NFAT4-dependent miR-324-5p regulates mitochondrial morphology and cardiomyocyte cell death by targeting Mtfr1

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Abstract Emerging evidence suggest that the abnormal mitochondrial fission participates in pathogenesis of cardiac diseases, including myocardial infarction and heart failure. However, the molecular components regulating mitochondrial network in heart remain largely unidentified. Here we report that NFAT4, miR-324-5p and mitochondrial fission regulator 1 (Mtfr1) function in one signaling axis that regulates mitochondrial morphology and cardiomyocyte cell death. Knocking down Mtfr1 suppresses mitochondrial fission, apoptosis and myocardial infarction. Mtfr1 is a direct target of miR-324-5p, and miR-324-5p attenuates mitochondrial fission, cardiomyocyte apoptosis and myocardial infarction by suppressing Mtfr1 translation. Finally, we show that transcription factor NFAT4 inhibits miR-324-5p expression. Knockdown of NFAT4 suppresses mitochondrial fission and protects cardiomyocyte from apoptosis and myocardial infarction. Our study defines the NFAT4/ miR-324-5p/Mtfr1 axis, which participates in the regulation of mitochondrial fission and cardiomyocyte apoptosis, and suggests potential new treatment avenues for cardiac diseases.

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[Citation: Cell Death and Disease (2015) 6, e2007; doi:10.1038/cddis.2015.348](http://dx.doi.org/10.1038/cddis.2015.348)
& 2015 Macmillan Publishers Limited All rights reserved 2041-4889/15

[www.nature.com/cddis](http://www.nature.com/cddis)

# NFAT4-dependent miR-324-5p regulates mitochondrial morphology and cardiomyocyte cell death by targeting Mtfr1

K Wang* ,1,5, D-l Zhang 2,5, B Long 3,5, T An 4, J Zhang 4, L-Y Zhou 1, C-Y Liu 1 and P-F Li* ,1

Emerging evidence suggest that the abnormal mitochondrial fission participates in pathogenesis of cardiac diseases, including
myocardial infarction and heart failure. However, the molecular components regulating mitochondrial network in heart remain
largely unidentified. Here we report that NFAT4, miR-324-5p and mitochondrial fission regulator 1 (Mtfr1) function in one signaling
axis that regulates mitochondrial morphology and cardiomyocyte cell death. Knocking down Mtfr1 suppresses mitochondrial
fission, apoptosis and myocardial infarction. Mtfr1 is a direct target of miR-324-5p, and miR-324-5p attenuates mitochondrial
fission, cardiomyocyte apoptosis and myocardial infarction by suppressing Mtfr1 translation. Finally, we show that transcription
factor NFAT4 inhibits miR-324-5p expression. Knockdown of NFAT4 suppresses mitochondrial fission and protects cardiomyocyte
from apoptosis and myocardial infarction. Our study defines the NFAT4/ miR-324-5p/Mtfr1 axis, which participates in the regulation
of mitochondrial fission and cardiomyocyte apoptosis, and suggests potential new treatment avenues for cardiac diseases.
[Cell Death and Disease (2015) 6, e2007; doi:10.1038/cddis.2015.348; published online 3 December 2015](http://dx.doi.org/10.1038/cddis.2015.348)

Cardiac disease remains the leading cause of death for people
worldwide. Heart pumps oxygen and nutrient-rich blood
throughout the body to sustain life and has an extensive
requirement for energy production. Many studies unveil that
cardiomyocytes possess large amounts of mitochondria.
Mitochondria ensure the continuous aerobic respiration and
production of ATP for cardiac function. Mitochondria forms a
complex network by fission and fusion, 1 and mitochondrial
network is associated with various cellular process, such as
embryo development, cell differentiation, apoptosis and
necrosis. 2–5 Mitochondrial network dynamics, particularly,
mitochondrial fission and fusion, participate in maintaining
mitochondrial function. 6 The disruption of mitochondrial
fission and fusion is related to several human diseases,
including neurodegeneration, cardiovascular diseases
and cancer. 7–9 Accumulating lines of evidence indicate that
cardiac diseases are associated with mitochondrial
dysfunction. 8,10,11 Mitochondrial fission leads to the formation
of small round mitochondria and promotes cell apoptosis,
whereas fusion results in mitochondria elongation and
have a protective role in cardiomyocytes maintenance. 8,11

These findings strongly suggest that mitochondrial dynamics
is important for cardiac function. Thus, unveiling the
mechanisms of mitochondrial network regulation will provide
a novel therapeutic strategy for cardiovascular diseases
and heart failure.

Mitochondrial fission regulator 1 (Mtfr1) is a mitochondrial
protein containing a short polyproline-rich region previously
named CHPPR (chondrocyte protein with a polyproline
region). Mtfr1 targets mitochondria and is mostly associated
with the mitochondrial inner membranes. 12 Recent finding
suggests that Mtfr1-expressing cells reveal the presence of a
number of small spheroid mitochondria which indicates that
Mtfr1 is able to promote mitochondrial fission. 12 In addition,
study also reports that deficiency of Mtfr1 results in oxidative
DNA damage. 13 However, it is not yet clear whether Mtfr1
participates in the regulation of mitochondrial dynamics in
cardiomyocytes.

MicroRNAs (miRNAs) are a class of short single-stranded
noncoding endogenous RNAs that suppress protein expression by binding to mRNAs. 14 MiRNAs were identified as
important transcriptional and post-transcriptional inhibitors of
gene expression. Numerous studies have suggested that
miRNAs are involved in several fundamental cellular
processes, such as cell survival, apoptosis, necrosis and
development. 3,4,15,16 Gain or loss‑of‑function studies have
uncovered important roles for miRNAs in cardiac diseases,
including myocardial infarction, cardiac hypertrophy and heart
failure. 17 During cardiac diseases, miRNA-expression profile
is significantly changed, indicating an extremely dynamic
regulation of miRNAs in the adult heart. 18 Although the
function of miRNAs has been widely studied, few works have

1Center for Developmental Cardiology, Institute for Translational Medicine, College of Medicine, Qingdao University, Qingdao, China; 2Department of Cardiology, Shanghai
Chest Hospital, Shanghai Jiaotong University, Shanghai, China; 3 Laboratory of Molecular Medicine, Central Research Laboratory, Peking Union Medical College Hospital,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China and 4 State Key Laboratory of Cardiovascular Disease, Heart Failure Center,
Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
*Corresponding author: K Wang or P-F Li, Center for Developmental Cardiology, Institute for Translational Medicine, College of Medicine, Qingdao University, No.38
[DengZhou Road, Qingdao 266021, China. Tel: +86 532 82991039; Fax: +86 532 83801449; E-mail: Wangk20150812@sina.com or peifengliqd@163.com](mailto:Wangk20150812@sina.com)
5These authors contributed equally to this work.

