Am J Physiol Heart Circ Physiol 308: H1194–H1204, 2015.
First published March 2, 2015; doi:10.1152/ajpheart.00002.2015.
Review
CALL FOR PAPERS
Autophagy in the Cardiovascular System
Myocardial autophagic energy stress responses—macroautophagy, mitophagy,
and glycophagy
Lea M. D. Delbridge,1 Kimberley M. Mellor,1,2 David J. R. Taylor,3 and Roberta A. Gottlieb3
1
Department of Physiology, University of Melbourne, Melbourne, Victoria, Australia; 2Department of Physiology, University
of Auckland, New Zealand; and 3Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California
Submitted 2 January 2015; accepted in final form 2 March 2015
autophagy; heart; cardiac; diabetes; ischemia; cardiomyocyte
SUGAR ISN’T ALWAYS SWEET,
When your heart is worn and skips a beat
Membranes keep it in or out
And insulin gives a special route
Glut transporters form a pore
Bringing sugar in the open door
Too much is bad-too little, too:
Cells need the proper fuel.
Inside the cell sugar’s stored
Glycogen the sweetest hoard.
Two enzymes live to break it down:
A neutral enzyme can be found
In cytosol where granules roam,
But in the acidic lysosome
Another waits on bended knee
To play its role in glycophagy.
Excess carbs are bad: this much is clear.
So consider maltose when quaffing beer!
Roberta A. Gottlieb
Address for reprint requests and other correspondence: L. M. D. Delbridge,
Dept. of Physiology, Univ. of Melbourne, Parkville, Victoria, Australia 3010
(e-mail: [email protected]).
H1194
Energy Stress and Autophagy Induction Overview
Autophagy is an intracellular degradation and recycling
function to support energy homeostasis and/or to eliminate
defective cell components. Autophagy is upregulated with
cellular stress and in the heart was first identified four decades
ago to be induced by ischemia (37) and starvation (8), and later
determined to be critical for neonatal cardiac tissues during the
period of temporary nutrient deprivation accompanying postnatal transition (45). Although upregulated expression of autophagy mediators can be associated with acute performance
improvement post-ischemia (24, 35), a sustained elevation of
autophagy has been linked with transition to cardiac failure in
vivo (112). The general consensus is that autophagy can be
beneficial and pro-survival as a short-term strategy to deal with
acute stress, but when chronically elevated or constitutive,
excess autophagic activity is linked to cell death, stimulating
cytokine-mediated collagen infill responses (31, 66, 81). The
signaling complexity regulating autophagy in acute and
chronic conditions under various trophic and endocrine settings
is yet to be fully elucidated. Involvement from PI3K/Akt/
mTOR (60, 77) and AMPK (100) pathways in regulating
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Delbridge LM, Mellor KM, Taylor DJ, Gottlieb RA. Myocardial autophagic
energy stress responses—macroautophagy, mitophagy, and glycophagy. Am J
Physiol Heart Circ Physiol 308: H1194 –H1204, 2015. First published March 2,
2015; doi:10.1152/ajpheart.00002.2015.—An understanding of the role of autophagic
processes in the management of cardiac metabolic stress responses is advancing rapidly
and progressing beyond a conceptualization of the autophagosome as a simple cell
recycling depot. The importance of autophagy dysregulation in diabetic cardiomyopathy and in ischemic heart disease - both conditions comprising the majority of cardiac
disease burden - has now become apparent. New findings have revealed that specific
autophagic processes may operate in the cardiomyocyte, specialized for selective
recognition and management of mitochondria and glycogen particles in addition to
protein macromolecular structures. Thus mitophagy, glycophagy, and macroautophagy
regulatory pathways have become the focus of intensive experimental effort, and
delineating the signaling pathways involved in these processes offers potential for
targeted therapeutic intervention. Chronically elevated macroautophagic activity in the
diabetic myocardium is generally observed in association with structural and functional
cardiomyopathy; yet there are also numerous reports of detrimental effect of autophagy
suppression in diabetes. Autophagy induction has been identified as a key component
of protective mechanisms that can be recruited to support the ischemic heart, but in this
setting benefit may be mitigated by adverse downstream autophagic consequences.
Recent report of glycophagy upregulation in diabetic cardiomyopathy opens up a novel
area of investigation. Similarly, a role for glycogen management in ischemia protection
through glycophagy initiation is an exciting prospect under investigation.
Review
H1195
CARDIOMYOCYTE STRESS AND MACRO/MITO/GLYCOPHAGY
1
2
tophagic breakdown of protein aggregates and some organelles
(including mitochondria). Identification of the machinery specific
to each autophagic pathway has enabled new tools for investigation of selective autophagy, and new insights are emerging in
relation to their differential roles in cardiac pathology.
