.↵
    1. Yamamoto A,
    2. Tagawa Y,
    3. Yoshimori T,
    4. Moriyama Y,
    5. Masaki R,
    6. Tashiro Y
    . Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct. 1998;23:33–42.
    OpenUrlCrossRefPubMed
  52. 52.↵
    1. Wu YC,
    2. Wu WK,
    3. Li Y,
    4. Yu L,
    5. Li ZJ,
    6. Wong CC,
    7. Li HT,
    8. Sung JJ,
    9. Cho CH
    . Inhibition of macroautophagy by bafilomycin A1 lowers proliferation and induces apoptosis in colon cancer cells. Biochem Biophys Res Commun. 2009;382:451–456. doi: 10.1016/j.bbrc.2009.03.051.
    OpenUrlCrossRefPubMed
  53. 53.↵
    1. Jahania SM,
    2. Sengstock D,
    3. Vaitkevicius P,
    4. Andres A,
    5. Ito BR,
    6. Gottlieb RA,
    7. Mentzer RM Jr.
    . Activation of the homeostatic intracellular repair response during cardiac surgery. J Am Coll Surg. 2013;216:719–26; discussion 726. doi: 10.1016/j.jamcollsurg.2012.12.034.
    OpenUrlCrossRefPubMed
  54. 54.↵
    1. Kaminskyy V,
    2. Abdi A,
    3. Zhivotovsky B
    . A quantitative assay for the monitoring of autophagosome accumulation in different phases of the cell cycle. Autophagy. 2011;7:83–90.
    OpenUrlCrossRefPubMed
  55. 55.↵
    1. Egan D,
    2. Kim J,
    3. Shaw RJ,
    4. Guan KL
    . The autophagy initiating kinase ULK1 is regulated via opposing phosphorylation by AMPK and mTOR. Autophagy. 2011;7:643–644.
    OpenUrlCrossRefPubMed
  56. 56.↵
    1. Kim J,
    2. Kundu M,
    3. Viollet B,
    4. Guan KL
    . AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 2011;13:132–141. doi: 10.1038/ncb2152.
    OpenUrlCrossRefPubMed
  57. 57.↵
    1. Chan NC,
    2. Salazar AM,
    3. Pham AH,
    4. Sweredoski MJ,
    5. Kolawa NJ,
    6. Graham RL,
    7. Hess S,
    8. Chan DC
    . Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy. Hum Mol Genet. 2011;20:1726–1737. doi: 10.1093/hmg/ddr048.
    OpenUrlAbstract/FREE Full Text
  58. 58.↵
    1. Katayama H,
    2. Kogure T,
    3. Mizushima N,
    4. Yoshimori T,
    5. Miyawaki A
    . A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem Biol. 2011;18:1042–1052. doi: 10.1016/j.chembiol.2011.05.013.
    OpenUrlCrossRefPubMed
  59. 59.↵
    1. Hernandez G,
    2. Thornton C,
    3. Stotland A,
    4. Lui D,
    5. Sin J,
    6. Ramil J,
    7. Magee N,
    8. Andres A,
    9. Quarato G,
    10. Carreira RS,
    11. Sayen MR,
    12. Wolkowicz R,
    13. Gottlieb RA
    . MitoTimer: a novel tool for monitoring mitochondrial turnover. Autophagy. 2013;9:1852–1861. doi: 10.4161/auto.26501.
    OpenUrlCrossRefPubMed
  60. 60.↵
    1. Ferree AW,
    2. Trudeau K,
    3. Zik E,
    4. Benador IY,
    5. Twig G,
    6. Gottlieb RA,
    7. Shirihai OS
    . MitoTimer probe reveals the impact of autophagy, fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age. Autophagy. 2013;9:1887–1896. doi: 10.4161/auto.26503.
    OpenUrlCrossRefPubMed
  61. 61.↵
    1. Laker RC,
    2. Xu P,
    3. Ryall KA,
    4. Sujkowski A,
