Supplementary Material
Supplementary Methods
Study design.
Four-to-six-week-old wild-type C57BL6/J mice were injected with 3.5x1010 viral
genomes (vg) encoding Luc, wild-type human PKP2, or the C-terminal deletion mutant
version R735X. Animals were divided into two groups: group A = trained, and group B
= sedentary. Training in group A started 2 weeks after injection and continued for 8
consecutive weeks. At the end of that period, Luc, PKP2 and R735X mice were imaged
by Cardiac Magnetic Resonance (CMR) and euthanized for heart sampling. Animals in
group B were analysed by CMR 6 and 10 months after infection with AAV particles.
Animals and endurance training protocol.
All animals were maintained and handled according to the recommendations of the
CNIC Institutional Ethics Committee. See Supplemental Methods for animal
procedures.
Wild-type 4-5-week-old male C57BL/6J mice were obtained from Charles River
Laboratories. Mice were individually housed in wire-bottomed cages in a temperaturecontrolled room (22±0.8°C) with a 12 h light–dark cycle and a relative humidity of
55±10%. The mice had free access to food and water.
All mice were adapted to water before beginning the eight-week endurance swimming
training program. Adaptation consisted of keeping the animals in shallow water at 30 to
32ºC, with the purpose of reducing the environmental stress without promoting any
physical training adaptations. Endurance training took place during the dark phase of
the light–dark cycle to coincide with the period of animals’ maximum activity, and
training intensity was increased gradually. Mice swam in water at a depth of 25 cm in a
glass container (60×30×45 cm) at 30 to 32ºC. The animals were progressively
familiarized with swimming during the first 2 weeks by increasing the swimming time
by 5 min per day, reaching 45 min per day at the end of the second week. Daily
swimming duration was then increased by 15 min each week, and held at 90 min per
day during the final 3 weeks. Mice were allowed to swim at their own pace, and the
water was gently bubbled to ensure that mice swam rather than floating.
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End
Random analysis of distance swum revealed a Gaussian distribution with a coefficient
of variation of 8.4% (Supplementary Figure 6), confirming homogeneous training
intensity across groups.
CMR imaging.
During CMR evaluation, animals were anaesthetized with isoflurane and were
monitored for core body temperature, cardiac rhythm and respiration rate using a CMR
compatible monitoring system. In vivo cardiac images were acquired using an Agilent
VNMRS DD1 7T MRI system (Santa Clara, California, USA). A k-space segmented
ECG-triggered cine gradient-echo sequence was used. After shimming optimization,
cardiac four-chamber and left two-chamber views were acquired and used to plan the
short axis sequence. Mice were imaged with the following parameter settings: number
of slices, 13; slice thickness, 0.8 mm; gap, 0 mm; matrix size, 256x256; field of view,
30x30 mm2; gating: ECG and respiratory triggered; cardiac phases, 20; averages, 4;
effectiveTE, ~1.8 ms, minimumTR, 7 ms; flip angle, 25º; trigger delay, 2 ms; trigger
window, 8 ms; dummy scans, 2.
CMR data analysis.
All CMR images were analysed using dedicated software (QMass MR v.7.5, Medis,
Leiden, the Netherlands). Images were analysed by two experienced observers blinded
to the study allocation. All CMR images were of good quality and could be analysed.
The short-axis data set was analysed quantitatively by manual detection of endocardial
borders in end-diastole and end-systole, with exclusion of papillary muscles and
trabeculae, in order to obtain both left and right end-diastolic volume, end-systolic
volume and ejection fraction.
The right ventricle was assessed qualitatively by evaluating the wall motion in the cine
modus in short- and long-axis views. The right ventricle was divided into 11
segments(1,2). Wall motion was classified as abnormal in the presence of akinesia,
dyskinesia (in ventricular systole), or bulging (in ventricular diastole).
Surface ECG. Mice were anaesthetized using isoflurane inhalation (0.8-1.0% volume in
oxygen), and efficacy of the anaesthesia was monitored by watching breathing speed.
Four-lead surface ECGs were recorded, for a period of 5 minutes, from subcutaneous
23-gauge needle electrodes attached to each limb using the MP36R amplificator unit
(BIOPAC Systems).
During offline analysis, lead II was used for QRS duration using AcqKnowledge 4.1
analysis software. A representative 30s segment of the recording was averaged to obtain
the signal-averaged ECG. Then, QRS duration was measured as the time interval
between the earliest moment of deviation from baseline and the moment when the Swave returned to the isoelectric line.
