Mitochondrial DNA--all things bad?

Mutations in mitochondrial DNA (mtDNA) are undoubtedly associated with a diverse spectrum of human disorders. More controversially, it has been claimed that they accumulate during ageing, and that they are responsible for an age-related decline in bioenergetic function and tissue viability. Here, we review the evidence for this assertion, concluding that claims for the age-accumulation of mtDNA mutations are based largely on non-quantitative methods, and that no clear, functional deficit of mitochondrial respiration has been shown to result from such lesions in aged individuals. The mitochondrial theory of ageing, however attractive in principle, is supported by very little hard evidence.     

Clonal Expansion of Early to Mid-Life Mitochondrial DNA Point Mutations Drives Mitochondrial Dysfunction during Human Ageing

Age-related decline in the integrity of mitochondria is an important contributor to the human ageing process. In a number of ageing stem cell populations, this decline in mitochondrial function is due to clonal expansion of individual mitochondrial DNA (mtDNA) point mutations within single cells. However the dynamics of this process and when these mtDNA mutations occur initially are poorly understood. Using human colorectal epithelium as an exemplar tissue with a well-defined stem cell population, we analysed samples from 207 healthy participants aged 17–78 years using a combination of techniques (Random Mutation Capture, Next Generation Sequencing and mitochondrial enzyme histochemistry), and show that: 1) non-pathogenic mtDNA mutations are present from early embryogenesis or may be transmitted through the germline, whereas pathogenic mtDNA mutations are detected in the somatic cells, providing evidence for purifying selection in humans, 2) pathogenic mtDNA mutations are present from early adulthood (<20 years of age), at both low levels and as clonal expansions, 3) low level mtDNA mutation frequency does not change significantly with age, suggesting that mtDNA mutation rate does not increase significantly with age, and 4) clonally expanded mtDNA mutations increase dramatically with age. These data confirm that clonal expansion of mtDNA mutations, some of which are generated very early in life, is the major driving force behind the mitochondrial dysfunction associated with ageing of the human colorectal epithelium.     

Mitochondrial DNA integrity is not dependent on DNA polymerase-beta activity

Mutations in mitochondrial DNA (mtDNA) are involved in a variety of pathologies, including cancer and neurodegenerative diseases, as well as in aging. mtDNA mutations result predominantly from damage by reactive oxygen species (ROS) that is not repaired prior to replication. Repair of ROS-damaged bases occurs mainly via base excision repair (BER) in mitochondria and nuclei. In nuclear BER, the two penultimate steps are carried out by DNA polymerase-beta (Polbeta), which exhibits both 5'-deoxyribose-5-phosphate (5'-dRP) lyase and DNA polymerase activities. In mitochondria, DNA polymerase-gamma (Polgamma) is believed to be the sole polymerase and is therefore assumed to function in mitochondrial BER. However, a recent report suggested the presence of Polbeta or a "Polbeta-like" enzyme in bovine mitochondria. Consequently, in the present work, we tested the hypothesis that Polbeta is present and functions in mammalian mitochondria. Initially we identified two DNA polymerase activities, one corresponding to Polgamma and the other to Polbeta, in mitochondrial preparations obtained by differential centrifugation and discontinuous sucrose density gradient centrifugation. However, upon further fractionation in linear Percoll gradients, we were able to separate Polbeta from mitochondria and to show that intact mitochondria, identified by electron microscopy, lacked Polbeta activity. In a functional test for the presence of Polbeta function in mitochondria, we used a new assay for detection of random (i.e., non-clonal) mutations in single mtDNA molecules. We did not detect enhanced mutation frequency in mtDNA from Polbeta null cells. In contrast, mtDNA from cells harboring mutations in the Polgamma exonuclease domain that abolish proofreading displayed a >or=17-fold increase in mutation frequency. We conclude that Polbeta is not an essential component of the machinery that maintains mtDNA integrity.     

Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids

Ever increasing evidence has been provided on the accumulation of mutations in the mitochondrial DNA (mtDNA) during the aging process. However, the lack of direct functional consequences of the mutant mtDNA load on the mitochondria-dependent cell metabolism has raised many questions on the physiological importance of the age-related mtDNA variations. In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids. The results show that the T414G mutation does not cause per se any detectable bioenergetic change. Moreover, three mtDNA mutations clustered in the 16S ribosomal RNA gene cosegregated together with the T414G in the same cybrid cell line. Two of them, namely T1843C and A1940G, are novel and associate with a negative bioenergetic phenotype. The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids.     

Altered Mitochondrial Function and Oxidative Stress in Leukocytes of Anorexia Nervosa Patients

Context

Anorexia nervosa is a common illness among adolescents and is characterised by oxidative stress.

Objective

The effects of anorexia on mitochondrial function and redox state in leukocytes from anorexic subjects were evaluated.

Design and setting

A multi-centre, cross-sectional case-control study was performed.

Patients

Our study population consisted of 20 anorexic patients and 20 age-matched controls, all of which were Caucasian women.

Main outcome measures

Anthropometric and metabolic parameters were evaluated in the study population. To assess whether anorexia nervosa affects mitochondrial function and redox state in leukocytes of anorexic patients, we measured mitochondrial oxygen consumption, membrane potential, reactive oxygen species production, glutathione levels, mitochondrial mass, and complex I and III activity in polymorphonuclear cells.

Results

Mitochondrial function was impaired in the leukocytes of the anorexic patients. This was evident in a decrease in mitochondrial O₂ consumption (P<0.05), mitochondrial membrane potential (P<0.01) and GSH levels (P<0.05), and an increase in ROS production (P<0.05) with respect to control subjects. Furthermore, a reduction of mitochondrial mass was detected in leukocytes of the anorexic patients (P<0.05), while the activity of mitochondrial complex I (P<0.001), but not that of complex III, was found to be inhibited in the same population.

Conclusions

Oxidative stress is produced in the leukocytes of anorexic patients and is closely related to mitochondrial dysfunction. Our results lead us to propose that the oxidative stress that occurs in anorexia takes place at mitochondrial complex I. Future research concerning mitochondrial dysfunction and oxidative stress should aim to determine the physiological mechanism involved in this effect and the physiological impact of anorexia.     

Precise determination of mitochondrial DNA copy number in human skeletal and cardiac muscle by a PCR-based assay: lack of change of copy number with age

Deletions in mitochondrial DNA (mtDNA) accumulate with age in humans without overt mitochondriopathies, but relatively limited attention has been devoted to the measurement of the total number of mtDNA molecules per cell during ageing. We have developed a precise assay that determines mtDNA levels relative to nuclear DNA using a PCR-based procedure. Quantification was performed by reference to a single recombinant plasmid standard containing a copy of each target DNA sequence (mitochondrial and nuclear). Copy number of mtDNA was determined by amplifying a short region of the cytochrome b gene (although other regions of mtDNA were demonstrably useful). Nuclear DNA content was determined by amplification of a segment of the single copy β-globin gene. The copy number of mtDNA per diploid nuclear genome in myocardium was 6970 ± 920, significantly higher than that in skeletal muscle, 3650 ± 620 (P = 0.006). In both human skeletal muscle and myocardium, there was no significant change in mtDNA copy number with age (from neonates to subjects older than 80 years). This PCR-based assay not only enables accurate determination of mtDNA relative to nuclear DNA but also has the potential to quantify accurately any DNA sequence in relation to any other.     

