Developmental changes in cardiac recovery from anoxia-reoxygenation.

The developing cardiovascular system is known to operate normally in a hypoxic environment. However, the functional and ultrastructural recovery of embryonic/fetal hearts subjected to anoxia lasting as long as hypoxia/ischemia performed in adult animal models remains to be investigated. Isolated spontaneously beating hearts from Hamburger-Hamilton developmental stages 14 (14HH), 20HH, 24HH, and 27HH chick embryos were subjected in vitro to 30 or 60 min of anoxia followed by 60 min of reoxygenation. Morphological alterations and apoptosis were assessed histologically and by transmission electron microscopy. Anoxia provoked an initial tachycardia followed by bradycardia leading to complete cardiac arrest, except for in the youngest heart, which kept beating. Complete atrioventricular block appeared after 9.4 +/- 1.1, 1.7 +/- 0.2, and 1.6 +/- 0.3 min at stages 20HH, 24HH, and 27HH, respectively. At reoxygenation, sinoatrial activity resumed first in the form of irregular bursts, and one-to-one atrioventricular conduction resumed after 8, 17, and 35 min at stages 20HH, 24HH, and 27HH, respectively. Ventricular shortening recovered within 30 min except at stage 27HH. After 60 min of anoxia, stage 27HH hearts did not retrieve their baseline activity. Whatever the stage and anoxia duration, nuclear and mitochondrial swelling observed at the end of anoxia were reversible with no apoptosis. Thus the embryonic heart is able to fully recover from anoxia/reoxygenation although its anoxic tolerance declines with age. Changes in cellular homeostatic mechanisms rather than in energy metabolism may account for these developmental variations.

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts.

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed. The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093 and the poly(C) tract at 302-309, whilst a complete A-G transition was confirmed at position 16129 in one hair. Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded. Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be associated with this technology. The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.

Mitochondrial uncoupling prevents cold-induced oxidative stress: a case study using UCP1 knockout mice.

The relationship between metabolism and reactive oxygen species (ROS) production by the mitochondria has often been (wrongly) viewed as straightforward, with increased metabolism leading to higher generation of pro-oxidants. Insights into mitochondrial functioning show that oxygen consumption is principally coupled with either energy conversion as ATP or as heat, depending on whether the ATP-synthase or the mitochondrial uncoupling protein 1 (UCP1) is driving respiration. However, these two processes might greatly differ in terms of oxidative costs. We used a cold challenge to investigate the oxidative stress consequences of an increased metabolism achieved either by the activation of an uncoupled mechanism (i.e. UCP1 activity) in the brown adipose tissue (BAT) of wild-type mice or by ATP-dependent muscular shivering thermogenesis in mice deficient for UCP1. Although both mouse strains increased their metabolism by more than twofold when acclimatised for 4 weeks to moderate cold (12°C), only mice deficient for UCP1 suffered from elevated levels of oxidative stress. When exposed to cold, mice deficient for UCP1 showed an increase of 20.2% in plasmatic reactive oxygen metabolites, 81.8% in muscular oxidized glutathione and 47.1% in muscular protein carbonyls. In contrast, there was no evidence of elevated levels of oxidative stress in the plasma, muscles or BAT of wild-type mice exposed to cold despite a drastic increase in BAT activity. Our study demonstrates differing oxidative costs linked to the functioning of two highly metabolically active organs during thermogenesis, and advises careful consideration of mitochondrial functioning when investigating the links between metabolism and oxidative stress.

HCV infection induces mitochondrial bioenergetic unbalance: causes and effects.

Cells infected by the hepatitis C virus (HCV) are characterized by endoplasmic reticulum stress, deregulation of the calcium homeostasis and unbalance of the oxido-reduction state. In this context, mitochondrial dysfunction proved to be involved and is thought to contribute to the outcome of the HCV-related disease. Here, we propose a temporal sequence of events in the HCV-infected cell whereby the primary alteration consists of a release of Ca(2+) from the endoplasmic reticulum, followed by uptake into mitochondria. This causes successive mitochondrial alterations comprising generation of reactive oxygen and nitrogen species and impairment of the oxidative phosphorylation. A progressive adaptive response results in an enhancement of the glycolytic metabolism sustained by up-regulation of the hypoxia inducible factor. Pathogenetic implications of the model are discussed.