QRS duration from endurance-trained R735X-infected mice was compared to those of
contemporaneous animals infected with wild type PKP2 and Luc pooled together.
Similarly, QRS duration from sedentary AAV-R735X mice at 10 months (long-term)
was compared to those of the 2 groups of controls pooled together.
AAV vector production and purification. AAV vectors were all produced by the triple
transfection method, using HEK 293A cells as described previously(3). AAV plasmids
were cloned and propagated in the Stbl3 E. coli strain (Life Technologies). Shuttle
plasmids pAAV-PKP2, pAAV-R735X and pAAV-Luc were derived from pAcTnT (a
gift from Dr B.A. French) and packaged into AAV-9 capsids with the use of helper
plasmids pAdDF6 (providing the three adenoviral helper genes) and pAAV2/9
(providing rep and cap viral genes), obtained from PennVector. Shuttle vectors were
generated by direct cloning (GeneScript) of synthesized fragments from NheI-SalI into
pAcTnT cut with the same restriction enzymes.
The AAV shuttle and helper plasmids were transfected into HEK 293A cells by
calcium-phosphate co-precipitation. A total of 840µg plasmid DNA (mixed in an
equimolar ratio) was used per Hyperflask (Corning) seeded with 1.2x108 cells the day
before. Seventy-two hours after transfection, the cells were collected by centrifugation
and the cell pellet was resuspended in TMS (50 mM Tris HCl, 150 mM NaCl, 2 mM
Cl2Mg) on ice before digestion with DNase I and RNaseA (0.1 mg/mL each; Roche) at
37 °C for 60 minutes. Clarified supernatant containing the viral particles was obtained
by iodixanol gradient centrifugation(4). Gradient fractions containing virus were
concentrated using Amicon UltraCel columns (Millipore) and stored at -70ºC.
Determination of AAV vector titer. Titers for the AAV vectors (vg per ml) were
determined by quantitative real-time PCR as described(5). See primers used below.
Known copy numbers (105–108) of the respective plasmid (pAAV-PKP2, pAAVR735X and pAAV-Luc) carrying the appropriate complementary DNA were used to
construct standard curves.
Quantitative real-time PCR. For mouse tissue analysis, total RNA was isolated from
mouse hearts and liver using the Direct-zol RNA Miniprep Kit (Zymo) and reverse
transcribed with the High Capacity cDNA Reverse Transcription Kit (Applied
Biosystems). Complementary DNAs were analyzed by real time PCR using the Power
SYBR® Green PCR Master Mix (Applied Biosystems). Amplification, detection and
data analysis were completed with the ABI PRISM® 7900HT Sequence Detection
System. The crossing threshold values for individual mRNAs were normalized to
GADPH. Changes in the expression of mRNA were expressed as the fold change
relative to the control. We used the following primers: PKP2 F, 5′
AGAGCAGCAGCACAGTACAG3′; PKP2 R,
5’ CGCCAGGCATACTGGTATTC3′; GapdhF, 5′ TTGATGGCAACAATCTCCAC 3′;
GapdhR, 5′ CGTCCCGTAGACAAAATGGT 3′; Pkp2 F, 5’
CAGAACAGGGCTTCCAGGTC 3’; Pkp2R, 5’ CTGCTCGCTCCAGAGTCATC3’;
eGFPF,5’ AGCTGGACGGCGACGTAAAC 3’; eGFPR 5’
AAGATGGTGCGCTCCTGGAC 3’
Immunofluorescence
At the end of exercise training, hearts were collected and fixed in 2% paraformaldehyde
in phosphate-buffered saline (PBS) for 1 hour at 4°C. After washing three times in PBS
for 5 min, hearts were equilibrated with 15% sucrose in PBS for 1 hour and incubated in
30% sucrose overnight at 4°C. Samples were included in OCT (Tissue-tek OCT
compound, Sakura-4583), and 5μm cryosections were prepared for
immunofluorescence. Tissues were permeabilized with PBS-0.2% Triton (Sigma,
T9284) for 10 min, and washed in PBS-0.1% Triton (PBST). Tissues were blocked with
10% goat serum in PBST for 1 hour. Primary antibodies against plakoglobin (610253,
BD) and PKP2 (651167, Progen) required antigen retrieval. Antibodies against
sarcomeric cardiac α-actinin (A7732, Sigma), Cx43 (C6219, Sigma), and eGFP
(632592, Clonthech) were diluted in PBST, and slides were incubated with antibodies
overnight at 4°C. After washes, slides were incubated with Cy3-conjugated or Alexa
Fluor 488-conjugated goat anti-mouse secondary antibodies for 45 min in the presence
of DAPI. Vectashield mounting medium without DAPI (Vectors Lab.; H-1000) was
applied to all slides. Fluorescence images were obtained with a Nikon A1-R inverted
confocal microscope. The percentage of positive cells in the total tissue area was
calculated using ImageJ after segmentation of the cells and measurement of signal
intensities.
