Download The Long Noncoding RNA CHRF Regulates Cardiac Hypertrophy

The Long Noncoding RNA CHRF Regulates Cardiac Hypertrophy by Targeting miR-489
Kun Wang, Fang Liu, Lu-Yu Zhou, Bo Long, Shu-Min Yuan, Yin Wang, Cui-Yun Liu, Teng
Sun, Xiao-Jie Zhang and Pei-Feng Li
Circ Res. 2014;114:1377-1388; originally published online February 20, 2014;
doi: 10.1161/CIRCRESAHA.114.302476
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Molecular Medicine
The Long Noncoding RNA CHRF Regulates Cardiac
Hypertrophy by Targeting miR-489
Kun Wang,* Fang Liu,* Lu-Yu Zhou,* Bo Long, Shu-Min Yuan, Yin Wang, Cui-Yun Liu,
Teng Sun, Xiao-Jie Zhang, Pei-Feng Li
Rationale: Sustained cardiac hypertrophy is often accompanied by maladaptive cardiac remodeling leading
to decreased compliance and increased risk for heart failure. Maladaptive hypertrophy is considered to be a
therapeutic target for heart failure. MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) have various
biological functions and have been extensively investigated in past years.
Objective: We identified miR-489 and lncRNAs (cardiac hypertrophy related factor, CHRF) from hypertrophic
cardiomyocytes. Here, we tested the hypothesis that miR-489 and CHRF can participate in the regulation of
cardiac hypertrophy in vivo and in vitro.
Methods and Results: A microarray was performed to analyze miRNAs in response to angiotensin II treatment,
and we found miR-489 was substantially reduced. Enforced expression of miR-489 in cardiomyocytes and
transgenic overexpression of miR-489 both exhibited reduced hypertrophic response on angiotensin II treatment.
We identified myeloid differentiation primary response gene 88 (Myd88) as a miR-489 target to mediate the
function of miR-489 in cardiac hypertrophy. Knockdown of Myd88 in cardiomyocytes and Myd88-knockout mice
both showed attenuated hypertrophic responses. Furthermore, we explored the molecular mechanism by which
miR-489 expression is regulated and found that an lncRNA that we named CHRF acts as an endogenous sponge of
miR-489, which downregulates miR-489 expression levels. CHRF is able to directly bind to miR-489 and regulate
Myd88 expression and hypertrophy.
Conclusions: Our present study reveals a novel cardiac hypertrophy regulating model that is composed of
CHRF, miR-489, and Myd88. The modulation of their levels may provide a new approach for tackling cardiac
hypertrophy. (Circ Res. 2014;114:1377-1388.)
Key Words: cardiomegaly
C
ardiac hypertrophy is an adaptive reaction of the heart
against cardiac overloading to maintain cardiac function
at the early stage. However, sustained cardiac hypertrophy
is often accompanied by maladaptive cardiac remodeling
leading to decreased compliance and increased risk for heart
failure and sudden death. Maladaptive hypertrophy is considered to be a therapeutic target for heart failure. Nevertheless,
the underlying molecular mechanisms of cardiac hypertrophy are still poorly understood. To prevent heart failure, it
is necessary to identify and characterize molecules that may
regulate hypertrophy.
Editorial see p 1362
In This Issue, see p 1361
MicroRNAs (miRNAs) are ≈22 nucleotides long and act as
negative regulators of gene expression by inhibiting mRNA
■
RNA, long noncoding
translation or promoting mRNA degradation.1,2 Growing evidence has demonstrated that miRNAs can play a significant
role in the regulation of development, differentiation, proliferation, and apoptosis.3–6 miRNAs can regulate cardiac function
including the conductance of electric signals, heart muscle
contraction, heart growth, and morphogenesis. They also participate in the regulation of cardiac hypertrophy.7,8 Given the
important role of miRNAs in the heart, it is necessary to identify those miRNAs that are able to regulate cardiac hypertrophy and to characterize their signal transduction pathways in
hypertrophic cascades.
miRNAs themselves are not hypertrophic executioners,
they exert their effect through targeting hypertrophic genes.
Although it has been reported that a variety of miRNAs can
be altered during cardiac hypertrophy,9,10 the molecular targets
of miRNAs still remain to be identified. Growing evidence has
Original received August 24, 2013; revision received February 20, 2014; accepted February 20, 2014. In January 2014, the average time from submission
to first decision for all original research papers submitted to Circulation Research was 14.35 days.
*These authors contributed equally.
From the Division of Cardiovascular Research, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese
Academy of Sciences, Beijing, China.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.
114.302476/-/DC1.
Correspondence to Pei-Feng Li, PhD, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of
Sciences, Beijing 100101, China. E-mail [email protected]
© 2014 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.114.302476
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by guest on June 24, 2014
1377
1378 Circulation Research April 25, 2014
Nonstandard Abbreviations and Acronyms
ANF
Ang-II
BNP
CHRF
lncRNA
miRNA
Myd88
NF-κB
atrial natriuretic factor
angiotensin II
brain natriuretic peptide
cardiac hypertrophy related factor
long noncoding RNA
microRNA
myeloid differentiation primary response gene 88
nuclear factor-κB
demonstrated that myeloid differentiation primary response
gene 88 (Myd88) has an impact on cardiac pathology. Myd88
is related to myocardial infarction induced by ischemia/reperfusion, and knockout of Myd88 is able to reduce myocardial
infarct sizes.11,12 There are a few reports showing that Myd88 is
involved in hypertrophy. The blockade of MyD88 by adenoviruses expressing dominant negative Myd88 significantly reduces
cardiomyocyte hypertrophy.13 Fibrinogen induces hypertrophic
response of cardiomyocytes partially through a ­toll-like receptor 4–mediated, Myd88-dependent nuclear factor-κB (NF-κB)
pathway.14 However, it is not yet clear whether Myd88 is a target of miRNAs in the hypertrophic machinery.
Long noncoding RNAs (lncRNAs) are transcribed RNA molecules >200 nucleotides in length but have no significant protein-coding potential. lncRNAs regulate the expression of genes
at epigenetic, transcriptional, and post-transcriptional levels and
play an important role in physiological processes. They have various functions such as RNA processing,15 structural scaffolds,16
modulation of apoptosis and invasion,17 marker of cell fate,18 reprogramming of induced pluripotent stem cells,19 and chromatin
modification.20 In addition, lncRNAs can act as antisense transcripts or as decoys for splicing factors leading to splicing malfunctioning,21,22 and as a competing endogenous RNA in mouse
and human myoblasts.23 However, it is not yet clear whether lncRNA is involved in the regulation of cardiac hypertrophy.
Our present work aims at finding out miRNAs and lncRNAs
that are able to regulate cardiac hypertrophy. miR-489 was
found to be altered substantially in response to hypertrophic
stimulation. In searching for downstream targets of miR489, we identified that Myd88 can be regulated by miR-489.
miR-489 affects cardiac hypertrophy through targeting
Myd88. In exploring the mechanism how miR-489 expression
is regulated, we identified that CHRF may act as an endogenous sponge that represses miR-489 activity. CHRF regulates Myd88 expression and consequent cardiac hypertrophy
through miR-489. Our results reveal a novel hypertrophic regulating model that is composed of CHRF, miR-489, and Myd88.
Methods
Cell culture, quantitative RT-PCR analyses, Western blot analyses,
adenoviral constructions and infection, transverse aortic constriction,
histological assessments, and immunochemistry were performed according to routine protocols. Details of materials and methods are
provided in the Online Data Supplement.
Statistical Analysis
Results are expressed as mean±SEM. Statistical comparison among
different groups was performed by 1-way ANOVA. Two groups
were evaluated by Student t test. P<0.05 was considered statistically
significant.
Results
miR-489 Is Able to Inhibit Hypertrophy in the
Cellular Model
Angiotensin II (Ang-II) has been well documented to induce cardiac hypertrophy. However, its underlying molecular mechanisms, including a possible role for miRNAs,
remain to be elucidated fully. miRNA microarray analysis
on cardiomyocytes identified significant downregulation of
9 miRNAs in response to Ang-II, including the evolutionary
conserved m
­ iR-489 (Figure 1A and 1B; Online Figure IA).
Ang-II–induced miR-489 downregulation in cardiomyocytes
was confirmed by qRT-PCR (Figure 1C). We also detected
the expression of miR-489 in different heart cell types. The
expression of miR-489 was no different among myocytes,
fibroblasts, and endothelial cells (Online Figure IB). The
expression of miR-489 was also significantly reduced in the
transverse aortic constriction model in mice and human heart
failure sample (Online Figure IC and ID). Because the cardiac
function of miR-489 was unknown, we investigated its role
in the heart under physiological and pathological conditions.
In vitro miR-489 knockdown in cardiomyocytes using antagomirs (anta-489; Online Figure IIA) promoted cardiomyocyte hypertrophy both at baseline and after Ang-II stimulation,
as evidenced by increased cell surface areas (Online Figure
IIB) and protein/DNA ratios as a measure of protein synthesis
(Online Figure IIC). In contrast, the enforced expression of
miR-489 (Online Figure IID) resulted in a reduction of hypertrophic responses, including cell surface area (Figure 1D),
protein/DNA ratio (Figure 1E), hypertrophic marker atrial
natriuretic factor (ANF), brain natriuretic peptide (BNP), and
β-myosin heavy chain (β-MHC) (Figure 1F), and sarcomere
organization (Figure 1G). These data suggest that miR-489
participates in antagonizing hypertrophy.
miR-489 Antagonizes Hypertrophy in the
Animal Model
To better understand the function of miR-489 in the heart, we
generated transgenic mice with cardiac-specific overexpressed
miR-489. Five lines of miR-489 transgenic mice demonstrated a high level of miR-489 in the heart (Figure 2A). miR-489
transgenic mice of Line#1 were used for the studies. We analyzed the phenotype of these mice under physiological conditions. These mice developed normally to adulthood without
significant alterations in terms of hypertrophy (Figure 2B) and
apoptosis (Online Figure IIIA). Subsequently, we detected the
hypertrophic responses of these mice in response to hypertrophic stimulation. miR-489 transgenic mice exhibited a reduced
hypertrophic phenotype on Ang-II treatment (Online Figure
IIIB; Figure 2C). We also observed attenuation of other hypertrophic responses including cross-sectional area (Figure 2D),
heart weight/body weight ratio (Figure 2E), and ANF, BNP, as
well as β-MHC (Figure 2F). Concomitantly, cardiac function
was ameliorated (Online Table I). We also analyzed cardiac fibrosis and observed that it was reduced in miR-489 transgenic
mice (Figure 2G). In addition, miR-489 mimic could increase
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Wang et al CHRF Regulates Hypertrophy 1379
B
A
miRNA
Fold Change
miR-489
miR-133a
miR-34a
miR-541
miR-1
miR-145
miR-378*
miR-130a
miR-125a-5p
-2.7
-1.8
-1.8
-1.6
-1.6
-1.5
-1.5
-1.5
-1.5
Intensity-Control
C
0.8
* *
0.4
0
* *
0 5 15 30 60 12 24
Time (min) (hour)
E
150
*
100
50
0
β-gal
miR-489
_
_
_
+
+
_
+
_
_
+
+
_
180
*
120
60
0
_
β-gal
miR-489
_
_
+ + +
_
_
+
_
+ _
G
ANF
BNP
β-MHC
200
mRNA levels
Protein/DNA ratio
(% of control)
F
miR-489
U6
Cell surface area
(% of control)
miR-489 relative
expression levels
D
1.2
Control
4
3
***
2
1
0
β-gal
miR-489
_
_
_
+
+
_
+
_
_
β-gal
+
+
_
Figure 1. miR-489 is able to inhibit hypertrophy in the cellular model. A, Microarray results depicting the log–log scatter plot of
intensity of microRNA (miRNA) expression from control vs angiotensin II (Ang-II) treatment. Neonatal mouse cardiomyocytes were
untreated (control) or treated with Ang-II. Twenty-four hours later, miRNAs were detected by microarray. Red dots and green dots indicate
1.5-fold up- or downregulated genes, respectively. Arrow indicates miR-489. B, Downregulated miRNAs on Ang-II treatment. C, ­qRT-PCR
analysis of miR-489. Cardiomyocytes were treated with Ang-II at the indicated time, and the expression of miR-489 was analyzed;
*P<0.05 vs control. D to G, Enforced expression of miR-489 reduces hypertrophic responses induced by Ang-II. Cardiomyocytes were
infected with adenoviral miR-489 or β-gal at multiplicities of infection of 80. Twenty-four hours after infection, cells were treated with
Ang-II. Hypertrophy was assessed by cell surface area measurement (D), protein/DNA ratio (E), and analysis of the transcripts for atrial
natriuretic factor (ANF), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC) by qRT-PCR (F); *P<0.05 vs Ang-II alone.
