SUPPLEMENTAL MATERIAL
(Zhaokang Cheng, et al. Mitochondrial Translocation of Nur77 Mediates
Cardiomyocyte Apoptosis)
Detailed Methods
Animals
All experiments were performed in 8-12-week-old male FVB/N mice unless otherwise
indicated. The Institutional Animal Care Committee of San Diego State University approved
all animal protocols.
Myocardial infarction, ischemia/reperfusion and trans-aortic constriction
Mice surgical procedures have been described previously1. Briefly, myocardial
infarction was produced by ligating the left anterior descending (LAD) branch of the
coronary artery using a 8-0 suture (Ethicon).
Ischemia/reperfusion injury was induced by ligating the LAD for 50 minutes
followed by reperfusion for indicated times.
For trans-aortic constriction, the aorta was ligated between the innominate artery
and the left common carotid artery by using a 7-0 polypropylene suture (Ethicon) with
an overlying 27-gauge needle to produce a discrete stenosis.
Neonatal rat cardiomyocyte cell culture and treatment
Primary neonatal rat cardiomyocytes were isolated and cultured as described
2
previously1. Cells were serum-starved in 0.5% FBS for 48 hours before treatment with
hydrogen peroxide (H2O2, 50μM or 10μM, Fisher BioReagents) for the indicated
times.
Plasmid construction
Nur77 cDNA and a Nur77 mutant lacking the DNA binding domain
(Nur77/ΔDBD-GFP) were kindly provided by Dr. Xiao-kun Zhang (Burnham
Institute, San Diego, CA). Nur77 cDNA was subcloned into the pShuttle-CMV vector
at the NotI site. Then GFP cDNA (HindIII/HpaI double digestion from pEGFP-N1)
was subcloned at the HindIII/EcoRV site. Nur77-GFP fusion gene was generated
through a T to G mutation of the Nur77 stop codon using the QuikChange XL site
directed mutagenesis kit (Stratagene, La Jolla, CA). Schematic diagrams of plasmids
used in this study were shown in Supplemental Figure I.
Adenoviral infection, plasmid and siRNA transfection
Nur77-GFP adenovirus was made by using the AdEasy XL Adenoviral Vector
System (Stratagene, La Jolla, CA). NRCM were infected with adenovirus at
multiplicity of infection (MOI) 50 for 2h, washed in PBS, and then refed with M199
with 0.5% FBS and antibiotics.
Plasmid transfection of NRCM was performed by using the Effectene
transfection reagent (Qiagen) following manufacturer's instructions. Briefly, 1μg DNA
was diluted in the DNA-condensation buffer to a total volume of 100 μl. Ten
3
microliter of Enhancer and 20μl of Effectene transfection reagent were added to the
DNA solution, respectively. The transfection complexes were finally added
drop-wisely onto the cells cultured in chamber slides.
NRCM were transfected with small interfering RNAs (siRNAs, 25nM) by using
HiPerfect transfection reagent (Qiagen) as per the manufacturer’s recommendations.
Briefly, 3μl siRNA and 12μl HiPerfect were diluted in 100μl serum-free M199
medium. After incubation for 5-10 minutes, transfection complexes were added to the
cells for 48h. The following siRNA sequences were used: rat Nur77 siRNA, 5’-UGG
CCC AGA GUU CCC UGA AGU UG UU-3’; The scrambled siRNA was obtained
from Ambion.
Real-time RT-PCR
Total RNA was isolated from frozen heart or cultured cells by using
Quick-RNA™
MiniPrep
(Zymo
Research)
and
reverse-transcribed
into
complementary DNA (cDNA) by using High Capacity cDNA Reverse Transcription
Kit (Applied Biosystems). Real-time PCR was performed on all samples in triplicate
using QuantiTect™ SYBR Green PCR Kit (Qiagen) according to the manufacturer’s
instructions. All primer sequences are shown in Online Table I of the Online Data
Supplement.
Subcellular fractionation
Heart tissues were snap-frozen in liquid nitrogen, pulverized, and homogenized
4
in isolation buffer (70mM sucrose, 190mM D-Mannitol, 20mM Hepes, 0.2mM EDTA)
by a Teflon-glass dounce homogenizer. NRCM were collected by scraping in isolation
buffer and immediately disrupted by homogenization in a Dounce apparatus with a
tight fitting pestle. Nuclear fractions were separated by centrifugation at 600g for
10min followed by discontinuous sucrose density centrifugation. Mitochondrial
fractions were separated by centrifugation at 5000g for 15min. Cytosolic fractions
were separated by centrifugation at 100,000g for 60min. Samples were resuspended in
SDS sample buffer (8 mol/L urea, 50 mmol/L DTT, 2% SDS, 150 mmol/L Tris-HCl
pH 6.8, 15% sucrose 2 mmol/L EDTA, 0.01% bromophenol blue), sonicated, boiled
for 5 minutes and stored at -80C until used for immunoblotting.
