Online Appendix for the following JACC article
TITLE: Transcriptome Characterization of Estrogen-Treated Human Myocardium Identifies MYLIP as
a Sex-Specific Element Influencing Contractile Function
AUTHORS: Georgios Kararigas, PhD, Virginie Bito, PhD, Hanna Tinel, PhD, Eva Becher, PhD, Istvan
Baczko, MD, PhD, Christoph Knosalla, MD, Barbara Albrecht, PhD, Karin R Sipido, MD, PhD, Vera
Regitz-Zagrosek, MD
APPENDIX
Supplementary Methods
Human cardiac tissue studies. All chemicals and reagents were purchased with the highest purity
grade available from Sigma-Aldrich unless otherwise indicated. Tissue harvest, culture and treatment
were recently described (1). The study was approved by the Charite University Hospital Ethics
Committee, it complies with the principles outlined in the Declaration of Helsinki and written consent
from patients (n = 6; 50% women) undergoing coronary bypass surgery was obtained. Collected atrial
tissues were incubated at 37°C in a conventional water bath for 24 h in the presence of 10−8 M E2 or
2-hydroxypropyl-β-cyclodextrin (HBC; vehicle – control for E2 treatment). This concentration was
chosen to obtain an E2 level of physiological range. Following the completion of the treatment, the
tissues were shock frozen in liquid nitrogen and stored at −80°C until further analysis.
In addition, ventricular samples from non-diseased male human hearts (n = 15) that were
technically unusable for transplantation (due to logistical, not patient-related, reasons) were obtained
from general organ donors. Before cardiac explantation, organ donor patients did not receive
medication except dobutamine, furosemide and plasma expanders. The investigations conform to the
principles outlined in the Declaration of Helsinki. Experimental protocols were approved by the
1
Scientific and Research Ethical Committee of the Medical Scientific Board at the Hungarian Ministry of
Health (ETT-TUKEB) under ethical approval No. 4991-0/2010-1018EKU (339/PI/010).
Hybridization and microarray profiling. Total RNA was isolated using the RNeasy Fibrous Tissue
Mini kit (Qiagen) following the manufacturer’s protocol. The RNA quality and quantity was established
using a 2100 Bioanalyzer (Agilent Technologies). Biotinylated complementary RNA (cRNA) from 3
male and 3 female individuals was prepared and hybridized to the GeneChip Human Genome U133
Plus 2.0 array (Affymetrix) according to the standard Affymetrix processing protocol. The array was
scanned in a GeneChip Scanner 3000. The quality of hybridization was assessed in all samples
following the manufacturer’s recommendations and raw data were analyzed using the Affymetrix
GeneChip Operating Software (GCOS 1.2). Microarray data have been deposited in the ArrayExpress
database (accession No. E-MEXP-2971).
Microarray data analysis. The computational and statistical analysis of the microarray data was
carried out using the R version 2.8.1 software (2) and the Bioconductor packages (3) as described
recently (4,5). Following background correction, expression data were normalized with the variance
stabilization and normalization algorithm (6) and log2 transformed using the median polish algorithm of
robust multi-array average (7). The quality of the data was assessed with the affy (8) and the
arrayQualityMetrics (9) packages. To detect differences in probe set expression between two
conditions, a moderated linear model was applied using the limma package (10). Gene lists were
generated selecting for candidates with an uncorrected P value threshold of 0.001. Microarrays from
CardioGenomics (http://cardiogenomics.org) and the E-GEOD-21610 set in the ArrayExpress
database were analyzed in the same manner. To adjust the resulting P values, the false discovery rate
was controlled. Statistical significance was considered at adjusted P ≤ 0.05.
Mouse experiments. A first set of middle-aged (11-14 months) male and female C57BL/6J mice
(Charles River) were randomly assigned to receive i.p. injections of vehicle (HBC; n = 4 male and 5
2
female) or 0.2 mg/kg HBC-encapsulated E2 (n = 6 male and 7 female) in a volume of 4 ml/kg for 5 h.