Photos show sarcomere organization (G). Bar, 20 μm.
the expression of miR-489 (Online Figure IIIC) and reduce
hypertrophic responses on Ang-II treatment in vivo (Online
Figure IIID–IIIG).
To understand the role of endogenous miR-489 in vivo, we
tested whether knockdown of miR-489 could influence hypertrophy. Knockdown of miR-489 using antagomirs potentiated hypertrophic responses as revealed by heart/body weight
(Online Figure IVA), cardiomyocyte size (Online Figure IVB),
BNP levels (Online Figure IVC), and β-MHC levels (Online
Figure IVD). Echocardiographic assessment demonstrated
an aggravated cardiac function in miR-489 antagomir-treated
mice (Online Figure IVE). Thus, it seems that miR-489 exerts
an antihypertrophic function in the animal model.
Myd88 Is a Downstream Target of miR-489
miRNAs negatively regulate gene expression by inhibiting
mRNA translation or promoting mRNA degradation. To find
out the target gene of miR-489, we screened some hypertrophic associated genes (Cyclin T, GSK3β, PKGI, Ras, MYL2,
CSRP3, MCIP1, Foxo3a, Calcineurin, NFAT, Myocardin,
Myd88) by luciferase assay. We analyzed the 3ʹUTR of each
mRNA sequence except Cyclin T (we analyzed the CDS region because of a lack of 3ʹUTR) by using the bioinformatics
program RNAhybrid software. Approximately 200 bp fragments, including the best predicted binding sites of miR-489
from each target mRNA, were cloned into the pGL3 vector.
Luciferase activity was detected. The results showed that
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1380 Circulation Research April 25, 2014
2
Saline
40
0
Tg
100
20
0
Tg
FS (%)
200
WT
Tg
Heart/Body
weight (mg/g)
0
D
Tg
WT
F
4
3
***
2
1
0
WT Tg
Saline
Cross-sectional
area (µm2)
G
ANF
BNP
β-MHC
WT
Tg
WT
WT
400
300
200
100
0
*
Tg
*
Saline
WT
Tg
Fibrotic
area (%)
4
0
Tg
*
6
2
60
WT
Cross-sectional
area (μm2)
Saline
Saline
8
WT
4
Tg
WT
mRNA levels
Heart/Body
weight (mg/g)
E
6
300
Saline
Saline
WT
Line#1
Line#2
Line#3
Line#4
Line#5
miR-489 relative
expression levels
C
7
6
5
4
3
2
1
0
Tg
WT
WT
B
A
*
6
Tg
*
4
2
0
Saline
Figure 2. miR-489 antagonizes hypertrophy in the animal model. A, Detection of miR-489 levels in miR-489 transgenic (Tg) mice.
The expression of miR-489 was analyzed by qRT-PCR from wild type (WT) and different lines of miR-489 Tg mice, and the results
were normalized to that of U6. B, miR-489 Tg mice developed normally without obvious phenotype alterations under basal conditions
(bar, 20 μm). Heart weight to body weight ratios (n=16). Histological sections were stained with wheat germ agglutinin–FITC conjugate
to determine cell size. Fractional shortening (n=10 per group). C to F, miR-489 Tg mice exhibit reduced hypertrophic responses to
angiotensin II (Ang-II) infusion. WT and miR-489 Tg mice were infused with Ang-II. C, Gross hearts (top; bar, 2 mm); heart sections
stained with hematoxylin and eosin (middle; bar=2 mm; bottom; bar, 20 μm). D, Cross-sectional areas analyzed by staining with T
­ RITCconjugated wheat germ agglutinin; *P<0.05. E, The ratios of heart weight to body weight; *P<0.05 vs Ang-II plus WT. F, Expression levels
of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC); *P<0.05 vs Ang-II plus WT. G, ­miR-489
Tg mice exhibited reduced cardiac fibrosis in response to Ang-II infusion. WT and miR-489 Tg mice were infused with Ang-II. Masson
trichrome staining for collagen; *P<0.05; bar, 20 μm.
­ iR-489 inhibited the luciferase activity of Myd88 (Online
m
Figure VA) and that it had no effect on other genes (data not
shown). Myd88 has been reported to be involved in cardiomyocyte hypertrophy.13,14 However, it is not yet clear whether
Myd88 is a target of miRNAs in the hypertrophic machinery.
The inhibitory effect of miR-489 on Myd88 3ʹUTR led us to
consider if they are related in the hypertrophic pathway. We
analyzed the 3ʹUTR region of Myd88 by RNAhybrid and noticed that miR-489 has a complementary sequence with Myd88
3ʹUTR (Figure 3A). To find out the binding sites of miR-489
to the 3ʹUTR of Myd88, mutations were introduced to Myd88
3ʹUTR and a mutated (mut) form was obtained (Figure 3B).
Luciferase assay revealed that miR-489 was able to suppress
the luciferase activity of wild-type Myd88. However, the mutated form of Myd88 3ʹUTR demonstrated lesser response to
miR-489 (Figure 3C). We tested whether miR-489 can regulate Myd88 levels. Enforced expression of miR-489 led to a
reduction of Myd88 on Ang-II treatment (Figure 3D). The
knockdown of endogenous miR-489 induced an increase in
Myd88 expression (Figure 3E). In contrast, enforced expression of miR-489 resulted in a reduction of endogenous Myd88
(Figure 3F). In human cell line HEK293, we also got similar
results (Online Figure VB and VC). miR-489 transgenic mice
exhibited a low level of Myd88 (Figure 3G).
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Wang et al CHRF Regulates Hypertrophy 1381
Myd88-3’UTR
C
Binding Site (210-232)
5’-UCUGC-AUAUGUGUGUU-UCCUUUG-3’
..
miR-489 3’-C--GACGGUAUAUACACCACAGUAA-5’
B
5’-TCTGCATATGTGTGTTTCCTTTG-3’
3’UTR-wt
BS
Firefly luciferase
Myd88 3’UTR
5’-TCTGCATATccagtcccCCTTTG-3’
3’UTR-mut
BS
Firefly luciferase
Myd88 3’UTR
Luciferase activity
(% of control)
A
120
*
*
90
60
30
0
Myd88-3’UTR-wt + + + _ _
Myd88-3’UTR-mut _ _ _ + +
β-gal _ + _ + _
miR-489 _ _ + _ +
E
β-gal
miR-489
_
_
_
+
_
_
+ +
+ _
_
+
F
Myd88
Actin
β-gal (moi) _ 100 _ _
miR-489 (moi) _ _ 50 100
G
Myd88
Actin
#1 #2 #3
WT miR-489 Tg
Myd88
Actin
anta-489 (h) 0 _ 12 24 48
anta-NC (h) _ 48 _ _ _
H
Myd88
Actin
Cell surface
area
(% of control)
Myd88
Actin
Protein/DNA
ratio
(% of control)
D
180
*
120
60
0
200
*
150
100
50
0
_
miR-489
Myd88-TPmiR-489
Myd88-TPcontrol
_
_
_
+ + + +
_
+ + +
_ _
+ _
_ _ _
+
Figure 3. Myeloid differentiation primary response gene 88 (Myd88) is a downstream target of miR-489. A, miR-489 targeting
site in Myd88 3ʹUTR is shown. B, miR-489 binding site in Myd88 wild type (WT) 3ʹUTR and a mutated 3ʹUTR are shown. C, Luciferase
assay. HEK293 cells were infected with adenoviral miR-489 or β-gal and then transfected with luciferase constructs of WT Myd88 3ʹUTR
(Myd88-3ʹUTR-wt) or mutated Myd88 3ʹUTR (Myd88-3ʹUTR-mut). Luciferase activity was analyzed; *P<0.05. D, Overexpression of m
­ iR-489
reduces the increase in Myd88 levels on angiotensin II (Ang-II) treatment. Cardiomyocytes were infected with adenoviral miR-489 or
β-gal at multiplicities of infection (moi) of 80 and then treated with Ang-II. Cells were harvested 1 h after treatment for the analysis of
Myd88 by immunoblot. E, Knockdown of miR-489 induces an increase in Myd88 levels. Cardiomyocytes were transfected with miR-489
antagomir (anta-489) or the antagomir control (anta-NC). Cells were harvested at the indicated time for the analysis of Myd88 expression
by immunoblot. F, miR-489 suppresses the expression of Myd88 in the cellular model. Cardiomyocytes were infected with adenoviral
miR-489 or β-gal at indicated moi. Myd88 expression was analyzed by immunoblot 48 h after infection. G, miR-489 suppresses the
expression of Myd88 in the animal model. Myd88 in WT and lines 1, 2, or 3 of miR-489 transgenic mice was analyzed by immunoblot.
H, Myd88 target protector reduces the inhibitory effect of miR-489 on Myd88 expression and hypertrophy. Cardiomyocytes were infected
with adenoviral miR-489, transfected with the target protector (Myd88-TPmiR-489) or the control (Myd88-TPcontrol), and then exposed to ­AngII. Myd88 was analyzed by immunoblot (top). Hypertrophy was assessed by cell surface area measurement (middle), protein/DNA ratio
analysis (bottom); *P<0.05.
To verify the interaction between miR-489 and Myd88
3ʹUTR in vivo, we performed biotin-labeled miR-489 pulldown assay to test whether miR-489 could pull down Myd88
in vivo. Cardiomyocytes were transfected with biotinylated
wild-type miR-489 (Bio-489-wt) or its mutated form (Bio489-Myd88-mut; Online Figure VIA) and then harvested
for biotin-based pulldown assay. Myd88 was pulled down
by wild-type miR-489. The mutated form that disrupts basepairing between Myd88 3ʹUTR and miR-489 significantly reduced the ability to pull down Myd88 (Online Figure VIB),
indicating that the interaction between miR-489 and Myd88
3ʹUTR actually exists in vivo. We further explored the relationship between endogenous miR-489 and Myd88 in RNAinduced silencing complex in vivo. It is well known that Ago2
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1382 Circulation Research April 25, 2014
is the catalytic center of RNA-induced silencing complex,24
and it associates with small regulatory RNAs that silence
target RNAs through partial base-pairing.25 Also, Ago2 immunoprecipitation can pull down non–seed-pairing target as
previously reported.26,27 And the nonseed target is also regulated by miRNA.28 Thus, we used Ago2 to explore the interaction
between endogenous miRNAs and mRNAs in RNA-induced
silencing complex in vivo. First, we conducted immunoprecipitation in mouse heart lysates with Ago2 antibody. Next
we performed further affinity purification using biotin-labeled
probes that are complementary to the 3ʹUTR region of Myd88
to pull down endogenous miR-489 (Online Figure VIC).
We synthesized 3 probes, 1 of which is complementary to
the 3ʹUTR region of Myd88 and is also the binding site of
miR-489 (P-BS489). The other 2 probes are located outside the
binding site of miR-489 (P1 and P2). A random probe that
is not complementary to Myd88 was used as a negative control. Our results showed that probes outside the binding site of
miR-489 could pull down miR-489, whereas the probe in the
binding site could not (Online Figure VID). In addition, we
also detected miR-489 from argonaute complexes after Ang-II
treatment. The argonaute complexes were obtained by immunoprecipitation using the Ago2 antibody. As shown in Online
Figure VIE, Ang-II treatment led to a reduction in the enrichment of miR-489 from argonaute complexes. Taken together,
these results indicate that there is a direct interaction between
endogenous miR-489 and Myd88 in RNA-induced silencing
complex in vivo.
Furthermore, we attempted to investigate whether miR-489
and Myd88 are functionally related in hypertrophy. To this
end, we used the target protector technology in which a target
protector is able to disrupt the specific interaction of miRNA–
mRNA pairs.29 The target protector of Myd88 was able to
augment the expression levels of Myd88 in the presence of
miR-489 (Figure 3H, upper panel). Concomitantly, miR-489
could not significantly suppress hypertrophic responses in the
presence of Myd88 target protector (Figure 3H, lower panel).
Together, these data suggest that miR-489 exerts its effect
through Myd88.
Myd88 Conveys Hypertrophic Signal
Next, we tested Myd88 expression levels on Ang-II treatment and observed that there was an increase (Figure 4A).
