Citation: Cell Death and Disease (2014) 5, e1325; doi:10.1038/cddis.2014.

287
OPEN & 2014 Macmillan Publishers Limited All rights reserved 2041-4889/14
[Link]/cddis

Review

Small molecules, big effects: the role of microRNAs

in regulation of cardiomyocyte death
J Skommer1,3, I Rana1,3, FZ Marques1, W Zhu2, Z Du2 and FJ Charchar*,1

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranscriptional regulation of gene expression, and
exerting regulatory roles in plethora of biological processes. In recent years, miRNAs have received increased attention for their
crucial role in health and disease, including in cardiovascular disease. This review summarizes the role of miRNAs in regulation
of cardiac cell death/cell survival pathways, including apoptosis, autophagy and necrosis. It is envisaged that these miRNAs may
explain the mechanisms behind the pathogenesis of many cardiac diseases, and, most importantly, may provide new avenues
for therapeutic intervention that will limit cardiomyocyte cell death before it irreversibly affects cardiac function. Through an in-
depth literature analysis coupled with integrative bioinformatics (pathway and synergy analysis), we dissect here the landscape
of complex relationships between the apoptosis-regulating miRNAs in the context of cardiomyocyte cell death (including
regulation of autophagy–apoptosis cross talk), and examine the gaps in our current understanding that will guide future
investigations.
Cell Death and Disease (2014) 5, e1325; doi:10.1038/cddis.2014.287; published online 17 July 2014

Facts conditions such as myocardial infarction (MI),1 and occurs

through a variety of cell death pathways, induction of which
 There are altered cardiac microRNAs levels in various depends not only on the type of stress stimuli, but also on the
cardiovascular diseases states intracellular biochemical and genetic makeup.2 Most impor-
 MicroRNAs can regulate cardiac cell proliferation, hyper- tantly, the converging regulatory network of cell death
trophy, death and cardiac fibrosis pathways. pathways is potentially amenable for control and therapeutic
 MicroRNAs have long biological half-lives, hence can be a intervention.3
good diagnostic marker of disease state Over the last several years, efforts have been directed
toward elucidating the exact regulatory mechanisms that
govern cell death processes, including protein oligomerisa-
tion, protein conformational changes, transient protein–
Open Questions protein interactions and protein relocations (for example,
Vela et al.4–8), as well as non-genetic cell-to-cell variability.9–12
 What are the mechanisms leading to increase or decrease Recently, microRNAs (miRNAs) have emerged as an
in cardiac microRNAs under pathological and physiological important, perhaps critical, regulatory factor when it comes
state? to cell death signaling. This is not surprising considering that
 What are the mechanisms by which microRNAs contribute miRNAs, a class of small non-coding RNAs, posttranscrip-
to cardiac pathological and physiological state? tionally regulate messenger RNAs by inhibiting their transla-
 What are the cardiac microRNA changes associated with tion or promoting their degradation, and thus represent a new
aging? paradigm that regulates the expression of up to 50% of human
 How do microRNAs regulate the balance between pro- genes.13,14 According to the latest version of miRNA database
survival and death pathways in the heart? miRBase 20.0, there are over 2500 known mature miRNAs in
the human genome.15
Increasing evidence indicates that miRNAs are linked with
Cell death processes are highly regulated in order to ensure many pathological conditions, and play important roles in
proper tissue functioning and homeostasis. Loss of cardio- cardiovascular homeostasis and in initiation and progression
myocytes is an important factor in the pathogenesis of of disease states, such as hypertrophy and heart failure.16–18

1
Robert HT Smith Chair in Cardiovascular Science, School of Health Sciences, Federation University Australia, Ballarat, Victoria 3350, Australia and 2 Institute of Clinical
Pharmacology, The Second Affiliated Hospital of Harbin Medical University, Harbin, China
*Corresponding author: F Charchar, Robert HT Smith Chair in Cardiovascular Science, School of Health Sciences, Federation University Australia, PO Box 663, Ballarat,
Victoria 3353, [Link]: +61 3 5327 6098; Fax: +61 3 5327 9242; E-mail: [Link]/charchar-lab
3
These two authors contributed equally to this work.
Abbreviations: STEMI, ST-elevation myocardial infarction; MI, myocardial infarction; DAXX, death domain-associated protein; HIF1A, hypoxia inducible factor 1, alpha
subunit; MFF, mitochondrial fission factor; HSP, heat shock protein; PC, progenitor cells; PKC, protein kinase C; PDCD4, programmed cell death 4; SIRT1, sirtuin 1;
SNP, single-nucleotide polymorphism; FasL, Fas ligand
Received 14.3.14; revised 28.5.14; accepted 03.6.14; Edited by G Melino
 MicroRNAs in cardiomyocyte death
J Skommer et al
2

