NLRP3 Inflammasome in Acute Myocardial Infarction

INVITED REVIEW ARTICLE
NLRP3 Inflammasome in Acute Myocardial Infarction
Adolfo G. Mauro, PhD,*† Aldo Bonaventura, MD,*‡ Eleonora Mezzaroma, PhD,*†§
Mohammed Quader, MD,*¶║ and Stefano Toldo, PhD*†¶
Abstract: Acute myocardial infarction (AMI) is associated with the
induction of a sterile inflammatory response that leads to further
injury. The NACHT, leucine-rich repeat, and pyrin domain–
containing protein 3 (NLRP3) inflammasome is a macromolecular
structure responsible for the inflammatory response to injury or
infection. NLRP3 can sense intracellular danger signals, such as
ischemia and extracellular or intracellular alarmins during tissue
injury. The NLRP3 inflammasome is primed and triggered by locally
released damage-associated molecular patterns and amplifies the
inflammatory response and cell death through caspase-1 activation.
Here, we examine the scientific evidence supporting a role for
NLRP3 in AMI and the available strategies to inhibit the effects of
the inflammasome. Our focus is on the beneficial effects seen in
experimental models of AMI in preclinical animal models and the
initial results of clinical trials.
Key Words: NLRP3 inflammasome, acute myocardial infarction,
interleukin-1b, interleukin-18, caspase-1
(J Cardiovasc Pharmacol Ô 2019;74:175–187)
eventually causing cardiomyocyte necrosis and extensive myocardial damage.4,5 In addition, reperfusion therapy, achieved
through the use of thrombolytics or percutaneous coronary
intervention, is associated with further damage due to the
adverse effects of oxygen utilization in damaged mitochondria
[ie, production of reactive oxygen species (ROS)], leading to
the so-called reperfusion injury.5,6 Overall, the release of cytoplasmatic content into the myocardial interstitium and intracellular stress-associated pathways (ROS production and
oxidative stress, autophagy, and protein quality regulations)
promote the activation of the innate immune response.6 Innate
immunity constitutes the first line of the host defense in case of
infection or tissue injury to coordinate tissue healing.7 AMI
represents a prototypical example of sterile inflammatory
response, in absence of pathogen or antigen-dependent immunity.8,9 Although inflammation is essential for a prompt healing
of the wounded tissue, an unrestrained inflammatory activity
represents a process for further damage, especially in organs
with little regenerative properties such as the heart.
INTRODUCTION
Despite constant improvement of the patient prognosis,
patient education, and management of risk factors, acute
myocardial infarction (AMI) remains one of the most
common causes of morbidity, hospitalization, and mortality
worldwide.1–3 The occlusion of one of the epicardial coronary
arteries, most commonly after the rupture of the atherosclerotic plaque, results in an abrupt blockage of blood flow
leading to anoxia and ischemia. This set of events compromises the ability of the cell to maintain an adequate production of intracellular energy [Adenosine triphosphate (ATP)],
Received for publication March 8, 2019; accepted June 10, 2019.
From the *VCU Pauley Heart Center, Virginia Commonwealth University,
Richmond, VA; †Johnson Center for Critical Care Medicine Pulmonary
Research, Virginia Commonwealth University, Richmond, VA; ‡Department of Internal Medicine, First Clinic of Internal Medicine, University of
Genoa, Genoa, Italy; §Pharmacotherapy and Outcomes Sciences, Virginia
Commonwealth University, Richmond, VA; ¶Department of Cardiothoracic Surgery, Virginia Commonwealth University, Richmond, VA; and
║Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond,
VA.
S. Toldo has received research support from Olatec. A. G. Mauro is supported
by an American Heart Association pre-doctoral grant. M. Quader is
supported by a Scientist Development Grant from the American Heart
Association and from a Merit Review Award from the Veterans Health
Administration.
The authors report no conflicts of interest.
Reprints: Stefano Toldo, PhD, VCU Pauley Heart Center, Virginia Commonwealth University, Box 980281, Richmond, VA 23298 (e-mail:
[email protected]).
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ISCHEMIC INJURY AND
MYOCARDIAL INFLAMMATION
The initiation of the inflammatory reaction after AMI is
attributable to a loss of cell membrane integrity as a result of
the prolonged lack of oxygen and consequent cell starvation.5,6 The membrane’s permeability leads to the release of
mediators that are termed as damage-associated molecular
patterns (DAMPs).7–10 DAMPs are a group of heterogeneous
molecules that share specific chemical/physical properties that
act as danger signals on binding with protein receptors known
as pattern recognition receptors (PRRs). Many of the same
PRRs involved in the recognition of DAMPs also recognize
invading microbiological pathogens after interacting with distinct conserved molecule/domains, termed pathogen-associated molecular patterns.10 The interaction between the
DAMPs released by dying or injured cells and the PPRs on
(or in) surviving/alive cells is critical for the initiation of the
inflammatory response and the healing process.10–12 The latter can be divided into 3 interconnected phases. The first, the
inflammatory phase, leads to the recruitment of leukocytes to
“clean” the damaged tissue and sets the conditions for the
second phase, defined as proliferative phase. This is characterized by the proliferation of cardiac fibroblasts and endothelial cells in the necrotic area.5 The third phase is the
maturation, which progresses to the formation of a functional
scar, adapted to the organ needs.13 These 3 phases are
highly regulated, and a disequilibrium in either of these
phases can lead to the formation of a weak scar, prone to
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Mauro et al