The expression levels of Myd88 were also significantly increased in the heart of transverse aortic constriction mouse
model (Online Figure VIIA) and human heart failure sample
(Online Figure VIIB). The knockdown of Myd88 attenuated
hypertrophic responses revealed by cell surface area measurement (Figure 4B), analysis of protein/DNA ratio, and ANF,
BNP, as well as β-MHC (Figure 4C). Myd88 often has NF-kB
as downstream effector, and so we also detected the activity
of NF-κB. Our results showed that Ang-II treatment can active the NF-κB system (Figure 4A and 4B). To further explore
the role of Myd88 in hypertrophy, we used Myd88-knockout
mice. In response to Ang-II treatment, Myd88-knockout mice
exhibited a reduced hypertrophic phenotype (Figure 4D)
and heart weight/body weight ratio (Figure 4E). The crosssectional area (Figure 4F) and ANF, BNP, as well as β
­ -MHC
(Figure 4G) were attenuated in Myd88-knockout mice. We
observed a preserved cardiac function in M
­ yd88-knockout
mice (Figure 4H). Furthermore, cardiac fibrosis was reduced
in Myd88-knockout mice (Figure 4I). Myd88 is an inflammatory mediator, and inflammation is a known player in pressure
overload–driven cardiac hypertrophy.30 Our results showed
that Myd88-knockout mice also exhibit attenuated inflammatory response to proinflammatory Ang-II (Online Figure VIIC
and VIID). Taken together, the results suggest that Myd88 is
a prohypertrophic factor. To further explore whether miR-489
exerts its effect through Myd88 in vivo, we tested the effects
of miR-489 in Myd88-knockout mice. The administration of
miR-489 mimic or antagomir was able to alter miR-489 levels on Ang-II treatment (Online Figure VIIIA). However, the
hypertrophic phenotype (Online Figure VIIIB), heart weight/
body weight ratio (Online Figure VIIIC), and cross-sectional
area (Online Figure VIIID) were not influenced by altering
miR-489 levels in Myd88-knockout mice.
CHRF Is Able to Regulate miR-489 Expression
and Activity
How is miR-489 expression regulated under pathological conditions? Recent studies have suggested that lncRNAs may act
as endogenous sponge RNA to interact with miRNAs and influence the expression of miRNA.23,31,32 To understand which
lncRNA is involved in the hypertrophic pathway of Ang-II
treatment, we screened 100 lncRNAs with moderate expression level in heart from a result of lncRNA array performed
by Affymetrix Company. We performed real-time RT-PCR to
detect lncRNA levels in response to Ang-II treatment. Among
the lncRNAs, AK048451, which we named CHRF, was substantially elevated (Figure 5A). CHRF is located in the first
intron of Dcc (deleted in colorectal carcinoma) in mouse,
and the length is 1843 nt (Online Table II). The expression
level of CHRF is more than one third of miR-489 in mouse
heart (Online Figure IXA), and it is no different among myocytes, fibroblasts, and endothelial cells (Online Figure IXB).
CHRF is also widely expressed in various tissues (Online
Figure IXC), and it is conserved across species in the binding site of miR-489 (Online Figure XA). Ang-II treatment led
to a t­ime-dependent elevation of CHRF levels (Figure 5B).
CHRF was also significantly increased in the heart of transverse aortic constriction mouse model (Online Figure XB)
and human heart failure sample (Online Figure XC). miR489 levels were elevated in the cells on knockdown of endogenous CHRF (Figure 5C and 5D). To know whether CHRF
can affect ­miR-489 activity, we constructed a miR-489 sensor (Figure 5E, upper panel). The miR-489 sensor construct
contains a perfect miR-489 target, and a reduced luciferase
activity of the sensor indicates the induction of miR-489 activity. Our results showed that the lucifease activity of m
­ iR-489
sensor was decreased in cells treated with CHRF siRNA
(Figure 5E, lower panel), suggesting the induction of miR-489
activity. Enforced expression of CHRF (Figure 5F) induced
a reduction in miR-489 levels (Figure 5G) and miR-489 activity (Figure 5H). Furthermore, we wanted to know whether
CHRF may act as a sponge of miR-489. Cardiomyocytes were
transfected with the miR-489 sensor luciferase reporter, along
with adenoviral miR-489, CHRF, or β-gal. The luciferase activity showed that CHRF counteracted the effect of miR-489
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Wang et al CHRF Regulates Hypertrophy 1383
B
C
Protein/DNA
ratio
(% of control)
180
5 15 30 60
Time (min)
*
120
Myd88-siRNA
Myd88-sc
D
_
+ + +
_
+ _
_
_
+
_
_
Myd88 KO
WT
Heart/Body
weight (mg/g)
4
3
2
ANF
BNP
β-MHC
H
*
**
1
0
WT KO WT KO
Saline
FS (%)
mRNA levels
G
50
40
30
20
10
0
***
_
+
_
_
_
_
+
+
_
+
_
+
F
Myd88
Actin
Saline
ANF BNP
β-MHC
Myd88-siRNA
Myd88-sc
E
Saline
0
4
3
2
1
0
60
0
60
mRNA
levels
0
*
120
WT
Myd88 KO
*
6
4
2
0
400
300
200
100
0
Saline
WT
Myd88 KO
*
Saline
I
Fibrotic area (%)
Myd88
p-I κ Bα
p65
Actin
Cell surface
area
(% of control)
Myd88
p-I κ Bα
p65
Actin
180
Cross-sectional
area (μm2)
A
WT
Myd88 KO
*
Saline
WT
Myd88 KO
*
6
*
4
2
0
Saline
Figure 4. Myeloid differentiation primary response gene 88 (Myd88) participates in mediating the hypertrophic signals of
angiotensin II (Ang-II). A, Ang-II induces an elevation of Myd88 levels. Cardiomyocytes were treated with Ang-II and harvested at the
indicated time for the analysis of Myd88, p-IκBα, and p65 levels by immunoblot. B and C, Knockdown of Myd88 reduces hypertrophic
responses induced by Ang-II. Cardiomyocytes were infected with adenoviral Myd88-RNAi or its scramble form (Myd88-sc). Twenty-four
hours after infection, the cells were treated with Ang-II. B, Analysis of Myd88, p-IκBα, and p65 levels by immunoblot (top), cell surface
area measurement (bottom). C, Protein/DNA ratio (top), atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and β-myosin heavy
chain (β-MHC) levels (bottom); *P<0.05 vs Ang-II alone. D to G, Myd88-knockout (KO) mice exhibit attenuated cardiac hypertrophy. WT
and Myd88-KO mice were infused with Ang-II. D, Histological sections of hearts, gross hearts (top; bar, 2 mm), wheat germ agglutinin–
FITC conjugate to determine cell size (bottom; bar, 20 μm). E, The ratio of heart weight to body weight; *P<0.05 vs Ang-II plus WT.
F, Cross-sectional areas; *P<0.05 vs Ang-II plus WT. G, Expression levels of ANF, BNP, and β-MHC; *P<0.05 vs Ang-II plus WT. H, Analysis
of cardiac function. Fractional shortening (n=8 per group); *P<0.05 vs Ang-II plus WT. I, Myd88-KO mice exhibited reduced cardiac fibrosis
in response to Ang-II infusion. WT and Myd88-KO mice were infused with Ang-II. Masson trichrome staining for collagen; *P<0.05.
(Figure 5I), suggesting that CHRF is a functional sponge for
miR-489. Taken together, these data suggest that CHRF is
able to regulate miR-489 levels and activity.
CHRF Is Able to Directly Bind to miR-489
To understand the mechanism by which CHRF regulates the
levels of mature miR-489, we tested whether CHRF can interact with miR-489. We compared the sequence of CHRF with
that of miR-489 using RNAhybrid and noticed that CHRF
contains a target site of miR-489 (Figure 6A). We produced
a luciferase construct of CHRF RNA (Luc-CHRF-wt) and a
mutated form (Luc-CHRF-mut). Luciferase assay revealed that
miR-489 could suppress the luciferase activity of CHRF RNA,
but it had less effect on the mutated form of CHRF RNA compared with the wild type (Figure 6B). These results reveal that
CHRF may interact with miR-489 by this putative binding site.
Furthermore, we applied a biotin–avidin pulldown system to
test whether miR-489 could pull down CHRF. Cardiomyocytes
were transfected with biotinylated miR-489 and then harvested for biotin-based pulldown assay. CHRF was pulled down
by miR-489 as analyzed by real-time RT-PCR, but the introduction of mutations that disrupt base-pairing between CHRF
and miR-489 (Figure 6C) led to the inability of miR-489 to
pull down CHRF (Figure 6D), indicating that the recognition
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1384 Circulation Research April 25, 2014
1
0
E
D
*
1.5
1
0.5
0
Sensor
_
+
_
_
+
Luc activity
(% of control)
CHRF-sc
CHRF-siRNA
_
Firefly miR-489
luciferase target
U6
β-gal
CHRF
_
_
+
_
_
+
Luc activity
(fold change)
miR-489
80
*
40
0
miR-489 sensor + + +
CHRF-sc _ + _
CHRF-siRNA _ _ +
H
G
F
*
2
+
+
_
+
_
+
*
0.4
_
+
_
_
4
_
+
*
2
0
β-gal
CHRF
I
4
0
miR-489 sensor +
β-gal _
CHRF _
0.8
0
0 5 15 30 60
CHRF-sc
CHRF-siRNA
Time (min)
120
miR-489
U6
1.2
CHRF level
2
2.5
2
_
_
120
Luc activity
(% of control)
3
C
*
CHRF level
B
*
Control
CHRF level
Relative expression
A
80
_
+
_
+
*
40
0
miR-489 sensor + + + +
miR-489 _ + + +
β-gal _ _ + _
CHRF _ _ _ +
Figure 5. Cardiac hypertrophy related factor (CHRF) can regulate miR-489 expression and activity. A, Long noncoding RNA (lncRNA)
expression levels on treatment with angiotensin II (Ang-II). Cardiomyocytes were untreated (control) or treated with Ang-II. lncRNAs
expressed in the heart in lncRNA array (Affymetrix Company; http://www.noncode.org) were analyzed by qRT-PCR; *P<0.05 vs control.
B, qRT-PCR analysis of CHRF in cells treated with Ang-II. Cardiomyocytes were treated with Ang-II at the indicated time, and the expression
of CHRF was analyzed; *P<0.05 vs control. C and D, Knockdown of CHRF upregulates the expression levels of miR-489. Cardiomyocytes
were infected with adenoviral CHRF siRNA (CHRF-siRNA) and its scramble form (CHRF-sc). Twenty-four hours after infection, CHRF levels
were analyzed by real-time RT-PCR (C), and miR-489 levels were analyzed by Northern blot (D). E, Knockdown of CHRF induces miR-489
activity. miR-489 sensor construct. Mouse genomic sequences (200 bp) flanking pre-miR-489 were reverse-inserted into the downstream
of luciferase gene in pGL3 vector (top). Cardiomyocytes were infected with adenoviral CHRF-siRNA and its scramble and then transfected
with miR-489 sensor. Luciferase activity was analyzed (bottom); *P<0.05 vs miR-489 sensor alone. F and G, Enforced expression of CHRF
reduces the expression levels of miR-489. Cardiomyocytes were infected with adenoviral CHRF or β-gal. Twenty-four hours after infection,
CHRF levels were analyzed by real-time RT-PCR (F), and miR-489 levels were analyzed by Northern blot (G). H, CHRF decreases miR-489
activity. Cardiomyocytes were infected with adenoviral CHRF or β-gal and then transfected with miR-489 sensor. Luciferase activity was
analyzed; *P<0.05 vs miR-489 sensor alone. I, CHRF acts as a sponge for miR-489 activity. Cardiomyocytes were infected with adenoviral
miR-489, CHRF, or β-gal and then transfected with miR-489 sensor. Luciferase activity was analyzed; *P<0.05.
of miR-489 to CHRF is in a sequence-specific manner. We
also used inverse pulldown assay to test whether CHRF could
pull down miR-489, using a biotin-labeled specific CHRF
probe. miR-489 was precipitated as analyzed by the Northern
blot (Figure 6E). Taken together, it seems that CHRF is able to
directly bind to miR-489.
CHRF Regulates Hypertrophy Through miR-489
and Myd88
Because CHRF can interact with miR-489, we thus tested
whether CHRF is able to regulate hypertrophy. Knockdown
of CHRF reduced Myd88 levels (Figure 7A). The overexpression of CHRF resulted in the upregulation of Myd88 expression and the activation of NF-κB system (Figure 7B). CHRF
counteracted the effect of miR-489 on Myd88 expression
(Figure 7C). The luciferase reporter assay showed that CHRF
counteracts the inhibitory effect of miR-489 on Myd88
(Figure 7D). These results indicated that CHRF may act as
endogenous sponge antagomir of miR-489.
Enforced expression of CHRF induced hypertrophic responses, including sarcomere organization and increase in cell
surface area and protein/DNA ratio (Figure 7E). In the animal
model, enforced expression of CHRF increased the apoptosis of cardiomyocytes (Online Figure XIA). Knockdown of
CHRF by siRNA (Online Figure XIB) significantly increased
miR-489 levels (Online Figure XIC) and attenuated ANF and
β-MHC levels (Online Figure XID) as well as cross-sectional
areas (Online Figure XIE). These results indicate that CHRF
is able to regulate hypertrophy.
