Reduced Cytochrome C Oxidase Activity Research Articles

SDHAF2 facilitates mitochondrial respiration through stabilizing succinate dehydrogenase and cytochrome c oxidase assemblies

Succinate dehydrogenase (SDH) plays pivotal roles in maintaining cellular metabolism, modulating regulatory control over both the tricarboxylic acid cycle and oxidative phosphorylation to facilitate energy production within mitochondria. Given that SDH malfunction may serve as a hallmark triggering pseudo-hypoxia signaling and promoting tumorigenesis, elucidating the impact of SDH assembly defects on mitochondrial functions and cellular responses is of paramount importance. In this study, we aim to clarify the role of SDHAF2, one assembly factor of SDH, in mitochondrial respiratory activities. To achieve this, we utilize the CRISPR/Cas9 system to generate SDHAF2 knockout in HeLa cells and examine mitochondrial respiratory functions. Our findings demonstrate a substantial reduction in oxygen consumption rate in SDHAF2 knockout cells, akin to cells with inhibited SDH activity. In addition, in our in-gel activity assays reveal a significant decrease not only in SDH activity but also in cytochrome c oxidase (COX) activity in SDHAF2 knockout cells. The reduced COX activity is attributed to the assembly defect and remains independent of SDH inactivation or SDH complex disassembly. Together, our results indicate a critical role of SDHAF2 in regulating respiration by facilitating the assembly of COX.

Mitochondrial Genome Alterations, Cytochrome C Oxidase Activity, and Oxidative Stress: Implications in Primary Open-angle Glaucoma.

To evaluate mitochondrial genome alterations, cytochrome c oxidase (COX) activity, and oxidative stress in primary open-angle glaucoma (POAG). Whole mitochondrial genome was screened in 75 POAG cases and 105 controls by polymerase chain reaction (PCR) sequencing. COX activity was measured from peripheral blood mononuclear cells (PBMCs). A protein modeling study was done to evaluate the impact of G222E variant on protein function. Levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), 8-isoprostane (8-IP), and total antioxidant capacity (TAC) were also measured. A total of 156 and 79 mitochondrial nucleotide variations were found in the cohort of 75 POAG patients and 105 controls, respectively. Ninety-four (60.26%) variations spanned the coding region, and 62 (39.74%) variations spanned noncoding regions (D-loop, 12SrRNA, and 16SrRNA) of mitochondrial genome in POAG patients. Out of 94 nucleotide changes in coding region, 68 (72.34%) were synonymous changes, 23 (24.46%) non-synonymous, and three (3.19%) were found in the region coding for transfer ribonucleic acid (tRNA). Three changes (p.E192K in ND1, p.L128Q in ND2, and p.G222E in COX2) were found to be pathogenic. Twenty-four (32.0%) patients were positive for either of these pathogenic mitochondrial deoxyribonucleic acid (mtDNA) nucleotide changes. Majority of cases (18.7%) had pathogenic mutation in COX2 gene. Patients who harbored pathogenic mtDNA change in COX2 gene had significantly lower levels of COX activity (p < 0.0001) and TAC (p = 0.004), and higher levels of 8-IP (p = 0.01) as compared to patients who did not harbor this mtDNA. G222E changed the electrostatic potential and adversely impacted protein function of COX2 by affecting nonpolar interactions with neighboring subunits. Pathogenic mtDNA mutations were present in POAG patients, which were associated with reduced COX activity and increased levels of oxidative stress. POAG patients should be evaluated for mitochondrial mutations and oxidative stress and may be managed accordingly with antioxidant therapies. Mohanty K, Mishra S, Dada R, et al. Mitochondrial Genome Alterations, Cytochrome C Oxidase Activity, and Oxidative Stress: Implications in Primary Open-angle Glaucoma. J Curr Glaucoma Pract 2022;16(3):158-165.

