Circulation Research

REVIEW

Macrophages, Metabolism and Heterophagy in

the Heart
José A. Nicolás-Ávila , Laura Pena-Couso , Pura Muñoz-Cánoves, Andrés Hidalgo

ABSTRACT: The heart is a never-stopping engine that relies on a formidable pool of mitochondria to generate energy and propel
pumping. Because dying cardiomyocytes cannot be replaced, this high metabolic rate creates the challenge of preserving
organelle fitness and cell function for life. Here, we provide an immunologist’s perspective on how the heart solves this
challenge, which is in part by incorporating macrophages as an integral component of the myocardium. Cardiac macrophages
surround cardiomyocytes and capture dysfunctional mitochondria that these cells eject to the milieu, effectively establishing
a client cell–support cell interaction. We refer to this heterologous partnership as heterophagy. Notably, this process shares
analogies with other biological systems, is essential for proteostasis and metabolic fitness of cardiomyocytes, and unveils a
remarkable degree of dependence of the healthy heart on immune cells for everyday function.

Key Words:  exopher ◼ heart ◼ heterophagy ◼ macrophages ◼ myocardium

T
he heart features remarkable metabolic activity and examine paradigms that we propose can be translated
relies on a continuous supply of oxygen and nutrients to other organs.
to keep beating. A decline in heart function is broadly
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detrimental for the organism, not only due to direct loss

of tissue oxygenation but also because it compromises MACROPHAGES AS METABOLIC
the long-term function of other organs, such as the kid- GUARDIANS
neys. This creates secondary alterations in fluid homeo-
stasis that exacerbate cardiac stress, ultimately leading Tissue-Resident Macrophages
to a vicious cycle ending in heart failure.1 Identifying the Macrophages are a diverse group of immune cells
mechanisms that ensure long-term cardiac fitness is, broadly characterized by high phagocytic activity.6 Even
therefore, critical for global organismal health. in the absence of inflammation, populations of macro-
Similar to the heart, the immune system exerts global phages are present in virtually every tissue, where they
influence on daily tissue function. Immune cells, and par- adopt tissue-specific molecular features and functions
ticularly macrophages, are important constituents of most tailored to support organ homeostasis.3,7 For example,
tissues, including the heart.2 While macrophages are microglia in the brain remodel neuronal synapsis and
accountable for tissue injury (including inflammation and enable survival8,9 and bone marrow macrophages par-
fibrosis), they are now equally recognized as regulators ticipate in red blood cell maturation and maintenance
of key homeostatic processes across multiple tissues.3 of the hematopoietic stem cell niche.10,11 Depletion
Paradoxically, their actual contribution to heart homeo- studies in mouse models have revealed essential roles
stasis has remained enigmatic, with immunologists only of tissue-resident macrophages (TRMs) in normal tis-
recently starting to focus on the heart beyond the context sue function, as well as the description of pathological
of disease.4,5 Below, we discuss aspects of the biology of conditions caused by tissue-specific defects in TRMs
cardiac macrophages as they relate to maintenance of survival or function, such as alveolar proteinosis12 or
cardiomyocyte metabolism and contractile function and heart dysfunction.13,14
 
Correspondence to: Andrés Hidalgo, PhD, Centro Nacional de Investigaciones Cardiovasculares, Area of Cell and Developmental Biology, Calle Melchor Fernandez
Almagro, 3, Madrid 28029, Spain, Email ahidalgo@cnic.es or José A. Nicolás-Ávila, PhD, Centro Nacional de Investigaciones Cardiovasculares, Area of Cell and
Developmental Biology, Calle Melchor Fernandez Almagro, 3, Madrid 28029, Spain, Email joseangel.nicolas@cnic.es
For Sources of Funding and Disclosures, see page 428.
© 2022 American Heart Association, Inc.
Circulation Research is available at www.ahajournals.org/journal/res