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Wang et al CHRF Regulates Hypertrophy 1385
A
B
Luc-CHRF-wt GUGUUG
Luc-CHRF-mut CAACGC
Luciferase activity
(% of control)
120
miR-489 3’-CGACGGUAUAUACACCACAGUAA-5’
..
.. ..
CHRF 5’-CCUGCCUUGUAUUUUGUGUUG-3’
D
.. ..
3’-GUUGUGUUUUAUGUUCCGUCC-5’
Bio-489-wt 5’-AAUGACACCACAUAUAUGGCAGC-3’
CHRF
Bio-489-mut 5’-AAACCACACACCCUACUAAGUCC-3’
12
CHRF
enrichment
C
E
miR-489
I
P
I
P
8
4
0
β-gal
80
40
0
*
*
miR-489
Figure 6. Interaction between cardiac
hypertrophy related factor (CHRF)
and miR-489. A, CHRF RNA contains
a site complementary to miR-489.
B, Luciferase assay. miR-489–binding
site in CHRF RNA wild-type (WT) form
(Luc-CHRF-wt) and mutated form ­(LucCHRF-mut) is shown (top). HEK293
cells were infected with adenoviral m
­ iR489 or β-gal and then transfected with
luciferase constructs of Luc-CHRF-wt
or Luc-CHRF-mut. Luciferase activity
was analyzed; *P<0.05. C, WT and the
mutated form of miR-489 sequence are
shown. D, CHRF is associated with ­miR489. Cardiomyocytes were transfected
with biotinylated WT miR-489 (­Bio489-wt) or biotinylated mutant miR-489
(Bio-489-mut). A biotinylated microRNA
that is not complementary to CHRF was
used as a negative control (Bio-NC).
Forty-eight hours after transfection, cells
were harvested for biotin-based pulldown
assay. CHRF expression levels were
analyzed by real-time RT-PCR; *P<0.05
vs Bio-NC. E, miR-489 is associated with
CHRF. miR-489 was pulled down by the
CHRF probe or random probe. miR-489
levels were analyzed by Northern blot.
I indicates input (10% samples were
loaded); and P, pellet (100% samples
were loaded).
miR-21
We explored the downstream targets of CHRF in hypertrophy. The modulation of miR-489 (Figure 7F and 7G) or Myd88
levels (Figure 7H) affected hypertrophic responses induced by
CHRF, suggesting that Myd88 is a downstream target of CHRF.
The target protector of Myd88 attenuated the inhibitory effect
of CHRF knockdown on hypertrophic responses (Figure 7I and
7J). Taken together, these data suggest that CHRF targets the
miR-489/Myd88 axis in hypertrophic cascades.
Discussion
Cardiac hypertrophy is a common response to a variety of
physiological as well as pathological stimuli and will eventually lead to heart failure. Heart failure is one of the leading
causes of hospitalization and death worldwide. Maladaptive
hypertrophy is considered to be a therapeutic target for heart
failure. It is essential to discover impactful therapeutic targets
suppressing maladaptive hypertrophy and the consequent
heart failure. Our present work identified miR-489 to be an
antihypertrophic molecule. We produced miR-489 transgenic
mice, and these mice are resistant to hypertrophy. We found
that Myd88 is a target of miR-489, and Myd88-deficient mice
exhibit reduced hypertrophic responses. Our data further
showed that miR-489 inhibits hypertrophy through repressing Myd88. Moreover, we identified that CHRF regulates hypertrophy through miR-489. Our results provide new insights
into understanding the pathogenesis of cardiac hypertrophy.
Recent works about miRNAs renovate our understanding about the regulation of cardiac hypertrophy. It is of note
that miR-489 transgenic mice exhibit no obvious alterations
in phenotype under physiological conditions. However, these
mice are resistant to hypertrophy under pathological stimulation with Ang-II. This is consistent with other reports showing that some miRNAs function only under pathological
conditions.33 Because physiological cell growth and pathological hypertrophy can be regulated by the distinct cellular
machinery, the underlying mechanism by which a miRNA
regulates cell size under physiological condition is an interesting topic for future studies. We have identified through the
present work that Myd88 is a target of miR-489. Although
most ­miRNA-mediated suppression of target genes have a
seed-match sequence, there is no perfect seed-match sequence
between miR-489 and Myd88 3ʹUTR. Many reports also demonstrate that perfect seed-pairing is not mandatory for miRNA
target recognition.34,35 The 3ʹ pairing, especially the 3ʹ core (positions 13–16), is also effective when there is at least a 6mer
seed match.36 The 3ʹ pairing can help compensate for imperfect seed-pairing.37 In the present study, enforced expression
of miR-489 resulted in a reduction of Myd88 on both mouse
cardiac myocytes and human cell line HEK293. We also verified the interaction of Myd88 and miR-489 by miR-489 pulldown assay and Ago-2 pulldown experiment. This indicated
that the interaction between miR-489 and Myd88 3ʹUTR actually exists in vivo. Our present work uses Myd88-knockout
mice to study its role in cardiac hypertrophy and demonstrates
that Myd88 is a prerequisite for Ang-II to initiate hypertrophy.
It remains largely unknown as to the molecular mechanism by which Myd88 regulates cardiac hypertrophy. The activation of NF-κB has been documented to stimulate cardiac
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1386 Circulation Research April 25, 2014
_
+
E
*
80
_
_
+
*
150
*
120
_
_
0
CHRF
β-gal
miR-489
+ + +
_
+ _
_ _
+
180
Cell surface area
(% of control)
_
*
120
60
0
_
CHRF-sc _
CHRF-siRNA _
TPcontrol __
Myd88-TPmiR-489
60
+++++
_
+_ _ _
_ _
+ + +_
_ _ _
+
_ _ _ _
+
_
_
_
J
_
*
+
_
180
80
40
0
β-gal
CHRF
_
+
_
_
+
_
_
+
200
150
*
100
50
0
CHRF
Myd88-sc
Myd88-siRNA
+ + +
_
+ _
_ _
+
Protein/DNA ratio
(% of control)
miR-489
U6
_
_
H
180
+ + +
_
+ _
_ _
+
+
160
50
Protein/DNA ratio
(% of control)
β-gal
G
F
_
_
120
0
β-gal
CHRF
CHRF
0
Myd88-3’UTR + + + +
miR-489 _ + + +
β-gal _ _ + _
CHRF _ _ _ +
I
+
_
_
miR-489
β-gal
CHRF
_
100
40
CHRF
β-gal
miR-489
_
Myd88
Actin
Protein/DNA ratio
(% of control)
_
_
+
120
Luc activity
(% of control)
D
_
Cell surface area
(% of control)
CHRF-sc
CHRF-siRNA
C
Cell surface area
(% of control)
Myd88
Actin
Myd88
p-Ι κ Βα
p65
Actin
β-gal
CHRF
Control
B
A
_
_
_
+ + +
_
+ _
_ _
+
*
120
60
0
_
CHRF-sc _
CHRF-siRNA _
_
TPcontrol _
miR-489
Myd88-TP
+++++
_
+_ _ _
_ _
+ + +_
_ _ _
+
_ _ _ _
+
Figure 7. Cardiac hypertrophy related factor (CHRF) regulates hypertrophy through targeting miR-489 and myeloid differentiation
primary response gene 88 (Myd88). A, Knockdown of CHRF reduces the expression levels of Myd88. Cardiomyocytes were infected with
adenoviral CHRF-siRNA or CHRF-sc. Twenty-four hours after infection, Myd88 levels were analyzed. B and C, Enforced expression of CHRF
upregulates Myd88 levels. Cardiomyocytes were infected with adenoviral miR-489, CHRF, or β-gal. Twenty-four hours after infection, Myd88,
p-IκBα, and p65 levels were analyzed. D, CHRF counteracts the inhibitory effect of miR-489 on Myd88. Cardiomyocytes were infected with
adenoviral miR-489, CHRF, or β-gal and then transfected with Myd88 3ʹUTR luciferase construct. Luciferase activity was analyzed; *P<0.05.
E, CHRF induces hypertrophic responses. Cardiomyocytes were infected with adenoviral CHRF or β-gal. Forty-eight hours after infection,
hypertrophy was assessed by sarcomere organization (left; bar, 20 μm), cell surface area measurement (middle), and protein/DNA ratio
analysis (right); *P<0.05 vs control. F, Cardiomyocytes were infected with adenoviral CHRF, miR-489, or β-gal. Expression of miR-489 was
analyzed by Northern blot. G, miR-489 reduces hypertrophic responses induced by CHRF. Cardiomyocytes were infected with adenoviral
CHRF, miR-489, or β-gal. Hypertrophy was assessed by cell surface area measurement; *P<0.05 vs CHRF alone. H, Knockdown of Myd88
reduces hypertrophic responses induced by CHRF. Cardiomyocytes were infected with adenoviral CHRF, Myd88-siRNA, or Myd88-sc.
Hypertrophy was assessed by protein/DNA ratio analysis; *P<0.05 vs CHRF alone. I and J, Myd88 target protector attenuates the inhibitory
effect of CHRF knockdown on hypertrophic responses induced by angiotensin II (Ang-II). Cardiomyocytes were infected with adenoviral
CHRF-siRNA or CHRF-sc, transfected with the target protector (Myd88-TPmiR-489) or the control (Myd88-TPcontrol), and then exposed to Ang-II.
Hypertrophy was assessed by cell surface area measurement (I) and protein/DNA ratio analysis (J); *P<0.05.
Downloaded from http://circres.ahajournals.org/ by guest on June 24, 2014
Wang et al CHRF Regulates Hypertrophy 1387
hypertrophy. For example, MAFbx can convey the signal of cardiac hypertrophy in response to pressure overload. The downregulation of MAFbx inhibits cardiac hypertrophy through
inactivation of ­NF-κB.38 The interleukin-1 receptor–mediated
­MyD88-dependent signaling pathway predominately activates
NF-κB. Our present results also show that Ang-II treatment can
active the NF-κB system, and Myd88-knockout mice reduce the
inflammatory response to Ang-II. Thus, it can be speculated that
NF-κB may be a downstream target of Myd88 in hypertrophy.
Mammalian genomes encode numerous lncRNAs.39,40 lncRNAs have been defined to have important functions in specific cell types, tissues, and developmental conditions such as
chromatin modification,20 RNA processing,15 structural scaffolds,16 and modulation of apoptosis and invasion.17 Despite
the biological importance of lncRNAs, it is not yet clear
whether lncRNAs are involved in the regulation of hypertrophy. It has been shown that lncRNAs may act as endogenous
sponge RNAs to interact with miRNAs and influence the expression of miRNA target genes. A recent report showed that a
muscle-specific lncRNA, linc-MD1, governs the time of muscle differentiation by acting as a competing endogenous RNA
in mouse and human myoblasts.23 Highly upregulated liver
cancer may act as an endogenous sponge, which downregulates miR-372 leading to reduced translational repression of its
target gene, PRKACB.31 Transient knockdown and ectopic expression of HSUR 1 directs the degradation of mature ­miR-27
in a sequence-specific and binding-dependent manner.32 Our
results show that the expression of miR-489 is reduced by the
sponge CHRF, which is consistent with a previous report.31
We speculate that there may exist some mechanisms that can
degrade part of the binding miRNA; it is similar to the function of antagomirs that promote miRNA degradation.41–43 But
the exact mechanism is still unclear. It is also an interesting
research topic and we will focus on it in future.
In general, lncRNAs lack strong conservation; many
­well-described lncRNAs, such as Xist, are poorly conserved.44
The poor conservation of lncRNAs may be the result of recent
and rapid adaptive selection. One report indicates that thousands of poor conservation sequences at the primary sequence
level in the mammalian genome have shown evidence of conserved RNA secondary structures.45 Despite the low conservation of lncRNAs in general, it should be noted that many
lncRNAs still contain strongly conserved elements.46 lncRNAs
may have more plastic structure function constraints and conserve only short regions that are constrained by structure or sequence-specific interactions. Our present work reveals a novel
function of lncRNA (CHRF) in regulating cardiac hypertrophy.
CHRF serves as a sponge of miR-489 regulating the expression of Myd88, which activates hypertrophic responses. CHRF
is located in the first intron of Dcc in mouse, and the length is
1843 nt. The full length of CHRF is poorly conserved across
species, but it is conserved between species in the binding site
of miR-489 (Online Figure XA). In addition, we also observed
an increased expression of CHRF in human heart failure tissue. This indicates that the CHRF may have similar function
between the mouse and the human. Our results may provide a
new clue for the understanding of lncRNAs-controlled cellular
events. The discovery of an lncRNA in cardiac hypertrophy
may shed new light on the understanding the complex molecular mechanisms of cardiac hypertrophy.