The dose used has been previously shown to result in E2 levels in the physiological range (11). All
mice were given a soy-free diet for at least one week prior to treatment and water ad libitum. Prior to
treatment, a vaginal smear was taken to assure that none of the females would be in proestrus at the
time of injection. Five hours after treatment initiation, mice were injected i.p. with heparin (1000 U/kg)
and killed by an i.p. injection of pentobarbital overdose (0.1 mg/g). The heart was quickly excised and
weighed. Single CMs were obtained by enzymatic dissociation through retrograde perfusion of the
aorta as described previously (12). Following isolation, CMs were either used for functional
(contraction) measurements or they were snap frozen in liquid nitrogen in the presence of TRIzol
Reagent (Invitrogen) and stored at −80oC until used for RNA isolation. A second set of middle-aged
male and female C57BL/6J mice (n = 5/group) were treated in exactly the same manner and left
ventricles were collected for protein extraction. Unloaded cell shortening was measured with a videoedge detector (Ionoptix) at 1, 2 and 4 Hz using field-stimulation. Cells were superfused with normal
Tyrode’s solution (in mmol/L: 137 NaCl, 5.4 KCl, 0.5 MgCl2, 1 CaCl2, 11.8 HEPES and 10 glucose, pH
adjusted to 7.4 with NaOH). Data are presented as fractional shortening, i.e. the maximum change in
cell length is expressed as the percent change relative to the resting length (L/L 0). The study was
approved by the Ethics Committee on Animal Use of the University of Leuven.
Quantitative real-time RT-PCR. RNA from mouse CMs was extracted using TRIzol Reagent
following the manufacturer's protocol. RNA from human donor myocardial tissue was extracted using
the RNeasy Fibrous Tissue Mini kit (Qiagen) following the manufacturer’s protocol. Assessment of
RNA quality and quantity, reverse transcription and quantitative real-time PCR were performed as
described previously (1). Reactions where RNA or reverse transcriptase had previously been omitted
during reverse transcription were used as negative controls. Primer sequences used for amplification:
mouse Gapdh 5′-ATGGTGAAGGTCGGTGTGA-3′ and 5′-AATCTCCACTTTGCCACTGC-3′ (84 bp
product),
mouse
Mylip
AGGGACTCTTTAATGTGCAAGAA-3′
5′-TGTGGAGCCTCATCTCATCTT-3′
(69
bp
3
product),
human
and
5′-
RPLP0
5′-
ACGGGTACAAACGAGTCCTG-3′ and 5′-AGCCACAAAGGCAGATGGAT-3′ (104 bp product) and
human MYLIP 5′-ATGAGGAGCTCTGTGCCAAG-3′ and 5′-GGTCCCCTCCAACTCCTTAT-3′ (80 bp
product).
Immunoblotting and co-immunoprecipitation. Left ventricles were homogenized in a modified RIPA
buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate)
supplemented with protease inhibitor cocktail (Roche), 1 mM Na3VO4, 1 mM NaF and 5 μM ubiquitin
aldehyde (Santa Cruz). After sonication and centrifugation, proteins were quantified using the BCA
Assay (Pierce), resolved by SDS-PAGE, transferred to PVDF membranes and immunoblotted with the
indicated antibodies. For co-immunoprecipitation, 300 mg lysates were incubated with the appropriate
antibody (1:50 dilution) for 2 h at 4°C followed by overnight incubation with Protein G sepharose beads
(GE Healthcare). Lysates incubated with antibody-lacking beads were used as negative controls.
Immunocomplexes were washed five times with IP buffer (1 mM Hepes, 50 mM NaCl, 10% glycerol
and 0.1% triton) supplemented with protease inhibitor cocktail (Roche), 1 mM Na 3VO4 and 1 mM NaF
before being resolved by SDS-PAGE, transferred to PVDF membranes and immunoblotted with the
indicated antibodies. Antibodies against Mylip (abcam), Mrlc (Epitomics), Ub (P4D1; Santa Cruz),
tubulin (Sigma; loading control) and donkey anti-mouse or -rabbit secondary antibody conjugated with
horseradish peroxidase (Dianova) were used. Immunoreactive proteins were detected using ECL Plus
(GE Healthcare), quantified by the ImageJ 1.41 version software (http://rsbweb.nih.gov/ij/).
Statistical analysis. All data were analyzed statistically using the R version 2.11.0 software. Data are
shown as the mean ± SEM. Comparisons between two groups were performed with unpaired t-test
and between multiple groups using two-way ANOVA with Tukey’s post hoc test adjusting for multiple
comparisons considering P ≤ 0.05 significant.