Immunoblotting
Immunoblotting was performed as described previously2. Briefly, protein lysates
from ventricles or cultured cardiomyocytes were loaded onto a 4-12% NuPAGE
Novex Bis-Tris Gel (Invitrogen) for electrophoresis. Separated proteins were then
transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked with 5% skim
milk in Tris-Buffered Saline Tween-20 (TBST) for 1 h at room temperature, and
exposed to rabbit anti-Nur77 (sc-5569, Santa Cruz Biotechnology, 1:200), mouse
anti-β-actin (sc-47778, Santa Cruz Biotechnology, 1:1000) and mouse anti-GAPDH
(MAB374, Chemicon, 1:2000) overnight at 4ºC. Alkaline phosphatase (AP),
horseradish peroxidase (HRP) or Cy5-conjugated IgG (Jackson ImmunoResearch,
West Grove, PA) were used as secondary antibodies. Fluorescence signal was detected
5
and quantified by using a Typhoon 9400 fluorescence scanner together with
ImageQuant 5.0 software (Amersham Biosciences).
Immunohistochemistry and confocal microscopy
Immunolabeling was performed as described previously2. Briefly, cultured
cardiomyocytes were permeabilized in PBS containing 0.1M Glycine and 0.1%
Triton-X100 for 5min, blocked with 10% horse serum in PBS for 1h, and incubated
with rabbit anti-Nur77 (LS-B114, Lifespan Biosciences, 1:50), goat anti-heat shock
protein
60
(HSP60,
sc-1052,
Santa
Cruz
Biotechnology,
1:50),
mouse
anti-cytochrome c (556432, BD Pharmingen, 1:50), mouse anti-α-actinin (A7732,
Sigma-Aldrich, 1:100) at 4°C overnight. The next day, slides were incubated 1.5 h at
room temperature with FITC, Cy3, or Cy5-conjugated secondary antibodies (Jackson
ImmunoResearch Laboratories, 1:100). Nuclei were stained with To-pro-3 iodide or
Sytox Blue (Invitrogen).
Formalin-fixed paraffin-embedded mouse heart sections were deparaffinized,
rehydrated, and antigen-retrieved in 10 mmol/L citrate, pH 6.0. After blocking in TNB
buffer, slides were incubated with rabbit anti-Nur77 (LS-B114, Lifespan Biosciences,
1:100), mouse anti-tropomyosin (T9283, Sigma-Aldrich, 1:100) at 4°C overnight
followed by FITC-conjugated donkey anti-rabbit IgG and Cy3-conjugated donkey
anti-mouse IgG (Jackson ImmunoResearch Laboratories, 1:100) at room temperature
for 1.5h. Nuclei were stained with Topro-3 iodide (T3605, Invitrogen, 1:10000).
Confocal images were acquired by using a Leica TCS-SP2 or a Molecular Dynamics
6
CLSM 2010 confocal laser-scanning microscope (Leica).
TUNEL staining
TUNEL staining was performed by using the In Situ Cell Death Detection Kit,
TMR red (Roche Applied Science) according to the manufacturer’s directions. Briefly,
NRCM were fixed in 4%PFA and permeabilized with fresh permeabilization solution
(0.1%Triton X-100 and 0.1% sodium citrate in PBS). Cells were then incubated with
TUNEL reagent for 1h at 37°C and covered for examination by confocal microscopy.
Flow cytometry
NRCM were stained with Annexin V (BD Biosciences) and Sytox blue
(Invitrogen) according to the manufacturer’s instructions. Briefly, cell pellet was
collected by trypsinization and centrifugation, then incubated with Annexin V (1:50)
and Sytox blue (1:1000) for 15min. Annexin V has a high affinity for the membrane
phospholipid phosphatidylserine which is exposed to the external environment during
early apoptosis. Sytox Blue is a high-affinity nucleic acid stain that easily penetrates
cells with compromised plasma membranes but will not cross intact membranes in
viable cells. Early apoptotic cells are Sytox Blue-/Annexin V+; Late apoptotic cells are
Sytox Blue+/Annexin V+; Necrotic cells are Sytox Blue+/Annexin V-; and viable cells
are Sytox Blue-/Annexin V-. Flow cytometry was performed by using a BD FACSAria
Flow Cytometer (BD Biosciences).
7
Statistical analysis
Statistical analysis was performed with the Windows SPSS 13.0 software package. All
data are expressed as mean ± SEM. Comparisons were performed by using unpaired Student
t-test or one-way analysis of variance (ANOVA) with Tukey’s post-hoc test as appropriate. All
tests were two-sided and a value of P<0.05 was considered statistically significant.