Revisiting Kadenbach: Electron flux rate through cytochrome c‐oxidase determines the ATP‐inhibitory effect and subsequent production of ROS

Mitochondrial respiration is the predominant source of ATP. Excessive rates of electron transport cause a higher production of harmful reactive oxygen species (ROS). There are two regulatory mechanisms known. The first, according to Mitchel, is dependent on the mitochondrial membrane potential that drives ATP synthase for ATP production, and the second, the Kadenbach mechanism, is focussed on the binding of ATP to Cytochrome c Oxidase (CytOx) at high ATP/ADP ratios, which results in an allosteric conformational change to CytOx, causing inhibition. In times of stress, ATP‐dependent inhibition is switched off and the activity of CytOx is exclusively determined by the membrane potential, leading to an increase in ROS production. The second mechanism for respiratory control depends on the quantity of electron transfer to the Heme aa3 of CytOx. When ATP is bound to CytOx the enzyme is inhibited, and ROS formation is decreased, although the mitochondrial membrane potential is increased.     

The Plasma Mitochondrial DNA Is an Independent Predictor for Post-Traumatic Systemic Inflammatory Response Syndrome

Background and Purpose

Mitochondrial DNA (mtDNA), a newly identified damage-associated molecular pattern, has been observed in trauma patients, however, little is known concerning the relationship between plasma mtDNA levels and concrete post-traumatic complications, particularly systemic inflammatory response syndrome (SIRS). The aim of this study is to determine whether plasma mtDNA levels are associated with injury severity and cloud predict post-traumatic SIRS in patients with acute traumatic injury.

Patients and Methods

Eighty-six consecutive patients with acute traumatic injury were prospectively enrolled in this study. The plasma mtDNA concentration was measured by a real-time, quantitative PCR assay for the human ND2 gene. The study population’s clinical and laboratory data were analyzed.

Results

The median plasma mtDNA was higher in trauma patients than in healthy controls (865.196 (251.042-2565.40)pg/ml vs 64.2147 (43.9049-80.6371)pg/ml, P<0.001) and was independently correlated with the ISS score (r=0.287, P<0.001). The plasma mtDNA concentration was also significantly higher in patients who developed post-traumatic SIRS than in patients who did not (1774.03 (564.870-10901.3)pg/ml vs 500.496 (145.415-1285.60)pg/ml, P<0.001). Multiple logistic regression analysis revealed that the plasma mtDNA was an independent predictors for post-traumatic SIRS (OR, 1.183 (95%CI, 1.015-1.379), P=0.032). Further ROC analysis demonstrated that a high plasma mtDNA level predicted post-traumatic SIRS with a sensitivity of 67% and a specificity of 76%, with a cut-off value of 1.3185 µg/ml being established, and the area under the ROC curves (AUC) was 0.725 (95% CI 0.613-0.837).

Conclusions

Plasma mtDNA was an independent indictor with moderate discriminative power to predict the risk of post-traumatic SIRS.     

Leukocyte Mitochondrial DNA Copy Number Is Associated with Chronic Obstructive Pulmonary Disease

Background

Oxidative stress is known to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Evidence suggests that leukocytes mitochondria DNA (mtDNA) is susceptible to undergo mutations, insertions, or depletion in response to reactive oxidative stress (ROS). We hypothesize that mtDNA copy number is associated with the development of COPD.

Methodology/Principal Findings

Relative mtDNA copy number was measured by a quantitative real-time PCR assay using DNA extracted from peripheral leukocytes. MtDNA copy number of peripheral leukocytes in the COPD group (n = 86) is significantly decreased compared with non-smoker group (n = 77) (250.3± 21.5 VS. 464.2± 49.9, P<0.001). MtDNA copy number in the COPD group was less than that in the healthy smoking group, but P value nearly achieved significance (250.3± 21.5 VS. 404.0± 76.7, P = 0.08) MtDNA copy number has no significance with age, gender, body mass index, current smoking, and pack-years in COPD group, healthy smoker group and no smoker group, respectively. Serum glutathione level in the COPD group is significantly decreased compared with healthy smoker and non-smoker groups (4.5± 1.3 VS. 6.2± 1.9 and 4.5± 1.3 VS. 7.1±1.1 mU/mL; P<0.001 respectively). Pearson correlation test shows a significant liner correlation between mtDNA copy number and serum glutathione level (R = 0.2, P = 0.009).

