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Online Appendix for the following JACC article
TITLE: Phenyl-α-tert-butyl-nitrone and Benzonidazole Treatment Controlled the
Mitochondrial Oxidative stress and Evolution of Cardiomyopathy in Chronic Chagasic
Rats
AUTHORS: Jian-Jun Wen, PHD, Shivali Gupta, PHD, Zhangjun Guan, MD, Monisha
Dhiman, PHD, David Condon, Charles Lui, MD, Nisha Jain Garg, PHD
APPENDIX
MATERIALS AND METHODS
Animals: Sprague Dawley rats (4-5 weeks old, Harlan Labs) were adapted to animal
facility for one-month and then infected with T. cruzi (SylvioX10/4, 50,000
trypomastigotes/rat, i. p.). The animals were further kept for another 6-7 months up to
chronic stage after infection making a total of about 9 months. Aging is related to
oxidative stress and a decline in heart function. Studies have shown that a decline in heart
function in aging rats begins by 9 months of age (1). Thus, to be specific that the decline
in heart function was a result of T. cruzi infection, and not an age-related event, we chose
to start with young 4-5 week old rats. Rats were treated with PBN in drinking water. We
chose PBN, because it protects good oxygen, preventing it’s conversion to harmful forms
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(i.e. reactive oxygen). A PBN derivative (NXY-059) has been examined in Food and
Drug Administration (FDA) phase III clinical trials for treatment of stroke (2), and PBN
has been reported to improve cognitive performance, better than that observed by vitamin
E and vitamin C usage in rats (3).
Parasite Burden. Total DNA isolated from cardiac tissues was used as template for the
amplification of T. cruzi-specific 18SrDNA sequence by traditional and real-time PCR, as
described (4). PCR amplicons were visualized and densitometric analysis performed
using a Fluorchem 8800 Imaging System (Alpha Innotech). The real-time PCR
amplification of Tc18SrDNA was calculated using the formula fold change = 2
-∆Ct
, where
∆Ct represents the Ct (infected sample) - Ct (control) (5). All data were normalized with
host-specific GAPDH.
Isolation and purification of mitochondria. All solutions were prepared with highly
pure chemicals devoid of divalent cations (from Sigma) in HPLC grade H2O. Freshly
harvested heart ventricular tissues were washed with ice-cold HMSB medium (10 mM
HEPES pH 7.4, 225 mM mannitol, 75 mM sucrose and 0.2% fatty acid free BSA) and resuspended in HMSB containing 200 U/ml collagenase (tissue: buffer ratio, 1:20). Tissues
were homogenized in a Dounce homogenizer, incubated for 3 min, and 1 mM EGTA
added to stop collagenolysis and prevent mitochondrial Ca2+ uptake. Samples were
centrifuged sequentially at 480 g and 8100 g and pelleted mitochondria kept on ice
without dilution. The determination of glucose-6-phosphatase (endoplasmic reticulum
marker) (6) and acid phosphatase (peroxisome marker) (7) activities in isolated
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mitochondria versus heart homogenates was performed to verify that isolated
mitochondria were >95% pure before being used for different experiments. Protein
content was measured by the Bradford method.
In some experiments, mitochondria were further purified by Percoll density
gradient centrifugation (8). Briefly, mitochondrial pellet was suspended in 15% Percoll,
layered on top of 23% and 40% Percoll, and centrifuged at 30,700 g. The mitochondrial
fraction at the lower interface was suspended in HMSB buffer (1:20, v/v), and
centrifuged at 14,000 g. The pellet was washed again, centrifuged at 8100 g, and used for
various assays. All centrifugations were performed for 10 min at 4oC.
Oxygen consumption. We assessed the respiration rate of isolated mitochondria using a
Mitocell S200A Micro Respirometry System (Strathkelvin Instruments, Motherwell,
UK). Briefly, a microcathode oxygen electrode was calibrated and after baseline
measurements, mitochondria (500 µg), suspended in 0.5 ml MSP medium (225 mM
mannitol, 75 mM sucrose, 20 mM K2HPO4/KH2PO4 buffer, pH 7.4), were added to the
mitocell. The substrate-stimulated oxygen consumption (state 4) was measured in the
presence of 10 mM pyruvate/2.5 mM malate (pyr/mal), 5 mM succinate/6.25 µM
rotenone. The state 3 oxygen consumption was measured after addition of 230 μM ADP.
Respirometry software (Strathkelvin) was used to calculate the respiratory control ratio
(RCR).
