SI Materials and Methods
All experiments were carried out in accordance with the United Kingdom Home Office Animals
(Scientific Procedures) Act 1986, which conforms to the Guide for the Care
and Use of
Laboratory Animals published by the US National Institutes of Health (NIH publication No. 8523, revised 1996). Ethical approvals were granted by the Home Office for the animal study
(70/7399) and by the National Researcher Ethics for the human study (11/LO/1874) which was
performed to conform with the declaration of Helsinki. Informed consent was obtained for the
human samples. Human Research Ethics was approved by the University of Sydney (#2012/2814).
Human ventricular tissue and CM isolation
Human ventricular tissue was obtained from explanted hearts of patients with dilated
cardiomyopathy (DCM) at the time of transplant or from unused donor hearts (13). Failing human
ventricular tissue was transported in cold cardioplegic solution (Plegivex solution) within 1-2h
after the explants. Pieces of ventricles (100 mg- 3 g) were chopped quickly then incubated in a low
calcium medium and stirred by bubbling with 100% O2. The low calcium medium was removed by
straining with 300µm gauze. The chunks were then incubated at 350C for 45min in the above
solution with nitrilotriacetic acid omitted and 4U/ml (Sigma) type XXIV protease (pronase) and
30µM calcium added, followed with the protease omitted and 400IU/ml (BCL) collagenase added.
The medium was shaken gently throughout the incubation, and kept under an atmosphere of 100%
O2. At the end, the solution containing the dispersed cells was filtered through a 300µm gauze and
centrifuged at 300g for 1 min. The cells were washed twice by centrifugation in Krebs-Henseleit
medium with 95% O2, 5% CO2, and resuspended in the same medium. Cells were then used
immediately (25).
Adult rat ventricular tissue, post-MI-HF assessment and CM isolation
Rat HF Model: Adult male Sprague-Dawley rats (250–300 g) (n = 5) underwent proximal coronary
ligation.
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First, animals were anaesthetised using isoflurane 5%, reducing to 1.5% once intubated and
ventilated. Preoperative medication was administered as follows: 0.015-0.03mg buprenorphine
(0.05-0.1mg/kg), 1.25-1.5mg enrofloxacin (5mg/kg), and 2.5 ml 0.9% saline subcutaneously (sc).
The rat was connected via the endotracheal tube (16G intravenous cannula) to the Harvard
Ventilator (Harvard Apparatus, Massachusetts), and ventilated using volume-controlled ventilation
at a rate of 90/min, with a tidal volume of 2-2.5ml, giving a minute volume of 180-225ml/min.
After the operation, animals were ventilated until they showed signs of spontaneous movement at
which time they were extubated and placed in an oxygen-enriched, temperature controlled
recovery chamber for at least 2 hours. Post-operative pain management involved repetition of
buprenorphine administration if rats showed signs of distress such as piloerection or limited
mobility. Animals were then maintained for 16 weeks or until signs of heart failure were observed,
following which they were anaesthetized once more in the same way for final echocardiographic
analysis, followed by euthanasia by anaesthetic overdose.
Hearts were explanted at various stages between 4 and 16 weeks following myocardial infarction
(MI). MI was quantified by planimetry of a mid left ventricular (LV) section to ensure adequate
infarction. All MI hearts used had full-thickness scarring of >40% of the LV circumference with
the infarction involving the apex of the heart. Sham ligation was used as control (n = 4). Rats
underwent echocardiography using a Vevo 770 system with B-mode and M-mode imaging to
identify the left ventricular internal diameter (LVID) in systole and diastole at the level of the
papillary muscles. The percentage reduction in LVID during the cardiac cycle is the fractional
shortening (FS) which is a measure of overall cardiac function. There was progressive
deterioration of whole-heart function between 4 and 16 weeks (Fig. S1). Within a week of
echocardiography hearts were explanted and prepared for cell isolation (13). For microinjection, 4,
8, 16 week-groups after MI were used: 4w MI, 8w MI, 16w MI, respectively with their relevant
AMC (AMC 4w, AMC 8w, AMC 16w). Failing and non failing adult rat myocytes were isolated
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using a low-Ca2+ solution (see section adult human CM isolation in Methods), pre-oxygenated
with 100% O2 and collagenase and protease enzymes, as previously described (15).