In summary, our present work identified miR-489 to be an
antihypertrophic miRNA. miR-489 is able to influence hypertrophy in cellular and animal models. Our results further
reveal that Myd88 is a target of miR-489 in the hypertrophic
pathway. Moreover, we demonstrated that CHRF acts as an
endogenous sponge RNA and inhibits miR-489 expression
and activity. The modulation of miR-489 and CHRF may provide an intriguing approach for tackling cardiac hypertrophy.
Sources of Funding
This work was supported by National Natural Science Foundation
of China (31010103911, 81270160, 81000034) and National Basic
Research Program of China (973 Program, 2011CB965300).
Disclosures
None.
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Novelty and Significance
What Is Known?
• MicroRNA participates in the regulation of cardiac hypertrophy.
• Knockdown of myeloid differentiation primary response gene 88
(Myd88) attenuates hypertrophic responses.
• Long noncoding RNAs (lncRNAs) act as antisense transcripts or competing endogenous RNAs and have various biological functions.
What New Information Does This Article Contribute?
• miR-489 is a key regulator of cardiac hypertrophy.
• Myd88 is a downstream target of miR-489.
• lncRNA cardiac hypertrophy related factor (CHRF) performs as an endogenous sponge RNA to interact with miR-489 and regulate cardiac
hypertrophy.
Heart failure is one of the leading causes of mortality worldwide.
The sustained cardiac hypertrophy usually leads to heart failure.
Thus, figuring out the underlying mechanism of pathological hypertrophy is essential for discovering novel therapeutics to inhibit
or reverse maladaptive hypertrophy and heart failure. The present
work identifies miR-489 as a new modulator of cardiac hypertrophy.
Knockdown of miR-489 stimulated cardiac hypertrophy. Conversely,
transgenic overexpression of miR-489 reduced cardiac hypertrophy.
We found that miR-489 directly inhibited Myd88 in cardiomyocytes.
Furthermore, we also found an lncRNA, CHRF, that acts as an endogenous sponge of miR-489. CHRF is able to directly bind to ­miR-489
and regulate Myd88 expression and cardiac hypertrophy. These
findings suggest that CHRF and miR-489 could be 2 potential therapeutic targets for cardiac hypertrophy and heart failure.
Downloaded from http://circres.ahajournals.org/ by guest on June 24, 2014
Supplemental Material
Materials and Methods
Generation of transgenic mice with cardiac-specific overexpression of miR-489 and Myd88
knockout mice
For creating transgenic mice with cardiac-specific overexpressed miR-489, a 396 bp DNA
fragment containing murine miR-489 was cloned to the vector, pαMHC-clone26 (kindly
provided by Dr. Zhongzhou Yang), under the control of the α-myosin heavy chain (MHC)
promoter. The primers used to generate miR-489 transgenic mice include, forward primer:
5’-TCTGGTAACCCAAAAGCAAACTGAT-3’;
reverse
primer:
5’-TAAAGGTGACAATGACACACAAACA-3’. Microinjection was performed following
standard protocols. The primers for genotyping miR-489 transgenic mice include, forward primer
in the α-MHC promoter, 5’-CAGAAATGACAGACAGATCCCTCC-3’; the reverse primer in the
miR-489 DNA, 5’-ACTTGTTGTTCCATGTAACAG-3’.
Conventional Myd88 knockout mice were purchased from Nanjing University Model
Animal Research Center (MARC), China. Myd88+/- mice were interbred to give knockout mice
(Myd88-/-), which were used for the studies. Mice were genotyped by multiplex PCR (primers
and conditions are available from MARC). All experiments were performed on Myd88-/- mice
and their wild type littermates (Myd88+/+), and were approved by government authorities.
Cardiomyocyte culture and treatment
Cardiomyocytes were isolated from 1-2 days old mice as we described 1. Briefly, after dissection
hearts were washed, minced in HEPES-buffered saline solution. Tissues were then dispersed in a
series of incubations at 37°C in HEPES-buffered saline solution containing 1.2 mg/ml pancreatin
and 0.14 mg/ml collagenase. After centrifugation cells were re-suspended in Dulbecco’s
modified Eagle medium/F-12 (GIBCO) containing 5% heat-inactivated horse serum, 0.1 mM
ascorbate, insulin-transferring-sodium selenite media supplement (Sigma, St. Louis, MO), 100
1
U/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM bromodeoxyuridine. The dissociated cells
were pre-plated at 37°C for 1 h. The cells were then diluted to 1x106 cells/ml and plated in 10
μg/ml laminin-coated different culture dishes according to the specific experimental
requirements. Cells were treated with angiotensin II (Ang-II, Sigma) at 150 nM for 24 h, except
as otherwise indicated elsewhere.
Isolation of cardiac fibroblasts
Cardiac fibroblasts were isolated as described in previous reports 2. Briefly, the C57BL/6 mice
(8-10 weeks) hearts were rapidly removed, rinsed, and mounted via the aorta onto a 27-gauge
cannula attached to a Langendorff-type apparatus allowing retrograde perfusion of the coronary
arteries. Hearts were perfused at 80 mmHg for 5 min with 37°C sterile calcium-free
Krebs-Ringer bicarbonate buffer (KRB) containing (in mM) 110 NaCl, 2.6 KCl, 1.2 KH2PO4, 1.2
MgSO4, 25 NaHCO3, and 11 glucose. They were then perfused for 20–25 min with KRB enzyme
solution containing 0.5 mg/ml type II collagenase (Worthington Biochemical), 2.5 mM CaCl2,
and 1 mg/ml fatty-acid free albumin. After digestion, the ventricles were trimmed free and
minced in KRB enzyme solution containing 10 mg/ml albumin, filtered through sterile nylon
mesh, and centrifuged at 25 g for 5 min to remove cardiomyocytes, red blood cells, and debris.
The resultant supernatant was then centrifuged at 1,000 g for 8 min. The cell pellet was
resuspended in 5 ml RPMI 1640 medium with 5 mM glucose, 10% heat-inactivated FBS, and
antibiotics (pH 7.3) and plated into T25 tissue-culture flasks. Non-adherent cells were removed
by aspiration after 4 h and discarded.
Cultured cardiac microvascular endothelial cells (CMVECs)
Primary MMVEC were isolated as previously described 3. Briefly, C57BL/6 mice (8-10 weeks)
were anesthetized and heparinized. After thoracotomy, the heart and aorta were rapidly excised
and washed in ice-cold D-Hanks solution. After removal of the epicardial and endocardial
surfaces, the remaining myocardial tissue was cut into pieces of 1 mm3 without visible vessels.
2
Myocardial tissues were seeded on type I rat-tail tendon collagen-coated plastic disks (Corning).
After 30 min attachment period in the incubator, the tissues were cultured in DMEM (1000 mg/L
d-glucose) supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml
streptomycin.
Cell Death Assays
Apoptosis was determined by the terminal deoxyribonucleotidyl transferase–mediated TUNEL
using a kit from Roche. The detection procedures were in accordance with the kit instructions.
Determinations of cell surface areas, sarcomere organization and protein/DNA ratio
Cell surface area of F-actin stained cells or unstained cells was measured as we described 4.
100-200 cardiomyocytes in 30-50 fields were examined in each group. For staining of
filamentous actin, the cardiomyocytes were fixed in 3.7% formaldehyde in PBS. Cells were
dehydrated with acetone for 3 min and treated with 0.1% Triton X-100 for 20 min. They were
then stained with a 50 μg/ml fluorescent Phalloidin-TRITC conjugate (Sigma, St. Louis, MO) for
45 min at room temperature, and visualized by a laser confocal microscopy (Zeiss LSM 510
META). To measure protein/DNA ratio, total protein and DNA contents were analyzed as we
described 5.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Stem-loop qRT-PCR for mature miR-489 was performed as described
6
on an Applied
Biosystems ABI Prism 7000 sequence detection system. Total RNA was extracted using Trizol
reagent. After DNAse I (Takara, Japan) treatment, RNA was reverse transcribed with reverse
transcriptase (ReverTra Ace, Toyobo). The results of qRT-PCR were normalized to that of U6.
The sequences of U6 primers were forward: 5’-GCTTCGGCAGCACATATACTAA-3’; reverse:
5’-AACGCTTCACGAATTTGCGT-3’. The relative levels of miRNA were normalized to the
levels of U6 using the 2−ΔΔCt method as previously described 7. The detailed calculating method
3
was as follows: The relative levels of miRNA were quantified according to the formula of 2-ΔΔCT,
where ΔΔCT = ((CTmiRNA - CTU6)Treatment group - (CTmiRNA - CTU6)Control group).
qRT-PCR for ANF, β-MHC, IL-6, MCP1 and CHRF were performed as we described 5. The
sequences of ANF primers were forward: 5’-CTCCGATAGATCTGCCCTCTTGAA-3’; reverse:
5’-GGTACCGGAAGCTGTTGCAGCCTA-3’.
BNP
forward
primer:
5’-GCTCTTGAAGGACCAAGGCCTCAC-3’;
reverse:
5’-GATCCGATCCGGTCTATCTTGTGC-3’;
β-MHC
forward
primer:
5’-CAGACATAGAGACCTACCTTC-3’; reverse: 5’-CAGCATGTCTAGAAGCTCAGG-3’.
Mouse
CHRF
forward
primer:
5’-CAACTTTACCCATCTCTTCTC-3’;
5’-CTGAATTACTTCAGAGGAAAG-3’.
Human
CHRF
forward
reverse:
primer:
5’-
AGATTCACATGGTATCCTGAAC’; reverse: 5’- TAGTCTGGCCACATTTTGTCTC -3’. The
results were standardized to control values of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH).
GAPDH
forward
primer:
5’-TGTGTCCGTCGTGGATCTGA-3’;
reverse:
5’-CCTGCTTCACCACCTTCTTGA-3’. The specificity of the PCR amplification was
confirmed by agarose gel electrophoresis. The relative levels of mRNA or CHRF were
normalized to the levels of GAPDH. The formula is 2-ΔΔCT, where ΔΔCT = ((CTmRNA CTGAPDH)Treatment group - (CTmRNA - CTGAPDH)Control group).
For Bio-labeled pulldown assay, the bound levels were normalized to the input of each
group. The formula is 2-ΔΔCT, where ΔΔCT = ((CTmRNA – CTinput)Treatment
group
- (CTmRNA –
CTinput)Control group). The values for the control group were set to 1.
Absolute quantification Real time RT-PCR
We measured the absolute copy number using the standard curve method. The cDNA of CHRF
was cloned into pcDNA 3.1 vector. The vector was linearized and generated sense RNA
transcript using in vitro T7 promoter transcription system (Promega). After digested with
RNAse-free DNAse and purification, the transcript was quantified using a spectrophotometer
and converted to the number of copies as following formula: Copy number/μl
4

A260 × 40 × 10-9 × (6.02 × 1023 )
. The quantified
(n of A × 329.2) + (n of U × 306.2) + (n of C × 305.2) + (n of G × 345.2) + 159
RNA was used as the standard of CHRF. Synthetic miR-489 RNA was used as the standard of
miR-489. For detection of CHRF, the standard and total RNA were reverse transcribed with
ReverTra Ace (Toyobo) using the reverse primer. The standard of miR-489 were reverse
transcribed the same as endogenous miR-489. The standard cDNA was serially diluted in
nuclease-free water. Serial dilutions from 106 to 101 copies were used for standard in a final
volume of 20 μl alongside a negative control (RNA) and a non-template control. The SYBR®
Premix Ex Taq™ II Kit (Takara) was used for amplification. Quantitative PCR was performed on
a CFX96 Real-Time PCR Detection System (Bio-Rad). Absolute quantification determines the
actual copy numbers of target genes by relating the Ct value to a standard curve. The data were
analyzed by CFX96 software. The final data was expressed as the copy number per 10pg of total
RNA.
Adenoviral constructions and infection
Mouse Myd88 cDNA was from Origene. The adenoviruses harboring Myd88 were constructed
TM
using the Adeno-X
expression system (Clontech). Myd88 3’UTR mutants were generated
using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). The adenovirus containing
β-galactosidase (β-gal) is as we described elsewhere 8. To construct adenoviruses encoding
miR-489, mouse genomic sequence harboring the pre-miR-489 was amplified using the
following
primer
sets:
5’-TCTGGTAACCCAAAAGCAAACTGAT-3’;
5’-TAAAGGTGACAATGACACACAAACA-3’, and then subcloned into the adenoviral system.