This review examines the role of autophagy in myocardial
energy stress settings, most particularly in diabetic/insulin
resistant and ischemic states. We focus on current questions
relating to macroautophagy/glycophagy signaling pathways in
these contrasting settings, and consider the interpretation of disparate findings of autophagy occurrence in similar disease models.
Finally, prospects for targeted manipulation of specific autophagic
processes of potential therapeutic value are highlighted.
Diabetes: An Energy Access Denial in the Myocardium
In diabetes, cardiac complications are evident even in the
absence of vascular abnormalities and represent a primary
manifestation of the disease. Diastolic abnormality is an early
sign of diabetic cardiomyopathy, linked with altered metabolism and energetics (25, 51, 74, 91). At a molecular level,
suppression of the PI3K/Akt insulin signaling pathway, either
due to insulin ligand deficiency (type 1 diabetes, or T1D) or
due to tissue insulin resistance (type 2 diabetes, or T2D),
3
4
5
Export of
breakdown products
Autophagosome
Atg5-Atg12
Atg8
Autophagolysosome
Phagophore
Lysosome fuses with
autophagosome
Lysosome
Cargo-selective phagophores
with specific adaptor proteins
Fig. 1. Schematic of autophagy. 1) Formation of the nucleating membrane is initiated by Ulk1 followed by Beclin1 and Vps34. Atg12 is conjugated onto Atg5
by the action of ubiquitin ligase-like enzymes Atg7 and Atg10. Atg8 (e.g., LC3, GABARAP, GABARAPL1) is activated by Atg4 cleavage and then ligated onto
membrane phosphatidylethanolamine by the action of Atg3 and Atg7 with the participation of Atg5-12 and Atg16Lcomplex, to form a lipidated Atg8 moiety
(e.g., LC3B-II). 2) Specific cargo is tagged with bifunctional adaptor proteins that interact with Atg8 or homologs to facilitate engulfment. 3) Atg5-12 is released
from the outer membrane when the phagophore closes upon itself. 4) A lysosome fuses with the autophagosomal outer membrane and delivers its contents. 5)
The vacuolar proton ATPase lowers the pH, causing activation of proteases, lipases, nucleases, and glycohydrolases to degrade cargo to single amino acids, fatty
acids, nucleotides, and monosaccharides, which are exported across the membrane to the cytosol.
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autophagy has been identified in diabetic cardiomyopathy and
may play a role in other cardiac pathological settings.
The term autophagy has been considered synonymous with
the term macroautophagy (implying a single macromolecular
degradative pathway). Macro-autophagy machinery has been
relatively well characterized and involves assembly of a membrane structure and parallel enzymatic conjugations analogous
to ubiquitin ligation, resulting in conjugation of Atg12 onto
Atg5, and subsequent conjugation of Atg8 proteins (e.g. LC3,
GABARAPL1, GABARAP) onto phosphatidylethanolamine
in the phagophore membrane (additional details provided in
Fig. 1). Engulfment of selective cargo requires adaptor proteins, which bridge Atg8 proteins and the target. More recently,
a new understanding of selective autophagy processing has
emerged, with specific protein machinery recognized for autophagic degradation of mitochondria [mitophagy (22, 33)],
endoplasmic reticulum [reticulophagy or ERphagy (26)], peroxisomes [pexophagy (89)], lipid droplets [lipophagy (52)],
and glycogen [glycophagy (39, 76)]. In this context, the term
macroautophagy is best used to denote the process of general
autophagic degradation of heterogeneous macromolecules. A
case can now be made that the autophagic processes delineated
by the commonly used markers LC3B and p62 (and possibly
Beclin-1) are predominantly those involved in macroau-
Review
H1196
CARDIOMYOCYTE STRESS AND MACRO/MITO/GLYCOPHAGY
operational in the adult heart and is upregulated by diabetes in
vivo (61, 76). Glycophagy is well characterized in the newborn
liver and heart where a transient upregulation of lysosomal acid
␣-glucosidase mediates an increase in autophagic glycogen
degradation (43). In this setting autophagic glycogen breakdown is thought to provide glycolytic substrate support during
the placental-lactational transition. This bulk glycogen degradation pathway is understood to operate in parallel with the
well-described conventional kinase-regulated process of glycogen breakdown by glycogen phosphorylase. In that pathway,
Akt via GSK3␤ mediates glycogen synthase activation,
whereas PKA activation via glycogen phosphorylase kinase
mediates glycogen phosphorylase activation (as shown in
Fig. 3). Thus glycogenolysis may take place in the cytosol at
neutral pH or in the lysosome at acidic pH, mediated by
