RESUMEN
A non-synonymous mtDNA mutation, m.3395A > G, which changes tyrosine in position 30 to cysteine in p.MT-ND1, was found in several patients with a wide range of clinical phenotypes such as deafness, diabetes and cerebellar syndrome but no Leber's hereditary optic neuropathy. Although this mutation has already been described, its pathogenicity has not been demonstrated. Here, it was found isolated for the first time, allowing a study to investigate its pathogenicity. To do so, we constructed cybrid cell lines and carried out a functional study to assess the possible consequences of the mutation on mitochondrial bioenergetics. Results obtained demonstrated that this mutation causes an important dysfunction of the mitochondrial respiratory chain with a decrease in both activity and quantity of complex I due to a diminution of p.MT-ND1 quantity. However, no subcomplexes were found in cybrids carrying the mutation, indicating that the quality of the complex I assembly is not affected. Moreover, based on the crystal structure of p.MT-ND1 and the data found in the literature, we propose a hypothesis for the mechanism of the degradation of p.MT-ND1. Our study provides new insights into the pathophysiology of mitochondrial diseases and in particular of MT-ND1 mutations.
Asunto(s)
ADN Mitocondrial/genética , Sordera/clasificación , Sordera/patología , Mitocondrias/patología , Mutación , NADH Deshidrogenasa/genética , Adolescente , Adulto , Niño , Preescolar , ADN Mitocondrial/análisis , Sordera/genética , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Mitocondrias/genéticaRESUMEN
Some cases of maternally inherited isolated deafness are caused by mtDNA mutations, frequently following an exposure to aminoglycosides. Two mitochondrial genes have been clearly described as being affected by mutations responsible for this pathology: the ribosomal RNA 12S gene and the transfer RNA serine (UCN) gene. A previous study identified several candidate novel mtDNA mutations, localized in a variety of mitochondrial genes, found in patients with no previous treatment with aminoglycosides. Five of these candidate mutations are characterized in the present study. These mutations are localized in subunit ND1 of complex I of the respiratory chain (m.3388C>A [p.MT-ND1:Leu28Met]), the tRNA for Isoleucine (m.4295A>G), subunit COII of complex IV (m.8078G>A [p.MT-CO2:Val165Ile]), the tRNA of Serine 2 (AGU/C) (m.12236G>A), and Cytochrome B, subunit of complex III (m.15077G>A [p.MT-CYB:Glu111Lys]). Cybrid cell lines have been constructed for each of the studied mtDNA mutations and functional studies have been performed to assess the possible consequences of these mutations on mitochondrial bioenergetics. This study shows that a variety of mitochondrial genes, including protein-coding genes, can be responsible for nonsyndromic deafness, and that exposure to aminoglycosides is not required to develop the disease, giving new insights on the molecular bases of this pathology.
Asunto(s)
ADN Mitocondrial , Pérdida Auditiva/genética , Mutación , NADH Deshidrogenasa/genética , ARN de Transferencia de Serina/genética , Aminoglicósidos/efectos adversos , Línea Celular , Respiración de la Célula/genética , Citocromos b/genética , Citocromos b/metabolismo , Femenino , Pérdida Auditiva/inducido químicamente , Humanos , Masculino , Mitocondrias/genética , Mitocondrias/metabolismo , Madres , NADH Deshidrogenasa/química , NADH Deshidrogenasa/metabolismo , Linaje , Fosforilación , Polimorfismo de Longitud del Fragmento de Restricción , Conformación Proteica , ARN de Transferencia de Isoleucina/genética , ARN de Transferencia de Serina/metabolismoRESUMEN
The voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) have central roles in mitochondrial functions such as nucleotides transport and cell death. The interaction between VDAC, an outer mitochondrial membrane protein and ANT, an inner membrane protein, was studied in isolated mitochondria and in vitro. Both proteins were isolated from various mitochondrial sources and reconstituted in vitro using a biomimetic system composed of recombinant human VDAC isoform 1 (rhVDAC1) immobilized on a surface plasmon resonance (SPR) sensor chip surface. Two enriched-preparations of (H)ANT (ANT from heart, mainly ANT1) and (L)ANT (ANT from liver, mainly ANT2) isoforms interacted differently with rhVDAC1. Moreover, the pharmacological ANT inhibitors atractyloside and bongkrekic acid modulated this interaction. Thus, ANT-VDAC interaction depends both on ANT isoform identity and on the conformation of ANT.
