Phenyl--tert-Butyl Nitrone Reverses Mitochondrial Decay in Acute Chagas?? Disease

【摘要】  In this study, we investigated the mechanism(s) of mitochondrial functional decline in acute Chagas?? disease. Our data show a substantial decline in respiratory complex activities (39 to 58%) and ATP (38%) content in Trypanosoma cruzi-infected murine hearts compared with normal controls. These metabolic alterations were associated with an approximately fivefold increase in mitochondrial reactive oxygen species production rate, substantial oxidative insult of mitochondrial membranes and respiratory complex subunits, and >60% inhibition of mtDNA-encoded transcripts for respiratory complex subunits in infected myocardium. The antioxidant phenyl--tert-butyl nitrone (PBN) arrested the oxidative damage-mediated loss in mitochondrial membrane integrity, preserved redox potential-coupled mitochondrial gene expression, and improved respiratory complex activities (47 to 95% increase) and cardiac ATP level (40% increase) in infected myocardium. Importantly, PBN resulted twofold decline in mitochondrial reactive oxygen species production rate in infected myocardium. Taken together, our data demonstrate the pathological significance of oxidative stress in metabolic decay and energy homeostasis in acute chagasic myocarditis and further suggest that oxidative injuries affecting mitochondrial integrity-dependent expression and activity of the respiratory complexes initiate a feedback cycle of electron transport chain inefficiency, increased reactive oxygen species production, and energy homeostasis in acute chagasic hearts. PBN and other mitochondria-targeted antioxidants may be useful in altering mitochondrial decay and oxidative pathology in Chagas?? disease.
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Chagas?? disease is a pathological process induced by infections with the hemoflagellate protozoan Trypanosoma cruzi and is a major human health problem in the southern parts of the American continent.1 In >95% of acutely infected individuals, parasitemia is controlled by the immune system. After several years of a clinically silent but ongoing process of organelle and myocardial degeneration, >40% of seropositive patients develop chronic cardiomyopathy.
Since early findings of abnormal mitochondria in cardiac biopsies obtained from seropositive patients,2 mitochondrial impairment has been associated with cardiac dysfunction in Chagas?? disease. Quantitative light and electron microscopic analysis of the myocardial biopsy samples from human chagasic patients and experimental models has revealed that mitochondrial degenerative changes occur early in the course of T. cruzi infection and are exacerbated with progressive disease severity.2-5 The functional decline of cardiac mitochondria in experimental models of Chagas?? disease was shown by impaired activities of the respiratory complexes that contain subunits encoded by mtDNA and nDNA.6-8 The demonstration of inefficient ATP production in infected mice8 provided the first indication of the physiological effects of mitochondrial dysfunction in chagasic hearts.
Toward understanding the mechanism(s) of mitochondrial decay, it is important to note that mitochondria are exposed to consistent oxidative stress in chagasic hearts. T. cruzi infection elicits host immune responses.9 These immune mechanisms control parasite burden in infected host through release of reactive oxygen species (ROS) and reactive nitrogen species.9,10 In the heart, superoxide anions (O2) are produced as a by-product of mitochondrial electron transport chain.11 This mitochondrial reactive oxygen species (mtROS) generation can increase exponentially when the electron transport chain functions at a suboptimal level12,13 and might be the case in infected myocardium consisting of impaired activity of CI and CIII respiratory complexes.8 It is, thus, likely that ROS-mediated responses, through various interrelated mechanisms, may contribute to a mitochondrial functional decline in chagasic hearts. For example, free radical-mediated oxidation of mitochondria, as noted in infected myocardium,14 can affect mitochondrial biogenesis,15-17 integrity,18 and/or gene expression.19 Oxidative modifications of mitochondrial membranes can result in the opening of the mitochondrial permeability transition pores and dissipation of the proton gradient, essential for the efficient transfer of electrons through the electron transport chain and ATP formation.20,21 The mtDNA is particularly susceptible to oxidative damage and, if not repaired properly, can lead to mtDNA mutation and/or depletion that would affect the expression and assembly of respiratory complexes, and, subsequently, the electron transport chain efficiency.22-25 These mechanisms, however, remain to be delineated in chagasic myocarditis.
Phenyl-N-tert-butylnitrone (PBN), a nitrone-based compound, is a potent antioxidant. PBN has been shown to 1) trap or scavenge a wide variety of free radical species, including biologically relevant O2 and hydroxyl (OH?

【关键词】  phenyl--tert-butyl reverses mitochondrial chagas??

Materials and Methods

Mice and Parasites

Six- to 8-week-old male C3H/HeN mice were purchased from Harlan (Indianapolis, IN). T. cruzi trypomastigotes (SylvioX10/4 strain) were maintained and propagated by the continuous in vitro passage of parasites in monolayers of C2C12 cells28 and used for infection of mice (10,000/mouse, intraperitoneal). Mice were given PBN (50 mg/kg) by intraperitoneal injection on alternate days throughout the course of infection. Mice were sacrificed in acute infection phase (27 to 35 days after infection). Animal experiments were performed according to the National Institutes of Health Guide for Care and Use of Experimental Animals and approved by the University of Texas Medical Branch Animal Care and Use Committee.

