1222?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 122? ? ? Number 4? ? ? April 2012
MicroRNA-214 protects the mouse heart
from ischemic injury by controlling
Ca2+ overload and cell death
Arin B. Aurora,1 Ahmed I. Mahmoud,2 Xiang Luo,2 Brett A. Johnson,1 Eva van Rooij,3
Satoshi Matsuzaki,4 Kenneth M. Humphries,4 Joseph A. Hill,2 Rhonda Bassel-Duby,1
Hesham A. Sadek,2 and Eric N. Olson1
1Department of Molecular Biology and 2Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.
3miRagen Therapeutics, Boulder, Colorado, USA. 4Free Radical Biology and Aging Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA.
Cardiovascular disease affects more than 80 million people in the
United States and is the leading cause of death in the developed
world (1). Recent studies have revealed that microRNAs (miRNAs)
play an indispensable role in various facets of cardiac function
through their repression of target mRNAs (2). miRNAs exert their
repressive functions by binding to sequences in the 3ʹ-UTRs of tar-
get mRNAs that have complementarity to nucleotides 2–8 of the
miRNA, known as the seed region. miRNAs mediate numerous
cellular processes associated with cardiac remodeling and disease,
including myocyte hypertrophy (3–9), fibrosis (10–13), angiogen-
esis (14–16), and apoptosis (17–21).
Cardiac ischemia, typically as a consequence of vessel occlusion,
is often followed by a second set of stresses during restoration of
blood flow to the tissue, known as ischemia/reperfusion (IR) inju-
ry, which can account for up to half of total infarct size (22). The
factors contributing to IR injury are complex and include micro-
vascular dysfunction, inflammation, release of oxygen radicals,
disruption of Ca2+ homeostasis, and activation of mitochondrial
apoptosis and necrosis. Cardiac failure results from the cardiomyo-
cyte dropout brought about by these sequelae. Several miRNAs have
been implicated in IR injury (19–21, 23–25), but there have been no
genetic loss-of-function studies demonstrating the mechanism of
action of individual miRNAs in this pathological process.
Ca2+ is central to cardiac contraction and to the signaling net-
works that regulate pathological cardiac growth and remodeling.
Intracellular Ca2+ overload can occur in cardiomyocytes as a con-
sequence of ischemic injury or other stresses, leading to contrac-
tile dysfunction and ultimately cell death (26, 27). Ca2+ handling
is orchestrated by a set of proteins, including the L-type calcium
channel sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2)
pump, ryanodine receptor (RyR) channel, and sodium/calcium
exchanger 1 (NCX1). Attenuation of Ca2+ overload with therapeu-
tics targeting these proteins provides cardioprotection in some set-
tings of IR (28–30), but clinical trials are limited by variables such
as the effects of chronic inhibition of Ca2+ handling and timing of
administration, and therefore future studies are needed to justify
the usefulness of such treatments. The uncertainties surrounding
these therapies highlight the importance of understanding the
regulatory mechanisms that govern Ca2+ handling protein expres-
sion and function (31).
Ca2+ overload leads to cardiomyocyte death via signals trans-
mitted through downstream effectors of Ca2+ handling (32). One
intracellular sensor of Ca2+ ions, calmodulin, interacts through the
calcium/calmodulin-dependent protein kinases (CaMKs) to regu-
late cardiomyocyte function and control cardiac hypertrophy and
heart failure (32). Both apoptosis and necrosis can contribute to
cardiomyocyte loss in response to Ca2+ overload by activating pro-
death members of the Bcl2 family and opening the mitochondrial
permeability transition (MPT) pore, respectively (26).
By analyzing conserved miRNAs that were upregulated in mul-
tiple disease models of hypertrophy and heart failure, we identi-
fied miRNA-214 (miR-214) as a sensitive marker of cardiac stress
(5). Here we show that miR-214 plays a protective role against IR
injury by attenuating Ca2+ overload–induced cardiomyocyte death
through repression of NCX1 and downstream effectors of Ca2+ sig-
naling and cell death. These findings provide new insights into the
Conflict?of?interest: Eric N. Olson and Eva van Rooij are cofounders of miRagen
Therapeutics, a company focused on developing miRNA-based therapies for
Citation?for?this?article: J Clin Invest. 2012;122(4):1222–1232. doi:10.1172/JCI59327.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 4 April 2012
molecular basis of heart disease and point to miR-214 as a poten-
tial therapeutic target in this setting.
miR-214 genomic structure and expression. miR-214 is highly conserved
across vertebrates and is encoded within a larger non-coding RNA,
Dnm3 opposite strand (Dnm3os). It is transcribed together with
miR-199a-2 from the opposite strand of the Dnm3 gene on mouse
chromosome 1 (Figure 1A). miR-214 is upregulated in response to
a variety of cardiac stresses, including pressure overload, myocar-
dial infarction (MI), and overexpression of the calcium/calmodu-
lin-sensitive phosphatase calcineurin (5, 12). Since many genes
activated during cardiac stress are also expressed developmentally,
we examined the temporal expression pattern of miR-214. Robust
expression of miR-214 at early embryonic stages in the heart (Fig-
ure 1B) was downregulated by E15.5 and further decreased in
adult mice. Expression could be detected in several adult tissues
by Northern blot analysis (Figure 1C).
