CVR-2013-913R2
Supplementary material
Supplemental figure legend
Figure S1. Female WT-mice show higher running performance and higher cardiac
response after voluntary cage wheel running compared to males. A, Average daily
running speed and B running time of female WT- and ER-/--mice is significantly higher
compared with male mice. In contrast female ER-/--mice show significant lower daily running
speed and time compared with male counterparts and female WT-mice. C, Female WT-mice
show significant higher percent of increase in LVM/TL and D, cardiomyocyte diameter
compared with males after exercise. WT-mice: n= 20-22 per group; for cardiomyocyte
diameter: n=7-8 per group; ER-deleted mice: n= 9-12 per group. 2-way ANOVA: *p<0.05,
**p<0.01 and ***p<0.001. unpaired t-test:
§
p≤0.05. WT: wild-type mice; ER-/-/ER -/-:
estrogen receptor alpha/beta deletion.
Figure S2. No induction of the fetal gene programme and collagen content in WT-mice
after VCR. A, Relative NPPA-, B NPPB-mRNA expression and C collagen content in left
ventricular tissue of female and male sed and VCR WT-mice. D, Representative Sirius red
staining of left ventricular tissue; x20 magnification; scale bar: 100µm. Number of mice: n=
16-21 per group for gene expression; n= 5-8 for collagen content. WT: wild-type mice; VCR:
voluntary cage wheel running; sed: sedentary.
Figure S3: MAPK-ERK1/2, AMPK, GSK-3 and p70s6 kinase and s6 in WT-mice are not
induced after 8 weeks of VCR. A, p-ERK1/2 is significantly higher in female sed- and VCRmice compared to male counterparts, but not changed after VCR. B, p-AMPK, C p-GSK-3,
D p-p70s6 kinase and E p-s6 are not induced by 8 weeks of VCR in WT-mice. Number of
mice: n= 6-11 per group and sex. 2-way ANOVA: *p<0.05 and **p<0.01. WT: wild-type mice;
VCR: voluntary cage wheel running; sed: sedentary.
Figure S4: E2 and activated ER increase expression of mitochondrial key regulators
in AC16 cells. MEF2A, NRF-1 and -2 mRNA-expressions are significantly up-regulated in
AC16 cells after 12h E2- and ERβ-agonist treatment. HPRT was used as internal standard.
CVR-2013-913R2
Relative mRNA-expression levels of E2- and ERβ-agonist treated cells are expressed as fold
induction of vehicle-treated cells. n= 3-4, carried out in triplicates; 1-way ANOVA: *p<0.05
and **p<0.01.
Figure S5: ERα and ERβ expression display no sex differences and are not modulated
by VCR. A-B, ERβ and C-D, ERα mRNA and protein expression and representative western
blot images for E, ERβ and F, ERα in mouse left ventricular tissue. mRNA: n=5-6 and
protein: 5-11 per sex and group. VCR: voluntary cage wheel running; sed: sedentary.
CVR-2013-913R2
Supplemental tables
Table S1: Primers used for Real-Time polymerase chain reaction.
Species
Gene
Sequence (5`to 3`)
Mouse
Hprt
FW: GCT TTC CCT GGT TAA GCA GTA CA
RV: ACA CTT CGA GAG GTC CTT TTC AC
Nppa
FW: GGG GGT AGG ATT GAC AGG AT
RV: ACA CAC CAC AAG GGC TTA GG
Nppb
FW: GCA CAA GAT AGA CCG GAT CG
RV: CTT CAA AGG TGG TCC CAG AG
Mef2a
FW: AGA GTT TTC TGC AAG AAT CAA ACA
RV: TCC AAT CTC CTA ATG CAT CGT
Atp5k
FW: TCA GGT CTC TCC ACT CAT AAA GT
RV: TTC TTT TCC TCC GCT GCT AT
Human
HPRT
FW: TGT TGT AGG ATA TGC CCT TGA CT
RV: GGC TTT GTA TTT TGC TTT TCC A
NRF-1
FW: GGC ACT GTC TCA CTT ATC CAG GTT
RV: CAG CCA CGG CAG AAT AAT TCA
NRF-2
FW: CAG CCT GAA CTG GTT GCA CAG AAA
RV: TCA ACT CCG CTG CAC TGT ATC CAA
MEF2A
FW: GCT TCA ACT CGC CAG GAA T
RV: GAT AAC TGC CCT CCA GCA AC
CVR-2013-913R2
Table S2: Echocardiographic parameters and cardiomyocyte diameter of ER-/--mice 8
weeks after VCR.