418   February 4, 2022 Circulation Research. 2022;130:418–431. DOI: 10.1161/CIRCRESAHA.121.319812

 Nicolás-Ávila et al
Nonstandard Abbreviations and Acronyms
cMac cardiac-resident macrophage
CSF colony stimulating factor
ERK extracellular signal regulated kinase
EV extracellular vesicle
IGFBP7 insulin-like growth factor–binding protein 7
IL interleukin
LC3 light chain 3
LXR liver X receptor
NAD nicotinamide adenine dinucleotide
PPARγ peroxisome proliferator activated recep-
tor γ
TAM Tyro3/Axl/Mer
TNFα tumour necrosis factor α
TRM tissue-resident macrophage
v-ATPase vacuolar ATPase
Macrophages derived from yolk sac-erythromyeloid
progenitors or from fetal liver precursors seed several tis-
sues during early embryonic stages and are maintained
throughout life with little or no dependence on adult
hematopoiesis. In contrast, other tissues, such as the
intestine, feature continuous replenishment by monocyte-
derived macrophages through adult life.15,16 Under local32
or remote17 stress, however, embryo-derived TRMs are
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normally lost and are replenished through engraftment by
monocytes with yet-to-be-defined implications for tissue
function. Although this repopulation is not always effi-
cient,18 the new macrophages largely acquire the identity
of their embryonic counterparts,3,15,19 except in the central
nervous system.20,21 Further, TRMs retain plasticity and
can adapt to new environments, as illustrated by studies
showing that peritoneal macrophages can become alve-
olar-like macrophages upon intratracheal transfer, even if
they retain part of their original peritoneal identity.19
Irrespective of their cellular origin, macrophage den-
sity and tissue-specific identity are regulated by the so-
called niches, a functional scaffold of cells and matrix
that provides survival signals and further imprints tissue
identity onto TRMs.18 These niches exert part of these
survival effects by producing cytokines such as IL34
(interleukin-34) and CSF1 and CSF2 (colony stimulat-
ing factors 1 and 2) which are required for macrophage
maturation and proliferation.22 This niche model has
been illustrated by elegant recent studies in the liver
and spleen,23,24 which revealed prominent niche roles for
tissue-resident fibroblasts. In this light, it is not surprising
that macrophages and fibroblasts reciprocally regulate
their numbers through exchange of factors affecting the
other cell type.25 Immune niches represent a key emerg-
ing concept for immunologists, and while they will not be
discussed here in detail, they have broad implications for
Circulation Research. 2022;130:418–431. DOI: 10.1161/CIRCRESAHA.121.319812
Heterophagy in the Heart
understanding the true contributions of TRMs to organ
physiology and pathology.
In summary, the term macrophage encompasses a
broad array of cells with unique tissue-tailored identi-
Review
ties that are dictated by specific transcriptional networks,
dependent in part on their ontogeny (embryonic- versus
adult-derived) and are supported by distinct environ-
ments or niches.19,20 The diversity of cells and signals that
support the fate and maintenance of TRMs in the heart
remain poorly defined, yet these niche elements may
hold the key to support cardiac health throughout life.
Cardiac-Resident Macrophages
The primary task of the heart is to pump blood that deliv-
ers a continuous supply of oxygen and nutrients to the
rest of tissues. Pumping is a highly specialized function
and is energetically expensive. Hence, the heart contains
cells dedicated to contraction, the cardiomyocytes, which
account for 25% to 35% of all cardiac cells, as well as
other cell types that support the health and function-
ality of cardiomyocytes in one way or another. Among
the nonmyocyte cells, cardiac resident macrophages
(cMacs) represent 6% to 10% of the cells present in the
healthy adult myocardium.2 Although originally described
decades ago,26 it was not until 2012 that cMacs were
formally characterized.27
From an ontogenic perspective, several groups of
macrophages populate the heart: embryo-derived cMacs
arise from yolk-sac erythromyeloid progenitors and
enter the heart as early as  embryonic days E9.5-E10.5,
whereas fetal liver-derived macrophages infiltrate at
later developmental stages28 and are involved in cardiac
development.29,30 After birth, embryo-derived cMacs self-
replenish and persist in the adult heart longer than in
other tissues,28 but are progressively replaced by mono-
cyte-derived macrophages at different rates, depending
on the subset.28,31–33 Embryo-derived cMacs have been
proposed to participate in homeostatic processes,13,14,29,34
while those of monocytic origin would mediate inflamma-
tion.33,35 cMacs can be also classified by their expression
of different markers and location: embryo-derived cMacs
are Timd4+ CCR2neg, while monocyte-derived cMacs
are Timd4neg CCR2+.28,29,33,36,37 Further, CCR2neg cMacs
reside near the myocardial wall while CCR2+ cMacs pre-
dominate around trabecular projections of the endocar-
dium.29 Other classifications and distributions have been
reported, for example for Lyve1hi macrophages in perivas-
cular areas38 and Gata6+ cells in the pericardium.39 This
heterogeneity in origin, location, and marker expression
may reflect functional diversity in yet undefined ways.
A broad variety of functions have been assigned to
cMacs in the healthy heart, including coronary develop-
ment,29 electrical conduction,14 postnatal proliferation of
cardiomyocytes,40 and extracellular matrix remodeling.39
We recently described that cMacs additionally support
February 4, 2022   419
 Nicolás-Ávila et al
metabolic stability to cardiomyocytes, as discussed in
more detail below.13 Thus, cMacs are not passive or mere
immune elements of the heart, but rather cells with an
extensive toolkit of specialized functions; this notion has
Review
