Mitochondrial DNA dysfunction in oncocytic hepatocytes

Abstract: Hepatic oncocytes with abundant granular, eosinophilic cytoplasm due to mitochondrial hyperplasia are seen in various chronic liver diseases, particularly chronic hepatitis and cirrhosis. Increased mitochondria in oncocytes are thought to be a compensatory mechanism for deficiencies in the hepatocellular respiratory chain, although the pathogenesis of these deficiencies has been uncertain. We selected seven cases of cirrhosis (six with oncocytes, one without) for the following analysis: histoenzymatic and immunohistochemical staining of several mitochondrial DNA (mtDNA)‐ and nuclear DNA (nDNA)‐encoded respiratory chain enzymes; immuno‐staining using antibodies against double‐strand‐DNA (anti‐DNA) and against Ki‐67 (a cell proliferation marker); and Southern blot analysis for mtDNA and nDNA. Eighty percent of oncocytes showed histoenzymatic and immunohistochemical deficiencies of cytochrome c oxidase and the mtDNA‐encoded subunit I of complex IV, with preserved expression of nDNA‐encoded succinate dehydrogenase and the iron‐sulfur subunit of complex III (FeS). Cytoplasmic (but not nuclear) anti‐DNA staining was partially or completely absent in approximately 50% of oncocytes. Three cases with oncocytes studied by Southern blot showed mtDNA reductions of 66%, 71%, and 85%. In conclusion, hepatic oncocytes demonstrate significant deficiencies of mtDNA and mtDNA‐encoded respiratory chain enzymes. We propose that mtDNA depletion plays an important role in hepatocellular oncocytic transformation.     

Characterization and prognostic significance of mitochondrial DNA variations in acute myeloid leukemia

Recent studies have suggested that mutations in the mitochondrial genome (mtDNA) may play a role in the development and response to treatment for human cancer. The aim of this study was to investigate whether mtDNA variations have any prognostic relevance, to clarify the spectra of mtDNA variation and to determine whether there was any correlation to known prognostic factors in acute myeloid leukemia (AML). To elucidate this, we sequenced the entire mtDNA in 56 AML patients and 14 control subjects. When analyzing the biologic impact of the non‐synonymous variations in the mtDNA coding genes, we found an inferior disease‐free survival for patients exhibiting variations in the two most important catalytic genes of the complex IV of the oxidative phosphorylation complexes (OXPHOS), that is, the cytochrome c oxidase subunit I and the cytochrome c oxidase subunit II (hazard ratio 2.6, P = 0.03; multivariate analysis). In addition, the most frequent variation was the T16311C in the control region, which was found in 11 (20%) of the 56 patients. This observation was confirmed in another cohort of 173 diagnostic AML samples. In this expanded group, the T16311C variation tended to be associated with chromosomal abnormalities.     

Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants

We previously have shown that Nicotiana sylvestris cytoplasmic male sterile (CMS) mutants I and II present large mtDNA deletions and that the NAD7 subunit of complex I (the main dehydrogenase of the mitochondrial respiratory chain) is absent in CMS I. Here, we show that, despite a large difference in size in the mtDNA deletion, CMS I and II display similar alterations. Both have an impaired development from germination to flowering, with partial male sterility that becomes complete under low light. Besides NAD7, two other complex I subunits are missing (NAD9 and the nucleus-encoded, 38-kDa subunit), identified on two-dimensional patterns of mitochondrial proteins. Mitochondria isolated from CMS leaves showed altered respiration. Although their succinate oxidation through complex II was close to that of the wild type, oxidation of glycine, a priority substrate of plant mitochondria, was significantly reduced. The remaining activity was much less sensitive to rotenone, indicating the breakdown of Complex I activity. Oxidation of exogenous NADH (coupled to proton gradient generation and partly sensitive to rotenone) was strongly increased. These results suggest respiratory compensation mechanisms involving additional NADH dehydrogenases to complex I. Finally, the capacity of the cyanide-resistant alternative oxidase pathway was enhanced in CMS, and higher amounts of enzyme were evidenced by immunodetection.     