4
Online Table 1. E2-regulated probe sets
Probe set ID
205516_x_at
209515_s_at
211675_s_at
202173_s_at
203244_at
200011_s_at
202047_s_at
209821_at
227984_at
224777_s_at
201040_at
204235_s_at
203048_s_at
234971_x_at
224949_at
221998_s_at
225133_at
212638_s_at
216033_s_at
203008_x_at
214870_x_at
218197_s_at
208684_at
221773_at
224977_at
209630_s_at
212153_at
217766_s_at
1555854_at
203514_at
224068_x_at
222550_at
226797_at
205140_at
223000_s_at
224678_at
234656_x_at
239477_at
213062_at
202551_s_at
227947_at
203445_s_at
208818_s_at
212994_at
224549_x_at
222047_s_at
1569872_a_at
202123_s_at
213290_at
Gene symbol
CIZ1
RAB27A
MDFIC
VEZF1
PEX5
ARF3
CBX6
IL33
LOC650392
PAFAH1B2
GNAI2
GULP1
TTC37
PLCD3
YIPF5
VRK3
KLF3
WWP1
FYN
TXNDC9
LOC100132540
OXR1
COPA
ELK3
C6orf89
FBXW2
POGZ
TMEM50A
NA
MAP3K3
RBM22
ARMC1
MBTD1
FPGT
F11R
KIAA1219
NA
EFHB
NTAN1
CRIM1
PHACTR2
CTDSP2
COMT
THOC2
NA
ARS2
LOC650392
ABL1
COL6A2
Fold
change
1.204669556
1.19595811
1.347856359
1.221790351
1.189462401
1.224077834
1.296073798
1.329844424
1.181756674
1.247792614
1.291040225
1.37302846
1.204703071
1.184474667
1.28838304
1.209769462
1.295681713
1.203918536
1.271047999
1.214310894
1.206803239
1.24570691
1.316893283
1.310993003
1.220769884
1.174080041
1.198278775
1.184447282
1.217367947
1.211211594
1.235833722
1.201505443
1.202963776
1.167328581
1.173617046
1.15614342
0.872626654
0.866220509
1.202495026
1.337214083
1.205451681
1.230994164
1.178809084
1.213920906
1.203550342
1.180754814
1.169101702
1.241759475
1.2059043
Unadjusted P
value
0.000119131
0.000141845
0.00014385
0.000163247
0.000195892
0.00020345
0.000217938
0.00023698
0.000272227
0.000280792
0.000291281
0.000299457
0.000301086
0.000303792
0.00030475
0.000312952
0.000315706
0.000330722
0.000331787
0.000351247
0.000373739
0.000386798
0.000425892
0.000444339
0.000452687
0.00049062
0.000535582
0.000540043
0.000545016
0.000546064
0.000559216
0.000571214
0.000576745
0.000630027
0.000630548
0.000633539
0.000634665
0.00065675
0.00068263
0.000688486
0.000691584
0.000707929
0.000718698
0.000723663
0.000729672
0.000748816
0.000752195
0.000755729
0.000756845
5
211066_x_at
203233_at
243916_x_at
201051_at
224605_at
202506_at
214020_x_at
226620_x_at
227527_at
241469_at
201938_at
37012_at
217885_at
208723_at
219628_at
226347_at
223071_at
224880_at
226977_at
221474_at
209311_at
209489_at
224748_at
217939_s_at
224778_s_at
224806_at
218313_s_at
219988_s_at
PCDHGC3
IL4R
UBLCP1
ANP32A
LOC401152
SSFA2
ITGB5
DAZAP1
MLL2
HFM1
CDK2AP1
CAPZB
IPO9
USP11
ZMAT3
NA
IER3IP1
RALA
LOC492311
MRLC2
BCL2L2
CUGBP1
WDR68
AFTPH
NA
TRIM25
GALNT7
RNF220
1.283611642
1.153809907
1.258384038
1.256757236
1.297995064
1.236615728
1.186804238
1.142544878
1.136112612
0.864227324
1.188240011
1.203850682
1.202660543
1.221248951
1.222712333
1.232992813
1.235528389
1.194428006
1.225617282
1.146496774
1.186487853
1.207381593
1.158820306
1.168426767
1.189074594
1.198966323
1.24539627
1.14295782
0.000762081
0.000763542
0.000775447
0.000776091
0.000785655
0.000806358
0.000824247
0.000850681
0.000853209
0.000879182
0.000880961
0.000912465
0.000912674
0.000917033
0.00093146
0.000959675
0.000959862
0.000959973
0.000963565
0.000972674
0.000973224
0.000974811
0.000993848
0.00099853
0.001005384
0.001007237
0.001016621
0.001034255
6
Online Table 2. Sexually-dimorphic E2-regulated probe sets