Considering the role of cell death processes in cardiovascular Types of Cell Death
disease (CVD) as well as the emerging role of miRNAs in
Cell death is a group of fundamental processes in both normal
regulating cardiac physiological and pathological states, we
physiology and pathological states. The mammalian cell
will focus our review on the role of miRNAs in regulating
death network comprises of many distinct functional mod-
myocardial cell death processes.
ules,32–34 including apoptosis (also referred to as pro-
grammed cell death), macroautophagy (commonly referred
The World of miRNAs to as autophagy, and more appropriately as cell death
dependent on autophagy genes), necrosis and an inflamma-
Small non-coding RNA molecules of B22–23 nucleotides
tory form of cell death referred to as pyroptosis.35 Of these,
in length, known as miRNAs, regulate the expression of
apoptosis has been studied most extensively and its
protein-coding genes through sequence-specific recognition
molecular framework is relatively well defined.36 Often more
in the 30 or the 50 untranslated regions (UTRs) of mRNAs, thus
than one process of cell death is induced, and the particular
regulating mRNA levels by posttranscriptional mechan-
extracellular milieu, as well as genetic and biochemical
isms.14,19 Partial sequence complementarity between a
context of the intracellular environment, will determine which
miRNA and its target site in a specific mRNA is often sufficient
pathway is ultimately responsible for cell demise. Because of
for binding.20 In animals, the binding to the 30 UTR lowers
this, blocking one pathway of cell death may not prevent the
mRNA levels by preferentially decreasing the stability of the
final outcome of cell loss, as other simultaneously induced
mRNA, thus increasing its degradation, or by repressing
pathways may take over the executioner’s role. At the same
translation.14 miRNAs were also described to bind to the
time, genetic and pharmacological inhibitor studies can
promoter region in the 50 UTR of mRNAs.21,22 The binding of
identify the culprit pathway among all the signaling induced
miRNAs to 50 UTRs can either repress or, most likely, activate
by a particular stress cue. Thus, detailed knowledge of
translation (reviewed by Da Sacco and Masotti19 and Vasude-
multiple pathways, their regulators and cross talks between
van23). Since no examples of this type of miRNA–mRNA
them, is of paramount importance when it comes to interfering
interactions are known in CVD or cell death, this review will
with cell death process for therapeutic purposes.
explore mechanisms involving miRNAs that bind to the 30 UTR
of mRNAs only.
miRNA biogenesis has been extensively reviewed else-
Apoptotic Pathways in Cardiovascular Disease
where.24–26 Briefly, transcription of miRNA genes is usually
performed by RNA polymerase (RNA Pol) II, or occasionally by The process of apoptosis is driven by a network of proteins
RNA Pol III, resulting in a precursor miRNA, termed primary connected to each other in an intricate manner, forming two
miRNA or pri-miRNA.26 In the nucleus pri-miRNAs (B1000 bp) main signaling pathways activated by a wide variety of stress
fold into hairpin structures, which act as a substrate for DGCR8 signals that converge on mitochondria (the intrinsic pathway)
subunit of Drosha, a nuclear enzyme of the RNase III family, or upon ligation of cell surface death receptors (the extrinsic
resulting in the cleavage of the pri-miRNA into a pre-miRNA pathway) (Figure 1). Caspase-9 activation by mitochondrial
(B60—70-bp long). The pre-miRNA is then exported to the pathway and caspase-8 activation by extrinsic pathway lead
cytoplasm, where Dicer, another enzyme of the RNAse III to activation of the executioner caspases, such as caspase-3
family, processes the pre-miRNA to a B20-bp double-stranded or -7 which then directly degrade the proteome and commit
miRNA duplexes.24–26 The miRNA duplexes associate with cells to death (Figure 1).
specific members of the Argonaute (Ago) family of proteins, Cytochrome c released from damaged mitochondria act as
and together form the core of the RNA-silencing complex a trigger for the activation of the mitochondrial pathway. The
(RISC), which mediates the posttranscriptional regulation of mitochondrial pathway of apoptosis is heavily regulated by
gene expression by utilizing the guide strand (miR) of the multiple proteins acting upstream and downstream of the
miRNA duplex.24–26 There is now evidence that the second mitochondria, determining the efficiency of apoptotic cell
miRNA strand, often called passenger or minor miRNA (miR*), death (Figure 1). Control over the integrity of mitochondrial
before believed to be degraded, can also associate with Ago outer membrane is executed by the proteins of the B-cell
proteins and impact gene expression.27 lymphoma 2 (Bcl-2) family, divided into three subfamilies: anti-
Several algorithms are used for the discovery of miRNA– apoptotic (for example, Bcl-2, Bcl-Xl, Mcl-1, Bcl-w), pro-
mRNA interactions (reviewed by Witkos et al.28), with apoptotic effectors (Bax and Bak) and BH3-only proteins (for
computational predictions suggesting that a single miRNA example, Bid, Puma, Bim, Bad and so on), which form a
can potentially regulate multiple mRNAs.20,28 In vitro experi- complex network of interactions (discussed in more detail in
mentation, however, is always necessary to validate the several reviews, including.6,37–39 Downstream of mitochon-
predicted targets as well as the role of miRNAs in regulation of dria, activation of caspases is also heavily regulated, first by
cell signaling pathways and cellular phenotypes. Moreover, the low-probability event of apoptosome formation and
the general role of miRNAs in specific cellular or physiological second by inhibitor of apoptosis (IAP) proteins such as XIAP
processes can be examined by deleting or inhibiting miRNA- (Figure 1).
processing machinery. For instance, the cardiac-specific In the extrinsic pathway of apoptosis, the initial stress signal
knockout of Dicer leads to rapidly progressive dilated comes from the binding of a death domain-containing
cardiomyopathy, heart failure and postnatal lethality, empha- receptor, located at the plasma membrane, to its cognate
sizing the importance of miRNAs for heart development and ligand (for example, Fas ligand (FasL) or tumor necrosis
homeostasis.29–31 factor-a (TNF-a)).40 Upon binding, the receptor promotes the