revitalized the interest of immunologists and cardiolo-
gists for these cells.
Macrophages as Metabolic Regulators
Macrophages are intimately integrated to tissue and
organismal metabolism. Nutrient-derived metabolites
can act as drivers of immune reprogramming during
disease, thus modulating key effector functions.41 Mac-
rophages adopt distinct metabolic profiles and have
diverse requirement for nutrients upon activation.42 This
so-called metabolic reprograming enables macrophages
to fuel anabolic and catabolic pathways needed to sus-
tain effector functions, a process that is typically referred
to as immunometabolism (for recent reviews on this topic
please see41,43–45). Beyond their own metabolic programs,
macrophages help to maintain the metabolic homoeosta-
sis of several tissues, for example by participating in the
homeostatic regulation of essential metabolites, includ-
ing iron, bilirubin, calcium, lipids, and amino acids.46–49 By
both controlling the levels of metabolites and respond-
ing to them, immune cells—and macrophages in par-
ticular—are important hubs for organismal metabolism,
as summarized in Figure 1 (and discussed below). Not
surprisingly, rodent models that lack Csf1R (colony
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stimulating factor 1 receptor) (and therefore most TRM
populations), display global metabolic alterations.50
Primary regulation of organismal metabolism occurs
at the intestinal mucosa, where a dense network of mac-
rophages largely derived from circulating monocytes
control multiple aspects of intestinal homeostasis, from
gastrointestinal peristalsis51 and maintenance of the epi-
thelial barrier integrity,52 to dynamic scanning of absorbed
fluids for pathogenic substances.53 Macrophages also
influence global metabolic control by recycling metabo-
lites from dying cells. Indeed, billions of cells are elimi-
nated daily from a healthy organism, and the homeostatic
clearance of these involves their engulfment and pro-
cessing by macrophages (as well as other phagocytes54).
This process is fundamental for removing inflammatory
by-products of dying cells (damage-associated molecu-
lar patterns) that would otherwise cause inflammation,55
while at the same time allows the recycling of essential
lipids and bioelements, such as iron. Clearance of apop-
totic cells often involves massive uptake of cellular con-
stituents56 and triggers transcriptional programs devoted
to manage metabolite degradation and export. For exam-
ple, the vast amount of apoptotic cell-derived lipids and
cholesterol must be appropriately handled to avoid toxic-
ity57 and to redistribute these important components to
other cells. For lipids, this is partly achieved by LXR (liver
X receptors), which are transcription factors activated
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Heterophagy in the Heart
by oxysterols58 and which in turn activate cholesterol
efflux pathways.59 Critically, these receptors are also part
of a feedback system that stimulates phagocytosis by
upregulating the expression of key phagocytic receptors,
such as Mertk.60 Internalization, storage, and efflux of lip-
ids by macrophages contribute significantly to vascular,
organ and systemic metabolic homeostasis.61 Similarly,
iron homeostasis is supported by macrophages in the
liver, bone marrow, and spleen, mainly through uptake of
senescent erythrocytes. Disruptions of such mechanisms
lead to iron overload syndromes, immune activation, and
poor health, as recently reviewed.48
Macrophages can additionally regulate metabolite
availability and influence tissue metabolism by compet-
ing for and degrading specific substrates. This is particu-
larly relevant in nutrient-restrictive microenvironments,
in which they take up and compete with surrounding
cells for metabolites.42 For example, they compete with
endothelial cells for glucose, and the glycolytic versus
oxidative metabolism of macrophages dictates glucose
uptake by endothelial cells and angiogenesis.62 In the
adipose tissue, sustained PI3K activity in macrophages
increases lipid uptake, catabolism and expression of the
scavenger macrophage receptor MARCO (macrophage
receptor with collagenous structure); dual MARCO and
myeloid PTEN (phosphatase and tensin homolog) defi-
ciencies prevent the lipid-buffering properties of macro-
phages on adipose tissue and cause metabolic disease.63
Another example is the control of the nicotinamide ade-
nine dinucleotide (NAD) by CD38, a NADase exoenzyme
expressed by inflammatory macrophages. In this context,
organismal aging and cellular senescence induce expres-
sion of CD38 by macrophages, resulting in reduced NAD
levels and enhanced aging-like phenotypes in the white
adipose tissue and the liver.64 Finally, macrophages in the
brown adipose tissue regulate catecholamine and norepi-
nephrine levels directly by synthesis65 or degradation,66,67
ultimately regulating thermogenesis and lipid expenditure.
Another exciting mechanism by which macrophages
modulate organismal metabolism involves the release
of extracellular vesicles (EVs) loaded with microRNAs.
For example, macrophages in the white adipose tis-
sue of obese individuals release microRNA-155 and
microRNA-29a, which inhibit PPARγ (peroxisome pro-
liferator-activated receptor γ) in adipocytes, myocytes,
and hepatocytes, thereby increasing the levels of fast-
ing blood glucose and serum insulin.68,69 In contrast,
microRNA-690 released in exosomes by M2-polarized
bone marrow-derived macrophages can improve glu-
cose-insulin homeostasis in obese mice by inhibiting
expression of the NAD+ kinase Nadk.70
Macrophages are major producers of cytokines that
can exert local or systemic effects on target cells to regu-
late metabolic outputs. In the liver, macrophages produce
IGFBP7 (insulin-like growth factor–binding protein 7),
which binds the insulin receptor to induce lipogenesis and
 Nicolás-Ávila et al Heterophagy in the Heart