    5. Kenwood BM,
    6. Chain KH,
    7. Zhang M,
    8. Royal MA,
    9. Hoehn KL,
    10. Driscoll M,
    11. Adler PN,
    12. Wessells RJ,
    13. Saucerman JJ,
    14. Yan Z
    . A novel MitoTimer reporter gene for mitochondrial content, structure, stress, and damage in vivo. J Biol Chem. 2014;289:12005–12015. doi: 10.1074/jbc.M113.530527.
    OpenUrlAbstract/FREE Full Text
  62. 62.↵
    1. Seglen PO,
    2. Reith A
    . Ammonia inhibition of protein degradation in isolated rat hepatocytes. Quantitative ultrastructural alterations in the lysosomal system. Exp Cell Res. 1976;100:276–280.
    OpenUrlCrossRefPubMed
  63. 63.↵
    1. Klionsky DJ,
    2. Elazar Z,
    3. Seglen PO,
    4. Rubinsztein DC
    . Does bafilomycin A1 block the fusion of autophagosomes with lysosomes? Autophagy. 2008;4:849–850.
    OpenUrlCrossRefPubMed
  64. 64.↵
    1. Poole B,
    2. Ohkuma S
    . Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages. J Cell Biol. 1981;90:665–669.
    OpenUrlAbstract/FREE Full Text
  65. 65.↵
    1. Kawai A,
    2. Uchiyama H,
    3. Takano S,
    4. Nakamura N,
    5. Ohkuma S
    . Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells. Autophagy. 2007;3:154–157.
    OpenUrlCrossRefPubMed
  66. 66.↵
    1. Haspel J,
    2. Shaik RS,
    3. Ifedigbo E,
    4. Nakahira K,
    5. Dolinay T,
    6. Englert JA,
    7. Choi AM
    . Characterization of macroautophagic flux in vivo using a leupeptin-based assay. Autophagy. 2011;7:629–642.
    OpenUrlCrossRefPubMed
  67. 67.↵
    1. Munafó DB,
    2. Colombo MI
    . A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci. 2001;114:3619–3629.
    OpenUrlAbstract/FREE Full Text
  68. 68.↵
    1. Perry CN,
    2. Kyoi S,
    3. Hariharan N,
    4. Takagi H,
    5. Sadoshima J,
    6. Gottlieb RA
    . Novel methods for measuring cardiac autophagy in vivo. Methods Enzymol. 2009;453:325–342. doi: 10.1016/S0076-6879(08)04016-0.
    OpenUrlCrossRefPubMed
  69. 69.↵
    1. Tian Z,
    2. Wang C,
    3. Hu C,
    4. Tian Y,
    5. Liu J,
    6. Wang X
    . Autophagic-lysosomal inhibition compromises ubiquitin-proteasome system performance in a p62 dependent manner in cardiomyocytes. PLoS One. 2014;9:e100715. doi: 10.1371/journal.pone.0100715.
    OpenUrlCrossRefPubMed
  70. 70.↵
    1. Kaushik S,
    2. Cuervo AM
    . Methods to monitor chaperone-mediated autophagy. Methods Enzymol. 2009;452:297–324. doi: 10.1016/S0076-6879(08)03619-7.
    OpenUrlCrossRefPubMed
  71. 71.↵
    1. Jänen SB,
    2. Chaachouay H,
    3. Richter-Landsberg C
    . Autophagy is activated by proteasomal inhibition and involved in aggresome clearance in cultured astrocytes. Glia. 2010;58:1766–1774. doi: 10.1002/glia.21047.
    OpenUrlCrossRefPubMed
  72. 72.↵
    1. Shpilka T,
    2. Weidberg H,
    3. Pietrokovski S,
    4. Elazar Z
    . Atg8: an autophagy-related ubiquitin-like protein family. Genome Biol. 2011;12:226. doi: 10.1186/gb-2011-12-7-226.
    OpenUrlCrossRefPubMed