J Cardiovasc Pharmacol ä Volume 74, Number 3, September 2019
rupture, or an excessively fibrotic scar, leading to aneurysm
formation.5,14,15
Among the PRRs, the toll-like receptors (TLRs) and the
NOD-like receptors (NLRs) are the most characterized.16 TLRs
are type I integral membrane sensor organized as homodimers
or heterodimers with several leucine-rich repeats (LRRs) on the
extracellular domain. Ten human and 12 murine TLRs have
been described, identifying a plethora of host or microbial
molecules.17,18 On binding to DAMPs and/or pathogenassociated molecular patterns, the extracellular domains of 2
challenged TLRs interact together leading to the recruitment of
the C-terminal toll/interleukin-1 receptor (TIR) domain. The
TIR engages several intracellular adaptor proteins, such as
myeloid differentiation factor 88 (Myd88), TIR adaptor protein, Tumor Necrosis Factor receptor–associated factor 6
(TRAF-6), and TIR domain–containing adapter-inducing interferon-b (TRIF).18,19 This cascade of events culminates with the
activation of the mitogen activating protein kinases, the
interferon-regulated transcription factors, and the nuclear factor
kappa B (NF-kB), which ultimately fosters the transcription of
a multitude of both proinflammatory and anti-inflammatory
genes, therefore orchestrating the inflammatory response.19
Several TLRs are activated after AMI, and their function is
important for the initiation and completion of the inflammatory
response.18 The first type of DAMPS to be released on necrotic
cell death after AMI is a particular class of molecules called
“alarmins.” One of the most studied is the high-mobility group
box 1 (HMGB1), a nuclear factor that diffuses in a paracrine
fashion binding to the TLR-4 or the receptor for advanced
glycation end products.7,20 On interaction with these 2 receptors, HMGB1 mediates the activation of NF-kB.21 A similar
mechanism of action is consistent with other types of alarmins
such as S100 which elicits NF-kB activation after the binding
of TLR-4 and receptor for advanced glycation end products,20,22 the glucose-regulated proteins (GRPs) (eg, GRP94/
gp96 and GRP170), and heat shock proteins (eg, hsp70 and
hsp90).7,9,10,23–25 Interleukin-1a (IL-1a) is a proinflammatory
cytokine (as reviewed in detail later) and also an alarmin when
released by necrotic cells.20
Despite a recognized role of the TLRs in the response to
AMI, we will discuss the relevance of the TLR signaling as
part of the inflammasome signaling, a specific pathway that is
guided by PRRs of the family of the NLRs, and in particular
the NACHT, LRR, and pyrin domain (PYD)-containing
protein 3 (NLRP3).16,26
IL-18, pro-IL-33, and pro-IL-37.32 IL-1b and IL-18 are potent
proinflammatory cytokines involved in several acute and
chronic disorders, including cardiovascular disease [eg, AMI,
atherosclerosis, hypertension, and heart failure (HF)].32–34
The NLRP1, NLRC4, and AIM2 inflammasomes are
widely investigated for their role during pathogen infection.
Recently, they have been studied as mediators of some chronic
inflammatory diseases (eg, inflammatory bowel disease), but
their role in the development of cardiovascular diseases is
unknown.9,27,29 The NLRP3 inflammasome has emerged as an
almost omnipresent PRR that is activated in many different
cardiovascular and noncardiovascular diseases.9,35
NLRP3
Inflammasomes are formed upon the activation of
intracellular PRRs and provide immune surveillance in the
cytoplasm by producing and releasing cytokines of interleukin1 (IL-1) family.16,27 Different sensing components (NLRs,
AIM2-like receptors, and RIG-I-like receptors) can identify
diverse stimuli and oligomerize to form inflammasomes.9,10,28,29 This process culminates with the activation of
the cysteine protease caspase-1 in humans and in mice.30 Additional caspases (caspase-4 and -5 in humans and caspase-11 in
mice) can be activated in the setting of the conventional or
unconventional inflammasome signaling.31 The activation of
caspase-1 leads to a proteolytic cleavage of pro-IL-1b, pro-
The NLRP3 protein is composed of 3 different domains:
a domain of LRRs assembled at the C-terminal; a central
NATCH domain [also known as nucleotide-binding oligomerization domain (NOD)]; and an N-terminal effector domain,
termed PYD). NLRP3 is principally linked to the inflammasome
pathway. Some reports, however, have reported that NLRP3 has
functions that are independent from the inflammasome pathway.36,37 For instance, deletion of NLRP3 protein inhibits
the ischemic preconditioning in an NLRP3-inflammasome–
independent manner through an IL-6/STAT3-dependent mechanism.38 More about the inflammasome-independent role of
NLRP3 and inflammasome protective signaling in AMI is
described in detail in another review article.39
Within the inflammasome signaling, when the NLRP3
senses damage, it oligomerizes and, through its N-terminal
PYD, interacts with the PYD of ASC, also known as
apoptosis-associated speck-like protein containing a caspase
recruitment domain (CARD).9,40–42 Polymerization of ASC is
initiated by this PYD–PYD interaction and leads to the formation of filamentous, insoluble structures that are reflected
macroscopically as large specks localized near the nucleus.42
A report studying the AIM2 inflammasome microscopic
structure showed that active AIM2 and ASC form a filamentous structure where ASC represents the majority of the “filaments.”43 The same group of researchers reported that this
may be a common structure in those inflammasomes that use
ASC (such as NLRP1 and NLRP3).43 On polymerization of
ASC, pro-caspase-1 is recruited by the CARD domain of
ASC, completing the structure of the NLRP3 inflammasome.9,44 At microscopic level, the ASC “filament” functions
as a core for the polymerization of pro-caspase-1 “branches,”
leading to the formation of a stellate structure (Figs. 1, 2).