ERα-/--sed
sex
ERα-/--VCR
Male
Female
Male
Female
11
9
9
9
LVM (mg)
89.48±2.4
80.37±2.1§
97.39±2.7*
79.84±1.8§§§
LVM/TL (mg/mm)
5.18±0.08
4.56±0.13§§§
5.63±0.12**
4.66±0.09§§§
IVSd (mm)
0.63±0.01
0.62±0.01
0.66±0.01
0.64±0.01
Pwd (mm)
0.61±0.01
0.62±0.01
0.66±0.01*
0.62±0.01
LVIDd (mm)
4.16±0.08
3.90± 0.08
4.19±0.10
3.84±0.07§
EF (%)
51.7±1.9
58.6±2.9
54.0±2.6
58.7±2.3
508.3±13.8
502.8±17.0
490.6±12.7
491.8±10.5
11.19±0.25
12.19±0.18
11.69±0.33
12.01±0.48
n
HR (beats/min)
Cardiomyocyte
(µm)*
diameter
Values show means±SEM. All measurements were performed after 8 weeks of VCR. N:
number of mice in each group, *N for cardiomyocyte diameter: 5-9; ERα-/-: estrogen receptor
alpha deleted mice, sed: sedentary, VCR: voluntary cage wheel running, TL: tibia length,
LVM: left ventricular mass, IVSd: septum thickness during diastole, PWd: posterior wall
thickness during diastole, LVIDd: left ventricular internal diameter during diastole, HR: heart
rate and EF: ejection fraction. 2-way ANOVA: (*) intervention (sed vs. VCR) *p≤0.05 and
**
p≤0.01; (§) sex (female vs. male) §§§p≤0.001 and §p≤0.05.
CVR-2013-913R2
Table S3: Running performance and echocardiographic parameters of male and
female WT-mice after 1 week of VCR.
WT-sed
sex
WT-VCR
Male
Female
Male
Female
6
6
7
7
4.24±0.3
8.18±0.9§§§
Running speed (km/h/24h)
1.03±0.0
1.4±0.1§§§
Running time (h/24h)
3.53±0.5
5.16±0.7
n
Running
distance
(km/24h)
LVM (mg)
114.2±3.1
86.95±2.0§§§
107.3±3.0
89.29±2.7§§§
LVM/TL (mg/mm)
6.76±0.2
5.30±0.1§§§
6.24±0.2
5.48±0.2§§
IVSd (mm)
0.62±0.0
0.58±0.0§§
0.62±0.0
0.60±0.0
Pwd (mm)
0.61±0.0
0.56±0.0§§
0.62±0.0
0.59±0.0§
LVIDd (mm)
4.81±0.1
4.34±0.1§§§
4.6±0.0
4.28±0.1§
EF (%)
42.9±2.8
43.4±1.8
39.4±1.2
50.3±3.7§
427.7±14.8
444.7±17.8
429.3±17.6
421.7±13.3
HR (beat/min)
Values show means±SEM. All measurements were performed after 1 week of VCR. N:
number of mice in each group; WT: wild-type mice, sed: sedentary, VCR: voluntary cage
wheel running, TL: tibia length, LVM: left ventricular mass, IVSd: septum thickness during
diastole, PWd: posterior wall thickness during diastole, LVIDd: left ventricular internal
diameter during diastole, HR: heart rate and EF: ejection fraction. 2-way ANOVA: (§) sex
(female vs. male) §§§p≤0.001, §§p≤0.01 and §p≤0.05.