Comparative biochemical studies in fibroblasts from patients with different forms of Leigh syndrome

Summary We have compared respiratory chain enzyme activities, ATP synthesis, and ATP hydrolysis in cultured fibroblast mitochondria from patients with Leigh syndrome (LS) due to: (i) cytochrome oxidase (COX) deficiency (#6); (ii) pyruvate dehydrogenase complex (PDHC) deficiency (#4); and (iii) maternally inherited LS (MILS) with the T8993G mutation in the ATPase 6 gene of mtDNA (#5). Enzyme activities were normal in patients with MILS and variably decreased in those with COX and PDHC deficiency. ATP hydrolysis was normal or mildly decreased in all three groups. In contrast, ATP synthesis was decreased in all patients but more markedly in those with MILS, and especially with pyruvate/malate as substrate. These studies show that impaired ATP production is the common feature of all three forms of LS, but it is both more severe and more specific in MILS, consistent with the genetic defect.     

Effects of in utero antiretroviral exposure on mitochondrial DNA levels, mitochondrial function and oxidative stress

Objectives

HIV and antiretroviral (ART) exposure in utero may have deleterious effects on the infant, but uncertainty still exists. The objective of this study was to evaluate aspects of mitochondrial DNA (mtDNA) content, mitochondrial function and oxidative stress simultaneously in placenta, umbilical cord blood and infant blood in HIV/ART‐exposed infants compared with uninfected controls.

Methods

HIV‐1‐infected pregnant women and HIV‐1‐uninfected healthy pregnant controls were enrolled in the study prospectively. Placenta and umbilical cord blood were obtained at delivery and infant blood was obtained within 48 h of delivery. mtDNA content was determined for each specimen. Nuclear [subunit IV of cytochrome c‐oxidase (COX IV)]‐ and mitochondrial (COX II)‐encoded polypeptides of the oxidative phosphorylation enzyme cytochrome c‐oxidase were quantified in cord and infant blood. Placental mitochondria malondialdehyde (MDA) concentrations were measured as a marker of oxidative stress.

Results

Twenty HIV‐positive/HIV‐exposed and 26 control mother–infant pairs were enrolled in the study. All HIV‐infected women and their infants received ART. Placental MDA concentration and mtDNA content in placenta and cord blood were similar between groups. The cord blood COX II:IV ratio was lower in the HIV‐positive group than in the controls, whereas the infant peripheral blood mtDNA content was higher in the HIV‐exposed infants, but the infant peripheral blood COX II:IV ratio was similar. No infant had clinical evidence of mitochondrial disease or acquired HIV infection. In multivariable regression analyses, the significant findings in cord and infant blood were both most associated with HIV/ART exposure.

Conclusions

HIV‐exposed infants showed reduced umbilical cord blood mitochondrial enzyme expression with increased infant peripheral blood mitochondrial DNA levels, the latter possibly reflecting a compensatory mechanism to overcome HIV/ART‐associated mitochondrial toxicity.     

Inhibition of Mitochondrial Permeability Transition Pores by Cyclosporine A Improves Cytochrome c Oxidase Function and Increases Rate of ATP Synthesis in Failing Cardiomyocytes

Background: We previously showed that mitochondrial respiratory function is abnormal in dogs with chronic heart failure (HF). Mitochondrial permeability transition pores (MPTP) can affect mitochondrial inner membrane potential (Δ < eqid1 > _m) and mitochondrial function in normal cardiomyocytes. The potential impact of MPTP on Δ < eqid2 > _m and mitochondrial respiratory function in HF has not yet been determined. We tested the hypothesis that cyclosporine A, a potent blocker of the MPTP, can improve mitochondrial function in HF. Methods: Cardiomyocytes were isolated from the left ventricular myocardium of 7 dogs with HF produced by intracoronary microembolizations and from 7 normal dogs. Cardiomyocytes were treated for 24 hours with cyclosporine A. Δ < eqid3 > _m, cytochrome c oxidase protein expression, mitochondrial cytochrome c oxidase-dependent respiration (CDOR) and ATP synthesis were measured. Results: Δ < eqid4 > _m, protein expression of cytochrome c oxidase, CDOR and the rate of ATP synthesis were decreased in HF compared to normal controls. Inhibition of MPTP in failing cardiomyocytes with low dose of cyclosporine A (0.2 μM) increased Δ < eqid5 > _m, preserved expression of cytochrome c oxidase, improved CDOR and the rate of ATP synthesis. Conclusion: MPTP opening contributes to the loss of mitochondrial function observed in the failing heart. Inhibition of MPTP opening represents a potential therapeutic target for the treatment of HF. This study was supported by a grant from the National Heart, Lung, and Blood Institute PO1 HL074237-01.     