Conclusions/Significance

COPD is associated with decreased leukocyte mtDNA copy number and serum glutathione. COPD is a regulatory disorder of leukocytes mitochondria. However, further studies are needed to determine the real mechanisms about the gene and the function of mitochondria.     

Impairment of Myocardial Mitochondria in Viral Myocardial Disease and Its Reflective Window in Peripheral Cells

Background

Viral myocardial disease (VMD) is a common disease inducing heart failure. It has not been clear the roles of mitochondrial damage in the pathological changes of cardiomyocytes in VMD.

Methods

Myocardial tissues and lymphocytes were collected from 83 VMD patients. Control groups included 12 cases of healthy accidental death with myocardial autopsy and 23 healthy blood donors. The mouse model of viral myocarditis (VMC) was established by Coxsackie virus B₃ infection and myocardial tissues and skeletal muscle were collected. Mitochondrial DNA (mtDNA) deletion rate was quantitatively determined using polymerase chain reaction.

Results

There was significantly difference of myocardial mitochondrial DNA deletion rate between VMD or VMC group and control group (P<0.05). Moreover, the loss of mitochondrial membrane phospholipids was significantly different between VMD or VMC group and control group. In VMC mice, there were negative correlations between myocardial mtDNA³⁸⁶⁷ deletion rate and left ventricular peak systolic pressure (LVPSP) (r = −0.66, P<0.05), and between myocardial mtDNA³⁸⁶⁷ deletion rate and +dp/dt_max (r = −0.79, P<0.05), while there was positive correlation between myocardial mtDNA³⁸⁶⁷ deletion rate and −dp/dt_max (r = 0.80, P<0.05).

Conclusion

Mitochondrial damage is an important pathophysiological mechanism leading to myocardial injury and cardiac dysfunction. The mitochondrial damage in the skeletal muscle and lymphocytes reflect a “window” of myocardial mitochondrial damage.     

Morphine-Induced Preconditioning: Involvement of Protein Kinase A and Mitochondrial Permeability Transition Pore

Background

Morphine induces myocardial preconditioning (M-PC) via activation of mitochondrial large conductance Ca²⁺-sensitive potassium (mK_Ca) channels. An upstream regulator of mK_Ca channels is protein kinase A (PKA). Furthermore, mK_Ca channel activation regulates mitochondrial bioenergetics and thereby prevents opening of the mitochondrial permeability transition pore (mPTP). Here, we investigated in the rat heart in vivo whether 1) M-PC is mediated by activation of PKA, and 2) pharmacological opening of the mPTP abolishes the cardioprotective effect of M-PC and 3) M-PC is critically dependent on STAT3 activation, which is located upstream of mPTP within the signalling pathway.

Methods

Male Wistar rats were randomised to six groups (each n = 6). All animals underwent 25 minutes of regional myocardial ischemia and 120 minutes of reperfusion. Control animals (Con) were not further treated. Morphine preconditioning was initiated by intravenous administration of 0.3 mg/kg morphine (M-PC). The PKA blocker H-89 (10 μg/kg) was investigated with and without morphine (H-89+M-PC, H-89). We determined the effect of mPTP opening with atractyloside (5 mg/kg) with and without morphine (Atr+M-PC, Atr). Furthermore, the effect of morphine on PKA activity was tested in isolated adult rat cardiomyocytes. In further experiments in isolated hearts we tested the protective properties of morphine in the presence of STAT3 inhibition, and whether pharmacological prevention of the mPTP-opening by cyclosporine A (CsA) is cardioprotective in the presence of STAT3 inhibition.

