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J. Exp. Med. Vol. 208 No. 3 549-560
Myocardial infarction (MI), caused by acute
occlusion of the coronary artery, is one of the
leading causes of death and disability world-
wide (Mackman, 2008). Over seven million
people die each year from acute MI or progres-
sive cardiac dysfunction after coronary artery
occlusion. MI induces cardiac cell death and
ischemic stress in surviving myocytes bordering
the region of infarct (border zone [BZ]), which
triggers left ventricle (LV) remodeling, leading to
dilation, hypertrophy, and fibrosis (Swynghedauw,
1999). Scar formation and pathological LV
remodeling result in cardiac dysfunction and
eventually lead to heart failure (Swynghedauw,
1999). Apoptosis (programmed cell death) con-
tributes significantly to cardiomyocyte loss dur-
ing acute MI, particularly in the BZ, and during
subsequent remodeling events (Kang et al., 2000;
Kitsis et al., 2007). Because cardiomyocytes are
terminally differentiated and have little potential
for division, controlling the loss of cardiomyocytes
after injury holds potential therapeutic value.
Posttranscriptional regulation involving a
class of small noncoding RNAs known as
microRNAs (miRNAs; Ambros, 2003; Zhao
and Srivastava, 2007; Ruvkun, 2008; Bartel,
2009) has emerged as a major regulator of
numerous cellular processes, including those
involved in the heart. Through imperfect
sequence-specific binding to their messenger
RNA (mRNA) targets, miRNAs negatively
influence the expression of proteins by destabi-
lizing target mRNAs or inhibiting translation
(Ambros, 2003; Zhao et al., 2005; Zhao and
Srivastava, 2007; Ruvkun, 2008; Bartel, 2009).
miRNAs control various aspects of heart de-
velopment and function, including cell prolif-
eration (Zhao et al., 2005, 2007; Chen et al.,
2006), lineage differentiation (Kwon et al., 2005;
Sokol and Ambros, 2005; Chen et al., 2006;
Ivey et al., 2008), and cardiac conduction (Yang
et al., 2007; Zhao et al., 2007). Several miRNAs
are dysregulated during cardiac remodeling
after injury or stress (van Rooij et al., 2006,
2007, 2008; Carè et al., 2007), including
Abbreviations used: AAR, area
at risk; ANF, atrial natriuretic
factor; BNP, brain natriuretic
peptide; BZ, border zone; DZ,
distant zone; ECG, electrocardio-
graphy; EF, ejection fraction;
IZ, infarct zone; LV, left ven-
tricle; MI, myocardial infarc-
tion; miRNA, microRNA;
MRI, magnetic resonance
imaging; mRNA, messenger
RNA; PFA, paraformaldehyde;
PI, propidium iodide; qPCR,
quantitative PCR; siRNA, small
interfering RNA; SV, stroke
volume; TTC, triphenyltetrazo-
lium chloride; TUNEL,
nick end labeling; UTR, un-
L. Qian and L.W. Van Laake contributed equally to this
miR-24 inhibits apoptosis and represses Bim
in mouse cardiomyocytes
Li Qian,1,2,3 Linda W. Van Laake,1,2,3,5 Yu Huang,1,2,3 Siyuan Liu,4
Michael F. Wendland,4 and Deepak Srivastava1,2,3
1Gladstone Institute of Cardiovascular Disease, 2Department of Pediatrics, 3Department of Biochemistry and Biophysics,
and 4Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA 94143
5University Medical Center Utrecht, Utrecht University, 3508 GA Utrecht, Netherlands
Acute myocardial infarction (MI) involves necrotic and apoptotic loss of cardiomyocytes.
One strategy to salvage ischemic cardiomyocytes is to modulate gene expression to pro-
mote cell survival without disturbing normal cardiac function. MicroRNAs (miRNAs) have
emerged as powerful regulators of multiple cellular processes, including apoptosis, suggest-
ing that regulation of miRNA function could serve a cardioprotective function. In this
study, we report that miR-24 (miRNA-24) expression is down-regulated in the ischemic
border zone of the murine left ventricle after MI. miR-24 suppresses cardiomyocyte apop-
tosis, in part by direct repression of the BH3-only domain–containing protein Bim, which
positively regulates apoptosis. In vivo expression of miR-24 in a mouse MI model inhibited
cardiomyocyte apoptosis, attenuated infarct size, and reduced cardiac dysfunction. This
antiapoptotic effect on cardiomyocytes in vivo was partially mediated by Bim. Our results
suggest that manipulating miRNA levels during stress-induced apoptosis may be a novel
therapeutic strategy for cardiac disease.
© 2011 Qian et al. This article is distributed under the terms of an Attribution–
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The Journal of Experimental Medicine
miR-24 inhibits cardiomyocyte apoptosis | Qian et al.
after overexpression or inhibition of miR-24, we found that
miR-24 negatively regulated this marker of apoptosis (Fig. 1,
F–I). Quantification for activated Caspase 3–positive cardio-
myocytes further confirmed the results (Fig. 1, J and K). Cell
cycle patterns were similar among miR-24 mimic– and miR-24
inhibitor–expressing and control cells (Fig. S3, E and F).
miR-24 directly targets Bim for repression
Because miR-24 inhibited cardiomyocyte apoptosis, we
searched for direct downstream effectors/targets by which
miR-24 exerts its function. We used established computa-
tional algorithms to predict potential miR-24 target genes
(Krek et al., 2005; Rajewsky, 2006; Bartel, 2009) and performed
gene expression analyses. Among down-regulated genes, the
proapoptotic Bcl2 family protein Bim was a strong candidate
target based on the presence of predicted miR-24 binding
sites. Bim mediates mitochondrial and ER stress–induced apop-
tosis, and its levels are controlled by multiple mechanisms, in-
cluding transcriptional and posttranscriptional events such as
phosphorylation, ubiquitination, proteasomal degradation,
and miRNA regulation (i.e., miR-17-92; Dijkers et al., 2000;
Puthalakath and Strasser, 2002; Ley et al., 2005; Puthalakath
et al., 2007; Ventura et al., 2008; Xiao et al., 2008).
