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Materials and Methods
Animals.
All procedures involving animals were approved by the institutional animal care and use
committee. Transgenic mice overexpressing a constitutively activated form of calcineurin were
described and characterized previously (21).
The mouse model of ischemia/reperfusion by ligation of the left descending coronary
artery was performed as described previously (22). Briefly, Five to 6 week old wild type or
calcineurin transgenic mice were subjected to sham operation or ischemia/reperfusion. Mice
were pre-anesthetized with methoxyflurane and anesthetized with 0.5% isoflurane in 95% O2 /
5% CO2. The chest wall was closed in two layers with the ends of the suture externalized so that
after 45 minutes of ischemia the sutures could be released allowing reperfusion for 4 hours
before sacrifice.
The Gq transgenic model was described previously. Mice were given cyclosporine
starting at week 4 through week 12 (8 weeks) at a dosage of 20 mg/kg/day by sub-cutaneous
injection of Sandimmune (Novartis, Basel).
Cardiomyocyte cell culture
Primary cultures of neonatal rat cardiomyocytes were obtained as described previously
with slight modifications (23). Briefly, neonatal cardiac myocytes were obtained by enzymatic
dissociation of cardiac ventricles from 1-2 day old Sprague-Dawley rat neonates in a balanced
salt solution prior to enzymatic digestion (23). The ventricular tissue parts were subjected to
multiple rounds of enzymatic digestion using 0.05 % pancreatin (Sigma) and 84 U/mL of
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collagenase (Worthington). Cells were then collected by centrifugation at 800 rpm for 5 min at 4
ºC and resuspended in M199 media (Gibco BRL) supplemented with 15 % fetal bovine serum
(FBS), penicillin/streptomycin (100 U/mL) and L-glutamine (2 mmol/L). Subsequently, the cells
were differentially plated for 1 hr on uncoated cell culture dishes to remove contaminating
nonmyocytes. The enriched cardiomyocytes were then plated on gelatinized 6 cm cell culture
dishes at a pre-adherent density of 1 million or 0.75 million cells (high density and low density,
respectively). On the following day, the cells were washed three times with sterile phosphate
buffered saline (PBS) and cultured thereafter in serum-free M199 media containing a supplement
(Nutridoma; Boehringer Mannheim), 100 U/mL penicillin/streptomycin and 2 mmol/L Lglutamine.
Replication-deficient adenovirus production and infections.
E1a deleted, replication-deficient adenovirus expressing either a constitutively activated
form of mouse calcineurin A amino acids 1-398 (AdCnA), full length human Bcl-xL (AdBclxL), or a constitutively nuclear form of NFAT3 (AdNFAT3), were initially constructed in
pACCMV-pLpA and co-transfected into HEK293 cells with pJM17 as described earlier (24).
For the generation of the adenovirus expressing a calcineurin inhibitory peptide (Adcain), a 582
bp fragment corresponding to amino acids 1989-2182 of cain (25,26) was generated by PCR and
subcloned with a flag epitope as a HindIII fragment into pACCMVpLpA. All initial
recombinants were plaque purified, expanded, and titered by duplicate plaque assays in
monolayers of HEK293 using the agarose gel overlay method (27). Recombinants were tested for
appropriate expression in neonatal cardiomyocytes by semi-quantitative RT-PCR or western
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blotting when appropriate. Adenoviral infection of cardiomyocytes was performed at a
multiplicity of infection (moi) of 100 plaque forming units (pfu) in 2 mL (6 cm culture dishes) of
DMEM supplemented with 2 % FBS for 2 hours at 37 ºC in a humidified, 5 % CO2 incubator.
Under these infection conditions, approximately 99 % of the cells were positive for protein
expression by immunocytochemistry or stained -galactosidase positive after 24 hours. As a
control, 5 to 9 fold higher infection rates (MOI of 500-900) did not compromise the health of
cardiomyocytes under the conditions that were employed.
