Download Overexpression of microRNA-1 impairs cardiac

Cardiovascular Research (2012) 95, 385–393
doi:10.1093/cvr/cvs196
Overexpression of microRNA-1 impairs
cardiac contractile function by damaging
sarcomere assembly
Jing Ai 1*†, Rong Zhang 1†, Xu Gao 1,2, Hui-Fang Niu 1, Ning Wang 1, Yi Xu1, Yue Li 3,
Ning Ma 2, Li-Hua Sun1, Zhen-Wei Pan 1, Wei-Min Li 3, and Bao-Feng Yang 1*
1
Department of Pharmacology (the State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education),
Harbin Medical University, No. 157 Baojian Road, Nangang District, Harbin 150081, China; 2Department of Biochemistry, Harbin Medical University, No.157 Baojian Road, Nangang District,
Harbin 150081, China; and 3Department of Cardiology, The First Affiliated Hospital, Harbin Medical University, No.23 Youzheng Street, Nangang District, Harbin 150001, China
Received 13 April 2012; revised 23 May 2012; accepted 13 June 2012; online publish-ahead-of-print 18 June 2012
Time for primary review: 22 days
Aims
The purpose of the present study was to evaluate the effects of overexpression of microRNA-1 (miR-1) on cardiac
contractile function and the potential molecular mechanisms.
.....................................................................................................................................................................................
Methods
Transgenic (Tg) mice (C57BL/6) for cardiac-specific overexpression of miR-1 driven by the a-myosin heavy chain proand results
moter were generated and identified by real-time reverse-transcription polymerase chain reaction with left ventricular samples. We found an age-dependent decrease in the heart function in Tg mice by pressure –volume loop analysis.
Histological analysis and electron microscopy displayed short sarcomeres with the loss of the clear zone and H-zone
as well as myofibril fragmentation and deliquescence in Tg mice. Further studies demonstrated miR-1 posttranscriptionally down-regulated the expression of calmodulin (CaM) and cardiac myosin light chain kinase
(cMLCK) proteins by targeting the 3′ UTRs of MYLK3, CALM1, and CALM2 genes, leading to decreased phosphorylations of myosin light chain 2v (MLC2v) and cardiac myosin binding protein-C (cMyBP-C). Knockdown of miR-1 by
locked nucleic acid-modified anti-miR-1 antisense (LNA-antimiR-1) mitigated the adverse changes of cardiac function
associated with overexpression of miR-1.
.....................................................................................................................................................................................
Conclusion
miR-1 induces adverse structural remodelling to impair cardiac contractile function. Targeting cMLCK and CaM likely
underlies the detrimental effects of miR-1 on structural components of muscles related to the contractile machinery.
Our study provides the first evidence that miRNAs cause adverse structural remodelling of the heart.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
miR-1 † Transgenic mice † Heart failure † CaM † cMLCK
1. Introduction
Progressive impairment of the cardiac contractile function is an indicator of heart failure as a result of adverse structural remodelling.
However, how the structural remodelling is triggered and sustained
is still poorly understood. Evidence from recent studies has been
rapidly evolving for the critical roles of microRNAs (miRNAs) in regulating cardiac development and function.1 – 3 MicroRNA-1 (miR-1)
stands out as a most prominent one, not only because it is a musclespecific, cardiac-enriched miRNA, but also because it has diverse functions in the heart. The pioneer studies on miRNAs and heart showed
that miR-1 was related to myoblast differentiation during embryonic
development of the heart.4,5 Down-regulation of miR-1 has been
observed in certain circumstances such as cardiac hypertrophy and
chronic myocardial infarction (MI).6 – 8 These properties of miR-1 are
ascribed to relief of repression of some growth-related and
hypertrophy-associated genes. On the other hand, up-regulation of
miR-1 has also been frequently seen in a variety of cardiac conditions,
such as acute myocardial infarction (AMI),9 – 11 ischaemia/reperfusion
injuries,12,13 ischaemic preconditioning,14 diabetic cardiomyopathy,15
oxidative stress,12 heat-shock insult,13 and hyperglycaemia.15 Coinciding with the up-regulation of miR-1 in cardiac tissues of AMI, the level
* Corresponding author. Tel: +86 451 8667 1354; fax: +86 451 8666 7511, E-mail: [email protected] (B.-F.Y.)/[email protected] (J.A.)