To test whether Bim is also regulated by miR-24, qPCR
and Western blots of cardiomyocytes transfected with miR-24
mimic or inhibitor were performed. mRNA and protein
levels of Bim were down-regulated when miR-24 was over-
expressed, whereas inhibiting miR-24 function resulted in
up-regulation of Bim (Fig. 2, A and B). We identified two
conserved miR-24 binding sites in the Bim mRNA 3 un-
translated region (UTR; Fig. 2 C). To test whether miR-24
repressed Bim by binding to these sites, the Bim 3 UTR
was inserted into the 3 UTR of a luciferase reporter, down-
stream of the coding region. The constitutively active reporter
was cotransfected with miR-24 mimic, inhibitor, or miR-
24 cloned in an expression vector (pEF–miR-24) into
HeLa cells. Transfection of the Bim 3 UTR chimeric lucifer-
ase reporters with miR-24 (miR-24 mimic or pEF–miR-24)
resulted in a decrease in luciferase activity, whereas co-
transfection with miR-24 inhibitor resulted in increased lu-
ciferase activity (Fig. 2 C). Mutations of the miR-24 binding
sites in the Bim 3 UTR abolished miR-24 responsiveness
(Fig. 2 C), suggesting that miR-24 represses Bim by physically
binding to its 3 UTR. The same luciferase experiment was
performed in cardiomyocytes, confirming the results from
HeLa cells (Fig. S3 G).
We investigated whether miR-24 repression of Bim in
primary cardiomyocytes could account for some of the effects
of miR-24 on the apoptotic pathway. Bim knockdown led to
a reduction in the number of apoptotic cells (Fig. 2 D and not
depicted), phenocopying overexpression of miR-24. Most
importantly, the increase in apoptotic cell number caused by
inhibition of miR-24 could be reversed by knockdown of
Bim (Fig. 2 E and not depicted), suggesting an epistatic rela-
tionship between miR-24 and Bim.
miR-29 (miRNA-29) and miR-21 (Thum et al., 2008;
van Rooij et al., 2008; Dong et al., 2009).
In this study, we identify miR-24 as an antiapoptotic
miRNA that is down-regulated in the ischemic zones of the
LV after acute MI. We show that miR-24 directly targets the
proapoptotic protein Bim within cardiomyocytes for repres-
sion and inhibits apoptosis in vitro and in vivo. In vivo delivery
of miR-24 after MI reduced scar size and improved long-term
miR-24 suppresses apoptosis
In an effort to identify miRNAs dysregulated within hours
after MI, we found that miR-24 was highly down-regulated
in the ischemic BZ but not in the nonischemic distant zone
(DZ) of the LV 24 h after MI (Fig. 1, A–C). miR-23a and
miR-23b, encoded by the same cluster as miR-24, were also
down-regulated at the BZ but to a lesser extent; in contrast,
expression of miR-27a and miR-27b, which are also clus-
tered with miR-24, was not altered at the BZ (Fig. S1 A).
miR-24 expression was normally enriched in the adult mouse
heart, specifically in sorted cardiac myocyte and fibroblast
populations but not in endothelial cells (Fig. S1, B–H). Inter-
estingly, miR-24 down-regulation was attenuated over time,
and its expression was restored back to wild-type levels by
4 wk after MI (Fig. 1, A and B). We quantified apoptotic car-
diomyocytes in the BZ and DZ regions over time (Fig. 1,
D and E) and found a close correlation between down-
regulation of miR-24 and increase in apoptosis (Fig. 1, A–E).
To investigate miR-24 function, we transfected primary
cardiomyocytes with chemically synthesized double-stranded
oligonucleotides that mimic the function of endogenous ma-
ture miR-24 (miR-24 mimic) or modified antisense oligori-
bonucleotides that inhibit miR-24 function (miR-24 inhibitor).
Transfection efficiency (80%) was monitored by FACS
analysis of cotransfected Alexa Fluor 488–conjugated oligo-
nucleotides (Fig. S2, A and B), expression levels were evalu-
ated by real-time quantitative PCR (qPCR; Fig. S2, C and D),
and functional activity of transfected miR-24 oligonucle-
otides was measured by luciferase sensor experiments (Fig. S2 E).
Additional miR-24 mimic and inhibitor controls in which
the seed sequence of miR-24 was mutated were used (see
Materials and methods).
miR-24 expression in cardiomyocytes increased cell num-
ber by 53%, and inhibition of miR-24 function decreased cell
number by 45% 48 h after transfection. We did not observe a
difference in cell number upon introduction of control mimic,
control inhibitor, or mock transfection (Lipofectamine only).