Asunto(s)
Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Animales , Humanos , Proteínas Inmovilizadas/metabolismo , Inmunoprecipitación , Isoenzimas/química , Isoenzimas/metabolismo , Translocasas Mitocondriales de ADP y ATP/química , Conformación Proteica , Ratas , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
BACKGROUND: Local anesthetics offer the benefits of extended analgesia with greater patient satisfaction and faster rehabilitation compared with intravenous morphine. These benefits, however, can be offset by adverse iatrogenic muscle pain caused by bupivacaine. Here, the authors describe the mechanisms of local anesthetic-induced myotoxicity and a partial protective effect of recombinant human erythropoietin (rhEPO). METHODS: The authors developed a rat analgesia model with femoral nerve catheter and a cell culture model of human skeletal muscle myoblasts to study local anesthetic effects. Rats were randomly assigned to four different groups: daily intraperitoneal injection with 5,000 U/kg rhEPO or saline coupled to a perineural catheter injection with 1 ml/kg bupivacaine, 0.25%, or saline. In psoas rat muscle, oxygen consumption rates were measured using a Clark-type electrode in saponin-skinned fibers. Mitochondrial adenosine triphosphate synthesis rates were determined by bioluminescence. Enzymatic activity of mitochondrial respiratory chain complexes was measured on tissue homogenates using spectrophotometric procedures, and mitochondrial morphology was analyzed by transmission electron microscopy. In addition, the interaction between bupivacaine and rhEPO was investigated on human skeletal muscle myoblasts by fluorescence microscopy using mitotracker green and using the lipophilic cation JC-1. RESULTS: Bupivacaine caused impairment of mitochondrial structure and bioenergetics in rats. Human myoblasts treated with bupivacaine showed a dose-dependent decrease in mitochondrial membrane potential associated with unusual morphologies. Impairment of mitochondrial bioenergetics was prevented partially by the use of rhEPO coadministered with bupivacaine. CONCLUSIONS: The authors demonstrated a dose- and time-dependent protective effect of rhEPO against bupivacaine-induced myotoxicity in regional analgesia.
Asunto(s)
Anestesia de Conducción/efectos adversos , Anestésicos Locales/efectos adversos , Eritropoyetina/uso terapéutico , Músculo Esquelético/efectos de los fármacos , Dolor Postoperatorio/inducido químicamente , Dolor Postoperatorio/prevención & control , Anestésicos Locales/administración & dosificación , Animales , Células Cultivadas , Eritropoyetina/farmacología , Humanos , Masculino , Músculo Esquelético/patología , Ratas , Ratas Wistar , Proteínas RecombinantesRESUMEN
BACKGROUND: Regional blocks improve postoperative analgesia and postoperative rehabilitation in children and adult patients. Continuous peripheral nerve blocks have been proposed as safe and effective techniques for postoperative pain relief and chronic pain therapy, particularly in small children. Few clinical reports have described myotoxicity induced by bupivacaine in these young patients, in contrast with a larger number of observations in adults. Here, the authors addressed this issue by a comparative evaluation of bupivacaine-induced myotoxicity in young versus adult rats. METHODS: Femoral nerve block catheters were inserted in male Wistar rats. Young (3-week-old) and adult (12-week-old) rats were randomly assigned to received seven injections (1 ml/kg) of 0.25% bupivacaine (n = 6 per experiment) or isotonic saline (n = 6 per experiment) at 8-h intervals. Rats were killed 8 h after the last injection. Psoas muscle adjacent to the femoral nerve was quickly dissected. Oxygen consumption rates were measured in saponin-skinned fibers, mitochondrial adenosine triphosphate synthesis rates were determined by bioluminescence, and citrate synthase activity was determined by spectrophotometry. Muscle ultrastructural damage was also examined and scored as normal, focal disruption, moderate disruption, or extreme disruption of the sarcomeres. RESULTS: Bupivacaine caused a reduction of mitochondrial adenosine triphosphate synthesis rate, a decrease of citrate synthase activity, and muscle ultrastructural damages. Young rats treated with bupivacaine showed more severe alterations of mitochondrial bioenergetics and muscle ultrastructure. CONCLUSIONS: These findings demonstrate that bupivacaine-induced myotoxicity can be explained by mitochondrial bioenergetics alterations, which are more severe in young rats.