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 MicroRNAs in cardiomyocyte death
J Skommer et al
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EXTRINSIC PATHWAY

miR-21
TRAF2
INTRINSIC PATHWAY TRADD
miR-149,214, 21,24 RIP1
FADD RIP3
tBid Bid

miR-1, 15,21 Caspase 8

EndoG
AIF
MOMP
APOPTOSIS
miR-378
NECROPTOSIS
IAPs

miR-133a

Figure 1 A simplified schematic representation of the extrinsic and the intrinsic (mitochondrial) pathways of apoptosis and necroptosis. The mitochondrial (intrinsic)
pathway of cell death is regulated both upstream (Bcl-2 proteins) and downstream (e.g., IAP proteins) of mitochondria. In response to stress, mitochondria undergo
permeabilisation of the outer mitochondrial membrane (MOMP), which leads to a release of a number of pro-apoptotic factors such as cytochrome c, AIF or EndoG.
The extrinsic pathway of apoptosis is activated via a complex signal transduction from the plasma membrane, whereby the death receptors (e.g., Fas, TNFR) bind their
cognate ligands (e.g., FasL, TNF), oligomerize, activate their intracellular death domains and recruit a number of receptor-associated proteins such as RIP1 or TRADD.
The multiprotein complex then recruits the initiatior pro-caspase-8 leading to its activation. Active caspase-8 propagates the apoptotic signal either by direct activation
of executioner caspases, or by cleaving a BH3 protein Bid which then leads to MOMP. In addition, components of the protein machinery that regulates the extrinsic pathway of
apoptosis are also involved in regulation of necrosis. The figure also demonstrates apoptosis regulatory microRNA and their target genes in apoptotic pathways

formation of the death-inducing complex (DISC) that activates myocardial ischemia-reperfusion injury,53 whereas TNF-a, a
caspase-8 (Figure 1). In some cell types, caspase-8 cleaves trigger of the death receptor pathway, is rapidly released from
the BH3 protein Bid, forming truncated Bid (tBid) and resident mast cells and macrophages in response to
ultimately inducing the mitochondrial outer membrane per- myocardial ischemia,54 and can also be released from
meabilisation. Thus, in some contexts, there is a cross talk cardiomyocytes.55 Overexpression of FasL was also shown
between the extrinsic and the intrinsic pathway of apoptosis. to increase cardiomyocyte apoptosis in vitro and in vivo.56 The
Cardiomyocytes undergo apoptosis in response to a fact that multiple cell death pathways are detectable and occur
plethora of stimuli including hypoxia (especially followed by side-by-side is not surprising, and is a common phenomenon
reoxygenation), acidosis, oxidative stress, glucose depriva- observed in contexts ranging from retrovirus-induced immu-
tion and metabolic restriction, b1-adrenergic agonists, stretch, nosuppression57 to chemotherapy-induced cancer cell
angiotensin II (Ang II), TNF-a, Fas L and anthracyclines as death.58–60 Therefore, it is well recognized that activation of
reviewed in.41 Even though it is often postulated that necrosis a particular cell death pathway does not constitute sufficient
is the main pathway responsible for cardiomyocyte death evidence to support its physiological importance in the
following MI, several observations in human, rabbit, rat and pathological process, and instead needs to be supported by
mice hearts indicate that apoptotic cell death contributes to prevention of cell loss using pathway-specific pharmacologi-
the overall cardiomyocyte loss following MI, particularly during cal and/or genetic inhibition. In this regard, conflicting results
subacute and chronic stages of infarction and after reperfu- have been published. For example, it was observed that Fas-
sion,42–47 as well as in end-stage cardiomyopathy and in deficient Ipr mice exhibit reduced infarcts and diminished
terminally failing human myocardium.48,49 Cardiomyocyte apoptosis after myocardial ischemia-reperfusion (I/R) injury,
apoptosis has also been shown to contribute to left ventricle both ex vivo and in vivo,56,61 whereas in transgenic mouse
dysfunction following cardiac surgery,50 particularly after lines with cardiomyocyte-specific overexpression of Bcl-2 or
retrograde cardioplegia.51 Moreover, inhibition of post- dominant-negative Fas-associated death domain (FADD)53
ischemic cardiomyocyte apoptosis emerges as an effective only inhibition of the mitochondrial pathway provided a
therapeutic avenue to improve diastolic heart function after significant decrease in the size of infarction following coronary
ischemia.52 artery ligation. Of note, in the latter study, there was still a
Studies indicate the relevance of both the mitochondrial and substantial decrease in the number of apoptotic (TUNEL
the extrinsic pathway of apoptosis in cardiac pathologies. The positive) cardiomyocytes in FADD dominant-negative mouse
mitochondrial pathway of apoptosis is activated following line, suggesting that forms of cell death other than apoptosis