However, the number of live cells (propidium iodide [PI])
positive for AnnexinV, an early apoptosis marker, was in-
creased when miR-24 was inhibited. Conversely, fewer
AnnexinV+PI cells were seen when miR-24 was overex-
pressed (Fig. S3, A–D). During later apoptosis, DNA fragmen-
tation results from activation of endonucleases (Hengartner,
2000). Using TUNEL (Tdt-mediated dUTP-biotin nick end
labeling) staining to detect fragmented DNA in cardiomyocytes
JEM VOL. 208, March 14, 2011
Figure 1. miR-24 is down-regulated early after MI and inhibits apoptosis. (A and B) qPCR of miR-24 was performed on RNA extracted
from the BZ and DZ of hearts 24 h, 3 d, and 1 and 4 wk after coronary artery ligation (MI) or sham surgery. (C) In situ hybridization using miR-24
locked nucleic acid (LNA) probe on heart sections from MI and sham operated mice 24 h after surgery. (D) TUNEL staining of infarcted hearts 24 h
after coronary artery ligation. Cardiomyocytes were costained with antibody to -Actinin. DAPI was used for nuclear staining. (E) Percentage of
TUNEL-positive cardiomyocytes (CM) at BZ of hearts over time after MI. n = 3 for each time point. (F and G) TUNEL and -Actinin staining on pri-
mary cardiomyocytes transfected with mimic control, miR-24 mimic, or miR-24mut mimic (F) or transfected with inhibitor control, miR-24 inhibi-
tor, or miR-24mut inhibitor (G). (H and I) Quantification of percentage of TUNEL-positive cardiomyocytes in F and G. (J and K) Quantification of
percentage of Caspase 3–positive cardiomyocytes. All experiments were repeated three times (technical triplicates) with biological triplicates
(n = 3). Error bars indicate SEM (*, P < 0.05). Bars, 50 µm.
miR-24 inhibits cardiomyocyte apoptosis | Qian et al.
Figure 2. Bim is a direct target of miR-24. (A) qPCR of Bim mRNA from cardiomyocytes transfected with control (mock transfection with Lipo-
fectamine 2000 only), miR-24 mimic, or miR-24 inhibitor. (B) Western blot comparing protein levels of Bim in control (mock transfection) and miR-24
mimic– or miR-24 inhibitor–expressing cells. Quantification compared with control (set as 1) is shown above the panel. (C) Conserved miR-24 binding
sites in the Bim 3 UTR and relative luciferase activity (RLA) in HeLa cells expressing miR-24 mimic, miR-24 precursor (in expression vector, pEF–miR-24),
or inhibitor compared with control. Base pairs highlighted in color (miR-24 in red and Bim 3 UTR in green) are seed sequences that are complimentary
between miRNA and target. Mutations in miR-24 binding sites for luciferase assay in 3 UTR are indicated. Controls are set up as 1, indicated by the
dashed line. (D) Primary cardiomyocytes (CM) expressing control siRNA or Bim siRNA were stained with AnnexinV, PI, TUNEL, or activated Caspase 3. Dot
plots show representative staining by flow cytometry, and arrows indicate the quadrant of AnnexinV+PI apoptotic cells. (E) Primary cardiomyocytes co-
transfected with Bim siRNA and miR-24 inhibitor or control inhibitor were stained with AnnexinV, PI, TUNEL, or activated Caspase 3. Dot plots show rep-
resentative staining by flow cytometry, and arrows indicate the quadrant of AnnexinV+PI apoptotic cells. All experiments were repeated three times
(technical triplicates) with biological triplicates (n = 3). Bar graphs show mean ± SEM (*, P < 0.05).
JEM VOL. 208, March 14, 2011
mimic than in control mimic–treated mice (Fig. 3, A and B).
This result was confirmed by using another apoptotic marker,
activated Caspase 3, in combination with -Actinin (Fig. 3 C
and not depicted). In parallel, we assayed for phosphorylated
histone H3 (pH3) and -Actinin to compare the difference
in the progression of cardiomyocyte mitosis among the two
groups of mice. We detected no mitotic cardiomyocytes in
either miR-24 mimic– or control mimic–treated mice (un-
published data). To determine whether the inhibition of
apoptosis by miR-24 affected the degree of myocardial dam-
age, we performed Evans blue/triphenyltetrazolium chloride
(TTC) double staining to assess the area at risk (AAR) and
the infarct size of myocardium 24 h after coronary ligation.
Treatment of miR-24 led to a decrease in infarct size but no
change in AAR (Fig. 3, D and E), suggesting that miR-24 ex-
pression reduced cardiac damage shortly after MI.
In vivo delivery of miR-24 reduces cardiac dysfunction
in a mouse MI model
Acute MI in mice causes myriad hemodynamic stresses, which
trigger left ventricular remodeling and eventually lead to
functional decompensation and heart failure (Lutgens et al.,
1999; Bock-Marquette et al., 2004). We hypothesized that a
In vivo delivery of miR-24 inhibits apoptosis
in a mouse MI model
Apoptotic signals are propagated in response to ischemic stress
in the heart. Thus, we speculated that the extensive apoptosis
after MI may be enhanced by the decreased levels of miR-24.
We used a Lipofectamine-mediated in vivo transfection
method (Yang et al., 2007) to locally deliver miR-24 mimics
to the infarcted hearts of mice in an effort to restore miR-24
levels. To validate the delivery efficiency, fluorescently labeled
oligonucleotides mixed with Lipofectamine were injected. By
24 h after the procedure, strong fluorescence around the in-
jection area revealed efficient uptake (Fig. S4, A–C). qPCR
and in situ hybridization of miR-24 revealed high expression
levels in the injected areas (Fig. S4, D and E), although it is
difficult to directly assess the functionality of injected oligo-
nucleotides in vivo.