Abbreviations: miRNAs, microRNAs; A/R, Anoxia/reoxygenation; I/R, Ischemia/reperfusion; Mtfr1, Mitochondrial fission regulator 1; TUNEL, Terminal deoxynucleotidyl
transferase-mediated dUTP nick-end-labeling; EM, Electron microscopy; qRT-PCR, Quantitative reverse transcription polymerase chain reaction; TP, Target protector

Received 12.8.15; revised 10.10.15; accepted 02.11.15; Edited by Y Shi

NFAT4 regulates mitochondrial dynamics by miR-324-5p/Mtfr1

K Wang et al

been focused on miRNAs in the mitochondrial network
regulation.

NFATc3, also named NFAT4, is produced by NFAT4 gene
located on murine chromosome 8. 19 NFAT4 is modulated by
Ca 2+ /calmodulin signaling pathway and its stimulation
requires calcium oscillation. 20 NFAT4 contains a conserved
rel similarity domain and an SP repeat region. 19 Overexpression of NFAT4 activates NFAT site-dependent transcription
and regulates distinct subset of genes. 19 It has been proved
that NFAT4 are also expressed in the heart and is a
downstream target of calcineurin. 21 In the quiescent cells,
NFAT4 is retained in the cytoplasm. 22 After dephosphorylation
by calcineurin, NFAT4 is activated and imported into the
nucleus. 22 The previous work shows that NFAT4 has an
essential role in reducing voltage-gated K + currents after a
myocardial infarction. 23 However, the role of NFAT4 in
regulating cardiomyocyte mitochondrial fission and apoptosis
remains largely unveiled.

Our present study unveils that Mtfr1 is involved in the
regulation of mitochondrial network in cardiomyocytes.
Knockdown of Mtfr1 inhibits mitochondrial fission and
apoptosis in cardiomyocytes. Also, knockdown of Mtfr1 in
mice exhibit a reduced myocardial infarction sizes upon
myocardial ischemia/reperfusion (I/R) injury in vivo. In search
for the mechanisms by which Mtfr1 is upregulated under a
pathological condition, we identified miR-324-5p as a
translational inhibitor of Mtfr1. MiR-324-5p inhibits
mitochondrial fission, apoptosis and myocardial infarction
through downregulating Mtfr1. We further find that NFAT4
transcriptionally suppresses miR-324-5p expression and thus
regulates mitochondrial fission and apoptosis. Our results
reveal a novel pathway in which NFAT4 promotes Mtfr1
expression by suppressing miR-324-5p transcriptional activity,
which could contribute to the regulation of mitochondrial
dynamics and apoptosis.

Results

Mtfr1 regulates mitochondrial fission and apoptosis in
cardiomyocytes. To test whether Mtfr1 participates in the
regulation of mitochondrial fission in cardiomyocytes, we
treated cardiomyocytes with A/R to induce mitochondrial
fission and apoptosis. A/R treatment induced the increase of
Mtfr1 expression levels (Figure 1a). We then tested
whether Mtfr1 is involved in the occurrence of mitochondrial
fission. Knockdown of Mtfr1 by siRNA technology efficiently
attenuated A/R-induced Mtfr1 levels (Figure 1b). Reduced
mitochondrial fission as revealed by the analysis of
mitochondrial morphology (Figure 1c) and the counting
of the cells with fission (Figure 1d and Supplementary
Figure 1A) were also observed together with Mtfr1
downregulation. Further, knockdown of Mtfr1 attenuated
A/R-induced apoptosis analyzed by TUNEL assay
(Figure 1e and Supplementary Figure 1B) and Caspase 3
activity (Figure 1f). Taken together, these results suggest that
Mtfr1 participates in the regulation of mitochondrial fission
and apoptosis in cardiomyoctyes.

Mtfr1 regulates mitochondrial fission and apoptosis
in vivo. To understand the pathophysiological role of Mtfr1,
we detected whether Mtfr1 was involved in the pathogenesis
of myocardial infarction in the animal model. We employed
the myocardial I/R model and found that I/R induced an
elevation in Mtfr1 expression levels (Figure 2a). The
administration of Mtfr1 siRNA adenoviruses in vivo reduced
the elevated expression of Mtfr1 upon I/R injury (Figure 2b).
Knockdown of Mtfr1 also resulted in a reduction in
mitochondrial fission (Figure 2c), apoptosis (Figure 2d) and
infarct size (Figure 2e) upon I/R. Echocardiography showed
that the cardiac function was ameliorated in Mtfr1-knockdown
group in response to I/R injury (Supplementary Figures 1C
and D, and Figure 2f). These data suggest that Mtfr1
participates in mediating the signal for mitochondrial fission
and apoptosis in the heart.

miR-324-5p participates in the regulation of Mtfr1 expression. miRNAs are a class of small noncoding RNAs and act
as negative regulators of gene expression. To explore the
underlying mechanism by which Mtfr1 is upregulated upon
A/R and I/R, we tested whether Mtfr1 can be regulated by
miRNA. We first screened some cardiac-associated miRNAs
that had been reported in past several years. Among several
miRNAs, miR-324-5p levels were significantly downregulated
upon A/R (Figure 3a) and other miRNAs remain unchanged
(data not shown). We also analyzed the 3′UTR of Mtfr1 using
the RNAhybrid program and observed that Mtfr1 is a potential
target of miR-324-5p (Figure 3b), which promoted us to focus
on the function of miR-324-5p. We then explored whether
Mtfr1 expression can be regulated by miR-324-5p. We found
that miR-324-5p had no effect on the mRNA level of Mtfr1
(Supplementary Figures 2A and B), whereas knockdown of
endogenous miR-324-5p by antagomir induced an increase
in Mtfr1 protein levels (Figure 3c), and enforced expression of
miR-324-5p attenuated the increase of Mtfr1 protein levels in
response to A/R treatment (Figure 3d). These results indicate
that miR-324-5p can specifically regulate Mtfr1.

