Supplementary Methods
Cells culture, anoxia/reoxygenation, apoptosis and cell death assay. Cardiomyocytes were
isolated from 1-2 days old mice according the published protocol 1. Anoxia/reoxygenation was
performed as we described elsewhere 2. Briefly, cells were placed in an anoxic hamber with a
water-saturated atmosphere composed of 5% CO2 and 95% N2. After anoxia, the cells were
subjected to reoxygenation (95% O2 and 5% CO2). Cell death was determined 12h after
reoxygenation by Trypan Blue exclusion, and the numbers of Trypan Blue-positive and Trypan
Blue-negative cells were counted on a hemocytometer. Caspase-3 was determined by analyzing
its activity using an Apo-ONE® Homogeneous Caspase-3/7 assay kit from Promega according
to the manufacturer’s protocol.
Generation of cardiac specific miR-325 transgenic mice, ARC transgenic mice without
3’UTR, ARC transgenic mice with 3’UTR (ARC-UTR),
ARC knockout mice and E2F1
knockout mice. For creating miR-325 transgenic mice, a 492bp DNA fragment containing
murine miR-325 was cloned to the vector, pαMHC-clone26 (kindly provided by Dr. Zhong zhou
Yang), under the control of the α-myosin heavy chain (α-MHC) promoter. The primers used to
generate
miR-325
transgenic
mice
include,
forward
primer
5’-ATAAGTCTTTTGGGTTCTGCT-3’; reverse primer: 5’-CTCTCCTTTCCTTCCTAAGGT-3’.
Microinjection was performed following standard protocols.
For generation of ARC transgenic mice without 3’UTR, ARC coding sequence was
1
synthesized as we described 3, and cloned to the vector under the control of the α-myosin heavy
chain promoter. The transgenic mice were genotyped by PCR using forward primer
(5’-CACATAGAAGCCTAGCCCACA-3’) in the α-MHC region and the reverse primer
(5’-TTAGGTGTTCTCACAACCTTC-3’) in the inserted rat ARC cDNA sequence. For
generation of ARC transgenic mice with 3’UTR, ARC plus 3’UTR sequence was cloned to the
vector under the control of the α-myosin heavy chain promoter. The primers used to generate
ARC
with
3’-UTR
transgenic
mice
5'-ATGGGCAACGTGCAGGAGCGC-3';
include,
reverse
forward
primer
primer:
5’-GAGTAGAACTCAGGACCTCTG-3’. Microinjection was performed following standard
protocols.
ARC knockout (KO) mice were generated as described elsewhere 4. E2F1 knockout (KO)
mice were purchased from Mutant Mouse Regional Resource Center, USA. E2F1+/-mice were
interbred to give knockout mice (E2F1-/-), which were used for further studies. Mice were
genotyped by multiplex PCR (primers and conditions are available from Mutant Mouse Regional
Resource Center, USA). All experiments were performed on E2F1-/- mice and their wild type
littermates (E2F1 +/+), and were approved by government authorities.
All transgenic and knockout mice were born normally and appeared externally
indistinguishable from the wild type littermates in terms of survival to adulthood. Phenotypes of
the hearts from all transgenic and knockout mice did not differ from that of the wild type mice.
There were no differences in body weight and heart weight between groups.
2
Immunoblot. Immunoblot was performed as we described 5. The anti-ARC antibody (Santa
Cruz Biotechnology, Inc), anti-Bcl-2 antibody (Santa Cruz Biotechnology, Inc), anti-LC3
antibody (Abcam), anti-E2F1 antibody (Abcam), anti-p62 antibody (Abcam) and anti-Caspase 3
antibody (Abcam) were used in this study. The films were scanned, and results were analyzed
with NIH ImageJ. The levels of desired proteins were normalized with loading controls.
Target protector preparation and transfection. Target protector was designed and named as
others
and
we
described
1,
6
.
In
brief,
CCTCTCAGGTCCTCCCTGCAAGACTGT-3’.
