Circulating miRNAs Novel Biomarkers of Acute Coron

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Circulating miRNAs: Novel biomarkers of acute coronary syndrome?
Article in Biomarkers in Medicine · April 2013

DOI: 10.2217/bmm.13.8 · Source: PubMed
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Circulating miRNAs: novel biomarkers of acute

coronary syndrome?
Acute coronary syndrome refers to any group of clinical symptoms compatible with acute myocardial
infarction (AMI). AMI is a major cause of death and disability worldwide with the greatest risk of death
within the first hours of AMI onset. Therefore, delays in ‘ruling in’ AMI may increase morbidity and
mortality due to the time lag in initiating therapy. Likewise, since the majority of patients presenting with
acute chest pain do not have AMI, the rapid ‘ruling out’ of AMI in those patients would increase emergency
department triage efficiency, decrease medical costs, and reduce morbidity and mortality. Thus, the
identification of novel biomarkers that improve current strategies and/or accurately identify subjects who
are at risk of developing acute and chronic manifestations of cardiovascular disease are desperately needed.
This article discusses the potential of peripheral blood microRNAs as clinical biomarkers for the diagnosis
and prognosis of cardiovascular diseases such as AMI.
KEYWORDS: acute coronary syndrome n acute myocardial infarction n biomarkers Adam Pleister1,
n circulating miRNAs n miRNAs
Helina Selemon2,
Shane M Elton3
Acute coronary syndrome plasma cardiac biomarker analysis [4–7] . Of note, & Terry S Elton*1,2,4
Coronary artery disease (CAD) is a major women, diabetics and the elderly may not present 1
Department of Internal Medicine,
cause of mortality and morbidity in developed with a ‘typical’ chest pain syndrome and therefore Division of Cardiovascular Medicine,
nations. Despite declining death rates from other symptoms such as nausea or syncope may The Ohio State University, 473 West
12th Avenue, OH 43210, USA
this medical condition in the USA and several be their ‘chest pain equivalent’. 2
Davis Heart & Lung Research Institute,
other countries over the last four decades, CAD The 12-lead ECG should be obtained The Ohio State University, 473 West
12th Avenue, OH 43210, USA
still causes approximately a third of all deaths immediately upon presentation and, if not ini- 3
Midwestern University, Glendale,
in persons over the age of 35 [1,2] . In the USA, tially diagnostic, should be repeated at regular AZ, USA
4
College of Pharmacy, Division of
approximately a third of all middle-aged women 5–10 min intervals. The ECG will differentiate Pharmacology, The Ohio State
and half of all middle-aged men develop some the type of ACS: STEMI versus NSTEMI or University, 473 West 12th Avenue,
degree of CAD [3] . Acute coronary syndrome UAP. STEMI diagnosis requires new ST eleva- OH 43210, USA
*Author for correspondence:
(ACS) is the symptomatic, clinical presentation at two anatomically contiguous leads of at Tel.: +1 614 292 1400
tion of CAD. The etiology in well over half of least 0.1 mV (1 mm), with different criteria in [email protected]
patients presenting with ACS is acute myocar- leads V2 and V3 (Figure 1) . The ECG can also
dial infarction (AMI), commonly referred to as help to localize the region where the myocardial
a heart attack. The presenting symptom is most infarction occurred.
often chest pain radiating down the left arm and Cardiac biomarkers are obtained as part of the
up the angle of the jaw, associated with nau- initial laboratory evaluation of patients present-
sea, emesis and diaphoresis. The chest pain and ing with chest pain regarding ACS diagnoses and
associated symptoms result from a lack of blood help to further differentiate the type of ACS and
flow and oxygen delivery to the myocardium guide treatment decisions. Positive biomarkers,
due to blockage in the coronary arteries. ACS cardiac troponin (cTn) levels in particular, will
results from three causes: ST elevation myocar- differentiate NSTEMI from UAP, since ECGs
dial infarction (STEMI; ~30%); non-STEMI in both cases are often similar. Furthermore, the
(NSTEMI; ~25%); or unstable angina pectoris ECGs in NSTEMI and UAP differ from the
(UAP; ~38%). Initial diagnostic steps to dif- more serious, life-threatening STEMI, as noted
ferentiate ACS on patient presentation (often above (Figure 1) .
to an emergency department) are of critical NSTEMI is defined as chest pain with pos-
importance [4–7] . sible new ECG changes, but no ST elevation
The essential first steps in the evaluation of a in the presence of elevated cardiac biomarkers
patient who presents with chest pain and possible (F igur e 2) . STEMI, therefore, represents chest
ACS are: obtain a brief history and physical exam- pain in the presence of ST elevation on ECG
ination, perform a 12-lead ECG, and complete a and positive biomarkers. part of
10.2217/BMM.13.8 © 2013 Future Medicine Ltd Biomarkers Med. (2013) 7(2), 287–305 ISSN 1752-0363 287
Review Pleister, Selemon, Elton & Elton
comprehensive therapeutic approaches, includ-

ing initiation of oxygen, nitrate, morphine,
ST segment
aspirin, platelet P2Y12 receptor blocker and
Normal ECG
b-blocker therapy, as well as electrolyte replace-
ment and arrhythmia management (Figur e 2)
P T [10,11] . Advanced therapies for ACS patients are
QRS based on well-validated risk stratification calcu-

lators such as the Thrombolysis in Myocardial
Infarction Risk Score [12] . Early reperfusion and
revascularization are the goals of these thera-
STEMI
pies, which include either fibrinolytic therapy
ST elevation or coronary angiography with percutaneous
coronary intervention (PCI) [13] .
Cardiac biomarkers in ACS

ACS results from inadequate blood flow and
oxygen delivery to myocardial tissue, which
NSTEMI damages the cardiac myocyte membrane. As a
consequence, intracellular contents including
ST depression proteins are released into the extracellular space
and peripheral blood. Whether detectable release
of these proteins occurs in the setting of revers-
ible or irreversible cardiac injury continues to be
an area of debate [14–18] . Importantly, several of
the proteins (e.g., creatine kinase, lactate dehy-
NSTEMI
drogenase, myoglobin and cTn) released after
T inversion the necrosis of cardiomyocytes have been uti-
lized as biomarkers to detect and monitor myo-
cardial injury. Although CK, and in particular
the CK myocardial band isoform (CK‑MB), has
Figure 1. Schematic representations of ECG been used for years to diagnose AMI [19] , with
patterns. Normal ECG pattern with normal the increased specificity and sensitivity of cTn,
P wave, QRS complex, ST segment and T wave, CK-MB is no longer routinely utilized in the
followed by ECG changes consistent with STEMI clinical setting as a biomarker [20,21] .
(ST elevation) and NSTEMI (ST depression or cTnI and cTnT, structural proteins unique
T wave inversion).
NSTEMI: Non-ST elevation myocardial to the heart, currently stand as the preferred
infarction; STEMI: ST elevation myocardial biomarker for the diagnosis of AMI [20,22,23] .
infarction. cTn is monitored serially at approximately every
6 h; cTn elevation occurs approximately 80%
UAP is defined by chest pain with new of the time in ACS patients at 3 h after the ini-
ECG changes (e.g., ST segment depression or tial patient presentation [24] . As cTn levels can
new T-wave inversion), but no ST elevation remain elevated for up to 10 days after AMI,
or elevation in cardiac biomarkers (F igur e 2) . they can also be employed to make a late diag-
UAP- and ACS-related chest pain differs from nosis of a previous infarction or assist in evalu-
stable angina (SA)-related chest pain, which ation of reinfarction after a primary event [25] .
occurs with exertion and improves with rest. Finally, cTn levels correlate with both the size
UAP- and ACS-related chest pain may occur at of the myocardial infarction and the prognosis
rest, but can also have a different character or after AMI (Figure 3) [26–29] .
increased severity compared with earlier chest Although cTn is the biomarker of choice in
pain [8,9] . They may also last longer than past the evaluation of AMI, considerable variation
chest pain episodes. Finally, new-onset chest exists in the specificity and sensitivity of the
pain is defined as ACS, as it also implies a new different immunoassays used to detect cTnI
abnormality of the coronary circulation. and cTnT in the peripheral blood [30,31] . There-
Each of these diagnoses represents a medi- fore, to confirm the diagnosis of cardiac injury,
cal emergency requiring efficient diagnosis, tri- a 2012 joint task force of the major cardiology
age and treatment. All three require specific, societies suggested that all laboratories use a
288 Biomarkers Med. (2013) 7(2) future science group