Western blotting
Protein extracts were made from left ventricular free wall tissue derived from calcineurin
transgenic mice, their wild type litter mates or from culture infected neonatal rat cardiomyocytes
in extraction buffer (in mmol/L: NaPO4 20, NaCl 150, MgCl2 2, NaF 10, sodium orthovanadate
0.1, sodium pyrophosphotae 10, DTT 1 and 1.0 %NP-40, 10 % glycerol, 10 g/mL leupeptin, 10
g/mL aprotinin, 10 g/mL pepstatin, 10 g/mL TPCK and 10 g/mL TLCK). The extracts were
sonicated on ice, the supernatant was obtained following a 5 min spin at 4 ºC in a microfuge at
13,000 rpm and the protein concentration was measured by the Bradford method. The proteins
were then size-fractioned by SDS-PAGE electrophoresis, transferred to PVDF membranes
(Amersham) and blocked with Tris buffered saline containing 0.1 % Tween-20 (TBS-T) and 10
% non-dry fat milk. Antibody dilutions for anti-Bcl-2, anti-Bcl-xL, anti-Bax (Trevigen), antiphospho Akt, Akt, and anti-phospho BAD (New England Biolabs) were all 1:500 in TBS-T
containing 5 % non-dry fat milk. Reactive signals on the blots were detected by enhanced
chemifluorescence (Amersham) and quantified with a Storm 860 PhosphorImager (Molecular
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Dynamics).
TUNEL labeling of DNA fragments
In situ DNA fragmentation was detected using the terminal deoxyribonucleotide
transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) in paraffin-embedded tissue
sections. Hearts were collected form calcineurin transgenic and wild type litter mate mice,
rapidly fixed, paraffin embedded and sectioned. TUNEL labeling was performed using the
CardioTACS kit (Trevigen) using the manufacturer’s recommended conditions.
TUNEL labeling of cultured cardiomyocytes was performed with the CardioTACS kit
(Trevigen) with slight modifications. Briefly, cultures were fixed by incubation in 3.7 %
formaldehyde/PBS for 5 min. Following 3 washes with PBS/0.1 % NP-40 and a 5 min incubation
in proteinase K (20 g/mL), endogenous peroxidase activity was inactivated with 3 % H2O2 in
methanol. DNA fragments were labeled with biotin-conjugated dUTP and TdT for 1 hr at 37 ºC.
After the TUNEL procedure, labeled nuclei were visualized by FITC-ExtraAvidin (Sigma)
incubation. In at least 6 fields the total number of nuclei versus the TUNEL positive nuclei were
scored (20X objective). Cardiomyocytes were also identified by double labeling with a
monoclonal antibody to sarcomeric -actinin (Sigma). All measurements were performed
blinded.
Immunocytochemistry
Cardiomyocytes were prepared for immunocytochemistry as described previously (21).
Anti--actinin antibody was added at dilution of 1:800 while CM1 (rabbit) antiserum, specific
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for the activated form of caspase-3 (Gift of Bob Armstrong, Idun Pharmaceuticals) was used at
1:500 and ANF antibody was used at 1:300 (Peninsula Laboratories). Secondary antibodies
included an anti-mouse TRITC conjugated antibody, and an anti-rabbit FITC conjugated
secondary antibody each at a dilution of 1:800. Quantitation of cardiomyocyte cell area was
performed on -actinin stained cardiomyocytes using confocal laser microscopy and NIH Image
software on a Sun system workstation. At least 25 fields of 100 cardiomyocytes were measured
in three separate experiments.
DNA laddering assays.
For DNA laddering of cultured cardiomyocytes, three 10 cm plates were utilized under
each experimental condition. To isolate genomic DNA, cells were trypsinized for 5 min and
collected by a 1 minute spin in a microfuge at 13,000 rpm. Cells were lysed in 10 mmol/L Tris
(pH = 8.0), 25 mmol/L EDTA, 100 mmol/L NaCl and 0.5 % SDS and incubated overnight with 1
mg/ml proteinase K at 37 ºC. After incubation, proteins were precipitated by addition of an equal
volume of 2.4 mol/L NaCl and removed by centrifugation. The supernatant was extracted once
with phenol:chloroform, the DNA precipitated with isopropanol, washed with 70 % ethanol and
resuspended in 10 mmol/L Tris (pH = 8.0), 1 mmol/L EDTA and treated with 100 g/mL DNAse
free RNAse (Boehringer Mannheim) for 30 min at 37 ºC. The genomic DNA was reprecipitated
using isopropanol, rewashed with 70 % ethanol and dissolved in 10 mmol/L Tris (pH = 8.0), 1
mmol/L EDTA and its concentration was measured by spectrophotometry. Equal amounts of
DNA were loaded on 1.5 % agarose gels containing 1 g/mL ethidium bromide and visualized
under UV light.
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For analysis of DNA laddering in vivo, hearts were harvested and DNA was prepared as
described previously (22).
Statistical analysis
Data are expressed as means ±SEM. Differences between experimental groups were
evaluated for statistical significance using Student’s t-test for unpaired data or one-way ANOVA
followed by Bonferroni’s post-test when appropriate. P values < 0.05 were considered to be
statistically significant.
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