ARC-TPmiR-325
ARC-TPcontrol
sequence
sequence
is
5’is
5’-CCTCTTACCTCAGTTACAATTTATA-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.
Transfection of antagomir. miR-325 antagomir (anta-325) and the antagomir negative control
(anta-NC) were purchased from GenePharma Co. Ltd. The antagomir sequence is
5’-ACACUUACUGAGCACCUACUAGG-3’. All the bases were 2′-OMe modified, and the
3’-end was conjugated to cholesterol. 5’-CAGUACUUUUGUGUAGUACAA-3’ was used as a
negative control. Cells were transfected with the antagomir or antagomir negative control at 50
nM. The transfection was performed using Lipofectamine 2000 (Invitrogen) according to the
manufacturer's instruction.
3
Constructions of mouse miR-325 promoter and its mutant. The miR-325 promoter was
amplified
from
mouse
genome
using
5’-GCAATACACAGGTTAAATGAC-3’.
PCR.
The
The
reverse
forward
primer
primer
was
was
5’-AAATGGCCTAAGTACTATAGG-3’. The promoter fragment was finally cloned into the
vector pGL4.17 (Promega). The introduction of mutations in the putative E2F1 binding site was
performed with the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) using the wild
type vector as a template. The construct was sequenced to check that only the desired mutations
had been introduced.
Immunoprecipitation. Immunoprecipitation was carried out as we described 5. In brief, cells
were lysed for 1h at 4°C in a lysis buffer. To perform immunoprecipitation, the cell lysates were
precleared with 10% (vol/vol) protein A-agarose (Roche) for 1h on a rocking platform. Specific
antibodies were added and rocked for 1h. Immunoprecipitates were captured with 10% (vol/vol)
protein A-agarose for another hour. The agarose beads were spun down and washed twice with
NET buffer. The antigens were released and denatured by adding SDS sample buffer.
Transmission electron microscopy. Conventional electron microscopy was performed as
described previously 7. In brief, cells were fixed with 2.5% glutaraldehyde and then postfixed
with 1% osmium tetraoxide, dehydrated in a graded series of ethanol concentrations, and
embedded in Embed812 resin. The ultrathin sections were mounted on copper grids and then
double-stained with uranyl acetate and lead citrate. The number of autophagic vacuoles was
4
determined for a minimum of 100 cells. Heart ultrastructural analysis was also performed. The
samples were examined and photographed with a FEI Tecnai spirit transmission electron
microscope.
miR-325 antagomir delivery, Z-VAD-fmk treatment, intracoronary delivery of Beclin 1
siRNA adenoviruses, ischemia/reperfusion (I/R), preparations of area-at-risk and histology.
Male adult C57BL/6 mice (8 weeks old) were obtained from Institute of Laboratory Animal
Science of Chinese Academy of Medical Sciences (Beijing, China). All experiments were
performed according to the protocols approved by the Institute Animal Care Committee. For
miR-325 antagomir delivery, the mice received on three consecutive days, intravenous injections
of miR-325 antagomir, or its control at a dose of 30 mg/kg body weight in a small volume (0.2
ml) per injection. For Z-VAD-fmk treatment, the mice received daily i.p. injection of 5μg/g body
weight of the pan-caspase inhibitor Z-VAD-fmk (Sigma) for 3 days. The mice were then
subjected to I/R surgery. For intracoronary delivery of Beclin 1 siRNA adenoviruses, the mice
were anesthetized. The chest was then opened and 2×1010 moi adenoviruses of Beclin 1 siRNA
were injected with a catheter from the apex of the left ventricle into the aortic root while the
aorta and pulmonary arteries were cross-clamped. The clamp was maintained for 20s when the
heart pumped against a closed system. The chest was then closed and the mice were returned
back to cage for recovery.