Next, we employed the luciferase assay system to test
whether miR-324-5p can influence the translation of Mtfr1.
As shown in Figure 3f, the luciferase reporter assay revealed
that the wild-type (wt) 3′-UTR of Mtfr1 exhibited a low
translation level in the presence of miR-324-5p, whereas the
mutated 3′-UTR (Figure 3e) did not show a significant
response to miR-324-5p. We finally tested whether
miR-324-5p regulates Mtfr1 expression by targeting its 3′
UTR. Mtfr1 with wt 3′UTR was expressed at a low level in the
presence of miR-324-5p (Figure 3g). Introduction of mutations
into the miR-324-5p binding site abolished the inhibitory effect
of miR-324-5p on Mtfr1 expression (Figure 3h). Taken
together, these results suggest that Mtfr1 is a specific target
of miR-324-5p.

miR-324-5p inhibits mitochondrial fission and apoptotic
program. We explored the functional role of miR-324-5p in
mitochondrial fission and apoptosis. Enforced expression of
miR-324-5p efficiently increased the miR-324-5p levels in
response to A/R treatment (Supplementary Figure 2C), and
miR-324-5p also attenuated A/R-induced mitochondrial
fission (Figures 4a and b). Concomitantly, apoptosis was

Cell Death and Disease

NFAT4 regulates mitochondrial dynamics by miR-324-5p/Mtfr1
K Wang et al

kDa

Mtfr1

Actin

0 3 6 9 12
A/R
Time (hour)

kDa

A/R _ +
Mtfr1-sc _ _
Mtfr1-siRNA _ _

Mtfr1

Actin

_ + + +

_ _

+ _

_ + + +

Control A/R

_ + + +

A/R+ A/R+
Mtfr1-sc Mtfr1-siRNA

## ~~*~~

A/R _ +
Mtfr1-sc _ _
Mtfr1-siRNA _ _

_ _

## ~~*~~

+ _

_ + + +

A/R _ +
Mtfr1-sc _ _
Mtfr1-siRNA _ _

_ _

+ _

A/R _ +
Mtfr1-sc _ _
Mtfr1-siRNA _ _

Figure 1 Mtfr1 regulates mitochondrial fission and apoptosis in vitro. (a) A/R induces an increase in Mtfr1 levels. Cardiomyocytes were treated with A/R at indicated time.
Mtfr1 levels were analyzed by immunoblot. (b) Knockdown of Mtfr1 attenuates the increased Mtfr1 levels upon A/R treatment. Cardiomyocytes were infected with adenoviral
constructs of Mtfr1 siRNA or its scrambled form (sc) and then exposed to A/R. Mtfr1 levels were analyzed by immunoblot. (c and d) Knockdown of Mtfr1 attenuates mitochondrial
fission induced by A/R treatment. Cardiomyocytes were infected with adenoviruses harboring Mtfr1 siRNA or its sc form and then treated with A/R. Mitochondria were stained by
MitoTracker red and the nuclei were visualized by DAPI. (c) Scale bar, 20 μm (upper panel); scale bar, 5 μm (lower panel). The cells with fragmented mitochondria were counted.
(d) *Po0.05 versus A/R alone. (e and f) Knockdown of Mtfr1 reduces A/R-induced apoptosis. Cardiomyocytes were infected with adenoviruses harboring Mtfr1 siRNA or its sc
form and then were subjected to 2-h anoxia followed by 12-h reoxygenation. TUNEL was employed to analyze apoptotic cells. TUNEL-positive cells were counted and calculated.
(e) The caspase-3 activity was analyzed by using an Apo-ONE Homogeneous Caspase-3/7 assay kit (Madison, WI, USA) (f) *Po0.05 versus A/R alone

reduced in the presence of miR-324-5p as revealed
by TUNEL assay (Figure 4c) and Caspase 3 activity
(Supplementary Figure 2D). These data indicate that
miR-324-5p can inhibit mitochondrial fission and apoptosis
in cardiomyocytes.

Subsequently, we detected whether miR-324-5p is involved
in the pathogenesis of myocardial infarction in the animal
model. MiR-324-5p levels were reduced in response to an I/R
injury (Figure 4d). Enforced expression of miR-324-5p by
mimic delivery in vivo resulted in a reduction in Mtfr1
expression (Figure 4e, upper panel), mitochondrial fission
(Figure 4e, lower panel) and apoptosis (Figures 4f and g). We
then tested whether miR-324-5p affects the size of myocardial
infarction and cardiac function. Administration of miR-324-5p
mimics significantly reduced infarct sizes upon I/R injury
(Supplementary Figure 3A). Concomitantly, the heart function
was ameliorated when the mice were treated with miR-324-5p
mimics as assessed by echocardiography (Supplementary
Figures 3B and D).

miR-324-5p regulates mitochondrial fission and
apoptosis through targeting Mtfr1. We explored how
miR-324-5p exerts its effect on the mitochondrial network.
As miR-324-5p is able to suppress Mtfr1 expression, we thus
tested whether Mtfr1 is a mediator of miR-324-5p. Mtfr1 with
mutated 3′UTR, but not the wt Mtfr1, attenuates the inhibitory
effect of miR-324-5p on Mtfr1 expression (Figure 5a, upper
panel), mitochondrial fission (Figure 5a, lower panel) and
apoptosis (Figure 5b). To further confirm the relationship
between miR-324-5p and Mtfr1 in mitochondrial fission
machinery, we employed the target protector technology in
which a target protector is able to disrupt the specific
interaction of miRNA–mRNA pairs. To this end, we produced
Mtfr1 target protector and observed that the inhibitory effect
of miR-324-5p on mitochondrial fission and apoptosis was
reduced in the presence of the Mtfr1 target protector
(Figures 5c and d). These data suggest that miR-324-5p
targets Mtfr1 in the cascades of mitochondrial fission and
apoptosis.

