Download Supplement to: HSP60, Apoptosis and Myocardial Injury S. R.

Supplement to:
HSP60, Apoptosis and Myocardial Injury
S. R. Kirchhoff, S. Gupta, and A. A. Knowlton
Cardiology Research, VA Medical Center and Baylor College of Medicine, Houston, TX 77030
Methods
Isolation of Cardiac Myocytes - Adult cardiac myocytes were isolated from male Sprague
Dawley rats and cultured as previously described.1
The cells were incubated at 37
hours to allow adherence, and then treated with either AS to HSP60 or a scrambled sequence
(SCR) as a control for the effects of phosphorothioate oligonucleotide; both were used at a
concentration of 5.5 uM (25 ug/ml).
Cells without any treatment were used as a second control.
The animal protocol was approved by the Baylor College of Medicine Animal Research
committee in accordance with the NIH “Guide for the Care and Use of Laboratory Animals”
[DHEW Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports,
DRR/NIH, Bethesda, MD 20205]
The primary AS oligonucleotide used throughout this study corresponds to bases 109-123
(5’TAAGGCTCGAGCATC‘3; TriLink; San Diego, CA), and has a melting temperature of 48.2
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. A second AS sequence was also used which corresponds to bases 27-41 (melting
sequence (5’GCTCGTGGTCAATAC‘3) was used as a control for the effects of the
phosphorothioate compound (SCR). All of the oligonucleotide sequences were screened in
Genbank for unexpected matches.
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Western Blotting - Western blotting was performed as described.3 For HSP60, 2 ug of each
sample was separated by SDS-PAGE. An anti-HSP60 monoclonal antibody was used at a
concentration of 1:35,000 (clone:LK-2, StressGen, Canada). Westerns were developed with antiα-sarcomeric actin at 1:1000 concentration as a control (clone: 5C5, Sigma; St Louis, MO). The
bax (N-20), bak (G-23), bcl-2 (N-19), and caspase 3 (to the 11 and 17 kDa cleaved portions of
caspase 3) antibodies were used at a concentration of 1:500, 1:250, 1:500 and 1:500,
respectively, and for these antibodies anti-rabbit polyclonal IgG-horseradish peroxidase was
used as the secondary antibody at a concentration of 1:1000 (Santa Cruz Biotechnology, Santa
Cruz, CA and New England BioLabs, Beverly, MA). Blots were developed and analyzed as
described.3
Live/Dead Staining, LDH - After 24 hours of treatment, the viability of the cells and LDH
release was analyzed as previously described.1,3
Tunnel Assay - was performed using a kit from Oncogene following the manufacturer’s
protocol (Cambridge, MA).
A minimum of 20 to 50 cells were scored per slide by an
individual blinded as to treatment group. Experimental data represents the sum of five separate
experiments.
Immunoprecipitation - Untreated cardiac myocytes were washed twice with phosphatebuffered saline (PBS) and collected in ice-cold PBS buffer with 1% NP-40, 1 mmol/L PMSF,
and one ug/ml of each pepstatin A, aprotinin, leupeptin, and antipain. Immunoprecipitation (IP)
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was carried out as described previously.4 The samples were separated on a 14% SDS PAGE,
and transferred to nitrocellulose for analysis.
Cytochrome C Release - Pilot studies were done to determine the digitonin concentration that
would release LDH from the myocytes, but not cytochrome c. This identified a level of digitonin
that permeabilized the cell membrane, but not the mitochondria. The cells were treated with AS,
SCR, or C for 24 hours. A cytoplasm permeabilization solution (40 mM HEPES, 140 mM KCl,
20 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 0.566 mM CaCl2, 5 mM ATP, and 3.125 uM
digitonin) released the contents of the cytoplasm without damaging subcellular components
including the mitochondria.5 The cells were also collected in permeabilization media and
sonicated. 2 μg of each sample was separated on a 10% SDS-PAGE, and transferred to
nitrocellulose. The westerns were developed with anti-cytochrome c at 1:2,000 (clone:
7H8.2C12, Pharmingen, San Diego, CA) followed by anti-mouse IgG-HRP at 1:500.