†
Authors with equal contributions to this work.
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2012. For permissions please email: [email protected].
386
of miR-1 is also markedly elevated in the plasma of MI patients as
revealed by several research groups.12 – 20 Also, pharmacological
down-regulation of miR-1 appears to produce cardioprotective outcomes.21 – 23 These studies suggested the importance to elucidate
the effects of up-regulation of miR-1 on adult heart. However,
despite the fact that miR-1 has been found up-regulated in diverse
cardiac conditions, including those associated with prominent
changes of cardiac contractility, the consequences of up-regulation
are poorly studied with our current understanding limited to only
the pro-arrhythmic and pro-apoptotic actions of miR-1.9,11,12 The
purpose of this study was to evaluate whether overexpression of
miR-1 could induce an impairment of the cardiac contractile function
by having a direct effect on the sarcomeric function.
2. Methods
2.1 Animals
Adult male and female C57BL/6 mice and male Wistar rats were housed
on a regular chow under controlled temperature of 23 + 18C and humidity of 55 + 5%. Animals were maintained on 12 h dark– light artificial cycle
(lights on at 07:00 A.M.) with food and water available ad libium. Female
mice used for microinjection of eggs and male mice used for heart functional detection and tail vein injection were anaesthetized with sodium
pentobarbital (60 mg/kg, intraperitoneal) and maintained by administrating
0.5 –1.0% isoflurane. The depth of anaesthesia was monitored by detecting reflexes, heart rate, and respiratory rate. Samples for morphological
detection, real-time reverse-transcription polymerase chain reaction
(qRT– PCR), and western blot assay were obtained from the left ventricles of mice after anaesthesia with sodium pentobarbital (100 mg/kg)
and confirmation of death by exsanguination. Hearts for isolating ventricular myocytes were from neonatal Wistar rats after administration with
20% isoflurane and confirmation of death by cervical dislocation. All
animal procedures were approved by the Institutional Animal Care and
Use Committee at Harbin Medical University (No. HMUIRB-2008-06)
and the Institute of Laboratory Animal Science of China (A5655-01). All
procedures conformed to the Directive 2010/63/EU of the European
Parliament.
2.2 Generation of miR-1 transgenic (Tg) mice
miR-1 transgenic (Tg) mice were generated as previously reported.24
Sexually immature female C57BL/6 mice (4– 5 weeks of age) were used
to obtain sufficient quantity of eggs (.250) for microinjection. The Tg
mice used in this study were the fifth generation or later. Two-, 4-, and
6-month-old mice were investigated separately.
J. Ai et al.
2.5 Injection of LNA-modified miR-1 antisense
oligonucleotide (LNA-antimiR-1) into mice
LNA-antimiR-1 was delivered into control mice at 4 months of age and
age-matched miR-1 Tg mice (8 mice for each group) through tail vein injection at a dose of 1 mg/kg once every 2 weeks for 8 weeks (i.e. four
injections in 2 months). Measurements were made 15 days after the last
(or the forth) injection when the mice were at an age of 6 months.
2.6 Preparation of cardiac myosin light chain
kinase antibody (ARM-Mylk3 (150-164))
Polyclonal antibody was prepared. ‘CPGRKGSLADGPEENK’ was selected
as the antigen for immunization.
2.7 Evaluation of morphological remodelling
Transmission electron microscopy (TEM) detection and hematoxylin and
eosin (H&E) staining were performed as routing methods. TEM images
were examined by a Hitachi H-7650 electron microscope. The
heart-to-body-weight ratio (HW/BW) was calculated for each group.
The length of sarcomeres was evaluated from 30 sarcomeres per heart
sample (n ¼ 6 animals) by Image-Pro Plus 6.0 (Media Cybernetics,
Bethesda, MA, USA).
2.8 Culture of neonatal rat ventricular
cardiomyocytes (NRVCs)
The enzymatic dispersion technique was used to isolate single ventricle
myocyte from neonatal Wistar rats. The cultures with 90 + 5% myocytes,
as assessed by microscopic observation of cell beating, were used for
experiments.
2.9 Transfection procedures
Neonatal rat ventricular cardiomyocyte (NRVC) cells (2 × 105/well) were
transfected with 2.5 mg miR-1, AMO-1, ODNs, or NC siRNAs with
X-treme GENE siRNA transfection reagent (Cat.#04476093001,
Roche). Forty-eight hours after transfection, cells were used for total
RNA or protein purification.