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 MicroRNAs in cardiomyocyte death
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were contributing to the infarct size. In addition, in lpr mice Consistent with tissue-specific functions, many miRNAs
Fas-deficiency is not limited to cardiomyocytes. Therefore, have distinct biological effects depending on the cell type. For
more studies in models where apoptosis contributes sig- example, inhibition of miR-24 has been reported to promote
nificantly to the infarct, and where it is blocked specifically in cardiomyocyte apoptosis while decreasing the survival of
cardiomyocytes, are needed to provide more evidence as to endothelial cells.68 Considering this, more work is needed to
the role of specific apoptotic pathways. investigate the apoptosis-regulating effects of miR-17-92
cluster, which appears to be expressed at high level in the
heart and has been extensively reported to exert anti-
MiRNAs that Regulate Cardiovascular Sensitivity to
apoptotic effects in tumor cells.69 Only few studies, however,
Apoptosis
have been reported so far with regards to the cardiac
The role of miRNAs in regulating the expression of anti- and apoptosis regulation by this miRNA cluster (see Table 1). Of
pro-apoptotic genes and regulation of cardiomyocyte and note, the variability in the role of miRNAs is observed not only
endothelial cell survival is well documented (Table 1). miRNAs between the cell types, but also between cellular processes,
appear to regulate cardiomyocyte apoptosis through multiple particularly as they relate to the effect on tissue functioning.
mechanisms. When it comes to the mitochondrial pathway of For example, in cardiac myocytes, the signaling pathways that
apoptosis, several miRNAs target the BCL2 gene, including regulate hypertrophy impinge also on the balance between
miR-1, miR-15 family and miR-21, whereas PUMA and BIM, cell survival and cell death,70 and many miRNAs may be
which encode for the pro-apoptotic BH3 proteins, are targeted involved in regulation of this delicate balance.
by miR-149 and miR-214 (as well as miR-24), respectively The striking abundance of miRNAs involved in the regula-
(Table 1). In addition, mitochondrial fission machinery is tion of apoptosis in cardiomyocytes suggests the possibility of
regulated by miR-30 family, which targets the TP53 gene an extensive overlap of regulatory functions and effects on
encoding for p53 protein which in turn regulates the expres- intracellular signaling. Such synergy between miRNAs has
sion of mitochondrial fission protein dynamin-related protein-1 been recently investigated using receiver operating charac-
(Drp1), and by miR-761, which targets a gene encoding for teristic (ROC) analysis, which indicated that miR-21 scores
mitochondrial fission factor (Mff) (Table 1).62 Of note, a high for its synergistic effects. Of particular interest, the
particular recent study suggests that Mff is responsible for synergistic effect of miR-21 and miR-1 was detected, and
recruiting Drp1 to the foci at the outer mitochondrial functionally validated in the context of regulation of myocardial
membrane.63 Downstream of mitochondria, the expression apoptosis, cardiac hypertrophy and fibrosis.71 Here, we have
of CASP9, which encodes for the initiator caspase-9, is used the integrated parameter synergy score calculations
regulated by miR-133a, whereas CASP3, which encodes for (Figure 2), based upon the experimentally validated and
the executioner caspase-3, is regulated by miR-378 (Table 1). confidently predicted miRNA-target interaction data obtained
In addition, miR-21 regulates genes involved in the death from miRSel72 and TargetscanHuman 6.2, to calculate the
receptor pathway of apoptosis, including death domain- synergy scores for the miRNAs linked to cardiomyocyte
associated protein gene (DAXX) and FASLG, which encode apoptosis (Figure 3). In total, we identified 35 synergistic
for death receptor-associated protein and Fas L, respectively (synergy score of more than 2.0) pair-wise combinations of
(Table 1). miRNAs. This finding confirms that the synergistic action of
Genes encoding proteins that control ROS signaling are these miRNAs on cardiomyocyte apoptosis should not be
also enriched among the targets of apoptosis-regulating ignored. As observed before,71 miR-21 was most commonly
cardiac miRNAs. In particular, three miRNAs, that is, involved in synergistic interactions (n ¼ 10) (Figure 3a), with
miR-34a, miR-195 and miR-199a, target SIRT1 (Table 1), which particularly strong synergy between miR-21 and miR-1
encodes for a founding member of a family of NAD-dependent (Figure 3b), followed by two miR-15 family members miR-
deacetylases called sirtuins. When expressed at low levels, 15a and miR-16 (each of them synergized with nine other
SIRT1 has been reported to protect cardiomyocytes from miRNAs). In comparison, several miRNAs were not involved
oxidative stress and apoptosis, and stimulation of Sirt1 protein in synergistic interaction, as indicated by the synergy score
mimics ischemic preconditioning and protects heart from I/R o2.0. These miRNAs include miR-133a, miR-149, miR-19b,
injury (reviewed in greater detail by Sundaresan et al.64 and miR-210, miR-214, miR-30, miR-320, miR-378, miR-499 and
Yamamoto and Sadoshima65). Other ROS signaling genes miR-761 (example given in Figure 3c). Overall, this analysis
targeted by cardiac miRNAs include TXN (encoding for indicates that there is a selective synergism among endo-
thioredoxin) and SOD (encoding for superoxide dismutase). genous miRNAs in regulating complex biological processes
Interestingly, a study on mice with cardiac-specific over- such as apoptosis.
expression of Sirt1 revealed that it upregulates the expression In addition, we used the Ingenuity Pathway Analysis (IPA)
of both superoxide dismutase and thioredoxin 1.66 Consider- to determine network of connections between the identified
ing that multiple miRNAs associated with apoptotic regulation apoptosis-regulating miRNAs of reported relevance to cardi-
in cardiomyocytes impinge on the network of SIRT1-ROS ovascular disease (Figure 4). We observed that many
signaling, while some other miRNAs affect ROS-induced miRNAs are regulated through each other either directly or
signaling pathways, such as changes in free calcium (for indirectly, and many are indirectly regulated by the same
example, miR-14567), more research is required to delineate signaling molecules, for example, beta estradiol (Figure 4). In
the exact mechanism and causal role of miRNAs in maintain- this context, it is interesting to note that to the best of our
ing cardiac redox homeostasis as well as the cell death knowledge, no experimental studies have been published so
regulatory role of redox-sensitive miRNAs. far on estradiol-mediated regulation of cardiac miRNAs,