Review
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Figure 1. Metabolic regulation by macrophages.

Macrophages regulate local and organismal metabolism at different levels. At the organismal level, macrophages control metabolite availability by
regulating nutrient absorption in the intestine, hormone production in endocrine glands and activity of highly metabolic organs. At the tissue level,
macrophages control metabolism of other cells either directly, through production of cytokines and miRs, or indirectly, by regulating metabolite
availability through recycling, production, degradation, and competition. In metabolic syndrome, for example, many of these processes are altered
due to macrophage activation and inflammatory signaling, causing anomalies in the metabolite pool and loss of metabolic homeostasis in tissues.

gluconeogenesis via activation of ERK (extracellular sig-
nal regulated kinase) signaling, thereby impacting systemic
metabolism.71 In the brain, microglia can release proinflam-
matory cytokines, including TNF-α (tumour necrosis factor
α), IL-1β, and IL-10,72 which act locally and can influence
central responses to insulin and leptin in the hypothala-
mus73; this in turn controls anorexigenic and adipostatic
signals in peripheral tissues. In the adipose tissue, macro-
phages can also regulate lipid accumulation by endothe-
lial cells, and obesity-associated hypertension through the
secretion of VEGF-B (vascular endothelial growth factor
B).74 Finally, macrophages have been suggested to regu-
late lipolysis in adipocytes,46 at least in part through release
of GDF3 (Growth Differentiation Factor 3), a PPARγ-
dependent cytokine that suppresses ATGL (adipose tri-
glyceride lipase) expression and lipolysis in adipocytes,
resulting in fat accumulation and insulin resistance.66,75,76
TRMs residing in the pancreas and white adipose
tissue control central physiological aspects of these
organs; however, increased macrophage accumulation