Targeted deletion of miR-214 in mice. To explore the functions of
miR-214 in vivo, we used homologous recombination to generate
a conditional targeted deletion of the gene in mice. Our target-
ing strategy introduced loxP sites flanking the genomic region
encompassing the 106-bp pre-miR and a neomycin resistance
cassette (Supplemental Figure 1A; supplemental material avail-
able online with this article; doi:10.1172/JCI59327DS1). Chime-
ric mice obtained from targeted ES cells transmitted the mutant
allele through the germline, yielding mice heterozygous for
miR-214(neo) (Supplemental Figure 1B). We bred these mice to
CAG-Cre transgenic mice to remove the neomycin cassette and
targeted region of the miR-214 locus. Breeding of the offspring of
these crosses generated homozygous miR-214 KO mice, which were
born at Mendelian ratios and were fertile (Supplemental Table 1).
miR-214 KO mice displayed a minimal, but not statistically sig-
nificant, reduction in body weight compared with WT littermates
(Supplemental Figure 2A). Northern blot analysis confirmed the
absence of miR-214 expression in the heart (Figure 1D), while the
expression of miR-199a was preserved. Expression of miR-199a-2
in miR-214 KO mice was confirmed by quantitative real-time PCR
(qPCR). Expression of the Dnm3 host gene on the opposite strand
was also maintained (Supplemental Figure 1C). RT-PCR spanning
the entire Dnm3os transcript in WT and miR-214 KO mice veri-
fied that splicing of the long non-coding RNA was not disrupted
(Supplemental Figure 1D).
miR-214 mutant mice have normal cardiac structure and function
at baseline. Hearts of miR-214 KO mice at 8, 12, and 24 weeks
appeared normal by gross examination and histological analysis
(Figure 2A and data not shown). Heart weight/body weight and
heart weight/tibia length ratios, cardiomyocyte size, and cardiac
function were also indistinguishable between miR-214 KO and
WT littermates at 8–12 weeks of age (Figure 2A, Figure 3C, and
Supplemental Figure 2C). We examined expression of collagen
and fetal cardiac genes as an indication of underlying defects
in the hearts of adult miR-214 KO mice, but saw no significant
differences from WT (Figure 2B). Sarcomere structure and mito-
chondrial structure and volume also appeared normal in the
miR-214 KO hearts, as assessed by electron microscopy (Figure 2C
and Supplemental Figure 2B). We performed similar analyses of
hearts from 10- to 12-month-old miR-214 KO mice and WT lit-
termates and saw no significant differences in heart/body weight
ratios, fetal gene expression profiles, cardiac function, or cardio-
myocyte size upon aging (Supplemental Figure 3, A–D).
While the above data did not show differences between WT and
miR-214 KO mice, microarray analysis of hearts from miR-214 KO
and WT littermates at 2 and 8 weeks of age revealed that several
miR-214 genomic structure and genetic
deletion. (A) Schematic representa-
tion of the mouse miR-214 locus and
its host gene, Dnm3. Boxes represent
exons of the Dnm3 gene. miR-214 and
miR-199a-2 are clustered on the oppo-
site strand within the non-coding RNA
Dnm3os. Conservation of miR-214 is
shown, and the seed region is highlight-
ed. (B) miR-214 expression levels in the
heart at various embryonic and postna-
tal stages according to miR-214–specific
RT and qPCR. Data were normalized to
RNU6B and expressed relative to lev-
els at P1. (C) Northern blots show the
relative expression level of miR-214 in
WT adult mouse tissues. E10.5 heart
RNA is included as a reference. U6 is a
loading control. Blot is representative of
2 different sets of tissues analyzed. (D)
Absence of miR-214 and preservation
of miR-199a expression in KO hearts as
shown by representative Northern blot
from heart tissue of WT, heterozygous
(HET), or miR-214 KO mice. U6 is a
loading control. Sk, skeletal.
1224? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 4 April 2012
metabolic and Ca2+-handling genes were upregulated in miR-214–
deficient hearts (Supplemental Table 2).
miR-214 protects mice against IR injury. The altered metabolic and
Ca2+-handling microarray profiles of miR-214 KO hearts led us to
hypothesize that the mutant mice might respond differently to
ischemic cardiac injury. Indeed, permanent ligation of the left ante-
rior descending coronary artery (LAD) in mice, which induces MI,
resulted in a significant increase in mortality in miR-214 KO mice
compared with WT controls (Figure 3A). Transient ligation of the
LAD in mice causes cardiomyocyte loss and impaired cardiac func-
tion that mimics the pathology seen in IR injury in human hearts.
By Northern blot analysis, we observed upregulation of miR-214
in the hearts of WT mice subjected to 45 minutes of ischemia and
1 or 7 days of reperfusion (Figure 3B). We tested the response of
miR-214 KO mice to the same IR procedure. Indeed, miR-214 KO
mice displayed severely impaired cardiac function 7 days after
reperfusion, as measured by transmural echocardiography, while
WT controls showed a minimal functional deficit (Figure 3C).
The extent of myocyte loss, fibrosis, and impairment of car-
diac performance varies depending on the length of the isch-
emic period during IR. We found that 45 minutes of ischemia in
WT mice resulted in transient myocyte apoptosis, detectable by
TUNEL staining 24 hours later, but not at day 7 following IR. In
contrast, miR-214 KO hearts had higher numbers
of TUNEL-positive cells after both 24 hours and
7 days of reperfusion (Figure 3D). We used desmin
staining to visualize TUNEL-positive cardiomyo-
cytes (Figure 3D). Apoptotic myocytes disassemble
their sarcomeres and downregulate contractile
proteins; therefore, desmin staining in most of the
TUNEL-positive areas was relatively dim.