To determine whether miR-24 mimic treatment could
inhibit ischemia-induced apoptosis of cardiomyocytes in vivo,
we combined TUNEL labeling with the cardiomyocyte
marker -Actinin on infarcted mouse hearts treated with
miR-24 or control mimics. The percentage of TUNEL-positive
(apoptotic) cardiomyocytes in the BZs along the infarct area
was 62% (P < 0.05) lower in animals treated with miR-24
Figure 3. In vivo delivery of miR-24 inhibits apoptosis. (A and B) Immunohistochemistry of control mimic– or miR-24 mimic–treated heart sections
marking TUNEL-positive cardiomyocytes costained with -Actinin antibody within the BZ of infarcted hearts. Quantification is shown in B. (C) Quantifica-
tion for activated Caspase 3+ cardiomyocytes (CM) at BZ. (D) Representative pictures of Evans blue/TTC staining on four continuous slices of LV from rep-
resentative hearts of control mimic– or miR-24 mimic–injected mouse. (E) Blinded quantification of size of the AAR and infarct size as described in
Materials and methods. All staining in this figure was performed on hearts 24 h after MI. Measurements were repeated three times (technical triplicates),
with biological sample size indicated in each panel. The mean number from technical triplicates was used for statistical calculation. Error bars indicate
SEM (*, P < 0.05). Bars: (A) 50 µm; (D) 500 µm.
miR-24 inhibits cardiomyocyte apoptosis | Qian et al.
controls (Fig. 4 A). Moreover, cardiac output (the product of
SV and heart rate) was increased in the miR-24 recipients
(Fig. 4 A). This was attributable to better structure and con-
tractility of the LV because heart rate was unaltered (Fig. S5 A).
To monitor cardiac structure and function at several time
decrease in cardiac cell death by miR-24 would translate into
improved heart function. Therefore, we assessed cardiac func-
tion 12 wk after MI by using magnetic resonance imaging
(MRI). MRI revealed that the miR-24–treated mice had a
higher ejection fraction (EF) and stroke volume (SV) than
Figure 4. In vivo delivery of miR-24 blunts effects of MI. After MI, miR-24 or control mimic was injected along the BZ of infarct. (A) EF, SV, and
cardiac output (CO) of the LV were measured by MRI 12 wk after MI. (B) M-mode echocardiography of representative hearts 3 d after MI. (C) Fractional
shortening (FS), EF, and cardiac output at varying time points were measured using high-resolution echocardiography. Data were collected 1 d before and
3 d, 4 wk, and 12 wk after MI in a blinded fashion. (D) qPCR of ANF and BNP on RNA extracted from BZ of MI hearts 24 h after injection of control or
miR-24 mimic. (E) Stress-responsive miRNAs were measured by qPCR 24 h after MI. Data in D and E are shown relative to control, indicated by dashed
lines. (F and G) Azan staining was performed on heart sections 4 wk after MI with miR-24 or control mimic injected. RV, right ventricle. Bars, 500 µm.
(H and I) Quantification of scar size was calculated by measurement of both scar area (H) and scar circumference (I). (J) Western blots for activated Cas-
pase 3, Caspase 12, activated Caspase 12, Caspase 9, Bim, and GAPDH in protein extracts from control mimic– or miR-24 mimic–injected hearts 24 h after
MI, as well as hearts without MI (sham). Relative quantification of Western blots is shown to the right of each panel; comparison was made between
control mimic (set as 1) and miR-24 mimic after MI. The vertical black line indicates that intervening lanes have been spliced out. For all histograms, sam-
ple size (n) is indicated for each group. Each experiment was repeated three times (technical triplicates), and the mean number was used for statistical
analyses. Error bars indicate SEM (*, P < 0.05; **, P < 0.01).
JEM VOL. 208, March 14, 2011
in less apoptosis at both the IZ and BZ of MI hearts 24 h after
MI, as measured by TUNEL and activated Caspase 3 staining
(Fig. 5, A and B; and not depicted).
To further investigate the relationship of miR-24 and
Bim, we performed loss of function experiments of miR-24
by inhibiting endogenous miR-24 levels with Lipofectamine-
mediated in vivo transfection of a miR-24 inhibitor, as de-
scribed previously (Yang et al., 2007), with or without Bim
knockdown. The miR-24 knockdown was confirmed by
qPCR (Fig. S4 G). Inhibition of miR-24 resulted in increased
apoptosis in BZ cardiomyocytes, increased expression of the
downstream target Bim, and increased activity of Caspase 3,
Caspase 12, and Caspase 9 (Fig. 5, C–E; and not depicted).
Co-delivery of Bim siRNA and miR-24 inhibitor partially
rescued the proapoptotic effects of miR-24 inhibition (Fig. 5,
F and G; and not depicted).
In this study, we reveal a novel role for miR-24 in the regula-
tion of mammalian cardiomyocyte apoptosis. We found that
miR-24 inhibits apoptosis in cardiomyocytes in vitro and
in vivo using a mouse MI model. In addition, we identified Bim
as one of the direct targets of miR-24 and, through epistasis
analyses, found that Bim mediates a part of miR-24’s effect
on apoptosis. Finally, introduction of miR-24 in vivo did not
disturb the normal function of the heart but partially pro-
tected the heart from injury. The beneficial effects of miR-24
in MI hearts, while statistically significant by our measure-
ment, are quite small and may be below the true sensitivity of
the techniques being used. Future experimentation using more
robust and accurate assays with a large sample number is
needed for validation of applying this miRNA in treating
ischemic cardiac disease. In addition, the improvement in
heart function and reduction in scar size by overexpressing
miR-24 might be caused by other functions of miR-24 inde-
pendent of apoptosis or a combination of all. Further cardiac
function analyses and scar size measurements when inhibiting
the antiapoptotic function of miR-24 (i.e., knocking down
the downstream components in miR-24–mediated apoptosis
pathway) will hopefully provide some clues.