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 Nicolás-Ávila et al
associated with over-nutrition is considered a driv-
ing force in the development of insulin resistance and
the so-called metabolic syndrome.77,78 The central role
of macrophages in global metabolic homeostasis has
Review
been substantiated by multiple studies demonstrating
that (1) reduced infiltration and accumulation of mono-
cyte-derived macrophages blunts insulin resistance,79,80
(2) macrophages can control insulin production and
promote pancreatic β-cell apoptosis through secretion
of IL1β,81 and (3) macrophages can amplify inflamma-
tory circuits through release of inflammatory cytokines
(eg, TNF-α and IL-1b) that block insulin action on adi-
pocytes leading to insulin resistance.77,82,83
In summary, macrophages are major regulators of
organismal and local metabolism, a notion that mecha-
nistically links inflammation with metabolic dysregulation
and has prompted the search of additional mechanisms
through which these immune cells influence tissue ener-
getics in organs subjected to intense metabolic demands,
such as the heart.
HETEROPHAGY: OLD CONCEPTS AND
NEW FINDINGS
Certain types of cells are unique in that they are postmi-
totic, have low or no renewal capacity and feature very
long lifespans, including cardiomyocytes and neurons.
Maintaining the life-long health of these cells is chal-
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lenging, particularly as they are under strong metabolic
demands. A critical question is are there additional quality
control systems that support the survival and functional
properties of these cells? Canonical quality control sys-
tems such as autophagy or mitophagy (for mitochondria)
have crucial roles in preserving cellular heath and func-
tion.84–86 However, autophagy is energetically demanding
itself and requires the dynamic formation and intracel-
lular transport of organelles for degradation,87,88 which
is arguably complicated in tightly packed cells such as
cardiomyocytes. It is, therefore, reasonable to propose
that noncanonical mechanisms beyond autophagy are
in place for these cells, as hinted by studies examin-
ing mice lacking genes involved in different forms of
autophagy.89,90
We have recently discovered an alternative mecha-
nism that promotes cardiomyocyte fitness, and involves
the local network of cardiac TRMs (cMacs). In brief, our
studies found that healthy cardiomyocytes eject dys-
functional mitochondria (as well as other cellular com-
ponents) through large vesicles called cardiac exophers.
Ejected exophers are efficiently captured and eliminated
by neighbouring cMacs, thereby preventing inflamma-
some activation, accumulation of dysfunctional mito-
chondria in cardiomyocytes, and global alterations in
cardiac metabolism.13 It is likely that this phenomenon
is not restricted to cardiomyocytes, and we propose that
422   February 4, 2022 Circulation Research. 2022;130:418–431. DOI: 10.1161/CIRCRESAHA.121.319812
Heterophagy in the Heart
other parenchymal cells exist that eject damaged mate-
rial out of the cell to be collected and disposed by neigh-
bouring phagocytes, an extrinsic mechanism that may
support long-term cell fitness. We model this process by
defining client cells that are fully viable but choose to
eject their intracellular waste, and support cells endowed
with phagocytic capacity that are in charge of eliminat-
ing the heterologous material, in a process somewhat
comparable to the trash-collecting systems of modern
cities. We refer to this interplay of client and support
cells involving the exchange of material as heterophagy,
a term that extends the phenomenon of autophagy to
reflect the heterologous nature of the degraded material.
Quality Control Mechanisms and Metabolic
Fitness
Macromolecules and organelles in cells carry out key bio-
logical functions, and maintenance of cell fitness demands
a healthy pool of these structures. This is achieved
through several mechanisms. For example, quality control
mechanisms are in place to assist nascent polypeptides
to fold properly, clear misfolded molecules, respond to
accumulation of protein aggregates and deposit poten-
tially toxic conformers in designated sites to maintain
protein homeostasis (proteostasis).91 Two main protein
degradation systems have been described in eukary-
otes, the ubiquitin–proteasome system and autophagy.
In ubiquitin–proteasome system, ubiquitin-tagged pro-
teins are targeted by a multi-subunit protease complex,
the proteasome.85,92 Misfolded, damaged and short-lived
proteins undergo ubiquitination by a multi-step process
that requires several enzymes and consumes ATP.92,93
Ubiquitinated proteins are then targeted, unfolded, and
degraded in the proteolytic chamber of the proteasome.94
Autophagy (eating of the self) is an evolutionarily
conserved catabolic process in which excessive, defec-
tive or otherwise harmful intracellular components are
delivered to lysosomes for degradation and recycling
of metabolites.95 Autophagy provides a critical quality
control system that helps cells to maintain proteostasis
under basal conditions by removing damaged compo-
nents. In addition to its housekeeping function, autoph-
agy can be stimulated in response to various stresses
including nutrient deprivation, mitochondrial damage and
toxic protein aggregation, thus exerting a cytoprotective
role.96 Autophagy comes in different forms, depend-
ing on the nature and processing of the material to be
degraded. Typically, organelles, proteins, and other mac-
romolecules are sequestered within a double-membrane
vesicle termed autophagosome, whose formation relies
on autophagy-related components (encoded by Atg
genes) and that subsequently fuses with lysosomes. This
pathway is coined macroautophagy (herein, autophagy)
and can either proceed in a nonselective, bulk manner
or selectively eliminate individual targets, a process that
 Nicolás-Ávila et al
is highly dependent on the inducing factors.97 In other
instances, proteins and organelles are sequestered by
invaginations or protrusions of the limiting membrane
of late endosomes or lysosomes in a process known as
microautophagy.98,99 Lastly, proteins can simply be trans-
located into lysosomes, which is referred to as chaper-
one-mediated autophagy.100 Other types of autophagy
exist that degrade specific cellular components, such
as mitophagy, in which autophagosomes that only con-
tain mitochondria and the membrane-associated protein
MAP1 LC3 (light chain 3) are selectively formed and
then fused to lysosomes for degradation.101
Autophagy-mediated delivery for lysosomal degra-
dation is a major quality guardian that helps to main-
tain cellular homeostasis. Lysosomes are highly motile
membrane-bound organelles containing ≈60 types of
hydrolytic enzymes capable of breaking down proteins,
DNA, lipids and glycogen, and sit as the end point of
several pathways, including endocytosis, phagocytosis
and autophagy.102 Lysosomal hydrolases require an
acidic pH (4.5) to function optimally, a microenviron-
ment that is produced and maintained by the v-ATPase
(vacuolar ATPase), a membrane-spanning proton
pump.103 Within muscle fibers, lysosomes are predomi-
nantly involved in the removal of damaged or dysfunc-
tional cytoplasmic proteins and organelles, as large
myofibrillar proteins organized in sarcomeres cannot
be engulfed by lysosomes.104
Failure to maintain proteostasis leads to the accumu-
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lation of damaged organelles and structurally abnormal
proteins that become toxic and disrupt cell and tissue
function.105 This is a typical hallmark of aging in long-
lived and highly metabolically active cells such as car-
diomyocytes and neurons, which feature high oxidative
phosphorylation and mitochondrial content.106,107 One
rationale behind this is that mitochondria do not burn
substrates cleanly and therefore generate metabolic
waste than can damage proteins and organelles. Thus,
the maintenance of a healthy population of mitochon-
dria is key to preserve metabolic stability and requires
a finely tuned balance between mitochondrial biogen-
esis and degradation through different quality control
mechanisms.108
Quality Control Mechanisms in Cardiomyocytes
Accumulation of damaged mitochondria with age results
in metabolic imbalance and pathology, as seen in neu-
rodegenerative disorders and cardiac or skeletal muscle
dysfunction.109–111 Hence, it seems reasonable to think
that the massive mitochondria pool of cardiomyocyte
(estimated to represent up to 30% of the cell volume106)
demands specialized mechanisms to support its fitness.
For proliferating cells, this problem can be partly solved
by removal of cells with accumulated defects by cell
competition112 or dilution of the damaged material among
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Heterophagy in the Heart
daughter cells.113 But how do nonreplicative cells like
cardiomyocyte deal with this problem? Indeed, despite
evidence of low proliferative rates,114–116 cardiomyocytes
in mammals are largely quiescent and, therefore, rely
Review
heavily on quality control mechanisms such as autophagy
and mitophagy to ensure long-term mitochondrial fitness
and cell function.84,95,117,118 Mounting lines of evidence,
however, point to the existence of noncanonical quality
control pathways, as hinted by analyses of mutants that
lack key components of mitophagy, (eg, Parkin89) or gen-
eral autophagy (eg, Atg5/790), suggesting that there are
alternative mechanisms to recycle unfit mitochondria.
Recent studies in neurons,119,120 cardiomyocytes13 and
other cell types have described a phenomenon involv-
ing ejection to the extracellular milieu of morphologically
damaged mitochondria and other wasted components
packed within particles which are then captured by neigh-
bouring phagocytes (eg, astrocytes and macrophages).
Remarkably, in cardiomyocytes this mechanism has been
estimated to account for more than half of the degraded
mitochondrial pool, and to be important for mitochondrial
and metabolic fitness of the heart.13 Collectively, these
recent findings demonstrate the existence, at least in
neurons and cardiomyocytes, of mechanisms distinct
from canonical autophagy that enable elimination of
unwanted material.13,120–122 They also raise the critical
concept that cell proteostasis and tissue fitness demand,
under certain scenarios, the existence of a network of
phagocytes to degrade the ejected waste.
Heterophagy, a Different Mechanism for Quality
Control?
We define heterophagy (or eating of the nonself, in
contraposition to autophagy) as the (phago)lysosomal
degradation of material derived from another cell, either
taken up from the milieu or through direct contact with a
client cell. This term was previously used to refer to elimi-
nation of material from apoptotic cells through phago-
cytosis, endocytosis, micropinocytosis or trogocytosis,123
and analogous terms such as transmitophagy or trans-
cellular mitophagy have been proposed when it involves
transfer and degradation of mitochondria.120 Consistent
with this conceptual framework, it is now accepted that
viable cells can release considerable amounts of material
to the extracellular milieu, including protein aggregates
and organelles.13,119,122 Heterophagy would then repre-
sent a previously undefined mechanism that preserves
proteostasis yet critically differs from canonical pathways
in that it demands heterologous interactions with a sup-
port cell endowed with high phagocytic capacity.
Examples of cells engaged in heterophagy-like part-
nerships have been known for decades. Perhaps the most
prominent examples are the elimination of photoreceptor
outer segments from photoreceptor cells by the retinal
pigment epithelium,124,125 and the uptake of erythroblast
February 4, 2022   423
 Nicolás-Ávila et al Heterophagy in the Heart