  73. 73.↵
    1. Codogno P,
    2. Mehrpour M,
    3. Proikas-Cezanne T
    . Canonical and non-canonical autophagy: variations on a common theme of self-eating? Nat Rev Mol Cell Biol. 2012;13:7–12. doi: 10.1038/nrm3249.
    OpenUrlPubMed
  74. 74.↵
    1. Ni HM,
    2. Bockus A,
    3. Wozniak AL,
    4. Jones K,
    5. Weinman S,
    6. Yin XM,
    7. Ding WX
    . Dissecting the dynamic turnover of GFP-LC3 in the autolysosome. Autophagy. 2011;7:188–204.
    OpenUrlCrossRefPubMed
  75. 75.↵
    1. Kimura S,
    2. Noda T,
    3. Yoshimori T
    . Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy. 2007;3:452–460.
    OpenUrlCrossRefPubMed
  76. 76.↵
    1. Terada M,
    2. Nobori K,
    3. Munehisa Y,
    4. Kakizaki M,
    5. Ohba T,
    6. Takahashi Y,
    7. Koyama T,
    8. Terata Y,
    9. Ishida M,
    10. Iino K,
    11. Kosaka T,
    12. Watanabe H,
    13. Hasegawa H,
    14. Ito H
    . Double transgenic mice crossed GFP-LC3 transgenic mice with alphaMyHC-mCherry-LC3 transgenic mice are a new and useful tool to examine the role of autophagy in the heart. Circ J. 2010;74:203–206.
    OpenUrlCrossRefPubMed
  77. 77.↵
    1. Choi KM,
    2. Nam HY,
    3. Na JH,
    4. Kim SW,
    5. Kim SY,
    6. Kim K,
    7. Kwon IC,
    8. Ahn HJ
    . A monitoring method for Atg4 activation in living cells using peptide-conjugated polymeric nanoparticles. Autophagy. 2011;7:1052–1062.
    OpenUrlCrossRefPubMed
  78. 78.↵
    1. Chen HH,
    2. Mekkaoui C,
    3. Cho H,
    4. Ngoy S,
    5. Marinelli B,
    6. Waterman P,
    7. Nahrendorf M,
    8. Liao R,
    9. Josephson L,
    10. Sosnovik DE
    . Fluorescence tomography of rapamycin-induced autophagy and cardioprotection in vivo. Circ Cardiovasc Imaging. 2013;6:441–447. doi: 10.1161/CIRCIMAGING.112.000074.
    OpenUrlAbstract/FREE Full Text
  79. 79.↵
    1. Schicchi N,
    2. Valeri G,
    3. Moroncini G,
    4. Agliata G,
    5. Salvolini L,
    6. Gabrielli A,
    7. Giovagnoni A
    . Myocardial perfusion defects in scleroderma detected by contrast-enhanced cardiovascular magnetic resonance. Radiol Med. 2014;119:885–894. doi: 10.1007/s11547-014-0419-7.
    OpenUrlCrossRefPubMed
  80. 80.↵
    1. Frech T,
    2. De Domenico I,
    3. Murtaugh MA,
    4. Revelo MP,
    5. Li DY,
    6. Sawitzke AD,
    7. Drakos S
    . Autophagy is a key feature in the pathogenesis of systemic sclerosis. Rheumatol Int. 2014;34:435–439. doi: 10.1007/s00296-013-2827-8.
    OpenUrlCrossRefPubMed
  81. 81.↵
    1. Wong TC,
    2. Piehler KM,
    3. Zareba KM,
    4. et al
    . Myocardial damage detected by late gadolinium enhancement cardiovascular magnetic resonance is associated with subsequent hospitalization for heart failure. J Am Heart Assoc. 2013;2:e000416. doi: 10.1161/JAHA.113.000416.
    OpenUrlAbstract/FREE Full Text
  82. 82.↵
    1. Ren SY,
    2. Xu X
    . Role of autophagy in metabolic syndrome-associated heart disease. Biochim Biophys Acta. 2015;1852:225–231. doi: 10.1016/j.bbadis.2014.04.029.