Probe set
ID
1567985_at
206114_at
209911_x_at
205290_s_at
211919_s_at
206278_at
208790_s_at
222670_s_at
241669_x_at
214805_at
212859_x_at
211271_x_at
209201_x_at
200853_at
206697_s_at
209302_at
210461_s_at
213503_x_at
231973_s_at
206100_at
222728_s_at
213901_x_at
209616_s_at
209160_at
228009_x_at
201806_s_at
219534_x_at
209385_s_at
209699_x_at
212014_x_at
223129_x_at
216289_at
208470_s_at
213971_s_at
209485_s_at
210822_at
225241_at
230071_at
217028_at
235186_at
211457_at
Gene
symbol
Unknown
EPHA4
HIST1H2BD
BMP2
CXCR4
PTAFR
PTRF
MAFB
PRKD2
EIF4A1
MT1E
PTBP1
CXCR4
H2AFZ
HP
POLR2H
ABLIM1
ANXA2
ANAPC1
CPM
JOSD3
RBM9
CES1
AKR1C3
ZNRD1
ATXN2L
CDKN1C
PROSC
AKR1C2
CD44
MYLIP
GPR144
HPR
SUZ12P
OSBPL1A
LOC283345
CCDC80
SEPT11
CXCR4
LOC388692
GABARAPL3
Unadjusted P
value
6.68627E-05
0.000137942
0.000176101
0.000188541
0.00025816
0.000277273
0.00031371
0.000336043
0.000429898
0.000448605
0.000454872
0.000465455
0.000522754
0.000596782
0.000641216
0.000651098
0.000669243
0.000673571
0.000703671
0.000710651
0.000727484
0.000744506
0.000751828
0.000751894
0.000754031
0.000763505
0.000773481
0.000775094
0.000778273
0.0007828
0.000787882
0.000847366
0.000854118
0.000874322
0.000905284
0.000942029
0.000963476
0.000970372
0.001000757
0.001030749
0.001039306
7
Supplementary References
1. Kararigas G, Becher E, Mahmoodzadeh S, Knosalla C, Hetzer R, Regitz-Zagrosek V. Sex-specific
modification of progesterone receptor expression by 17beta-oestradiol in human cardiac tissues.
Biol Sex Differ 2010;1:2.
2. Ihaka R, Gentleman R. R: A language for data analysis and graphics. J Comput Graph Stat
1996;5:299-314.
3. Gentleman RC, Carey VJ, Bates DM et al. Bioconductor: open software development for
computational biology and bioinformatics. Genome Biol 2004;5:R80.
4. Kararigas G, Fliegner D, Gustafsson JA, Regitz-Zagrosek V. Role of the estrogen/estrogenreceptor-beta axis in the genomic response to pressure overload-induced hypertrophy. Physiol
Genomics 2011;43:438-46.
5. Toischer K, Rokita AG, Unsold B et al. Differential cardiac remodeling in preload versus afterload.
Circulation 2010;122:993-1003.
6. Huber W, von Heydebreck A, Sultmann H, Poustka A, Vingron M. Variance stabilization applied to
microarray data calibration and to the quantification of differential expression. Bioinformatics
2002;18 Suppl 1:S96-104.
7. Irizarry RA, Hobbs B, Collin F et al. Exploration, normalization, and summaries of high density
oligonucleotide array probe level data. Biostatistics 2003;4:249-64.
8. Gautier L, Cope L, Bolstad BM, Irizarry RA. affy--analysis of Affymetrix GeneChip data at the probe
level. Bioinformatics 2004;20:307-15.
9. Kauffmann A, Gentleman R, Huber W. arrayQualityMetrics--a bioconductor package for quality
assessment of microarray data. Bioinformatics 2009;25:415-6.
10.
Smyth GK. Linear models and empirical bayes methods for assessing differential expression in
microarray experiments. Stat Appl Genet Mol Biol 2004;3:Article3.
11.
Gresack JE, Frick KM. Effects of continuous and intermittent estrogen treatments on memory
in aging female mice. Brain Res 2006;1115:135-47.
8
12.
Antoons G, Mubagwa K, Nevelsteen I, Sipido KR. Mechanisms underlying the frequency
dependence of contraction and [Ca(2+)](i) transients in mouse ventricular myocytes. J Physiol
2002;543:889-98.
9