2.10 RNA isolation and qRT– PCR for mRNAs
Total RNA samples were isolated from the left ventricles of mice using
phenol/chloroform. The SYBR Green PCR Master Mix Kit was used in
qRT– PCR to quantify target genes. GAPDH was used as an internal
control. For ODNs and LNA-antimiR-1 experiments, total RNA
samples were extracted using Trizol from NRVCs or the left ventricles
of mice. The miR-1 level was quantified by the TaqManw MicroRNA
Reverse Transcription Kit and the TaqManw MicroRNA Assay, and U6
was used as an internal control. The qRT– PCR was performed on
7500 FAST Real-Time PCR System (Applied Biosystems) for 40 cycles.
2.11 Western blot analysis
2.3 Detection of heart function
Heart functional parameters were measured in C57BL/6 male wild-type
(WT) littermate mice at 2, 4, and 6 months of age and age-matched
miR-1 Tg mice in the anaesthetized state, using a pressure–volume (PV)
loops (Scisense, Ontario, Canada).25
2.4 Synthesis of various oligonucleotides
miR-1, anti-miRNA-1 oligonucleotide (AMO-1), and the miRNA-masking
antisense oligodeoxynucleotides (ODNs) were synthesized by Shanghai
GenePharma Co., Ltd. Locked-nucleic-acid (LNA)-modified oligonucleotides (methylene bridge between the 2′ -O and the 4′ -C atoms) were
synthesized by Exiqon (Denmark).
Protein samples extracted from the left ventricles of mice or cultured
NRVCs were used for the immunoblotting analysis. GAPDH was used
as an internal control. The final results were expressed as fold changes
compared with the control values.
2.12 Luciferase reporter assay
The 3′ UTRs of mouse MYLK3, CALM1, and CALM2 genes were obtained
by PCR amplification. These fragments were cloned separately into the
multiple cloning sites in the psi-CHECKTM-2 luciferase miRNA expression
reporter vector. HEK293T cells were transfected with 20 mmol/L miR-1,
AMO-1, or negative control siRNAs (NC) as well as 0.5 mg
psi-CHECKTM-2-target DNA (firefly luciferase vector) and 1 mL blank
plasmid, using Lipofectamine 2000 transfection reagent (Invitrogen)
according to the manufacturer’s instructions. Luciferase activities were
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Overexpression of miR-1 damages heart function
Figure 1 Damaged cardiac function in the miR-1 Tg mice. (A) The relative levels of miR-1 in the Tg and the age-matched WT hearts at varying ages.
*P , 0.05 vs. age-matched WT; mean + SEM; n ¼ 6. (B – H) Demonstration of reduced systolic and diastolic functions of the hearts in the miR-1 Tg
mice relative to the age-matched wild-type (WT) animals. *P , 0.05 vs. age-matched WT; mean + SEM; n ¼ 8. EF, ejection fraction; ESP, end-systolic
pressure; EDP, end-diastolic pressure; dp/dt max, maximum derivative of change in systolic pressure over time; dp/dt min, maximum derivative of
change in diastolic pressure over time; PRSW, preload recruitable stroke work; t, monoexponential time constant of relaxation.
measured with a dual luciferase reporter assay kit. Nucleotide-substitution
mutations were carried out using direct oligomer synthesis for the 3′ UTRs
of MYLK3, CALM1, and CALM2. All constructs were sequence verified.
2.13 Data analysis
Data are presented as mean + SEM. The ANOVA test was performed for
statistical comparisons among multiple groups. Two-tailed Student’s t-test
was applied for comparisons between the two groups. P , 0.05 was considered significant. SPSS13.0 was used for all statistical analyses.
For more detailed information, see Supplementary material online.
3. Results
3.1 Impairment of cardiac contractile
and diastolic functions in miR-1 Tg mice
The miR-1 subfamily consists of miRNAs encoded by distinct genes
that share nearly the same sequence and is designated miR-1-1 and
miR-1-2. Since mature or functional miR-1-1 and miR-1-2 have an identical sequence and miR-1-2 has been used to generate both overexpression and knockout transgenic (Tg) mice in previous studies,5,26
we generated the Tg mouse line for cardiac-specific overexpression
of miR-1-2 driven by the a-myosin heavy chain (a-MHC) promoter
(see Supplementary material online, Figure S1A). The cardiac-specific
overexpression of miR-1 in our Tg mice was verified (Figure 1A; see
Supplementary material online, Figure S1B–E).