    OpenUrl
  83. 83.↵
    1. Umezawa R,
    2. Ota H,
    3. Takanami K,
    4. Ichinose A,
    5. Matsushita H,
    6. Saito H,
    7. Takase K,
    8. Jingu K
    . MRI findings of radiation-induced myocardial damage in patients with oesophageal cancer. Clin Radiol. 2014;69:1273–1279. doi: 10.1016/j.crad.2014.08.010.
    OpenUrlCrossRefPubMed
  84. 84.↵
    1. Sridharan V,
    2. Tripathi P,
    3. Sharma S,
    4. Moros EG,
    5. Zheng J,
    6. Hauer-Jensen M,
    7. Boerma M
    . Roles of sensory nerves in the regulation of radiation-induced structural and functional changes in the heart. Int J Radiat Oncol Biol Phys. 2014;88:167–174. doi: 10.1016/j.ijrobp.2013.10.014.
    OpenUrlCrossRefPubMed
  85. 85.↵
    1. Huang ML,
    2. Sivagurunathan S,
    3. Ting S,
    4. Jansson PJ,
    5. Austin CJ,
    6. Kelly M,
    7. Semsarian C,
    8. Zhang D,
    9. Richardson DR
    . Molecular and functional alterations in a mouse cardiac model of Friedreich ataxia: activation of the integrated stress response, eIF2α phosphorylation, and the induction of downstream targets. Am J Pathol. 2013;183:745–757. doi: 10.1016/j.ajpath.2013.05.032.
    OpenUrlCrossRefPubMed
  86. 86.↵
    1. Raman SV,
    2. Phatak K,
    3. Hoyle JC,
    4. Pennell ML,
    5. McCarthy B,
    6. Tran T,
    7. Prior TW,
    8. Olesik JW,
    9. Lutton A,
    10. Rankin C,
    11. Kissel JT,
    12. Al-Dahhak R
    . Impaired myocardial perfusion reserve and fibrosis in Friedreich ataxia: a mitochondrial cardiomyopathy with metabolic syndrome. Eur Heart J. 2011;32:561–567. doi: 10.1093/eurheartj/ehq443.
    OpenUrlAbstract/FREE Full Text
  87. 87.↵
    1. Satoh H,
    2. Sano M,
    3. Suwa K,
    4. Saitoh T,
    5. Nobuhara M,
    6. Saotome M,
    7. Urushida T,
    8. Katoh H,
    9. Hayashi H
    . Distribution of late gadolinium enhancement in various types of cardiomyopathies: Significance in differential diagnosis, clinical features and prognosis. World J Cardiol. 2014;6:585–601. doi: 10.4330/wjc.v6.i7.585.
    OpenUrlCrossRefPubMed
  88. 88.↵
    1. Chévrier M,
    2. Brakch N,
    3. Céline L,
    4. Genty D,
    5. Ramdani Y,
    6. Moll S,
    7. Djavaheri-Mergny M,
    8. Brasse-Lagnel C,
    9. Annie Laquerrière AL,
    10. Barbey F,
    11. Bekri S
    . Autophagosome maturation is impaired in Fabry disease. Autophagy. 2010;6:589–599. doi: 10.4161/auto.6.5.11943.
    OpenUrlCrossRefPubMed
  89. 89.↵
    1. Kassiotis C,
    2. Ballal K,
    3. Wellnitz K,
    4. Vela D,
    5. Gong M,
    6. Salazar R,
    7. Frazier OH,
    8. Taegtmeyer H
    . Markers of autophagy are downregulated in failing human heart after mechanical unloading. Circulation. 2009;120:S191–S197. doi: 10.1161/CIRCULATIONAHA.108.842252.
    OpenUrlAbstract/FREE Full Text
  90. 90.↵
    1. Garcia L,
    2. Verdejo HE,
    3. Kuzmicic J,
    4. Zalaquett R,
    5. Gonzalez S,
    6. Lavandero S,