Fatal dilated cardiomyopathy associated with a mitochondrial DNA deletion

A 27-year-old man was admitted to hospital because of severe cardiac failure. Investigation revealed dilated cardiomyopathy with a left ventricular ejection fraction of 15-20%. During adolescence the patient had been investigated for growth retardation and he also had progressive external ophthalmoplegia. There had been no symptoms of cardiac disease until 2 weeks before admittance. An endomyocardial biopsy showed cardiomyocytes deficient in cytochrome c oxidase (COX) in a mosaic pattern. A skeletal muscle biopsy showed mitochondrial myopathy with COX-deficient ragged-red fibers. Molecular genetic analysis revealed a heteroplasmic, 3.8-kb, mitochondrial DNA (mtDNA) deletion in heart and muscle. PCR-based quantification of the proportion of mtDNA with deletion showed 47% mutated mtDNA in the myocardial biopsy and 68% in muscle. In spite of treatment, the condition deteriorated and the patient died 5 days after admittance. This case demonstrates that mtDNA deletions may occasionally be the cause of severe dilated cardiomyopathy, and that morphological and molecular genetic diagnosis may be obtained by endomyocardial biopsy.     

Palmitate oxidation and cytochromes in human and rat heart

Palmitate oxidation rates were determined in mitochondria and homogenates of human and rat heart muscle. Oxidation rates based on the sum of the production of ¹⁴CO₂ and ¹⁴C-labeled acid-soluble productsfrom [¹⁴C]palmitatewere in good agreement with those calculated from the oxygen consumption during palmitate oxidation. With limited amounts of substrate palmitate was completely oxidized to CO₂, but with non-limiting palmitate concentrations acetyl-carnitine and citric acid cycle intermediates accumulated. Palmitate oxidation rates per mg protein were about equal for human and rat heart muscle. Palmitate oxidation rates by homogenates and mitochondria were about equal, when expressed on the basis of cytochrome c oxidase activity for each species, but they differed between rat and man. This difference is caused by the lower activity of cytochrome c oxidase in human heart mitochondria. Difference spectrophotometric measurements showed, however, higher concentrations of cytochrome aa₃ and c + c₁ in human heart mitochondria than in those of the rat.     

Five TGA “stop” codons occur within the translated sequence of the yeast mitochondrial gene for cytochrome c oxidase subunit II

A mitochondrial mutation that genetically maps in the middle of the gene coding cytochrome c oxidase subunit II has been found to be a single-base-pair deletion. Three independently isolated spontaneous revertants of this mutant have different single-base-pair insertions within 15 nucleotides of the mutation. These findings clearly identify the location of the gene and suggest that the mutation causes a frame-shift. The sequence of about 900 base pairs surrounding the mutation has been determined and found to have several chain termination codons in every possible reading frame. The sequence can, however, be translated in one frame by assuming that the codon TGA does not cause chain termination in yeast mitochondira, as was recently suggested for the human organelle [Barrell, B. G., Bankier, A. T. & Drouin, J. (1979) Nature (London), in press]. If TGA codes for tryptophan residues, as is apparently the case in human mitochondria, a polypeptide can be read from the yeast mtDNA that is identical to bovine cytochrome oxidase subunit II at 37.8% of its residues. Furthermore, the DNA sequences of the frame-shift revertants discussed above predict relative isolectric point differences between the wild-type and various revertant forms of the polypeptide. The detection of these isolectric point differences by two-dimensional electrophoresis of subunit II from the various strains independently confirms the presumed reading frame of the gene. It is concluded that TGA is translated in yeast mitochondria, most probably as tryptophan.     