NLRP3 can also activate, as noncanonical pathway, 2 other
caspases: caspase-8 and caspase-11. Although caspase-11 is
strictly an inflammatory caspase, caspase-8 is also involved in
the apoptotic pathway.31,45 Although the role of these 2 caspases in the response to tissue injury that follows heart ischemia has not been yet clarified, several studies report their
involvement in the pathogenesis of ischemic injury in different organs (eg, brain and kidney).46–48 Caspase-11 is upregulated in cultured primary astrocytes after simulated ischemic
condition. Ischemic brain injury induces caspase-1 activation
and IL-1b production with consequent cell death.46 Caspase11 is also increased after renal ischemia-reperfusion injury in
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The Inflammasomes
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J Cardiovasc Pharmacol ä Volume 74, Number 3, September 2019
Inflammasome in AMI
FIGURE 1. Role of ischemia as promoter of the
NLRP3 pathway. A wealth of molecules, deriving
from either microbe invasion or host tissue/cell
damage, is responsible for the activation of the
innate immune response through TLRs and NODlike receptors (NLRs). After tissue injury, the DAMP
molecules, including alarmins [high-mobility
group box 1 (HMGB1), GRPs, heat shock proteins
(HSPs), ROS, and nucleotides], promote the activation of the immune response. The amplification
of the inflammatory response is mediated by the
inflammasome, leading to the production and
release of active interleukin-1b (IL-1b) and/or
interleukin-18 (IL-18).
rats.47 Caspase-8 has been found upregulated in a human
stroke subjects and in a model of cerebral artery occlusion.48
Caspase-1 was first described in 1989 as a cysteine
protease, responsible for the conversion of the pro-IL-1b to the
active form IL-1b, earning the name IL-1b–converting
enzyme.49,50 Caspase-1 is a zymogen, which is then cleaved
on inflammasome oligomerization, reaching its active form.9,50
Thirty years later, this enzyme is recognized as a regulator of
the release of several cytokines and a mediator of cell death by
cleaving several substrates involved in pivotal metabolic
pathways and by promoting the formation of membrane
pores.9,51 In particular, 5 glycolysis enzymes were found to be
caspase-1 enzymatic substrates.52 The downregulation of cellular metabolism can undermine the cells’ viability by directing
the fate of the cell toward death instead of survival.
Caspase-1 is also associated with a highly regulated
inflammatory-linked cell death, named pyroptosis.53 Morphologically, pyroptosis shares most of the features of both
necrosis and apoptosis. Apoptosis leads to a noninflammatory
form of cell death characterized by shrinkage of the cells and
fragmentation into apoptotic bodies. By contrast, pyroptosis
serves as an immune signal to generate an inflammatory
response. Pyroptosis is characterized by membrane blebbing,
nuclear condensation, cellular swelling, and ultimately pore
formation with spillage of the intracytoplasmic contents.53
The role of caspase-1 in myocardial ischemia-reperfusion
injury is described with greater details elsewhere.54 The activation of caspase-1 on inflammasome oligomerization fosters
the cleavage of the Asp275 and Asp276 residues present on
the protein gasdermin D (GSDMD) generating an N-terminal
GSDMD product (GSDMD-NT). The GSDMD-NT oligomerizes and binds to the phosphatidylinositol phosphate and
phosphatidylserine on the cell membrane, forming pores
through the cytoplasmic membrane (Fig. 2).51,55–58 The pore
formation perturbs the intracellular ionic gradients, causing
sodium to enter into the cells and therefore leading to
water influx, osmotic swelling, and membrane rupture.51,56
However, in macrophages with an activated inflammasome
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and intact lipidic membrane, GSDMD-NT pores have been
found having an active role in IL-1b (and IL-18) release
(Fig. 2).58,59
Pyroptosis can also be triggered, in a noncanonical
fashion, on caspase-11 (and caspase-4/5 in human cells)
activity after stimulation with lipopolysaccharide (LPS).56,60
Caspase-1–mediated inflammatory cell death contributes to
the reperfusion injury after AMI (Figs. 2, 3).
Signaling that Regulates the Formation of the
Inflammasome in the Heart
The formation of the inflammasome is a finely regulated process. Depending on the cell type, there is a need for
the convergence of 2 parallel pathways, defined as inflammasome priming and triggering.61,62 The priming refers to
a very heterogeneous set of signals that regulate the
expression/degradation of the inflammasome components
(NLRP3, ASC, and caspase-1) and cytokines (IL-1b and
IL-18). Triggering refers to the signals that contribute to the
activation of NLRP3 (Fig. 2). These 2 signals are often linked
and are intrinsic in the nature of tissue damage. In fact, AMI
is responsible for both these signals (Fig. 3).
Priming of the NLRP3 Inflammasome
Alarmins and DAMPs are the principal key factors that
control the priming phase. During AMI, TLRs/IL-1 receptor
is among the first challenged PRRs, leading to NF-kB activation. NF-kB controls the transcription of hundreds of proinflammatory genes, including the NLRP3 inflammasome
components and substrates.32,63,64 However, other cytokines,
hormones, and metabolites (eg, angiotensin II, glucose, and
fatty acids) can work as priming agents, especially during
chronic conditions, such as obesity, diabetes, and hypertension, which may then exaggerate the inflammasome response
to AMI. The translation of all the inflammasome constituents
is critical to generate a significant mass to foster the formation
of the macromolecular complex.9,65
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Mauro et al
J Cardiovasc Pharmacol ä Volume 74, Number 3, September 2019
FIGURE 2. Regulation of the NLRP3
inflammasome. The activation of the
NLRP3 inflammasome depends on 2
independent steps. During tissue
injury, DAMPs activate the PRRs, such
as the TLRs or the IL-1 receptor, and
lead to the translocation of the NF-kB
into the nucleus. After this event, the
gene transcription of hundreds of
proinflammatory
genes—including
the components of the inflammasome
pathway—occurs. This process is
defined as inflammasome “priming.”
A moderate translation of the inflammasome components (NLRP3,
ASC, and pro-caspase-1) is needed for
the inflammasome formation but does
not coincide with its activation. Extracellular ATP (eATP) binding to the
P2X7, or intracellular DAMPs can
stimulate the NLRP3 activation (inflammasome trigger) through different mechanisms involving the K+
efflux. Once active, NLRP3 oligomerizes into a platform for recruitment
of the apoptosis-associated spec-like
protein containing a carboxy-terminal
CARD (ASC) and pro-caspase-1. After
the formation of the macromolecular
structure, the activation caspase-1
mediates the cleavage of the prointerleukin-1 b (pro-IL-1b) pro-interleukin-18 (pro-IL-18) and gasdermin-D
(GSDMD). The oligomerization of the
N-terminal fragment of GSDMD into
a plasma membrane pore is responsible for the secretion of the active IL-1b and IL-18 for further autocrine, paracrine, and endocrine
amplification of the immune responses. Caspase-1 and GSDMD mediate also a form of regulated cell death known as pyroptosis.