nuclei by macrophages in bone marrow126,127 (for a list of
heterophagy-like behaviours reported in the literature see
Table). These examples illustrate that heterophagy events
take place in defined anatomic structures and serve to dif-
Review
mechanism, and we reserve the term heterophagy to those
that involve degradation of the cargo. Indeed, there are
examples of material exchange between cells that involve
subcellular particles but whose final purpose is not degra-

ferent purposes, from maintaining organelle fitness and dation (Table, bottom). These examples of cell cooperation
cell function (photo-reception,128) to functional matura- may not fit the concept of heterophagy but are still relevant,
tion (erythrocyte enucleation and male germ cell matura- since particles mediating the transfer could have the same
tion129,130). The evidence that similar heterologous interplay origin as those observed in heterophagic partnerships.
also occurs under physiological conditions in defined areas
of the brain120 and heart13 suggests that this mechanism Heterophagy in the Heart
is multipurpose, and that it is a broadly adopted biological
Although cardiomyocytes can beat autonomously in
strategy when large elements of a client cell must be elim-
culture,131 nonmyocyte cells are essential for optimal
inated. An appealing analogy of heterophagy with canoni-
contractile function of the myocardium. These include
cal autophagy is one in which cardiomyocyte-surrounding
fibroblasts, vascular, and immune cells.132 Macrophages
macrophages function as support lysosomes recruited ad
comprise the majority of immune interstitial cells in the
hoc around the client cells, such that the overall process
heart in the steady-state,2 and their relevance in normal
and purpose would remain equivalent for autophagy or
cardiac physiology is highlighted by depletion studies
heterophagy (Figure 2).
showing that elimination of macrophages leads to func-
Heterophagy in the mammalian heart was identified
tional impairment, including defective electrical con-
through the presence of cardiomyocyte-derived parti-
duction and metabolic derailment.13,14 Additionally, the
cles of large size that contained damaged mitochondria,
finding that in vivo interference with phagocytic activity
which accumulated in phagolysosomes of the surround-
(by removing only the involved receptors, as discussed
ing cMacs.13 While exophers are prominent in the heart,
below) causes similar dysfunction of parenchymal cells
equivalent elements may differ from tissue to tissue and
provides strong evidence of active heterophagy in myo-
not necessarily appear as free particles in the extracellular
cardium. We summarize below emerging mechanistic
space. Along this line, and although the exact origin and
insights on heterophagy in heart, and delineate the roles
name of these structures may vary depending on the tissue
of each cellular or subcellular element in this process.
(Table), we propose that structures analogous to exophers
are found in each context and reflect the transport func-
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The Client Cell (Cardiomyocytes)

tion of these structures. Hence, various processes across In the myocardium, the presence of a quantifiable
tissues may be manifestations of the same physiological number of exophers as transfer particles13 provides a

Table.  Examples of Cells Engaged in Heterophagy-Like Partnerships

Examples of heterophagy-type pairs

Tissue Client cell Transferred particle Support cell Reference

Heart Cardiomyocyte Exopher Macrophage 13

Retina Photoreceptor Photoreceptor outer segment particles Retinal pigment epithelium 121,124,125

Retina Ganglion cell Axonal evulsion Astrocyte 120

Central nervous system Neuron Exopher/axonal evulsion Astrocyte 119,120

Central nervous system Neuron Synaptosome Astrocyte/microglia 9,156,157

WAT Adipocyte Mitochondrial loaded EV Macrophage 152,153

Tumor Cancer cell TRAP; cytoplast Macrophage 158,159

Bone marrow, fetal liver, spleen Erythroblast Pyrenocyte Macrophage 11,126,127

Testis Germ cell Residual body Sertoli cell 160–163

Embryo Primordial germ cell Lobes Endodermal cell 164

Cell cooperation involving material transfer but no degradation

Tissue Support cell Transferred particle Client cell Reference
Skin/hair Melanocyte Melanosome-containing Keratinocyte 165

vesicle
Muscle Body wall Muscle cell Exopher Oocyte 137

Lung Mesenchymal stem cell Mitochondrial loaded EV Macrophage 166

Top: Examples of heterophagy across tissues. Client cells release material within EVs that are transferred to support cells for degradation. Bottom: Other examples of
cell cooperation involving material transfer for cellular support, but whose purpose is not degradation. EV indicates extracellular vesicle; TRAP, tumor released autophago-
some; and WAT, white adipose tissue.

424   February 4, 2022 Circulation Research. 2022;130:418–431. DOI: 10.1161/CIRCRESAHA.121.319812

 Nicolás-Ávila et al Heterophagy in the Heart

Review
Figure 2. Heterophagy vs autophagy.
Top, Autophagy (macroautophagy) occurs in a series of steps that include: (1) tagging dysfunctional proteins and organelles for degradation;
Downloaded from http://ahajournals.org by on July 12, 2023

(2) formation of a double-membrane structure, the so-called phagophore or isolation membrane, that sequesters cytoplasmic material tagged for
degradation; (3) expansion of the phagophore around the cargo and formation of a sealed, double-membrane vesicle known as the autophagosome;
(4) fusion of the autophagosome with the lysosomes (auto-phagolysosome) that leads to the degradation and recycling of the cargo.154,155 Bottom,
Modeling of the same process in the context of heterophagy. Although the pathways involved in exopher production (exophergenesis) remain
unknown, it is conceivable that they involve the release of maturing autophagosomes, or particles extruded directly from plasma membrane.