For I/R injury model, mice were subjected to 45 min ischemia, then 3 h or 1 week
reperfusion as described 2. Sham-operated group experienced the same procedure except the
5
snare was left untied. Evans blue dye (1 ml of a 2.0% solution; Sigma-Aldrich) was injected into
jugular vein into the heart for delineation of the ischemic zone from the nonischemic zone. The
heart was rapidly excised. The heart slices were incubated in 1.0% 2,3,5-triphenyltetrazolium
chloride (Sigma-Aldrich) for 15 minutes at 37°C for demarcation of the viable and nonviable
myocardium within the risk zone. The staining was stopped by ice-cold sterile saline and the
slices were fixed in 10% neutral buffered formaldehyde and individually weighed. Both sides of
each slice were photographed. The areas of infarction (INF), area at risk (AAR), and
nonischemic left ventricle (LV) were assessed with computer-assisted planimetry (NIH Image
1.57) by an observer blinded to the sample identity. The ratio of AAR/LV, INF/AAR and
INF/LV were calculated. AAR in the center of the territory of the left anterior descending
coronary artery and the remote area in the posterior part of the left ventricle far from the AAR
were prepared as described 8.
Echocardiographic assessment. Transthoracic echocardiographic analysis was performed on
mice after the sham or I/R surgery as we described1. Echocardiographic parameters such as
systolic left ventricular internal diameters (LVIDs) and diastolic left ventricular internal
diameters (LVIDd) were measured. Fractional shortening (FS) of left ventricular diameter was
calculated as [(LVIDd –LVIDs)/LVIDd] × 100. After in vivo evaluation of cardiac function the
mice were euthanized and the hearts were harvested, weighted and used for histological
examination.
6
References
1.
Lin Z, Murtaza I, Wang K, Jiao J, Gao J, Li PF. Mir-23a functions downstream of nfatc3 to regulate cardiac
hypertrophy. Proc Natl Acad Sci U S A. 2009;106:12103-12108
2.
Wang JX, Jiao JQ, Li Q, Long B, Wang K, Liu JP, Li YR, Li PF. Mir-499 regulates mitochondrial dynamics
by targeting calcineurin and dynamin-related protein-1. Nat Med. 2011;17:71-78
3.
Li PF, Li J, Muller EC, Otto A, Dietz R, von Harsdorf R. Phosphorylation by protein kinase ck2: A
signaling switch for the caspase-inhibiting protein arc. Mol. Cell. 2002;10:247-258
4.
Donath S, Li P, Willenbockel C, Al-Saadi N, Gross V, Willnow T, Bader M, Martin U, Bauersachs J,
Wollert KC, Dietz R, von Harsdorf R. Apoptosis repressor with caspase recruitment domain is required for
cardioprotection in response to biomechanical and ischemic stress. Circulation. 2006;113:1203-1212
5.
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
6.
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
7.
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T.
Lc3, a mammalian homologue of yeast apg8p, is localized in autophagosome membranes after processing.
EMBO J. 2000;19:5720-5728
8.
Kim CH, Cho YS, Chun YS, Park JW, Kim MS. Early expression of myocardial hif-1alpha in response to
mechanical stresses: Regulation by stretch-activated channels and the phosphatidylinositol 3-kinase
signaling pathway. Circ Res. 2002;90:E25-33
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Supplementary figure legends
Supp. Figure 1. Anoxia/reoxygenation induces autophagy and cell death. (A) ARC levels are
reduced upon anoxia/reoxygenation (A/R). Cardiomyocytes were exposed to A/R, and harvested
at the indicated time for the analysis of ARC levels by immunoblot. (B) A/R induces punctate
accumulations of GFP-LC3. Cardiomyocytes were infected with adenoviral GFP-LC3 and then
exposed to A/R. The percentage of cells with GFP-LC3 puncta was quantified. *p<0.05 vs
control.
Supp. Figure 2. ARC inhibits autophagy induced by anoxia/reoxygenation. (A and B)
Cardiomyocytes were infected with adenoviral ARC (80moi) and β-gal, and then exposed A/R.