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To measure myocardial oxygen consumption in vivo, mice were anesthetized, and
heart perfused to remove blood. The left ventricle was dissected longitudinally such that
each section contained a portion of the septum, free wall, and apex. The LV tissue slices
were incubated for 2 h at 37°C in Kreb’s buffer (20 mM HEPES, 1.03 mM KH2PO4, 99
mM NaCl, 4.69 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3 and 5.6 mM
glucose) equilibrated with 10% CO2 (pH 7.4), transferred to mitocell containing 1 ml
Kreb’s bicarbonate solution/10 mmol/1 HEPES (pH 7.4), and oxygen consumption
measured as above. The in vivo rate of mitochondrial respiration in LV tissue slices was
determined in presence of 0.5 mM succinate /10 μM rotenone (9).
Enzymatic activity of respiratory complexes. Cardiac mitochondria were isolated and
purified by differential centrifugation (10) or Percoll density gradient centrifugation, and
enzymatic activity of respiratory complexes was monitored by spectrophotometric
methods, as we have described previously (11).
ROS content and rate of ROS production. To determine myocardial and mitochondrial
ROS levels, heart (25-g protein) and isolated mitochondrial (25-µg protein)
homogenates were incubated with 30 M dihydroethidium (DHE, oxidized to fluorescent
ethidium that binds nuclear DNA, Ex498nm/Em585nm) or 33 M amplex red/0.1 U HRP
(oxidized to fluorescent resorufin, Ex563nm/Em587nm) and fluorometry was performed
using a SpectraMax M5 microplate reader (Molecular Devices) (12). Frozen heart tissue
sections (10-µm) were equilibrated in Kreb’s buffer, and then incubated in dark for 30
min with 5-µM DHE to detect intracellular/intra-mitochondrial ROS. Fluorescence was
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detected on an Olympus BX-15 microscope and images captured by using a mounted
digital camera.
To measure the rate of ROS production, freshly isolated mitochondria were
incubated with 5 mM pyruvate/5 mM malate (pyr/mal) or 5 mM succinate/6.5 µM
rotenone to energize complex I and complex II, respectively, and the rate of ROS
formation determined using amplex red or DHE probes (as above). Standard curves were
prepared with purified H2O2 (50 nM - 5 µM) and ethidium bromide (0-15 µM). With
intact mitochondria, DHE was used to detect intra-mitochondrial ROS, and amplex
red/HRP assay was employed to detect extra-mitochondrial/released H2O2.
Lipid and protein oxidation. We employed a TBARS assay (ε532nm=1.56 x 105 M-1cm-1)
to measure the lipid peroxidation products (malonyldialdehyde) by spectrophotometry
(13). Protein carbonyls, derivatized with 2,4-dinitrophenylhydrazine (DNPH), were
estimated by ELISA (14) using a polyclonal anti-DNP antibody (Chemicon). To detect
hydroxynonenal (HNE) adducts, paraffin-embedded tissue sections (5-µm) were
dewaxed, blocked with 1.5% H2O2/5% BSA, and sequentially incubated at 4°C with
rabbit
anti-HNE antibody (Alpha Diagnostic, 1:200 dilution) for 16 h, and HRP-
conjugated goat-anti-rabbit IgG (Bio-Rad, 1:500 dilution) for 1 h. Color was developed
using diaminobenzidine tetrahydrochloride substrate, and images analyzed by light
microscopy. Oxidative adducts were semi-quantitatively scored on a scale of 0-4 (0negative, 1-weak, 2-moderate, 3-strong, and 4-diffused throughout the tissue section).
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Inflammation and tissue pathology. Myocardial sections were fixed in 10% formalin,
embedded in paraffin, and stained with hematoxylin/eosin or Masson’s trichrome (4).
Tissue sections were scored for myocarditis, viewing 4-6 serial H&E stained slides of
each biopsy. The myocarditis score was determined by a semi-quantitative analysis of the
presence of inflammatory cells as (0) - absent/none, (1) - focal or mild myocarditis with
≤1 foci, (2) - moderate with ≥ 2 inflammatory foci, (3) - extensive with generalized
coalescing of inflammatory foci or disseminated inflammation with minimal necrosis and
retention of tissue integrity, and (4) - severe with diffused inflammation, severe tissue
necrosis, interstitial edema, and loss of tissue integrity. The foci of necrosis, fibrosis, and
pseudocysts (parasite nests) were scored as (0) absent, (1) 0-1 foci, (2) 1-5 foci, and (3) >
5 foci.
The enzymatic activities of xanthine oxidase (XOD) and NADPH oxidase (NOX)
in heart homogenates were monitored using amplex red method via determination of
H2O2 production in presence of xanthine and NADPH substrate, respectively (15). We
measured NOX activity in presence of 100 µM oxypurinol (to prevent XOD interference)
and XOD activity in presence of 600 µM apocynin (to prevent NOX interference).