To further assess the response of miR-214 KO
mice to IR, we examined cardiomyocyte size and
fibrosis. Wheat germ agglutinin staining of heart
sections after 7 days of reperfusion showed a
slight increase in cardiomyocyte size in miR-214
KO mice compared with WT controls (Supple-
mental Figure 4A). Masson’s trichrome staining
of hearts at day 7 of reperfusion revealed small
areas of fibrosis in WT hearts, while miR-214 KO
hearts contained larger fibrotic regions (Figure
3E). These findings suggest that increased and
sustained apoptosis in miR-214 KO mice after IR triggers exten-
sive fibrosis and impaired cardiac function.
IR injury initiates a complex cycle of hypoxia and cell death that
is perpetuated by inflammation, production of oxygen radicals,
Ca2+ overload, and activation of mitochondrial apoptosis (22).
Histological examination of miR-214 KO and WT hearts after
24 hours of reperfusion revealed no differences in the numbers
of infiltrating leukocytes (Supplemental Figure 4B). At baseline,
mitochondrial morphology and number also appeared unchanged
in the KO mice (Figure 2C). Enzymatic assays for electron trans-
port chain activity and superoxide production showed no differ-
ences between WT and miR-214 KO mitochondria at baseline or
at multiple time points after reperfusion (Supplemental Figure 5,
A–D). We therefore investigated the possibility that Ca2+ homeo-
stasis was regulated by miR-214.
Regulation of NCX1 by miR-214. While Ca2+ is a central mediator
of excitation-contraction coupling of cardiomyocytes, ischemic
injury causes intracellular Ca2+ overload, leading to contractile
dysfunction and ultimately cell death (26, 27). To identify candi-
date miR-214 targets involved in Ca2+ regulation, we used the pre-
diction algorithm TargetScan (http://www.targetscan.org/). One
of the top predicted targets of miR-214 was the sodium/calcium
exchanger Slc8a1 (i.e., Ncx1) mRNA. Under normal conditions,
Normal cardiac structure and function in miR-214 KO
mice at baseline. (A) Heart sections stained with H&E
from WT and miR-214 KO mice at 8 weeks of age.
Heart weight/body weight (HW/BW) and heart weight/
tibia length (HW/TL) ratios shown are representative
of adult mice at 3 different ages. Mean values ± SEM;
n = 3. Scale bar: 2 mm. (B) qPCR expression analy-
sis of cardiac stress response genes in miR-214 KO
hearts relative to WT. n = 3 per group; data are repre-
sentative of 2 separate experiments. Data are shown
as fold induction of gene expression normalized to
18S and expressed as mean ± SEM. (C) Representa-
tive electron microscopy images from a miR-214 het-
erozygote and 2 miR-214 KO hearts at 8 weeks of age
highlighting sarcomeric structure and mitochondria.
Scale bars: 1 μm (top row) and 200 nm (bottom row).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 4 April 2012
miR-214 protects the heart against MI and IR. (A) Survival curve following MI (permanent LAD ligation) in miR-214 KO mice or WT littermates.
n = 13–15, data represent mice from 3 different experiments. *P < 0.05. (B) Northern blotting and quantification of miR-214 expression in
WT hearts at baseline and following IR. miR-214 levels were normalized to U6 loading control and expressed relative to baseline. *P = 0.04,
**P < 0.01. (C) Cardiac function in WT and miR-214 KO mice before and after IR (7 days reperfusion). Quantification of left ventricular inter-
nal diameter in systole or diastole (LVIDs or LVIDd), fractional shortening (FS) and ejection fraction (EF), is shown. n = 6; data represent
mean ± SEM of 3 independent experiments. **P < 0.01, ***P < 0.001. (D) TUNEL staining in heart sections following IR. Representative images
at 24 hours and 7 days of reperfusion are shown. Scale bar: 200 μm. The percentage of TUNEL-positive nuclei was calculated. For each mouse,
sections at 3 different levels (6–8 fields per section) were counted. Bottom: Simultaneous TUNEL (green) and desmin staining (red) from
miR-214 KO heart sections following IR. Scale bar: 40 μm. (E) Masson’s trichrome staining on transverse heart sections after 7 days of reper-
fusion. Representative images shown at two different magnifications. Scale bars: 2 mm (top), 100 μM (bottom). The area of blue staining was
quantified (3 section levels per heart) and expressed as a percentage of total area. For D and E, mean ± SEM; n = 3; *P < 0.05.
1226? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 4 April 2012
NCX1 is the primary pump by which Ca2+ is extruded from car-
diomyocytes during relaxation; but during stress, the exchanger
contributes to Ca2+ overload by operating in reverse mode, result-
ing in an increased concentration of intracellular Ca2+ (33).
The 3ʹ-UTR of Ncx1 mRNA is greater than 15 kb in length and
contains 3 conserved miR-214 binding sites (sites 1–3) (Figure 4A).