distinct enzymes. Lysosomal glycogen degradation is mediated
by the acid ␣-glucosidase, which possesses glycogen, maltose,
and isomaltose hydrolyzing activities (43). Glucose liberated
from glycogen degradation exits lysosomes via the lysosomal
membrane glucose carrier (55). The glycogen hydrolyzing
activity of acid ␣-glucosidase is enhanced by Ca2⫹, which in
the setting of increased cytosolic Ca2⫹ in the ischemic myocardium, in concert with cAMP-mediated augmented lysosomal Ca2⫹ uptake, may prime lysosomes for accelerated
glycogen degradation (41, 79). There is strong evidence from
glycogen storage diseases that autophagic/lysosomal glycogen
breakdown is important for maintaining normal cardiac glycogen levels and does not simply constitute a redundant alternative breakdown route for glycogen (68). Deficiency of functional lysosomal acid ␣-glucosidase as observed in Pompe
disease results in the accumulation of glycogen-filled lysosomes in multiple tissues including the heart (75), indicating
that lysosomal degradation of glycogen is essential in normal
physiology. Glycophagy may provide a quantitatively different route of glycogen breakdown in comparison with
phosphorylase-mediated degradation, facilitating large glucose release in settings of high energy demand. Glycogen
destined for autophagic/lysosomal degradation may also be
qualitatively different to that degraded in the cytosol, thus
necessitating a different mode of degradation. For example,
abnormally branched, insoluble, and/or hyperphosphorylated glycogen may impede phosphorylase action and favor
recruitment to the glycophagosome.
Starch binding domain protein 1 (STBD1) is a likely candidate for facilitating glycogen sequestration into the glycophagosome. STBD1 contains both a carbohydrate binding domain
and an Atg8 interacting motif (39). Proteomics analysis has
demonstrated that STBD1 is localized to the glycogen particle
(86). In cell lines overexpressing HA-tagged STBD1, colocalization (via immunostaining) and interaction (via co-immunoprecipitation) of STBD1 and the autophagy (Atg8) protein,
GABARAPL1, has been observed (38, 39). STBD1 colocalization with the lysosomal protein, LAMP1, has also been
reported (38). Thus STBD1 may play a role in intracellular
trafficking of glycogen to the autophagosome by binding to
GABARAPL1 on the autophagosome membrane, constituting
specific autophagy machinery for glycophagy. We have demonstrated that STBD1 does not colocalize with the macroautophagy Atg8 protein, LC3B, in cultured cardiomyocytes,
suggesting that the glycophagy process is distinct from conventional macroautophagy (Fig. 4) (61). Visualization of
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results in reduced GLUT4 glucose transporters in the plasma
membrane and limited glucose uptake (10). These glucose
handling abnormalities may underlie the observed metabolic shift
decreasing carbohydrate oxidation with associated increase in
fatty acid oxidation for energy supply (95). Paradoxically, diabetic
cardiomyocytes exhibit an accumulation of glycogen, despite
suppressed glucose uptake. In contrast with other tissues (i.e.,
skeletal muscle, liver), the heart uniquely increases glycogen
content in periods of energy deprivation (e.g., fasting) (76),
perhaps as a protective mechanism to mobilize glucose from
nonessential tissues and elevate cardiac stores. In diabetes, the
intracellular glucose deprivation conferred by impaired glucose
transport (albeit in the context of extracellular fuel overabundance) may trigger a pseudo fasted heart phenotype. Energy stress
is also a potent activator of cardiac autophagy in vivo and in
isolated cardiomyocytes (3, 42, 76). The role of autophagy in the
diabetic heart is emerging as an important research question.
Reports to date provide interesting and discrepant findings, and
these are evaluated in detail below.
New insights into the role of autophagy in the diabetic heart.
Since our first report of increased cardiac autophagy in the type
2 diabetic fructose-fed mouse (59), the experimental literature
in this field has expanded considerably, but it is not yet
possible to synthesize a comprehensive understanding. Relying
on a variable selection of molecular tools, states of increased
(4, 12, 49, 50, 59, 61, 78, 93, 102), unchanged (47, 48, 57, 62),
and decreased (6, 23, 28, 29, 73, 82, 101, 103, 104, 110) basal
cardiac autophagy activity have all been reported in diabetic/
insulin resistant contexts (see Table 1). These discrepancies are
not necessarily attributable to the different models of diabetes,
since contrasting findings have been observed within the same
diabetic model [e.g., STZ-mouse: increased LC3BII (12) vs.