Like most miRNAs, miR-24 functions in many other bi-
ological processes and pathways. miR-24 was first reported to
negatively regulate erythroid differentiation through inhibi-
tion of human activin type I receptor ALK4 (Wang et al.,
2008). In addition, miR-24 appears to affect the tumor sup-
pressor p16 (Lal et al., 2008), the DNA repair process, and cell
cycle regulation (Lal et al., 2009a,b; Rogler et al., 2009). Par-
ticularly relevant to this study, miR-24 is required to prevent
apoptosis during normal development of the retina in frogs
(Walker and Harland, 2009), suggesting evolutionary conser-
vation of miR-24 function, although the mechanisms may
be divergent. These findings support the idea that a single
miRNA, like miR-24, can regulate multiple independent
pathways that may converge on a common biological out-
come such as regulation of apoptosis. Other miRNAs, such
as miR-21, may also play a role in regulating the cardiac
points after MI, we used high-resolution echocardiography
(Fig. 4, B and C; and Fig. S5, B and C). miR-24– and control-
treated mice underwent serial imaging 1 d before and 3 d,
4 wk, and 12 wk after MI. All mice showed a reduction in left
ventricular function after coronary artery ligation, confirm-
ing successful induction of MI (Fig. 4, B and C; and Fig. S5 B).
However, in mice treated with miR-24, the initial loss of
contractility was significantly attenuated compared with con-
trols (Fig. 4, B and C; and Fig. S5 B).
As a molecular readout of cardiac dysfunction, we per-
formed qPCR to monitor the expression levels of atrial natri-
uretic factor (ANF) and brain natriuretic peptide (BNP) from
miR-24–injected and control hearts. Both ANF and BNP
were up-regulated 24 h after MI in controls as expected
(Fig. 4 D). The up-regulation of ANF and BNP was attenuated
by injection of miR-24 mimic in infarcted hearts (Fig. 4 D). We
also assessed the expression level of a battery of stress-responsive
miRNAs by qPCR. miR-29, miR-195, and miR-208 levels,
which are dramatically reduced upon MI (van Rooij et al.,
2006, 2007, 2008), were partially restored by overexpression
of miR-24 (Fig. 4 E).
Consistent with the improvement of cardiac function, in-
jection of miR-24 resulted in a smaller scar size 4 wk after MI
(Fig. 4, F–I; and Fig. S5, D–F). Mice treated with miR-24 had
a 33% reduction in scar circumference and a 46% reduction
in scar area (P < 0.05; Fig. 4, F–I; and Fig. S5, D–F). Because
stimulation of neovascularization could be another mecha-
nism by which miR-24 decreased infarct size and improved
cardiac function (van Laake et al., 2006), we quantified vascu-
lar density in the infarcted area, BZ, and distant myocardium.
We found no difference in vessel formation between miR-24–
and control-injected hearts (unpublished data).
miR-24 modulates apoptosis and suppresses Bim in vivo
Because our cell culture experiments (Figs. 1 and 2) demon-
strated that miR-24 inhibited apoptosis and targeted the pro-
apoptotic Bcl2 family protein Bim, we tested whether miR-24
regulated the same apoptosis pathway in vivo. Protein was ex-
tracted from the infarct zone (IZ) and BZ of mouse hearts
24 h after coronary ligation and injection with miR-24 mimic
or control mimic. Consistent with its inhibitory role in apop-
tosis, miR-24 led to reduced protein levels of the final effec-
tor Caspase, activated Caspase 3, compared with controls after
MI (Fig. 4 J). Caspase 8 was not significantly affected by
miR-24 expression (not depicted); however, the increases in
Caspase 12 and Caspase 9 were significantly attenuated in in-
farcted mouse hearts treated with miR-24 (Fig. 4 J). In addi-
tion, the increase in protein level of Bim after MI was
dampened in these hearts compared with controls (Fig. 4 J).
Next we determined whether a decrease in Bim could
mimic the in vivo effect of miR-24 expression on cardio-
myocyte apoptosis. We delivered Bim small interfering RNA
(siRNA) cocktails to the IZ and BZ of mouse hearts after
coronary artery ligation. RNA and protein levels of Bim were
measured to ensure efficient knockdown of Bim (Fig. S5,
G and H). We found that inhibition of Bim expression resulted
miR-24 inhibits cardiomyocyte apoptosis | Qian et al.
Figure 5. In vivo inhibition of Bim reduces miR-24 inhibitor–induced apoptosis. (A and B) After MI, control or Bim siRNA was injected along the
BZ of infarct. Immunohistochemistry of heart sections labeling TUNEL-positive cardiomyocytes, marked by -Actinin antibody within the BZ. DAPI indi-
cates nuclei. Quantification of TUNEL+ or activated Caspase 3+ cells is shown in B. (C and D) Cardiomyocyte (CM) apoptosis was determined by TUNEL and
activated Caspase 3 stainings on heart sections at the BZ of infarcted hearts with miR-24 or control inhibitor injected. (E) Western blots for activated
Caspase 3, Caspase 12, activated Caspase 12, Caspase 9, Bim, and GAPDH in protein extracts from control inhibitor– or miR-24 inhibitor–injected hearts
24 h after MI, as well as hearts without MI (sham). Relative quantification of Western blots is shown to the right of each panel; comparison was made
JEM VOL. 208, March 14, 2011
5-CCCTGCAGTGGGAACGAAGACA GCTGATTTATAAGGC-3 and
and 5-GTTTCACTCGTCAACGAAGACAAATGTCTCTGTGC-3 and
5-GCACAGAGACATTTGTCTTCGTTGACGAGTGAAAC-3 (the un-
derlined nucleotides are mutated ones).