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 MicroRNAs in cardiomyocyte death
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Table 1 Summary of miRNAs linked to apoptotic regulation in cardiomyocytes

miRNAs Relevance to CVD Targets relevant to Refs

apoptosis

miR-1 Serum expression upregulated in human AMI and in patients after open-heart surgery with PRKCE 147–159
cardiopulmonary bypass (protein kinase C)
Overexpression enhances and inhibition attenuates apoptosis and infarct area after cardiac I/R HSPD1 (HSP60)
injury in mice
Overexpression inhibits apoptosis in a rat model of cardiac hypertrophy induced by pressure BCL2
overload
Ischemic post-conditioning upregulates miR-1 and inhibits cardiomyocyte apoptosis in rats
Inhibition in vitro in cardiomyocytes reduces H2O2-induced and high-glucose-induced
apoptosis
Upregulation has pro-apoptotic effect in H9c2 cells exposed to oxidative stress
miR-1 transfected ES cells protect host myocardium from MI-induced apoptosis
overexpression enhances the angiogenic differentiation of human cardiomyocyte PC
Upregulated in rat cardiomyocytes exposed to high glucose
Downregulated in response to Tanshinone IIA
miR-15 Upregulated in response to MI BCL2 160–163
family Silencing in vitro renders cardiomyocytes resistant to hypoxia-induced cell death
Regulates angiogenic activity of endothelial cells
miR-133a Upregulation of miR-133a following ischemic post-conditioning CASP9 155,156,164,165
miR-133a mimic attenuated IR-induced apoptosis in rats
Anti-apoptotic effect in H9c2 cells exposed to oxidative stress
Elevated levels of miR-133a in patients with ST-elevation myocardial infarction (STEMI) linked
to more severe injury
Increased expression in Tanshinone IIA-treated hypoxic neonatal cardiomyocytes
miR-17-92 Overexpression of the cluster results in lethal cardiomyopathy 166–169
cluster Expression decreases in aging mice hearts
miR-20a is upregulated in mechanically stretched neonatal rat cardiomyocytes and exerts anti-
apoptotic effect
Overexpression of miR-19b inhibits apoptosis in P19 cells
miR-21 Myocardial upregulation of miR-21 reduces MI size and apoptotic rate by increasing Bcl-2 levels BCL2 FASLG (FASL) 170–176
Expression declines in cardiac myocytes upon exposure to hypoxia, and increases after PDCD4
ischemic preconditioning
Overexpression diminishes murine coxsackievirus B3-induced myocardiatis ANXA2
Overexpression in transgenic mouse heart results in smaller infarct following ischemia SOD2 TXNDAXX
Expression elevated in circulating endothelial progenitor cells from diabetic patients and
protective from high-glucose-induced apoptosis
Expressed in cardiac valve endothelium, where it regulates the development of AV valve
miR-24 Expression lower in peri-infarct tissue in mouse model of MI BIM 68,177
Inhibition induces cardiomyocyte apoptosis
In vivo overexpression inhibits cardiomyocyte apoptosis and attenuates infarct size
Inhibition enhances EC survival
miR-30 Inhibits mitochondrial fission and apoptosis in cardiomyocytes TP53 178
miR-150 Upregulated in cardiac myocytes treated with H2O2 MYB (c-myb) 147,179
Silencing protects from H2O2-induced apoptosis
Dysregulated in human MI
miR-210 Upregulated in hypoxic cardiomyocytes ? 147,180
Overexpression reduces cell death in response to oxidative stress
Deregulated in human MI
miR-199a Downregulated to undetectable levels during cardiac ischemia in vitro and in vivo HIF1A SIRT1 181
Overexpression inhibits hypoxia-induced expression of several pro-apoptotic genes (e.g.
CASP3 and CASP9)
miR-320 Downregulated in murine hearts following I/R HSPB6 (Hsp20) 182
Overexpression enhanced cardiomyocyte apoptosis
miR-149 Overexpression decreases apoptotic sensitivity BBC3 (PUMA) 183
G-allele of A4G SNP in pre-miR-149 decreases production of miR-149 and influences cardiac
function in mouse model of MI
miR-761 Inhibits mitochondrial fission MFF 62
Knockdown diminished H2O2-induced and I/R-induced cardiomyocyte apoptosis and infarct
size in mice
miR-499 Inhibits cardiomyocyte apoptosis PPP3CA, PPP3CB 184
miR-214 Protects cardiomyocytes from H2O2-induced apoptosis in vitro PTEN BIM 185,186
Genetic deletion in mice increases cardiac apoptosis
miR-145 Circulating levels reduced in patients with coronary artery disease CAMK2G 67,187
Ameliorates ROS-induced apoptosis in cardiomyocytes
miR-378 Downregulated in a rat model of myocardial ischemia CASP3 188
Overexpression in H9c2 cardiomyocytes reduces apoptosis and necrosis
Inhibition increases H2O2-induced apoptosis
miR-195 Inhibition leads to decreased ROS production and apoptosis in palmitate-treated mouse SIRT1 189
cardiomyocytes
miR-34a Expression increases with aging (in mice) SIRT1 PNUTS 125,190,191
Inhibition reduces cardiomyocyte apoptosis
Levels higher in endothelial progenitor cells from coronary artery disease patients
Regulates SIRT1 expression in endothelial progenitor cells and contributes to endothelial
senescence

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 MicroRNAs in cardiomyocyte death
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Considering that autophagy can potentially exert both pro-

and anti-survival function, it is not surprising that its role in
cardiovascular disease remains relatively poorly understood,
with some studies pointing toward its beneficial effects,94–99
while others suggest its detrimental role.100 Nevertheless, the
preponderance of evidence supports the notion of beneficial
role for autophagy in the heart.