To construct adenoviruses encoding CHRF, mouse genomic sequence harboring the CHRF was
amplified
using
the
following
primer
sets:
5’-GCTCTTCTAAATTCATAGCCCC-3’;
5’-ATATGAGATGGCAGATGCCATC-3’, and then subcloned into the adenoviral system.
Constructions of mouse Myd88 RNAi and mouse CHRF RNAi
5
The mouse Myd88 RNAi target sequence is 5′-CGATATCGAGTTTGTGCAG-3′. A nonrelated,
scrambled RNAi without any other match in the mouse genomic sequence was used as a control
(5’-TATTGCGGTACTGATGCAG-3’).
The
mouse
CHRF
RNAi
target
sequence
is
5’-TGCCTCTCTAGAGAGCAGC-3’. A nonrelated, scrambled RNAi without any other match
in the mouse genomic sequence was used as a control (5’-CCGATCTGACATGACTGCG-3’).
The adenoviruses harboring these RNAi constructs were generated using the pSilencer™ adeno
1.0-CMV System (Ambion) according to the Kit’s instructions. Adenoviruses were amplified in
HEK293 cells. Adenoviral infection of cardiomyocytes was performed as we described
previously 4.
Pull-down assay with biotinylated miRNA
Cardiomyocytes were transfected with biotinylated miRNA (50 nM), harvested 72h after
transfection. The cells were washed with PBS followed by brief vortex, and incubated in a lysis
buffer [20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2, 0.05% Igepal, 60 U/mL Superase-In
(Ambion), 1 mM DTT, protease inhibitors (Roche)] on ice for 10 min. The lysates were
precleared by centrifugation, and 50μl of the samples were aliquoted for input. The remaining
lysates were incubated with M-280 streptavidin magnetic beads (Sigma). To prevent non-specific
binding of RNA and protein complexes, the beads were coated with RNase-free BSA and yeast
tRNA (both from Sigma). The beads were incubated at 4℃ for 3h, washed twice with ice-cold
lysis buffer, three times with the low salt buffer (0.1%SDS, 1%Trition X-100, 2 mM EDTA, 20
mM Tris-HCl pH8.0, 150 mM NaCl), and once with the high salt buffer (0.1%SDS, 1%Trition
X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl). The bound RNAs were purified
using Trizol for the analysis.
Pull-down assay with biotinylated DNA probe
The biotinylated DNA probe complementary to CHRF RNA was synthesized and dissolved in
500 μl of wash/binding buffer (0.5M NaCl, 20 mM Tris-HCl, pH 7.5, and 1 mM EDTA). The
6
probes were incubated with streptavidin-coated magnetic beads (Sigma) at 25 °C for 2 h to
generate probe-coated magnetic beads. Cardiomyocyte lysates were incubated with probe-coated
beads, and after washing with the wash/binding buffer, the RNA complexes bound to the beads
were eluted and extracted for Northern blot analysis. The following primer sequences were used:
CHRF pull-down probe, GTTGATGGATGATACTAACTATG; and random pull-down probe,
TGATGTCTAGCGCTTGGGCTTTG.
Ago2 IP followed by biotin-labeled probe pulldown assay
Adult C57BL/6 mouse hearts were cut into small pieces, and cross-linking with 1.5%
formaldehyde at room temperature for 15 minutes. Stop the cross-linking reaction by adding
glycine to a final concentration of 0.125 M at room temperature for 5 minutes. After wash with
PBS, the tissues were homogenated in lysis buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM
MgCl2, 0.05% Igepal, 60 U Superase-In/ml (Invitrogen), 1 mM DTT, 1×Pefabloc (Roche)).
AGO2-specific antibody was added into the lysates. After incubation and rotation at 4°C
overnight, the immune complexes were pulled down with protein A-agarose beads (Roche) and
washed with the lysis buffer. Following the last wash, elute the beads with 0.2 M glycine pH 2.6
(1:1) by incubating the sample for 10 minutes with frequent agitation before gentle
centrifugation. Pool the eluate and neutralize by adding equal volume of lysis buffer (20 mM Tris,
pH 8.0, 200 mM NaCl, 2.5 mM MgCl2, 0.05% Igepal, 60 U Superase-In/ml (Invitrogen), 1 mM
DTT, 1×Pefabloc (Roche)). The eluate was ready for next biotin-labeled probe pulldown assay.
Three biotin-labeled DNA probes complementary to Myd88 3’UTR were synthesized, Probe-1
(GGCTGGGAGGAAAGGCAGTCCTAGT)
and
Probe-2
(GCAAAGGCACCCCACTTTTGTCCAG) are outside the miR-489 binding site, and the
Probe-BS489 (CCAAAGGAAACACACATATGCAGAT) targets to the binding site of miR-489.
A random probe (TGATGTCTAGCGCTTGGGCTTTG) that is not complementary to Myd88
was used as a negative control. These probes were dissolved in 500 μl of wash/binding buffer
(0.5M NaCl, 20 mM Tris-HCl, pH 7.5, 60 U Superase-In/ml and 1 mM EDTA). The probes were
7
separately incubated with streptavidin-coated magnetic beads (Sigma) at 25 °C for 2 h to
generate probe-coated magnetic beads. The neutralized eluate after Ago2 IP was separately
incubated with the probe-coated beads, and after washing with the wash/binding buffer, the RNA
complexes bound to the beads were eluted and extracted for northern blot analysis.
Luciferase constructs and transfection of Myd88 3’UTR, CHRF and miR-489 sensor
reporter
Myd88
3’UTR
was
amplified
by
PCR.
5’-GGAAGATGAGACTGATGCGGA-3’.
The
The
forward
reverse
primer
primer
was
was
5’-TCACTTTCTTGGGGACTCAGG-3’. To produce mutated 3’UTR, the mutations were
generated using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). The constructs
were sequence verified. Wild type and mutated 3’UTRs were subcloned into the pGL3 vector
(Promega) immediately downstream the coding region of luciferase gene. Mouse CHRF wild
type (CHRF-wt) and the mutant derivative devoid of miR-489 binding site (CHRF-mut) were
cloned downstream the coding region of luciferase gene. The forward primer was
5’-GCTCTTCTAAATTCATAGCCCC-3’;
the
reverse
primer
was
5’-ATATGAGATGGCAGATGCCATC-3’. miR-489 sensor reporter was constructed according to
the method previously described
9
. Briefly, mouse genomic sequence (200bp) flanking
pre-miR-489 was reversely inserted into the pGL3 vector, downstream of the coding region of
luciferase gene.
HEK293 cells (Fig. 4G and Fig. 6B) or cardiomyocytes (Fig. 5E, Fig. 5H, Fig. 5I and Fig.
7D) were infected with the indicated adenoviruses, then transfected with the indicated luciferase
constructs as described in the corresponding figure legends. The transfection was performed
using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. The
luciferase activity was analyzed as we described elsewhere 10.
Transfection of antagomir and mimic
8
miR-489-3p antagomir, antagomir negative control (antagomir-NC), miR-489-3p mimic and the
mimic negative control (mimic-NC) were purchased from GenePharma Co. Ltd. All the bases
were 2′-OMe modified, and the 3’-end was conjugated to cholesterol. Cells were transfected with
the antagomir or mimic at 50 nM. The transfection was performed using Lipofectamine 2000
(Invitrogen) according to the manufacturer's instruction.
Immunoblot
Immunoblot was performed as we described 11. In brief, cells were lysed for 1 h at 4°C in a lysis
buffer (20 mM Tris [pH 7.5], 2 mM EDTA, 3 mM EGTA, 2 mM DTT, 250 mM sucrose, 0.1 mM
PMSF, 1% Triton X-100 and a protease inhibitor cocktail). Samples were subjected to 12%
SDS-PAGE and transferred to nitrocellulose membranes. Equal-protein loading was controlled
by Ponceau red staining of membranes. Blots were probed using antibodies. The anti-Myd88
antibody was from Abcam, the p-IκBα antibody and p65 antibody were from Cell Signaling
technology, the Ago2 antibody was from Abcam and horseradish peroxidase-conjugated goat
anti-rabbit or rabbit anti-goat IgG were purchased from Santa Cruz Biotechnology. Western blot
bands were quantified using the ImageJ software.
Microarray analysis
Total RNA was extracted from cardiomyocytes by using Trizol reagent. For the miRNA
microarray experiments, low-molecular-weight RNA was isolated with the described method 12,
and then used for miRNA microarray by using the Affymetrix Arrays (CapitalBio Corp.). The
miRNA microarray analysis identified 224 miRNAs, and the Significance Analysis of
Microarrays (SAM) software was used for data analysis. 19 miRNAs were identified to have at
least a 1.5 fold change [q-value < 5%] in expression.
Northern blot analysis
Northern blot was performed as described
13
. In brief, the samples were run on a 15%
9
polyacrylamide-urea gel, transferred to positively charged nylon membranes (Millipore)
followed by cross-linking through UV irradiation. The membranes were subjected to
hybridization with 100 pmol 3’-digoxigenin (DIG)-labeled probes for miR-489 overnight at
42°C. miR-489 probes were labeled with DIG using a 3′-End DIG Labeling Kit (Roche). The
detection was performed using a DIG luminescent detection kit (MyLab) according to the
manufacturer’s
instructions.
The
probe
sequence
for
miR-489
was
5’-GCTGCCATATATGTGGTGTCATT-3’. DIG-labeled U6 probe was used as an internal
control, and its sequence was 5′-TGGAACGCTTCACGAATTTG-3’.
Target protector preparation and transfection
Target protector was designed and named as others and we described
10, 14
. In brief,
Myd88-TPmiR-489 sequence is 5’-CAAAGGAAACACACATATGCAGATG-3’. Myd88-TPcontrol
sequence is 5’-TGACAAATGAGACTCTCTCCTCTCC-3’. They were synthesized by Gene
Tools, and transfected into the cells using the Endo-Porter kit (Gene Tools) according to the kit’s
instructions.
Transverse aortic constriction
For pressure-overload, transverse aortic constriction was carried out as described 15. Sham mice
were subjected to a comparable operation without tightening of the suture encircling the aorta.
Three weeks after the surgery, the hearts were harvested for real time RT-PCR analysis.
Echocardiographic assessment of cardiac dimensions and function
Transthoracic echocardiography was performed on lightly anesthetized mice by using a Vevo 770
high-resolution system (Visualsonics, Toronto, Canada) equipped with a 40-MHz RMV 704
scanhead. Two-dimensional guided M-mode tracings were recorded in both parasternal long and
short axis views at the level of papillary muscles. End-diastolic interventricular septum thickness
(IVSd), left ventricular internal diameters at end-diastole (LVIDd), left ventricular posterior wall
10
thickness at end-diastole (LVPWd), end-systolic interventricular septum thickness (IVSs), left
ventricular internal diameters at end-systole (LVIDs), left ventricular posterior wall thickness at
end-systole (LVPWs), and fractional shortening (FS) were calculated with the established
standard equation. All the measurements were made from more than three beats and averaged.
Animal experiments
C57BL/6 mice (wild type mice) were purchased from Institute of Laboratory Animal Science,
Chinese Academy of Medical Sciences (Beijing, China). The adult male mice (8-10 weeks old)
were used in the study. Wild type mice, transgenic mice and knockout mice were infused with
Ang-II (Sigma, 0.6mg/kg/day dissolved in 0.9% NaCl), saline-infused mice served as controls.
All mice were infused with Ang-II for 2 weeks. The infusions were executed with implanted
osmotic minipumps (Alzet model 1002, Alza Corp.) as we described 10.
We infused the adult male C57BL/6 mice or Myd88 knockout mice with Ang-II (0.6
mg/kg/day dissolved in 0.9% NaCl), along with miR-489 antagomir (30mg/kg/day), miR-489
mimic (30mg/kg/day) or their negative control (anta-NC or mimic-NC) for 2 weeks. All the
bases of antagomir and mimic were 2′-OMe modified. The 5’ terminal two and 3’ terminal four
nucleotides contained phosphorothioate modification and the 3’-end was conjugated to
cholesterol. Saline-infused mice served as controls. We executed all of the infusions with
implanted osmotic minipumps (Alzet model 1002; Alza Corp.).
Adenoviral CHRF siRNA (2X1011 moi) was administered by direct injection to the jugular
vein before Ang II infusion using an implanted osmotic minipump. After 14 d, mice were
subjected to hypertrophic analysis.
Histological analysis
Histological analysis of the hearts was carried out as we described
10
. Briefly, hearts were
excised, fixed in 10% formalin, embedded in paraffin and sectioned into 7 μm slices, and stained
with hematoxyline-eosin (HE). To measure the cross-sectional area of the cardiomyocytes, the
11
sections were stained with FITC-conjugated or TRITC-conjugated wheat germ agglutinin (Sigma,
St. Louis, MO) according to the method previously described 16. To determine cardiac fibrosis,
we stained the heart sections with standard Masson trichrome staining according to
manufacturer’s instructions (Sigma).