Protein Transduction Domain-Mediated Delivery of Recombinant Proteins and In Vitro Transcribed mRNAs for Protein Replacement Therapy of Human Severe Genetic Mitochondrial Disorders: The Case of Sco2 Deficiency

Mitochondrial disorders represent a heterogeneous group of genetic disorders with variations in severity and clinical outcomes, mostly characterized by respiratory chain dysfunction and abnormal mitochondrial function. More specifically, mutations in the human SCO2 gene, encoding the mitochondrial inner membrane Sco2 cytochrome c oxidase (COX) assembly protein, have been implicated in the mitochondrial disorder fatal infantile cardioencephalomyopathy with COX deficiency. Since an effective treatment is still missing, a protein replacement therapy (PRT) was explored using protein transduction domain (PTD) technology. Therefore, the human recombinant full-length mitochondrial protein Sco2, fused to TAT peptide (a common PTD), was produced (fusion Sco2 protein) and successfully transduced into fibroblasts derived from a SCO2/COX-deficient patient. This PRT contributed to effective COX assembly and partial recovery of COX activity. In mice, radiolabeled fusion Sco2 protein was biodistributed in the peripheral tissues of mice and successfully delivered into their mitochondria. Complementary to that, an mRNA-based therapeutic approach has been more recently considered as an innovative treatment option. In particular, a patented, novel PTD-mediated IVT-mRNA delivery platform was developed and applied in recent research efforts. PTD-IVT-mRNA of full-length SCO2 was successfully transduced into the fibroblasts derived from a SCO2/COX-deficient patient, translated in host ribosomes into a nascent chain of human Sco2, imported into mitochondria, and processed to the mature protein. Consequently, the recovery of reduced COX activity was achieved, thus suggesting the potential of this mRNA-based technology for clinical translation as a PRT for metabolic/genetic disorders. In this review, such research efforts will be comprehensibly presented and discussed to elaborate their potential in clinical application and therapeutic usefulness.

Is Type 2 Diabetes a Primary Mitochondrial Disorder?

Diabetes mellitus is the most common endocrine disturbance in inherited mitochondrial diseases. It is essential to increase awareness of the correct diagnosis and treatment of diabetes in these patients and screen for the condition in family members, as diabetes might appear with distinctive clinical features, complications and at different ages of onset. The severity of mitochondrial-related diabetes is likely to manifest on a large scale of phenotypes depending on the location of the mutation and whether the number of affected mitochondria copies (heteroplasmy) reaches a critical threshold. Regarding diabetes treatment, the first-choice treatment for type 2 diabetes (T2D), metformin, is not recommended because of the risk of lactic acidosis. The preferred treatment for diabetes in patients with mitochondrial disorders is SGLT-2i and mitochondrial GLP-1-related substances. The tight relationship between mitochondrial dysfunction, reduced glucose-stimulated insulin secretion (GSIS), and diabetes development in human patients is acknowledged. However, despite the well-characterized role of mitochondria in GSIS, there is a relative lack of data in humans implicating mitochondrial dysfunction as a primary defect in T2D. Our recent studies have provided data supporting the significant role of the mitochondrial respiratory-chain enzyme, cytochrome c oxidase (COX), in regulating GSIS in a rodent model of T2D, the Cohen diabetic sensitive (CDs) rat. The nutritionally induced diabetic CDs rat demonstrates several features of mitochondrial diseases: markedly reduced COX activity in several tissues, increased reactive oxygen production, decreased ATP generation, and increased lactate dehydrogenase expression in islets. Moreover, our data demonstrate that reduced islet-COX activity precedes the onset of diabetes, suggesting that islet-COX deficiency is the primary defect causing diabetes in this model. This review examines the possibility of including T2D as a primary mitochondrial-related disease. Understanding the critical interdependence between diabetes and mitochondrial dysfunction, centering on the role of COX, may open novel avenues to diagnose and treat diabetes in patients with mitochondrial diseases and mitochondrial dysfunction in diabetic patients.

Retrieval of allocentric spatial memories is preserved up to thirty days and does not require higher brain metabolic demands

Spatial orientation is a cognitive ability that is indispensable for survival. Several visual distal cues present in the context can be integrated, establishing a cognitive map. Although there is cumulative evidence about the neural substrate involved in spatial memory acquisition, the brain networks mediating the processes involved in the retrieval of allocentric spatial memories have been studied less. Here, we aimed to explore the role of neuronal oxidative metabolism in the retrieval of allocentric spatial memories through cytochrome c oxidase (CCO) histochemistry seven, 15, 30, 45, and 60 days after task acquisition. Our behavioural results show that spatial memory retrieval in male and female rats is preserved seven, 15, and 30 days post-acquisition, but there is forgetfulness after this time, with subjects not being able to remember the position of the hidden platform after 45 and 60 dfearays. Regarding the study of male brain metabolism, we observed reduced CCO activity in the medial prefrontal cortex, the parietal, retrosplenial, rhinal cortex, and the hippocampal regions in all the groups that failed to solve the task. Similar results were found for female brain oxidative metabolism, in addition to certain differences between succefearssful-retrieval female groups. In conclusion, our work adds information about the behavioural retrieval of an allocentric spatial reference task, suggesting that recovering spatial information seven, 15, and 30days after acquisition is a simple task that does not require a high metabolic demand, in both male and female rats.