Circulating miRNAs: novel biomarkers of acute coronary syndrome? Review
cut-off value of the 99th percentile of a normal infarction from other emergent clinical syn-
population (Figure 3) [32] . dromes that require vastly different treatment
Furthermore, merely detectable cTns (but strategies, such as stress-induced cardiomyopa-
not ‘positive’ per the 99th percentile defini- thy, acute myocarditis, pulmonary embolism,
tion) in otherwise stable patients increases the heart failure without CAD (nonischemic cardio
likelihood of future morbidity and mortality myopathy), trauma or implantable cardioverter
[33–35] . When cTn levels were used with another defibrillator firings [41,42] . In addition, since
validated cardiac biomarker (B-type natriuretic cTnT is also expressed in skeletal muscle tissue
peptide) in otherwise healthy adults, a two- [43] , cTnT levels may be elevated in the blood
fold increase in mortality from cardiovascular after skeletal muscle tissue injury, and could
causes was noted [36] . lead to a false-positive diagnosis of cardiac injury
Recently, the introduction of ‘high-sensi- [44] . Finally, cTnT levels have been shown to be
tivity’ cTnT (hs-cTnT) assays showed up to elevated in patients with advanced renal fail-
ten-times more sensitive results than previous ure, which may also result in false diagnosis [45] .
‘sensitive’ cTn assays. These newer laboratory Therefore, the need for more specific biomark-
tests allow for a quicker and more precise diag- ers that can differentiate between cardiac injury
nosis of ACS in patients that present concerning from ACS and noncardiac conditions still exists.
symptoms [37,38] . Similar to their predecessors,
high sensitivity values in the normal range (not Overview of miRNA biology
above the 99th percentile) can predict cardiac Since their discovery almost 20 years ago [46] ,
disease and mortality in otherwise healthy miRNAs have emerged as key negative post-
adults [39] . However, no consensus exists as to transcriptional regulators of gene expression by
the appropriate cut-off levels and the clinical promoting the degradation and/or inhibiting
implication of false positives [40] . the translation of specific mRNA targets [47,48] .
Although cTns are excellent biomarkers, miRNAs are an endogenous family of small
one major limitation of cTns is their inability nonprotein-coding ssRNAs (20–25 nucleo-
to differentiate between ACS and myocardial tides) [49,50] ; 2042 miRNAs processed from
Clinical presentation
consistent with ACS
Initial therapies
(aspirin, oxygen, nitrate and
morphine, among others)
ST elevation ST depression or T inversion

ECG
No Elevated Yes
Normal
troponin?
Consider alternative
diagnoses
STEMI UAP NSTEMI
Figure 2. Diagnostic algorithm for acute coronary syndrome. When a patient presents with
signs and symptoms raising concern for ACS, the ECG will differentiate between STEMI versus
NSTEMI or UAP. Positive cardiac biomarkers such as cardiac troponin levels will differentiate between
NSTEMI and UAP.
ACS: Acute coronary syndrome; NSTEMI: Non-ST elevation myocardial infarction; STEMI: ST elevation
myocardial infarction; UAP: Unstable angina pectoris.
future science group www.futuremedicine.com 289

50
Troponin (large MI)
20
Multiples of the AMI cut-off limit

10
Troponin (moderate MI)

5
2
Troponin (small MI)
99th percentile
0
0 1 2 3 4 5 6 7 8 9
Days after initial presentation for AMI
Figure 3. Time course of cardiac troponin elevation as it relates to the size of the
myocardial infarction. The time–concentration/activity curves for troponin after large, moderate
and small MIs are shown.
AMI: Acute myocardial infarction; MI: Myocardial infarction.
Adapted with permission from [116] .
1600 precursors have so far been annotated in pre-miRNAs are recognized and endonucleo-
humans [201] . Computational predictions sug- lytically cleaved near the base of the stem by the
gest that miRNAs may regulate the expression nuclear microprocessor complex (Figure 4) [55] . In
of 60% of all human protein-coding genes [49] . animals, pre-miRNAs are exported from the
The expression of these molecules varies widely; nucleus to the cytoplasm where they are sub-
some miRNAs are ubiquitously expressed, sequently processed by Dicer and its cofactor
while others are expressed in a tissue- and/or TRBP into RNA duplexes of approximately
cell-specific manner, and many show spatiotem- 22 nucleotides (Figure 4) [55] .
poral expression patterns [50] . Although miR- Following cleavage, an Ago protein and a
NAs have the primary responsibility of ‘fine- glycine–tryptophan repeat-containing protein
tuning’ gene expression in the regulation of of 182 kDa (GW182) bind to the short RNA
development and tissue homeostasis [51] , recent duplexes, forming the core of a multisubunit
studies suggest that miRNA function becomes complex called the miRNA-induced silencing
more pronounced in response to physiologic complex (miRISC) (Figure 4) [56] . In a manner
and pathophysiologic stresses [52,53] . similar to siRNA duplexes, one of the two
strands, the ‘passenger miRNA’ (also referred
miRNA biogenesis & function to as the ‘complementary star-form miRNA’
The majority of mammalian miRNAs are tran- strand or ‘miRNA*’), is released and degraded
scribed by RNA polymerase II as pri-miRNAs while the other strand, designated the ‘guide
that are subsequently matured in a multistep strand’ or ‘mature miRNA,’ is retained within
biogenesis process to generate the single- the miRISC (Figure 4) [47,48] .
stranded 20–25 nucleotide mature functional It has been widely assumed that passenger
form (Figure 4) [47,48] . miRNA expression levels strands or miRNA* species simply promote
are tightly controlled by both transcriptional the accurate processing of their guide strand
and post-transcriptional mechanisms (i.e., partners [57] . However, recent reports have sug-
processing of miRNA precursors and active gested that passenger strands are evolutionarily
degradation of mature miRNAs) [54] . conserved and have inhibitory activity similar to
As the pri-miRNAs are transcribed, hairpin that of guide strands in both cultured cells and
structures (~60–80 nucleotides) containing transgenic animals [58–60] . Given the increasing
imperfectly base-paired stem loops known as number of cases where two functional mature

miRNAs get processed from opposite arms of regulatory effects of miRNAs/miRISCs occur
the same RNA duplexes, these products are now through mRNA decay rather than translational
denoted with the suffix -5p (from the 5´ arm) repression [63] . Recent data suggest that in order
(e.g., miR‑155-5p) or -3p (from the 3´ arm) for efficient repression of target gene expression
(e.g., miR‑155-3p) following their name [61] . to occur, a threshold level of miRNA expression
The guide strand subsequently targets the seems to be required (typically >100 copies per
miRISC to specific mRNAs by base pairing cell) [64] . Therefore, most miRNAs within a cell
interactions between nucleotides two and eight that are expressed at low levels may have little
of the miRNA (the seed region) and complemen- or no role in regulating gene expression [65] .
tary nucleotides predominantly localized to the Finally, pseudogenes or competing endogenous
3´-untranslated region of mRNAs [62] . With the RNAs have also been implicated in regulating
miRNA acting as an adaptor for the miRISCs, miRNA activity by acting as decoys or sponges
complex-bound mRNAs are subjected to trans- to sequester miRNAs and thus prevent binding
lational repression (i.e., inhibition of transla- to their mRNA targets [66] .
tion initiation) and/or degradation following
deadenylation by the CCR4–NOT complex Circulating miRNAs
and decapping by Dcp1/2 enzymes (Figure 4) [56] . Several studies have demonstrated that miR NAs
Currently, it is believed that the major- are present in the blood; sites of detected
ity of the negative post-transcriptional gene miRNAs include erythrocytes, peripheral
miRNA gene or intron
RNA Pol
II/III Transcription
5´
3´ pri-mRNA
DGCR8
Processing Drosha Exportin-5
5´
3´
pre-miRNA TRBP
5´
o2
3´ Ag
Dicer
Nucleus
5´ 3´
3´ 5´
Cytoplasm Maturation Ago2
TRBP
5´ 3´ Strand selection
3´ 5´
Dicer
PABP
3´
CCR4-NOT
182
5´
5´
GW
PAN2-PAN3
miRISC formation
3´
CCR4-NOT
CP2 miRISC complex
1-D 182PAN2-PAN3
D CP GW PABP
Decreased miRNA mRNA 3´ 5´
5´ AAAA
target protein levels 3´
Translation repression
Biomarkers Med. © Future Science Group (2013) and/or deadenylation
Figure 4. Mechanisms involved in miRNA biogenesis. This diagram includes miRNA transcription, maturation of miRNA/mRNA and
two potential mechanisms for miRNA/mRNA silencing. The specific details describing these processes have recently been extensively
reviewed [55–57] and are briefly discussed in the text.