Human heart samples
Samples of human heart failure were collected from patients with end-stage heart failure. Control
samples were obtained from the left ventricles of the patients who died without cardiac disease.
Written informed consent was obtained from the family of prospective heart donors. The samples
were obtained according to the regulations of the Institute of Zoology, Chinese Academy of
Sciences.
Supporting Information References:
1.
Wang K, Lin ZQ, Long B, Li JH, Zhou J, Li PF. Cardiac hypertrophy is positively regulated by microrna
mir-23a. The Journal of biological chemistry. 2012;287:589-599
2.
Fan D, Takawale A, Lee J, Kassiri Z. Cardiac fibroblasts, fibrosis and extracellular matrix remodeling in
heart disease. Fibrogenesis & tissue repair. 2012;5:15
3.
Wang XH, Chen SF, Jin HM, Hu RM. Differential analyses of angiogenesis and expression of growth
factors in micro- and macrovascular endothelial cells of type 2 diabetic rats. Life sciences. 2009;84:240-249
4.
Murtaza I, Wang HX, Feng X, Alenina N, Bader M, Prabhakar BS, Li PF. Down-regulation of catalase and
oxidative modification of protein kinase ck2 lead to the failure of apoptosis repressor with caspase
recruitment domain to inhibit cardiomyocyte hypertrophy. J. Biol. Chem. 2008;283:5996-6004
5.
Tan WQ, Wang K, Lv DY, Li PF. Foxo3a inhibits cardiomyocyte hypertrophy through transactivating
catalase. The Journal of biological chemistry. 2008;283:29730-29739
6.
Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR,
Andersen MR, Lao KQ, Livak KJ, Guegler KJ. Real-time quantification of micrornas by stem-loop rt-pcr.
Nucleic Acids Res. 2005;33:e179
7.
Peltier HJ, Latham GJ. Normalization of microrna expression levels in quantitative rt-pcr assays:
Identification of suitable reference rna targets in normal and cancerous human solid tissues. RNA (New
York, N.Y.). 2008;14:844-852
8.
Li PF, Dietz R, von Harsdorf R. P53 regulates mitochondrial membrane potential through reactive oxygen
species and induces cytochrome c-independent apoptosis blocked by bcl-2. EMBO J. 1999;18:6027-6036
12
9.
Wang J, Liu X, Wu H, Ni P, Gu Z, Qiao Y, Chen N, Sun F, Fan Q. Creb up-regulates long non-coding rna,
hulc expression through interaction with microrna-372 in liver cancer. Nucleic Acids Res.
2010;38:5366-5383
10.
Lin Z, Murtaza I, Wang K, Jiao J, Gao J, Li PF. Mir-23a functions downstream of nfatc3 to regulate cardiac
hypertrophy. Proceedings of the National Academy of Sciences of the United States of America.
2009;106:12103-12108
11.
Li YZ, Lu DY, Tan WQ, Wang JX, Li PF. P53 initiates apoptosis by transcriptionally targeting the
anti-apoptotic protein arc. Mol Cell Biol. 2008;28:564-574
12.
Thomson JM, Parker J, Perou CM, Hammond SM. A custom microarray platform for analysis of microrna
gene expression. Nat Methods. 2004;1:47-53
13.
Lau P, Verrier JD, Nielsen JA, Johnson KR, Notterpek L, Hudson LD. Identification of dynamically
regulated microrna and mrna networks in developing oligodendrocytes. J Neurosci. 2008;28:11720-11730
14.
Choi WY, Giraldez AJ, Schier AF. Target protectors reveal dampening and balancing of nodal agonist and
antagonist by mir-430. Science. 2007;318:271-274
15.
Rockman HA, Ross RS, Harris AN, Knowlton KU, Steinhelper ME, Field LJ, Ross J, Jr., Chien KR.
Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo
murine model of cardiac hypertrophy. Proceedings of the National Academy of Sciences of the United
States of America. 1991;88:8277-8281
16.
Dolber PC, Bauman RP, Rembert JC, Greenfield JC, Jr. Regional changes in myocyte structure in model of
canine right atrial hypertrophy. Am J Physiol. 1994;267:H1279-1287
13
Online Figures
Online Figure I
A
Species
miR-489 sequence
Position
Human
5’ - GAGUGACAUCACAUAUACGGCAGCUA-3’
Chr7:93113248-93113331(-)
Mouse
5’ - GAAUGACACCACAUAUAUGGCAGCUA-3’
Chr6:3721897-3722003(-)
Rat
5’ - GAAUGACAUCACAUAUAUGGCAGCUA-3’
Chr4:28564161-28564266(-)
Chimpanzee
5’ - GAGUGACAUCACAUAUACGGCAGCUA-3’
Chr7:94020205-94020287(-)
Rhesus Monkey
5’ - GAGUGACAUCACAUAUACGGCAGCUA-3’
Chr3:123746812-123746895(+)
Bornean Orangutan
5’ - GAGUGACAUCACAUAUACGGCAGCUA-3’
Chr7:82622602-82622685(+)
Cow
5’ - GAGUGACAUCACAUAUAUGGCGACUA-3’
Chr4:10654196-10654277(-)
Dog
5’ - GAGUGACAUCACAUAUACGGCGGCUA-3’
Chr14:21927484-21927567(-)
Horse
5’ - GAGUGACAUCACAUAUACGGCGGCUA-3’
Chr4:37128192-37128276(-)
Zebra fish
5’ - GAGUGACAUCAUAUGUACGGCUGCUA-3’
Chr19:41741794-41741897(-)
C
0.8
0.4
0
Fi
br
ob
la
st
s
s
lls
te
e
y
c
lc
ia
yo
l
e
M
th
o
d
En
1.2
0.8
*
0.4
0
Sham
TAC
_
+
_
_
_
+
miR-489 relative
expression levels
1.2
D
miR-489 relative
expression levels
miR-489 relative
expression levels
B
1.2
0.8
0.4
0
Normal +
Heart failure _
*
_
+
Online Figure I. The expression of miR-489. A. miR-489 conservation in various
species and the position of miR-489 in the genome in various species. B. The
expression of miR-489 in myocytes, fibroblast and endothelial cells in mouse heart
detected by real time RT-PCR. C. The expression of miR-489 in the heart of TAC
mouse modal. D. The expression of miR-489 in human normal and heart failure
tissue.
Online Figure II
B
Cell surface area
(% of control)
A
Pre-miR-489
miR-489
anta-NC
anta-489
_
+
_
_
_
+
C
Protein/DNA ratio
(% of control)
50
_
_
_
+
_
_
_
_
+
_
_
+
_
_
+
+ + +
*
miR-489
U6
120
60
0
anta-NC
anta-489
AngⅡ
100
D
*
180
*
150
0
anta-NC
anta-489
AngⅡ
U6
*
200
β-gal (moi)
miR-489 (moi)
_
_
_
+
_
_
_
_
+
_
_
+
_
_
100 _
_
_
50 100
_
_
+
+ + +
Online Figure II. Knockdown of miR-489 induces hypertrophic responses in
cardiomyocytes. A. Cardiomyocytes were transfected with antagomir (anta-489) or
its negative control (anta-NC). 24h after transfection miR-489 expression levels
were assessed by northern blot. B and C. Knockdown of miR-489 induces
hypertrophic responses. Cardiomyocytes were transfected with antagomir (anta-489)
or its negative control (anta-NC). 24h after transfection cells were treated with AngII (100nM). Hypertrophy was assessed by cell surface area measurement (B) and
protein/DNA ratio analysis (C), *p<0.05. D. Overexpression of miR-489 by
adenovirus harboring miR-489. Cardiomyocytes were infected with adenovirus
harboring miR-489 or β-gal control. 24h after infection cells were harvested and
analyzed by northren blot.
2
miR-489
miR-489
U6
1
U6
_
WT +
Tg _ +
_
_
+
E
8
6
*
4
2
0
Saline + _ _ _
AngⅡ _ + + +
mimic-NC _ _ + _
mimic-489 _ _ _ +
mRNA levels
F
4
mimic-NC
mimic-489
_
_
+
_
_
+
Saline AngⅡ
+
Heart/Body
weight (mg/g)
+
_
ANF
BNP
G
3
**
2
1
0
Saline +
AngⅡ _
mimic-NC _
mimic-489 _
_
_
_
+
+
+
+
_
_
_
_
+
400
*
300
200
100
0
Saline + _ _ _
AngⅡ _ + + +
mimic-NC _ _ + _
mimic-489 _ _ _ +
50
*
40
30
20
10
0
Saline + _ _ _
AngⅡ _ + + +
mimic-NC _ _ + _
mimic-489 _ _ _ +
FS (%)
0
WT +
miR-489 Tg _
D
C
B
Cross-sectional
area (m2)
A
Apoptotic cells
(%)
Online Figure III
Online Figure III. miR-489 mimic attenuates Ang-II induced cardiac hypertrophy.
A. miR-489 transgenic mice developed normally without obvious apoptotic alterations
under basal conditions. Apoptosis of cardiomyocytes were detected by TUNEL staining.
B. WT or miR-489 transgenic mice were infused with Ang-II. The expression levels of
miR-489 were analyzed by northern blot. C. Adult male C57BL/6 mice were infused
with miR-489 mimic (mimic-489) or mimic negative control (mimic-NC) as described in
the section of Material and Methods. The expression levels of miR-489 were analyzed by
northern blot. D-F. Adult male C57BL/6 mice were infused with Ang-II along with
mimic-489 or mimic-NC as described in the section of Material and Methods. The ratios
of heart/body weight (D), cross-sectional areas (E) and the expression levels of ANF and
BNP (F) were analyzed. *p<0.05 vs Ang-II alone. G. Echocardiographic analysis of
cardiac function. Fractional shortening (FS) was analyzed. *p<0.05 vs Ang-II alone.
Online Figure IV
9
6
*
3
0
Saline + _ _ _ _ _
anta-NC _ + _ _ + _
anta-489 _ _ + _ _ +
AngⅡ _ _ _ + + +
D
BNP mRNA
levels
*
5
4
*
3
2
1
0
Saline + _ _ _ _ _
anta-NC _ + _ _ + _
anta-489 _ _ + _ _ +
AngⅡ _ _ _ + + +
*
500
*
400
300
200
100
0
Saline + _ _ _ _ _
anta-NC _ + _ _ + _
anta-489 _ _ + _ _ +
AngⅡ _ _ _ + + +
*
5
4
*
3
2
1
0
Saline + _ _ _ _ _
anta-NC _ + _ _ + _
anta-489 _ _ + _ _ +
AngⅡ _ _ _ + + +
-MHC
mRNA levels
C
B
E
50
*
40
*
30
20
10
0
Saline + _ _ _ _ _
anta-NC _ + _ _ + _
anta-489 _ _ + _ _ +
AngⅡ _ _ _ + + +
FS (%)
*
Cross-sectional
area (m2)
Heart/Body
weight (mg/g)
A
Online Figure IV. The mice upon knockdown of miR-489 are susceptible to
undergoing hypertrophy. A-D. Adult male C57BL/6 mice were infused with or
without Ang-II along with miR-489 antagomir (anta-489) or antagomir negative
control (anta-NC) for 2 weeks. The ratios of heart/body weight (A), cross-sectional
areas (B), the expression levels of BNP (C) and β-MHC (D) were analyzed.
*p<0.05. E. Echocardiographic analysis of cardiac function. Fractional shortening
(FS) was analyzed. *p<0.05.
A
Luciferase activity
(% of control)
Online Figure V
B
120
C
90
60
*
30
0
Myd88-3’UTR +
β-gal _
miR-489 _
HEK293
HEK293
+
+
_
+
Myd88
Actin
Myd88
Actin
anta-NC
anta-489
_
+
_
_
_
+
β-gal
miR-489
_
+
_
_
_
+
_
+
Online Figure V. Myd88 is a downstream target of miR-489 in HEK293 cell line.
A. Luciferase assay. HEK293 cells were infected with adenoviral miR-489 or β-gal,
then transfected with Myd88-3’UTR. The luciferase activity was analyzed. *p<0.05
vs Myd88-3’UTR alone. B. HEK293 cells were transfected with miR-489 antagomir
(anta-489) or the antagomir control (anta-NC). Myd88 expression was analyzed by
immunoblot 48h after transfection. C. HEK293 cells were infected with adenoviral
miR-489 or β-gal at a moi of 80. Myd88 expression was analyzed by immunoblot
48h after infection.
Online Figure VI
Relative Myd88
bound levels
B
A
..