Compared with WT mice, ejection fraction (EF) was reduced by
25.8 and 35.6% in Tg mice at 4 and 6 months of age, respectively
(Figure 1B). Both of the impaired cardiac contractile and diastolic functions were indicated by decreased end-systolic pressure (ESP) and
+dp/dtmax, as well as increased end-diastolic pressure (EDP)
(Figure 1C – F; see Supplementary material online, Table S1). In addition, preload recruitable stroke work (PRSW) was also decreased
in the Tg mice at 4 and 6 months of age, indicating depressed LV
contractility (Figure 1G; see Supplementary material online, Table S1).
Furthermore, the monoexponential time constant of relaxation
(t, Weiss), an index of LV relaxation, was found prolonged in the
Tg mice, implying reduced diastolic function (Figure 1H). Qualitatively
similar changes were observed in the 2-month-old Tg mice over the
age-matched WT mice, although statistical significance of these
changes were not attained.
3.2 Cardiac structural remodelling
in miR-1 Tg mice
Although the miR-1 Tg mice survived well before 6 months of age,
decreases in body weight and increases in the HW/BW ratios were
observed compared with age-matched WT mice (Figure 2A and B).
H&E staining showed increased heart size in the Tg mice at 4 and 6
months of age (Figure 2C). We did not find increases in cell surface
area and myocardial fibrosis in the Tg mice compared with control
(Figure 2D; see Supplementary material online, Figure S2). TEM
images displayed shortened and uneven sarcomeric length as well as
loss of clear zone and H-zone in the Tg mouse hearts (Figure 2E).
This observation was confirmed by quantification of the length and
variation of sarcomeres (Figure 2F), suggesting a participation of
remodelled sarcomeric assembly in the detrimental alterations of
heart function in the Tg mice. In addition, severe myofibrillar fragmentation and deliquescence of cardiomyocytes were found in
6-month-old Tg mice hearts but not in 2-month- or 4-month-old
Tg mice (Figure 2E), suggesting age-dependent damages of cardiac
structure in miR-1 Tg mice.
3.3 Repression of MYLK3, CALM1,
and CALM2 genes by miR-1
To understand how miR-1 could damage cardiac contractile function,
we searched the potential target genes relevant to the issue by computational prediction from miRNA database. We found that miR-1 has
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J. Ai et al.
Figure 2 Cardiac morphological remodelling in the miR-1 Tg mice. (A) Decreased body weight in the miR-1 Tg mice compared with age-matched
WT mice. (B) The increased ratio of heart weight to body weight in the miR-1 Tg mice compared with age-matched WT mice. (C) Longitudinal sections of hearts stained by H&E from mice at 2, 4, and 6 months of age (2, 4, and 6 months, respectively), scale bar: 5 mm. (D) Comparison of cell area
between WT and the Tg mice at 2, 4, and 6 months of age showing no hypertrophy phenotype in the Tg mice. (E) TEM examination of cardiac muscles
from mice at 2, 4, and 6 months. Left panels: direct magnification ×4000 (HV ¼ 80.0 KV, scale bar: 10 mm) and right panels: direct magnification
×20 000 (HV ¼ 80.0 KV, scale bar: 2 mm). (F ) Comparison of length and variation of sarcomeres between WT and Tg mice at 2, 4, and 6
months. For (A), (B), (D): *P , 0.05 vs. age-matched WT; mean + SEM; n ¼ 8. For (F): *P , 0.05; mean + SD; n ¼ 180 sarcomeres from six mice
for each group.
a binding site in the 3′ UTRs of MYLK3, CALM1, and CALM2 mRNAs
containing highly conserved miR-1 seed-matching sequences (see Supplementary material online, Figure S3). MYLK3 encodes the cardiac
myosin light chain kinase (cMLCK), and CALM1 and CALM2 encode
the cardiac calmodulin (CaM) protein. We then cloned the miR-1binding sites from the 3′ UTRs of MYLK3, CALM1, and CALM2 into
the luciferase reporter plasmid separately, which contains a constitutively active promoter to determine the effects of miR-1 on reporter
activities in HEK293T cells (see Supplementary material online,
Figure S4). Co-transfection of miR-1 with the plasmid into HEK293T
cells consistently produced less luciferase activities than transfection
of the plasmid alone, whereas miR-1 failed to reduce the luciferase
activity of the mutant 3′ UTRs of MYLK3, CALM1, and CALM2. Transfection of AMO-1 into the cells eliminated the silencing effects of
miR-1 on the activities of the luciferase-wild-type 3′ UTR chimeric
vectors (Figure 3A).