Skeletal Muscle Mitochondrial and Lipid Droplet Volume Density: Validity of Electron Microscopy Point-counting Measurements

Mitochondrial (M) and lipid droplet (L) volume density (vd) are often used in exercise research. Vd is the volume of muscle occupied by M and L. The means of calculating these percents are accomplished by applying a grid to a 2D image taken with transmission electron microscopy; however, it is not known which grid best predicts these values. PURPOSE: To determine the grid with the least variability of Mvd and Lvd in human skeletal muscle. METHODS: Muscle biopsies were taken from vastus lateralis of 10 healthy adults, trained (N=6) and untrained (N=4). Samples of 5-10mg were fixed in 2.5% glutaraldehyde and embedded in EPON. Longitudinal sections of 60 nm were cut and 20 images were taken at random at 33,000x magnification. Vd was calculated as the number of times M or L touched two intersecting grid lines (called a point) divided by the total number of points using 3 different sizes of grids with squares of 1000x1000nm sides (corresponding to 1µm2), 500x500nm (0.25µm2) and 250x250nm (0.0625µm2). Statistics included coefficient of variation (CV), 1 way-BS ANOVA and spearman correlations. RESULTS: Mean age was 67 ± 4 yo, mean VO2peak 2.29 ± 0.70 L/min and mean BMI 25.1 ± 3.7 kg/m2. Mean Mvd was 6.39% ± 0.71 for the 1000nm squares, 6.01% ± 0.70 for the 500nm and 6.37% ± 0.80 for the 250nm. Lvd was 1.28% ± 0.03 for the 1000nm, 1.41% ± 0.02 for the 500nm and 1.38% ± 0.02 for the 250nm. The mean CV of the three grids was 6.65% ±1.15 for Mvd with no significant differences between grids (P>0.05). Mean CV for Lvd was 13.83% ± 3.51, with a significant difference between the 1000nm squares and the two other grids (P<0.05). The 500nm squares grid showed the least variability between subjects. Mvd showed a positive correlation with VO2peak (r = 0.89, p < 0.05) but not with weight, height, or age. No correlations were found with Lvd. CONCLUSION: Different size grids have different variability in assessing skeletal muscle Mvd and Lvd. The grid size of 500x500nm (240 points) was more reliable than 1000x1000nm (56 points). 250x250nm (1023 points) did not show better reliability compared with the 500x500nm, but was more time consuming. Thus, choosing a grid with square size of 500x500nm seems the best option. This is particularly relevant as most grids used in the literature are either 100 points or 400 points without clear information on their square size.

Species identification in mammals from mixed biological samples based on mitochondrial DNA control region length polymorphism.

The ability to identify the species origin of an unknown biological sample is relevant in the fields of human and wildlife forensics. However, the detection of several species mixed in the same sample still remains a challenge. We developed and tested a new approach for mammal DNA identification in mixtures of two or three species, based on the analysis of mitochondrial DNA control region interspecific length polymorphism followed by direct sequencing. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and is not based on a pre-defined panel of species. Amplicons can be separated either on agarose gels or using CE. The advantages and limitations of the assay are discussed under different conditions, such as variable template concentration, amplicon sizes and size difference among the amplicons present in the mixture. For the first time, this protocol provides a simple, reliable and flexible method for simultaneous identification of multiple mammalian species from mixtures, without any prior knowledge of the species involved.

Biogeographic origin and radiation of the Old World crocidurine shrews (Mammalia: Soricidae) inferred from mitochondrial and nuclear genes.

The crocidurine shrews include the most speciose genus of mammals, Crocidura. The origin and evolution of their radiation is, however, poorly understood because of very scant fossil records and a rather conservative external morphology between species. Here, we use an alignment of 3560 base pairs of mitochondrial and nuclear DNA to generate a phylogenetic hypothesis for the evolution of Old World shrews of the subfamily Crocidurinae. These molecular data confirm the monophyly of the speciose African and Eurasian Crocidura, which also includes the fossorial, monotypic genus Diplomesodon. The phylogenetic reconstructions give further credit to a paraphyletic position of Suncus shrews, which are placed into at least two independent clades (one in Africa and sister to Sylvisorex and one in Eurasia), at the base of the Crocidura radiation. Therefore, we recommend restricting the genus Suncus to the Palaearctic and Oriental taxa, and to consider all the African Suncus as Sylvisorex. Using molecular dating and biogeographic reconstruction analyses, we suggest a Palaearctic-Oriental origin for Crocidura dating back to the Upper Miocene (6.8 million years ago) and several subsequent colonisations of the Afrotropical region by independent lineages of Crocidura.

Mitochondrial targeting adaptation of the hominoid-specific glutamate dehydrogenase driven by positive Darwinian selection.