LC3 (A) and p62 (B) levels were analyzed by immunoblot. The densitometric analysis of
western blot of LC3-II is shown. (C) ARC transgenic mice present markedly preserved cardiac
function after I/R. ARC transgenic mice (Tg) or wild type mice (WT) were subjected to
sham-operation or 45 min of ischemia followed by 1 week of reperfusion (I/R). Transthoracic
echocardiographic analysis was performed. LVIDd, diastolic left ventricular internal diameters;
LVIDs, systolic left ventricular internal diameters; FS, fractional shortening of left ventricular
diameter. n=6, *p<0.05.
Supp. Figure 3. The effect of Beclin 1 and ATG5 on cell death. (A and B) Beclin 1
knockdown
reduces
autophagy
and
cell
8
death
in
cardiomyocytes
subjected
to
anoxia/reoxygenation (A/R). Cardiomyocytes were infected with adenoviral Beclin 1-siRNA
(1moi) or Beclin 1-sc, and then exposed to A/R. Beclin 1 expression level was analyzed by
immunoblot. Quantification of autophagic vacuoles is shown (A). Quantitation of cell death is
shown (B), *p<0.05 vs A/R alone. (C) Cardiomyocytes were infected with adenoviral Beclin 1
(50moi), ARC (80moi) or β-gal, or exposed A/R in the presence or absence of BFA. BFA was
preincubated with cells for 1h. Quantitation of cell death is shown. *p<0.05. (D) Knockdown of
ATG5 reduces cell death in cardiomyocytes subjected to A/R. Cardiomyocytes were infected
with adenoviral ATG5-siRNA (50moi) or ATG5-sc, and then exposed to A/R. Quantitation of
cell death is shown, *p<0.05 vs A/R alone. (E) Knockdown of ATG5 reduces the Beclin 1
induced cell death. Cardiomyocytes were infected with adenoviral Beclin 1 (50moi) or β-gal, and
then infected with adenoviral ATG5-siRNA (50moi) or ATG5-sc. Quantitation of cell death is
shown, *p<0.05. (F) Overexpression (upper panel) or knockdown (lower panel) of ARC does not
affect the interaction of Beclin 1 and Bcl-2. Cardiomyocytes were infected with adenoviral ARC
(80moi), β-gal, ARC-siRNA, or its scramble form (ARC-sc). Immunoprecipitation was
performed using Bcl-2 antibody. Beclin 1 levels were analyzed by immunoblot.
Supp. Figure 4. ARC has an impact with Beclin 1. (A and B) Beclin 1-induced autophagy and
cell death are inhibited by ARC. Cardiomyocytes were infected with adenoviral Beclin 1 (50moi),
ARC (80moi) or β-gal, and then exposed to 30min anoxia followed by 20min reoxygenation.
Z-VAD (20µM/L) was administrated 1h before A/R. The analysis of autophagy (A) and cell
death (B) are shown, *p<0.05. (C) Cardiomyocytes were treated as described above. The active
9
caspase-3 levels (upper panel) and the caspase-3 activity (lower panel) were analyzed. *p<0.05.
(D) The inhibitory effect of ARC on autophagic cell death. Cardiomyocytes were infected with
adenoviral ARC (80moi), β-gal or treated with Z-VAD (20µM/L) alone, 3-MA (5mM) alone and
their combination, then exposed to A/R. Quantitation of cell death is shown, *p<0.05. (E) ARC
transgenic mice exhibit less myocardial infarction than the mice treated with Beclin 1
knockdown and Z-VAD. Wild type mice (WT) were treated with Z-VAD-fmk or Beclin 1
siRNA as described in methods, and then subjected to I/R. ARC transgenic mice (ARC Tg) were
also subjected to I/R. Myocardial infarct sizes were analyzed. Area-at-risk (AAR), left ventricle
(LV), infarct area (INF). n=9, *p<0.05.
Supp.