Nucleus
Cytoplasm Ago2
?
5´
3´
pre-miRNA
5´
pre-miRNA High-density
lipoproteins
3´
TRBP
5´
3´
Dicer
TRBP
T RBP
5´ 3´ Exosomes
3´ 5´
Dicer
MVB
Ago
2
Microvesicles
5´
3´ Ago2
Apoptotic bodies
Biomarkers Med. © Future Science Group (2013)
Figure 5. Mechanisms protecting circulating miRNAs from degradation. miRNAs can be protected from degradation by
several distinct types of lipid vesicles including exosomes, microvesicles and apoptotic bodies. The exact proportion of mature and
pre‑miRNAs in the different extracellular compartments is not known. miRNAs can also be protected when they are bound to
specific proteins (e.g., NPM1 and Ago2), to form miRNA–protein complexes, and/or HDL to form HDL/miRNA particles. Currently, it is
not known how these miRNA–protein complexes are released from the cell; however, it is hypothesized that they may be released
passively (i.e., as by‑products of dead cells) or actively (i.e., through interactions with specific membrane channels or proteins). It remains
to be determined which, if any, of these sources of circulating miRNAs can serve as an appropriate biomarker for acute coronary
syndrome.
blood mononuclear cells (PBMCs), plate- degradation, thus miRNAs may be useful as
lets, plasma and serum [67–69] . Interestingly, blood-based biomarkers.
plasma/serum miRNAs display remarkable Recent reports have revealed that circulating
stability even under harsh conditions, includ- miRNAs can be packaged into several distinct
ing endogenous circulating RNase activity, types of lipid vesicles, thereby protecting them
boiling, low or high pH, long-term storage at from blood RNase activity. Vesicles proposed
room temperature and multiple freeze–thaw as carriers of circulating miRNAs include: exo-
cycles [67–69] . Taken together, these observa- somes, membranous vesicles of 30–100 nm in
tions suggest that plasma miRNAs are pro- size that arise inside many cells from endo-
tected by mechanism(s) that shield them from somal compartments called multivesicular

Table 1. Studies of circulating miRNAs in acute coronary syndrome.
Study subjects/ Circulating Correlation miRNA Time of blood draw (mi)RNA miRNA detection Ref.
subject pool miRNAs source isolation and normalization
method/kit method
30 healthy controls, 1, 133a, 208a, miR‑208a shows highest Plasma AMI: onset to emergency Tri Reagent® TaqMan® probes. [89]
future science group

16 CAD, 17 other CVD, 499-5p ↑ sensitivity and specificity, time = within 12 h (4.8 ± 3.5 h) cel-miR‑39
33 AMI comparable with cTnI
36 atypical chest pain 1, 133, 208b, 499 ↑ miR‑208b and miR‑499 Plasma Mechanical reperfusion, stenting mirVana™ SYBR®-green based, [94]
(non-AMI), 32 AMI correlate with cTnT of infarcted artery within 12 h of PARIS™ 3 Caenorhabditis elegans
chest pain
11 healthy controls, 1, 133, 208b, miR‑208b correlates with cTnT Plasma Collection at 24, 48 and 72 h, and TRIzol® LS, SYBR-green based, [92]
25 STEMI 499-5p ↑ 3 and 6 months after discharge miRNeasy none
17 healthy controls, 122, 375 ↓, 1, 133a, Time course of upregulated Plasma 156 ± 72 min, 3, 9, 15, 21, 33, 45, mirVana TaqMan probes, [91]
33 STEMI 133b, 499-5p ↑ miR‑associated with cTnl 69 h after T0 PARIS miR‑17‑5p
10 controls, 5 UAP, 499 ↑ Correlation with CK-MB Plasma ACS: within 48 h of last onset mirVana TaqMan probes, [93]
15 CHF, 9 AMI chest pain; CHF: NYHA class II PARIS synthetic miRNA
or III
42 non-AMI, 8 UAP, 1, 133a ↑ Correlation with cTnT Serum <3, 3–9, 9–15, 15–21, >21 h after TRIzol LS TaqMan probes, none [95]
3 NSTEMI, 18 STEMI onset of chest pain
20 controls, 31 AMI 1↑ Association with CK-MB levels Serum AMI: within 24 h (8.5 ± 3.82 h) miRNA SYBR-green based, [90]
isolation none
www.futuremedicine.com
7 no CAD, 31 stable CAD, 126, 133, 208a, 499 Transcoronary gradients Plasma – Tri Reagent TaqMan probes, [97]
19 ACS ↑ document cardiac release of cel-miR‑39 spike-in
miR‑133 and miR‑499
87 healthy controls, 208b, 499 ↑ miR‑499 showed diagnostic Plasma 1 h, <3 h after onset chest pain mirVana SYBR-green, [98]
113 NSTEMI, 397 STEMI accuracy comparable with PARIS 3 C. elegans
hs-cTnT
66 healthy controls, 1↑ Association with QRS duration Plasma – mirVana SYBR-green based, [99]
93 AMI PARIS RNU6
99 healthy controls, 1, 21, 133a, 423-5p, miR‑499 showed diagnostic Plasma 4–9 h after onset of symtons mirVana TaqMan probes, [83]
81 CHF, 92 AMI 499-5p ↑ accuracy comparable with PARIS cel-miR‑39
hs-cTnT
20 controls, 20 AMI 30c, 145 ↑, 663b, miR‑30c and miR‑145 correlated Whole blood – miRNeasy kit Microarray, none [102]
1291 ↓ with hs-cTnT
↑: Concentration increases; ↓: Concentration decreases; ACS: Acute coronary syndrome; AMI: Acute myocardial infarction; CAD: Coronary artery disease; CHF: Congestive heart failure; CK‑MB: Creatine kinase myocardial
band; CPS: Chest pain syndrome; cTn: Cardiac troponin; CVD: Cardiovascular disease; hs-cTnT: High-sensitivity cardiac troponin T; NSTEMI: Non-ST elevation myocardial infarction; NYHA: New York Heart Association;
Circulating miRNAs: novel biomarkers of acute coronary syndrome?
PBMC: Peripheral blood mononuclear cell; SAP: Stable angina pectoris; STEMI: ST elevation myocardial infarction; T0: Onset of acute myocardial infarction symptoms; UAP: Unstable angina pectoris.
Review
293
Ref.
[103]
[85]
[86]
↑: Concentration increases; ↓: Concentration decreases; ACS: Acute coronary syndrome; AMI: Acute myocardial infarction; CAD: Coronary artery disease; CHF: Congestive heart failure; CK‑MB: Creatine kinase myocardial
bodies [70,71] , microvesicles or microparticles
band; CPS: Chest pain syndrome; cTn: Cardiac troponin; CVD: Cardiovascular disease; hs-cTnT: High-sensitivity cardiac troponin T; NSTEMI: Non-ST elevation myocardial infarction; NYHA: New York Heart Association;
(MPs), relatively large (100–1000 nm) plasma
membrane-derived particles that are released
and normalization
miRNA detection
SYBR-green based,
SYBR-green based,
SYBR-green based,
into the extracellular space by outward bud-
ding and fission of the plasma membrane (ecto-
cytosis) of almost all cell types under physi-
PBMC: Peripheral blood mononuclear cell; SAP: Stable angina pectoris; STEMI: ST elevation myocardial infarction; T0: Onset of acute myocardial infarction symptoms; UAP: Unstable angina pectoris.
method
ological and pathological conditions [72] ; and
5S rRNA
5S rRNA
RNU6
apoptotic bodies (ABs), even larger lipid vesi-
cles in size (up to 4000 nm) that are released
during the terminal steps of programmed cell
death (Figure 5) [73] .
method/kit
miRNeasy kit
miRNeasy kit
Although little is known regarding the
isolation
(mi)RNA
TRIzol LS
export mechanisms, protein-bound miRNAs
have also been detected in both cell culture
supernatants and in plasma samples (Figure 5) .
For example, a recent study by Wang et al.
reported that the majority of miRNAs in cell
<24 h after onset chest pain
<24 h after onset chest pain culture supernatants were in fact not vesicle-
miR‑133 and miR‑328 correlated Whole blood AMI: 24 h and 7 days after
encapsulated, but rather were bound by RNA-