Results

Morphine reduced infarct size from 64±5% to 39±9% (P<0.05 vs. Con). H-89 completely blocked preconditioning by morphine (64±9%; P<0.05 vs. M-PC), but H-89 itself had not effect on infarct size (61±10%; P>0.05 vs. Con). Also, atractyloside abolished infarct size reduction of morphine completely (65±9%; P<0.05 vs. M-PC) but had no influence on infarct size itself (64±5%; P>0.05 vs. Con). In isolated hearts STAT3 inhibitor Stattic completely abolished morphine-induced preconditioning. Administration of Stattic and mPTP inhibitor cyclosporine A reduced infarct size to 31±6% (Stat+CsA, P<0.05 vs. Con). Cyclosporine A alone reduced infarct size to 26±7% (CsA P<0.05 vs. Con). In cardiomyocytes, PKA activity was increased by morphine.

Conclusion

Our data suggest that morphine-induced cardioprotection is mediated by STAT3-activation and inhibition of mPTP, with STA3 located upstream of mPTP. There is some evidence that protein kinase A is involved within the signalling pathway.     

Mitochondrial DNA 10609T Promotes Hypoxia-Induced Increase of Intracellular ROS and Is a Risk Factor of High Altitude Polycythemia

Hypobaric hypoxia is the primary cause of high altitude polycythemia (HAPC). Mitochondria are critical organelles that consume high levels of oxygen and generate ATP. We hypothesize that the mitochondrion may be at the center of HAPC, and mitochondrial DNA (mtDNA) SNPs may be involved in its development. First, we conducted a case-control study to investigate the association of mtDNA variants with HAPC in Han Chinese migrating to the Qinghai-Tibetan Plateau. Pearson’s chi-square tests revealed that mtDNA 8414T (MU) frequency (19.5%) in the HAPC group was significantly higher than that of the control (13.0%, P = 0.04, OR = 1.615, 95%CI: 1.020–2.555). The multivariate logistic regression analysis, after adjustment for environmental factors, revealed that mtDNA 10609T (WT) was significantly associated with an increased risk of HAPC (P<0.01, OR = 2.558, 95%CI: 1.250–5.236). Second, to verify the association, in vitro experiments of transmitochondrial cybrids was performed and revealed that the mtDNA 10609 variant promoted hypoxia-induced increase of intracellular ROS, but the mtDNA 8414 variant did not. Our findings provide evidence that, in Han Chinese, mtDNA 10609T promotes hypoxia-induced increase of intracellular ROS and is a HAPC risk factor.     

Effect of H. pylori Infection on Cytokine Profiles and Oxidative Balance in Subjects with Chronic Alcohol Ingestion

Different amounts of ingested alcohol can have distinct effects on the human body. However, there is limited research on chronic alcohol consumption with Helicobacter pylori infection. We sought to investigate the relationship between the cytokine profile, oxidative balance and H. pylori infection in subjects with chronic alcohol consumption. A total of 142 subjects were divided into three groups: 59 subjects with chronic alcohol ingestion and H. pylori infection (group A); 53 subjects with chronic alcohol ingestion without H. pylori infection (group B); and 30 control subjects (group C). The serum levels of CagA, interleukin (IL)-10, E-selectin, TNF-α, malondialdehyde (MDA) and superoxide dismutase (SOD) activity were measured by enzyme-linked immunosorbent assay (ELISA). We found that the ages and serum H. pylori CagA levels among the three groups, as well as both the mean drinking age and the mean daily alcohol consumption between groups A and B, were matched and comparable. Comparing the BMIs among the three groups, the BMI differences were found to be statistically significant (F=3.921, P<0.05). Compared with group C, the BMIs in groups A and B were significantly higher (P<0.001 and P<0.01, respectively); however, the BMI differences between group A and group B were not statistically significant (P>0.05). Additionally, no differences in the serum CagA levels were found in comparisons among the groups (all P>0.05). The serum IL-10 and E-selectin levels in group A were significantly lower than those in group B (serum IL-10: P<0.05; E-selectin: P<0.05). The serum IL-10 in group A was significantly higher than that in group C (P<0.01); the serum E-selectin levels in group A did not significantly differ compared with those in group C (P>0.05). Furthermore, the serum IL-10 and E-selectin levels in group B were significantly higher than those in group C (serum IL-10: P<0.001; E-selectin: P<0.05); however, the serum TNF-α levels did not differ among groups (all P>0.05). Although the serum levels of MDA and SOD in groups A and B were slightly lower than those in group C, there were no significant differences among groups (all P>0.05). In conclusion, we believe that H. pylori infection might cause a significant inhibition of certain cytokine profiles in subjects with chronic alcohol ingestion. Moreover, chronically ingested alcohol may exert an adjusted inflammatory effect, but there was no association between H. pylori infection, chronic alcohol consumption and oxidative balance.     