Cell Microinjection and confocal microscopy
Adult CM on laminin-coated mateck dishes were transferred to a low-Ca2+ solution 370C (see
section adult human cardiomyocyte isolation in Methods), supplemented with 0.5% BSA, and 20
mmol/L 2,3-butanedione monoxime (BDM) used to transiently block contractions during
microinjection as it acts as an inhibitor of crossbridge interaction of the contractile cytoskeletal
apparatus (19). The microinjection is performed under a Leica SP5 Resonant inverted confocal
microscope (FILM facility). Microinjection of the cells occurs when a glass capillary, prefilled
with an appropriate solution impale the cells and a gentle pressure is applied to allow the injectants
to flow out of the needle. By use of glass capillaries with thin walls and small tips, mechanical
damage to the cell membrane can be reduced. For our experiments we used pre-pulled, sterile glass
capillaries with 0.5 ± 0.2 μm tip diameter (Eppendorf). We load the microcapillaries (Femtotips)
using sterile microloaders by avoid the formation of air bubbles that can clog the capillary (19).
Glass capillaries were filled with a fluorescent substrate, the ALEXA-BSA NLS import fluorescent
substrate, prepared in our lab but designed by the “Grant N Pierce” Laboratory by conjugating
BODIPY-BSA to a SV40 large T antigen NLS (CGGGPKKKRKVED) (7,9,20-22). The glass
capillaries were attached to a nanometerprecision microinjector (Eppendorf 5242) coupled to a
micromanipulator (Eppendorf 5171) set at a compensation pressure of 40hPa, an injection pressure
of 225hPa and an injection time of 0.7sec (19). Images were taken of pre- and post-injection cells
followed by set time points to observe the rate of nuclear import (represented as a ratio nucleus/
cytoplasm) for each cell over time (up to 15min). The signal strength in the nucleus is proportional
to the amount of substrate present in the nucleus. Transport of the import protein into the nucleus
from the cytoplasm was analyzed on a Leica LAS AF confocal (FILM facility) and the ImageJ
softwares. For nuclear fluorescence, the value was obtained from both total nuclear areas. An
average of the nuclear fluorescence was then calculated. For cytosolic fluorescence, an area that
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surrounded each nucleus and equivalent to the nuclear area was used. Again an average of
cytosolic fluorescence of both areas was calculated. The ratio (nuclear fluorescence
(N)/cytoplasmic fluorescence (C)) was further calculated from these values.
Immunocyto- and immunohistochemistry:
Cells and cryosections were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X100 (Sigma). Then only frozen sections were incubated with L.A.B. Solution (Polysciences) for 10
min to enable the liberation of primary antibody binding sites (23). Cells and cryosections were
blocked with 0.1% Triton X-100 (Sigma) and 5% normal goat serum in PBS for 1 h at room
temperature. They were then labelled with primary antibodies directed against the nucleoporin Nup
62, (p62, BD, 1:200 dilution), transport receptors Importin-α (Imp α, BD, 1:100 dilution),
Importin-β (Imp β, Abcam, 1:100 dilution), Exportin-1 (CRM1, Abcam, 1;200), and transport
driving force protein (RanBP1, Abcam, 1:200 dilution), overnight at 4oC. Primary antibodies were
detected with Alexa 488- (Invitrogen) and Alexa 546- (Invitrogen) conjugated secondary
antibodies (all 1:400). DNA was visualised with DAPI (0.5 μg/ml; Sigma). The sections were
enclosed by mounting agent (ProLong Gold antifade reagent, Molecular Probes). The
immunostained sections and cells were observed and photographed with a Leica SP5 inverted
confocal microscope (FILM facility). Signal intensity by each antibody was measured by analysis
on the ImageJ software.