Cell Death and Disease

NFAT4 regulates mitochondrial dynamics by miR-324-5p/Mtfr1

K Wang et al

kDa

Mtfr1

Actin

kDa

+ _ _ _

_ + + +

_ _ + _

_ _ _ +

_ _

Sham

12 0 4 8 12

Sham I/R (hour)

I/R

## ~~*~~

Mtfr1

Actin

Sham
I/R

Mtfr1-sc

Mtfr1-siRNA

## ~~*~~

I/R+Mtfr1-sc I/R+Mtfr1-siRNA

Sham + _

_ _ Sham + _ _ _

_ + + +

I/R _ +
Mtfr1-sc _ _
Mtfr1-siRNA _ _

_ + + +

_ _

_ + _
+

+ _

_ _

_ + _
+

+ _

## ~~*~~

50
## 40 *

0
Sham + _ _ _

_ _

0
+ _ _ _

_ + + +

_ _

_ + _
+

+ _

Sham + _

_ + + +

I/R _ +
Mtfr1-sc _ _
Mtfr1-siRNA _ _

_ _

_ + _
+

+ _

Figure 2 Mtfr1 regulates mitochondrial fission and apoptosis in vivo. (a) Mtfr1 is upregulated in response to ischemia/reperfusion. Mice were subjected to various time periods
of ischemia, and Mtfr1 levels were detected by immunoblot. (b) Mtfr1 siRNA can efficiently knockdown Mtfr1 in the animal model. After intracoronary delivery of adenoviruses
harboring Mtfr1 siRNA or the scrambled form, the mice were subjected to I/R injury. Mtfr1 levels were detected by immunoblot. (c and d) Knockdown of Mtfr1 attenuates
I/R-induced mitochondrial fission and apoptosis. After intracoronary delivery of adenoviruses harboring Mtfr1 siRNA or the scrambled form, the mice were subjected to 45-min
ischemia, then 3-h reperfusion. Mitochondrial fission was analyzed. (c) TUNEL assay was employed to detect apoptotic cells. (d) *Po0.05 versus I/R alone. (e) Knockdown of
Mtfr1 decreases myocardial infarct sizes in response to I/R. Mice were treated as described for (b) and infarct sizes were calculated. The upper panels are myocardial
representative photos of mid-ventricular myocardial slices. Scale bar, 2 mm. *Po0.05 versus I/R alone. (f) Echocardiographic analysis. Mice were treated as described for (b)
and echocardiography was employed to test heart function. Fractional shortening (FS) were calculated. *Po0.05 versus I/R alone

NFAT4 regulates mitochondrial fragmentation and
apoptosis by affecting miR-324-5p transcription. We
explored how miR-324-5p expression is regulated under a
pathological condition. MiRNA could be regulated at the
transcriptional level as we described previously. 3 Thus, we
analyzed the promoter region of mouse miR-324-5p and
observed that there was a potential binding site of NFAT4
(Figure 6a). The luciferase assay demonstrated that NFAT4
attenuated the wt miR-324-5p promoter activity (Figure 6b).
However, mutations in the NFAT4 binding site abolished the
effect of NFAT4 on miR-324-5p promoter activity (Figure 6b).
Enforced expression of NFAT4 (Supplementary Figure 4A)
induced a decrease in miR-324-5p levels (Supplementary
Figure 4B), whereas NFAT4 knockdown (Supplementary
Figure 4C) exhibited an increased expression levels of
miR-324-5p (Supplementary Figure 4D). The ChIP assay

Cell Death and Disease

revealed that NFAT4 is bound to the miR-324-5p
promoter under the physiological condition. A/R treatment
led to an increase in the association levels of NFAT4
with miR-324-5p promoter (Figure 6c), suggesting that
miR-324-5p is a potential transcriptional target of NFAT4.
Furthermore, A/R induced a decrease in miR-324-5p
promoter activity in cardiomyocytes (Figure 6d), and
knockdown of NFAT4 attenuated the inhibitory effect of A/R
on miR-324-5p promoter activity (Figure 6d). These data
indicate that miR-324-5p can be transcriptionally inhibited
by NFAT4.

We investigated the role of NFAT4 in mitochondrial fission
and apoptosis in cardiomyocytes and in vivo. Enforced
expression of NFAT4 induced an increase in Mtfr1 levels
(Supplementary Figure 5A), whereas NFAT4 knockdown
exhibited a decreased Mtfr1 expression (Supplementary

1.2

0.8

0.4

NFAT4 regulates mitochondrial dynamics by miR-324-5p/Mtfr1
K Wang et al

_miR-324-5p_

_Mtfr1 3’UTR_

~~*~~

0 6 9 12
A/R

Time (hours)

kDa

Mtfr1

Actin

kDa

Mtfr1

Actin

_ 12 24 A/R

_ +

anta-324-5p (h) 0 _ 12 24

anta-NC (h) _ 24 _ _

mimic-324-5p _ _ _ +

_ _ +

+ + +

_ _

anta-NC (h) _ 24 _ _

mimic-NC

* ~~*~~

5’-GGCCAGAAGCACCU UGGGGGA G-3’

_BS_

_3’UTR-wt_
_Firefly luciferase_ _Mtfr1 3’UTR_

5’-GGCCAGAAGCACCU Ucaaaac G-3’

120

_BS_

_3’UTR-mut_

_Firefly luciferase_ _Mtfr1 3’UTR_

Mtfr1-3’UTR-mut _ _ _ + +

Mtfr1-3’UTR-wt + + + _ _

_Firefly luciferase_

+ +

_ _

_ +

+ +

+ _

kDa

+ +

mimic-NC

mimic-324-5p

Mtfr1

Actin

Mtfr1

Actin

kDa

+ _

+ _ _

+
+ +

Mtfr1-mut-UTR _ +

mimic-NC

mimic-NC __ __ + _ _ mimic-324-5pmimic-NC

mimic-324-5p

Mtfr1-wt-UTR

Figure 3 MiR-324-5p participates in the regulation of Mtfr1 expression. (a) MiR-324-5p levels were analyzed by qRT-PCR. *Po0.05 versus control. (b) Analysis of Mtfr1 3′
UTR potential binding site for miR-324-5p. (c) Knockdown of miR-324-5p induces an elevated Mtfr1 levels. Cardiomyocytes were transfected with antagomir miR-324-5p
(anta-324-5p) or antagomir-negative control (anta-NC). The expression of Mtfr1 was detected by immunoblot. (d) miR-324-5p attenuates A/R-induced Mtfr1 upregulation.
Cardiomyocytes were transfected with miR-324-5p mimic (mimic-324-5p) and its negative control (mimic-NC), and then were treated with A/R. Mtfr1 levels were detected by
immunoblot. (e) miR-324-5p binding site was mutated in the Mtfr1 3′UTR. (f) miR-324-5p inhibits the luciferase activity of Mtfr1 with wild-type 3′UTR. HEK293 cells were
transfected with Mtfr1 with a wild-type 3′UTR or mutated 3′UTR, miR-324-5p mimic or its negative control, and then the cells were harvested, and luciferase activity was
measured; *Po0.05. (g and h) miR-324-5p inhibits the expression of Mtfr1 with wild-type 3′UTR. Cardiomyocytes were infected with adenoviral Mtfr1 with a wild-type 3′UTR or
mutated 3′UTR, and then were transfected with miR-324-5p mimic. The expression of Mtfr1 was assayed by immunoblot

