Download SOD activity: Total SOD activity of homogenates was determined

Extracellular SOD deficiency exacerbates pressure overload induced
left ventricular hypertrophy and dysfunction
Zhongbing Lu*1,2, Xin Xu*1,2, Xinli Hu2, Guangshuo Zhu1,2, Ping Zhang2, Elza D. van Deel1,4,
Joel P. French2, John J. Fassett2,Tim D. Oury3, Robert J Bache2, Yingjie Chen1,2
Running Title: SOD3 protects the pressure overloaded heart
1 Center
for Vascular Biology and 2 Cardiovascular Division, Department of Medicine, University
of Minnesota Medical School, Minneapolis, MN55455
3 Department of Pathology, University of Pittsburgh Medical Center, University of Pittsburgh,
Pittsburgh, Pennsylvania 15261, USA.
4 Division of Experimental Cardiology, Department of Cardiology, Cardiovascular Research
School COEUR, Erasmus MC, University Medical Center Rotterdam, The Netherlands
*These authors contributed equally.
Address for correspondence:
Yingjie Chen, MD, PhD
University of Minnesota
Mayo Mail Cod 508
420 Delaware St. SE
Minneapolis, MN55455
Tel: (612) 624-8970
Fax: (612) 626-4411
email: [email protected]
Supplementary data
Western blots. Protein content was analyzed using Western blots as previously described
(n=5 samples each group) 1. Primary antibodies against SOD1, SOD2, SOD3, ANP (atrial
natriuretic peptide), nitrotyrosine, MMP2, MMP9, collagen-I and collagen-III were purchased from
Transduction Laboratories, Santa Cruze Inc, and Sigma, respectively.
SOD activity: Total SOD activity of homogenates was determined with a Superoxide Anion
Detection kit (Calbiochem Company), according to the manufacturer’s instructions, and expressed
as scavenging ability of the homogenates on superoxide anion (n=5 samples each group).
Measurement of superoxide anion: Relative superoxide anion production was determined
by chemiluminescence of coelenterazine (4μM, Molecular Probes) as previously described2,3. To
assess ROS production with coelenterazine chemiluminescence, flash-frozen myocardium
(~40mg) was minced into fine pieces (about ~1mm3) on ice, and added to 500μl ice cold Krebs
bicarbonate buffer containing the following reagents, in mmol/l: NaCl, 118; KCl, 4.7; CaCl2, 1.5;
MgSO4, 1.1; KH2PO4, 1.2; glucose, 5.6; and NaHCO3, 25. Coelenterazine (4μM) was added to the
tissue suspension and chemiluminescence was assessed for 60 seconds at 5 min intervals over a
60 min interval at room temperature in a Lumat LB 9507 tube luminometer. Data were normalized
by sample weight (n=5 each group).
Ratio of GSSG/GSH. The ratio of GSSG/GSH was determined as previous described (n=5
each group) 4. Briefly, 20 μl homogenate was mixed with 180 μl phosphate-EDTA (pH 8.3) and
100 μl 20% trichloroacetic acid for 10 min and the mixture was then centrifuged at 12,000 × g for
10 min to pellet the protein. For the GSH assay, the final assay mixture contained 50 μl
supernatant, 0.9 ml phosphate-EDTA buffer and 50 μl O-phthaldehyde (OPT, 1mg/ml). After
allowing the mixture to react for 40 min, fluorescence was recorded at 350 nm excitation and 425
nm emission on a CytoFluor 4000 fluorescence spectrophotometer. For the GSSG assay, the final
assay mixture contained 40 μl supernatant, 16 μl 0.04M N-ethylmaleimide, 0.9 ml 0.1 M NaOH,
and 50 μl OPT and fluorescence was determined at 338 nm excitation and 422 nm emission.
TBARS content: TBARS was determined by the method of Ohkawa et al (n=5 each group)
5.
First, 0.2ml of LV homogenate was mixed with 1 ml 20% (w/v) trichloroacetic acid, 0.7 ml DD
water, 1 ml 0.67% (w/v) 2-thiobarbituric acid, and 0.1 ml 0.2% (w/v) butylated hydroxytoluene.
After concussing, the mixture was incubated for 60 min in boiling water, and TBARS extracted in 2
ml of n-butanol. After centrifugation at 4400 × g for 10 min, the absorption of the butanol layer was
measured at 532 nm.
Histological staining and measurement of myocardial fibrosis. Tissue sections (8µm) from
the central portion of the LV were stained with H&E (Sigma) for overall morphology, Sirius Red
(Sigma) for fibrosis, and FITC-conjugated wheat germ agglutinin (AF488, Invitrogen) to evaluate
myocyte size. For mean myocyte size, the cross section sectional area of at least 120 cells/sample
(from 4 areas) and at least 4 samples of each group were averaged 2. The percent volume fibrosis
was determined using the method described in Unbiased Stereology 6.
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