Bio-489-wt 5’-AAUGACACCACAUAUAUGGCAGC-3’
Myd88
3’-GUUUCCU-UUGUGUGUAUA-CGUCU-5’
C
RISC
Myd88
ORF
12
6
0
Bi
oBi
B
N
o48 io-4 C
98
M 9-w
yd
88 t
-m
ut
Bio-489-Myd88-mut 5’-AAUGACCCACACACGCGGGCAGC-3’
*
*
18
miR-489
AAAAA
3’UTR
Ago2 IP
D
miR-489
miR-489
ORF
AAAAA
U6
ra
nd Inp
om ut
pr
o
pr be
ob
e
pr -1
o
b
pr
ob e-2
eBS
48
9
Biotin-labeled
Myd88 probe P1 P-BS489 P2
Pull down
miR-489
AAAAA
ORF
P1
E
Input
P2
Extract RNA
Northern blot
(detect miR-489)
P
IP
2I
o
IgG
Ag
miR-489
U6
AngⅡ
_
+
_
+
_
+
Online Figure VI. The interaction between miR-489 and Myd88 in vivo. A. The wild
type biotin labeled miR-489 (Bio-489-wt) and its mutated form (Bio-489-Myd88-mut) are
shown. B. Biotin labeled miR-489 could pull down Myd88 in vivo. Cardiomyocytes were
transfected with biotinylated wild-type miR-489 or its mutated form. Cells were harvested
for biotin-based pull-down assay 24 hours after transfection. The bound levels of Myd88
were analyzed by real-time RT-PCR. *p<0.05. C. The diagram of Ago2 IP followed by
biotin-labeled probe pulldown. D. Endogenous miR-489 can directly bind to Myd88 in
RISC complex in vivo. Ago2 IP and biotin-labeled probe pulldown assay were conducted
in C57BL/6 mouse heart lysates as described in the method. The levels of miR-489 were
analyzed by northern blot. E. Ang-II reduces the levels of miR-489 in argonaute
complexes. Wild type C57BL/6 mice were infused with Ang-II for 2 weeks. And then,
mouse heart lysates were immunoprecipitated using the Ago2 antibody, and the levels of
miR-489 were analyzed by northern blot. IgG was used as a negative control.
Online Figure VII
A
B
Myd88
Actin
Myd88
Actin
IL-6 mRNA levels
C
16
_
+
_
_
_
+
Normal Heart failure
D
WT
Myd88 KO
12
8
4
*
0
Saline
AngⅡ
MCP-1 mRNA levels
Sham
TAC
24
WT
Myd88 KO
18
12
*
6
0
Saline
AngⅡ
Online Figure VII. A. Expression levels of Myd88 are increased in mouse TAC
model. Mouse hearts were harvested after 3 weeks of TAC. The expression of
Myd88 was analyzed by immunoblot. B. The expression levels of Myd88 were
analyzed by immunoblot in human heart failure tissue. C and D. Myd88 knockout
mice and wild type mice were infused with Ang-II for 2 weeks, hearts were removed
and the inflammatory factors, IL-6 (C) and MCP-1 (D) were analyzed by real-time
RT-PCR. *p<0.05 vs Ang-II plus WT.
Online Figure VIII
WT
Myd88 KO
miR-489 relative
expression level
A
1.2
*
*
0.9
0.6
0.3
0
AngⅡ
mimic-NC
mimic-489
anta-NC
anta-489
_
_
_
_
_
+ + + + + +
_ _
+ _ _ _
_ _ _
+ _ _
_ _ _ _
+ _
_ _ _ _ _
+
AngⅡ
B
Myd88 KO
Heart/Body
weight (mg/g)
C
Myd88 KO mimic-NC mimic-489
WT
Myd88 KO
8
*
6
4
2
0
AngⅡ
mimic-NC
mimic-489
anta-NC
anta-489
_
_
_
_
_
+ + + + + +
_ _
+ _ _ _
_ _ _
+ _ _
_ _ _ _
+ _
_ _ _ _ _
+
D
anta-NC
anta-489
WT
Myd88 KO
Cross-sectional
area (μm2)
WT
WT
*
400
200
0
AngⅡ
mimic-NC
mimic-489
anta-NC
anta-489
_
_
_
_
_
+ + + + + +
_ _
+ _ _ _
_ _ _
+ _ _
_ _ _ _
+ _
_ _ _ _ _
+
Online Figure VIII. miR-489 exerts its effect through Myd88. Wild type (WT)
and Myd88 knockout mice were infused with Ang-II along with miR-489 antagomir
(anta-489), antagomir negative control (anta-NC), miR-489 mimic (mimic-489) or
mimic negative control (mimic-NC) for 2 weeks. A. Administration of miR-489
mimic and antagomir affected miR-489 expression levels upon treatment with AngII, *p<0.05. B. Gross hearts (bar=2mm). C and D. The ratios of heart/body weight
(C) and cross-sectional areas (D) were analyzed. *p<0.05.
Online Figure IX
B
1,200
900
600
300
m
89
-4
iR
RF
0
C
1.2
0.8
0.4
0
s
ls
te
el
y
c
c
l
lia
yo
e
M
h
ot
d
En
Fi
br
ob
la
st
s
CHRF relative
expression levels
1,500
CH
Copy number
per 10pg heart RNA
A
1.2
0.8
0.4
0
He
Sp art
lee
Liv n
e
B r
St rain
om
ac
Lu h
n
kid g
ne
y
CHRF levels
(fold)
1.6
Online Figure IX. The expression of CHRF. A. The copy numbers of CHRF and
miR-489 in C57BL/6 mouse heart were determined by qRT-PCR using standard
curve method. B. The expression levels of CHRF were analyzed by qRT-PCR in
different heart cell types (myocytes, fibroblasts and endothelial cells). C. qRT-PCR
analysis of CHRF expression levels in different organs or tissues isolated from wild
type mice.
Online Figure X
A
Conservation of CHRF in the binding site of miR-489
miR-489 binding site
C
*
CHRF levels
4
3
2
1
0
Sham
TAC
_
+
_
_
_
+
5
4
3
2
1
0
Normal +
Heart failure _
CHRF levels
B
*
_
+
Online Figure X. A. Conservation of CHRF in the binding site of miR-489. This is
a snapshot from mouse genome (2011 assembly) in UCSC Genome Browser. B.
CHRF expression levels are increased in mouse TAC model. Mouse hearts were
harvested after 3 weeks of TAC. The expression levels of CHRF were analyzed by
qRT-PCR. C. The expression levels of CHRF were analyzed by qRT-PCR in human
heart failure tissue.
Online Figure XI
B
8
6
4
2
0
Saline +
β-gal _
CHRF _
_
_
+
_
_
+
2
1
0
Saline +
CHRF-sc _
CHRF-siRNA _
Saline
*
0.4
_
_
+
_
_
+
_
_
+
_
_
+
ANF
BNP
D
*
3
0.8
0
Saline +
CHRF-sc _
CHRF-siRNA _
4
3
**
2
1
0
Saline +
AngⅡ _
CHRF-sc _
CHRF-siRNA _
_
_
_
+
_
+
+
+
_
_
+
_
AngⅡ
AngⅡ+
AngⅡ+
CHRF-sc CHRF-siRNA
Cross-sectional
area (m2)
miR-489 relative
expression levels
C
E
CHRF levels
*
1.2
mRNA levels
Apoptotic cells
(%)
A
400
*
200
0
Saline +
AngⅡ _
CHRF-sc _
CHRF-siRNA _
_
_
_
+
_
+
+
+
_
_
+
_
Online Figure XI. Cardiac hypertrophy can be attenuated by knockdown of CHRF.
A. CHRF induces apoptotic responses. Adenoviral CHRF or β-gal was injected into
mice. Apoptosis was detected by TUNEL staining. *p<0.05 vs saline. B and C.
Adenoviral CHRF-siRNA or CHRF scramble form was injected into mice. The
expression levels of CHRF (B) and miR-489 expression levels (C) were analyzed by
qRT-PCR. *p<0.05 vs saline. D and E. Adenoviral CHRF-siRNA or CHRF scramble
form was injected into mice with or without Ang-II for 2 weeks. The expression levels of
ANF and BNP (D) and cross-sectional areas (E) were analyzed. *p<0.05 vs Ang-II alone.
Online Table I
Echocardiographic data in mice treated with saline or angiotensinⅡ(Ang II)
Saline
Parameters Analyzed
IVSd (mm)
WT
0.75±0.07
AngⅡ
miR-489 Tg
WT
miR-489 Tg
0.73±0.07
1.08±0.03*
0.87±0.06#
*
2.93±0.11#
LVIDd (mm)
3.69±0.06
3.95±0.12
1.69±0.08
LVPWd (mm)
0.67±0.05
0.64±0.04
0.96±0.05*
0.75±0.02#
IVSs (mm)
1.29±0.05
1.27±0.06
1.46±0.03*
1.35±0.04#
LVIDs (mm)
2.03±0.17
2.09±0.12
1.18±0.16*
1.71±0.09#
LVPWs (mm)
1.23±0.03
1.21±0.07
1.49±0.05*
1.32±0.04#
FS (%)
45.1±2.6
47.3±3.1
30.2±2.1*
41.6±1.9#
Heart rate (beats/min)
436 ±12
423±10
447±13
438 ±17
IVSd, end-diastolic interventricular septum thickness; LVIDd, left ventricular internal diameters at
end-diastole; LVPWd, left ventricular posterior wall thickness at end-diastole; IVSs, end-systolic
interventricular septum thickness; LVIDs, left ventricular internal diameters at end-systole; LVPWs,
left ventricular posterior wall thickness at end-systole; FS, fractional shortening. Data are expressed as
mean±SEM, (n=8).
*P < 0.05 vs WT saline; #P <0.05 vs WT AngⅡ.
Online Table II
CHRF sequence
>AK048451.1（1843nt）
gctcttctaaattcatagcccccttttcatgttgttatatatctttctctccacatatatggtcttacaataaaatgctgcatatacag
ctttgtatactttcacttcagtctgtgagcagttttctcatacaacctactcaactttacccatctcttctcaatttggtcctttattgtc
tttattaagtcactatgttaaaaaaaaatcctgccttgtattttgtgttggtttatgaccccctcacattcctctttcctctgaagtaat
tcagtgtaccaggtgaatcatcctcacctttacatcttcagtggccccatctttgatccttgttgagagggcaagattttttttatgt
aatgcctctctagagagcagctgttcccgatgcagtgacctacttgtcttggcatatgtcctctctagtaacttcttcctcttctttt
ttttcctttctcttaaaaaaaaaaagcctagtaacttcttataaacatgcatttccttttaaccttggttgtaaattctttcatagcattg
gttttctcttttccctttgcatattactgaaagtataggacatagctgactataaataaacacattagtgaatgaatgaatgaatga
atgaatgaatgcattaatgaatgatgcatagttagtatcatccatcaactaagctgacaaacatacagtgaaaaaatttaaagt
attttttctttctttttttattagatattttcttcatttacatttcaaatgttatccctaatgccccctatatcctttccctgccatgctcccc
aacccacccactcctgcttcctggccctggccttcccctgtactggggcatatgaccttcacaggaccaagggcctctcctc
ccattgatggctgacttcttcagaaacaaatgcgatttttttgtttttgttttgtttgtttgtttgtttgtttgagacagggtttctctgtg
tagccctgtctgtcctggaactcactctgtagaccaggctgtcattgaacttagaaatccacctgcctctgcctaccaagccct
gggattaaaggtgttagccaccactacccagctaagaactcttaagttagtgaatggttatacagatgacttttcatttagaaca
aaattggtctctacattaagcacagatcttgaaagttgtgggctgtggcagcacaaagctggactataaaacaaaccaatact
acttcttccttggggtcagccaatagcataaattctctaactgtgtttccttaaacataaatttgagctgtcctgagttctcttagga
tcctcccaattccctgactgctaaattagattggtttaatagattaaaatattattctggtaccaccctttattattttctatttgccaat
ctcatgtttacgaagaaattttcttaaatgcagaaacactgattggactgaattattcctatggtaatttcttccacagccttagta
agaaagtaattattgcatactagatatatggtgaatgagcaagaggcaccaccattgccttgtcaaaaatagcaggcaaact
gagaaaaagaggcctgctcaaaattgatttccagagctaccaaattctagaagccctgatggattagatctcaggattagaa
ctcaggtgatatgcatcaaaaattggtttgttcatgggccttcaggctgttgacttttgaatccatgtgagctcaataaatttctttt
catagagcctgtctttggttattgttggatggcatctgccatctcatataaaaaaaaaatgaaagagagaaagaaagaaagag
agaaagagagaaag