Transmission electron microscopy
Immediately after excision, tissues were fixed in 4% formahaldehyde: 1%
glutaraldehyde in cacodylate buffer, and postfixed in 1% osmium tetroxide. Tissues
were then washed in PBS, dehydrated through graded alcohols followed by acetone, and
then infiltrated with Durcupan ACM Fluka resin and polymerized at 60 ºC for 48h.
Blocks were cut with a Leica ultracut UCT ultramicrotome (Leica, Heerbrugg,
Switzerland) and sections (60–70 nm) were mounted onto 200-mesh grids. Sections
were stained with a 2% solution of aqueous uranyl acetate for 10 min, followed by lead
citrate staining for 10 min. Stained sections were viewed with a JEOL JEM-1010
transmission electron microscope (Tokyo, Japan) operating at 100 kV. Images were
acquired with a GATAN (Orius 200SC) digital camera.
Statistics.
Experiments were designed to use the minimum number of mice needed to give
sufficient statistical power, and the numbers of animals used are comparable to
published literature for the same assays(6). No animals were excluded from the
analyses. Data were analysed by one-way ANOVA, two-way ANOVA and Student’s t
test. Relative risk analysis was assessed by two-tailed Fisher’s exact test. Reliability of
CMR measures was assessed by inter-observer intraclass correlation coefficient
(absolute agreement) and mean bias. Error bars represent SEM. In all corresponding
figures, ∗ p< 0.05, ∗∗ p< 0.01, and ∗∗∗ p< 0.001, and ns p> 0.05.
Supplementary Video 1. CMR ventricular short axis (SAX) slices covering from apex
to base in a control mouse expressing wild-type PKP2.
Supplementary Video 2. CMR ventricular short axis (SAX) slices covering from apex
to base in an R735X mutant mouse. Note the dyskinesis in the basal portion of the right
ventricle (RV).
Supplementary Video 3. CMR ventricular short axis (SAX) slices covering from apex
to base in an R735X mutant mouse. Note the severe dyskinesis in the right and left
ventricle.
Supplementary Figure 1. Efficient expression of target genes in heart. qRT-PCR
analysis of total mRNA isolated from heart and liver 10 weeks after AAV infection.
Elevations of EGFP, PKP2 and R735X mRNA expression levels were evident in hearts
relative to the levels in liver.
Supplementary Figure 2. Long-term expression of target genes in heart. qRT-PCR
analysis of total mRNA isolated from heart and liver 10 months after AAV infection.
Elevations of PKP2 and R735X mRNA expression levels were evident in hearts relative
to the levels in liver.
Supplementary Figure 3. Electrocardiographic findings. QRS complex duration in
R735X mice and controls (PKP2 and Luc infected). Panel A shows the results of longterm sedentary mice while panel B corresponds to mice after finishing the endurance
training. ms= miliseconds.
Supplementary Figure 4. Desmosomal structure and protein components in which
mutation is linked to ARVC: Desmoplakin (DSP) ARVC8, Plakophilin-2 (PKP2)
ARVC9, Desmoglein-2 (DSG2) ARVC10, Desmocollin-2 (DSC2) ARVC11, and
Plakoglobin (JUP) ARVC12.
Supplemetary Figure 5. Cx43 mRNA and total protein levels remain constant in
AAV transduced hearts. (A) Hearts were lysed after swimming training, and protein
extract was probed by western blot for Cx43 and tubulin. Each lane represents a lysate
from an individual mouse. (B) Quantitative PCR of hearts infected with AAV
expressing Luciferase, PKP2 and R735X.
Supplementary Figure 6. Uniformity of training intensity. Analysis of distance
swum, calculated from randomly taken time-lapse images, including 1000 consecutive
frames. The data fit a Gaussian distribution (passed DÁgostino & Pearson omnibus
normality test; alpha=0.05).
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