Many new gene copies emerged by gene duplication in hominoids, but little is known with respect to their functional evolution. Glutamate dehydrogenase (GLUD) is an enzyme central to the glutamate and energy metabolism of the cell. In addition to the single, GLUD-encoding gene present in all mammals (GLUD1), humans and apes acquired a second GLUD gene (GLUD2) through retroduplication of GLUD1, which codes for an enzyme with unique, potentially brain-adapted properties. Here we show that whereas the GLUD1 parental protein localizes to mitochondria and the cytoplasm, GLUD2 is specifically targeted to mitochondria. Using evolutionary analysis and resurrected ancestral protein variants, we demonstrate that the enhanced mitochondrial targeting specificity of GLUD2 is due to a single positively selected glutamic acid-to-lysine substitution, which was fixed in the N-terminal mitochondrial targeting sequence (MTS) of GLUD2 soon after the duplication event in the hominoid ancestor approximately 18-25 million years ago. This MTS substitution arose in parallel with two crucial adaptive amino acid changes in the enzyme and likely contributed to the functional adaptation of GLUD2 to the glutamate metabolism of the hominoid brain and other tissues. We suggest that rapid, selectively driven subcellular adaptation, as exemplified by GLUD2, represents a common route underlying the emergence of new gene functions.

Mitochondrial tRNA(Leu(UUR)) mutation m.3302A > G presenting as childhood-onset severe myopathy: threshold determination through segregation study.

Mitochondrial tRNA(Leu(UUR)) mutation m.3302A > G is associated with respiratory chain complex I deficiency and has been described as a rare cause of mostly adult-onset slowly progressive myopathy. Five families with 11 patients have been described so far; 5 of them died young due to cardiorespiratory failure. Here, we report on a segregation study in a family with an index patient who already presented at the age of 18 months with proximal muscular hypotonia, abnormal fatigability, and lactic acidosis. This early-onset myopathy was rapidly progressive. At 8 years, the patient is wheel-chair bound, requires nocturnal assisted ventilation, and suffers from recurrent respiratory infections. Severe complex I deficiency and nearly homoplasmy for m.3302A > G were found in muscle. We collected blood, hair, buccal swabs and muscle biopsies from asymptomatic adults in this pedigree and determined heteroplasmy levels in these tissues as well as OXPHOS activities in muscle. All participating asymptomatic adults had normal OXPHOS activities. In contrast to earlier reports, we found surprisingly little variation of heteroplasmy levels in different tissues of the same individual. Up to 45% mutation load in muscle and up to 38% mutation load in other tissues were found in non-affected adults. The phenotypic spectrum of tRNA(Leu(UUR)) m.3302A > G mutation seems to be wider than previously described. A threshold of more than 45% heteroplasmy in muscle seems to be necessary to alter complex I activity leading to clinical manifestation. The presented data may be helpful for prognostic considerations and counseling in affected families.

Inflammation-induced alteration of astrocyte mitochondrial dynamics requires autophagy for mitochondrial network maintenance.

Accumulating evidence suggests that changes in the metabolic signature of astrocytes underlie their response to neuroinflammation, but how proinflammatory stimuli induce these changes is poorly understood. By monitoring astrocytes following acute cortical injury, we identified a differential and region-specific remodeling of their mitochondrial network: while astrocytes within the penumbra of the lesion undergo mitochondrial elongation, those located in the core-the area invaded by proinflammatory cells-experience transient mitochondrial fragmentation. In brain slices, proinflammatory stimuli reproduced localized changes in mitochondrial dynamics, favoring fission over fusion. This effect was triggered by Drp1 phosphorylation and ultimately resulted in reduced respiratory capacity. Furthermore, maintenance of the mitochondrial architecture critically depended on the induction of autophagy. Deletion of Atg7, required for autophagosome formation, prevented the reestablishment of tubular mitochondria, leading to marked reactive oxygen species accumulation and cell death. Thus, our data reveal autophagy to be essential for regenerating astrocyte mitochondrial networks during inflammation.

Glutamate Transport Decreases Mitochondrial pH and Modulates Oxidative Metabolism in Astrocytes.

During synaptic activity, the clearance of neuronally released glutamate leads to an intracellular sodium concentration increase in astrocytes that is associated with significant metabolic cost. The proximity of mitochondria at glutamate uptake sites in astrocytes raises the question of the ability of mitochondria to respond to these energy demands. We used dynamic fluorescence imaging to investigate the impact of glutamatergic transmission on mitochondria in intact astrocytes. Neuronal release of glutamate induced an intracellular acidification in astrocytes, via glutamate transporters, that spread over the mitochondrial matrix. The glutamate-induced mitochondrial matrix acidification exceeded cytosolic acidification and abrogated cytosol-to-mitochondrial matrix pH gradient. By decoupling glutamate uptake from cellular acidification, we found that glutamate induced a pH-mediated decrease in mitochondrial metabolism that surpasses the Ca(2+)-mediated stimulatory effects. These findings suggest a model in which excitatory neurotransmission dynamically regulates astrocyte energy metabolism by limiting the contribution of mitochondria to the metabolic response, thereby increasing the local oxygen availability and preventing excessive mitochondrial reactive oxygen species production.