To test whether miR-214 could repress these predicted sequences,
we co-transfected cells with constitutively active luciferase reporter
constructs containing regions of the Ncx1 3ʹ-UTR and miR-214
expression plasmid. Since site 3 is almost 15 kb away from sites
1 and 2 in the 3ʹ-UTR of the Ncx1 mRNA, we made two different
pmiR reporter constructs: construct 1 contained sites 1 and 2, and
construct 2 contained site 3. We observed dose-dependent repres-
sion of luciferase activity by miR-214 for the Ncx1-UTR reporter
containing site 3, and repression was abolished by mutagenesis of
the seed-binding region (Figure 4B). miR-214 also repressed activ-
ity of the reporter containing sites 1 and 2 but was not affected by
mutation of either or both sites (Supplemental Figure 6A). How-
ever, this construct contained several non-conserved sequences
that have 6-nucleotide complementarity to the miR-214 seed and
therefore likely act as additional sites for repression.
At baseline, we observed a significant increase in NCX1 protein
expression in miR-214 KO hearts (Figure 4C). NCX1 protein levels
in miR-214 KO hearts were also greater than in WT littermates at
24 hours and 7 days of reperfusion (Figure 4C), suggesting that
NCX1 is a direct target of miR-214 repression. To assess abso-
lute changes in NCX1 levels after IR, we performed a side-by-side
analysis of littermate samples from miR-214 KO and WT mice at
baseline and at 24 hours and 7 days after IR (Supplemental Figure
6, B–D). In WT mice, NCX1 levels rose slightly at 24 hours after
IR and then significantly dropped by 7 days to levels below base-
line. In contrast, in miR-214 KO hearts, NCX1 levels (which were
increased at baseline compared with WT) significantly increased at
24 hours after IR and did not significantly drop at day 7 (Supple-
mental Figure 6D). Together, these findings suggest that in the
absence of miR-214, uncontrolled increases in NCX1 lead to greater
myocyte apoptosis and injury during IR.
miR-214 regulation of Ca2+ signaling and cell death. Many miRNAs
mediate stress responses by fine-tuning multiple target mRNAs
that function in the same or parallel pathways within a cell. There-
fore, we assessed other potential miR-214 targets that might
contribute to cardiomyocyte death following IR by sensing Ca2+
overload or mediating apoptosis. Three additional miR-214 tar-
get candidates were identified. Bcl2l11 (BCL2-like 11 or Bim) is a
proapoptotic Bcl2 family member predicted to have four miR-214
binding sites: one highly conserved (site 4), one conserved through
primates (site 3), and two non-conserved sites (sites 1 and 2)
(Figure 5A). The mRNA encoding CaMKIIδ, which can drive car-
diac hypertrophy (34) and has been implicated in apoptosis and
IR injury (35, 36), contains one highly conserved predicted miR-
214 site (Figure 5A). The mRNA encoded by Ppif (the gene encod-
ing cyclophilin D) contains one predicted miR-214 site (Figure
5A). Cyclophilin D (CypD) is a major regulator of the MPT pore
required for mediating Ca2+- and oxidative damage–induced cell
death independent of the Bcl2 family pathway (37–40).
To test whether miR-214 could repress protein expression through
these predicted binding sites, we co-transfected cells with constitu-
tively active luciferase reporter constructs containing the 3ʹ-UTR
sequences for Ppif, Bim, or CamkIId and miR-214 expression plasmid.
miR-214 significantly repressed the activity of the CamkIId and Ppif
3ʹ-UTRs (Figure 5D), though we did not observe repression of lucif-
erase activity with the Bim 3ʹ-UTR construct in this assay (data not
shown). Baseline protein levels of CypD, BIM, and CaMKIIδ were
significantly elevated in miR-214 KO compared with WT hearts
(Figure 5B). Following IR, we detected increased levels of BIM and
CaMKIIδ in the miR-214 KO samples compared with controls
miR-214 regulates NCX1. (A) Predicted miR-214 binding sites in the 3ʹ-UTR of Ncx1 mRNA. Ncx1 contains 3 conserved sites. Site position
relative to beginning of the 3ʹ-UTR is indicated above. Seed and target sequences are highlighted in red, and base pairing between miR-214 and
target site marked by vertical lines. (B) Ability of miR-214 to directly repress activity of the luciferase reporter construct that contains the portion of
the Ncx1 3ʹ-UTR that includes site 3 (pmiR-Ncx1 site 3). WT and mutant Ncx1 3ʹ-UTR sequences were tested. Black triangles indicate increas-
ing amounts of transfected miR-214 expression plasmid (0, 50, 100, and 200 ng). Luciferase activity was normalized to β-galactosidase activity
and compared with empty vector measurements (pmiR empty). Luciferase assays were performed in triplicate and are representative of 2–3
independent experiments. Data are mean ± SEM. **P < 0.01, #P < 0.001. (C) NCX1 protein levels measured by immunoblotting in whole heart
lysates from miR-214 KO mice compared with WT at baseline and after IR (24 hours and 7 days). Quantification was normalized to tubulin as a
loading control and then compared with WT. Data are representative of 2 independent experiments. Mean ± SEM; n = 3. *P < 0.04, **P < 0.01.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 4 April 2012
(Figure 5C), but no changes at this time point for CypD (data not
shown). Together, the data suggest that miR-214 may further pro-
tect the heart against IR injury by directly attenuating target genes
that transmit Ca2+ overload signals and mediate cell death.