Detection of "deleted" mitochondrial genomes in cytochrome-c oxidase-deficient muscle fibers of a patient with Kearns-Sayre syndrome

Using in situ hybridization and immunocytochemistry, we studied a muscle biopsy sample from a patient with Kearns-Sayre syndrome (KSS) who had a deletion of mitochondrial DNA (mtDNA) and partial deficiency of cytochrome-c oxidase (COX; EC 1.9.3.1). We sought a relationship between COX deficiency and abnormalities of mtDNA at the single-fiber level. COX deficiency clearly correlated with a decrease of normal mtDNA and, conversely, deleted mtDNA was more abundant in COX-deficient fibers, especially ragged-red fibers. The distribution of mtRNA had a similar pattern, suggesting that deleted mtDNA is transcribed. Immunocytochemistry showed that the nuclear DNA-encoded subunit IV of COX was present but that the mtDNA-encoded subunit II was markedly diminished in COX-deficient ragged-red fibers. Because the mtDNA deletion in this patient did not comprise the gene encoding COX subunit II, COX deficiency may have resulted from lack of translation of mtRNA encoding all three mtDNA-encoded subunits of COX.     

Deletion of the mitochondrial NADH kinase increases mitochondrial DNA stability and life span in the filamentous fungus Podospora anserina

In the filamentous fungus Podospora anserina, aging is systematically associated with mitochondrial DNA (mtDNA) instability. A causal link between deficiency of the cytochrome respiratory pathway and lifespan extension has been demonstrated. Knock out of the cytochrome respiratory pathway induces the expression of an alternative oxidase and is associated with a reduction in free radical production. The question of the links between mtDNA stability, ROS generation and lifespan is therefore clearly raised in this organism. NADPH lies at the heart of many anti-oxidant defenses of the cell. In Saccharomyces cerevisiae, the mitochondrial NADPH is largely provided by the Pos5 NADH kinase. We show here that disruption of PaNdk1 encoding the potential mitochondrial NADH kinase of P. anserina leads to severe somatic and sexual defects and to hypersensitivity to hydrogen peroxide and paraquat. Surprisingly, it also leads to a spectacular increase of mtDNA stability and lifespan. We propose that an adaptative metabolic change including the induction of the alternative oxidase can account for these results.     

Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion: a slip-replication model and metabolic therapy.

The muscle mitochondria of a patient with Kearns-Sayre/chronic external ophthalmoplegia plus syndrome were found to be completely deficient in respiratory complex I activity and partially deficient in complex IV and V activities. Treatment of the patient with coenzyme Q10 and succinate resulted in clinical improvement of respiratory function, consistent with the respiratory deficiencies. Restriction enzyme analysis of the muscle mtDNA revealed a 4.9-kilobase deletion in 50% of the mtDNA molecules. Polymerase chain reaction analysis demonstrated that the deletion was present in the patient's muscle but not in her lymphocytes or platelets. Furthermore, the deletion was not present in the muscle or platelets of two sisters. Hence, the mutation probably occurred in the patient's somatic cells. Direct sequencing of polymerase chain reaction-amplified DNA revealed a 4977-base-pair deletion removing four genes for subunits of complex I, one gene for complex IV, two genes for complex V, and five genes for tRNAs, which paralleled the respiratory enzymes affected in the disease. A 13-base-pair direct repeat was observed upstream from both breakpoints. Relative to the direction of heavy-strand replication, the first repeat was retained and the second repeat was deleted, suggesting a slip-replication mechanism. Sequence analysis of the human mtDNA revealed many direct repeats of 10 base pairs or greater, indicating that this mechanism could account for other reported deletions. We postulate that the prevalence of direct repeats in the mtDNA is a consequence of the guanine-cytosine bias of the heavy and light strands.     

Stochastic mitochondrial DNA changes: bioenergy decline in type I skeletal muscle fibres correlates with a decline in the amount of amplifiable full-length mtDNA