Figure 5B). A/R induced an elevation in NFAT4 expression
level (Supplementary Figure 6A). Knockdown of NFAT4
attenuated NFAT4 expression (Supplementary Figure 6B),
mitochondrial fission (Figure 6e and Supplementary Figure
6C) and apoptosis (Figure 6f and Supplementary Figure 6D)
upon A/R. Furthermore, NFAT4 knockdown in vivo
(Supplementary Figure 7A) reduced Mtfr1 expression
(Figure 6g, upper panel) and induced the increase of
miR-324-5p levels (Supplementary Figure 7B). Knockdown
of NFAT4 attenuated mitochondrial fission (Figure 6g, lower
panel), apoptosis (Figure 6h) and myocardial infarction size
(Figure 6i) upon I/R injury. These data suggest that NFAT4

participates in mediating the signal for mitochondrial fission
and apoptosis in the heart.

Discussion

Recently, it has been found that mitochondria has an important
role in regulating cell death. Mitochondria constantly fuse and
divide to form a dynamic network. 24 Mitochondria fragmentation during cell death has been shown to have a key role in cell
death progression, including release of the mitochondrial
apoptotic proteins. Other morphological changes, such as
cristae remodeling, swelling and outer membrane rupturing,

Cell Death and Disease

NFAT4 regulates mitochondrial dynamics by miR-324-5p/Mtfr1

K Wang et al

_ + + +

Control A/R

A/R+ A/R+
mimic-NC mimic-324-5p

### ~~*~~

A/R _ +

_ _

I/R _ +

_ + + +

+ _

kDa

mimic-NC
mimic-324-5p

remote area
area-at-risk

### ~~*~~ ~~*~~

60 0 30 60

Sham Ischemia (min)

Mtfr1

Actin

50
40
30

20
10

### ~~*~~

1.2

0.8

0.4

### ~~*~~

_ + + +

A/R _ +

mimic-NC _
mimic-324-5p _

_ _

Sham + _ _ _

+ _

_ _

_ + _
+

+ _

Sham I/R I/R+ I/R+
mim-NC miR-324-5p

mimic-NC
mimic-324-5p

### ~~*~~

Sham + _ _ _

_ + + +

I/R _ +

_ _

_ + _
+

+ _

mimic-NC
mimic-324-5p

Figure 4 MiR-324-5p inhibits mitochondrial fission and apoptosis in cardiomyocytes and in vivo. (a and b) miR-324-5p suppresses A/R-induced mitochondrial fission.
Cardiomyocytes were transfected with miR-324-5p mimic or NC. After that, they were treated with A/R. Mitochondrial fission was assayed. (a) Scale bar, 20 μm (upper panel);
scale bar, 5 μm (lower panel). *Po0.05 versus A/R alone. (c) MiR-324-5p suppresses A/R-induced apoptosis. Cardiomyocytes were transfected with miR-324-5p mimic or NC
and were treated with A/R. Apoptosis was assayed by TUNEL. *Po0.05 versus A/R alone. (d) Analysis of miR-324-5p expression levels. Mice were subjected to I/R at indicated
time and the expression of miR-324-5p was assayed by qRT-PCR. *Po0.05 versus sham or 0-min group. (e) MiR-324-5p attenuates Mtfr1 expression and mitochondrial fission
upon I/R injury. Adult C57BL/6 mice received miR-324-5p mimic or mimic-NC for 3 days. They were then subjected to I/R. Mtfr1 levels were analyzed by immunoblot (upper panel).
The percentage of mitochondrial fission was calculated (lower panel). *Po0.05 versus I/R alone. (f and g) MiR-324-5p attenuates apoptosis upon I/R injury. Mice were treated as
described in e. Apoptosis was detected by TUNEL assay. TUNEL-positive cardiomyocyte nuclei (apoptotic cells) are green. Nuclei stained by DAPI showed blue. Cardiomyocytes
were labeled with α-actinin. *Po0.05 versus I/R alone

are also involved in cell death initiation. 24 Here we present
evidence for a critical role of Mtfr1 in regulating mitochondrial
dynamics. Mtfr1 can regulate mitochondrial fission and
apoptosis in cardiomyocytes and knockdown of Mtfr1 in mice
showed a reduced myocardial infarction sizes upon ischemia
injury in vivo. In addition, we demonstrated that miR-324-5p is
responsible for the downregulation of Mtfr1, and modulation of
miR-324-5p levels also affects mitochondrial fission, apoptosis
and myocardial infarction. MiR-324-5p exerts its effect on
mitochondrial fission and apoptosis through targeting Mtfr1.
We further identify that NFAT4 regulates mitochondrial fission
and apoptosis by suppressing miR-324-5p in transcriptional

Cell Death and Disease

level. Our results provide a novel evidence demonstrating that
NFAT4, miR-324-5p and Mtfr1 constitute an axis in the
regulated machinery of mitochondrial network and apoptosis.

The present work shows that Mtfr1 participates in the
regulation of mitochondrial fission in cardiomyocytes. This is in
consistence with other reports demonstrating the promotive
effect of Mtfr1 on mitochondrial fission. The function of Mtfr1 in
apoptosis remains unknown. Our work for the first time
suggests that Mtfr1 participates in the regulation of apoptosis
in cardiomyocytes. Knockdown of Mtfr1 in mice exhibits a
reduced mitochondrial fission, apoptosis and myocardial
infarction sizes upon I/R injury. Whether Mtfr1 can also act