Triggering of the NLRP3 Inflammasome
AMI promotes both the priming and the triggering
signal. In fact, the inflammasome components and cytokines
are increased at the transcriptional level and at the protein
level.9,65 In the heart of healthy mice, the triggering alone, in
absence of priming, is insufficient to promote the formation of
the inflammasome.65 Therefore, in the heart, a simultaneous
activation of priming and triggering signals are needed for the
NLRP3 inflammasome activation.9,65 This feature of the
NLRP3 inflammasome formation in the heart is of particular
importance because inhibition of triggering or priming may
be equally beneficial in AMI. However, this requires additional investigation, especially to define whether the presence
of chronic diseases (eg, obesity, diabetes, hypertension,
aging, arthritis, or gout) establishes a priming effect that precedes the ischemic insult. Recent findings have shown that in
aged rats, the ischemia-reperfusion injury induces greater
plasma levels of IL-1b.72 Similarly, diabetes increases the
basal expression of NLRP3, ASC, and pro-caspase-1. On
ischemia-reperfusion injury, the activation of the inflammasome (caspase-1 cleavage and mature IL-1b) was significantly increased compared with normoglycemic rats with
AMI.73 These data support the notion that comorbidities
increase the priming and enhance the effect of inflammasome
activation.
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The priming signaling, by itself, may be sufficient to
induce the NLRP3 inflammasome only in few cell types (ie,
monocytes), but in most cells, the priming alone is insufficient
to lead to an active NLRP3 inflammasome.66 The triggering
signal becomes necessary and often occurs intracellularly due
to the generation of ROS or the impairment of the autophagy/
mitophagy processes or the increase in extracellular ATP. All
these signals trigger the activation of the inflammasome by
inducing potassium (K+) efflux (Fig. 2).16,67–70
After AMI, the extracellular ATP released by necrotic
cells mediates the activation of the ligand-gated cation channel
purinoreceptor P2X7. P2X7 channels open in response to ATP
binding, leading to K+ efflux, which finally triggers the NLRP3
activation cascade (Fig. 2).9 Another important mechanism
activated by ischemia-reperfusion injury is the stimulation of
the redox-sensitive thioredoxin-interacting protein (TXNIP),
necessary for the activation (trigger) of NLRP3.71
Necessity of Priming and Triggering in the Heart
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Inflammasome in AMI
FIGURE 3. The effect of the NLRP3 inflammasome activation in the ischemia-reperfusion injury. Prolonged ischemia leads to
cardiomyocyte death through necrosis. The initial area of necrosis represents only a part of the mature infarct evaluated after
reperfusion. In the initial phase of reperfusion (less than 3 hours), the activity of the inflammasome is very low, but the priming
activity is promoted by DAMPs released by necrotic cells, increasing the expression of the inflammasome components. When the
inflammasome protein levels reach the activation threshold (after the first few hours), the intensity of inflammasome activity
increased and promotes a rapid growth of the infarct through pyroptosis, for example, the inflammatory cell death mediated by
the inflammasome. Based on these findings, the ideal time window for a successful therapeutic intervention is in the very first
hours following ischemia.
NLRP3 INFLAMMASOME ACTIVATION CONTRIBUTES TO INFARCT SIZE
THROUGH PYROPTOSIS
The activity of the inflammasome after AMI was
reported in a thorough study by Kawaguchi et al74 that revealed the presence of ASC aggregates in myocardial autoptic
samples from patients with ischemic heart disease. In addition, after AMI induced by experimental ischemia-reperfusion
injury, the same group of researchers reported preserved cardiac function, a smaller infarcted area, and a reduction of IL1b synthesis in mice lacking ASC or caspase-1.74
The cardioprotective effect of caspase-1 deletion/
inhibition in the heart was already known even before it was
defined as part of the inflammasome pathway.75 Genetic deletion of caspase-1 was found to reduce the onset of early mortality and left-ventricular dilatation after AMI.75,76 Human
myocardial strips cultured in vitro and exposed to ischemia
in the presence of a caspase-1 inhibitor showed improved contractility.76 As of today, there are no data confirming that inhibition of GSDMD-NT pore formation could reduce infarct size
and myocardial damage after AMI.
