Supplementary Materials for
Canopy 2 Attenuates the Transition from Compensatory Hypertrophy to Dilated Heart
Failure in Hypertrophic Cardiomyopathy
Jian Guo, Anton Mihic, Jun Wu, Yuemei Zhang, Kaustabh Singh, Sanjiv Dhingra, Richard D.
Weisel, Ren-Ke Li
Division of Cardiovascular Surgery and Toronto General Research Institute, University Health
Network and Department of Surgery, Division of Cardiac Surgery, University of Toronto,
Toronto, ON, Canada
Address for correspondence:
Ren-Ke Li, MD, PhD
MaRS Centre, Toronto Medical Discovery Tower, Room 3-702
101 College Street, Toronto, Ontario, Canada, M5G 1L7
Phone: 1-416-581-7492
Fax: 1-416-581-7493
E-mail: renkeli@uhnres.utoronto.ca
Supplementary Methods
Transgenic Mice
All animal studies were approved by the Animal Care Committee of the University
Health Network and were performed in accordance with the “Guide for the Care and Use of
Laboratory Animals, 8th edition” (NIH, revised 2011). We generated cardiac-specific transgenic
(TG) mice that carry human CNPY2 driven by the α-myosin heavy chain (α-MHC) promoter
(Supplementary Figure 1 and Supplementary Tables 1–3). The transgenic construct also
expressed EGFP, and human CNPY2 and endogenous mouse Cnpy2 could be differentiated by
species-specific PCR primers. The final construct was linearized and microinjected into one-cell
C57BL6 embryos that were transferred into the oviduct of pseudopregnant CD1 mice. In total,
six founders were obtained and bred with wild-type C57BL6 mice, and four independent
transgenic lines were used for experiments.
Transverse Aortic Constriction (TAC) model
Pressure overload of the left ventricle (LV) was induced by TAC in wild-type (WT) and
TG mice as previously described with minor modifications (1). Briefly, under general
anaesthesia, the aorta was exposed and ligated between the innominate and left common carotid
arteries by tying a 7-0 silk suture around a 27-gauge needle placed parallel to the aorta. The
needle was then removed, leaving the ligature in place, causing constant and permanent aortic
constriction. The incision was closed and buprenorphine (0.05mg/kg) was administered for
analgesia.
Echocardiography and Cardiac Function
Cardiac systolic function was evaluated by echocardiography prior to (0) and 1, 2, 4, 8
and 12 weeks after TAC. Mice were sedated with a 2% isoflurane (Pharmaceutical Partners of
Canada) nosecone. Echocardiographic examinations were performed using a GE Vivid 7
ultrasound system (GE Healthcare Canada) with an i13L transducer. Depth and frequency were
set at 1cm and 14MHz, respectively. Short-axis views were obtained from the parasternal
approach. LV dimensions [left ventricular end-diastolic internal diameter (LVIDd) and endsystolic internal diameter (LVIDs)] and posterior wall thickness (PWT) were measured in Mmode at short-axis views of the LV at mid-level. Fractional shortening was calculated as follows:
[(LVIDd−LVIDs)/LVIDd]×100.
Cardiac diastolic function was also evaluated using a pressure–volume (P–V) catheter at
1, 4 and 12 weeks after TAC. Briefly, under general anesthesia with 2% isoflurane and positive
pressure ventilation, a calibrated Millar and conductance P–V catheter (Millar Instruments) was
inserted into the LV cavity through the right carotid artery. Real-time P–V loops were
constructed and adjusted for parallel conductance. The diastolic functional parameters dp/dtmin
(maximal rate of LV pressure decrease) and Tau (time constant of LV pressure decay) were
calculated using PVAN 3.3 software (Millar Instruments) as previously described (2).
Histology and immunohistochemistry
The heart morphology of WT and TG mice was evaluated histologically at 4 and 12
weeks after initial surgery. Briefly, hearts were excised and arrested in diastole with 1M KCl
buffer, followed by washing and perfusion fixation with 10% neutral buffered formalin (Sigma).
After 72h, hearts were sectioned into 1mm thick rings and photographed. Myocardial crosssections were embedded in paraffin and 5μm serial sections were cut.
To confirm the expression of CNPY2 and EGFP protein in TG mice, sections were
deparaffinized and rehydrated, and underwent antigen retrieval in 10mM sodium citrate (pH 6.0),
0.05% Tween 20. Samples were boiled in a microwave pressure cooker for 20min, followed by
permeabilization with 0.25% Triton X-100 at room temperature for 5min. Cross-sections were
blocked with 10% normal donkey serum for 20min, and then probed with anti-rabbit CNPY2
(1:100, custom-made polyclonal antibody) and anti-goat GFP (1:100, Santa Cruz Biotechnology
sc-5385) primary antibodies for 2h at room temperature. Secondary antibodies were donkey antirabbit Alexa Fluor 568 (1:400, Life Technologies) and donkey anti-goat Alexa Fluor 647 (1:400,
Life Technologies) for 1h at room temperature. Tissues were also stained with 4',6-diamidino-2phenylindole (DAPI) to identify cell nuclei (1:4000, 5min, Sigma). Whole slide scans were
performed at 20× using a fluorescence slide scanner. GFP signal was obtained in the CY5 far-red
channel as there was noticeable autofluorescence caused by fixation in formalin, but the final
image was pseudo-coloured green. Imaging parameters were the same for all samples including
exposure, gain, threshold and contrast.