Time of blood draw
binding proteins. These investigators identified

NPM1, a known nucleolar RNA-binding pro-
tein, in the supernatant of serum-deprived cells
by mass spectrometry and demonstrated that
symptoms
the direct binding of NPM1 protected some

miRNAs from RNase A degradation in vitro
[74] . Arroyo et al. extended these observations
by utilizing differential centrifugation and
size exclusion chromatography to systemati-
miRNA
source
PBMCs
PBMCs
Table 1. Studies of circulating miRNAs in acute coronary syndrome (cont.).
cally characterize circulating miRNA com-

plexes in human plasma and serum [75] . Their
study established that up to 90% of miRNAs
in the circulation were present in a nonvesicle-
encapsulated form that was specifically desta-
bilized by proteinase K digestion of plasma,
indicating the existence of a miRNA–protein
complex as a mechanism for their stability in
the RNase-rich circulation [75] . These inves-
Correlation
tigators subsequently determined that a sig-

with cTnI
nificant portion of circulating miRNAs were

associated with Ago2, a key component of the
–
miRISC (Figure 4) [75] . In support of this study

Turchinovich et al. also reported that most
extracellular miRNAs in blood plasma and cell
Circulating
culture are bound to Ago2 and not associated

miR‑146a ↑
miR‑146a ↑
133, 328 ↑
miRNAs
with exosomes [76] . It was hypothesized that

Ago2–miRNA complexes result from byprod-
ucts of dead cells that remain in the extra-
cellular space due to the high stability of the
Ago2 protein and the Ago2–miRNA complex
18 CPS, 22 SAP, 25 UAP,
18 CPS, 15 SAP, 15 UAP,
[76] . However, it is also possible that specific

28 controls, 51 AMI
release of Ago2–miRNA complexes results

Study subjects/
from unidentified cell membrane-associated

subject pool
channels or receptors.
Finally, Vickers et al. established that
26 AMI
16 AMI
high-density lipoprotein (HDL) particles

(comprising of lipids and apolipoprotein A-I,

Table 2. Studies of prognostic circulating miRNAs in acute coronary syndrome.

Study Changes in Correlation miRNA Time of blood (mi)RNA Normalizing/ miRNA Ref.
subjects/ circulating source draw isolation housekeeping detection
subject pool miRNAs method/kit probe method
117 UAP 1, 133a, miR‑133a and Plasma Plasma samples Master-Pure™ cel-miR‑54 TaqMan® [100]
131 NSTEMI 208b ↑ miR‑208b obtained on spike-in probes
196 STEMI associated with admission
risk of death
820 197, 223 ↑ miR‑197, -223 Plasma Prospective miRNeasy U6 TaqMan [87]
participants 126 ↓ and -126 plasma samples Human
were associated obtained from all Microarray
with the inhabitants age
incidence of MI 40–80 years of
Bruneck, Italy
21 SV group 18a, 134, 155, miR‑155 and Serum Serum samples mirVana™ Validation study, TaqMan [108]
19 CD group 192, 380† ↑ miR‑380† obtained on PARIS™ normalization Human
93†, 125a-5p, associated with survival discharge probe unknown Microarray,
190b, 212, risk of death A and B
223†, 331-3p ↓
†
Mature miRNA that originates from the minor arm.
↑: Concentration increases; ↓: Concentration decreases; ACS: Acute coronary syndrome; CD: Cardiac death; MI: Myocardial infarction; NSTEMI: Non-ST elevation
myocardial infarction; STEMI: ST elevation myocardial infarction; SV: Survival group; UAP: Unstable angina pectoris.
averaging 8–12 nm in size) transport endog- pathology-specific manner; and detectable

enous miRNAs and deliver them to recipient with high sensitivity and specificity using
cells with functional targeting capabilities (Fig - real-time PCR, next-generation sequencing
ure 5) [77] . They also determined that the HDL– technologies and microarrays, the use of cir-
miRNA profile was distinct from the exosome– culating miRNAs as clinical biomarkers has
miRNA profile and that Ago2 and NPM1 generated great interest. However, as circu-
proteins were not present in the purified HDL lating miRNAs have been detected in whole
fractions [77] . In addition, when reconstituted blood, erythrocytes, PBMCs, platelets, plasma,
HDL was injected into mice, these particles serum, lipid vesicles (i.e., exosomes, MPs and
retrieved distinct miRNA profiles from nor- ABs), miRNA–protein complexes (i.e., NPM1
mal and atherogenic mouse models suggesting and Ago2), and HDL/miRNA particles, it
that HDL can bind circulating miRNAs [77] . remains to be determined which, if any, of
Although the process by which miRNAs load these sources of circulating miRNAs can serve
onto HDLs is not known, biophysical studies as an appropriate biomarker for ACS.
suggest that HDL can bind miRNAs through
divalent cation bridging [77] . Interestingly, Circulating miRNAs as biomarkers
HDLs isolated from familial hypercholester- for ACS
olemia subjects had a higher concentration of miRNAs have emerged as important regula-
miRNAs (e.g., miR‑223) and contained more tors of cardiac developmental and pathological
individual miRNAs than HDL from healthy processes, including AMI, cardiac arrhythmia,
subjects [77] . fibrosis, hypertrophy, heart failure and vascular
atherosclerosis [79] . AMI potentially may pro-
Circulating miRNAs as biomarkers vide the ideal scenario to test whether or not
An ideal biomarker for any disease must ful- miRNAs can be utilized as biomarkers, given
fill a number of criteria, including accessibility that the loss of blood flow to the heart can
through minimally invasive techniques, reli- cause ischemia, injury and eventual necrosis
ability as an early indicator of disease before of cardiomyocytes, which results in the release
clinical symptoms appear, specificity to the of cellular components into the bloodstream
disease or pathology of interest, sensitivity to (e.g., cTnI). It is now well established that
changes in the pathology, ability to remain miR‑208a, hosted by the a-myosin heavy chain
within the sample (i.e., have a long half-life) gene (MYH6 ), is exclusively expressed in car-
and capability of rapid and accurate detec- diac myocytes [80] , and that miR‑1 (i.e., con-
tion [78] . Given that miRNAs are stable in the sisting of two distinct miRNAs that share an
plasma/serum; often regulated in a tissue- and identical mature sequence, designated miR‑1-1

