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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 1 (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 2 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). 3 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 4 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). 5 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 6 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 7 1. Tanabe S, Bunag RD. Age-related central and baroreceptor impairment in female Sprague-Dawley rats. Am J Physiol 1989;256:H1399-406. 2. 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; studies on dose, plasma concentration and therapeutic time window. Br J Pharmacol 2002;135:103-12. 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. 6. Harper A. Glucose-6-phospate. Methods of Enzymatic Analysis 1963:788-792. 7. Lui NS, Roels OA, Trout ME, Anderson OR. Subcellular distribution of enzymes in Ochromonas malhamensis. J Protozool 1968;15:536-42. 8. Sims NR, Anderson MF. Isolation of mitochondria from rat brain using Percoll density gradient centrifugation. Nat Protoc 2008;3:1228-39. 8 9. 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. 10. 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. 9 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. 19. Wen J-J, Vyatkina G, Garg N. Oxidative damage during chagasic cardiomyopathy development: Role of mitochondrial oxidant release and inefficient antioxidant 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). 11 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). 12 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. 13 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). 14