Aberrant Ca2+ handling in miR-214–deficient cardiomyocytes. The
enhanced expression of NCX1 suggested that miR-214 main-
tains Ca2+ homeostasis in cardiomyocytes under conditions of
stress. To further explore the influence of miR-214 on Ca2+ regu-
lation, we isolated cardiomyocytes from adult miR-214 KO mice
or WT controls and examined their Ca2+ handling activity at the
single-cell level using Fura-2. At physiological extracellular Ca2+
concentrations (1.8 mM), a condition wherein NCX1 operates
in Ca2+ efflux mode, the miR-214 KO cells had lower levels of
intracellular Ca2+ transients compared with controls (Figure 6,
A and B). We pulsed cardiomyocytes with 5 mM extracellular
Ca2+ to reflect the environment in the heart during IR injury.
In the presence of high extracellular Ca2+, a condition wherein
NCX1 operates in reverse mode, intracellular Ca2+ transients were
increased in the miR-214 KO cells (Figure 6, A and B). Under both
conditions, decay rates were similar between KO and WT cardio-
myocytes. These findings suggest that cardiomyocytes deficient
for miR-214 are sensitized to Ca2+ overload following IR injury as
a consequence of elevated reverse mode NCX1.
miR-214–depleted cardiomyocytes are sensitized to IR-induced cell death.
The Ca2+ abnormalities that occur during IR are complex and can
include intracellular release from mitochondria. To further reca-
pitulate these effects, we used an in vitro model of IR to confirm
that miR-214 protects cardiomyocytes from Ca2+ overload and sub-
sequent cell death by directly regulating NCX1 and other targets.
First, isolated neonatal rat cardiomyocytes in culture were trans-
fected with 15-nucleotide locked nucleic acid (LNA)–modified anti-
miRNAs against miR-214 (antimiR-214) or a 15-mer oligonucle-
otide control (against a Caenorhabditis elegans miRNA) (100 nM).
Real-time PCR showed efficient knockdown of miR-214 expression
in the antimiR-214 group compared with the 15-mer control at 72
hours after transfection (Figure 7A). We used two different mod-
els of in vitro IR to test the effects of miR-214 knockdown on IR-
mediated apoptosis in cardiomyocytes by TUNEL staining: 2 hours
of ischemia (5% CO2, 1% O2) followed by 24 hours of reperfusion
(mild IR) and 1 hour of anoxia (5% CO2, 0% O2) followed by 4 hours
of reperfusion (severe IR). Both IR conditions increased the per-
centage of TUNEL-positive cells among control transfected cardio-
myocytes, while antimiR-214 treatment further increased apoptosis
relative to control anti-miRNA (Figure 7, B and C). Knockdown of
miR-214 expression also resulted in significant increases in Ncx1,
Bim, Ppif, and CamkIId mRNAs, measured by qPCR (Figure 7D),
miR-214 regulation of Ca2+ signaling and cell death genes. (A) Predicted miR-214 binding sites in the 3ʹ-UTR of Bim, CamkIId, and Ppif mRNA.
Bim contains 4 predicted sites, while CamkIId and Ppif each contain one. Site position relative to beginning of the 3ʹ-UTR is indicated above each
panel. Seed and target sequences are highlighted in red and base pairing between miR-214 and target site marked by vertical lines. (B and C)
Target protein levels measured by immunoblotting in whole heart lysates from miR-214 KO mice compared with control at baseline (B) and after
IR (7 days) (C). Quantification is normalized to indicated loading control and then compared with WT. Data are representative of 2 independent
experiments. Mean ± SEM; n = 3. *P < 0.05, **P < 0.01. (D) Ability of miR-214 to directly repress the activity of luciferase reporter constructs that
contain the 3ʹ-UTR for CamkIId and Ppif as indicated. Transfection with or without miR-214 expression plasmid is indicated. Luciferase activity
was normalized to β-galactosidase activity and compared with empty vector measurements. Luciferase assays were performed in triplicate and
are representative of 2–3 independent experiments. Data are mean ± SEM. *P < 0.05.
1228? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 4 April 2012
supporting the in vivo evidence that they are direct miR-214 targets
in cardiomyocytes. The target regulation we observed in vitro and
subsequent sensitization to IR-induced apoptosis are supportive
of both the in vivo IR phenotype and Ca2+ handling data showing
enhanced reverse mode NCX1 activity in miR-214 KO cardiomyo-
cytes subjected to high extracellular Ca2+.
The results of this study reveal miR-214 as a central, stress-respon-
sive protector against excessive Ca2+ uptake and cardiomyocyte
cell death both in vivo and in vitro. Mice deficient for miR-214 are
sensitized to IR injury, evidenced by increased cardiac apoptosis
and fibrosis and loss of pump function. miR-214 directly inhib-
its Ncx1 mRNA such that elevated NCX1 expression in miR-214
KO mice causes increased Ca2+ overload, consistent with reverse
mode activity of the exchanger during IR. By inhibiting effectors
of Ca2+ overload signaling pathways such as CaMKIIδ, CypD, and
BIM, miR-214 can diminish the degree of cardiomyocyte death sus-
tained during IR. Cardiomyocytes lacking miR-214 have impaired
Ca2+ handling and an increased sensitivity to IR-induced apoptosis.
A model to account for the role of miR-214 in mediating Ca2+ han-
dling and cell death during ischemic injury is shown in Figure 8.
miR-214 protects the heart against IR injury. At baseline, miR-214
KO mice appear histologically and functionally normal, consis-
tent with an increasing number of reports that individual miRNAs
can be deleted in mice with minimal consequences under normal
conditions (41). While studies in vitro and in zebrafish suggest
that miR-214 controls skeletal muscle development (42, 43), we
observed no skeletal muscle abnormalities in miR-214 KO mice.