The silencing of the NLRP3 gene in mice with
permanent ligation of the left anterior descending coronary
artery or in mice undergoing ischemia-reperfusion injury
further supported the role of NLRP3 inflammasome in
exacerbating the damage after AMI.77
These reports were further strengthened by the work of
Sandanger et al78 that used an ex vivo Langendorff model of
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ischemia-reperfusion and showed a reduced infarct size in
Nlrp32/2 mice. Furthermore, knocking out ASC in a model of
in vivo transient coronary ligation exerted protective effects on
both infarct size and ventricular function.74 However, it is worth
noting that ASC-deficient mice did not show a reduction in infarct
size after ischemia-reperfusion injury in an ex vivo Langendorff
perfusion system.78 This discrepancy may be explained through
the differences in the models used for the experiments. In vivo, all
the cellular components are present together within the organism,
while ex vivo, the blood is substituted with a physiological crystalloid solution which lacks the cell component of the blood and
may even harm the heart tissue due to the lower oxygen carrier
capability of crystalloid solutions at 378C compared with the
blood, which contains hemoglobin in red blood cells.79
The NLRP3 inflammasome activation mediates different responses depending in which cell type is activated after
AMI.80 In fibroblasts, the activation of NLRP3 promotes the
release of IL-1b and IL-18.74,78 Endothelial cells are also able
to upregulate NLRP3 as well as caspase-1 activity and secrete
of IL-1b and IL-18 in response AMI.71 Leukocyte infiltration
also sustains the NLRP3 inflammatory response.77 Monocytes and macrophages produce large amounts of IL-1b and
IL-18, and the presence of the inflammasome can be observed
in these cells in the infarct and peri-infarct areas.77 In cardiomyocytes instead, the activation of NLRP3 induces little
amounts of IL-1b, despite a sustained activation of caspase1, which primarily mediates pyroptotic cell death.74,77 Furthermore, the effects of the inflammasome cytokines can
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Mauro et al
J Cardiovasc Pharmacol ä Volume 74, Number 3, September 2019
produce a biological effect on the cardiac resident cells and on
the systemic inflammatory response. IL-1 and IL-18 reduce
myocardial contraction and can promote apoptosis, fibrosis,
and endothelial dysfunction.9,26,34,41,81 The specific deletion
of NLRP3 in all cell compartments here described may help
to better understand the pathophysiological role of the inflammasome in each cell type and the dynamics of inflammasomemediated acute and chronic injury. A more detailed report of
the cell-specific signaling of NLRP3 in AMI can be found
elsewhere.82
OVERVIEW OF THE MECHANISMS OF REGULATION OF NLRP3 IN AMI
As oft-reported, NLRP3 is influenced by a wealth of
different triggers. Extensive research has clarified some
pivotal pathways and events involved in the activation of
NLRP3. Here follows a summary of some key pathways that
are induced by ischemia-reperfusion injury.
Reactive Oxygen Species and Mitochondrial
Dysfunction
Mitochondrial dysfunction sets in at the moment of
reperfusion and is responsible for oxidative stress and
inflammasome activation.14,70 ROS are a potent trigger of
NLRP3 mediating detachment of thioredoxin from TXNIP
or lysosomal damage.9,69,71
Intramyocardial delivery of a small interfering RNA
against TXNIP after ischemia-reperfusion injury was shown
to decrease inflammasome formation in cardiac microvascular
endothelial cells and reduced the infarct size while preserving
cardiac function.71 TXNIP is a regulator of cell metabolism
important for shifting from aerobic to anaerobic metabolism.
TXNIP has also a critical role in the inhibition of the antioxidant thioredoxin protein that actively reduces oxidized protein thiols. TXNIP knock out mouse hearts had an overall
preserved cardiac function and a lower infarct size after
ischemia-reperfusion injury.83
Cardiolipin is a mitochondrion-specific lipid and, when
exposed into the cytoplasm, binds and activates NLRP3.84
Cardiolipin together with an ineffective autophagic clearance
of damaged mitochondria can activate caspase-1 and the subsequent production of IL-1b through NLRP3. An impaired
mitochondrial fission activates NLRP3 by deleting the
dynamin-related protein 1 (Drp1).85 However, in hypoxic
neonatal rat cardiomyocytes, mitochondrial fission promotes
production of ROS and leak of mitochondrial DNA in the
cytoplasm, promoting the activation of NLRP3.86 This set
of data suggests that an efficient mitochondrial fission is
needed to limit the activation of the inflammasome, but during ischemia, this pathway may become inefficient and promote the inflammasome pathway. An impaired clearance of
damaged mitochondria fosters the release of oxidized mitochondrial DNA (ox-mtDNA) into the cytosol. Ox-mtDNA
then forms a complex with NLRP3 mediating its activation.87
in myocardial ischemia to clear the cell from damaged
proteins and organelles (eg, mitochondria) so as to reduce
myocardial damage after AMI. However, a dysfunctional
autophagic flux is associated with inflammasome activation.88–90 An efficient autophagic process prevents the activation of the NLRP3 inflammasome by removing damaged
mitochondria. This, in turn, might limit the secretion of
mature IL-1b in macrophages, although this has not been
confirmed in the heart.88,91,92
Post-translational Regulation of NLRP3
Inflammasome’s Activity
Although ROS and autophagic/lysosomal dysfunction
have been demonstrated to play roles in the pathophysiology
of AMI,93 there are mechanisms that control NLRP3 and
inflammasome activation by post-translational modifications
(Fig. 4).
In a stroke model, the Bruton’s tyrosine kinase has been
shown to interact with both NLRP3 and ASC, suggesting
a potential role in the phosphorylation of ASC.94 Also, the
spleen tyrosine kinase and c-Jun N-terminal kinases can
enhance the ASC oligomerization through post-translational
modification.95 An additional kinase, NEK7, a member of the
never in mitosis gene A–related kinases family, acts downstream of P2X7 and binds to NLRP3 modulating its activation
and oligomerization and may represent a therapeutic target in
the heart.96 However, the role of spleen tyrosine kinase– and
Jun N-terminal kinase–mediated regulation of ASC, as well
as the NEK7 modulation of NLRP3, have not been assessed
in ischemic models.
Recently, in an in vitro model of hypoxia-induced
myocardial injury, the NLR family member X1 (NLRX1) has
been found to inhibit NLRP3 activation through the mitochondrial antiviral signaling protein,97 which is essential for
the recruitment of NLRP3 to the mitochondrial outer membrane (Fig. 4).97,98
Specific nutrients and the products of cell metabolism
can influence the activation of the inflammasome. Although
these mechanisms have not been fully described in the
context of AMI, a detailed description of the metabolic
regulation of NLRP3 inflammasome is reported elsewhere.99
INFLAMMASOME-ASSOCIATED CYTOKINES
The inhibition of the NLRP3 in a preclinical setting
proves to be beneficial in reducing inflammatory injury after
myocardial ischemia-reperfusion. Caspase-1 also plays a pivotal role in the function of the inflammasome. An additional
important aspect of this pathway is the release of the
inflammasome-associated cytokines IL-1b and IL-18 (Fig.