and miR‑1-2, found on chromosomes 20 and after coronary artery ligation [88] . Similarly,
18, respectively), -133a/b (i.e., three known Cheng et al. utilizing the same animal model
miR‑133 genes: miR‑133a-1, miR‑133a-2 and of AMI, and only measuring miR‑1, found
miR‑133b, found on chromosomes 18, 20 and that serum levels of this miRNA increased
6, respectively), -208b (i.e., a miR‑208 fam- 200-fold and peaked at 6 h post-AMI, return-
ily member encoded by an intron of a-myosin ing to basal levels 3 days later [89] . Further-
heavy chain gene [MYH7]) and -499 are car- more, serum miR‑1 level in rats with AMI had
diac and skeletal muscle cell-enriched miRNAs a strong positive correlation with myocardial
[81] . Therefore, many studies have been initi- infarct size [89] .
ated to test the hypothesis that heart-specific D’Alessandra et al. reported that in the
and/or muscle-enriched miRNAs leak into the mouse coronary artery ligation model, miR‑1,
circulation during AMI and can be used to -133a/b and -208a circulating plasma levels
detect and monitor myocardial injury (Table 1) . followed the same general time courses as
In addition, heart-specific and/or muscle- described above in rats after AMI [90] . By
enriched miRNAs may be useful in monitor- contrast, however, they demonstrated that
ing the development of CAD, beginning with mouse miR‑499-5p exhibited a slower time
patients having symptoms of chest pain but course and circulating plasma levels peaked
with normal coronary angiograms (that is, at 24 h after AMI [90] . Their results also sug-
no visible CAD) and progressing to patients gested that miR‑499-5p was an extremely sen-
with UAP, to patients with stable CAD and sitive indicator of cardiac damage, given that
finally to patients with STEMI or NSTEMI the expression of this miRNA closely paralleled
[82] . Alternatively, since inflammation plays a the increase in cTnI from 15 min to 6 h after
critical role in the development of atherosclero- coronary ligation [90] .
sis and the onset of ACS [83] , immune cells may In addition, Gidlof et al. utilized an occlu-
also potentially provide an important source sion–reperfusion protocol in a closed-chest pig
of circulating miRNAs that can be utilized as model (n = 6) to investigate whether or not
biomarkers (Table 1) [84,85] . Finally, endothelial circulating miRNA plasma levels were modu-
cell or platelet released specific miRNAs may lated after AMI [91] . They found that circu-
also be diagnostic and/or prognostic, given lating plasma levels of miR‑1, -133a, -208a,
that these miRNAs may reflect systemic dis- -208b and -499-5p were not detectable during
turbances in endothelial cell and/or platelet the 40-min occlusion period [91] . However, all
function in patients with ACS (Table 2) [86] . of these miRNAs, except miR‑208a, were sig-
nificantly increased in the plasma 20 min after
Time course of circulating miRNA reperfusion [91] . The circulating levels of miR‑1,
release after AMI -133 and -208b peaked at 2 h, followed by a fast
To begin to test the utility of miRNAs as bio- reduction of 25–50% at 2.5 h [91] . By contrast,
markers, several studies investigated the time the levels of miR‑499-5p were still increasing
course of miRNA release after AMI. The first at the last time point taken (i.e., 2.5 h).
published study investigating circulating miR- D’Alessandra et al. also investigated the time
NAs levels before and after AMI was performed course of miRNA and cTnI release in a small
by Ji et al. utilizing a rat isoproterenol-induced number of patients with acute STEMI (n = 8;
myocardial injury model [87] . These investiga- four treated with thrombolysis and four with
tors found that miR‑208a was not detectable PCI) starting at 156 ± 72 min after the onset
in the plasma of control rats; however, circu- of AMI symptoms (T0 ) and at different times
lating miR‑208a level significantly increased thereafter [90] . Plasma miR‑1, -133a and -133b
at 3 and 12 h after isoproterenol treatment and cTnI expression levels were already at their
and was highly correlated with plasma cTnI peak at T0, whereas miR‑499-5p exhibited a
levels [87] . In support of this study, Wang et al. slower time course and peaked approximately
established that in a rat coronary artery ligation 12 h after STEMI [90] . Finally, Adachi et al.
model of AMI, circulating miR‑208a levels in measured the plasma miR‑499 concentra-
the plasma were undetectable before surgery or tions in blood samples obtained within 48 h
in sham-operated control rats, but were signifi- of the last onset of chest pain from 14 indi-
cantly elevated as early as 1 h after ligation and viduals with ACS (nine patients with AMI and
peaked at 3 h [88] . They also demonstrated that five patients with UAP, all of which under-
miR‑1, -133a and -499 circulating plasma levels went PCI) [92] . Importantly, in patients with
peaked at 6 h and were still elevated at 24 h AMI, plasma miR‑499 concentrations were

measurably increased and became undetect- coronary angiogram) or patients with other
able before hospital discharge. By contrast, cardiovascular diseases (n = 17). Importantly,
miR‑499 was not detected in the plasma of receiver operating characteristic curve analysis
patients with UAP [92] . established that miR‑208a detection separated
Taken together, these time course stud- AMI patients from other cardiovascular disease
ies suggest that miR‑1, -133a/b, -208a/b and patients better than miR‑1, -133a and -499 [88] .
-499 may be useful biomarkers of myocar- Finally, miR‑208a demonstrated superior sen-
dial injury in mammals since they display a sitivity in detecting AMI when compared with
very fast time course relative to the onset of cTnI, given that this miRNA was detected in
AMI and appear to correlate well with cTnI. 100% of AMI patients within 4 h of the onset
However, there is some controversy regarding of symptoms, while cTnI was detected in only
the usefulness of miR‑208a and/or -208b as 85% of these patients [88] .
plasma biomarkers in humans. For example, In support of these findings, Corsten et al.
several reports have demonstrated that plasma reported that the circulating plasma levels of
miR‑208a and/or -208b expression levels were miR‑1, -133a, -208b and -499 were elevated
increased in patients with AMI [88,91,93] . By by three-, four-, 1600- and 100-fold, respec-
contrast, other investigators have found that tively, in patients with AMI (n = 32, presence
miR‑208a and/or -208b were not detectable or of chest pain <12 h with STEMI and increased
were present at very low levels in plasma sam- CK and cTnI) as compared with patients with
ples obtained from AMI patients [90,92] , or only
chest pain but normal angiograms (n = 36)
detectable in some patients with myocardial [93] . Both miR‑208b and -499 correlated with
injury [94] . The inconsistency between these cTnI levels, suggesting that these miRNAs
studies may result from the relatively late time
are released from injured cardiomyocytes [93] .
points at which plasma samples were collected Receiver operating characteristic curve analysis
in the three reports that were unable to rou- established that miR‑208b and -499 showed
tinely detect miR‑208 [90,92,94] , given that the
a superior performance in separating AMI
level of this miRNA increases rapidly, peak- patients from controls with atypical chest pain
ing within hours of the presentation of symp- and no cardiac disease when compared with
toms [90] . In addition, circulating miR‑208a miR‑1 and -133a [93] . Importantly, similar
and/or -208b concentrations are rather low and results were obtained by Gidlof et al. utilizing
therefore this miRNA may be more difficult to plasma samples obtained <12 h from the onset
detect. Thus, researchers may have to utilize of symptoms from patients with STEMI (n = 9)
larger sample sizes to reliably measure miR‑208[91] . Finally, D’Alessandra et al. found that the
plasma levels. Finally, Fichtlscherer et al. have
circulating plasma levels of miR‑1, -133a, -133b
also established that measuring miR‑208 by the and -499-5p were elevated in STEMI patients
TaqMan® (Life Technologies Ltd, UK) PCR (n = 33), and returned to baseline 5 days later
methodology is more difficult compared with [90] . In this study, miR‑208a/b was undetectable
other miRNAs [95] . in STEMI patients [90] .
In addition to the more comprehensive
Plasma/serum miRNAs as biomarkers studies described above, several small cohort
in small patient ACS cohorts AMI studies have also been performed where
Wang et al. performed the first human study the investigators examined only one or two
to begin evaluating whether or not heart- of the heart-specific and/or muscle-enriched
specific and/or muscle-enriched miRNAs leak miRNAs. For example, Adachi et al. measured
into the circulation during AMI and can be the circulating levels of miR‑208a/b and -499
used to detect and monitor myocardial injury in patients with AMI (n = 9), with UAP (n = 5),
[88] . They reported that the expression lev- with congestive heart failure (n = 15) and with-
els of miR‑1, -133a, -208a and -499 in the out cardiovascular diseases (n = 10) [92] . They
plasma of patients clinically diagnosed with reported that plasma miR‑499 and not -208a/b
AMI (n = 33; acute ischemic-type chest pain, concentrations increased measurably in all AMI
ECG change, coronary angiography and cTnI individuals [92] . Kuwabara et al. measured the
0.1 ng/ml) were significantly increased when circulating serum levels of miR‑1, -133a and
compared with healthy controls (n = 30; nor- -208a in patients with STEMI (n = 18), with
mal ECG finding and no history of cardiovas- NSTEMI (n = 3), with UAP (n = 8) and non-
cular disease), patients with non-AMI coronary ACS (n = 42) and established that miR‑1
heart disease (n = 16; determined by cTnI and and -133a, yet not -208a levels, significantly