Synergistic Synergistic
MiRNAs and Cardiovascular Autophagy: The Causality
Dilemma
The role of miRNAs in regulation of autophagy was first
suggested in 2009, when BECN1, a gene encoding for Beclin
1 which is an important factor controlling vesicle nucleation
(see Figure 5), was shown to be posttranscriptionally
regulated by miR-30a.101 Since then, multiple miRNAs have
been linked to the autophagic pathway and have been
associated with disease states, including cardiac pathologies
(Table 2). In addition, there are several miRNAs that have
been reported to modulate autophagy in other tissues, and
Non-synergistic Non-synergistic have also known roles in cardiac function. For example, in
Figure 2 A graphic representation for quantitative assessment of miRNA hematopoietic cells, miR-17 has been shown to regulate the
synergy. (a) Targeting more common genes creates synergy between two miRNAs. expression of SQSTM1 (p62), an ubiquitin-binding protein and
(b) Denser functional association between proteins encoded by individual miRNA- regulator of autophagy-mediated protein degradation,102 and
target genes makes two miRNAs more likely to act synergistically the expression of ATG7 in glioblastoma cells.103 It also
appears to modulate cardiac remodeling following MI.104
despite a well-recognized cardioprotective (via an anti-apoptotic Whether miR-17 regulates cardiac autophagy remains to be
mechanism) function of estrogen.73–86 investigated.
We conducted pathway analysis of miRNAs linked to cardiac
autophagy, using the Ingenuity Pathway Analysis (IPA)
Autophagy, a Double-Edged Sword in Cardiovascular
software (Ingenuity Systems, Qiagen, Redwood City, CA,
Disease
USA), and discovered two main nodes of interaction, that is
Autophagy is an evolutionarily conserved mechanism of p53 and STAT3 (signal transducer and activator of transcription
cellular self-digestion in which long-lived proteins and entire 3) (Figure 6). Interestingly, both p53 and STAT3 are known as
organelles are degraded through delivery to lysosomes critical regulators of autophagy. Specifically, STAT3 inhibitors or
(Figure 5). It is characterized by the formation of autophago- genetic ablation stimulate autophagic flux,105,106 and reduced
somes, cellular structures that encapsulate cytoplasmic levels of STAT3 are reported in patients with end-stage heart
cargoes. Autophagosomes ultimately fuse with lysosomes to failure.107,108 Moreover, the activation of STAT3 by angiotensin
form autolysosomes in which the contents are degraded into (Ang)II-induced Janus-activated kinase 2 (JAK-2) is impaired in
their constituent parts and delivered back into the cytosol for failing human cardiomyocytes.109 With regards to p53, its
further biocatalysis or to be utilized in biosynthetic pathways. inhibition is cardioprotective against ischemic injury, and an
It is well documented that basal levels of autophagy, increase in autophagic flux has been observed in p53 (  /  )
operating in the majority, if not all, cells, serve to maintain heart under ischemic conditions.110 This effect is suggested to
homeostasis by removing misfolded or aggregated proteins, occur through the p53-TIGAR axis,110 with TIGAR (TP53-
and by clearance of damaged organelles such as mitochon- induced glycolysis and apoptosis regulator) being involved in
dria and endoplasmic reticulum.87,88 Both the rate of regulation of the glycolysis and pentose phosphate pathway,
autophagy and the selection of cargoes destined for degrada- ROS-levels, and inhibition of apoptosis and autophagy.111,112
tion can, however, change in response to specific external and The p53-TIGAR axis has also been specifically shown to
internal cues. In this context, excessive autophagy, induced in regulate cellular energy homeostasis and cell death in
response to stress signals, can have dual effects, acting either cardiomyocytes under ischemic stress.113 There is a need for
to maintain cell survival (for example, by providing essential more detailed studies on the role of miRNAs in regulation of
energy for sustaining physiological function during the times of such complex signaling pathways, and their role in the context of
catabolic defects) or contributing to cell death (e.g., by myocardial cell death.
diminishing cell volume or providing energy for the execution
of apoptosis).
At the Crossroad of Apoptosis and Autophagy
Defects in the process of autophagy have been implicated
in numerous human diseases, including cardiovascular It is a hackneyed expression that simultaneous induction of
diseases such as hypertrophy and heart failure (reviewed in multiple cell signaling pathways occurs in biological systems,
Marzetti et al.89–92). Moreover, induction of autophagy by but it is nonetheless true, even in the context of concurrent
perioperative ischemia/reperfusion has been observed.93 induction of cell death and cell survival pathways. Concomitant

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 MicroRNAs in cardiomyocyte death
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Figure 3. miRNA synergy in cardiac apoptosis. (a) Synergy score calculation for random combinations of validated miRNAs that are involved in cardiac apoptosis.
Synergy scores of more than 2.0 are highlighted as thick red-dash lines. (b) Network location of apoptosis-related proteins encoded by target genes of miR-1 (red nodes),
miR-21 (green nodes) and both (yellow nodes). (c) Network location of apoptosis-related proteins encoded by target genes of miR-1 (red nodes), miR-30 (blue nodes) and
both (purple nodes). Synergistic apoptosis regulation should be expected for the pair of miR-1 and miR-21 instead of miR-1 and miR-30, due to more common target genes
and denser functional association between gene-encoded products. Synergy scoremiR-1:miR-21 ¼ 3.23; Synergy scoremiR-1:miR-30 ¼ 1.13; Box shows the network core area

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 MicroRNAs in cardiomyocyte death
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8