P.102 Long-term efficacy of lurasidone in antipsychotic-naïve vs. antipsychotic-exposed adolescents with schizophrenia: Post-hoc analysis of a two year, open-label study

Spatial orientation is a cognitive ability that is indispensable for survival. Several visual distal cues present in the context can be integrated, establishing a cognitive map. Although there is cumulative evidence about the neural substrate involved in spatial memory acquisition, the brain networks mediating the processes involved in the retrieval of allocentric spatial memories have been studied less. Here, we aimed to explore the role of neuronal oxidative metabolism in the retrieval of allocentric spatial memories through cytochrome c oxidase (CCO) histochemistry seven, 15, 30, 45, and 60 days after task acquisition. Our behavioural results show that spatial memory retrieval in male and female rats is preserved seven, 15, and 30 days post-acquisition, but there is forgetfulness after this time, with subjects not being able to remember the position of the hidden platform after 45 and 60 dfearays. Regarding the study of male brain metabolism, we observed reduced CCO activity in the medial prefrontal cortex, the parietal, retrosplenial, rhinal cortex, and the hippocampal regions in all the groups that failed to solve the task. Similar results were found for female brain oxidative metabolism, in addition to certain differences between succefearssful-retrieval female groups. In conclusion, our work adds information about the behavioural retrieval of an allocentric spatial reference task, suggesting that recovering spatial information seven, 15, and 30days after acquisition is a simple task that does not require a high metabolic demand, in both male and female rats.

APOPT1/COA8 assists COX assembly and is oppositely regulated by UPS and ROS.

Loss‐of‐function mutations in APOPT1, a gene exclusively found in higher eukaryotes, cause a characteristic type of cavitating leukoencephalopathy associated with mitochondrial cytochrome c oxidase (COX) deficiency. Although the genetic association of APOPT1 pathogenic variants with isolated COX defects is now clear, the biochemical link between APOPT1 function and COX has remained elusive. We investigated the molecular role of APOPT1 using different approaches. First, we generated an Apopt1 knockout mouse model which shows impaired motor skills, e.g., decreased motor coordination and endurance, associated with reduced COX activity and levels in multiple tissues. In addition, by achieving stable expression of wild‐type APOPT1 in control and patient‐derived cultured cells we ruled out a role of this protein in apoptosis and established instead that this protein is necessary for proper COX assembly and function. On the other hand, APOPT1 steady‐state levels were shown to be controlled by the ubiquitination–proteasome system (UPS). Conversely, in conditions of increased oxidative stress, APOPT1 is stabilized, increasing its mature intramitochondrial form and thereby protecting COX from oxidatively induced degradation.

Sco2 deficient mice develop increased adiposity and insulin resistance

Cytochrome c oxidase (COX) is an essential transmembrane protein complex (Complex IV) in the mitochondrial respiratory electron chain. Mutations in genes responsible for the assembly of COX are associated with Leigh syndrome, cardiomyopathy, spinal muscular atrophy and other fatal metabolic disorders in humans. Previous studies have shown that mice lacking the COX assembly protein Surf1 (Surf1−/− mice) paradoxically show a number of beneficial metabolic phenotypes including increased insulin sensitivity, upregulation of mitochondrial biogenesis, induction of stress response pathways and increased lifespan. To determine whether these effects are specific to the Surf1 mutation or a more general effect of reduced COX activity, we asked whether a different mutation causing reduced COX activity would have similar molecular and physiologic changes. Sco2 knock-in/knock-out (KI/KO) mice in which one allele of the Sco2 gene that encodes a copper chaperone required for COX activity is deleted and the second allele is mutated, have previously been shown to be viable despite a 30–60% reduction in COX activity. In contrast to the Surf1−/− mice, we show that Sco2 KI/KO mice have increased fat mass, associated with reduced β-oxidation and increased adipogenesis markers, reduced insulin receptor beta (IR-β levels in adipose tissue, reduced muscle glucose transporter 4 (Glut4) levels and a impaired response to the insulin tolerance test consistent with insulin resistance. COX activity and protein are reduced approximately 50% in adipose tissue from the Sco2 KI/KO mice. Consistent with the increase in adipose tissue mass, the Sco2 KI/KO mice also show increased hepatosteatosis, elevated serum and liver triglyceride and increased serum cholesterol levels compared to wild-type controls. In contrast to the Surf1−/− mice, which show increased mitochondrial number, upregulation of the mitochondrial unfolded protein response (UPRMT) pathway and no significant change in mitochondrial respiration in several tissues, Sco2 KI/KO mice do not upregulate the UPRMT, and tissue oxygen consumption and levels of several proteins involved in mitochondrial function are reduced in adipose tissue compared to wild type mice. Thus, the metabolic effects of the Sco2 and Surf1−/− mutations are opposite, despite comparable changes in COX activity, illuminating the complex impact of mitochondrial dysfunction on physiology and pointing to an important role for complex IV in regulating metabolism.