Mitochondrial DNA sequence variation within the remnant populations of the endangered numbat (Marsupialia: Myrmecobiidae: Myrmecobius fasciatus).

The numbat has been reduced to two populations in Western Australia. To better understand the effects of range reduction on gene flow and genetic variation, and to address questions crucial for the species' management, we analysed mitochondrial DNA (mtDNA) sequences of free-ranging individuals and museum specimens. The results suggest recent connectivity between the remnant populations, although one of those may have lost significant amounts of genetic diversity during the recent population size reduction. We propose that for management purposes the remnant populations should be treated as a single historical lineage and that, subject to certain caveats, consideration should be given to population augmentation by translocation.

Rapidly fatal poisoning with an insecticide containing rotenone.

Introduction: Rotenone is a botanical pesticide derived from extracts of Derris roots, which is traditionally used as piscicide, but also as an industrial insecticide for home gardens. Its mechanism of action is potent inhibition of mitochondrial respiratory chain by uncoupling oxidative phosphorylation by blocking electron transport at complex-I. Despite its classification as mild to moderately toxic to humans (estimated LD50, 300-500 mg/kg), there is a striking variety of acute toxicity of rotenone depending on the formulation (solvents). Human fatalities with rotenone-containing insecticides have been rarely reported, and a rapid deterioration within a few hours of the ingestion has been described previously in one case. Case report: A 49-year-old Tamil man with a history of asthma, ingested 250 mL of an insecticide containing 1.24% of rotenone (3.125 g, 52.1-62.5 mg/kg) in a suicide attempt at home. The product was not labeled as toxic. One hour later, he vomited repeatedly and emergency services were alerted. He was found unconscious with irregular respiration and was intubated. On arrival at the emergency department, he was comatose (GCS 3) with fixed and dilated pupils, and absent corneal reflexes. Physical examination revealed hemodynamic instability with hypotension (55/30 mmHg) and bradycardia (52 bpm). Significant laboratory findings were lactic acidosis (pH 6.97, lactate 17 mmol/L) and hypokalemia (2 mmol/L). Cranial computed tomography (CT) showed early cerebral edema. A single dose of activated charcoal was given. Intravenous hydration, ephedrine, repeated boli of dobutamine, and a perfusor with 90 micrograms/h norepinephine stabilized blood pressure temporarily. Atropine had a minimal effect on heart rate (58 bpm). Intravenous lipid emulsion was considered (log Pow 4.1), but there was a rapid deterioration with refractory hypotension and acute circulatory failure. The patient died 5h after ingestion of the insecticide. No autopsy was performed. Quantitative analysis of serum performed by high-resolution/accurate mass-mass spectrometry and liquid chromatography (LC-HR/AM-MS): 560 ng/mL rotenone. Other substances were excluded by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS/MS). Conclusion: The clinical course was characterized by early severe symptoms and a rapidly fatal evolution, compatible with inhibition of mitochondrial energy supply. Although rotenone is classified as mild to moderately toxic, physicians must be aware that suicidal ingestion of emulsified concentrates may be rapidly fatal. (n=3): stridor, cyanosis, cough (one each). Local swelling after chewing or swallowing soap developed at the earliest after 20 minutes and persisted beyond 24 hours in some cases. Treatment with antihistamines and/or steroids relieved the symptoms in 9 cases. Conclusion: Bar soap ingestion by seniors carries a risk of severe local reactions. Half the patients developed symptoms, predominantly swellings of tongue and/or lips (38%). Cognitive impairment, particularly in the cases of dementia (37%), may increase the risk of unintentional ingestion. Chewing and intraoral retention of soap leads to prolonged contact with the mucosal membranes. Age-associated physiological changes of oral mucosa probably promote the irritant effects of the surfactants. Medical treatment with antihistamines and corticosteroids usually leads to rapid decline of symptoms. Without treatment, there may be a risk of airway obstruction.

Hepatitis C virus proteins promote mitochondrial bioenergetic dysfunction and nitro-oxidative stress: insights into pathogenesis

HCV-infection induces a state of oxidative stress more pronounced than in many other inflammatory diseases. Here we propose a temporal sequence of events in the HCV-infected cell whereby the primary alteration consists in release of Ca2+ from the ER followed by uptake into mitochondria. This triggers successive mitochondrial dysfunctions leading to generation of ROS and to a progressive metabolic adaptive response. Pathogenetic implications of the model and new opportunities for therapeutic intervention are discussed.