DNA and RNA Isolation

Heart tissue sections (50 mg) were finely chopped and incubated for 4 hours at 55??C in 250 µl of lysis buffer . Tissue lysates were extracted twice with an equal volume of phenol/chloroform/isoamylalcohol (24:24:1), and total DNA was purified by ethanol precipitation. All DNA samples were suspended in 100 µl of distilled H2O.5

For RNA isolation, freshly excised hearts were cut into pieces, blotted onto paper towels to remove excess blood, and flash-frozen in liquid nitrogen. The frozen tissues were individually transferred into guanidine-phenol solution and processed with a tissue homogenizer, and the total RNA was isolated as described.29,30 Total RNA was resolved by denaturing formaldehyde gel electrophoresis, quantitated, and stored at C80??C.

Mitochondria Isolation

Heart tissue sections were homogenized in 20 mmol/L Tris-HCl at pH 7.4, containing 250 mmol/L sucrose, 2 mmol/L K2EGTA , and cardiac mitochondria isolated by differential centrifugation.8 When tissue samples were processed for estimation of protein carbonyls and thiobarbituric acid-reactive substances, extraction buffer included 2% ß-mercaptoethanol and 500 µmol/L butylated hydroxytoluene, respectively.

Parasite Detection

For parasite detection, polymerase chain reaction (PCR) was performed for 28 cycles, using T. cruzi 18S rDNA-specific oligonucleotides5 with 2 µl of the total DNA as template. Denaturation, annealing, and elongation steps were performed for 1 minute each at 94??C, 58??C, and 72??C, respectively. A 5-µl aliquot of each PCR reaction was resolved on a 1.2% agarose gel. The ethidium bromide-stained gels were visualized using long-wave UV light and densitometric analysis performed on a FluorChem Imaging System (Alpha Innotech, San Leandro, CA).

Mitochondrial DNA and mRNA Levels

The mtDNA content in infected murine hearts was examined by Southern blot analysis.8 In brief, total DNA samples were digested with BamHI restriction enzyme, resolved on 1% agarose gel, and transferred to Zeta-Probe membrane (Bio-Rad, Hercules, CA). Membranes were hybridized with 32P-labeled CO2 cDNA probe and exposed to a phosphorimaging screen. The images were captured using a Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and densitometric analysis was performed. After stripping off the CO2 probe, the same membranes were hybridized with a ß-actin probe.

We examined mitochondrial gene expression by Northern blot analysis.8 Total RNA samples were resolved on 1% agarose gel containing 2 mol/L formaldehyde and transferred to the Zeta-Probe membrane. Membranes were hybridized with 32P-labeled cDNA probes (ND4L, CO1, CO2, ATP6, ATP8)8 and the images captured and analyzed, as above. All cDNAs used as probe were amplified by reverse transcriptase (RT)-PCR using gene-specific primer pairs and total RNA isolated from C57BL/6 mouse as template. The cDNA amplicons were cloned in Topo (T) vector and confirmed by restriction digestion and sequencing at a University of Texas Medical Branch core facility.8

Real-Time RT-PCR

First-strand cDNA was synthesized from total RNA (2 µg) using 2.5 U of Superscript II reverse transcriptase and 1 µmol/L poly(dT)18 oligonucleotide at 42??C for 50 minutes in a 20-µl reaction volume. Real-time PCR reactions (25-µl volume) were performed in an iCycler thermal cycler (Bio-Rad) using SYBR-Green Supermix (Bio-Rad) with 2 µl of the 10-fold diluted cDNA and 5 µl of 1 µmol/L primer pair specific for antioxidant cDNAs31 and mitochondria-encoded genes.8 The PCR base line subtracted curve fit mode was applied for threshold cycle (Ct) determination, using iCycler iQ real-time detection system software, version 3.0a (Bio-Rad). For each target gene, the Ct values were normalized to GAPDH expression and represent the mean of triplicate samples in two independent experiments. The relative expression level of each target gene in infected mice was calculated using the formula fold change = 2CCt, where Ct represents the Ct (infected sample) C Ct (control). To visualize better the mRNA levels in all groups, bar graphs were generated by plotting the 1/2Ct values on the y axis.32

Respiratory Complex Activities

Cardiac mitochondria were isolated in 5 mmol/L HEPES at pH 7.2, containing 210 mmol/L mannitol, 70 mmol/L sucrose, 1 mmol/L EGTA, and 0.1% fatty acid-free bovine serum albumin. The activities of the respiratory complexes (CI, CII, and CIII) and citrate synthase were monitored by spectrophotometric methods as described.8,33 Total protein was measured using the Bradford assay34 and specific activities evaluated using extinction coefficients: CI, 5.5 mmol/LC1 ?

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作者单位：From the Departments of Microbiology and Immunology* and Pathology, University of Texas Medical Branch, Galveston, Texas