Figure 4 Ingenuity Pathway Analysis of the miRNAs identified as involved in regulation of cardiomyocyte apoptosis (see Table 1). The analysis revealed several regulatory
nodes, with one particularly interesting being the oestradiol regulation (highlighted in red) of several of the miRNAs

induction of autophagic and apoptotic signaling, and the and autophagy. Secondly, any miRNA regulating the expres-
extensive cross talk between these two pathways have been sion of genes that encode the autophagy–apoptosis node
reported extensively in cancer cells114 and also in cardiac proteins listed above can contribute to the switch between the
cells.115 In general, the nodes of cross talk include Beclin-1–Bcl-2 two pathways. Finally, miRNAs that target histone deacety-
interaction (reviewed in Marquez and Xu116), Beclin-1–Bim lases (such as sirtuins), which regulate the expression of both
interaction,117,118 caspase-mediated Beclin 1 cleavage,119,120 autophagic and apoptotic genes, are likely to impact the
caspase- and calpain-mediated Atg5 cleavage,121 UVRAG–BAX dynamic relationship between these two pathways.
interaction,122 ATG12–Mcl-1 and ATG12–Bcl-2 interaction,123 As yet, the role of miRNAs that potentially affect the cross
ATG5–FADD interaction,124 p53-mediated cross regulation,111 talk between apoptosis and autophagy on cardiac cell survival
as well as regulation of Akt signaling that has differential effects and tissue homeostasis remains largely unknown. Recent
on autophagy and apoptosis (Figure 7). studies identified that genetic deletion of aging-associated
Several models can be put forward to suggest the possible miR-34a reduces cardiomyocyte cell death and improves
role of miRNAs in regulation of autophagic–apoptotic cross functional recovery after MI, which is attributed to the function
talk. Firstly, different transcripts can share a common miRNA- of a novel miR-34a target gene, PNUTS.125 One of the cellular
binding site and compete for the same miRNA. It can be effects of Pnuts protein is nuclear sequestration of Pten,126 a
envisaged that overexpression of one of the transcripts, for negative regulator of Akt, which in turn is a master regulator of
example, one that regulates apoptosis, could leave less both apoptosis and autophagy (Figure 7). Moreover, another
miRNAs free to bind the autophagy-related transcript (a miR-34a target, SIRT1, is known to regulate both apoptosis
buffering effect) and thus shift the balance between apoptosis (via a p53 pathway and ROS signaling) and autophagy

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 MicroRNAs in cardiomyocyte death
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(via p53 and the activation of FoxO transcription factor family trigger of cellular senescence and has also been associated
members).127,128 Thus, miR-34a-mediated regulation of with several CVDs, including MI and the onset and progres-
PNUTS and SIRT1 expression in cardiac cells could contribute sion of arterial hypertension.129 Contrary to cell death,
to changes in apoptosis–autophagy switch in response to senescence is a process of irreversible cell cycle arrest which
stress and during cardiac recovery and remodeling. allows cells to remain viable and metabolically active for a long
Interestingly, increased levels of the miRNA miR-34a time (at least in in vitro cell culture). Cells undergoing
appear to be also associated with telomere shortening, with senescence exhibit several phenotypic changes, including
overexpression of PNUTS in human cardiomyocytes leading enhanced autophagy, the role of which, according to a recent
to telomere attrition.125 Telomere shortening is a well-known study, is to process cytoplasmic chromatin fragments budded
off nuclei.130 Another miR-34a target gene, SIRT1, is also
known to influence certain aspects of vascular ageing,
INDUCTION
including senescence.131,132 This suggests another mechan-
(cargoselection)
LC3-I ism for miRNA-regulated cardiomyocyte autophagy involving
senescence signaling pathways.

LC3-II
miRNAs in Cardiovascular Necrosis
cytosol COMPLETION
Necrosis is a type of cell death which is morphologically
EXPANSION characterized by increase of cell volume, dilation of orga-
Isolation nelles, rupture of the plasma membrane and subsequent loss
autophagosome
NUCLEATION
membrane of intracellular contents.34 For a long time, necrosis was
FUSION
considered to be a passive, accidental and unregulated form
Bafilomycin A of cell death, and as such was sharply contrasted against
heavily regulated and tightly orchestrated apoptosis. This
Atg12-Atg5-Atg16L
view has changed over the last few years, with increasing
Beclin1 number of reports describing the regulatory network that
cargo material
BREAKDOWN
governs necrotic cell death (reviewed in Henriquez et al.133
and Wu et al.134). Many authors suggest that myocardial
ischemia results predominantly in necrotic cell death,135 due
EFFLUX to depletion of ATP, increased calcium load, acidosis and
Biosynthesis amino-acids severe oxidative stress, with apoptotic cell death playing a role
(ATP) fatty-acids
following reperfusion. Accordingly, inhibitors of necrotic cell
Figure 5 Key steps in the autophagic pathway. The process is initiated by the death appear to be effective at reducing myocardial cell death
formation of autophagosomes, double-membrane vesicles that engulf fractions of and infarct size in animal models.136 In addition, myocardial
the cytosol. Autophagosomes undergo a step-wise maturation associated with necrosis is relatively common among infants who die as a
expansion and completion of the sequestering vesicle, which is regulated by many result of congenital heart disease, perinatal asphyxia, sepsis
ATG proteins, particularly Beclin-1 and two ubiquitin-like conjugation systems
or coronary artery abnormalities.137
Atg12-Atg5-Atg16L and Atg8(LC3)-PE. Autophagosomes then fuse with lysosomes
to form single-membrane autolysosomes. In this process, autophagosomes acquire So far, only limited evidence has been gathered on the role
hydrolytic enzymes and are able to degrade the sequestered material. Recycling of of miRNAs in regulation of cardiac necrosis. A recent study
the basic components, such as amino or fatty acids, helps the cell to maintain has reported that miR-874 inhibits cardiomyocyte necrosis,
homeostasis in vitro as well as in animal model of MI, via downregulation of