MNRR1 (formerly CHCHD2) is a bi-organellar regulator of mitochondrial metabolism

Our understanding of stress-associated regulatory mechanisms for mitochondria remains incomplete. We now report a new regulator of mitochondrial metabolism, the coiled-coil–helix–coiled-coil–helix domain-containing protein 2 (CHCHD2) which, based on the functionality described here, is renamed MNRR1 (Mitochondria Nuclear Retrograde Regulator 1). It functions in a novel way by acting in two cellular compartments, mitochondria and nucleus. In normally growing cells most MNRR1 is located in mitochondria; during stress most MNRR1 is now located in the nucleus. MNRR1 is imported to the mitochondrial intermembrane space by a Mia40-mediated pathway, where it binds to cytochrome c oxidase (COX). This association is required for full COX activity. Decreased MNRR1 levels produce widespread dysfunction including reduced COX activity, membrane potential, and growth rate, and increased reactive oxygen species and mitochondrial fragmentation. In the nucleus, MNRR1 acts as a transcription factor, one of whose targets is the COX subunit 4 isoform, COX4I2, which is transcriptionally stimulated by hypoxia. This MNRR1-mediated stress response may provide an important survival mechanism for cells under conditions of oxidative or hypoxic stress, both in the acute phase by altering mitochondrial oxygen utilization and in the chronic phase by promoting COX remodeling.

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX9C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX9C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.

IL-1β hampers glucose-stimulated insulin secretion in Cohen diabetic rat islets through mitochondrial cytochromecoxidase inhibition by nitric oxide

A high-sucrose, low-copper-diet (HSD) induces inhibition of glucose-sensitive rats (CDs) but not Cohen diabetes-resistant rats (CDr). Copper-supplemented HSD increased activity of the copper-dependent mitochondrial respiratory chain enzyme cytochrome c oxidase (COX) and reversed hyperglycemia. This study examined the mechanism by which interleukin-1β modulates GSIS and the role of COX in this process. We measured COX activity, ATP content, GSIS, iNOS expression, and nitrite production with and without IL-1β, N(ω)-nitro-l-arginine, copper, or potassium cyanide in isolated islets of CDs and CDr fed different diets. We found reduced COX activity, ATP content, and GSIS in isolated islets of CDs rats fed a regular diet. These were severely reduced following HSD and were restored to regular diet levels on copper-supplemented HSD (P < 0.01 vs. CDr islets). Potassium cyanide chemically reduced COX activity, decreasing GSIS and thus reinforcing the link between islet COX activity and GSIS. Interleukin-1β (2.5 U/ml) reduced GSIS and COX activity in CDs islets. Exposure to 10 U/ml interleukin-1β decreased GSIS and COX activity in both CDs and CDr islets, inducing a similar nitrite production. Nevertheless, the effect on GSIS was more marked in CDs islets. A significant iNOS expression was detected in CDs on the HSD diet, which was reduced by copper supplementation. N(ω)-nitro-l-arginine and copper prevented the deleterious effect of interleukin-1β on COX activity and GSIS. We conclude that reduced islet COX activity renders vulnerability to GSIS inhibition on low-copper HSD through two interrelated pathways: 1) by further reducing the activity of COX that is essential for β-cell ATP-production and insulin secretion and 2) by inducing the expression of iNOS and nitric oxide-mediated COX inhibition. We suggest that islet COX activity must be maintained above a critical threshold to sustain adequate GSIS with exposure to low-copper HSD.