Asunto(s)
Anestésicos Locales/toxicidad , Bupivacaína/toxicidad , Músculo Esquelético/efectos de los fármacos , Bloqueo Nervioso/efectos adversos , Factores de Edad , Animales , Metabolismo Energético/efectos de los fármacos , Masculino , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/metabolismo , Ratas , Ratas WistarRESUMEN
Mitochondria play a central role in the intrinsic pathway of apoptosis. In response to many pro-apoptotic stimuli, mitochondria undergo an irreversible process called mitochondrial membrane permeabilization (MMP). The detection of MMP in isolated mitochondria is most often based on assays that monitor either the loss of the inner transmembrane potential (DYm; classically with Rhodamine 123), permeability transition (PT, cyclosporin A-sensitive matrix swelling), or the release of critical pro-apoptotic intermembrane space effectors. To gain complementary information on MMP mechanisms, we have systematically used three additional assays optimized for the 96-well microplate format: (1) inner membrane permeability, (2) VDAC-associated NADH reductase activity, and (3) ATP/ADP translocase activity. We report that ad hoc combinations of ANT and VDAC ligands, carbonyl cyanide m-chlorophenylhydrazone (CCCP), mastoparan and Vpr52-96 peptide and PT inhibitors, permit to explore relationships between enzymatic functions of sessile mitochondrial proteins (i.e. ANT, VDAC) and MMP. These assays should be useful tools to investigate mitochondrial apoptosis, decipher the implication of inner and outer membrane permeabilization and provide a multi-parametric approach for drug discovery.
Asunto(s)
Apoptosis/efectos de los fármacos , Animales , Calcio/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ratones , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/ultraestructura , Dilatación Mitocondrial/efectos de los fármacos , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/farmacologíaRESUMEN
An immunodetection study of protein tyrosine phosphatase 1B (PTP-1B), SHP-2, and Src in isolated mitochondria from different rat tissues (brain, muscle, heart, liver, and kidney) revealed their exclusive localization in the brain. Given this result, we sought whether mitochondria respond to ATP and to the general tyrosine phosphatase inhibitor orthovanadate and found little or no change in the tyrosine phosphorylation profile of mitochondria from muscle, heart, liver, and kidney. In contrast, ATP induced an enhancement in the tyrosine-phosphorylated protein profile of brain mitochondria, which was further greatly enhanced with orthovanadate and which disappeared when Src was inhibited with two inhibitors: PP2 and PP1. Importantly, we found that in brain mitochondria, ATP addition induced Src autophosphorylation at Tyr-416 in its catalytic site, leading to its activation, whereas the regulatory Tyr-527 site remained unphosphorylated. Functional implications were addressed by measurements of the enzymatic activity of each of the oxidative phosphorylation complexes in brain mitochondria in the presence of ATP. We found an increase in complex I, III, and IV activity and a decrease in complex V activity, partially reversed by Src inhibition, demonstrating that the complexes are Src substrates. These results complemented and reinforced our initial study showing that respiration of brain mitochondria was partially dependent on tyrosine phosphorylation. Therefore, the present data suggest a possible control point in the regulation of respiration by tyrosine phosphorylation of the complexes mediated by Src auto-activation.