Mtfr1

Actin

A/R

NFAT4 regulates mitochondrial dynamics by miR-324-5p/Mtfr1
K Wang et al

kDa

~~*~~

+ _ +_ +_ ++ ++

_ _ _

+ _

+ + + +

+ _ _ _

+ +
_ _ _

+ _

A/R

miR-324-5p _

Mtfr1-mut-UTR
Mtfr1-wt-UTR

miR-324-5p _

_ _ + _ +

_ _ _ + _ +

Mtfr1-mut-UTR
Mtfr1-wt-UTR

A/R

_ _ + _ +

_ _ _ + _ +

+_ +_ ++ ++ miR-324-5pA/R __ + _ +_ +_

+ _ + _ Mtfr1-mut-UTR _ _ + _

+ _

## ~~*~~

Mtfr1-TP miR-324-5p

+ + + A/R _ + + +

A/R

miR-324-5p _ _ +

+ +

Mtfr1-TP control

miR-324-5p _ _ +

+ +

Mtfr1-TP miR-324-5p

_ _

Mtfr1-TP control

_ _ _ +

_ _ + _ Mtfr1-TP miR-324-5p _ _ _ +

_ _ _ Mtfr1-TP control _ _ _ _

_ _

Figure 5 MiR-324-5p regulates mitochondrial fission and apoptosis through Mtfr1. (a and b) MiR-324-5p suppresses the expression of Mtfr1, mitochondrial fission and
apoptosis in the presence of Mtfr1 with wild-type 3′UTR rather than mutated 3′UTR. Cardiomyocytes were infected with adenoviral Mtfr1-wt-UTR or Mtfr1-mut-UTR and were
transfected with miR-324-5p mimic. After transfection, cells were treated with A/R. Mtfr1 levels were detected by immunoblot (a, upper panel). Mitochondrial fission
(a, lower panel) and apoptosis (b) were assayed. *Po0.05. (c and d) The inhibitory effect of miR-324-5p on mitochondrial fission and apoptosis is abolished by Mtfr1 target
protector. Cardiomyocytes were co-transfected with miR-324-5p mimic, Mtfr1 target protector (Mtfr1-TP miR-324-5p ) or the protector control (Mtfr1-TP control ), and then were treated
with A/R. Mitochondrial fission (c) and apoptosis (d) were detected. *Po0.05

as a mediator of mitochondrial fission and apoptosis in other
cell types need to be further clarified in our future research.

Some miRNAs have been reported to regulate apoptosis in
cardiomyocytes. 25–27 However, few works have been focused
on miRNAs in the mitochondrial network regulation. Our
present work for the first time demonstrates that miR-324-5p
regulates mitochondrial fission and apoptosis both in vitro and
in vivo by targeting Mtfr1. MiR-324-5p and Mtfr1 have
complementary expression patterns during cardiomyocytes
apoptosis and myocardial infarction. Overexpression of
miR-324-5p resulted in decreased Mtfr1 expression levels,
mitochondrial fission, cardiomyocytes apoptosis and myocardial infarction. Accordingly, miR-324-5p might be a potential
therapeutic target for myocardial infarction. Our results further
support the notion that miRNAs are important modifiers of
gene expression program in cardiovascular diseases.

Many scientific works report that NFAT4 has an important
role in cardiomyocytes. For example, the nuclear occupancy of
NFAT4 was increased in heart failure. 28 Inhibiting the
calcineurin-NFAT pathway protects the heart from excessive
cardiac remodeling. 29 NFAT4 activation also contributes to
hypertension and increased wall thickness in the heart. 30 Our
present work for the first time reveals the function of NFAT4 in
cardiomyocyte mitochondrial fission and apoptosis. Our
results show that knockdown of NFAT4 inhibits mitochondrial

fission and apoptosis in cardiomyocytes. Knockdown of
NFAT4 in mice also demonstrate reduced mitochondrial
fission and myocardial infarction size upon I/R injury. NFAT4
affects the mitochondrial dynamics and apoptotic pathway by
regulating the expression of miR-324-5p. It would be interesting to study the role of other targets of NFAT4 in the pathway of
mitochondrial fission and apoptosis. Modulation of their levels
may provide a new approach for tackling myocardial infarction.

Hitherto, extraordinary efforts have been devoted to
determining the molecular and pathophysiological characteristics of the diseased heart, aiming to develop novel diagnostic
and therapeutic strategies to combat cardiac diseases. Our
study suggests a potential signal pathway of miR-324-5p and
Mtfr1 for cardiac diseases and they will be intriguing targets for
therapeutic intervention.

Materials and methods
Adenoviral constructions and infection. The mouse Mtfr1 was
synthesized by PCR using mouse cDNA as the template. The adenovirus
harboring the Mtfr1 was constructed using the Adeno-X expression system
(Clontech, Otsu, Japan). The adenovirus containing β-galactosidase (β-gal) is as
we described elsewhere. 31 The mouse Mtfr1 RNA interference (siRNA) target
sequence is 5′-CCATATGGTTCATCTCGAA-3′. A scramble form was used as a
control, 5′-TACTATCGTCACTGACTAG-3′. The mouse Mtfr1-siRNA-B target
sequence is 5′-TGAGCTTGCTGCCCTTAGA-3′. A scramble form was used as a
control, 5′-GCTGAGTTGACCTCTATCG-3′. The NFAT4 RNA interference target

Cell Death and Disease

NFAT4 regulates mitochondrial dynamics by miR-324-5p/Mtfr1

K Wang et al

pGL4.17
wt
mutant ~~*~~

_ + + +

(-1438 ~ -1432)