decreased LC3BII (103)]. Duration/severity of diabetes is also
unable to explain the variable findings [e.g., high fat-fed mouse
18 –20 wk duration: increased LC3BII (102) vs. decreased
LC3BII (82)]. New work involving evaluation of autophagic
flux will be important in gaining mechanistic understanding to
resolve these variable findings, and we have recently highlighted the importance of flux measurement in relation to
interpreting data from a dietary intervention model (21). Suppressed insulin signaling through the PI3K/Akt/mTOR pathway is a common observation in most diabetic models due to
either lower insulin ligand stimulation of the receptor (T1D) or
tissue insulin resistance (T2D). Downregulation of this pathway would be expected to be linked to upregulated autophagy
as inhibition via mTOR would be relieved (40). Conversely,
downregulation of AMPK signaling, also commonly observed
in the diabetic heart, would be expected to suppress autophagy
activity (11, 27, 108). Thus the autophagy outcome may reflect
the net effect of these opposing autophagy regulators and
depend on the extent of downregulation of Akt versus AMPK
signaling (as shown in Fig. 2). As summarized in Table 1,
suppressed cardiac autophagy in diabetes is often observed
coincident with decreased AMPK activity, despite variable
findings relating to Akt (29, 82, 103, 110). The absence of both
Akt and AMPK data in many of these diabetes-autophagy
studies limits these conclusions, and new studies are required
to elucidate the role of autophagy in the diabetic heart.
A role for glycophagy in the heart. Recently we have
demonstrated that the autophagic/lysosomal glycogen degradation pathway (glycogen-specific autophagy, ‘glycophagy’) is
Review
H1197
CARDIOMYOCYTE STRESS AND MACRO/MITO/GLYCOPHAGY
Table 1. Literature reports of autophagy in the insulin resistant or diabetic heart
Autophagy
Model
Autophagy Measurement
AMPK
Activity
Reference
12 wk
18 wk old
2
nr
↔*
1
59
50
16 wk
nr
nr
93
8 wk
4 wk
2
nr
nr
nr
61
49
24 wk
18 wk
nr
nr
nr
nr
4
78
7 wk
nr
nr
12
7–20 yr
10 wk
8 wk
10–12 wk
nr
nr
nr
nr
nr
nr
nr
nr
62
57
57
47
Fructose-fed mouse
Goto-Kakizaki rat
T2D
T2D
1
STZ-mouse (x5 inj)
T1D
1
1
STZ rat (x1 inj)
STZ rat (x1 inj)
T1D
T1D
1
1
High-fat fed mouse
High-fat fed mouse
ins res
ins res
1
STZ mouse (x1 inj)
T1D
↔
↔
↔
↔
T2D
ins res
T2D
ins res
↔
↔
↔
↔
↔
T2D humans
High-fat fed rat
High fat-STZ rat
High-fat fed mouse
(female)
pancreatectomy rats
T1D
↔LC3II:I ratio#, p62 protein
11 wk
↔
nr
48
2
High-fat fed mouse
ins res
20 wk
1
nr
104
2
High-fat fed mouse
ins res
22 wk
nr
2
23
2
2
2
High-fat fed mouse
High-fat fed mouse
db/db mouse
ins res
ins res
T2D
8 wk
12 wk
14–18 wk old
nr
nr
2
↔
nr
↔
6
28
73
2
2
High-fat fed mouse
STZ mouse (x1 inj)
ins res
T1D
18–20 wk
6, 9, 12 wk
↔
1
2
2
82
103
2
2
2
2
STZ mouse (x5 inj)
STZ mouse (x5 inj)
STZ mouse (x5 inj)
OVE26 mouse
T1D
T1D
T1D
T1D
8 wk
16 wk
16 wk
40 wk old
2
2
nr
nr
2
2
2
2
110
29
101
101
2
db/db mouse
T2D
2 LC3BII:I ratio, LC3BII, Beclin1 protein
1 p62 protein
1 pULK1/ULK1, ↔ LC3BI
2 LC3BII:I ratio, LC3BII protein
1 p62 protein
↔ LC3BI, Beclin1 protein
2 LC3II:I ratio#
2 LC3II:I ratio (ns)#, 1 p62 protein
2 LC3II:I ratio#
↔Beclin1, Atg12-Atg5 protein
2 LC3II# protein
2 LC3II#, Atg12, Atg12-Atg5 protein
2 LC3 protein (Baf-Con)
2 LC3 puncta (Baf-Con)
2 LC3II#, Beclin1 protein
2 LC3BII protein
2 LC3II protein#
2 LC3II protein#
2 autophagic vacuoles (EM)
2 Beclin1 staining (IHC)
2 LC3II# (⫹Baf, sample blot)
10–14 wk old
nr
nr
57
High-fat fed mouse
ins res
1tAkt2
2
102
LC3II:I
LC3II:I
LC3II:I
LC3II:I
ratio#
ratio#, Beclin1, LAMP1 protein
ratio#, Beclin1, LAMP1 protein
ratio#, ↔ Atg5 mRNA
1 LC3BI, LC3BII, p62 protein
1 LC3, p62 mRNA
1 double membrane-bound vacuole/cell
↔LC3BII:I ratio, Beclin1 protein
CQ-induced LC3BII increase abrogated
20 wk
nr, not reported; STZ, streptozotocin; T2D, type 2 diabetes; T1D, type 1 diabetes; Atg, autophagy; ns, not significant; ins res, insulin resistance; LAMP,
lysosome associated membrane protein 1; Baf-Con, bafilomycin-control; EM, electron microscopy; CQ, chloroquine. 1, Diabetes-induced increase; 2,
diabetes-induced decrease; ↔, no effect of diabetes. *Personal communication; #LC3 isoform nonselective antibody used. All data presented are obtained in the
absence of lysosomal inhibition unless otherwise noted.