The repressive effects of miR-1 on cMLCK (Figure 3B) and CaM
(Figure 3C) protein levels were confirmed by immunoblotting analyses
in cultured NRVCs. Since the anti-cMLCK-specific antibody was generated in our lab, we also verified the specificity of the antibody (see
Supplementary material online, Figures S5 –S8). In all cases, AMO-1
rescued the down-regulation of proteins elicited by miR-1 transfection
and scrambled negative control (NC) miRNA failed to affect the
protein levels, indicating that the observed changes were specifically
ascribed to miR-1 actions. The successful uptake of miR-1 into
NRVCs after transfection was verified (see Supplementary material
online, Figure S9A).
Consistently, we found that both mRNA and protein levels of
cMLCK in the Tg mice were significantly decreased, relative to the
control values from age-matched WT mice (Figure 3D; see Supplementary material online, Figure S9B). Similarly, the protein level of
CaM was also decreased in the Tg mice (Figure 3E), but mRNA
levels of CALM1 and CALM2, both encoding CaM, were similar in
the Tg mice and age-matched WT mice (see Supplementary material
online, Figure S9C).
In order to further confirm that the observed changes of CaM and
cMLCK in both Tg mice and NRVCs were the direct results of miR-1
actions, we employed the miRNA-masking antisense ODN
(miR-Mask) techniques previously developed.27 The anti-miRNA
action of a miR-Mask is gene-specific because it is designed to be
fully complementary to the target mRNA sequence of a miRNA
(Figure 3F). It is important to note that this miR-Mask only acts on
this gene but minimally affects the effects of the miRNA on other
target genes.27 In our study, we designed three miR-1-Masks that
can base pair the miR-1-binding sites in the 3′ UTRs of MYLK3,
CALM1, or CALM2 genes labelled MYLK3-ODN, CALM1-ODN, and
Overexpression of miR-1 damages heart function
389
Figure 3 Validation of miR-1 target genes. (A) Luciferase reporter gene assay for measuring interactions between miR-1 and its binding sites in the
3′ UTRs of the MYLK3, CALM1, or CALM2 mRNAs in HEK293T cells. Cells were transfected with luciferase-target motif chimeric vector alone (Ctl) or
with miR-1 or NC using lipofectamine 2000. n ¼ 3. (B and C) Effects of miR-1 on protein levels of cMLCK and CaM, respectively, in primary cultured
neonatal rat ventricular cells (NRVCs), using western blot analysis. Cells were transfected with miR-1 alone, miR-1, and AMO-1 together or NC. n ¼ 8.
(D) An age-dependent decrease in the cMLCK protein levels in Tg mice. n ¼ 6. (E) Examples of western blot bands and mean values of band densities
for CaM. n ¼ 6. (F) Schematic of miR-1 silencing using the miRNA-masking antisense ODNs. Gene-specific ODNs (designed as 22 oligonucleotides
fully complementary to the complete sequence of miR-1 target sites in the 3′ UTRs of target mRNAs) with high binding affinity completely mask the
target sites of miR-1 in the 3′ UTRs of target mRNAs, which block the repression of target mRNAs. ORF, open reading frame; AGO, argonaute. And
lack of effects of gene-specific ODNs on miR-1 expression in NRVCs. The miR-1 level was quantified by qPCR. (G and H ) Derepression of target genes
of miR-1 by MYLK3-ODN, CALM1-ODN, or CALM2-ODN, respectively, determined by western blot analysis. n ¼ 6. *P , 0.05 vs. Ctl or NC;
#
P , 0.05 vs. miR-1; mean + SEM.
CALM2-ODN. As expected, these miR-Masks, unlike AMO-1, failed to
affect the miR-1 level when co-transfected with miR-1 (Figure 3F).