_NFAT4 BS_ _Pre-miR-324_
_Wild type (wt)_

NFAT4

M CHIP: NFAT4
250bp
100bp

0 30 60 60
_ _ _ _

GAGGAAA

GcaaccA

_Pre-miR-324_

_Mutant_

A/R (min) _ 0 30

+ _ _

Input

NFAT4 _ + + + _

_ +

_ + + +

_ _

_ _ _

_ + _

+ _ +

negative

_ _ _

___ +__ +__ ++_

+_ + +_

~~*~~

pGL4.17

+ _ _ _ _

_ _ _

+ + +

_ _

~~*~~

_ _

~~*~~

NFAT4-sc

+ _

A/R _ +
NFAT4-sc _ _
NFAT4-siRNA _ _

NFAT4-siRNA

A/R

Mtfr1

Actin

0
Sham + _ _ _

_ _ +

+ +

kDa

~~*~~

0
Sham + _ _ _

_ + + +

_ _

_ + _
+

+ _

I/R _ +
NFAT4-sc _ _
NFAT4-siRNA _ _

_ _

_ + _
+

+ _

Sham + _ _ _

_ + + +

I/R _ +
NFAT4-sc _ _
NFAT4-siRNA _ _

_ _

_ + _
+

+ _

Figure 6 miR-324-5p is a transcriptional target of NFAT4. (a) Mouse miR-324-5p promoter region contains a potential NFAT4 binding site. (b) NFAT4 attenuates miR-324-5p
promoter activity. Cardiomyocytes were treated with the adenoviral β-gal or NFAT4, the constructs of the empty vector (pGL-4.17), the wild-type promoter (wt) or the promoter with
mutations in the binding site (mutant), respectively. Luciferase activity was assayed. *Po0.05. (c) ChIP analysis of NFAT4 binding to the promoter of miR-324-5p. (d) Knockdown
of NFAT4 inhibits the decrease of miR-324-5p promoter activity induced by A/R. Cardiomyocytes were treated with the adenoviral NFAT4-siRNA or NFAT4-sc, the constructs of the
empty vector (pGL-4.17), the wt promoter, then were treated with A/R. Luciferase activity was assayed. *Po0.05. (e and f) Knockdown of NFAT4 reduces mitochondrial fission
and apoptosis. Cardiomyocytes were infected with adenoviral NFAT4-siRNA or NFAT4-sc, and then exposed to A/R, mitochondrial fission (e) and apoptosis (f) were analyzed.
*Po0.05 versus A/R alone. (g and h) Knockdown of NFAT4 attenuates mitochondrial fission and apoptosis upon I/R. After intracoronary delivery of adenoviruses harboring
NFAT4 siRNA or the scrambled form, the mice were subjected to I/R injury. Mtfr1 levels were analyzed by immunblot (g, upper panel). Mitochondrial fission (g, lower panel) and
apoptosis (h) were analyzed. *Po0.05 versus I/R alone. (i) Knockdown of NFAT4 decreases myocardial infarct sizes in response to I/R. Mice were treated as described for (g),
and infarct sizes were calculated. *Po0.05 versus I/R alone

sequence is 5′-AAATGTCAAGGGGCTCACA-3′. A scramble form was used as a
control, 5′-ACTCACGTGACGAGAGATA-3′. The mouse NFAT4-siRNA-B target
sequence is 5′-CCCTTTGAGTGCCCAAGTA-3′. A scramble form was used as a
control, 5′-ATTCGCCTAGCTGACTGAC-3′. The adenoviruses harboring siRNA or
their scramble form were constructed using the pSilencer adeno 1.0-CMV System
(Ambion, Grand Island, NY, USA) according to the Kit’s instructions. All constructs
were amplified in HEK293 cells. Adenoviral infection of cardiomyocytes was
performed as we described previously. 32

Transfection of the antagomir. The chemically modified antagomir
complementary to miR-324-5p designed to inhibit endogenous miR-324-5p
expression, the antagomir-negative control (antagomir-NC) were obtained from
GenePharma Co. Ltd. (Shanghai, China). The miR-324-5p antagomir sequence was
5′-ACACCAAUGCCCUAGGGGAUGCG-3′. The antagomir-NC sequence was
5′-CAGUACUUUUGUGUAGUACAA-3′. Cells were transfected with the antagomirs

Cell Death and Disease

or the antagomir-NC using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA)
according to the manufacturer's instruction.

Target protector preparation and transfection. Target protector was
designed and named as others and we described, 31 In brief, Mtfr1-TP miR-324-5p

sequence is 5′- CTCCCCCAAGGTGCTTCTGGCCATG-3′. Mtfr1-TP control sequence
is 5′-TGACAAATGAGACTCTCTCCTCTCC-3′. They were synthesized by Gene
Tools, and were transfected into the cells using the Endo-Porter kit (Gene Tools,
Philomath, OR, USA) according to the kit’s instructions.

Constructions of mouse miR-324-5p promoter. The promoter of
miR-324-5p was amplified from mouse genome using PCR. The forward primer was
5′-GCTATCACAGAGCATTTTCTCAT-3′. The reverse primer was 5′-TGCACCAAAC
ACGACTTTTAACC-3′. The promoter fragment was finally cloned into the vector
pGL4.17 (Promega, Madison, WI, USA). The introduction of mutations in the
putative NFAT4 binding site was performed with the QuikChange II XL

Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) using the wt vector
as a template.

Animal experiments. Male adult C57BL/6 mice (8 weeks old) were obtained
from Institute of Laboratory Animal Science of Chinese Academy of Medical Sciences
(Beijing, China). All experiments were performed according to the protocols approved
by the Institute Animal Care Committee. The mice received on three consecutive days,
intravenous injections of miR-324-5p mimic, or its control at a dose of 35 mg/kg body
weight in a small volume (0.2 ml) per injection. To perform intracoronary delivery of
adenovirus, 5 days before the I/R operation, the mice were anesthetized. The chest
was opened and Mtfr1-siRNA adenoviruses (2 × 10 11 MOI) or NFAT4-siRNA
adenoviruses (2 × 10 10 MOI) were injected with a catheter from the apex of the left
ventricle into the aortic root while the aorta and pulmonary arteries were cross-clamped.
The clamp was maintained for 20 s when the heart pumped against a closed system.
The chest was closed and the animal was transferred back to its cage for recovery.

To perform I/R surgery, the mice were treated as we previously described. 25 All
mice were subjected to 45-min ischemia and then 3-h reperfusion. Sham-operated
group experienced the same procedure except the snare was left untied. Cardiac
function of these groups of animals was evaluated by echocardiographic analysis
14 days after the surgery. Evans blue dye was treated as described, 25 The areas of
infarction (INF) and nonischemic left ventricle (LV) were assessed with computerassisted planimetry (NIH Image 1.57) by an observer blinded to the sample identity.
The ratio of INF/LV was calculated as described. 25

Statistical analysis. Data are expressed as the mean ± S.E.M. of at least
three independent experiments. We used a one-way analysis of variance for
multiple comparisons. A value of Po0.05 was considered significant.

Conflict of Interest
The authors declare no conflict of interest.

Acknowledgements. This work was supported by National Natural Science
Foundation of China (81270160 and 81470522), Beijing Municipal Natural Science
Foundation (7142103).