Quantitative real-time PCR. RNA was extracted by the TRIZOL
method (Invitrogen). RT-PCR was performed using the Superscript III first-
strand synthesis system (Invitrogen). qPCR was performed using the ABI
7900HT (TaqMan; Applied Biosystems) per the manufacturer’s protocols.
Optimized primers from TaqMan Gene Expression Array were used. miRNA
RT was conducted using the TaqMan MicroRNA Reverse Transcription kit
(Applied Biosystems). miRNA real-time PCR (quantitative RT-PCR) was
performed per the manufacturer’s protocols by using primers from TaqMan
MicroRNA assays (Applied Biosystems). Expression levels were normalized
to Gapdh expression and RNU6 (miRNA qPCR).
Cell culture, transfection, and luciferase assay. Primary cardiomyocytes
from mouse neonatal hearts were isolated and maintained as described previ-
ously (Ieda et al., 2009). Lipofectamine 2000 (Invitrogen)–mediated transfec-
tion was performed according to Invitrogen’s protocol. miR-24 mimic
(5-UGGCUCAGUUCAGCAGGAACAG-3), mimic control (5-UUCU-
CCGAACGUGUCACGUTT-3), miR24mut mimic (5-UGGCUCAGU-
UCAGUAAGAACCG-3), miR-24 inhibitor (5-ACCGAGUCAAGUCG-
UCCUUGUC-3), inhibitor control (5-UCUACUCUUUCUAGGAG-
GUUGUGA-3), and miR24mut inhibitor (5-ACCGAGUCAAGUCAU-
UCUUGGC-3; the underlined nucleotides are mutated ones) were purchased
from Thermo Fisher Scientific and Shanghai GenePharma Co. For each
transfection in 1 well of a 6-well plate, 40 pmol of mimic or inhibitor was
used. For plasmid transfection, 100 ng of plasmid was transfected in 1 well of
a 6-well plate. Luciferase assays were performed as described previously (Zhao
et al., 2005) with the Dual-Luciferase reporter assay system (Promega).
Western blots and immunocytochemistry. Western blots were per-
formed as described previously (Zhao et al., 2005). Mouse monoclonal anti–
Caspase 8 (Sigma-Aldrich), mouse monoclonal anti–Caspase 9 (Sigma-Aldrich),
rabbit anti–Caspase 3 (Sigma-Aldrich), rat monoclonal anti–Caspase 12
(Sigma-Aldrich), and rabbit polyclonal antibody against Bim (aa 4–195 of
BimEL form) were all used at a 1:1,000 dilution for Western blots. All Western
blots were quantified using AlphaImager software (Alpha Innovations). Im-
munocytochemistry was performed according to a standard protocol. In
brief, cells were fixed in 10% formalin (vol/vol) for 15 min on ice and washed
with PBS twice. Cells were then incubated with primary antibody for 1 h at
room temperature and washed with PBS three times and then incubated
with secondary antibody for 0.5 h. After washing with PBS, cells were
mounted in Vectashield with DAPI (Vector Laboratories). Mouse anti–-
Actinin was used at 1:400; guinea pig anti-Vimentin (Progen) was used at
1:200; rabbit anti–Caspase 3 (Sigma-Aldrich) was used at 1:200 for immuno-
cytochemistry. Alexa Fluor 546 or Alexa Fluor 488 anti–mouse (Invitrogen)
was used at 1:200 as secondary antibody. TUNEL staining was performed
using the In Situ Cell Death Detection kit, Fluorescein (Roche) per the
Flow cytometry. For FACS analysis to detect early apoptotic cells
(AnnexinV+PI), 5 × 105 dissociated cells were washed twice in PBS and resus-
pended in 1× binding buffer (BD). Then the cells were stained with 1 µl
AnnexinV-FITC and 0.5 µl PI (BD) for 30 min in the dark and followed
by FACS analysis with FACSCalibur (BD). For FACS analysis of cell cycle, dis-
sociated cells were fixed and permeabilized by cold 70% ethanol (overnight
response to ischemic injury (Thum et al., 2008; Dong
et al., 2009).
miR-24 is expressed in both cardiomyocytes and fibro-
blasts. Our experiments demonstrate that miR-24 can exert
antiapoptotic effects in cardiomyocytes in a cell-autonomous
fashion. We and others have shown that cardiac fibroblasts can
function as major signaling centers to affect the neighboring
cardiomyocytes (Ieda et al., 2009; Takeda et al., 2010), and it is
quite possible that miR-24 also functions in cardiac fibroblasts
to promote cardiomyocyte survival. In this case, miR-24 would
likely regulate pathways leading to paracrine secretion of sur-
vival factors that impinge on neighboring myocytes. Future
studies of fibroblast-specific expression of miR-24 could help
resolve this issue.
It is curious that an miRNA that represses an apoptotic
factor such as Bim in the setting of ischemia would be down-
regulated. One might expect that such an miRNA would be
up-regulated to protect the cells from ischemic damage, par-
ticularly in the BZ which is attempting to survive in hypoxic
conditions. However, the down-regulation may reflect the
need to remove cells that are under ischemic stress. It will be
interesting to determine whether the ischemia-induced miR-24
down-regulation has other more beneficial consequences
through yet unknown targets.
The finding that miR-24 targets the Bcl2 family member
Bim for repression highlights an important facet of miRNA-
mediated regulation of critical cellular events. The dosage of Bim
is apparently very important, as cells have engineered numer-
ous mechanisms to quantitatively regulate its protein levels.