CVR-2013-913R2
Supplemental methods:
Monitoring and evaluation of daily running distance. Each VCR mouse was kept in an
individual cage supplied with a running wheel (11.5 cm diameter) and equipped with a
tachometer (ML Solution GmbH, Germany) recording daily running distance, time and speed,
which then were averaged over the 1- and 8-week time period.
Echocardiography. Mice were anesthetized with isoflurane (1.5%) and measurements were
carried out using a Vevo 770 High-Resolution Imaging system with an RMV 707 Scanhead
(Visual Sonics). MH was assessed by the ratio of left ventricular mass (LVM) to tibia length
(TL). To better compare the magnitude of cardiac mass adaptation between sexes in WTmice, increase of LVM/TL compared to each respective sed group was calculated and are
given as percent of increase.
Histology. For histology, LV was paraffin-embedded and cut in 2µm sections.
Hematoxylin/eosin staining was carried out to determine cardiomyocyte diameter. At least
ten pictures with an x20 magnification of each section were taken (AxioStar, Zeiss) and the
diameter of at least 100 cardiomyocytes was measured using AxioVision Rel.4.6 (Zeiss).
Sirius Red staining was used to obtain collagen content. At least ten pictures of each section
with an x20 magnification were taken (AxioStar, Zeiss) and quantified using ImageJ 1.38 &
software. Collagen content was calculated: [collagen area (red)/total section area (yellow)] x
100.
Western blot. Used primary antibodies are as followed: phospho-AKT1/2/3 (Ser 473-R,
Santa Cruz); AKT1/2/3 (H-136, Santa Cruz); phospho-p38-MAPK (Thr180/Tyr182) (#4511,
Cell Signaling Technology); p38-MAPK (#9212, Cell Signaling); phospho-p44/p42 ERK1/2
(Thr202/Tyr204) (#4370, Cell Signaling Technology); p44/p42 ERK1/2 (#4695, Cell Signaling
Technology); phospho-s6 ribosomal protein (Ser240/244) (#5364, Cell Signaling technology);
NRF-1 (ab34682, abcam); NRF-2 (21542-1-AP, proteintech); MitoProfile® total OXPHOS
Rodent WB Antibody Cocktail (#MS604, MitoSciences); Mfn-2 (#9482, Cell Signaling
Technology); phospho-AMPK (Thr172) (#2535, Cell Signaling Technology); AMPK (#2532,
Cell Signaling Technology); phospho-GSK-3β (Ser9) (#5558, Cell Signaling Technology);
CVR-2013-913R2
GSK-3β (#9315, Cell Signaling Technology); phospho-p70s6k (T389) (AF8963, R&D
systems) p70s6k (AF8962, R&D systems); ERα (G-20, Santa Cruz); ERβ (1531, Santa
Cruz); α-tubulin (clone DM1A, Sigma) and GAPDH (#MAB374, Millipore).
Transmission electron microscopy. Mitochondrial area and number evaluations were
accomplished by taking Transmission electron microscopy-micrographs from different areas
and estimated using AxioVision Rel.4.6 (Zeiss). Five to ten micrographs were taken for each
heart (n=4-6 per group and sex) to measure mitochondrial number (x6.000 magnification) or
mitochondrial area (x16.700 magnification) per unit area in a blinded fashion. To determine
the effects of treatment, sex and genotype on mitochondrial size, mitochondrial profiles were
categorized as small (≤0.5µm2), medium (0.5-1µm2) and large (≥1µm2) as described
elsewhere.59
Supplemental references:
59.
Sohal RS, Bridges RG. Associated changes in the size and number of mitochondria
present in the midgut of the larvae of the housefly, Musca domestica and phospholipid
composition of the larvae. J Cell Sci 1978;34:65-79.