Table 2 Autophagy-modulating miRNAs with relevance to CVD

miRNAs Relevance to CVD Targets relevant to Function Refs

autophagy

miR-30 Downregulated in a model of cardiac hypertrophy and by BECN1 Vesicle nucleation 192
angiotensin II
Circulating miR-30 elevated in patients with left-ventricular
hypertrophy
miR-204 Reduces cardiomyocytes autophagy in response to hypoxia- 193,194
reoxygenation
Concomitant downregulation of miR-204 and induction of MAP1LC3A Vesicle maturation and 195
autophagy following cardiac ischemia-reperfusion in rats fusion with the lysosome
Downregulated in pulmonary arterial smooth muscle cells from
patients with PAH
miRNA-212/132 Impaired autophagy in starved cardiomyocytes FOXO3 PTEN Pro-autophagic transcrip- 196
tion factor
Induced cardiac hypertrophy
miR-21 Cell treatment with anti-miR-21 induced autophagy BECN1 MAP1LC3A 197
PIK3C3 (VPS34)
Not yet investigated with regards to cardiomyocte autophagy

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 MicroRNAs in cardiomyocyte death
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reperfusion dramatically reduced infarct size after I/R njury.144

These results indicates important role of necroptosis in
regulation of cardiac cell death. Involvement of necroptosis
in myocardia injury after Mi and in other cardiovascular
conditions has not been investigated in detail. Further
investigation on this pathway may hold promise for finding
further clinical targets for treating heart diseases.

Can miRNAs Regulate Cell-to-Cell Variability in Cardiac

Cell Death?
The performance of the apoptotic network determines the
individual cell’s probability to die, which, when assessed over
a large population of cells, can be translated into the percent of
cell death at a given time point. The apoptotic pathway
consists of many processes that are stochastic in nature, that
is, occur with certain probability rather than in a deterministic
fashion. These processes include protein translocation (e.g.,
Figure 6 Ingenuity network analysis of miRNAs identified as regulators of translocation of Bax from the cytosol to mitochondria) or
autophagy in cardiomyocytes (and miR-21)
formation of multiprotein complexes (e.g., the apoptosome).
The architecture of the network includes also positive and
negative feedback loops, which may buffer or enhance the
PTEN miR-212/132
inherent noise in the apoptotic signaling. Therefore, the net
PNUTS balance between the amount of pro- and anti-apoptotic
miR-34a
proteins, the spatial and temporal availability of protein
PIP3 PIP2 partners for interaction and functioning of the feedback loops
are all critical for determining the performance of the apoptotic
network and thus the threshold of stress signal required for
Akt successful execution of cell death at the level of cell
population.
Gene expression is subject to stochastic variability even
mTOR caspase
Bcl-2 among the cells with the same genotype and environment.145
Bad
The inherent stochastic nature of transcriptional regulation
affects cell ability to control gene expression levels in
AUTOPHAGY APOPTOSIS response to extracellular stimuli, including stress cues. In
Figure 7 MiR-34a as potential regulator of the cross talk between autophagic
the context of cell death, such cell-to-cell variability translates
and apoptotic signaling in cardiac cells. PNUTS is a PTEN-associated protein that to dramatic differences in the time between exposure to stress
sequesters PTEN to the nucleus127 and the execution of cell demise.9,12 The differences in
kinetics of cell death are particularly pronounced when critical
events involve small numbers of molecules, for example,
the expression of caspase-8,138 which normally serves not apoptosome formation. Importantly, for many genes, the
only as an element of the death receptor pathway, but also as noise in the expression pattern increases as their expression
a pro-survival regulator that supresses necrosis.139 Apart decreases. As miRNAs affect cell homeostasis by regulating
from caspase-8, other components of death receptor path- gene expression, it is conceivable that they inherently
way, for example, receptor interacting protein 1 (RIP1), are contribute to the cell-to-cell variability in cell signaling as well
also involved in regulation of necrosis (see Figure 1). In this as to the robustness of cell phenotypes and responses to
regard, overexpression of miR-155 was reported to attenuate stimuli, including pro-apoptotic stimuli. This field of research
necrotic cell death in cardiomyocyte progenitor cells exposed remains, as of now, largely unexplored, with only a few
to hydrogen peroxide.140 Considering the continuum and attempts to analyze the stochastic properties of regulatory
interdependence of cell death pathways, as well as the clinical clusters involving miRNAs.146 It is, nevertheless, and exciting
importance of necrosis in conditions such as myocardial avenue with potentially far-reaching implications when it
infarction, more work is needed to decipher the exact role of comes to regulation of cardiomyocyte cell death, particularly
miRNAs in necrotic cell death. in the contexts of a delayed and slow-progressing response
Necroptosis is a specialized pathway of programmed outcome of which can be substantially affected by changes in
necrosis (Figure 1), targeting the kinase RIP1 involved in cell-to-cell variability. This line of research will require not only
extrinsic pathway of apoptosis.141 Necroptosis shares char- knowledge of the effects of specific miRNAs on oscillations in
acteristics of both necrosis and apoptosis.142,143 A study on gene expression patters but also stochastic version of
mice model has showing that a bolus dose of necroptosis deterministic models employed traditionally to study cell
inhibitor, necrostatin-1, in mice at the time of coronary signaling pathways.

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