Addition of enzyme-specific inhibitors (XOD: oxypurinol, NOX: apocynin) resulting in
>90% inhibition of change in absorbance validated the specificity of reaction.
The XOD and NOX activities were also determined by catalytic staining (16,17).
Briefly, heart homogenates were resolved on 6% native polyacrylamide gels. Catalytic
staining for NOX was performed by incubating gels with 2 mM nitroblue tetrazolium
(NBT) for 20 min at 37oC and then with 1 mM NADPH/100 µM oxypurinol at room
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temperature until the appearance of blue formazan bands was observed (± 600 µM
apocynin). Catalytic staining for XOD was performed by incubating gels at room
temperature in 50 mM Tris-HCl (pH 7.6), 0.5 mM xanthine, 0.25 mM NBT, and 630 mM
TEMED/600 µM apocynin (± 100 µM oxypurinol). Myeloperoxidase (MPO) activity
was determined by a dianisidine-H2O2 method (18), modified for 96-well plates (± 10
µM 4-aminobenzoic acid, MPO inhibitor).
The myocardial expression level of mRNAs for proinflammatory cytokines and
hypertrophy markers was determined by a real-time RT-PCR (5). Briefly, total RNA
from tissue samples (50 mg each) was isolated by guanidinium thiocyanate-phenolchloroform extraction (19), treated with DNase (Ambion) to remove contaminating DNA,
and analyzed for quality and quantity with SPECTRAmax PLUS 384 and for integrity
with an Agilent 2100 Bioanalyzer. Total RNA (2.5 µg) was reverse transcribed with 2.5
U of Moloney murine leukemia virus reverse transcriptase (New England BioLabs) and 1
µM poly(dT)18 oligonucleotide, and the first-strand cDNA was used as a template in a
real-time PCR on an iCycler thermal cycler (Bio-Rad) with SYBR Green Supermix (BioRad) and specific oligonucleotides (Table 1). The threshold cycle (Ct) values for each
target mRNA were normalized to -actin mRNA, and the relative expression level of
each target gene was calculated with the formula n-fold change = 2-ΔCt, where ΔCt
represents Ct (infected sample) – Ct (control).
REFERENCES
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1.
Tanabe S, Bunag RD. Age-related central and baroreceptor impairment in female
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Sydserff SG, Borelli AR, Green AR, Cross AJ. Effect of NXY-059 on infarct
volume after transient or permanent middle cerebral artery occlusion in the rat;
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3.
Sack CA, Socci DJ, Crandall BM, Arendash GW. Antioxidant treatment with
phenyl-alpha-tert-butyl nitrone (PBN) improves the cognitive performance and
survival of aging rats. Neurosci Lett 1996;205:181-4.
4.
Garg N, Popov VL, Papaconstantinou J. Profiling gene transcription reveals a
deficiency of mitochondrial oxidative phosphorylation in Trypanosoma cruziinfected murine hearts: implications in chagasic myocarditis development.
Biochim Biophys Acta 2003;1638:106-20.
5.
Garg N, Bhatia V, Gerstner A, deFord J, Papaconstantinou J. Gene expression
analysis in mitochondria from chagasic mice: Alterations in specific metabolic
pathways. Biochemical J. 2004;381:743-752.
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Harper A. Glucose-6-phospate. Methods of Enzymatic Analysis 1963:788-792.
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Lui NS, Roels OA, Trout ME, Anderson OR. Subcellular distribution of enzymes
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Sims NR, Anderson MF. Isolation of mitochondria from rat brain using Percoll
density gradient centrifugation. Nat Protoc 2008;3:1228-39.
8
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Loke KE, Curran CM, Messina EJ, et al. Role of nitric oxide in the control of
cardiac oxygen consumption in B(2)-kinin receptor knockout mice. Hypertension
1999;34:563-7.
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Wen J-J, Garg N. Oxidative modifications of mitochondrial respiratory complexes
in response to the stress of Trypanosoma cruzi infection. Free Radic Biol Med
2004;37:2072-81.
11.
Rustin P, Chretien D, Bourgeron T, et al. Biochemical and molecular
investigations in respiratory chain deficiencies. Clin Chim Acta 1994;228:35-51.
12.
Wen J-J, Bhatia V, Popov VL, Garg NJ. Phenyl-alpha-tert-butyl nitrone reverses
mitochondrial decay in acute Chagas disease. Am J Pathol 2006;169:1953-64.