increased in patients with AMI and UAP [94] . may be useful biomarkers of myocardial
Finally, Cheng et al. measured the circulating injury in the plasma of AMI patients, these
serum levels of miR‑1 in patients with AMI studies are underpowered (too low numbers
(n = 31) and age- and sex-matched healthy con- of patients) to definitively access the diag-
trols (n = 20) and found the miR‑1 level sig- nostic importance of these miRNAs. Recent
nificantly increased in patients with AMI and publications using larger cohort studies have
had a positive correlation with serum CK-MB further tested this hypothesis. For example,
levels [89] . Devaux et al. utilized a very large, well-defined
Given that expression levels of miR‑1, patient population with documented times of
-133a/b, -208a/b and -499 may be useful bio- blood draw after onset of acute ongoing chest
markers of myocardial injury in the plasma pain and measured miR‑208b and -499 lev-
of patients clinically diagnosed with AMI, De els in plasma samples isolated from STEMI
Rosa et al. investigated whether or not myocar- (n = 397), NSTEMI (n = 113) and healthy
dial injury resulted in the release of miRNAs controls (n = 87) [97] . They found that circu-
from the heart into the coronary circulation lating levels of miRNA-208b and -499 were
[96] . They simultaneously obtained plasma nearly undetectable in healthy controls and
from the aorta and the coronary sinus of were highly elevated in both STEMI and
patients with hs-cTnT-positive ACS (n = 19), NSTEMI patients [97] . In addition, plasma
with stable CAD (n = 31; angiographically miRNA-208b and -499 levels were signifi-
documented CAD but normal hs-cTnT lev- cantly higher in STEMI versus NSTEMI
els), and without CAD (n = 7; angiographic patients [97] . Both miRNAs correlated with
absence of CAD and normal hs-cTnT levels) peak concentrations of CK and cTnT; however,
[96] . Circulating plasma levels of miR‑133a, fewer patients were positive for miR‑208b or
-208a and -499 were found in higher quanti- cTnT than for miR‑499 or hs-cTnT. In this
ties in both the aorta and the coronary sinus cohort, miR‑499 showed a diagnostic accu-
in patients with hs-cTnT-positive ACS com- racy comparable with hs-cTnT and superior to
pared with patients with or without stable miR‑208b [97] . Unfortunately, the combined
CAD [96] . miR‑133a and -499 showed posi- determination of miR‑499 and hs-cTnT did
tive transcoronary concentration gradients in not significantly improve the diagnostic value
patients with hs-cTnT-positive ACS, suggest- of single biomarkers [97] .
ing that these muscle-derived miRNAs were In another large cohort study, Ai et al. mea-
indeed released into the coronary circulation sured miR‑1 and -133 in plasma samples iso-
during myocardial injury [96] . Finally, De Rosa lated from AMI patients (n = 93) and healthy
et al. also established that minute elevations in control subjects (n = 66) [98] . This study dem-
hs-cTnT levels were associated with increased onstrated that miR‑1 and not -133 levels were
miR‑499 plasma concentrations, suggesting significantly higher in plasma isolated from
that miR‑499 may be a useful biomarker to AMI patients compared with control subjects
detect even minor myocardial injury [96] . [98] . Interestingly, miR‑1 levels were well-cor-
Taken together these studies suggest that related with QRS by both univariable linear
heart-specific and/or muscle-enriched miRNAs and logistics regression analyses; however, no
(e.g., miR‑1, -133a/b, -208a/b and -499) may significant correlations were found between
serve as useful biomarkers of myocardial injury miR‑1 and ST segment or cTnI/CK-MB [98] .
in the plasma of patients clinically diagnosed In addition, Widera et al. measured the levels
with AMI. However, these experiments were of miR‑1, -133a/b, -208a/b and -499 in plasma
performed using small patient cohorts and need samples isolated from 444 patients with ACS
to be validated using larger patient cohorts. It (UAP, n = 117; NSTEMI, n = 131; STEMI,
is important to note that plasma and serum n = 196) [99] . Patients with NSTEMI or STEMI
sample miRNA profiling studies demonstrated presented with higher plasma levels of miR‑1,
identical results suggesting that it did not mat- -133a and -208b compared with patients with
ter whether or not plasma or serum was utilized UAP [99] . By contrast, the circulating plasma
as the starting material [90] . concentrations of miR‑133b, -208a and -499 did
not increase in patients with AMI when com-
Plasma miRNAs as biomarkers in pared with those with UAP [99] . Unfortunately,
large patient ACS cohorts the diagnostic usefulness of these miRNAs for
Although the reports described above sug- the discrimination of patients with AMI from
gest that miR‑1, -133a/b, -208a/b and -499 those with UAP may be limited given that all six

miRNAs showed a large overlap between these based on the release of miRNAs, but rather
types of patients [99] . from the necrosis of cardiac cells, as well as on
Finally, Olivieri et al. investigated whether active processes involved in the pathogenesis of
or not miRNAs could serve as sensitive and AMI (e.g., inflammation, plaque rupture and
specific early biomarkers of NSTEMI in a large vascular injury). To test this hypothesis, Meder
geriatric cohort study [82] . This was an impor- et al. investigated the expression of miRNAs on
tant study given that the diagnosis of AMI in a whole-genome level in total peripheral blood
geriatric patients is often challenging since of patients with STEMI (n = 20) and control
the elderly frequently present with atypical subjects without ACS (n = 20) [101] . These
NSTEMI symptoms and nondiagnostic ECGs investigators determined that 121 miRNAs
[100] . Furthermore, the diagnostic value of cTnT (45 upregulated, 76 downregulated) were
in these patients can also be flawed since the significantly dysregulated in whole-blood
modest increase observed in this biomarker samples isolated from AMI patients in com-
upon presentation at the hospital cannot read- parison with controls [101] . Noticeably, miR‑1,
ily be differentiated from non-cTn sources due -133a/b, -208a/b and -499 were not among
to other pathological conditions (e.g., acute the 20 most dysregulated miRNAs detected
decompensated heart failure [ADHF] in the in this study, even though the blood was
absence of CAD or pulmonary embolism) [100] . drawn within 3 h after the reported onset of
Therefore, Olivieri et al. measured the plasma symptoms [101] . These investigators found that
levels of miR‑1, -21, -133a, -208a, -423-5p miR‑1291 and -663b were the most predictive
and -499-5p in samples obtained from elderly for AMI, with the area under the curve val-
NSTEMI patients (n = 92; 82.6 ± 6.9 years old; ues 0.91 and 0.94, respectively [101] . They also
AMI complicated by ADHF in 74% of cases), identified two upregulated miRNAs in AMI
elderly patients with acute congestive heart patients, miR‑30c and -145, which showed
failure without AMI (n = 81; 81.3 ± 6.8 years high correlation with hs-cTnT as a measure
old; patients with ADHF due to noncoronary of infarct size [101] . Importantly, a combina-
etiology, termed nonischemic cardiomyopa- tion of 20 miRNAs (miR‑142-5p, -498, -492,
thy) and healthy age-matched control subjects -1281, -497*, -151-5p, -802, -23b*, -455-3p,
(n = 99) [82] . These investigators reported that -1250, -380*, -135b*, -345, -566, -631, -1254,
circulating miR‑1, -21 -133a, -423-5p and -139-5p, -892b and -146b-3p) exhibited an
-499-5p levels were significantly increased in area under the curve value of 0.99, indicating
NSTEMI patients versus control subjects and that a miRNA signature may serve as a superior
miR‑21 and -499-5p levels were also higher in diagnostic biomarker for AMI [101] .
NSTEMI versus ADHF without AMI patients To date, only one additional study examin-
[82] . Interestingly, miR‑208a was undetectable ing miRNA expression in whole-blood samples
in the patient plasma samples isolated from isolated from AMI patients (n = 51) has been
individuals representing each group (n = 10) published [102] . Even though these investigators
and therefore it was excluded from additional only measured circulating miR‑133 and -328
analyses [82] . Importantly, miR‑499-5p was (this miRNA may contribute to adverse electri-
comparable with cTnT in discriminating cal remodeling in atrial fibrillation [103]) levels
NSTEMI versus ADHF without AMI patients in these patients, they found that the expres-
and control subjects and its diagnostic accuracy sion levels of both miRNAs were increased in
was higher than cTnT and hs-cTnT in differen- the whole blood and in plasma samples from
tiating NSTEMI (n = 31) versus ADHF with- AMI patients compared with those in control
out AMI (n = 32) patients with modest cTnT subjects [102] . These results suggest that circu-
elevation at presentation [82] . lating miR‑133 and miR‑328 may be promising
biomarkers for diagnosing AMI. In addition,
Whole-blood miRNAs as biomarkers since miR‑133 and miR‑328 levels remained
in small ACS patient cohorts unchanged in the presence of cardiac dys-
Given that the circulating miRNAs detected rhythmias, and cTns are known to be elevated
in whole blood can originate from a number of in the presence of dysrhythmias (even in the
sources (e.g., erythrocytes, PBMCs, platelets, absence of CAD), these miRNAS may have
exosomes, MPs, ABs, miRNA–protein com- a benefit beyond what cTns currently offer;
plexes and HDL/miRNA particles), it may be that is, these miRNAs may help differentiate
possible to identify a miRNA signature for the cardiac myocyte damage from CAD versus
diagnosis and/or prognosis of AMI not solely dysrhythmia [102] .