Nuclei isolation
Adult rat CM nuclei were isolated as published previously with some modifications (39). At the
end of the treatment, after several washes with cold PBS 1X, CM were suspended in a Hypoosmotic buffer (10 mM Tris, 1 mM MgCl2, 10 mM NaCl, 5mM CaCl2, adjusted to pH 7.4 with
HCl) for 30 min at 4°C. Following sedimentation at 1000 x g cells were resuspended in hypoosmotic buffer and sonicated in the buffer solution containing triton X-100. This was then
centrifuged at 1000 x g at 4oC. The supernatant (Layer 1) containing the membrane and
cytoplasmic fractions was kept on ice for subsequent protein dosage and immunoblotting
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experiments. The resulting pellet was resuspended in MC buffer (2.2 M sucrose, 10 mM Tris, 1
mM MgCl2, 0.1 mM phenylmethylsulfonyl fluoride, adjusted to pH 7.4 with HCl). This
suspension was transferred to high resistance centrifugation tubes underlain with 1 ml of MC
buffer and centrifuged at 100,000x g for 60 min (Sorvall Discovery Ultracentrifuge M120 SE:
rotor: S100-AT6). The pellet containing the nuclear fraction (Layer 2) was resuspended in Buffer
X (20 mM Na- HEPES, 25% (volume/ volume) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM
EDTA, 0.2 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 25 µg/ml of
leupeptin, 0.2 mM Na3VO4, with a final pH of 7.4 (NaOH). The supernatant was added to layer 1.
Both layers 1 and 2 were used freshly or aliquoted, snap frozen with liquid nitrogen, and stored at 80 °C.
Protein Quantification and Western Blot Analysis
Cultured adult rat CM and isolated nuclei were homogenized in a buffer containing 150mM
Sodium Chloride, 1% Triton-X 100, 0.5% Sodium Deoxycholate, 0.1% SDS, 50mM Tris pH 8.0,
1mM NaF, and protease inhibitors (Sigma P8340). Protein quantification was measured using the
BioRad quantification assay (BioRad Laboratories, CA) with absorbance spectrophotometry. For
SDS-PAGE analysis, lysates were diluted in Laemmeli buffer and electrophoresed on a 15% (w/v)
acrylamide running gels with 3.5% (w/v) stacking gels and separated proteins transferred onto
activated nitrocellulose or PDVF membranes. Membranes were placed in blocking buffer then
incubated with primary antibodies to Nup 62 1:1000 (610498, BD Transduction Lab), Importin-α
1:2500 dilution (610486, BD Transduction Lab), Importin-β 2µg/ml dilution (Ab45938, Abcam),
Exportin- 1 CRM1 2µg/ml (Ab24189, Abcam), transport driving force protein RanBP1 1:1000
(Ab97659, Abcam) in TBS-Tween and 1% skimmed milk). GAPDH antibody (SC32233 Santa
Cruz Biotechnology) was used as a loading control for cytoplasmic extracts and Histone H3 (9715
Cell signalling) as a loading control for nuclear extracts (39). HRP-conjugated anti-mouse IgG and
anti-rabbit IgG (Dako) were used as secondary antibodies. To ensure equal protein loading, and for
comparing loading between nuclear and cytoplasmic extracts, blots were stripped and stained using
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Ponceau S (39) or gels were stained with Coomassie blue. Band densities were quantified on a
Gene Genius bio-imaging system (Syngene) using ECL detection reagents (GE Healthcare) (24).
Isolation of RNA
Cultured Adult rat CM and human LV tissue (healthy or DCM) were lysed in RLT buffer for total
RNA extraction as per manufacturer's protocol (Qiagen, CA) as reported previously (16). The
RNA was purified using RNeasy columns (Qiagen, CA), quantified, and checked for quality on a
denaturing 1% agarose gel. To generate double stranded cDNA, 1 μg of total RNA was used for
RT2 First Strand Kit (SABiosciences, MD) or High Capacity cDNA Reverse Transcription Kit
(Applied Biosystems, CA), according to published protocols.
Quantitative RT-PCR and PCR array
For quantifying mRNA levels of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP),
and p62, real-time PCR analyses were performed with TaqMan Gene Expression Assays (Rat:
Rn00561661_m1, Rn00580641_m1, Rn02586441_s1, respectively; Human: Hs00383230_g1,
Hs00173590_m1, Hs02621445_s1, respectively, Applied Biosystems, CA). GAPDH Endogenous
Control (FAM / MGB probe) (Rn01775763_g1 and Hs03929097_g1) was used as a housekeeping
control. For PCR array, the cDNA was hybridized in a 96-well format against the Gene Array
PAHS-018 with RT2 qPCR Master Mix which contained SYBR green dye (RT2 Profiler™ PCR
Array System, SABiosciences) as per the manufacturer's instructions (16). The PCR was
performed with ABI 5700 (Applied Biosystems) and Rotor-Gene 3000 (Corbett Research) realtime PCR instruments and by ΔΔCt method in which fold increase = 2-ΔΔCt was used to determine
the relative expression (16).