    7. Corbalan R
    . Impaired cardiac autophagy in patients developing postoperative atrial fibrillation. J Thorac Cardiovasc Surg. 2012;143:451–459. doi: 10.1016/j.jtcvs.2011.07.056.
    OpenUrlCrossRefPubMed
  91. 91.↵
    1. Gedik N,
    2. Thielmann M,
    3. Kottenberg E,
    4. Peters J,
    5. Jakob H,
    6. Heusch G,
    7. Kleinbongard P
    . No evidence for activated autophagy in left ventricular myocardium at early reperfusion with protection by remote ischemic preconditioning in patients undergoing coronary artery bypass grafting. PLoS One. 2014;9:e96567. doi: 10.1371/journal.pone.0096567.
    OpenUrlCrossRefPubMed
  92. 92.↵
    1. Singh KK,
    2. Yanagawa B,
    3. Quan A,
    4. Wang R,
    5. Garg A,
    6. Khan R,
    7. Pan Y,
    8. Wheatcroft MD,
    9. Lovren F,
    10. Teoh H,
    11. Verma S
    . Autophagy gene fingerprint in human ischemia and reperfusion. J Thorac Cardiovasc Surg. 2014;147:1065–1072.e1. doi: 10.1016/j.jtcvs.2013.04.042.
    OpenUrlCrossRefPubMed
  93. 93.↵
    1. Weng TP,
    2. Huang SC,
    3. Chuang YF,
    4. Wang JS
    . Effects of interval and continuous exercise training on CD4 lymphocyte apoptotic and autophagic responses to hypoxic stress in sedentary men. PLoS One. 2013;8:e80248. doi: 10.1371/journal.pone.0080248.
    OpenUrlCrossRefPubMed
  94. 94.↵
    1. Wu G,
    2. Wei G,
    3. Huang J,
    4. Pang S,
    5. Liu L,
    6. Yan B
    . Decreased gene expression of LC3 in peripheral leucocytes of patients with coronary artery disease. Eur J Clin Invest. 2011;41:958–963. doi: 10.1111/j.1365-2362.2011.02486.x.
    OpenUrlCrossRefPubMed
  95. 95.↵
    1. Wu G,
    2. Liu L,
    3. Huang J,
    4. Pang S,
    5. Wei G,
    6. Cui Y,
    7. Yan B
    . Alterations of autophagic-lysosomal system in the peripheral leukocytes of patients with myocardial infarction. Clin Chim Acta. 2011;412:1567–1571. doi: 10.1016/j.cca.2011.05.002.
    OpenUrlCrossRefPubMed
  96. 96.↵
    1. Wu G,
    2. Wang X,
    3. Feng X,
    4. Zhang A,
    5. Li J,
    6. Gu K,
    7. Huang J,
    8. Pang S,
    9. Dong H,
    10. Gao H,
    11. Yan B
    . Altered expression of autophagic genes in the peripheral leukocytes of patients with sporadic Parkinson’s disease. Brain Res. 2011;1394:105–111. doi: 10.1016/j.brainres.2011.04.013.
    OpenUrlCrossRefPubMed
  97. 97.↵
    1. Chan LL,
    2. Shen D,
    3. Wilkinson AR,
    4. Patton W,
    5. Lai N,
    6. Chan E,
    7. Kuksin D,
    8. Lin B,
    9. Qiu J
    . A novel image-based cytometry method for autophagy detection in living cells. Autophagy. 2012;8:1371–1382. doi: 10.4161/auto.21028.
    OpenUrlCrossRefPubMed
  98. 98.↵
    1. Eng KE,
    2. Panas MD,
    3. Karlsson Hedestam GB,
    4. McInerney GM
    . A novel quantitative flow cytometry-based assay for autophagy. Autophagy. 2010;6:634–641. doi: 10.4161/auto.6.5.12112.
    OpenUrlCrossRefPubMed
View Abstract
Back to top
Previous ArticleNext Article