Extra-long PCR (XL-PCR) was used to assess the relative concentration of functional full-length mitochondrial DNA (mtDNA) in single type I human vastus lateralis muscle fibres of defined cytochrome c oxidase (COX)activity. Type I muscle fibres rely more on mitochondrial oxidative phosphorylation for their energy demands, compared to the other common fibre types (IIa, IIab and IIb) that principally depend on glycolysis for their energy requirements. A total of 60 single type I fibres were analyzed from 15 individuals (8males and 7 females) of various ages. COX positive muscle fibres were shown to contain amplifiable full-length mtDNA together with a small number of mtDNA rearrangements. By contrast, COX negative fibres did not contain detectable full-length mtDNA, but did contain aheterogeneous mixture of rearranged mtDNA species with the frequency and occurrence of each deletion varying considerably from fibre to fibre. These data lead us to the conclusion that the level of COX activity in type I muscle fibres is reflected by the amount of amplifiable full-length mtDNA. It is proposed that the amount of amplifiable full-length mtDNA constitutes the functional fraction of the total mtDNA. A comprehensive hypothesis that relates the dynamics of mtDNA turnover, mtDNA mutations, mtDNA damage and repair to the ageing process is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Polyunsaturated fatty acid deficiency reverses effects of alcohol on mitochondrial energy metabolism

BACKGROUND/AIMS: Polyunsaturated fatty acids (PUFA) deficiency is common in patients with alcoholic liver disease. The suitability of reversing such deficiency remains controversial. The aim was to investigate the role played by PUFA deficiency in the occurrence of alcohol-related mitochondrial dysfunction. METHODS: Wistar rats were fed either a control diet with or without alcohol (control and ethanol groups) or a PUFA deficient diet with or without alcohol (PUFA deficient and PUFA deficient+ethanol groups). After 6 weeks, liver mitochondria were isolated for energetic studies and fatty acid analysis. RESULTS: Mitochondria from ethanol fed rats showed a dramatic decrease in oxygen consumption rates and in cytochrome oxidase activity. PUFA deficiency showed an opposite picture. PUFA deficient+ethanol group roughly reach control values, regarding cytochrome oxidase activity and respiratory rates. The relationship between ATP synthesis and respiratory rate was shifted to the left in ethanol group and to the right in PUFA-deficient group. The plots of control and PUFA deficient+ethanol groups were overlapping. Phospholipid arachidonic over linoleic ratio closely correlated to cytochrome oxidase and oxygen uptake. CONCLUSIONS: PUFA deficiency reverses alcohol-related mitochondrial dysfunction via an increase in phospholipid arachidonic over linoleic ratio, which raises cytochrome oxidase activity. Such deficiency may be an adaptive mechanism.     

Heme-copper terminal oxidase using both cytochrome c and ubiquinol as electron donors

The cytochrome c oxidase Cox2 has been purified from native membranes of the hyperthermophilic eubacterium Aquifex aeolicus. It is a cytochrome ba₃ oxidase belonging to the family B of the heme-copper containing terminal oxidases. It consists of three subunits, subunit I (CoxA2, 63.9 kDa), subunit II (CoxB2, 16.8 kDa), and an additional subunit IIa of 5.2 kDa. Surprisingly it is able to oxidize both reduced cytochrome c and ubiquinol in a cyanide sensitive manner. Cox2 is part of a respiratory chain supercomplex. This supercomplex contains the fully assembled cytochrome bc₁ complex and Cox2. Although direct ubiquinol oxidation by Cox2 conserves less energy than ubiquinol oxidation by the cytochrome bc₁ complex followed by cytochrome c oxidation by a cytochrome c oxidase, ubiquinol oxidation by Cox2 is of advantage when all ubiquinone would be completely reduced to ubiquinol, e.g., by the sulfide∶quinone oxidoreductase, because the cytochrome bc₁ complex requires the presence of ubiquinone to function according to the Q-cycle mechanism. In the case that all ubiquinone has been reduced to ubiquinol its reoxidation by Cox2 will enable the cytochrome bc₁ complex to resume working.     

Genomic analysis of mutations in platelet mitochondria in a case of benzene-induced leukaemia: A case report