5).50,51 Levels of these cytokines correlate with the progression of atherosclerosis, predict the outcome after AMI, and
correlate with HF severity.100,101 Finally, IL-1 and IL-18
affect the contractility of cardiomyocytes.100,101
Autophagy
Interleukin-1b
IL-1b plays an integral role in the initiation and perpet-
Autophagy is a critical pathway necessary for the
maintenance of the cell homeostasis. As such, it is activated
uation of physiologic and pathologic inflammation. As the
original member of the IL-1 family of cytokines, it has been
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J Cardiovasc Pharmacol ä Volume 74, Number 3, September 2019
FIGURE 4. Molecular signaling regulating the NLRP3 inflammasome during tissue damage. Several stimuli regulate
the activation of NLRP3 and the function of ASC in the formation of the inflammasome. Mitochondria promote NLRP3
activation through the generation of ROS. Indeed, the mitochondrial origin of ROS is predominant during the reperfusion
phase of injury. ROS also cause the dissociation of the thioredoxin interacting protein (TNXIP) from thioredoxin, a step
responsible for the activation of NLRP3. Damaged mitochondria also release cardiolipin, a phospholipid present of the
inner mitochondrial membrane, which can be released within
the cytoplasm thus activating NLRP3. The mitochondrial
antiviral signaling protein is also an important regulator of
NLRP3 during viral infection and tissue damage. The interaction of mitochondrial antiviral signaling protein with the
nucleotide-binding oligomerization domain, leucine-rich
repeat-containing X1 (NLRX1) protein blocks NLRP3 activation. Damaged mitochondria are cleared by autophagic vesicles. Effective autophagy and mitophagy leading to complete
removal of defective proteins and intracellular organelles
prevents the activity of the inflammasome. On the contrary,
defective autophagy and mitophagy cause the leakage of the
proteolytic enzyme cathepsin B inside the cytoplasm, leading
to NLRP3 activation. NEK7, a member of the never in mitosis
gene A–related kinase family, stimulates NLRP3 activation after
potassium efflux. The Bruton’s tyrosine kinase also triggers the
inflammasome by interacting with NLRP3 and ASC. Other 2
kinases, the spleen tyrosine kinase and the c-jun N-terminal
kinase, enhance the oligomerization of ASC.
intensely studied due to the important role playing in the
inflammatory process.33,63,102 IL-1b is typically transcribed
and translated in the cytosol at low basal levels in its inactive
proform, although transcription and translation increase
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Inflammasome in AMI
dramatically within 15 minutes after TLRs or other cytokine
receptors are stimulated.33 IL-1b secretion in its active form,
as noted previously, requires caspase-1. IL-1b lacks a signal
peptide for the classic endoplasmic reticulum–Golgi apparatus secretion pathway.53,59 Therefore, the rate limiting steps in
IL-1b processing and secretion are the inflammasome activation and the formation of the GSDMD-NT pore.33 IL-1b
acts in paracrine, autocrine, and endocrine way once secreted
into the extracellular space, initiating and sustaining proinflammatory activity.33,59
The IL-1 signaling begins through the binding of the
IL-1b and/or IL-1a to the IL-1 receptor type I (IL-1RI). The 2
IL-1 isoforms leads to the heterodimerization between the
IL-1R1 and IL-1 receptor accessory protein (IL-1RAcP).103
The TIR of IL-1RI interacts in the cytoplasmatic side with the
TIR domain present on MyD88, which induces the intracellular signaling through a cascade of activated kinases.32 As
a result of this intracellular cascade, a multitude of chemokines, cytokines, and adhesion molecules are then released
mediating the recruitment and activation of immune cells.67,77
IL-1 signaling orchestrates the immunity response by altering
protein expression, cellular function and metabolism.102
A second type II receptor, the IL-1RII, which is
membrane-bound and lacks the intracellular TIR, acts as
a scavenger receptor preventing the binding of the 2 IL-1
isoforms to their own receptors.32,104 IL-1b and IL-1a share
approximately 26% amino acid homology with the IL-1 receptor antagonist (IL-1Ra), an endogenous protein that strategically occupies IL-1RI. IL-1Ra inhibits the binding between IL1RAcP and IL-1RI, therefore impeding the receptor heterodimerization and the intracellular signal transduction.9 Several
proinflammatory stimuli, including LPS—a component of the
wall of Gram-negative bacteria known to activate TLR4,
induce the IL-1Ra secretion. Therefore, IL-1Ra assumes a strategic role in the balancing of a perpetual cycle of proinflammatory signaling.105,106 Intracellular antiapoptotic functions of
IL-1Ra have also been noted in cardiomyocytes.107
Interleukin-18
IL-18 is another cytokine, member of the IL-1 family,
requiring caspase-1 cleavage to become active.50 The priming
signaling promotes the production of pro-IL-18. However,
IL-18 is readily available and stored in the cytoplasm also
in absence of TLR or any other inflammatory stimuli. IL-18
processing and release depend on inflammasome activation
or, in case of passive necrotic release, on the activity of extracellular proteases.9,101 IL-18 interaction with its receptor resembles the one of IL-1 with IL-1RI. A heterodimeric
complex of the IL-18Ra and IL-18Rb culminates in an intracellular cascade of events leading to proinflammatory gene
transcription.101,108 The sodium-chloride cotransporter (NCC)
is a solute carrier symporter and is mainly present in the distal
tubule of the kidney glomeruli.109 NCC has been identified as
a second putative receptor for IL-18.110
An endogenous circulating IL-18–binding protein (IL18BP) neutralizes IL-18. In healthy humans, IL-18BP is found
in a range of concentrations about 25–50 times higher than IL18.111 IL-18 is produced by fibroblasts and macrophages. A
recent study proved that adult mouse cardiomyocytes produce
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J Cardiovasc Pharmacol ä Volume 74, Number 3, September 2019
Mauro et al
FIGURE 5. Caspase-1 activation has a double
function in myocardial infarction. After the activation of the inflammasome, caspase-1 activates
promotes 2 effects: (1) pyroptotic cell death,
which in the heart promotes the growth of the
infarct size, and (2) release of cytokines, for
example, interleukin-1b (IL-1b) and interleukin-18
(IL-18), responsible for contractile dysfunction
and regulated cell death through apoptosis.