MYLK3-ODN blocked the repressive effects of miR-1 on cMLCK
but did not affect the effects on CaM (Figure 3G). On the other
hand, when cells were co-transfected with miR-1 and CALM1-ODN
or CALM2-ODN, downregulation of CaM by miR-1 was prevented
(Figure 3H).
3.4 Reduction in p-MLC2v, p-CaMKIId,
and cardiac myosin binding protein-C
in miR-1 Tg mice
cMLCK is known to be critical to the phosphorylation of myosin light
chain 2v (MLC2v),28 which has a unique function in the maintenance
of cardiac contractility and sarcomeric assembly.29 Decreased phosphorylation of MLC2v can result in reduced cardiac diastolic function
by defected assembly of the myosin thick filaments.30 We thought
that MLC2v might be involved in this process. As displayed in
Figure 4A, the protein levels of MLC2v were in the same range in
Tg and WT mice at younger ages, and became lower in the Tg
mice after 6 months of age, relative to the age-matched WT
animals. However, the phosphorylation status of MLC2v in hearts
from the Tg mice was reduced to 83.6 (2 months), 65 (4 months),
and 57% (6 months) of the values from control mice, for all age
groups tested. These results suggested that miR-1 directly affects
MLC2v function via a mechanism independent of gene expression
regulation.
CaM is an upstream activator of Ca2+-dependent CaM-activated
protein kinase II (CaMKII)31 which is responsible for phosphorylation
of cardiac myosin binding protein-C (cMyBP-C),32 a sarcomeric
thick filament protein that interacts with titin, myosin, and actin
to regulate sarcomeric assembly, structure, and function.33,34
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J. Ai et al.
3.5 Knockdown of miR-1 reverses structural
remodelling and improves contractile
function
To verify the specificity and the reversibility of miR-1 actions on
cardiac contractile function, we treated the Tg mice with LNAmodified miR-1 antisense oligonucleotide (LNA-antimiR-1), a technique that has been shown to efficiently knockdown target miRNAs
with long-lasting efficacy under in vivo condition.36 The results
showed that all aforementioned adverse changes were abolished in
the Tg mice after tail vein injection with LNA-antimiR-1 (Figure 5A–D;
see Supplementary material online, Figure S10A –I). The inhibitory
effect of LNA-antimiR-1 on expression of miR-1 in Tg mice was verified and displayed in Supplementary material online, Figure S10J.
4. Discussion
Figure 4 Regulation of expression of proteins key to cardiac contractile function by miR-1 overexpression, determined by western
blot analysis. (A) Reduction in p-MLC2v without changing MLC2v
total protein levels in the miR-1 Tg mice. (B) Examples of western
blot bands and mean values of band densities for CaMKIId and
p-CaMKIId. (C) Decreased cMyBP-C and p-cMyBP-C levels in the
miR-1 Tg mice compared with age-matched WT mice. *P , 0.05
vs. age-matched WT; mean + SEM; n ¼ 6.
Reduced phosphorylation accompanied by increased cleavage of
cMyBP-C has been reported to induce malformation of actomyosin
cross-bridges so as to initiate changes in myofibril thick filament structure and, thereafter depart the interaction of myosin heads with actin
thin filaments.35 In the present study, the phosphorylation level of
CaMKIId was significantly decreased in the Tg mice at 4 and 6
months compared with age-matched WT mice (Figure 4B), although
the level of the CaMKIId total protein was not changed at younger
ages. Consistent with the reduced phosphorylation of CaMKII, the
phosphorylated protein levels of cMyBP-C were decreased in hearts
of Tg mice, compared with age-matched WT mice (Figure 4C).
Although the total protein levels of cMyBP-C were also decreased,
the changes were smaller than the phosphorylated cMyBP-C levels
(Figure 4C).
In the present study, we used the Tg mouse line to achieve overexpression of miR-1 under the control of cardiac-specific a-MHC promoter for studying the possible remodelling process in adult hearts.
We found that overexpression of miR-1 in Tg mice induces reduction
in the heart function. The molecular mechanism may be that miR-1
post-transcriptionally inhibits the expression of cMLCK and CaM,
which are structural components of muscles related to the contractile
machinery.