Mice lacking Dnm3os, a large non-coding RNA (lncRNA) that con-
tains miR-214, display severe skeletal defects and die within the
first month of birth, though the functions of Dnm3os have not
been reported (44). Any potential role of miR-214 in this pheno-
type is difficult to discern, since the Dnm3os deletion also elimi-
nates much of the lncRNA including miR-199a.
In contrast, miR-214 KO mice show impaired cardiac function
and susceptibility to death following MI and IR injury. Multiple
initiators in IR injury including inflammation, oxygen radicals,
and Ca2+ overload contribute to the loss of cardiomyocytes and the
progression to heart failure (27). The cardioprotective effects of
miR-214 correlate with the repression of NCX1, CaMKIIδ, CypD,
and BIM. However, miR-214 may have additional targets that con-
tribute to its function during cardiac stress.
Ca2+ homeostasis during cardiac stress: regulation by miR-214 and NCX1.
In WT mice, we found that NCX1 expression increased at 24 hours
of reperfusion and then declined below baseline levels by 7 days
(Supplemental Figure 6, B–D). Elevated miR-214 expression (Figure
3B) presumably counteracts upregulation of NCX1 and continues
to increase such that by 7 days of reperfusion, miR-214 downregu-
lation of NCX1 is sufficient to reestablish Ca2+ homeostasis in car-
diomyocytes. In miR-214 KO hearts, NCX1 expression was elevated
at baseline, predisposing cardiomyocytes to Ca2+ overload. Further-
more, without upregulation of miR-214 following IR, NCX1 levels
continued to increase unchecked (Supplemental Figure 6, B–D).
NCX1 counter-transports sodium and Ca2+ across the sarco-
lemmal membrane and, at baseline conditions in the heart, is one
of the major pathways through which intracellular Ca2+ effluxes
out of cardiomyocytes. However, it is well documented that dur-
ing various forms of cardiac stress, including IR injury, the NCX1
transporter works in reverse mode to pump Ca2+ back into the cell.
Furthermore, reverse mode NCX1 can induce Ca2+-induced Ca2+
release from the SR, leading to additional Ca2+ overload, injury,
and cardiac dysfunction (45, 46).
Consistent with our model, transgenic mice that overexpress
NCX1 in the heart phenocopy the exacerbated response to IR
observed with miR-214 KO mice, ultimately resulting in reduced
cardiac function and survival (47–49). NCX1 upregulation has also
been observed in cardiac hypertrophy (50–52) in which miR-214 is
also upregulated. Together, these findings suggest that repression
of reverse mode NCX1 by miR-214 following IR attenuates Ca2+
overload and apoptosis in the heart and that a similar mechanism
may be critical in other settings of cardiac stress.
High extracellular Ca2+ levels in vitro mimic conditions of car-
diac stress such as IR, where cardiomyocytes are exposed to sig-
nificant increases in Ca2+ from surrounding tissue. Interestingly,
induced overexpression of NCX1 in cardiomyocytes leads to simi-
lar or lower levels of intracellular Ca2+ transients in the presence
of physiological extracellular Ca2+, but higher transients in the
presence of high extracellular Ca2+ (53). In miR-214 KO myocytes,
we observed similar results, suggesting that the increased expres-
miR-214 is required to maintain efficient Ca2+ handling. (A) Rep-
resentative Ca2+ traces obtained from isolated WT or miR-214 KO
cardiomyocytes. Myocytes were imaged in the presence of 1.8 mM
or 5.0 mM extracellular Ca2+. n = 15–20 cells, with 3–5 animals per
group for all experiments. (B) Quantitative analyses of intracellular
Ca2+ levels shown as corrected average peak Ca2+ transients in WT
and miR-214 KO cardiomyocytes in 1.8 mM or 5.0 mM extracellular
Ca2+ (top). Ca2+ transient decay shown as τ (seconds) comparing WT
and KO at 1.8 mM and 5.0 mM extracellular Ca2+ (bottom). All data
are mean ± SEM; ***P < 0.0001.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 4 April 2012
sion of NCX1 in KO cells working in reverse mode contributes to
Ca2+ overload. Since NCX1 can contribute to Ca2+ efflux during
relaxation, one might expect to see an increased rate of transient
decline with increased NCX1 expression. However, in our study
and others, changes in NCX1 expression do not impact the rate
of Ca2+ decline (refs. 53, 54, and Figure 6B). The variable effects on
decline rate observed with NCX1 overexpression seem to depend
on the species studied, differences in temperature, and the phase
of the decline that was evaluated (55–57).
miR-214 regulation of Ca2+ signaling and cardiomyocyte death. Intra-
cellular Ca2+ levels are modulated by the activity of numerous
channels, pumps, and exchangers and then transduce signals to the
cell through downstream effectors. Elevation in intracellular Ca2+
activates CaMKIIδ, the main isoform of CaMKII expressed in the
heart, allowing it to modulate many other Ca2+-handling proteins
including PLB, RyR, and L-type Ca channels (58). CaMKIIδ plays a
key role in several types of heart disease including IR, and CaMKII
inhibition is protective against IR-induced cell death and contrac-
tile dysfunction (35, 59). In miR-214 KO mice, elevated CaMKIIδ
levels could therefore contribute to additional cardiomyocyte loss.