simple metric to explore mechanisms of production and
heterophagy. Using this criterion in combination with
genetic mouse models, the contribution of the autopha-
gic machinery of cardiomyocytes for exopher produc-
tion (exophergenesis) became evident. Indeed, exophers
contain LC3, a protein involved in cargo selection during
the formation of autophagosomes.133 Importantly, phar-
macological stimulation of autophagy with rapamycin
increases exopher numbers whereas genetic ablation
of Atg7 specifically in cardiomyocytes halts their pro-
duction.13 Although other elements of the autophagy or
mitophagy machinery of cardiomyocytes have yet to be
explored, these findings reveal marked commonalities
between heterophagy and autophagy in the heart. Fur-
ther, these findings suggest that cardiomyocytes have
developed mechanisms to tag dysfunctional mitochon-
dria but may use different strategies for routing them into
autophagosomes for fusion with internal lysosomes, or to
expel them out of the cell. Whether this model is accurate
and which are the molecular drivers of the distinct routes
are currently unknown. Identification of the mechanisms
involved in exopher production will be essential to define
bona fide client cells in the future.
Cardiomyocytes can exchange mitochondria and
mitochondrial components among each other using dif-
ferent EVs and nanotubes.134 EV-mediated transfer of
mitochondria from progenitor cells to cardiomyocytes
has been also reported and is associated with therapeu-
tic effects in the ischemic myocardium, where it restores
bioenergetic levels.135 In the context of heterophagy, how-
ever, heterologous transfer of material appears to have
the unique purpose of degradation, since cardiomyocyte-
derived mitochondrial proteins and DNA become rapidly
undetectable in cMacs when these cells are isolated
from the tissue and tracked ex vivo.13 Degradation of the
transferred material is also consistent with the observa-
tion that the mitochondria present in exophers feature
aberrant morphology, reduced membrane potential and
impaired ATP generation, and are, therefore, unlikely to
be usable for the recipient cells.
The Transfer Vesicles (Exophers)
The first description of exophergenesis was made in
neurons from C. elegans, in which similar structures to
the ones identified in the myocardium of mice mediate
the extrusion of protein aggregates and mitochondria

Circulation Research. 2022;130:418–431. DOI: 10.1161/CIRCRESAHA.121.319812 February 4, 2022   425

 Nicolás-Ávila et al Heterophagy in the Heart

from these cells (Melentijevic et al119). These findings
suggested that there are functional and structural simi-
larities of these particles across tissues and species.
While an accurate definition of exophers beyond func-
Review
the location of tagged mitochondria in lysosomes in
cardiomyocytes or cMacs suggest that heterophagy
accounts for the recycling of up to 60% of all mito-
chondria in the healthy myocardium,13 which high-

tion or cargo will require a more precise knowledge of
the molecular processes leading to their formation and
ejection, comparison with other types of EVs highlights
the unique properties of exophers in terms of size (in the
range of microns), cargo (large organelles), mechanism
of formation (nonendosomal origin), and purpose (inter-
cellular transfer).13,119,136,137 This differentiates them from
other EVs such as exosomes,138–141 microvesicles,138–141
and apoptotic bodies142 (Figure 3).
Heterophagy in the mammalian heart is unique in
that exophers are phagocytosed and degraded by
proximal cMacs, thus coupling the cell-autonomous
phenomenon of cargo selection and ejection from
the cell with macrophage-dependent degradation.
This is important because mitochondrial proteins and
DNA are well-known damage-associated molecular
patterns that are highly inflammogenic.143,144 Consis-
tently, depletion of cMacs leads to accumulation of
free mitochondria in the extracellular space and acti-
vation of the inflammasome.13 Estimations based on
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lights the magnitude of this clearance mechanism and
its relevance for the silent removal of mitochondria
in healthy hearts. These findings further underscore
what could be canonical properties of client cells in
the context of heterophagy.
Another salient finding was that exopher numbers
increase in mice upon isoproterenol treatment (which
induces reactive oxygen species production by mito-
chondria145), but that this increase is blunted when
mitochondria are genetically protected from oxidative
damage. Myocardial infarction also elicits increased
levels of exophers, but this appears to be restricted
to the viable myocardium adjacent to the dead zone.13
Importantly, functional anomalies associated with cMac
depletion are reversible and diastolic parameters return
to normal when cMacs repopulate the tissue. These
findings suggest that exophergenesis by cardiomyo-
cytes is adaptable to stress, requires actual organelle
damage and may represent a strategy to recover cell
fitness after an insult.

Figure 3. Features of exophers vs other extracellular vesicles (EVs).

Exophers have properties that differentiate them from classical extracellular vesicles, including size (in the range of microns), cargo (large
organelles), mechanism of formation (nonendosomal origin), and purpose (transport cargo out of living cells for disposal). These properties
differentiate them from other EVs such as exosomes, microvesicles, or apoptotic bodies. ER indicates endoplasmic reticulum; and ESCRT,
endosomal sorting complexes required for transport.