IL-18 in response to pressure overload.112 Similarly, mechanical stretch induces IL-18 cleavage in rabbit cardiomyocytes
and isoproterenol induces the mature form of IL-18 in neonatal
mouse cardiomyocytes.113,114 However, such evidence in
response to myocardial ischemia is missing.
Interleukin-1a, a Cytokine and an Alarmin
IL-1a is produced as a precursor.115 Pro-IL-1a is stored
in the cell and, differently than IL-1b and IL-18, is functionally active in its precursor form.20,63 Therefore, pro-IL-1a can
act as an alarmin beginning proinflammatory paracrine responses soon after the release from the cytosol after cellular
necrosis and before the full activation of IL-1b.20,116,117 ProIL-1a is not cleaved by caspase-1 and thus is not activated
through the inflammasome.116 However, the inflammasome
can participate in its secretion outside of the cell.20 Whether
GSDMD mediates the inflammasome dependent release of
IL-1a is unknown. The initial inflammatory signal after
necrosis and neutrophil recruitment has been proven to
depend on IL-1a, whereas the recruitment of macrophages
is due to IL-1b.32,118
NLRP3 BLOCKADE IN AMI
Several pharmacological approaches to inhibit the
NLRP3 inflammasome have been tested in preclinical setting
in order to reduce ischemia-reperfusion damage after AMI. A
detailed description of the biochemistry and pharmacology of
the inflammasome inhibitors has been reviewed in detail
elsewhere.119,120
Colchicine, a nonspecific inhibitor of NLRP3, was
tested in mice showing a decreased infarct size and ventricular remodeling after AMI.121–125
Similar results were obtained using a derivative of
glyburide, 4-[2-(5-Chloro-2-methoxybenzamido) ethyl] benzenesulfamide (known also as 16,673-34-0), lacking the moiety
responsible for insulin secretion.126–129 Bay 11-7082 (a NF-kB
and NLRP3 inhibitor) reduced infarct size, cardiac fibrosis, and
improved left ventricle fraction of shortening in rats subjected
to ischemia-reperfusion.130,131 MCC950, another potent inflammasome inhibitor, reduced the myocardial damage in
pigs.132 INF4E, a specific inhibitor of the ATPase activity of
NLRP3 was found protective in a model of ischemiareperfusion reducing the infarct size and improving the
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developed pressure.133 OLT1177 (Dapansutrile), the only
NLRP3 inhibitor currently in clinical phase trials, has shown
significant infarct size reduction and preservation of the systolic function in the mouse after reperfusion.134,135
NLRP3 INFLAMMASOME INHIBITION BEYOND
AMI: POSSIBLE USE IN DONATION AFTER CIRCULATORY DEATH ORGAN TRANSPLANTATION
Organ transplantation is associated with a period of
ischemia between the organ procurement and the transplantation. Cold storage is used to lower cell metabolism and reduce
the impact of ischemia. Machine perfusion system (MPS)
preservation is an alternative to cold storage developed to
support the organ with oxygen and nutrients.136 The ischemicrelated injury becomes prominent in the setting of donation
after circulatory death (DCD), an organ donation protocol that
requires the heart to go through spontaneous arrest. The DCD
process implicates a short to medium time of warm ischemia
before organ procurement.136 He et al studied the rat DCD liver
preservation with hypothermic oxygenated perfusion (HOPE)
MPS compared with cold storage. They noticed better graft
performance in the HOPE MPS group and attributed this to
the less activation of NLRP3-mediated inflammasome. Tissue
levels of NLRP3, caspase-1, IL-1b, and IL-18 were significantly lower in the HOPE group.137 In a DCD pig liver transplantation study, hypothermic MPS in combination with
MCC950 resulted in decreased activation of the inflammasome
and improved parameters that are predictive of early and late
allograft function.138 The DCD process also induces caspase-1
in the human heart.139 A recent study in the mouse proved that
NLRP3 increases in the DCD heart reanimated ex vivo
together with increased caspase-1 activity. Deletion of NLRP3
or pharmacologic inhibition of NLRP3 with 16,773-34-0 in
this model reduced the caspase-1 activity, myocardial dysfunction, and myocardial damage.140 The above-reported evidence
suggests the possible role of NLRP3 blocker in future organ
transplantation interventions.
BLOCKADE OF IL-1a AND IL-1b IN ACUTE
MYOCARDIAL INFARCTION
The IL-1R1 mediates post-AMI inflammation, ventricular dysfunction, and greater scar formation.141,142 The IL-1
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J Cardiovasc Pharmacol ä Volume 74, Number 3, September 2019
isoforms (IL-1b or IL-1a) can be blocked using antibodies,
recombinant IL-1Ra (anakinra), or a chimeric protein made
by the ectodomains of IL-1RI and IL-1RAcp (IL-1Trap).143,144
Anakinra prevents the binding of IL-1a and IL-1b to the IL1RI and has received a Food and Drug Administration indication to treat rheumatoid arthritis and cryopyrin-associated periodic syndromes (CAPS), distinguished by enhanced IL-1
activity.145
Anakinra prevents the adverse left ventricular remodeling and dysfunction in mice subjected to AMI.142 The release
of IL-1b on ischemia-reperfusion injury affects the recruitment of the immune cells from the bone marrow to the
infarcted area.146 An anti–IL-1b treatment lowered monocyte
and neutrophil infiltration in the myocardium after ischemiareperfusion injury, preventing adverse ventricular remodeling
but not reducing the infarct size.146 A mouse equivalent of
canakinumab, an IL-1b blocking antibody, did not reduce the
infarct size, but improved long-term survival after AMI, inhibited myocardial apoptosis, and prevented ventricular
enlargement when given at reperfusion and repeated 1 week
later.146–148 The administration of Gevokizumab, another
monoclonal antibody against IL-1b in diabetic rats, limited
the oxidative stress and the progression of HF after AMI. This
translated in improved ventricular remodeling, lower scar
size, and coronary endothelium-dependent relaxation, independent of infarct size.149,150 Conversely, the administration
of a polyclonal antibody against IL-1a after ischemiareperfusion injury reduced inflammasome activity and
decreased the infarct size, with a beneficial effect on the overall left ventricular function.151 This highlights how IL-1a and
b isoforms play a different role during injury after AMI.