Bim mediates both mitochondrial and ER stress–induced
apoptosis and is regulated through multiple cellular modalities,
including phosphorylation, ubiquitination, and proteosome-
mediated degradation (Dijkers et al., 2000; Puthalakath and
Strasser, 2002; Ley et al., 2005; Puthalakath et al., 2007). Our
findings reveal yet another layer of posttranscriptional Bim
regulation involving miR-24 and suggest that the cell uses
numerous back-up mechanisms and regulatory pathways to
carefully titrate the dose of this central regulator of apoptosis.
MATERIALS AND METHODS
Plasmid construction. Bim 3 UTR was cloned from mouse genomic DNA
and cardiomyocyte cDNA (Fig. S3 H) with the primers 5-AGCGCTCTG-
CACTGTGTCGATGTGAACGG-3 and 5-ATGGCAGGGCTGTCA-
GGGATAGGGATGTC-3. Amplified DNA was cloned into pCRII vector
and subsequently cloned into pMIR-REPORT vector (Applied Biosystems). To
express miR-24 in HeLa cells for luciferase assay, the genomic sequence contain-
ing premiR-24-2 plus flanking sequence (total 300 bp) were inserted into
pEF-DEST51 (Invitrogen) with the primers 5-CACCATCTCCTCAGGCC-
GCTGCTG-3 and 5-CTATCTGCTTTGGGGAACCACAG-3. Site-
directed PCR-mediated mutagenesis was performed using the QuikChange II
XL Site-Directed Mutagenesis kit (Agilent Technologies). The following two
pairs of primers were used to mutate both miR-24 binding sites in Bim 3 UTR:
between control inhibitor (set as 1) and miR-24 inhibitor after MI. The vertical black line indicates that intervening lanes have been spliced out. (F) TUNEL
stainings on MI heart sections coinjected with Bim siRNA or control siRNA and miR-24 inhibitor. (G) Quantification of TUNEL+ or activated Caspase 3+
cardiomyocytes. All experiments were repeated three times (technical triplicates), with biological duplicates indicated in each panel. Error bars indicate
SEM (*, P < 0.05; **, P < 0.01). All data in this figure were collected 24 h after MI. Bars, 50 µm.
miR-24 inhibits cardiomyocyte apoptosis | Qian et al.
oxygen administered via an MRI-compatible mobile inhalation anesthesia
system (VetEquip) and maintained at 37°C. Two electrocardiography (ECG)
leads were inserted into the right front and left rear leg. ECG waveforms
were monitored with a small animal monitoring and gating system (SA In-
struments). The mouse was then placed into a homemade 1H birdcage coil
with an inner diameter of 32 mm. A group of ECG (R-wave rising edge)-
triggered spin echo scout images were acquired first to define the oblique
plane of the short axis. Then an ECG-triggered two-dimensional gradient
echo sequence with an echo time of 2.75 ms, repetition time of 200 ms, and
a flip angle of 45° was used to obtain cine short-axis images at 12 or 13
phases per cardiac cycle. Each scan consisted of eight to nine contiguous
slices spanning LV from apex to base with 1-mm thickness, a matrix size of
128 × 128, a field of view of 25.6 × 25.6 mm, and four means.
Immunohistochemistry on mouse hearts. After perfusion fix, the heart
was removed and fixed by immersion in 4% PFA in PBS (diluted from 20%
PFA stock; Electron Microscopy Sciences) and routinely processed and paraf-
fin embedded. 5-µm sections were stained with hematoxylin and eosin and
analyzed for regular morphology. To measure the infarct size at 4 wk after MI,
LVs were cut from apex to base; sections from four equally distributed levels
were Azan or Masson’s trichrome stained. Scar size was calculated as the per-
centage of the LV circumference, or total scar area divided by total LV area,
and was summed from four transverse sections per heart. Adjacent sections
were used to quantify vascular density after staining with PECAM-1 anti-
body (rat; 1:20; BD) as described previously (van Laake et al., 2006). To quan-
tify apoptotic cardiomyocytes, mouse hearts were removed 24 h after
coronary artery ligation, fixed with 0.5% PFA in 5% sucrose, and routinely
frozen embedded in OCT and processed for sectioning and staining with
Caspase 3 (1:200) or TUNEL and -Actinin (mouse; 1:800; Sigma-Aldrich)
as described previously (van Laake et al., 2008). TUNEL was performed using
an In Situ Cell Death Detection kit, Fluorescein (Roche) per the manufac-
turer’s protocol. DAPI was used for nuclear counterstaining. Frozen heart
sections were also stained with pH3 (rabbit; 1:100; Millipore), -Actinin, and
DAPI to quantify proliferating cardiomyocytes.
Statistics. For echocardiography, MRI, TUNEL, Azan, Masson’s trichrome,
Evans blue/TTC, and PECAM-1 staining quantification, statistical analysis
was performed using SPSS version 15. Comparisons between groups were
made by one-way analysis of variance or Mann–Whitney U test, as applica-
ble. Sample numbers were indicated in corresponding figures. For quantita-
tive RT-PCR, FACS, and luciferase assay, we used biological triplicates and
technical triplicates; data were analyzed by unpaired Student’s t test. Error
bars indicate SEM. *, P < 0.05; **, P < 0.01.