It has been shown that overexpression of miR-1 in a Tg mouse
model resulted in a phenotype characterized by thin-walled ventricles,
attributable to premature differentiation and early withdrawal of cardiomyocytes from the cell cycle.5 This overexpression was under the
control of the b-MHC which is dominant in the late foetal life but is
almost completely replaced after birth by a-MHC and the influences
of miR-1 on the cardiac function in the postnatal life to adulthood
cannot be investigated with this model. In another study, adenoviralmediated overexpression of miR-1 suppressed sarcomeric a-actin
organization that is normally observed in serum-deprived cultured
neonatal cardiac myocytes in vitro.37 However, these studies were
not performed in adult hearts. Because, in mouse hearts, a-MHC is
the dominant isoform over b-MHC in the adult, we performed the
Tg mouse line to achieve overexpression of miR-1 under the
control of cardiac-specific a-MHC promoter for studying the possible
remodelling process in adult hearts. With this model, we demonstrated that overexpression of miR-1 in mouse hearts caused agedependent decline in the cardiac function, as indicated by decreases
in EF%, ESP, +dp/dtmax, and PRSW, as well as increases in EDP and
t. Notably, the decreased PRSW, an index of modification of
Frank –Starling law for left ventricular contractile function,38 suggested that miR-1 repressed heart function by damaging sarcomeric
assembly. Indeed, our data also showed that miR-1 induced adverse
structural remodelling of the heart, as revealed by electron microscopic examination showing shortened and uneven sarcomeric
length, loss of clear zone and H-zone, and severe myofibrillar fragmentation and deliquescence of cardiac muscles in the Tg mice
heart. Our findings suggest that, in addition to its recognized role in
cardiac development, overexpression of miR-1 can cause adverse
structural remodelling to impair cardiac contractile function directly
by damaging sarcomere assembly.
Previous studies reported that fine tuning of phosphorylation of
MLC2v was necessary for maintaining the normal structure of the
sarcomere in vivo.39 The reduced phosphorylation of MLC2v directly
Overexpression of miR-1 damages heart function
391
Figure 5 LNA-antimiR-1 alleviates structural remodelling and improves heart function of Tg mice at an age of 6 months. (A) Elevated EF% in miR-1
Tg mice treated with LNA-antimiR-1, compared with age-matched Tg mice. n ¼ 8. (B) Improved cMLCK protein levels in Tg mice treated with
LNA-antimiR-1. n ¼ 6. (C) Examples of western blot bands and mean values of band densities for CaM. n ¼ 6. (D) EM examination of cardiac
muscles from Tg mice. Upper panels: direct magnification ×10 000 (HV ¼ 80.0 KV, scale bar: 4 mm) and lower panels: direct magnification
×25 000 (HV ¼ 80.0 KV, scale bar: 1 mm). *P , 0.05 vs. WT + NC; #P , 0.05 vs. Tg + NC; mean + SEM.
led to unstable, short myofilaments following defective assembly of
the myosin thick filaments, which interrupted the basic structure for
the Frank –Starling law and resulted in impairment of diastolic and systolic functions. Our study showed that phosphorylation of MLC2v
was substantially abrogated in the miR-1 Tg mice. cMyBP-C was also
reported to play a critical role in sarcomeric structure and function
and in protecting the heart from ischaemia/reperfusion injury.33 The
decreases in expression or dephosphorylation of cMyBP-C either
could induce loss of H-zone in sarcomeres.35 In the present study,
both decrease in dephosphorylation of cMyBP-C and loss of the
sarcomeric H-zone were observed in the Tg mice.
cMLCK has been demonstrated to regulate sarcomeric structure in
adult mice by phosphorylating MLC2v40 and activation of cMLCK is
controlled by CaM.41 Moreover, CaMKII,42 which is automatically activated by CaM, phosphorylates MyBP-C.33 These proteins together
form a network to finely regulate cardiac function. Dysfunction of
these proteins can lead to the reduction in cardiac contraction.
Thus, cMLCK serves as a specific kinase for MLC2v in a Ca2+/CaMdependent manner and regulates sarcomere assembly through the
phosphorylation of MLC2v.28 Here, we found that the levels of
both cMLCK and CaM were decreased in the miR-1 Tg mice and verified MYLK3 and CALM1/CALM2 coding for cMLCK and CaM, respectively, as targets for miR-1. It is conceivable that down-regulation of
cMLCK and CaM may underlie the observed decreases in phosphorylations of MLC2v and cMyBP-C with overexpression of miR-1. Of
particular, it has been reported that overexpression of miR-1 attenuated cardiomyocyte hypertrophy in vitro and in vivo by negatively
regulating CaM and Ca2+/CaM-CN-NFAT signalling.43 This result provided positive evidence not only for the post-transcription of miR-1 on
CaM expression, but also for the absence of hypertrophic remodelling
of the heart in miR-1 Tg mice displayed in the present study.