Introduction:As a hematopoietic carcinogen, benzene induces human leukemia through its active metabolites such as benzoquinone, which may cause oxidative damage to cancer-related nuclear genes by increasing reactive oxygen species (ROS). Mitochondrion is the main regulatory organelle of ROS, genetic abnormality of mitochondrion can impede its regulation of ROS, leading to more severe oxidative damage. Mutations have been related to certain types of cancer in several mitochondrial genes, but they have never been completely analyzed genome-wide in leukemia.Patient concerns:The patient was a 52-year-old female who had chronic exposure to benzene for several years. Her symptoms mainly included recurrent dizziness, fatigue, and they had lasted for nearly 8 years and exacerbated in recent weeks before diagnosis.Diagnosis:Samples of peripheral blood were taken from the patient using evacuated tubes with EDTA anticoagulant on the second day of her hospitalization. At the same time blood routine and BCR/ABL genes of leukemic phenotype were tested. Platelets were isolated for mitochondrial DNA (mtDNA) extraction. The genetic analysis of ATP synthase Fo subunit 8 (complex V), ATP synthase Fo subunit 6 (complex V), cytochrome c oxidase subunit 1 (complex IV), cytochrome c oxidase subunit 2 (complex IV), cytochrome c oxidase subunit 3, Cytb, NADH dehydrogenase subunit 1 (complex I) (ND) 1, ND2, ND3, ND4, ND5, ND6, 12S-RNA, 16S-RNA, tRNA-Cysteine, A, N, tRNA-Leucine, E, displacement loop in platelet mtDNA were performed. All the detected gene mutations were validated using the conventional Sanger sequencing method.Interventions:The patient received imatinib, a small molecule kinase inhibitor, and symptomatic treatments.Outcomes:After 3 months treatment her blood routine test indicators were restored to normal.Conclusion:A total of 98 mutations were found, and 25 mutations were frame shift. The ND6 gene mutation rate was the highest among all mutation points. Frame shifts were identified in benzene-induced leukemia for the first time. Many mutations in the platelet mitochondrial genome were identified and considered to be potentially pathogenic in the female patient with benzene-induced leukemia. The mutation rate of platelet mitochondrial genome in the benzene-induced leukemia patient is relatively high, and the complete genome analysis is helpful to fully comprehend the disease characteristics.     

Cardiac mitochondrial dysfunction and DNA depletion in children with hypertrophic cardiomyopathy

Abnormalities in specific mitochondrial respiratory enzymes and DNA (mtDNA) have been reported in cardiomyopathy. In this study, we report 4 cases of severe hypertrophic cardiomyopathy (HCM) in which specific cardiac mitochondrial enzyme activity defects were found, including complex I (n = 2), complex III (n = 2), complex IV (n = 2) and complex V (n = 1). Other abnormalities were also noted including a marked depletion of mtDNA (n = 1) and decreased content of subunit 2 of cytochrome c oxidase (n = 1). None of the mtDNA point mutations and common deletions previously found in association with cardiomyopathy were detected in these patients. These data indicate that specific respiratory enzyme activity defects are frequently present in HCM. Also, our finding of a marked depletion of mtDNA in 1 patient suggests that cardiac mtDNA depletion, previously unreported in HCM, needs further examination in order to establish whether it plays a primary role in its pathogenesis.     

Variation in mitochondrial DNA levels in muscle from normal controls. Is depletion of mtDNA in patients with mitochondrial myopathy a distinct clinical syndrome?

Summary Recent studies have identified a group of patients with cytochrome oxidase (COX) deficiency presenting in infancy associated with a deficiency of mtDNA in muscle or other affected tissue (Moraes et al 1991). We used a novel approach to compare the level of mitochondrial (mtDNA) compared to nuclear DNA in skeletal muscle from a group of patients and controls, based on dot blots that were hybridized with a mtDNA probe labelled with³⁵S[dCTP] and a reference nuclear DNA probe labelled with [³²P]dCTP.The ratio of mtDNA to nuclear DNA varied in samples from different muscles of the same individual. Secondly, fetal muscle had very low levels of mtDNA compared to nuclear DNA, and data from older controls (cross-sectional rather than sequential) suggest that this increases rapidly over the first 3 months after birth and thereafter more slowly. Four patients with COX deficiency had levels of mtDNA that were below the age-specific range defined by normal quadriceps muscle. The clinical features of two of these patients were similar to earlier case reports of mtDNA depletion. In three patients the clinical course was relatively benign compared to cases that have previously been described.Levels of mtDNA in skeletal muscle from some patients with other forms of muscle disease were also found to be low, suggesting that mtDNA depletion, possibly related to depletion of mitochondria, may be a relatively non-specific response of muscle to various pathological processes. However, there does appear to be a distinctive group of young patients with reduced cytochrome oxidase activity in muscle, in whom marked mtDNA depletion reflects the primary defect.