Hematoxylin and eosin staining was carried out as previously described (2). Stained
sections were scanned with a whole-slide scanner (Olympus VS-120, Olympus). Comparison of
cross sections was made possible by landmarking with the papillary muscles so that the same
longitudinal plane could be measured. To quantify cardiac morphology, Cellsens software
(Olympus) was used to accurately quantify whole-heart cross-sectional area and circumference.
To assess LV parameters, LV free wall was defined as a 180° portion of the cross section
containing the papillary muscle structures, but not including the wedge-shaped thickening and
bifurcating portion of the myocardium bordering the ventricular septum. This measurement
facilitated the quantification of LV circumference and LV free wall area. To measure LV free
wall thickness, an average of five evenly spaced measurements was taken at 90° to the
endocardial wall, and not through the papillary muscles. As these measurements were simple
representations of cardiac morphology for one anatomical location in fixed hearts, further
measurements were obtained from the working heart through echocardiography at various time
points throughout the study.
Assessment of cardiac vasculature
Capillary and arteriolar structure density was assessed at 4- and 12-weeks post-TAC
using GS-Isolectin-B4 (isolectin) and α-smooth muscle actin (α-SMA) staining, respectively.
Cardiac sections were deparaffinized and rehydrated, followed by permeabilization with 0.25%
Triton X-100 for 5min at room temperature. Tissues were blocked with 10% normal donkey
serum for 20min, and subsequently incubated with 30μg/ml GS-Isolectin-B4-conjugated Alexa
Fluor-568 (Life Technologies) and 1:400 anti-α-SMA (Sigma A2547) for 2h at room
temperature. Slides were then washed and incubated with donkey anti-mouse Alexa Fluor-647
secondary antibody (1:400, 1h, Life Technologies). Nuclei were stained with DAPI (1:4000,
5min). To ensure that samples could be directly compared for quantification, all staining was
carried out simultaneously under identical conditions. Additionally, all microscopy was
performed using the same excitation, exposure, gain and contrast settings.
Imaging was performed using a whole-slide fluorescence scanner (Olympus VS-120) and
images were obtained at 20× in four channels corresponding to DAPI (blue), autofluorescence
(green), isolectin (red) and α-SMA (far-red). For capillary density, isolectin-positive
representative images were obtained for capillaries in the transverse (cross-sectional) and
longitudinal axes. Quantification was carried out using Cellsens software by thresholding the red
channel for signal detected within the LV free wall and expressed as percentage area. The total
number of distinct capillary structures was also quantified and normalized/mm2. Similarly,
arteriolar structures (pseudo-coloured red) were also quantified as a percentage of total LV free
wall area. We manually counted the total number of distinct α-SMA-positive structures
possessing a clearly defined lumen/mm2 of LV free wall. This parameter allowed for the
differentiation of thickening or enlargement of arteriolar structures versus a change in the overall
density in the vasculature.
Cardiac apoptosis
Programmed cell death was assessed using TUNEL, which labels DNA stand breaks,
following the manufacturer’s instructions (Roche). Positive and negative control slides were used
to confirm the sensitivity of the assay and preclude the possibility of false negative signals.
Cardiac sections were deparaffinized and rehydrated as above, but did not undergo antigen
retrieval. Slides were incubated in 5μg/ml Proteinase K working solution at 37°C for 10min,
followed by permeabilization in 0.1% sodium citrate, 0.1% Triton X-100 for 10min at room
temperature. Tissues were labeled with enzyme solution and fluorescein following the
manufacturer’s guidelines for 1h at 37°C in the dark. Nuclei were stained with DAPI so TUNEL
co-localization could be confirmed (1:4000, 5min). The total number of nuclei in the LV free
wall, as well as the total number of co-localized TUNEL-positive nuclei were counted using
Cellsens software (Olympus), and expressed as a percentage at 4 and 12 weeks following TAC.
For caspase-9 and -3 colourimetric assays, 100µg of myocardial tissue lysate was used and
absorbance was measured at OD405nm according to the manufacturer’s instructions (BioVision).
Cardiac fibrosis
Assessment of cardiac fibrosis was performed by staining cardiac cross sections with
picrosirius red (PSR) as reported previously (3). Whole-slide bright field images were assessed
using Cellsens software (Olympus) and RGB thresholding allowed for the specific quantification
of PSR-positive staining. This quantification was only performed for the LV free wall as defined
above. Total PSR-positive area was normalized to the total area of the LV free wall and
expressed as a percentage. We also used circularly polarized light microscopy of the same slides
to visualize the relative abundance of thin (green) and thick (bright yellow/orange) collagen
fibers, a well-characterized birefringence property of collagen. Total collagen (thin + thick)
composition was measured and normalized to total LV free wall area. The ratio of thick/thin
collagen fibers was obtained by thresholding specifically against the red light component of the
polarized images and normalizing it to the green light component.