426   February 4, 2022 Circulation Research. 2022;130:418–431. DOI: 10.1161/CIRCRESAHA.121.319812

 Nicolás-Ávila et al
The Support Phagocytes
cMacs have important features that are relevant for their
role as support phagocytes in the myocardium. One is
their pervasive distribution in the tissue, where they form a
dense network that surrounds cardiomyocytes, and estima-
tions based on imaging studies revealed that, on average,
each cardiomyocyte is served by ≈5 macrophages.13 Fur-
ther, although cMacs are in a low proliferative state under
homeostatic conditions, we have found that they rapidly
enter into cycle and display fast recovery kinetics after
depletion, revealing the presence of active cues and niches
for the maintenance of these cells in the healthy myocar-
dium (Nicolás-Ávila and Hidalgo unpublished, and28). It will
now be critical to define the actual nature of the cellular
niches and the cytokines that dictate the distribution and
number of cMacs to understand whether and how altera-
tions associated with age or disease (eg, cardiomyopathies)
compromise the network of cMacs during the natural or
pathological decline of cardiac function. Although different
subsets of cMacs co-exist in the heart,28 and major special-
ization has been described across anatomic regions of the
same tissue,146 it is currently unclear whether specific sub-
sets of cMacs are differentially involved in exopher uptake.
Finally, studies that tracked the fate of exophers and
mitochondria in the myocardium demonstrated that
absence of the receptor Mertk was sufficient to reca-
pitulate cardinal features seen in hearts depleted of
cMacs, including accumulation of dysfunctional mito-
chondria inside and outside of cardiomyocytes, exag-
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gerated glucose uptake and loss of cardiac contractile
function.13 Of note, Mertk also mediates debris clear-
ance in the infarcted myocardium.147 Mertk is a tyrosine
kinase receptor on the surface of phagocytes that medi-
ates recognition of phosphatidylserine on target apop-
totic cells and their subsequent internalization.54 Similar
to apoptotic cells, exophers present phosphatidylserine
on their surface13; thus, these findings demonstrate that
phagocytosis is the key process mediated by cMacs that
regulates mitochondrial and metabolic fitness in cardio-
myocytes, rather than any other of the multiple functions
assigned to macrophages (see section Macrophages
as metabolic regulators above). It is noteworthy that the
same receptor, Mertk, either alone or in combination with
other TAM (Tyro3/Axl/Mer) receptors, plays a major role
in the uptake of photoreceptor outer segments in the
retina (by epithelial cells128), pyrenocytes in the marrow
(by central macrophages148), and residual bodies in testis
(by Sertoli Cells130,149) hinting to the presence of recep-
tors dedicated to heterophagy.
PERSPECTIVES AND OUTSTANDING
QUESTIONS
While the discovery that phagocytosis is a major homeo-
static process in the heart is conceptually striking and
exciting, it is clear that the unknowns still far outnumber
Circulation Research. 2022;130:418–431. DOI: 10.1161/CIRCRESAHA.121.319812
Heterophagy in the Heart
the knowns. We briefly comment here on 2 specific
directions, highlighting questions and hurdles that will be
important to address as the field moves forward.
(1) Crosstalk between the immune system and the
Review
heart. Macrophages are global regulators of tissue physi-
ology, yet the discovery of these immune cells as central
players of mitochondrial homeostasis in the myocardium
opens remarkable opportunities for research and therapy
in numerous fronts. Conceptually, it spurs the question
of whether derailment of the immune pool, for example
during senescence and age,150 is mechanistically associ-
ated with anomalies in cardiac metabolism and function
that are typically seen in aged individuals.110 For devel-
opmental immunologists, it raises the fascinating issue
of when and how this partnership is established, as well
as the identification of putative niches for cMacs in the
myocardium, particularly in light of the marked switch in
metabolism that this organ undergoes after birth.110 Ulti-
mately, focusing on immune cells may enrich the diag-
nostic palette for cardiomyopathies of unknown origin,
and allow the design of new types of therapies targeting
the immune system.
(2) Heterophagy beyond the heart. It will be particu-
larly important to explore whether similar client-support
phagocyte partnerships occur in other tissues. Evidence
for mitochondria transfer from white adipocytes to adja-
cent macrophages151–153 and imaging-based demon-
stration of transfer between neurons and neighbouring
astrocytes120 suggest that heterophagy may be a broad
phenomenon. Thus, it will be important to search for het-
erophagy-type partnerships in other tissues, particularly
those subjected to a high metabolic demand and that
have high mitochondria turnover, such as brown adipose
tissue and skeletal muscle. Nonetheless, we urge caution,
as even in the aforementioned examples the presence of
structures similar to exophers have not been reported,
and the functional impact for putative client cells has not
been examined. This raises the critical question of what
defines bona fide heterophagy (transfer of material, deg-
radation of cargo, type of cells involved, etc). Even the
definition of what is an exopher is currently under debate,
since degradation of cargo or the presence of support
phagocytes have not yet been described in the C. ele-
gans studies.119,137 Clearly, identification of the molecular
drivers of exophergenesis136 will be needed to provide a
rigorous response to these questions.
ARTICLE INFORMATION
Affiliations
Centro Nacional de Investigaciones Cardiovasculares Carlos III, Spain (J.A.N.-A.,
L.P.-C., P.M.-C., A.H.). Department of Experimental & Health Sciences, Universitat
Pompeu Fabra, CIBERNED, Spain (P.M.-C.). ICREA, Spain (P.M.-C.).
Acknowledgments
We are indebted to members of our labs for discussion and previous experimental
work. We are grateful to J.A. Enriquez and many researchers at Centro Nacional
de Investigaciones Cardiovasculares (CNIC) with whom we performed critical ex-
February 4, 2022   427
 Nicolás-Ávila et al
periments that led to the concept of heterophagy. We thank Monica Driscoll for
kind and open discussion on exophers. The CNIC is supported by the Ministerio
de Ciencia e Innovación (MICINN)  and the Pro-CNIC Foundation and is a Severo
Ochoa Center of Excellence (MICINN). Illustrations were created by adapting
templates from Servier Medical Arts (https://smart.servier.com/).
Review
Disclosures
A. Hidalgo is a paid consultant for Flagship Pioneering, Inc on topics related to the
content of this review article. The other authors report no conflicts.
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