Citrate Synthase - activity was measured on the samples used for cytochrome c release, as an
index of mitochondrial function.6 The cells were collected and sonicated in 10mM Tris-HCl,
pH 8.1. For the assay, cell lysate or media were added to a reaction mixture containing 1mM
DTNB in 1 M Tris-HCl, pH 8.1, and 10mM Acetyl-CoA in H20. The reaction was initiated by
adding 10mM oxaloacetate in 0.1 M Tris-HCl, pH 8.1, and linear values for acetyl CoA
deacylase activity were obtained for three min. at 415 nm.
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Cell Fractionation - Separation of cytosol and mitochondria – Cytosol and mitochondria
were prepared from the myocytes according to Bilan et al. 7 Briefly, cells were homogenized
with a Dounce homogenizer in buffer I (250 mM Sucrose, 20 mM HEPES-NaOH, pH 7.4, 2 mM
EGTA, 3 mM NaN3, and protease inhibitors). The homogenate was centrifuged at 700 g for 5
min and the supernatant was centrifuged at 31,000 g for 60 min. The supernatant from this step
was saved as the cytosol, and the pellet was resuspended in buffer I and subjected to
ultracentrifugation on a discontinuous sucrose density gradient (32, 40, and 50% w/w, in 20 mM
HEPES-NaOH, pH 7.4) for 2h at 65,000 g. The 32/40 % interface is a plasma membrane
fraction while the mitochondrial fraction pellets at the bottom of tube.
Citrate synthase activity
was used to verify the integrity of the fractions. Cytosolic levels of citrate synthase were trivial
- average mitochondrial and cytosolic citrate synthase activities for the fractions were 8,047 ±
365 and 128 ± 12 mU/ug protein, respectively.
Co-Immunoprecipitation Studies on Cell Cytosol - The cytosolic fraction, generated by cell
fractionation as described above, was used for IP studies. The initial cytosol from untreated
cells (i.e. normal myocytes or controls) was divided into 3 fractions, and an aliquot was set
aside for later comparison. An equal amount of protein from the cytosol (divided into 3 parts for
simultaneous immunoprecipitation) was subjected to exhaustive immunoprecipitation (IP).
Thus, the starting protein concentration for the 3 IPs was the same. ¼ volume of ice-cold PBS
buffer with 1% NP-40, 1mmol/L PMSF, and one μg/ml of each pepstatin A, aprotinin, leupeptin,
and antipain was added to the cytosolic fractions. The cytosol was immunoprecipitated with
either anti-HSP60, anti-bax, or a nonspecific mouse IgG1 (MOPC 21, Sigma). Three rounds of
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IP were preformed on each sample. Each IP was washed as follows: 1. high stringency - 0.1%
SDS, 0.1% deoxycholic acid, 0.5% tritonx100, 20 mM Tris HCl, 120 mM NaCl, 25 mM KCl, 5
mM EDTA, 5 mM EGTA, 0.1 mM DTT, pH 7.5; 2. High Salt - high stringency plus 1 M NaCl,
pH 7.5; 3. Low Salt Buffer - 2 mM EDTA, 0.5 mM DTT, 10 mM Tris HCl, pH 7.5. The samples
were resuspended in sample buffer, separated on a 10% SDS PAGE, and transferred to
nitrocellulose for western analysis with bax antibody and then with HSP60 antibody. For each
study 40 ug of initial starting protein and 40 ug of the remaining supernatant after completion of
the IPs was also loaded on the same SDS PAGE.
ATP Levels - ATP was measured using a kit (Sigma) based on measuring the change in
absorbance at 340 nm by the oxidation of NADH. Briefly, the protein from the sample was
precipitated out by adding equal volume of 12% TCA. The initial absorbance was measured at
340 nm by adding the known volume of the protein free supernatant to the vial containing
NADH and 3-phosphoglyceric acid. Then GAPD/PGK Enzyme mixture was added and
absorbance was recorded vs water as a reference until the minimum absorbance was reached
(approx. 10 min). ATP concentrations were estimated by the calculating from the change in the
absorbance and using 6.22 as the millimolar absorptivity of NADH at 340nm .
Turnover Studies - 1) mRNA - total RNA was isolated as previously described.3 Levels of
mRNA for bcl-2 and bax were compared by RT-PCR followed by PCR for 25 and 35 cycles on
5 to 10 ug of the RT-PCR product. These studies were performed with a kit (Maxim Biotech,
Inc., So. San Francisco, CA), which allowed simultaneous detection of rat bcl-2, bax, GAPDH,
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caspase 3, bcl-xl, and bcl-xs. Samples were separated on a 2.5% agarose gel, and ethidium
bromide staining used to detect DNA. 2) New synthesis - At 24h, immediately after 4 to 8h of
labeling with 10 uCi/ml of S35-methionine (Amersham), cells were collected in lysis buffer,
sonicated, and applied to an affinity column containing anti-bcl-2 or anti-bax (Pierce, Seize-X).