Online supplemental material. Fig. S1 shows qPCR for miR-23 and
miR-27 in MI heart and the expression pattern of miR-24. Fig. S2 shows
validation for miR-24 mimic and inhibitor transfection in primary cardio-
myocytes. Fig. S3 shows that miR-24 regulates apoptosis but not cell cycle
and miR-24 regulates Bim in cardiomyocytes. Fig. S4 shows validation for
miR-24 mimic and inhibitor in vivo transfection. Fig. S5 shows additional
MRI and echo data, Masson’s trichrome staining on hearts 4 wk after MI,
and validation for Bim siRNA knockdown. Online supplemental material is
available at http://www.jem.org/cgi/content/full/jem.20101547/DC1.
We are grateful for expert technical assistance from the Gladstone Stem Cell Core
(J. Arnold and K.N. Ivey), Flow Cytometry Core (S. Elmes), and Genomics Core (L. Ta,
Y. Hao, and C.S. Barker). We thank all of the members of the Srivastava laboratory
for helpful discussion.
L. Qian is a postdoctoral scholar of the California Institute for Regenerative
Medicine. D. Srivastava was supported by grants from the National Heart, Lung and
Blood Institute/National Institutes of Health, the California Institute for
Regenerative Medicine, and the Younger Family Foundation. L.W. van Laake was
supported by an Interuniversity Cardiology Institute of the Netherlands fellowship
grant. This work was supported by National Institutes of Health/National Center for
Research Resources grant C06 RR018928 to the Gladstone Institute of
The authors have no conflicting financial interests.
at 4°C). Subsequently, 10 µl of 500 µg/ml RNase and 10 µl of 1 mg/ml PI
were added to resuspended cells and incubated at 37°C for 30 min in the
dark. After washing with PBS twice, stained cells were analyzed by FACS-
Calibur. For FACS sorting of endothelial cells, dissociated cells from mouse
hearts were stained with FITC rat anti–mouse CD31 (BD) for 30 min at
room temperature. After washing with PBS twice, stained cells were sorted
by Aria (BD).
Mouse MI model. The protocol was approved by institutional guidelines
(University of California, San Francisco Institutional Animal Care and Use
Committee). All surgeries and subsequent analyses were performed in a
blinded fashion for genotype and intervention. Mice were anesthetized with
2.4% isoflurane/97.6% oxygen and placed in a supine position on a heating
pad (37°C). Animals were intubated with a 19-gauge stump needle and ven-
tilated with room air with a MiniVent Type 845 mouse ventilator (Hugo
Sachs Elektronik-Harvard Apparatus; SV, 250 µl; respiratory rate, 120 breaths/
min). MI was induced by permanent ligation of the left anterior descending
artery with a 7-0 Prolene suture as described previously (Bock-Marquette
et al., 2004). Sham-operated animals served as surgical controls and were
subjected to the same procedures as the experimental animals with the ex-
ception that the left anterior descending artery was not ligated. All surgical
procedures were performed under aseptic conditions. 4 wk or 24 h after oc-
clusion, the heart was removed for perfusion fix (4% paraformaldehyde
[PFA]; paraffin sections for structural analysis and immunohistochemistry) or
immersion fix (0.5% PFA in 5% sucrose; cryo sections for immunofluores-
cent staining), or the tissues within the IZ, BZ, and nonischemic zone distal
to the IZ (DZ) were dissected for RNA or protein isolation.
Determination of the AAR and myocardial infarct size. 24 h after
coronary ligation, the mice were anesthetized and cannulated with tubing.
2% Evans blue (Sigma-Aldrich) was perfused into the aorta; thus, all myocar-
dial tissue was stained blue except the AAR. The LV was isolated and cut into
four 1-mm pieces with the first cut at the ligation level. LV slices were
stained in 1.5% TTC for 30 min at 37°C and then fixed in 4% PFA overnight
at 4°C. The area of infarction was demarcated as a white area, whereas viable
myocardium was stained red. Photographs were taken for both sides of each
section. The AAR and the infarct area were determined via planimetry by
using the computer software ImagePro (Bio-Rad Laboratories). Infarct size
was calculated as the percentage of MI compared with the AAR using a pre-
viously described methodology (Kurrelmeyer et al., 2000).
In vivo delivery of miR-24 mimic, inhibitor, and Bim siRNA. With
the chest open, oligonucleotides stabilized with 2-O-methyl modification
pretreated with 20 µl Lipofectamine 2000 were injected into the myocar-
dium through an insulin syringe with incorporated 29-gauge needle (BD)
into the myocardium. The dosages used per mouse were 40 ng miR-24
mimic, 40 ng control mimic, 80 ng miR-24 inhibitor, 80 ng inhibitor control
(Shanghai GenePharma Co.), 3 nmol Bim siRNAs (a pool of three target-
specific 20–25 nt siRNAs; Santa Cruz Biotechnology, Inc.), or 3 nmol nega-
tive control siRNAs (Santa Cruz Biotechnology, Inc.). For each type of
oligonucleotide, one injection with a full dosage was used along the bound-
ary between the IZ and BZ (based on the blanched infarct area) after coro-
nary artery occlusion. After injection, the chest was closed with sutures, and
the mouse was allowed to recover with mouse ventilator and heating pad.
Echocardiography. Echocardiography was performed by the Vevo 770
High-Resolution Micro-Imaging System (VisualSonics) with a 15-MHz lin-
ear array ultrasound transducer. The LV was assessed in both parasternal long-
and short-axis views at a frame rate of 120 Hz. End systole or end diastole
was defined as the phase in which the smallest or largest area of LV, respec-
tively, was obtained. Left ventricular end systolic diameter and left ventricular
end diastolic diameter were measured from the LV M-mode tracing with a
sweep speed of 50 mm/s at the papillary muscle level.
MRI. MRI was performed on a small animal scanner (DirectDrive 7T;
Varian). Each mouse was anesthetized by inhalation of 2% isoflurane/98%
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