1. Li Q, Zhou LY, Gao GF, Jiao JQ, Li PF. Mitochondrial network in the heart. Protein Cell 2012;
3: 410–418.
2. Ishihara N, Nomura M, Jofuku A, Kato H, Suzuki SO, Masuda K et al. Mitochondrial fission
factor Drp1 is essential for embryonic development and synapse formation in mice. Nat Cell
Biol 2009; 11: 958–966.
3. Wang JX, Jiao JQ, Li Q, Long B, Wang K, Liu JP et al. miR-499 regulates
mitochondrial dynamics by targeting calcineurin and dynamin-related protein-1. Nat Med
2011; 17: 71–78.
4. Wang K, Liu F, Zhou LY, Ding SL, Long B, Liu CY et al. miR-874 regulates myocardial
necrosis by targeting caspase-8. Cell Death Dis 2013; 4: e709.
5. Wang X, Winter D, Ashrafi G, Schlehe J, Wong YL, Selkoe D et al. PINK1 and Parkin target
Miro for phosphorylation and degradation to arrest mitochondrial motility. Cell 2011; 147:
893–906.
6. Dorn GW 2nd. Mitochondrial dynamics in heart disease. Biochim Biophys Acta 2013; 1833:
233–241.
7. Cho D-H, Nakamura T, Fang J, Cieplak P, Godzik A, Gu Z et al. S-nitrosylation of Drp1
mediates β-amyloid-related mitochondrial fission and neuronal injury. Science 2009; 324:
102–105.
8. Ong S-B, Subrayan S, Lim SY, Yellon DM, Davidson SM, Hausenloy DJ. Inhibiting
mitochondrial fission protects the heart against ischemia/reperfusion injury. Circulation 2010;
121: 2012–2022.
9. Wang J-X, Li Q, Li P-F. Apoptosis repressor with caspase recruitment domain contributes to
chemotherapy resistance by abolishing mitochondrial fission mediated by dynamin-related
protein-1. Cancer Res 2009; 69: 492–500.
10. Lesnefsky EJ, Moghaddas S, Tandler B, Kerner J, Hoppel CL. Mitochondrial dysfunction in
cardiac disease: ischemia–reperfusion, aging, and heart failure. J Mol Cell Cardiol 2001; 33:
1065–1089.

NFAT4 regulates mitochondrial dynamics by miR-324-5p/Mtfr1
K Wang et al

11. Chen Y, Liu Y, Dorn GW 2nd. Mitochondrial fusion is essential for organelle function and
cardiac homeostasis. Circ Res 2011; 109: 1327–1331.
12. Tonachini L, Monticone M, Puri C, Tacchetti C, Pinton P, Rizzuto R et al. Chondrocyte protein
with a poly-proline region (CHPPR) is a novel mitochondrial protein and promotes
mitochondrial fission. J Cell Physiol 2004; 201: 470–482.
13. Monticone M, Tonachini L, Tavella S, Degan P, Biticchi R, Palombi F et al. Impaired
expression of genes coding for reactive oxygen species scavenging enzymes in testes of
Mtfr1/Chppr-deficient mice. Reproduction 2007; 134: 483–492.
14. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small
RNAs with antisense complementarity to lin-14. Cell 1993; 75: 843–854.
15. Brennecke J, Hipfner DR, Stark A, Russell RB, Cohen SM. bantam encodes a
developmentally regulated microRNA that controls cell proliferation and regulates the
proapoptotic gene hid in Drosophila. Cell 2003; 113: 25–36.
16. Ambros V. MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell
2003; 113: 673–676.
17. Mendell JT, Olson EN. MicroRNAs in stress signaling and human disease. Cell 2012; 148:
1172–1187.
18. Small EM, Frost RJ, Olson EN. MicroRNAs add a new dimension to cardiovascular disease.
Circulation 2010; 121: 1022–1032.
19. Ho SN, Thomas DJ, Timmerman LA, Li X, Francke U, Crabtree GR. NFATc3, a lymphoidspecific NFATc family member that is calcium-regulated and exhibits distinct DNA binding
specificity. J Biol Chem 1995; 270: 19898–19907.
20. Kar P, Parekh AB. Distinct spatial Ca2+ signatures selectively activate different NFAT
transcription factor isoforms. Mol Cell 2015; 58: 232–243.
21. Liu J, Koyano-Nakagawa N, Amasaki Y, Saito-Ohara F, Ikeuchi T, Imai S et al. Calcineurindependent nuclear translocation of a murine transcription factor NFATx: molecular cloning
and functional characterization. Mol Biol Cell 1997; 8: 157–170.
22. Chow CW, Rincon M, Cavanagh J, Dickens M, Davis RJ. Nuclear accumulation of NFAT4
opposed by the JNK signal transduction pathway. Science 1997; 278: 1638–1641.
23. Rossow CF, Minami E, Chase EG, Murry CE, Santana LF. NFATc3-induced reductions in
voltage-gated K+ currents after myocardial infarction. Circ Res 2004; 94: 1340–1350.
24. Archer SL. Mitochondrial dynamics–mitochondrial fission and fusion in human diseases.
N Engl J Med 2013; 369: 2236–2251.
25. Wang JX, Jiao JQ, Li Q, Long B, Wang K, Liu JP et al. miR-499 regulates
mitochondrial dynamics by targeting calcineurin and dynamin-related protein-1. Nat Med
2011; 17: 71–78.
26. Matkovich SJ, Van Booven DJ, Eschenbacher WH, Dorn GW 2nd. RISC RNA
sequencing for context-specific identification of in vivo microRNA targets. Circ Res 2011;
108: 18–26.
27. Kim HW, Haider HK, Jiang S, Ashraf M. Ischemic preconditioning augments survival of stem
cells via miR-210 expression by targeting caspase-8-associated protein 2. J Biol Chem 2009;
284: 33161–33168.
28. Walther S, Awad S, Lonchyna VA, Blatter LA. NFAT transcription factor regulation by
urocortin II in cardiac myocytes and heart failure. Am J Physiol Heart Circ Physiol 2014; 306:
H856–H866.
29. Tokudome T, Horio T, Kishimoto I, Soeki T, Mori K, Kawano Y et al. Calcineurin-nuclear
factor of activated T cells pathway-dependent cardiac remodeling in mice deficient in
guanylyl cyclase A, a receptor for atrial and brain natriuretic peptides. Circulation 2005; 111:
3095–3104.
30. de Frutos S, Caldwell E, Nitta CH, Kanagy NL, Wang J, Wang W et al. NFATc3 contributes to
intermittent hypoxia-induced arterial remodeling in mice. Am J Physiol Heart Circy Physiol
2010; 299: H356–H363.
31. Lin Z, Murtaza I, Wang K, Jiao J, Gao J, Li PF. miR-23a functions downstream of NFATc3 to
regulate cardiac hypertrophy. Proc Natl Acad Sci USA 2009; 106: 12103–12108.
32. Tan WQ, Wang K, Lv DY, Li PF. Foxo3a inhibits cardiomyocyte hypertrophy through
transactivating catalase. J Biol Chem 2008; 283: 29730–29739.

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