Ca2+ overload activates both apoptotic and necrotic pathways of
cell death. Ischemic injury in the heart is accompanied by decreased
expression of Bcl-2 and increased expression of proapoptotic Bcl-2
family members that modulate mitochondria-dependent apoptosis
(26, 60). For example, mice lacking BNIP3, a proapoptotic family
member, show decreased apoptosis and left ventricular remodel-
ing following IR injury (61). Our data suggest that another pro-
apoptotic Bcl-2 family member, BIM, is elevated in the miR-214 KO
heart and may be a target of miR-214. While the role of BIM has not
been extensively studied in the heart, data suggest it is regulated
during ischemic injury and that its decreased expression hallmarks
the rescue of cardiomyocyte apoptosis following IR (62, 63).
Ca2+ overload also stimulates necrotic cell death through open-
ing of the MPT pore, which has recently been shown to play a
major role in heart failure (39, 40). CypD (the Ppif gene product),
a prolyl isomerase, is a key regulatory component of the MPT
pore. Ppif KO mice are protected from Ca2+ overload and oxidative
induced cell death and therefore are resistant to IR-induced cardi-
ac injury (39). In contrast, mice that overexpress CypD show spon-
taneous cell death. We show increased CypD expression in miR-
214 KO hearts at baseline and in cardiomyocytes with depleted
miR-214, suggesting together with our luciferase data that Ppif is
a direct miR-214 target and may sensitize KO cardiomyocytes to
Ca2+ overload–mediated cell death. We did not observe elevated
protein levels of CypD in miR-214 KO hearts at 7 days after IR.
We speculate that a miR-214–independent mechanism allows for
repression of CypD following IR to protect the heart from further
cell death and loss of contractility.
miR-214 protection of cardiomyocytes and regulation of target genes in vitro. (A) RNA isolated from neonatal rat cardiomyocytes transfected
with antimiR-214 or 15-mer control (100 nM) was analyzed by miR-214–specific RT-PCR and qPCR to assess levels of miR-214 suppres-
sion. **P < 0.01. (B and C) TUNEL staining of neonatal rat cardiomyocytes transfected with control or antimiR-214 and then subjected to in
vitro simulation of IR. (B) Representative images of total nuclei (blue) and apoptotic nuclei (red) from normoxic and both IR conditions are
shown for control and antimiR-214 transfected samples. Scale bar: 100 μm. (C) Quantification of percent TUNEL-positive nuclei. Samples
were assayed in triplicate, and results are representative of 2–3 independent experiments. Data are mean ± SEM; **P < 0.01. (D) Levels of
miR-214 target mRNAs in cardiomyocytes transfected with antimiR-214 or control antimiR as indicated. Data were normalized to ribosomal
18S and expressed relative to control. Samples were run in triplicate, and results are representative of 2 independent experiments. All data
are mean ± SEM; *P = 0.02, **P < 0.01.
1230?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 4 April 2012
Significance of miRNAs in cardiac IR. Several miRNAs are regulated
in response to IR injury (reviewed in ref. 23) and are reported to
modulate cardiomyocyte cell death and contractility (20). However,
the studies reported here are the first to our knowledge to address
miRNA regulation of IR using both in vitro models and genetic
deletion in mice. miR-21 has also been postulated to play a protec-
tive role in the heart during IR or hypoxic injury, based on stud-
ies performed with cardiomyocytes in vitro or using antagomiR
against miR-21 (17, 64). In contrast, genetic deletion of miR-21 or
knockdown with LNA-antimiRs has no affect on ischemic injury
(65), highlighting the need to investigate the biology of miRNAs
using genetic models.
In conclusion, the results of this study suggest that miR-214
protects the heart against IR injury by blunting Ca2+ overload and
cell death in response to injury through its repression of NCX1,
CaMKIIδ, CypD, and BIM. To our knowledge, these findings pro-
vide the first evidence for an important role of a miRNA in direct
modulation of cardiac Ca2+ handling. Given that overexpression of
NCX1 and intracellular Ca2+ overload also underlie cardiac hyper-
trophy and other forms of heart disease (66, 67), it is likely that
miR-214 plays a cardioprotective role in a variety of stress settings.
Thus, boosting miR-214 levels to attenuate Ca2+ overload and car-
diac cell death may provide therapeutic benefit.
Northern blot analysis. Total RNA was isolated from mouse tissues, and
miRNAs were detected as previously described (5). 32P-labeled StarFire oli-
gonucleotide probes (IDT) against mature miR-214 and miR-199a were
used in the hybridization. U6 was used as a loading control.
Western blot analysis. Western blotting was performed according to stan-
dard protocols. See Supplemental Methods for details.
RT-PCR and qPCR analysis. qPCR for miR-214 and miR-199a was performed
according to the manufacturer’s protocol using the TaqMan miRNA assay
kits (ABI). The relative quantities of miRNAs were normalized to RNU6B.
RT-PCR was performed using random hexamer primers with the Super-
script III kit (Invitrogen). qPCR was performed using TaqMan probes (ABI).
Qualitative RT-PCR to assess Dnm3os splicing. RNA isolated from miR-214
KO hearts and WT littermates was subjected to RT-PCR, and then cDNA
was amplified by PCR with 5 different primer sets spanning the Dnm3os
transcript. Amplified products from WT and KO hearts were separated by
gel electrophoresis side-by-side to visualize differences in size. See Supple-
mental Methods for primer sets used.