Our results allow us to establish a novel signalling mechanism by
which overexpression of miR-1 impairs cardiac contractile function,
as illustrated in Figure 6. Overexpression of miR-1 produces posttranscriptional repression of cMLCK and CaM, which attenuates
phosphorylations of MLC2v, CaMKII, and cMyBP-C leading to destruction of sarcomeric assembly. These changes trigger an adverse
structural remodelling process causing impairment of heart function.
This is, to our knowledge, the first to study the regulation of the
cardiac contractile function by miRNAs in the context of structural
remodelling in adult hearts.
Our results provided evidence that chronic overexpression of
miR-1 can produce progressive increases in damages of heart function
over time. Intriguingly, overexpression of miR-1 has been identified in
a number of cardiac conditions associated with the pathogenesis of
the heart. In patients with AMI, the miR-1 level was found upregulated
by 2.8-fold in one study9 and 3.8-fold in another with the samples of
infarcted tissue and remote myocardium from 24 patients with AMI.10
A greater degree (22 folds) of expression up-regulation of miR-1
was reported in a rat model of MI, also measured by qPCR.11 In a
rat model of ischaemia/reperfusion injuries, the miR-1 level was elevated by more than six times.12 In diabetic cardiomyopathy of a rat
model of type I diabetes mellitus, miR-1 transcript was increased up
to six times.15 In our Tg mice, the miR-1 level was 4-fold higher
392
J. Ai et al.
Figure 6 Schematic illustrations explaining the possible targeting and signalling mechanisms by which miR-1 causes impairment of cardiac contractility. Increased miR-1 represses CaM expression by targeting CALM1 and CALM2 leading to decreases in the activation of both CaMKIId and cMLCK.
Increased miR-1 also directly suppresses cMLCK expression by targeting MYLK3. Reduction in cMLCK results in lower MLC2v phosphorylation and
suppression of CaMKIId activation diminishes cMyBP-C phosphorylation. Decreased phosphorylations of MLC2v and cMyBP-C then cause failure of
movement of actin filament along myosin filament. CaM, calmodulin; CaMKII, calmodulin-dependent protein kinase II; cMLCK, cardiac specific myosin
light chain kinase; MLC2v, myosin regulatory light chain 2; cMyBP-C, myosin binding protein-C; TnT, troponin T.
than that in WT animals. This amount of overexpression falls well
within the range of up-regulation of miR-1 under various diseased
states described above. This coincidence points to a possibility that
the structural remodelling mechanisms leading to impairment of
cardiac function observed in our miR-1 Tg mouse hearts may well
operate in the real pathological conditions mentioned above. A
recent study displayed that down-regulation of miR-1 can induce
heart failure via sorcin-dependent calcium homeostasis by the
tamoxifen-inducible cardiac-specific Dicer knockdown model in
adult mice.44 These results suggested that both up- and downregulation of miR-1 might result in abnormal heart function, although
the molecular mechanisms were different. Moreover, the fact that
LNA-antimiR-1 is able to rescue the damaging actions of miR-1 in
this study suggests knockdown of miR-1 using the
miRNA-loss-of-functional approach to be a rational strategy to
reverse the adverse changes of cardiac function associated with aberrant up-regulation of miR-1. The findings in the present study also
provide mechanistic explanations for the beneficial effects of pharmacological inhibition of miR-1 expression.
Supplementary material
Supplementary material is available at Cardiovascular Research online.
Acknowledgements
The authors would like to thank Dr Zhi-Guo Wang for valuable suggestions for the design and manuscript revision. We also thank
Dr Fang Xie for drawing the schematic diagram used in Figure 6.
Conflict of interest: none declared.
Funding
This work was supported by the Funds for Creative Research Groups of
The National Natural Science Foundation of China (81121003), Key
Project of National Science Foundation of China (81130088), Natural
Science Foundation of China (81070882, 81102434), and Outstanding
Youth Foundation of Heilongjiang Province (JC200904).
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