STBD1 and GABARAPL1 colocalization has been demonstrated in a noncardiac cell line, and further information is
required regarding the direct/indirect interaction of these two
proteins in vitro in cardiomyocytes. It is likely that, in pathological settings, selective autophagy pathways play differential
roles and identification/characterization of these specific pathways may lead to more targeted intervention opportunities.
Glycogen flux in the diabetic myocardium. Glycogen pathology in human diabetic myocardium was first reported over 80
years ago (96). This is a replicated finding in many diabetic
experimental settings (2, 46, 84) and occurs in parallel to a
depletion of glycogen stores in the liver and skeletal muscle
(30, 85, 87). The fundamental bases for differential glycogen
handling responses in distinct muscle types are still not yet
understood. Advances in the understanding of the gene/protein
origins of various genetically determined glycogen storage
diseases have provided considerable new insight into the role
of disrupted glycogen handling in the etiology of cardiac
pathology. In these diseases, glycogen disturbance results in
contractile dysfunction, derangement of myofibrillar and cytoskeletal proteins, arrhythmia, and hypertrophy (63). Presentation of vacuole-filled glycogen granules in cardiomyocytes
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Akt
Activity
1
1
Ambiguous
1 LC3BII:I ratio, Beclin1, p62 protein
1 LC3BI, LC3BII, LC3BII:I ratio,
Beclin1, Atg12-Atg5, Atg7 protein
1 LC3II:I ratio#, p62, Beclin1 protein
1 autophagosomes (EM)
1 Atg5, Atg12, Beclin1 mRNA
↔ LC3#, p62 mRNA
1 LC3BII:I ratio
1LC3# puncta
1 LC3II:I ratio#, Beclin1 protein
1 Beclin1, LAMP2A mRNA
1 LC3B, Beclin1 mRNA
1 LC3B puncta
1LC3BII, CathepsinD protein
2 p62 protein
Duration of
Diabetes
Review
H1198
CARDIOMYOCYTE STRESS AND MACRO/MITO/GLYCOPHAGY
Insulin
IR
Energy stress
IRS1/2
AMP/ATP
PI3K
mTORC1
P
Akt
ULK1
AUTOPHAGY
Fig. 2. Key signaling regulation of autophagy in the heart. Energy stress
increases the AMP-to-ATP ratio and activates AMPK. AMPK has dual action
to promote autophagy via inhibition of mTORC1 and activation of ULK1 via
phosphorylation. The insulin signaling pathway inhibits autophagy via activation of PI3K/Akt/mTORC1 to inhibit ULK1.
supports a role for glycophagy in these settings (32). Glycogen
handling dysregulation in diabetes may similarly reflect a
storage anomaly.
In diabetes, disturbances in insulin signaling and glucose
handling may underlie cardiac glycogen pathology. In cultured
neonatal rat cardiomyocytes, protein expression of the glycophagy marker STBD1 was increased by extracellular insulin
concentration linked with activation of the insulin-regulated
PI3K/Akt signaling pathway (61). Similarly, fasting-induced
upregulation of STBD1 and GABARAPL1 protein content was
associated with activation of Akt in vivo (76). In cultured
cardiomyocytes, it has been demonstrated using chromatin
immunoprecipitation that FoxO1 and FoxO3, which are inhibited by Akt, directly bind to the GABARAPL1 promoter in the
nucleus and regulate transcription of this glycophagy marker
(83). Thus Akt activation may lead to inhibition of FoxOmediated GABARAPL1 transcription, but such a response is
difficult to reconcile with the experimental observations that
there is parallel upregulation of glycophagy and Akt activity with
fasting (76). A more detailed dissection of the signaling nexus of
PI3K/Akt and AMPK pathways in mediating glycophagy induction is required to resolve these contrasting observations, in both
physiological and pathophysiological settings.