Generation of miR-214 KO mice. The targeting vector for generating a con-
ditional allele of miR-214 mutation was constructed using the pGKneo-
F2L2dta vector. The miR-214 targeting strategy was designed to replace
the pre-miR-214 sequence with the neomycin resistance cassette flanked
by loxP sites. See Supplemental Methods for details.
Histology and immunohistochemistry. H&E and Masson’s trichrome stain-
ings were performed using standard procedures. Wheat germ agglutinin
staining was done on heart sections to assess cardiomyocyte size, and
TUNEL staining was performed using the In Situ Cell Death Detection kit
(Roche) according to the manufacturer’s instructions. See Supplemental
Methods for details.
Transthoracic echocardiography. Cardiac function and heart dimensions
were evaluated by two-dimensional echocardiography using a Visual
Sonics Vevo 2100 Ultrasound on conscious mice. See Supplemental
Methods for details.
Electron microscopy. Samples were processed by the University of Texas
Southwestern Medical Center Electron Microscopy Core facility. See Sup-
plemental Methods for details.
Microarray analysis. For the microarray, P14 or adult heart RNA was
pooled from 3 wild-type and 3 miR-214 KO animals. Microarray analy-
sis was performed by the University of Texas Southwestern Microarray
Core Facility using the Mouse Genome Illumina Mouse-6 V2 BeadChip.
Data were deposited in NCBI’s Gene Expression Omnibus (accession
#GSE35421). See Supplemental Methods for details.
Cell culture, transfection, and luciferase assays. Cell culture, transfection, and
luciferase studies were performed as previously described (7). See Supple-
mental Methods for details.
Neonatal rat cardiomyocyte culture and in vitro IR assays. See Supplemental
Methods for cardiomyocyte isolation. To simulate IR in vitro, cardiomyo-
cytes plated on coverslips were transfected with LNA-modified antimiRs,
washed, and placed in DMEM containing no serum or supplements. Cells
were placed in the hypoxia chamber for either 2 hours of ischemia (5% CO2,
1% O2) followed by 24 hours of reperfusion (mild IR) or 1 hour of anoxia
(5% CO2, 0% O2) followed by 4 hours of reperfusion (severe IR). Coverslips
were processed for TUNEL staining as described above and counterstained
with Hoechst to visualize nuclei. Samples were run in triplicate, and 5–6
10× fields were imaged per coverslip.
Mouse model of MI and IR. Eight- to 12-week-old miR-214 KO male mice or
WT controls were subjected to permanent (MI) or transient (IR) ligation of
the LAD. See Supplemental Methods for details.
Adult mouse cardiomyocyte isolation and intracellular calcium ([Ca2+]i) mea-
surements. Cardiomyocytes from 8- to 10-week-old male mice were isolated
by using enzymatic digestion and mechanical dispersion methods as
described in detail in Supplemental Methods.
Mitochondrial respiratory activity and superoxide production. Mitochondrial
electron transport activity and superoxide production were assessed as
described in detail in Supplemental Methods.
Model demonstrating miR-214 cardioprotection against Ca2+ overload
injury and cell death. Ischemic injury leads to Ca2+ overload, causing a
switch to reverse mode NCX1 activity in cardiomyocytes that enhances
Ca2+ overload and leads to cell death via downstream effectors of Ca2+
signaling. miR-214, also induced by ischemic injury, protects the myo-
cyte from damage by attenuating NCX1 levels to prevent excessive
Ca2+ influx into the cytoplasm. Additional protection by miR-214 occurs
through suppression of the Ca2+ effector kinase CaMKII and the cell
death mediators CypD and BIM. In the absence of miR-214 expression
in the heart, higher levels of reverse mode NCX1 and Ca2+ effectors
further perpetuate Ca2+ overload and cell death during IR, resulting in
greater impairment of cardiac function.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 4 April 2012
Statistics. Results are expressed as the mean ± SEM. We used a 2-tailed,
unpaired Student’s t test for all pairwise comparisons (GraphPad Prism
version 5). P values less than 0.05 were considered significant.
Study approval. All animal procedures were approved by the Institu-
tional Animal Care and Use Committee of University of Texas South-
western Medical Center.
We are grateful to Christopher Gilpin and the University of Texas
Southwestern Medical Center (UTSW) Electron Microscopy Core
for the EM images. We thank the UTSW Microarray Core Facility
for Illumina microarray data and analysis. We thank Gaile Vitug
and John Shelton for technical assistance and Jose Cabrera for
graphics. Work in the laboratory of E.N. Olson was supported by
grants from the NIH, the Donald W. Reynolds Center for Clinical
Cardiovascular Research, the Robert A. Welch Foundation (grant
I-0025), the Foundation Leducq’s Transatlantic Network of Excel-
lence in Cardiovascular Research Program, the American Heart
Association–Jon Holden DeHaan Foundation, and the Cancer
Prevention & Research Institute of Texas (CPRIT). A.B. Aurora
was supported by an American Cancer Society Fellowship (grant
Received for publication June 2, 2011, and accepted in revised form
February 1, 2012.
Address correspondence to: Eric N. Olson, Department of Molec-
ular Biology, 5323 Harry Hines Blvd, Dallas, Texas 75390-9148,
USA. Phone: 214.648.1187; Fax: 214.648.1196; E-mail: eric.olson@
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