COX10Mutations Resulting in Complex Multisystem Mitochondrial Disease That Remains Stable Into Adulthood

Isolated cytochrome-c oxidase (COX) deficiency is one of the most frequent respiratory chain defects seen in human mitochondrial disease. Typically, patients present with severe neonatal multisystem disease and have an early fatal outcome. We describe an adult patient with isolated COX deficiency associated with a relatively mild clinical phenotype comprising myopathy; demyelinating neuropathy; premature ovarian failure; short stature; hearing loss; pigmentary maculopathy; and renal tubular dysfunction. Whole-exome sequencing detected 1 known pathogenic and 1 novel COX10 mutation: c.1007A>T; p.Asp336Val, previously associated with fatal infantile COX deficiency, and c.1015C>T; p.Arg339Trp. Muscle COX holoenzyme and subassemblies were undetectable on immunoblots of blue-native gels, whereas denaturing gels and immunocytochemistry showed reduced core subunit MTCO1. Heme absorption spectra revealed low heme aa3 compatible with heme A:farnesyltransferase deficiency due to COX10 dysfunction. Both mutations demonstrated respiratory deficiency in yeast, confirming pathogenicity. A COX10 protein model was used to predict the structural consequences of the novel Arg339Trp and all previously reported substitutions. These findings establish that COX10 mutations cause adult mitochondrial disease. Nuclear modifiers, epigenetic phenomenon, and/or environmental factors may influence the disease phenotype caused by reduced COX activity and contribute to the variable clinical severity related to COX10 dysfunction.

Contribution of common deletion to total deletion burden in mitochondrial DNA from inner ear of d-galactose-induced aging rats

Mitochondrial DNA (mtDNA) mutations, especially deletions, have been suggested to play an important role in aging and degenerative diseases. In particular, the common deletion in humans and rats (4977 bp and 4834 bp deletion, respectively) has been shown to accumulate with age in post-mitotic tissues with high energetic demands. Among numerous deletions, the common deletion has been proposed to serve as a molecular marker for aging and play a critical role in presbyacusis. However, so far no previous publication has quantified the contribution of common deletion to the total burden of mtDNA deletions in tissues during aging process. In the present study, we established a rat model with various degrees of aging in inner ear induced by three different doses of d-galactose ( d-gal) administration. Firstly, multiple mtDNA deletions in inner ear were detected by nested PCR and long range PCR. In addition to the common deletion, three novel mtDNA deletions were identified. All four deletions, located in the major arc of mtDNA, are flanked by direct repeats and involve the cytochrome c oxidase (COX) subunit III gene, encoded by mtDNA. Additionally, absolute quantitative real-time PCR assay was used to detect the level of common deletion and total deletion burden of mtDNA. The quantitative data show that the common deletion is the most frequent type of mtDNA deletions, exceeding 67.86% of the total deletion burden. Finally, increased mtDNA copy number, reduced COX activity and mosaic ultrastructural impairments in inner ear were identified in d-gal-induced aging rats. The increase of mtDNA replication may contribute to the accelerated accumulation of mtDNA deletions, which may result in impairment of mitochondrial function in inner ear. Taken together, these findings suggest that the common deletion may serve as an ideal molecular marker to assess the mtDNA damage in inner ear during aging.

Novel insights into the assembly and function of human nuclear-encoded cytochromecoxidase subunits 4, 5a, 6a, 7a and 7b

Mammalian CcO (cytochrome c oxidase) is a hetero-oligomeric protein complex composed of 13 structural subunits encoded by both the mitochondrial and nuclear genomes. To study the role of nuclear-encoded CcO subunits in the assembly and function of the human complex, we used stable RNA interference of COX4, COX5A and COX6A1, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)-293 cells. Knockdown of Cox4, Cox5a and Cox6a resulted in reduced CcO activity, diminished affinity of the residual enzyme for oxygen, decreased holoCcO and CcO dimer levels, increased accumulation of CcO subcomplexes and gave rise to an altered pattern of respiratory supercomplexes. An analysis of the patterns of CcO subcomplexes found in both knockdown and overexpressing cells identified a novel CcO assembly intermediate, identified the entry points of three late-assembled subunits and demonstrated directly the essential character as well as the interdependence of the assembly of Cox4 and Cox5a. The ectopic expression of the heart/muscle-specific isoform of the Cox6 subunit (COX6A2) resulted in restoration of both CcO holoenzyme and activity in COX6A1-knockdown cells. This was in sharp contrast with the unaltered levels of COX6A2 mRNA in these cells, suggesting the existence of a fixed expression programme. The normal amount and function of respiratory complex I in all of our CcO-deficient knockdown cell lines suggest that, unlike non-human CcO-deficient models, even relatively small amounts of CcO can maintain the normal biogenesis of this respiratory complex in cultured human cells.