Marked increase in the number and variety of mitochondrial DNA rearrangements in aging human skeletal muscle.

Several reports have shown that individual mitochondrial DNA (mtDNA) deletions accumulate with age. However, the overall extent of somatic mtDNA damage with age remains unclear. We have utilized full-length PCR to concurrently screen for multiple mtDNA rearrangements in total DNA extracted from skeletal muscle derived from physiologically normal individuals (n = 35). This revealed that both the number and variety of mtDNA rearrangements increases dramatically between young and old individuals (P < 0.0001). We further examined the mtDNA from both the younger and older subjects by Southern blot analysis and observed an age-related increase in mtDNA(s) comparable in size to mtDNA products unique to patients with known mtDNA deletions. These data imply that a wide spectrum of mtDNA rearrangements accumulate in old individuals, which correlates with the marked age related decrease in OXPHOS capacity observed in post-mitotic tissues.     

Eicosapentaenoic Acid and Oxypurinol in the Treatment of Muscle Wasting in a Mouse Model of Cancer Cachexia

Cancer cachexia is a wasting condition, driven by systemic inflammation and oxidative stress. This study investigated eicosapentaenoic acid (EPA) in combination with oxypurinol as a treatment in a mouse model of cancer cachexia. Mice with cancer cachexia were randomized into 4 treatment groups (EPA (0.4 g/kg/day), oxypurinol (1 mmol/L ad-lib), combination, or control), and euthanized after 29 days. Analysis of oxidative damage to DNA, mRNA analysis of pro-oxidant, antioxidant and proteolytic pathway components, along with enzyme activity of pro- and antioxidants were completed on gastrocnemius muscle. The control group displayed earlier onset of tumor compared to EPA and oxypurinol groups (P<0.001). The EPA group maintained body weight for an extended duration (20 days) compared to the oxypurinol (5 days) and combination (8 days) groups (P<0.05). EPA (18.2±3.2 pg/ml) and combination (18.4±3.7 pg/ml) groups had significantly higher 8-OH-dG levels than the control group (12.9±1.4 pg/ml, P≤0.05) indicating increased oxidative damage to DNA. mRNA levels of GPx1, MURF1 and MAFbx were higher following EPA treatment compared to control (P≤0.05). Whereas oxypurinol was associated with higher GPx1, MnSOD, CAT, XDH, MURF1, MAFbx and UbB mRNA compared to control (P≤0.05). Activity of total SOD was higher in the oxypurinol group (32.2±1.5 U/ml) compared to control (27.0±1.3 U/ml, P<0.01), GPx activity was lower in the EPA group (8.76±2.0 U/ml) compared to control (14.0±1.9 U/ml, P<0.05), and catalase activity was lower in the combination group (14.4±2.8 U/ml) compared to control (20.9±2.0 U/ml, P<0.01). There was no change in XO activity. The increased rate of weight decline in mice treated with oxypurinol indicates that XO may play a protective role during the progression of cancer cachexia, and its inhibition is detrimental to outcomes. In combination with EPA, there was little significant improvement from control, indicating oxypurinol is unlikely to be a viable treatment compound in cancer cachexia.