13.
Ohkawa H, Ohishi N, Kunio T. Assay for lipid peroxides in animal tissues by
thiobarbituric acid reaction. Analytical Biochem 1979;95:351-358.
14.
Buss IH, Winterbourn CC. Protein carbonyl measurement by ELISA. Methods
Mol Biol. 2002;186:123-128.
15.
Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP. A stable nonfluorescent
derivative of resorufin for the fluorometric determination of trace hydrogen
peroxide: applications in detecting the activity of phagocyte NADPH oxidase and
other oxidases. Anal Biochem 1997;253:162-8.
16.
Carter C, Healy R, O'Tool NM, et al. Tobacco nectaries express a novel NADPH
oxidase implicated in the defense of floral reproductive tissues against
microorganisms. Plant Physiol 2007;143:389-99.
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17.
Wang Q, Pfeiffer GR, 2nd, Stevens T, Doerschuk CM. Lung microvascular and
arterial endothelial cells differ in their responses to intercellular adhesion
molecule-1 ligation. Am J Respir Crit Care Med 2002;166:872-7.
18.
Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous
inflammation: estimation of neutrophil content with an enzyme marker. J Invest
Dermatol 1982;78:206-9.
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Wen J-J, Vyatkina G, Garg N. Oxidative damage during chagasic cardiomyopathy
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defense. Free Radic Biol Med 2004;37:1821-33.
Table 1: Oligonucleotides used in this study
Accession #
Gene name
(Genbank)
Forward primer (5’->3’)
Rat
Inflammatory cytokines
Interleukin 1 (IL-1)
NM_031512
5’-ggctgacagaccccaaaaga-3’
NM_138880
5’-ctgccaaggcacactcattg-3’
Interferon  (IFN-)
S40199
5’-accaccaagcagaggagcag-3’
Tumor necrosis factor 
(TNF-)
Hypertrophy markers
Atrial natriuretic peptide
M60731
5’-actccggcttctgactctgc-3’
(ANP)
Brain natriuretic peptide
M25297
5’-tgcagcatggatctccagaa-3’
(BNP)
NM_019212
5’-cacggcattatcaccaactg-3’
Skeletal  -Actin
(sk-Actin)
GAPDH
BC161847
5’-ccatgttggtcatgggtgtg-3
T. cruzi 18S rDNA
X53917
5‘-ttgtttggttgattccgtca-3’
Reverse primer (5’->3’)
Amplicon
size (bp)
5’-gtgcttgggtcctcatcctg-3’
5’-gctgatggcctggttgtctt-3’
5’-gaagtggcaaatcggctgac-3’
201
205
199
5’-tggcaagtttgtgctggaag-3’
221
5’-agggccttggtcctttgaga-3’
204
5’-ccggaggcatagagagacag3’
5’-cgccagtccttctttgatgg-3’
5‘-cccagaacattgaggagcat-3’
221
10
208
200
RESULTS
Figure 1. Benzonidazole has no effect on myocardial respiration in rats. Normal rats
were treated with 0.7 mM benzonidazole in drinking water for 3 weeks. LV cardiac tissue
slices (dissected longitudinally) were incubated with the indicated substrates to energize
mitochondria, and respiration rate determined by oxygraphy. The data (mean ± SD) are
representative of three independent experiments (n = 3 animals/group).
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Figure 2. Catalytic staining. Heart homogenates were resolved on native
polyacrylamide gels. Catalytic staining for (A) NADPH oxidase (NOX) and (B) xanthine
oxidase (XOD) was performed as described in Supplement file-1/Materials and Methods.
(C&D) Shown are densitometric analysis of NOX (C) and XOD (D) catalytic staining
after subtraction of background signal obtained in absence of substrate. Data are
presented as mean ± SD (n=9). Abbreviations: NH-normal heart, AH-acutelyinfected/untreated (25-dpi), AHP-acutely-infected/PBN-treated, CH-chronicallyinfected/untreated (>150-dpi), CHP-chronically-infected/PBN-treated, CHB-chronicallyinfected/benzonidazole-treated, CHBP-chronically-infected/PBN+BZ-treated. The level
of significance between normal versus infected/untreated rats is shown by *; and between
infected versus PBN and/or BZ-treated rates by # (*, #p<0.05; **, ##p<0.01, ***p<0.001).