PBMC miRNAs as biomarkers in discriminatory ability of hs-cTnT between

small ACS patient cohorts survivors and nonsurvivors [99] .
It is well known that immune cells, including Given that comparisons between STEMI,
monocytes and T cells, play a critical role in the NSTEMI, and/or UAP patients and/or healthy
development of atherosclerosis and the onset of controls are potentially confounded by issues
ACS [83] . T cells in intimal lesions recognize such as age, sex, medication, comorbidities and
local antigens and mount Th1 responses with common cardiovascular risk factors, a prospec-
secretion of proinflammatory cytokines, thus tive experimental design where plasma samples
contributing to local inflammation and growth for measurement of miRNAs were drawn prior to
of the plaque [83] . Given that enhanced Th1 lym- ACS would optimally determine whether or not
phocyte activation occurs in patients with ACS miRNAs could be used as prognostic biomarkers
[104] and that miR‑146a is involved in innate of AMI. Importantly, a recent prospective study
immune responses [105] , Guo et al. hypothesized published by Zampetaki et al. [86] explored the
that this miRNA may play a role in the onset association between plasma miRNA baseline
of ACS [84] . To test this hypothesis, they mea- levels (1995) and incident AMI (1995–2005)
sured miR‑146a levels in PBMCs isolated from in 820 participants of the Bruneck, Italy cohort
patients with AMI (n = 26), UAP (n = 25), defined in [106] . These investigators quantified
SA (n = 22) and chest pain syndrome (CPS; 19 candidate miRNAs, of which baseline levels
n = 18). Importantly, they found that miR‑146a (1995 evaluation) of miR‑126 showed a positive
expression levels were significantly increased in association (multivariable statistical analysis),
patients with AMI and UAP versus the SA and and miR‑197 and -223 an inverse association
CPS individuals [84] . Finally, these investigators with subsequent myocardial infarctions over a
demonstrated that ACS-purified patient PBMCs 10-year observation period (1995–2005) [86] .
or the overexpression of miR‑146a in PBMCs Interestingly, in a subanalysis, miR‑223 showed
resulted in enhanced Th1 differentiation [84] . a stronger association with fatal than nonfa-
In support of this study, Yao et al. tested the tal myocardial infarctions and miR‑126 and
hypothesis that inflammation-related miRNAs -197 were predominantly associated with early
(i.e., miR‑155, -146a, -21, -125a-5p, -125b and cardiovascular events. Finally, the data from
-31) were dysregulated in PBMCs isolated from Zampetaki et al. [86] suggest that platelet MPs
patients with AMI (n = 16), UAP (n = 15), SA containing miR‑197 and -223, and endothelial
(n = 15) and CPS (n = 18) [85] . These investiga- cell MPs harboring miR‑126, are the probable
tors found that miR‑21 and -146a levels were sources for circulating miRNAs measured in this
higher and miR‑155 levels were lower in patients study [86] .
with AMI and UAP versus patients with SA Recently, Matsumoto et al. investigated the
and CPS [85] . Importantly, the expression pat- prognostic capabilities of circulating miRNAs
terns for miR‑21 and -155 in plasma samples in patients who survived AMI by creating a
correlated with those observed in PBMCs iso- miRNA signature of banked serum samples of
lated from these patients [85] . Taken together, these individuals at the time of discharge [107] .
these studies suggest that inflammation-related In the discovery phase, serum samples from
miRNAs in patients with ACS may be useful patients who died of a cardiac cause within
as biomarkers. 1 year after discharge (n = 7) and those who
did not experience any cardiovascular events
Prognostic circulating miRNAs for during the 3-year follow-up period (n = 7) were
ACS patients analyzed by a high-throughput array consist-
As described above, Widera et al. demonstrated ing of 667 miRNAs [107] . Eleven miRNAs were
that although the plasma levels of miR‑1, found to be differentially expressed in the serum
-133a and -208b were higher in patients with among patients at high-risk for cardiac death
NSTEMI and STEMI compared with patients with five miRNAs upregulated (miR‑18a, -134,
with UAP, they were diagnostically limited -155, -192 and -380*) and six downregulated
[99] . However, importantly, this was the first (miR‑93*, -125a-5p, -190b, -212, -223* and
study to establish that miRNAs can also pro- -331-3p) [107] . Importantly, in the validation
vide prognostic information in ACS given phase, only two (miR‑155 and -380*) of the
that circulating concentrations of miR‑133a eleven miRNAs were confirmed by quantitative
and -208b were significantly associated with PCR (qPCR) to be augmented in the cardiac
all-cause mortality (n = 34 ) at 6 months [99] . death group (n = 19) as compared with the sur-
Nevertheless, neither miRNA enhanced the vival group (n = 21) [107] . Although this is a small