Ischemia-Reperfusion: An Energy Supply Crisis
Role of autophagy in the ischemic and reperfused heart.
Autophagy is powerfully upregulated as part of the response to
ischemic stress, and it has been suggested that macroautophagy
induction is beneficial during ischemia but deleterious during
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AMPK
reperfusion (58). We have shown that autophagy is essential
for cardioprotection observed with ischemic preconditioning
(35), and others have shown its importance in postconditioning
(98). In a Langendorff model of ischemia/reperfusion, inhibition of autophagy with the cell-permeable inhibitor (TATAtg5K130R) neither increased nor reduced infarct size, suggesting that in the absence of pre- or post-conditioning, the
beneficial and deleterious effects of autophagy may be balanced (34). Inhibition of autophagic flux increases hypoxia/
reoxygenation injury (109) and blocks the beneficial effects of
sevoflurane postconditioning (107). Ischemia/reperfusion injury disrupts autophagosome-lysosome fusion (54), accompanied by a significant increase in Beclin-1. Beclin-1 effects are
complex. Beclin-1 plays an essential role in autophagosome
initiation for both canonical and noncanonical macroautophagy
pathways, but it also partners with Rubicon to interfere with
autophagosome-lysosome fusion (111), and binds to Bcl-2,
thereby fostering mitochondria-dependent apoptosis (36). The
preponderance of evidence supports the notion that autophagy
is a protective response to acute ischemia/reperfusion injury,
and new studies are required to elucidate the mechanism by
which autophagy confers benefit in this setting.
Mitochondrial clearance (mitophagy) and mitochondrial sequestration (lysosomal regulation). Mitochondria represent
both a target of injury during ischemia, and a source of tissue
damage, via production of reactive oxygen species (ROS),
during reperfusion. Clearance of damaged mitochondria by
autophagy (mitophagy) may play a beneficial role in the setting
of myocardial ischemia/reperfusion. We have demonstrated
that ischemic preconditioning ameliorates reperfusion injury
by triggering Parkin-dependent mitophagy (33). Given the role
of autophagy in liberating metabolic substrates (amino acids,
carbohydrates, lipids) during nutrient deprivation, it has been
generally assumed that both autophagic flux and lysosomal
function are essential for autophagy-mediated cardioprotection. We have provided evidence that sequestration of mitochondria in autophagosomes may mediate the cardioprotective
effects of chloramphenicol in the absence of lysosomal function. Chloramphenicol-induced infarct size reduction was preserved when the lysosomal inhibitor chloroquine was administered in isolated ischemia/reperfused rat hearts (19). Although autophagic sequestration of nutrient sources without
lysosomal degradation during an episode of metabolic stress
seems counterintuitive, it is also a process by which regulated
lysosomal function participates in protective energy management.
In contrast, lysosomal function appears to play an important
role in cardioprotection in other contexts. Enhanced lysosome
biogenesis was shown to attenuate apoptosis mediated by
overexpression of BNIP3 in neonatal rat cardiomyocytes (53).
Increased lysosome biogenesis (via cobalt protoporphyrin
treatment) contributes to autophagy-mediated cardioprotection
in sepsis (90). It is noteworthy that mTOR is associated with
the lysosome, and during starvation, mTOR is inactivated until
amino acids derived from the lysosome restore mTORC1
signaling (106), enabling new protein synthesis required for
cellular repair processes. It may be that sequestration of mitochondria (or other cellular components) into autophagosomes
is protective as a short-term strategy to minimize cellular injury
in ischemia-reperfusion, and delayed/inhibited lysosomal degradation slows the negative feedback on autophagy via mTOR.
Review
CARDIOMYOCYTE STRESS AND MACRO/MITO/GLYCOPHAGY
Akt
PKA
GSK3β
GP kinase
H1199
Glycogen
Synthase
P
Glucose
G1P
STBD1
Lysosome
GABARAPL1
STBD1
Glycophagosome
The serine/threonine kinase GSK3␤ has been extensively
investigated in the past decade with respect to its role in
cardioprotection. Ser9 phosphorylation of GSK3␤ is considered to exert its cardioprotective effects through inhibition of
mitochondrial permeability transition pore (mPTP) opening.
The precise nature of the GSK3␤-mPTP interaction is unclear;
however, several mechanisms have been proposed including
preservation of hexokinase II in the mPTP complex (72),
suppression of the interaction between adenine nucleotide
translocase and cyclophilin D (67), and inhibition of p53 (97).