Figure
5.
miR-325
is
upregulated
upon
anoxia/reoxygenation
and
ischemia/reperfusion. (A) miR-325 levels upon anoxia/reoxygenation. Cardiomyocytes were
exposed to anoxia at indicated time and harvested 2h after the reoxygenation for qRT-PCR
analysis of miR-325 levels. *p<0.05 vs control. (B) miR-325 levels during myocardial
ischemia/reperfusion. Mice were induced to undergo cardiac ischemia/reperfusion. Area-at-risk
and the remote area were prepared at the indicated time for qRT-PCR analysis of miR-325 levels.
n=5, *p<0.05 vs 0 min or sham. (C) The miR-325 potential binding sites in 3’UTR regions of
human and rat ARC.
Supp. Figure 6. miR-325 transgenic mice genotyping assay. (A) Schematic map showing the
transgenic construct of miR-325 and the primers for genotyping miR-325 transgenic mice. (B)
10
Genotyping of miR-325 transgenic mice. Genomic DNA was isolated from mice tail biopsies
and analyzed by PCR using primers described in the section of Supplementary Methods. The
positive miR-325 transgenic mice (Line 1-3) but not wild type mice (WT) express a 461 bp
genomic fragment. The positive control plasmid contains miR-325 cDNA as templates for RCR,
and the negative control does not contain miR-325 cDNA. (C) qRT-PCR analysis of miR-325
expression levels in different organs or tissues isolated from individual miR-325 transgenic
mouse (Tg) and wild type mouse (WT).
Supp. Figure 7. miR-325 transgenic mice exhibit more severe cardiac dysfunction upon I/R.
(A) Knockdown of miR-325. Cardiomyocytes were transfected with antagomir-325 (anta-325) or
antagomir-NC (anta-NC). 24h after transfection cells were treated with A/R. miR-325 levels
were analyzed by qRT-PCR. *p<0.05 vs A/R alone. (B) p62 levels assessments. Cardiomyocytes
were transfected with antagomir-325 (anta-325) or antagomir-NC (anta-NC). 24h after
transfection cells were treated with A/R. p62 levels were analyze by immunoblot. (C) miR-325
transgenic mice exhibit more severe cardiac dysfunction upon I/R. Mice were treated as
described in Supp. Figure 2C. Transthoracic echocardiographic analysis was performed at 1
week after sham or I/R. LVIDd, diastolic left ventricular internal diameters; LVIDs, systolic left
ventricular internal diameters; FS, fractional shortening of left ventricular diameter. n=5-6,
*p<0.05.
Supp. Figure 8. E2F1 induces autophagy and cell death. (A) E2F1 potential binding sites in
11
the promoter regions of human and rat miR-325. Human miR-325 promoter region contains two
potential E2F1 binding sites between -4963～-4958 (BS1), and -2923～-2918 (BS2). Rat
miR-325 promoter region contains one potential E2F1 binding site between -4105～-4100 (BS).
(B) E2F1 levels are elevated upon A/R. Cardiomyocytes were exposed to A/R, and harvested at
the indicated time for the analysis of E2F1 levels by immunoblot. (C) Knockdown of E2F1 leads
to a reduction of miR-325 levels. Cardiomyocytes were infected with adenoviral E2F1-siRNA or
its scramble form (E2F1-sc). miR-325 levels were analyzed by qRT-PCR. *p<0.05 vs control.
E2F1 levels were analyzed by immnoblot (lower panel). (D) Autophagic flux assay.
Cardiomyocytes were infected with adenoviral GFP-LC3 and adenoviral E2F1-siRNA or its
scramble form (E2F1-sc), and then exposed to A/R in the presence or absence of bafilomycin A1
(BFA). Cells was preincubated with BFA for 1h. The percentage of cells with GFP-LC3 puncta
was quantified. *p<0.05. (E and F) Enforced expression of E2F1 induces autophagy and cell
death. Cardiomyocytes were infected with adenoviral GFP-LC3, E2F or β-gal. GFP-LC3
staining (E) and cell death (F) were analyzed. *p<0.05 vs control.
12