Characterization of the Cytochrome c Oxidase Assembly Factor Cox19 of Saccharomyces cerevisiae

Cox19 is an important accessory protein in the assembly of cytochrome c oxidase in yeast. The protein is functional when tethered to the mitochondrial inner membrane, suggesting its functional role within the intermembrane space. Cox19 resembles Cox17 in having a twin CX(9)C sequence motif that adopts a helical hairpin in Cox17. The function of Cox17 appears to be a Cu(I) donor protein in the assembly of the copper centers in cytochrome c oxidase. Cox19 also resembles Cox17 in its ability to coordinate Cu(I). Recombinant Cox19 binds 1 mol eq of Cu(I) per monomer and exists as a dimeric protein. Cox19 isolated from the mitochondrial intermembrane space contains variable quantities of copper, suggesting that Cu(I) binding may be a transient property. Cysteinyl residues important for Cu(I) binding are also shown to be important for the in vivo function of Cox19. Thus, a correlation exists in the ability to bind Cu(I) and in vivo function.

Protein Kinase A-mediated Phosphorylation Modulates Cytochrome c Oxidase Function and Augments Hypoxia and Myocardial Ischemia-related Injury

We have investigated the effects of hypoxia and myocardial ischemia/reperfusion on the structure and function of cytochrome c oxidase (CcO). Hypoxia (0.1% O(2) for 10 h) and cAMP-mediated inhibition of CcO activity were accompanied by hyperphosphorylation of subunits I, IVi1, and Vb and markedly increased reactive O(2) species production by the enzyme complex in an in vitro system that uses reduced cytochrome c as an electron donor. Both subunit phosphorylation and enzyme activity were effectively reversed by 50 nm H89 or 50 nm myristoylated peptide inhibitor (MPI), specific inhibitors of protein kinase A, but not by inhibitors of protein kinase C. In rabbit hearts subjected to global and focal ischemia, CcO activity was inhibited in a time-dependent manner and was accompanied by hyperphosphorylation as in hypoxia. Additionally, CcO activity and subunit phosphorylation in the ischemic heart were nearly completely reversed by H89 or MPI added to the perfusion medium. Hyperphosphorylation of subunits I, IVi1, and Vb was accompanied by reduced subunit contents of the immunoprecipitated CcO complex. Most interestingly, both H89 and MPI added to the perfusion medium dramatically reduced the ischemia/reperfusion injury to the myocardial tissue. Our results pointed to an exciting possibility of using CcO activity modulators for controlling myocardial injury associated with ischemia and oxidative stress conditions.

Mitochondrial Myopathy and Rhabdomyolysis Associated with a Novel Nonsense Mutation in the Gene Encoding CytochromecOxidase Subunit I

Mitochondrial DNA (mtDNA) mutations associated with rhabdomyolysis are rare but have been described in sporadic cases with mutations in the cytochrome b and cytochrome c oxidase (COX) genes and in 3 cases with tRNALeu mutation. We report a novel heteroplasmic G6708A nonsense mutation in the mtDNA COI gene encoding COX subunit I in a 30-year-old woman with muscle weakness, pain, fatigue, and one episode of rhabdomyolysis. Histochemical examination of muscle biopsy specimens revealed reduced COX activity in the majority of the muscle fibers (approximately 90%) and frequent ragged red fibers. Biochemical analysis showed a marked and isolated COX deficiency. Analysis of DNA extracted from single fibers revealed higher levels of the mutation in COX-deficient fibers (> 95%) compared with COX-positive fibers (1%-80%). The mutation was not detected in a skin biopsy, cultured myoblasts, or blood leukocytes. Nor was it identified in blood leukocytes from the asymptomatic mother, indicating a de novo mutation that arose after germ layer differentiation. Western blot analysis and immunohistochemical staining revealed that reduced levels of COX subunit I were accompanied by reduced levels of other mtDNA encoded subunits, as well as nuclear DNA encoded subunit IV, supporting the concept that COX subunit I is essential for the assembly of complex IV in the respiratory chain.