Supplemental Table I. Primer sequences for real-time PCR
Species
Gene
Forward
Reverse
rat
Nur77
5’-TGTTGATGTTCCTGCCTTTGC-3’
5’-TGCGGTTCTGCAGCTCCT-3’
β-actin
5’-GAAGATCAAGATCATTGCTCCTCCT-3’
5’-GAAGGTGGACAGTGAGGCCA-3’
GAPDH
5’- GACATGCCGCCTGGAGAAAC-3’
5’-AGCCCAGGATGCCCTTTAGT-3’
Nur77
5’-GTCCGCTCTGGTCCTCATCA-3’
5’-CCATGTGCTCCTTCAGACAGC-3’
ANP
5’-TCTGATGGATTTCAAGAACCTGC-3’
5’-CTGCTTCCTCAGTCTGCTCACTC-3’
BNP
5’-GCAATTCAAGATGCAGAAGCTG-3’
5’-CTGCCTTGAGACCGAAGGACT-3’
β-MHC
5’-GAGCCTTGGATTCTCAAACG-3’
5’- GTGGCTCCGAGAAAGGAAG-3’
β-actin
5’-CATGAAGATCAAGATCATTGCTCCT-3’
5’-GCTGATCCACATCTGCTGGAA-3’
GAPDH
5’- CATGGCCTTCCGTGTTCCTA-3’
5’- CCTGCTTCACCACCTTCTTGAT-3’
mice
8
Supplemental Figures and Figure Legends
Supplemental Figure I. Construction and confirmation of Nur77 mutants. (A)
Schematic diagrams of Nur77-GFP and Nur77/ΔDBD-GFP. (B-C) Confirmation of
protein expression. GFP-, Nur77-GFP-, or Nur77/ΔDBD-GFP-transfected cells were
immunolabeled for Nur77 (B) or GFP (C), respectively, with non-transfected cells as
a negative control. Both Nur77 and GFP bands appeared at the same size, confirming
expression of the fusion protein. β-actin served as a loading control.
9
Supplemental Figure II. Myocardial Nur77 mRNA declines during postnatal
development. qRT-PCR results of Nur77 mRNA levels in ventricular tissues of 2-day
and 2-month-old mice, with 18S rRNA as an internal control (n=3). Values are mean ±
SEM. ***P<0.001.
10
Supplemental Figure III. Nur77 expression is induced by myocardial infarction
and pressure overload-induced hypertrophy. (A) Confocal images of myocardial
sections at 3 days after infarction, with sham-operated animal as a control. Nur77
(green) localized in the nuclei (Topro-3, blue) of cardiomyocytes (tropomyosin, red)
within the border zone adjacent to the infarct. Boxed regions in left are shown at
higher magnification at right. Scale bar = 40μm (left) and 10μm (right); (B)
Immunoblot shows cardiac Nur77 peaks at 3 days after infarction (n=3). * P<0.05 vs
sham; (C) Confocal images of myocardial sections at 4 days after trans-aortic
constriction, with sham-operated animal as a control. Nur77 (green) localized in the
nuclei (Topro-3, blue) of cardiomyocytes (tropomyosin, red). Boxed regions in left are
shown at higher magnification at right. Scale bar = 40μm (left) and 10μm (right); (D)
Immunoblot shows myocardial Nur77 increased at 4 days after trans-aortic
constriction (n=3). * P<0.05 vs sham; (E) Real-time RT-PCR showing Nur77 mRNA
is increased by TAC together with the hypertrophy markers ANP, BNP and β-MHC
(n=4). * P<0.05 vs sham; *** P<0.001 vs sham.
11
Supplemental Figure IV. Subcellular localization of Nur77 was not affected by
ischemia. Mice were subjected to ischemia for 65 min or 170min. (A) Confocal
images of sham and ischemic heart sections stained for Nur77 (green), HSP60 (red)
and nuclei (blue). Nur77 localized to nuclei of cardiomyocyte in both sham and
ischemic heart sections (arrows). Colocalization of Nur77 with HSP60 was not
significant. Scale bar = 20μm; (B-E) Subcellular fractionation performed in
sham-operated and ischemic hearts (n=3). Whole cell (B), nuclear (C), cytosolic (D)
12
and mitochondrial (E) lysates were probed for Nur77 and normalized to β-actin, H3
(nuclear marker), GAPDH (cytosolic marker) and VDAC (mitochondrial marker).
*P<0.05, **P<0.01.
Supplemental Figure IV. Ischemia Reperfusion cause oxidative DNA damage.
Mice were subjected to ischemia for 50min and then reperfusion for 15min. Confocal
images of sham and I/R treated hearts stained for 8-Hydroxy-2'-deoxyguanosine (red)
and desmin (blue). 8OH-dG localized to the nuclei of cardiomyocytes in IR heart
sections. Inlets are shown on the right. Bar 40M.
13
REFERENCES
1.
Muraski JA, Rota M, Misao Y, Fransioli J, Cottage C, Gude N, Esposito G,
Delucchi F, Arcarese M, Alvarez R, Siddiqi S, Emmanuel GN, Wu W, Fischer
K, Martindale JJ, Glembotski CC, Leri A, Kajstura J, Magnuson N, Berns A,
Beretta RM, Houser SR, Schaefer EM, Anversa P, Sussman MA. Pim-1
regulates
cardiomyocyte
survival
downstream
of
Akt.
Nat
Med.
2007;13:1467-1475.
2.
Tsujita Y, Muraski J, Shiraishi I, Kato T, Kajstura J, Anversa P, Sussman MA.
Nuclear targeting of Akt antagonizes aspects of cardiomyocyte hypertrophy.
Proc Natl Acad Sci U S A. 2006;103:11946-11951.