GSK3␤ inhibition has been implicated in the cardioprotection
mediated by a multitude of agents and therapeutic strategies
(13, 20, 70, 92); however, little attention has been given to the
Glycophagolysosome
original process from which it derives its name - in regulating
glycogen turnover. GSK3␤ inhibition during ischemia should
favor glycogen synthesis and decreased degradation, which in
turn may reduce the availability of glycolytic substrate, the
oxidation of which promotes ischemic acidosis and worsened
reperfusion injury. Although a role for GSK3␤ in regulating
glycophagy is speculative at this time, two separate reports
have recently demonstrated that GSK3␤-inhibition promotes
lysosome biogenesis (56, 71). If GSK3␤ inhibition in preconditioned hearts promotes lysosomal biogenesis, this may hypothetically prime the cell for enhanced autophagic glycogen
disposal. Similarly GSK3␤ has been shown to regulate autophagic activity in both tumor and nontumor cell lines (18).
10 µm
Fig. 4. Distinct cardiomyocyte immunohistologic localization
of glycogen autophagic trafficking cargo in vitro. STBD1
(green) and LC3B (red) do not colocalize in neonatal ventricular rat myocytes. Confocal image, x63 mag: DAPI (blue)
shows nuclei. Reprinted from Ref. 53 with permission.
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Fig. 3. Regulation of glycogen synthesis and
degradation in the heart. Akt signaling promotes
glycogen synthesis via relieving the GSK3␤mediated inhibition of glycogen synthase. PKA
signaling promotes glycogen degradation via activation of glycogen phosphorylase kinase and
subsequent activation of glycogen phosphorylase. Phosphorylase removes a glycosyl residue
from the glycogen strand to release glucose-1phosphate. STBD1 tags glycogen for degradation
via glycophagy. Glycogen is engulfed by the
glycophagosome, which may involve STBD1
binding to GABARAPL1 in the forming glycophagosome membrane. The glycophagosome
fuses with a lysosome, and the glycogen is degraded to free glucose by acid ␣-glucosidase
(black dots).
Phosphorylase
Review
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CARDIOMYOCYTE STRESS AND MACRO/MITO/GLYCOPHAGY
chloro-N6-cyclopentyladenosine) has been shown to increase
pre-ischemic glycogen content and suppress glycogen utilization in the first 2–5 min of ischemia, but total glycogen
utilization over the ischemic period was increased relative to
the ischemic preconditioning group (7). These findings must
also be considered in the context of studies showing that
adenosine or adenosine A1 agonists inhibited glycolysis and
glycolytic proton production yet had no effect on glucose
oxidation, resulting in tighter coupling of glycolysis to glucose
oxidation (14). In a related study it was observed that A1
agonists promoted glycogen synthesis without affecting glycogen degradation; yet glycogen turnover was accelerated (16).
Little information is available to address the question of
whether stimulated glycogenolysis occurred in the cytosol or
the lysosome. We have demonstrated that cardioprotection by
CCPA is dependent on autophagy (105), which would be
consistent with lysosomal glycogen breakdown. Although no
clear answers have emerged, it may be speculated that autophagic engulfment of glycogen particles (i.e., glycophagy)
might sequester glycogen and prevent its utilization during
acute ischemic stress, thereby limiting lactic acidosis and risk
of Ca2⫹ overload.
Glycophagy in the ischemic heart. An issue of major importance relating to ischemic glycogen turnover that has been
largely overlooked is the contribution of autophagic (i.e.,
glycophagic) sequestration of glycogen. Cytosolic glycogen
degradation is normally regulated via the actions of the cytosolic phosphorylase and debranching enzymes, and it could be
also assumed that some glycogen is constitutively degraded via
lysosomes. The advantage of sequestering glycogen into autophagosomes for subsequent delivery to the lysosomes for
degradation is not yet understood. Cytosolic glycogen is localized within distinct myocyte subcellular regions, including
intermyofibrillar, intramyofibrillar, and subsarcolemmal compartments (65). In stimulated skeletal muscle fibers, rates of
glycogen depletion differ depending upon subcellular localization (64). It is conceivable, therefore, that in the setting of local
high rates of glycogenolysis, glycophagy may function to
supplement the cytosolic machinery and that localized glycophagic hotspots provide important metabolic support under
m
Fig. 5. Electron micrograph of glycogen and a glycophagosome
in an adult rat ventricular cardiomyocyte. Free glycogen particles (short arrowhead) are aligned between myofibrils. Some
glycogen particles are seen sequestered in a glycophagosome
(arrow). m, Mitochondria. Bar indicates 500 nm.
m
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Thus it should be noted that pharmacologic agents that target
GSK3␤ (some of which have been proposed as cardioprotective drugs) may at least partially act through a mechanism
involving glycogen action.