This Issue

Circulation Research
January 30, 2015, Volume 116, Issue 3
  • Table of Contents
Previous ArticleNext Article

Jump to

  • Article
    • Abstract
    • Overview of Autophagy
    • Examples of Confusing Autophagy Studies and Confounding Variables
    • Methods to Measure Autophagy and Mitophagy
    • Conclusions
    • Sources of Funding
    • Disclosures
    • Footnotes
    • References
  • Figures & Tables
  • Info & Metrics

Article Tools

  • Print
  • Citation Tools
    Untangling Autophagy Measurements
    Roberta A. Gottlieb, Allen M. Andres, Jon Sin and David P.J. Taylor
    Circulation Research. 2015;116:504-514, originally published January 29, 2015
    https://doi.org/10.1161/CIRCRESAHA.116.303787

    Citation Manager Formats

    • BibTeX
    • Bookends
    • EasyBib
    • EndNote (tagged)
    • EndNote 8 (xml)
    • Medlars
    • Mendeley
    • Papers
    • RefWorks Tagged
    • Ref Manager
    • RIS
    • Zotero
  •  Download Powerpoint
  • Article Alerts
    Log in to Email Alerts with your email address.
  • Save to my folders

Share this Article

  • Email

    Thank you for your interest in spreading the word on Circulation Research.

    NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

    Enter multiple addresses on separate lines or separate them with commas.
    Untangling Autophagy Measurements
    (Your Name) has sent you a message from Circulation Research
    (Your Name) thought you would like to see the Circulation Research web site.
  • Share on Social Media
    Untangling Autophagy Measurements
    Roberta A. Gottlieb, Allen M. Andres, Jon Sin and David P.J. Taylor
    Circulation Research. 2015;116:504-514, originally published January 29, 2015
    https://doi.org/10.1161/CIRCRESAHA.116.303787
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Related Articles

Cited By...

Subjects

  • Imaging and Diagnostic Testing
    • Imaging
  • Basic, Translational, and Clinical Research
    • Metabolism
    • Cell Signaling/Signal Transduction
    • Cell Biology/Structural Biology
    • Animal Models of Human Disease

Circulation Research

  • About Circulation Research
  • Editorial Board
  • Instructions for Authors
  • Abstract Supplements
  • AHA Statements and Guidelines
  • Permissions
  • Reprints
  • Email Alerts
  • Open Access Information
  • AHA Journals RSS
  • AHA Newsroom

Editorial Office Address:
3355 Keswick Rd
Main Bldg 103
Baltimore, MD 21211
CircRes@circresearch.org

Information for:
  • Advertisers
  • Subscribers
  • Subscriber Help
  • Institutions / Librarians
  • Institutional Subscriptions FAQ
  • International Users
American Heart Association Learn and Live
National Center
7272 Greenville Ave.
Dallas, TX 75231

Customer Service

  • 1-800-AHA-USA-1
  • 1-800-242-8721
  • Local Info
  • Contact Us

About Us

Our mission is to build healthier lives, free of cardiovascular diseases and stroke. That single purpose drives all we do. The need for our work is beyond question. Find Out More about the American Heart Association

  • Careers
  • SHOP
  • Latest Heart and Stroke News
  • AHA/ASA Media Newsroom

Our Sites

  • American Heart Association
  • American Stroke Association
  • For Professionals
  • More Sites

Take Action

  • Advocate
  • Donate
  • Planned Giving
  • Volunteer

Online Communities

  • AFib Support
  • Garden Community
  • Patient Support Network
  • Professional Online Network

Follow Us:

  • Follow Circulation on Twitter
  • Visit Circulation on Facebook
  • Follow Circulation on Google Plus
  • Follow Circulation on Instagram
  • Follow Circulation on Pinterest
  • Follow Circulation on YouTube
  • Rss Feeds
  • Privacy Policy
  • Copyright
  • Ethics Policy
  • Conflict of Interest Policy
  • Linking Policy
  • Diversity
  • Careers

©2018 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited. The American Heart Association is a qualified 501(c)(3) tax-exempt organization.
*Red Dress™ DHHS, Go Red™ AHA; National Wear Red Day ® is a registered trademark.

  • PUTTING PATIENTS FIRST National Health Council Standards of Excellence Certification Program
  • BBB Accredited Charity
  • Comodo Secured