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Supplemental Figure Legends
Fig. S1: Remodelling of the rat heart over time following myocardial infarction (MI). a.
Fractional Shortening (FS); b. Left ventricular end diastolic dimension; c. left ventricular posterior
wall thickness in diastole; d. Interventricular septal width in diastole; e. Heart weight to tibial
length ratio measured by echocardiography from AMC (4 weeks and 16 weeks) rats and 4 weeks
after MI and 16 weeks after MI rats. Results are mean ± SEM (n=3- 5); Two-way ANOVA
followed by a Bonferroni post-hoc test for multiple comparisons (effects of MI and/or age) *
p<0.05, *** p<0.001.
Fig S2: Cellular remodelling in the rat heart following myocardial infarction (MI). Length,
width (µm) and area (µm2) of freshly isolated ventricular CM from AMC rats (4W, n=75 cells;
8W, n=51; 16W, n=427) and MI rat CM (4W, n=60 cells; 8W, n=187; 16W, n=680). Mean ±SD,
***P<0.001.
Fig S3: Nuclear remodelling in the rat heart following myocardial infarction (MI). Increase
in nuclear area (µm2) in adult rat ventricular CM, 4, 8, and 16 weeks after MI compared to agedmatched controls (AMC 4w and AMC 12w). Mean ± SEM (n= 3 preparations, 32 cells per
condition); *** p<0.001.
Fig. S4: Increase in ANF mRNA levels in PE-treated adult rat CM. Failing rat and human
CM and ventricular tissue have similarly increased levels. (A) and (B) Bar graphs showing
ANF mRNA levels in healthy AMC 16w rats CM and MI 16w rat CM and failing human CM, as
well as in rat control CM (CT 8w) exposed to PE (PE 48h 8w) in the absence (A) or presence for
48h of SB, TSA, LMB,AZA (C). Results are normalised to GAPDH and expressed in fold changes
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via Control ; mean ± SEM of triplicate wells (n=3); NS: Non significant; ♦p<0.05, ** and
♦♦p<0.01, *** and ♦♦♦ p<0.001; ♦vs CT.
Fig. S5: Increase in BNP mRNA levels in PE-treated adult rat CM. Failing rat and human
CM and ventricular tissue have similar increased levels. Bar graphs showing BNP mRNA
levels in healthy and failing rat and human CM and in rat CM exposed to PE. Results are
expressed in fold changes via Control group and as a ratio of mRNA to GAPDH and shown as
mean ± SEM of triplicate wells (n=3); ♦♦p<0.01, **** and ♦♦♦ p<0.001; ♦vs CT.
Fig. S6. Decrease relative density of Nucleoporin p62 and the nucleocytoplasmic trafficking
of transport receptors (imp-α, imp-β) and transport driving force protein (RanBP1) in
Failing rat ventricular tissue (VT); (A) Bar graphs showing mean fluorescence levels
(%Control) of p62 levels in 16w AMC (white bar) and 16w MI: rICM (grey bar) rat VT. (n=4);
(B) (C) Bar graphs showing the ratio Nuclear/Cytoplasmic (N/C) mean fluorescence intensity
(%Control) of Importin- β (B) and the ratio Cytoplasmic/Nuclear (C/N) mean fluorescent intensity
of RanBP1 (C) in 16w AMC (white bar) and 16w MI: rICM (grey bar) rat VT. Results are shown
as mean ± SEM (n=4 hearts);
* p<0.05 and ** p<0.01.
Fig. S7: Absence of effect of p38MAPK, HDAC CL II, CRM1, and GSK-3β pathways on the
expression of CRM1 export in adult rat CM. Analysis by densitometry of western blots
(%Control) of CRM1 expression in the nuclear and cytosolic fractions of rat adult control CM (CT
8w) in the presence of LMB (CT 8w LMB) SB (CT 8w SB), TSA (CT 8w TSA), AZA (CT 8w
AZA); All results are shown as mean ± SEM.
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Supplemental References.
39. Tadevosyan A, et al. (2010) Nuclear-delimited angiotensin receptor mediated
signaling regulates cardiomyocyte gene expression. J Biol Chem 285(29): 22338-49.
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