Pharmacological IL-1 blockade has been tested successfully in pilot clinical trials in patients with reperfused ST
elevation myocardial infarction and primary percutaneous
coronary intervention, where it significantly limited the
increase of C-reactive protein, lowering the rate of newonset HF.152–155
Phase II clinical trials in patients with HF also support
a beneficial role for IL-1 blockade with anakinra.156–158 However, no clinical trial of selective IL-1b or IL-1a blockade to
treat AMI has yet to be completed. Canakinumab, a human
monoclonal antibody that inhibits IL-1b, was tested in a randomized, double-blinded study involving 10,061 patients that
had a previous AMI .30 days before enrollment with a serum
C-reactive protein $2 mg/L. Canakinumab, by the end of the
study, decreased the incidence of nonfatal AMI, nonfatal
stroke, or cardiovascular death.159
BLOCKADE OF IL-18 IN ACUTE
MYOCARDIAL INFARCTION
IL-18 has been less studied in myocardial ischemia and
infarction. The expression and plasma levels of IL-18 increase
after reperfusion in animals with AMI, with myocardial levels
peaking at 3 hours and serum levels at 6 hours after
reperfusion.160 Infarct size was significantly decreased pretreating mice with an IL-18 neutralizing antibody 1 hour
before ischemia-reperfusion.160 Gu et al161 investigated the
cardioprotective effects of recombinant IL-18BP using
Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.
Inflammasome in AMI
a syngeneic heterotopic heart transplantation. IL-18BP
increased graft survival, reduced myocardial damage, leukocyte infiltration, and lowered the expression of proinflammatory cytokines after the ischemia-reperfusion injury in this
model.161
EVALUATION OF NLRP3 INFLAMMASOME
ACTIVATION IN EXPERIMENTAL
ANIMAL MODELS
The expression of the NLRP3 inflammasome mRNA
levels and protein upregulation can be easily assessed in
animal tissue by quantitative polymerase chain reaction and
Western blot.65 However, the increased or reduced expression
of the inflammasome components cannot be taken as proof of
NLRP3 inflammasome activation. As described above, the
inflammasome activation can go through 2 phases, the priming (in tissue or cells that express low levels of the inflammasome components) and the NLRP3 activation, or trigger.
Measuring the priming alone is insufficient to determine
whether the inflammasome is active. Therefore, the measurement of inflammasome activity needs to be incorporated into
the investigational process. The activity of the inflammasome
can be measured by assessing the appearance of the inflammasome products. Cleaved caspase-1, IL-1b, IL-18, and
GSDMD can be measured by Western blot or enzymelinked immunosorbent assay for active IL-1b and IL-18.162
However, enzyme-linked immunosorbent assay may not discriminate between active and nonactive form of the proteins,
which is of utmost importance for IL-1b to be distinguished
from its inactive pro-IL-1b. The release of the proforms of the
inflammasome cytokines that follows cell death may therefore
affect data interpretation.
The enzymatic activity of caspase-1 can be measured
using enzymatic assays performed on tissue or cell extracts.
The formation of the characteristic “specks” after ASC polymerization can be measured using microscopy.77,151 ASC
polymerization forms dense specks in the cytosol and can
be measured by immunostaining or by flow cytometry.77,163
Size-exclusion chromatography can also be used to detect
NLRP3 inflammasome polymerization. This is a chromatographic method in which molecules are separated by their size
or molecular weight and is especially suitable for large molecules or macromolecular complexes, such as the NLRP3
inflammasomes.164,165
Recently, Ulke-Lemée et al166 described and validated
a multiple reaction monitoring mass spectrometry assay to
accurately quantify ASC in human biospecimens, for which
authors claimed about the critical importance of both sample
collection and storage conditions on the assay reliability.
Lastly, when measuring the effects of a specific therapy
or protein on the inflammasome pathway, it is important to
define whether the inflammasome is directly affected or not.
In fact, an inflammasome-independent reduction/increase of
the injury can mitigate/amplify the activation of the inflammasome pathway. To overcome this potential confounding
factor, it is important to couple the in vivo study with in vitro
assays, where the therapy or protein of interest is studied in
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J Cardiovasc Pharmacol ä Volume 74, Number 3, September 2019
Mauro et al
the setting of specific inflammasome activation, like cell
stimulation with LPS and nigericin or LPS and ATP.77
CONCLUSION
The injury consequent to AMI activates the innate
immune response. The injured tissue promotes the activation
of the NLRP3 inflammasome through the release of DAMPS
to promote healing, but at the same time exacerbates the
reperfusion injury. Several preclinical studies proved the
importance of inhibiting the inflammasome and its cytokines.
This has been proven to be beneficial, attenuating the
myocardial injury and preventing the deterioration of the
ventricular function and consequent HF onset. However,
there are data that describe a possible protective effect of
NLRP3, which seems independent of the inflammasome
activation. Today, the only therapy aimed at lowering the
impact of this pathway in AMI and HF in the setting of
clinical testing relies on IL-1 blockade. Two different
inhibitors, anakinra and canakinumab, have been shown to
reduce the cardiovascular events. Early testing of an NLRP3
inhibitor in human patients is now ongoing.167,168 If proven
safe for patients and effective, and with the support of the
accumulating preclinical data, NLRP3 inhibition should be
tested in the clinical setting to reduce AMI injury and prevent
the development of HF.
ACKNOWLEDGMENTS
The authors thank Julia Bashore, BS, and William del
Castillo Reyes, BS, MBA, for the careful revision of the
article.
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