At day 0–21 following TAC, the activity of myocardial Mmp9 was quantified using
gelatin zymography as described previously (4). Briefly, LV tissues were homogenized in MMP
extraction buffer [1% SDS, 50mM Tris-HCl (pH 7.6), 150mM NaCl, 20mM ZnCl2, 1.5mM
NaN3, and 10mM cacodylic acid]. After centrifugation, 20μg of total protein was added to 5×
sample buffer [10% glycerol, 2% SDS, 0.5% bromophenol blue, 0.5M Tris-HCl (pH 6.8)] and
resolved by 10% SDS-PAGE) containing 0.1% gelatin (Invitrogen). After electrophoresis, the
gels were rinsed twice in 2.5% Triton X-100 renaturing buffer for 30 min, followed by
incubation in developing buffer [50mM Tris (pH 8.8), 5mM CaCl2, 0.02% NaN3] at 37°C for
24h. Gels were stained in Coomassie Brilliant Blue followed by destaining, and gelatinase
activity was identified as clear bands on a blue background. Densitometry of the target bands
was quantified using AlphaImager 2200 software and was expressed as the relative fold change
compared to the WT sham control group.
Western blot and ELISA
Total protein was extracted from mouse hearts or other tissues in lysis buffer [20mM Tris
(pH 7.4), 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton, 2.5mM sodium pyrophosphate,
1mM -glycerolphosphate, 1mM Na3VO4, 1µg/mL leupeptin, 1µg/mL pepstatin, 1mM
phenylmethylsulfonyl fluoride]. The rabbit-anti-CNPY2 polyclonal antibody was raised and
purified as previously described (5). Antibody for CNPY2, p53 or Hif-1α (Abcam) was
incubated with the blots overnight at 4°C. After washing three times for 5min in Tris-buffered
saline, 0.1% Tween-20, the blots were incubated with horseradish peroxidase-conjugated
secondary antibody (1:5000) for 1h at room temperature. Visualization was performed with
enhanced chemiluminescence. For quantification, densitometry of the target bands was divided
by the corresponding densitometry of the Gapdh band using AlphaImager 2200 software. The
values were expressed as the relative fold change of densitometry of gene/Gapdh compared to
the sham control group.
The CNPY2 ELISA kit was purchased from MybioSource. For measurement of secreted
CNPY2 in WT or TG mouse blood, serum was obtained after 2h of clotting and centrifuged at
2000×g for 20min at room temperature. 100µl of serum was used. The absolute amount of
CNPY2 was expressed as pg/mL, according to the manufacturer’s instructions.
RNA extraction, RT-PCR, and real-time PCR
Total RNA was isolated from whole heart tissues from WT or TG mice using Trizol
reagent (Sigma) according to the manufacturer’s instructions. Reverse transcription was
performed using SuperScript III (Invitrogen) at 50°C for 60 min. For PCR amplification, samples
were heated at 95°C for 5min, followed by 30 cycles of 95°C for 30s, 55°C for 30s, 72°C for
1min, and 72°C for an extra 10min at the end of amplification. The RT-PCR products were
separated on a 1.5% agarose gel containing ethidium bromide. Mouse Gapdh served as a loading
control. The sequences of the primers used in RT-PCR are provided in Supplemental Table 5.
Taqman probe-based real-time PCR was performed using a GeneAmp PCR 9600
Thermocycler (Life Technologies) for 40 cycles at 95°C for 15s, 60°C for 1min. Mouse β-actin
was used as the internal loading control. The sequences of the primers and probes used in realtime PCR are provided in Supplemental Table 6. Relative expression was calculated by the
comparative threshold cycle method, and the relative fold change in the experimental group
compared to sham controls was expressed as 2-[ (CT of Gene of Interest-CT of control) in experiment-(CT of Gene of
Interest-CT of control) in sham
.
Statistics
Data are presented as mean±standard deviation (SD). The sample size of 5
mice/experimental group was statistically determined, using an α probability of 0.05, a β of 0.8
and an expected SD of 1.5 (based on previous studies from our group and others, 6–9). The
echocardiographic measures, which evaluated the same animals at different timepoints, were
analyzed by linear regression models adjusted for repeated measures using SAS v9.3 (SAS
Institute). For each outcome, a compound symmetry covariance structure was used to generate
general estimating equations with the maximum likelihood estimate method for parameter
estimation. Two sets of regression models were created, one directly comparing average values
between the 2 groups and one comparing the slope of change over time. All other statistical
analyses were performed using GraphPad Prism 5 (GraphPad). A two-tailed Student’s t-test was
used to compare CNPY2 levels in blood serum in WT and TG mice. One-way ANOVAs
followed by Tukey post-hoc tests were used to evaluate differences in gene expression over time
in WT mice. All other comparisons were analyzed by two-tailed ANOVAs followed by
Bonferroni post-hoc tests. Differences were considered statistically significant at P<0.05.
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