Each column contained 200 ug of antibody (same as above) covalently linked. Samples were
eluted and separated on a 10% SDS-PAGE. The gels were treated with Amplify (Amersham),
dried, and either exposed to film or analyzed with a phospho-imager (Molecular Dynamics).
Levels of newly synthesized bcl-2 and bax were compared based on the intensity of their
respective signals. 3) Degradation - The myocytes were labeled as above from hour 4 to 16.
Media was changed, and cells were cultured for another 8 h in the presence of respective
treatments. At 24 h, cells were collected and analyzed.
Immunocytochemistry/EPR - Myocytes were fixed as previously described.8 After blocking
for 1h with 5% BSA, cells were incubated with anti-HSP60 1:200 (as above) and anti-bax 1:200
(rabbit polyclonal, Pharmingen). Anti-mouse IgG-TRITC (Southern Biotechnology Associates,
Birmingham, AL ) and anti-rabbit IgG-FITC (Molecular Probes, Eugene, Oregon) were each
used at 1:500. Control slides developed with second antibodies alone had no significant signal.
Exhaustive photon reassignment (EPR) was carried out as previously described.9 Cellview
software (Scanalytics, CSPI., Billerica, MA ) was used to compare localization of the two
antibodies. Both composite images showing co-localization of signal from the multiple sections
made with this method, and subtraction images were analyzed, subtracting bax signal from
HSP60.
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Results
A TUNEL assay was done, and 28.7% of AS-treated cells were positive for DNA
fragmentation (figure 9A, p < 0.05 compared to SCR and C). Representative cells are shown in
figure 9B.
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References
1. Sun,L., J. Chang, S.R. Kirchhoff, and A.A. Knowlton. 2000. Activation of HSF and Selective
Increase in Heat Shock Proteins by Acute Dexamethasone Treatment. Am. J. Physiol.
278:H1091-H1096.
2. Venner,T.J. and R.S. Gupta. 1990. Nucleotide sequence of rat hsp60 (chaperonin, GroEl
homolog) cDNA. Nucleic Acid Res. 18:5309.
3. Nakano,M., D.L. Mann, and A.A. Knowlton. 1997. Blocking the endogenous increase in
HSP72 increases susceptibility to hypoxia and reoxygenation in isolated adult feline
cardiocytes. Circulation. 95:1523-1531.
4. Knowlton,A.A., M. Grenier, S.R. Kirchhoff, and M. Salfity. 2000. Phosphorylation at
tyrosine 524 influences nuclear concentration of HSP72 with stress. Am. J. Physiol.
278:H2143-H2149.
5. Sparagna,G.C., D.L. Hickson-Bick, L.M. Buja, and J.B. McMillin. 2000. A metabolic role
for mitochondria in palmitate-induced cardiac myocyte apoptosis. Am. J. Physiol.
279:H2124-H2132.
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6. Srere,P.A. 1969. The citric acid cycle. Methods in Enzymology. 13:3-11.
7. Bilan,P.J., Y. Mitsumoto, T. Ramlal, and A. Klip. 1992. Acute and long-term effects of
insulin-like growth factor I on glucose transports in muscle cells. Translocation and
biosynthesis. FEBS Letters. 298:285-290.
8. Knowlton,A.A. and L. Sun. 2001. Heat shock factor-1, steroid hormones, and regulation of
heat shock protein expression in the heart. Am. J. Physiol. 280:H455-H464.
9. Knowlton,A.A. 1999. Mutation of amino acids 246-251 alters nuclear accumulation of
human heat shock protein (HSP) 72 with stress, but does not reduce viability. J. Mol.
Cell. Cardiol. 31:523-532.
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Figure Legends
Figure 9 - A) The graph summarizes results of 5 experiments showing increased DNA
fragmentation by TUNEL assay in AS-treated cells. Total cell counts for these experiments
were: C: 502, 72 positive; SCR 425, 58 positive; AS - 299, 99 positive. B) Representative cells
as labeled. The arrows indicate the labeled DNA fragments in the nuclei. Cells are
counterstained with methyl green.
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