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Table-2. Benzonidazole has no effect on respiratory chain efficiency of isolated
mitochondria from rat myocardium
Substrate
Animals
Rate of respiration (n mol atom O/min/mg protein)
State 4 (- ADP)
State 3 (+ ADP)
RCR
(state3/state4)
Pyruvate +
Malate
Succinate+
Rotenone
Normal
7.29 ± 0.78
35.26 ± 5.50
4.82 ± 0.31
+ BZ
7.23 ± 0.76
30.86 ± 4.80
4.25 ± 0.39
Normal
14.61 ± 1.53
61.41 ± 13.50
4.17 ± 0.57
+ BZ
15.06 ± 0.77
68.72 ± 5.80
4.57 ± 0.33
Rats were treated with 0.7 mM benzonidazole in drinking water for 3-weeks. Cardiac
mitochondria were isolated from normal rats (treated and untreated) as described in
Materials and Methods. Mitochondria were incubated with the indicated substrates and
the rate of oxygen consumption was determined by oxygraphy. RCR: Respiratory control
ratio. The data (mean ± S.D.) are representative of three independent experiments (n = 3
animals/group/experiment).
Note that state 4 respiration supported by pyr/mal (complex I substrates) and succinate
(complex II substrate) were not statistically different in cardiac mitochondria of
normal/untreated and normal/BZ-treated rats, indicating that mitochondrial oxygen
uptake and substrate-dependent respiration were not compromised by BZ-treatment. No
significant effect of BZ treatment was observed on ADP-stimulated state 3 respiration
(indicates proton gradient for ATP synthesis). RCR values >4.0 indicated that electron
transport chain and oxidative phosphorylation were coupled in mitochondria isolated
from normal/untreated and normal/BZ-treated rats.
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Table-3. Hemodynamic measurements and indices of systolic and diastolic function derived from pressure-volume
(P-V) relations in chagasic rats (± PBN/BZ)
Parameters
NH
Baseline measurements
Heart rate
(HR, beats/min)
367.2 ± 33.4
End systolic
volume (ESV, µl)
53.6 ± 2.8
End diastolic
volume (EDV, µl)
197.5 ± 7.1
Stroke volume
(SV, µl)
157.7 ± 4.5
Cardiac output
(CO, ml/min)
57.8 ± 1.0
Ejection fraction
(EF, %)
72.9 ± 1.1
dp/dt max
(mm Hg/sec)
5255 ± 133
- dp/dt max
(mm Hg/sec)
4642 ± 777
CH
CHP
CHB
CHBP
228.5 ± 55.5***
349.8 ± 47.7##
249.8 ± 24.8
352.7 ± 47.1##
146.8 ± 22***
55.1 ± 9.2##
115.9 ± 25.5
58.9 ± 9.6##
284 ± 14.8***
193.8 ± 4.3#
248.9 ± 27.5
201.6 ± 27.9##
137.2 ± 20.3***
142.5 ± 5.9##
133 ± 19.7#
160.2 ± 13.2##
30.9 ± 1.59***
49.8 ± 5.2##
32.9 ± 0.9
56.6 ± 2.6##
48.4 ± 7***
71.2 ± 4.8##
53.7 ± 7.3
70.7 ± 5.7##
3794 ± 128***
4494 ± 137##
3890 ± 485
4551 ± 358##
2645 ± 386***
3828 ± 1157##
2856 ± 336
3708 ± 466##
Measurements after inferior vena cava occlusion
ESPVR slope
(mm Hg/µl)
EDPVR slope
0.143 ± 0.008
0.074 ± 0.005***
0.129 ± 0.023##
0.081 ± 0.006#
0.134 ± 0.011##
(mm Hg/µl)
0.025 ± 0.003
0.042 ± 0.004***
0.029 ± 0.002##
0.041 ± 0.002
0.024 ± 0.003##
+dp/dt max: peak rate of pressure rise, -dp/dt max: peak rate of pressure decline, ESPVR: end systolic P-V relation slope,
EDPVR: end diastolic P-V relation slope, E SV/EDV: blood volume in left ventricle at the end of contraction/filling,
SV=EDV-ESV, CO (volume of blood pumped by the heart/min) = SV × HR, EF (fraction of blood ejected by LV during a
contraction) = (SV / EDV) × 100%. Abbreviations: NH-normal heart, AH-acutely-infected/untreated (25-dpi), AHPacutely- infected/PBN-treated, CH-chronically-infected/untreated (>150-dpi), CHP-chronically-infected/PBN-treated, CHBchronically-infected/benzonidazole-treated, CHBP-chronically-infected/PBN+BZ-treated. Statistical analysis was done
using Mann Whitney and Kruskal-Wallis test. The level of significance between normal versus infected/untreated rat is
shown by *; and between infected versus PBN and/or BZ-treated mice by # (*, #p<0.05; **, ##p<0.01, ***p<0.001).
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