Glycogen turnover in the preconditioned heart. Glycogenolysis contributes significantly to glycolytic substrate supply
during ischemia, and a number of studies have sought to
determine a relationship between ischemic glycogen consumption and infarct severity (5, 15, 80). Ischemic glycogenolysis
may have beneficial effect through increased glycolytic ATP
supply or detrimental effect due to enhanced cellular lactate
and proton production. Interventions that increase pre-ischemic
glycogen content have been shown to be both beneficial and
detrimental to the ischemic heart (80). One study demonstrated
that separate cardioprotective interventions, which deplete or
enhance pre-ischemic glycogen, can confer a similar level of
cardioprotection (7). Because any intervention intended to
manipulate glycogen levels before ischemia will likely involve
parallel activation of signaling pathways related to cardioprotection, delineating the precise contribution of glycogen depletion to ischemic injury is problematic. Indeed, it has recently
been reported that ischemic preconditioning and insulin administration independently protected against ischemia/reperfusion
injury while having differential impacts on glycogen turnover
(17). When both ischemic preconditioning and insulin administration were combined, cardioprotection was abolished.
The role of myocardial glycogen breakdown in the cardioprotection observed with preconditioning is controversial: one
study reported that ischemic preconditioning attenuated glycogenolysis in rat hearts (99), whereas another study showed that
24-h in vivo fasting resulted in increased glycogen content and
greater glycogen utilization during ischemia/reperfusion in a
Langendorff model (80), attributed to activation of phosphorylase a (94). Interestingly, it was also observed that insulin
pretreatment increased glycogen content to a similar extent but
suppressed glycogen utilization during ischemia/reperfusion,
resulting in increased injury and function (80). Fasting upregulates autophagy, whereas insulin treatment suppresses both
autophagy and glycogen utilization, which might explain the
differential effects. The adenosine A1 agonist CCPA (2-
Review
CARDIOMYOCYTE STRESS AND MACRO/MITO/GLYCOPHAGY
Conclusions and Directions
An understanding of the role of autophagic processes in the
management of cardiac metabolic stress responses is advancing
rapidly and progressing beyond a conceptualization of the
autophagosome as a simple cell recycling depot. The importance of autophagy dysregulation in diabetic cardiomyopathy
and in ischemic heart disease - both conditions comprising the
majority of cardiac disease burden - has now become apparent.
New findings have revealed that specific autophagic processes
may operate in the cardiomyocyte, specialized for selective
recognition and management of mitochondria and glycogen
particles in addition to protein macromolecular structures.
Thus mitophagy, glycophagy, and macroautophagy regulatory
pathways have become the focus of intensive experimental
effort, and delineating the signaling pathways involved in these
processes offers potential for targeted therapeutic intervention.
A body of evidence is emerging that suggests that cytosolic
autophagosomal sequestering of cellular structures has a role in
acute energy management and cell viability preservation. Subsequent metabolic access to sequestered stores appears to rely
on more complex lysosomal-autophagosomal regulatory mechanisms yet to be delineated. Chronically elevated macroautophagic activity in the diabetic myocardium is generally
observed in association with structural and functional cardiomyopathy; yet there are also numerous reports of detrimental
effect of autophagy suppression in diabetes. Autophagy induction has been identified as a key component of protective
mechanisms that can be recruited to support the ischemic heart,
but in this setting benefit may be mitigated by adverse down-
stream autophagic consequences. New studies are required in
both these disease contexts to undertake detailed mapping of
convergent AMPK and PI3K/Akt signaling pathways to identify metabolic leverage points to optimize macroautophagic
responses. Recent report of glycophagy upregulation in diabetic cardiomyopathy opens up a new area of investigation.
Similarly, a role for glycogen management in ischemia protection through glycophagy initiation is an exciting prospect under
investigation.
GRANTS
L. M. D. Delbridge acknowledges research funding from the National
Health and Medical Research Council of Australia and Diabetes Australia
Research Trust. R. A. Gottlieb is supported by National Heart, Lung, and
Blood Institute Grant NIH P01HL-112730 and the Dorothy and E. Phillip Lyon
Chair in Molecular Cardiology. K. M. Mellor is supported by the Rutherford
Foundation Postdoctoral Fellowship, New Zealand. Figure 5 has been contributed by Maryam Ghochani.3
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: L.M.D., K.M.M., D.J.T., and R.A.G. prepared figures; L.M.D., K.M.M., D.J.T., and R.A.G. drafted manuscript; L.M.D.,
K.M.M., D.J.T., and R.A.G. edited and revised manuscript; L.M.D., K.M.M.,
D.J.T., and R.A.G. approved final version of manuscript.
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