Supplemental Ascorbate or α-Tocopherol Induces Cell Death in Cu-Deficient HL-60 Cells

Cytochrome c oxidase (CCO) is the Cu-dependent, terminal respiratory complex of the mitochondrial electron transport chain. Inhibition of CCO can promote oxidative stress by increasing mitochondrial production of reactive oxygen species (ROS). Because mitochondria have an important role in apoptosis as both a target and source for ROS, enhanced ROS production resulting from inhibition of CCO by Cu deficiency may trigger apoptosis. The present study focuses on the mitochondrial effects of N,N'-bis(2-aminoethyl)-1,3-propanedi-amine (TET), which inhibits CCO by causing cellular Cu deficiency, and the antioxidants ascorbate and alpha-tocopherol in a human promyelocytic leukemia cell line (HL-60). The following effects were observed: (i) TET reduced both cell growth and viability only in the presence of ascorbate or alpha-tocopherol; (ii) TET reduced CCO activity and increased mitochondrial ROS production as indicated by increased expression of Mn super-oxide dismutase, but the induction of Mn superoxide dismutase was not affected by ascorbate or alpha-tocopherol; (iii) TET acted independently of ascorbate or alpha-tocopherol in disrupting mitochondrial membrane potential; (iv) TET did not increase caspase-8 activity in the absence of ascorbate or alpha-tocopherol; and (v) TET did not increase transfer of cytochrome c from mitochondria to the cytosol unless alpha-tocopherol was present. These findings indicate that reduction in CCO activity by TET-induced Cu deficiency increased oxidative stress in HL-60 cells sufficiently to disrupt the electrochemical gradient of the inner mitochondrial membrane but did not trigger cell death. Also, ascorbate and alpha-tocopherol did not alleviate oxidative stress but may have become pro-oxidants, adding to the oxidant burden sufficiently to trigger cell death in TET-treated cells.

Reduction of mitochondrial DNA content and respiratory chain activity occurs in adipocytes within 6-12 months of commencing nucleoside reverse transcriptase inhibitor therapy.

Mitochondrial enzyme activity was assessed in adipocytes from seven patients initiating nucleoside reverse transcriptase inhibitor (NRTI) regimens. After 6-12 months of therapy, adipocytes from six patients demonstrated cytochrome C oxidase (COX) activity reduced from baseline, correlating positively with mitochondrial DNA levels, concomitant with evidence of cellular toxicity. Three patients also showed reduced expression of COX subunit I. This is the first study to demonstrate that NRTI-associated mtDNA depletion in adipocytes impacts functionally on mitochondrial respiratory proteins in vivo.

Mitochondrial impairment in p53-deficient human cancer cells.

The mechanism linking p53 inactivation to human cell malignancy remains unclear. Studies have indicated that mitochondrial dysfunction is involved in carcinogenesis. In this study we investigated the role of p53 in mitochondrial DNA (mtDNA) mutation and maintenance of proper mitochondrial function. We measured mtDNA mutation and found no difference in frequency of mutation between the p53(+/+) and p53(-/-) cell lines. However, mitochondrial cytochrome c oxidase (COX) activity was significantly diminished in p53(-/-) cells. This decrease in COX activity was attributed to decreased protein levels of the COXII subunit encoded by the mitochondrial genome and was not due to mutation in the mitochondrial COXII gene. Further investigation revealed no concomitant decrease in COXII mRNA levels in p53(-/-) cells and the stability of mRNA in p53(-/-) cells was unaffected. This study suggests that decreased COX activity is likely due to post-transcriptional regulation of the COXII subunit by p53. COX is a critical enzyme in the mitochondrial electron transport chain and reduced COX activity may affect mitochondrial structure. However, examination of mitochondrial ultrastructure revealed no obvious differences between p53(+/+) and p53(-/-) cell lines. Together, our study suggests that p53 is involved in regulation of COXII at the protein level but not at the mRNA level. p53 does not affect mtDNA mutation or mitochondrial ultrastructure.