study requiring independent confirmation, this the circulating levels of miRNA biomarkers.
study suggests that circulating miRNAs might Given that 90% of miRNAs in the circulation
be predictive for cardiac death in post-AMI are present in nonvesicle-encapsulated forms
patients. [75] , qPCR and miRNA array results may be
more consistent if only miRNA–protein com-
Future perspective plexes are utilized as starting material. Alter-
The potential of circulating miRNAs as blood- natively, it may be more appropriate in certain
based biomarkers for cardiovascular disease is disease states to utilize exosomes, MPs or APs
promising because they offer several advan- as the starting material to investigate the abil-
tages over protein biomarkers. First, circulating ity of circulating lipid vesicle miRNA sources
miRNAs are relatively stable in human blood. as biomarkers [111] . Then again, it may be
Second, miRNAs can be amplified and detected more useful to use circulating cellular miRNA
with high sensitivity and specificity. Third, many sources as biomarkers, such as PBMCs [84,85] ,
miRNAs can be quantified in a single experiment platelets [112] or endothelial progenitor cells [113] ,
by qPCR and/or miRNA array methodology. dependent upon the disease state being investi-
That being said, miRNA biomarkers also have gated. Finally, a recent report suggests utilizing
several inherent disadvantages. First, the concen- plasma/serum miRNAs as biomarkers may be
trations of most circulating miRNAs are typically problematic since these sources may be con-
very low, which makes reliable quantification and taminated with miRNAs that originated from
normalization very challenging. Second, the blood cells [114] .
amplification and detection methodologies uti- Total RNA isolation from any circulating
lized in qPCR and miRNA array platforms have miRNA source and subsequent quantification
thermodynamic biases [108] . Third, quantifica- by qPCR or miRNA array methodology, as
tion of circulating miRNAs is greatly influenced currently performed, is quite time-consuming.
by the sources of these miRNAs (e.g., erythro- Therefore, to allow for the broad utilization of
cytes, PBMCs, platelets, exosomes, MPs, ABs, miRNAs as biomarkers in the future, new tech-
miRNA–protein complexes and HDL/miRNA nologies must be developed that would more
particles). Fourth, qPCR and microarray tech- quickly provide results, ideally in the form of
nologies are still time-consuming compared with, a bedside test.
for example, the cTn biomarker test. In conclusion, the studies reviewed above
What can be done to overcome these disad- (Ta bl e s 1 & 2) support the hypothesis that
vantages? The variability in the sources of start- circulating cardiac and skeletal muscle cell-
ing material, amount of starting material, RNA enriched miRNAs might be useful as diagnos-
extraction protocols, miRNA detection methods tic and prognostic biomarkers for AMI. How-
and normalization procedures can lead to incon- ever, these studies need to be validated using
sistencies in the identification of the miRNAs appropriately designed studies with much larger
that may be used as biomarkers for ACS. It is patient cohorts, properly matched cases and
critically important that these technical param- controls, and data subjected to multivariable
eters be standardized. Furthermore, the utiliza- statistical analyses that takes into account mul-
tion of new miRNA detection technologies that tiple potentially confounding parameters (e.g.,
eliminate thermodynamic biases (next-genera- age, sex, medication and comorbidities). Finally,
tion sequencing platforms) need to be incorpo- recent studies demonstrate that the evaluation
rated into ACS biomarker studies. Importantly, of individual miRNA changes in the context
next-generation sequencing has successfully of the overall miRNA network improves their
been utilized to investigate the miRNA profiles diagnostic and prognostic value [86,115] .
of serum samples isolated from patients with
cancer [109,110] . Financial & competing interests disclosure
More thought must be devoted to investi- The authors have no relevant affiliations or financial
gating the origins of circulating miRNAs in involvement with any organization or entity with a finan‑
biomarker studies since they can be harbored in cial interest in or financial conflict with the subject matter
any circulating cell type, several unique catego- or materials discussed in the manuscript. This includes
ries of lipid vesicles or protein-protected com- employment, consultancies, honoraria, stock ownership or
plexes [70–77] . Therefore, qPCR and miRNA options, expert testimony, grants or patents received or
array results will vary greatly depending upon pending, or royalties.
the origin of circulating miRNAs in either No writing assistance was utilized in the production of
healthy or sick people, or the factors influencing this manuscript.

Executive summary
Acute coronary syndrome
Acute coronary syndrome (ACS) is defined as the symptomatic, clinical presentation of coronary artery disease.
The etiology of over 50% of patients with ACS is acute myocardial infarction (AMI).
ACS results from three causes: ST elevation myocardial infarction, non-ST elevation myocardial infarction or unstable angina pectoris.
Delays in ‘ruling in’ AMI increase morbidity and mortality due to the time lag in initiating therapy.
Delays in ‘ruling out’ AMI decreases emergency department triage efficiency, increasing overall medical costs and most importantly
augments morbidity and mortality.
Cardiac biomarkers in ACS
AMI results in myocardial tissue damages and as a consequence intracellular contents including proteins are released into the
extracellular space and peripheral blood.
Currently cardiac troponin I and cardiac troponin T are the preferred biomarker for the diagnosis of AMI.
miRNA biogenesis & function
miRNAs are transcribed as pri-miRNAs that are subsequently matured in a multistep biogenesis process to generate the functional
nonprotein-coding ssRNA (20–25 nucleotides).
Most miRNAs are embedded within the introns of protein-coding genes and cotranscribed with the host.
With the miRNA acting as an adaptor for the miRNA-induced silencing complex, complex-bound mRNAs are subjected to translational
repression and/or degradation following deadenylation.
Circulating miRNAs as biomarkers
miRNAs found circulating in the peripheral blood display remarkable stability and therefore may be useful as biomarkers.
miRNAs found circulating in the peripheral blood can be accurately and rapidly detected.
Circulating miRNAs are detectable in all circulating cell types (e.g., endothelial progenitor cells, erythrocytes, peripheral blood
mononuclear cells and platelets).
Circulating miRNAs can also be packaged and protected by several distinct types of lipid vesicles including exosomes, microparticles
and apoptotic bodies.
The majority of circulating miRNAs are nonvesicle-encapsulated (i.e., bound and therefore protected from degradation by RNA-binding
proteins).
Circulating miRNAs as biomarkers for ACS
Heart-specific and/or muscle-enriched miRNAs (e.g., miR‑1, -133a/b, -208a and -499) leak into the circulation during AMI, therefore
these miRNAs can be utilized to detect and monitor myocardial injury.
miR‑1, -133a/b, -208a and -499 leak into the circulation during AMI in a time-dependent manner.
miR‑1, -133a/b, -208a and -499 levels are elevated in the plasma/serum isolated from patients with ST elevation myocardial infarction
and non-ST elevated myocardial infarction, but not in patients with unstable angina pectoris.
In patients with AMI, miR‑133a and -208b levels have been shown to be significantly associated with all-cause mortality.
miR‑126 has shown a positive association (multivariable statistical analysis), and miR‑197 and -223 an inverse association with
subsequent AMIs.
Future perspective
miRNA isolation and assay standardization must be achieved.
The origin of circulating miRNAs must be closely scrutinized.
miRNA biomarker candidates must be validated in large, well-characterized patient cohorts.
All miRNA biomarker data need to be subjected to multivariable statistical analysis that account for confounding parameters (e.g., age,
sex, medication and comorbidities).
miRNAs biomarkers need to be evaluated in the context of overall miRNA networks to improve their diagnostic and prognostic value.
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Circulating miRNAs: Novel biomarkers of acute coronary syndrome?

Article in Biomarkers in Medicine · April 2013

Helina Selemon Shane M Elton

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Circulating miRNAs: novel biomarkers of acute

comprehensive therapeutic approaches, includ-

QRS based on well-validated risk stratification calcu-

Cardiac biomarkers in ACS

288 Biomarkers Med. (2013) 7(2) future science group

ST elevation ST depression or T inversion

STEMI UAP NSTEMI

future science group www.futuremedicine.com 289

Multiples of the AMI cut-off limit

Troponin (moderate MI)

290 Biomarkers Med. (2013) 7(2) future science group

miRNA gene or intron

Processing Drosha Exportin-5

future science group www.futuremedicine.com 291

292 Biomarkers Med. (2013) 7(2) future science group

future science group

encapsulated, but rather were bound by RNA-

binding proteins. These investigators identified

the direct binding of NPM1 protected some

cally characterize circulating miRNA com-

tigators subsequently determined that a sig-

nificant portion of circulating miRNAs were

miRISC (Figure 4) [75] . In support of this study

culture are bound to Ago2 and not associated

with exosomes [76] . It was hypothesized that

18 CPS, 15 SAP, 15 UAP,

[76] . However, it is also possible that specific

release of Ago2–miRNA complexes results

from unidentified cell membrane-associated

high-density lipoprotein (HDL) particles

294 Biomarkers Med. (2013) 7(2) future science group

Table 2. Studies of prognostic circulating miRNAs in acute coronary syndrome.

averaging 8–12 nm in size) transport endog- pathology-specific manner; and detectable

future science group www.futuremedicine.com 295

296 Biomarkers Med. (2013) 7(2) future science group

future science group www.futuremedicine.com 297

298 Biomarkers Med. (2013) 7(2) future science group

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 PBMC miRNAs as biomarkers in discriminatory ability of hs-cTnT between

300 Biomarkers Med. (2013) 7(2) future science group

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302 Biomarkers Med. (2013) 7(2) future science group

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