RESUMEN
The total amount of cellular mitochondrial DNA (mtDNA) varies widely and seems to be related to the nature and metabolic state of tissues and cells in culture. It is not known, however, whether this variation has any significance in vivo, and to which extent it regulates energy production. To better understand the importance of the cellular mtDNA level, we studied the influence of a gradual reduction of mtDNA copy number on oxidative phosphorylation in two models: (a) a control human cell line treated with different concentrations of 2', 3'-dideoxycytidine, a nucleoside analogue that inhibits mtDNA replication by interfering with mitochondrial DNA polymerase gamma, and (b) a cell line derived from a patient presenting mtDNA depletion. The two models were used to construct biochemical and phenotypic threshold curves. Our results show that oxidative phosphorylation activities are under a tight control by the amount of mtDNA in the cell, and that the full complement of mtDNA molecules are necessary to maintain a normal energy production level.
Asunto(s)
Fenómenos Fisiológicos Celulares , ADN Mitocondrial/genética , Metabolismo Energético/genética , Enfermedades Mitocondriales/genética , Modelos Genéticos , Animales , Simulación por Computador , HumanosRESUMEN
BACKGROUND: Long-acting local anesthetics cause muscle damage. Moreover, long-acting local anesthetics act as uncoupler of oxidative phosphorylation in isolated mitochondria and enhance sarcoplasmic reticulum Ca(2+) release. The aim of the study was to evaluate effects of perineural injections of local anesthetics on mitochondrial energetic metabolism and intracellular calcium homeostasis in vivo. METHODS: Femoral nerve block catheters were inserted in adult male Wistar rats. Rats were randomized and received seven injections (1 ml/kg) of bupivacaine, levobupivacaine, ropivacaine, or isotonic saline at 8-h intervals. Rats were killed 8 h after the last injection. Psoas muscle was quickly dissected from next to the femoral nerve. Local anesthetic concentrations in muscle were determined. Oxidative capacity was measured in saponin-skinned fibers. Oxygen consumption rates were measured, and mitochondrial adenosine triphosphate synthesis rate was determined. Enzymatic activities of mitochondrial respiratory chain complexes were evaluated. Local calcium release events (calcium sparks) were analyzed as well as sarcoplasmic reticulum calcium content in saponin-skinned fibers. RESULTS: Eight hours after the last injection, psoas muscle concentration of local anesthetics was less than 0.3 microg/g tissue. Adenosine triphosphate synthesis and adenosine triphosphate-to-oxygen ratio were significantly decreased in the muscle of rats treated with local anesthetics. A global decrease (around 50%) in all of the enzyme activities of the respiratory chain was observed. Levobupivacaine increased the amplitude and frequency of the calcium sparks, whereas lower sarcoplasmic reticulum calcium content was shown. CONCLUSION: Bupivacaine, levobupivacaine, and ropivacaine injected via femoral nerve block catheters induce a deleterious effect in mitochondrial energy, whereas only levobupivacaine disturbs calcium homeostasis.
Asunto(s)
Amidas/farmacología , Anestésicos Locales/farmacología , Bupivacaína/farmacología , Calcio/metabolismo , Metabolismo Energético/efectos de los fármacos , Nervio Femoral/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Músculos Psoas/efectos de los fármacos , Animales , Bupivacaína/análogos & derivados , Transporte de Electrón/efectos de los fármacos , Nervio Femoral/fisiología , Homeostasis/efectos de los fármacos , Levobupivacaína , Masculino , Mitocondrias/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Músculos Psoas/metabolismo , Ratas , Ratas Wistar , RopivacaínaRESUMEN
OBJECTIVE: The present study was designed to characterize mitochondrial adaptation to chronic hypoxia (CH) in the rat heart. Mitochondrial energy metabolism was differentially examined in both left and right ventricles since CH selectively triggers pulmonary hypertension and right ventricular hypertrophy. METHODS: Rats were exposed to a hypobaric environment for 2 or 3 weeks and compared with rats maintained in a normoxic environment. Oxidative capacity (oxygen consumption and ATP synthesis) was measured in saponin-skinned fibers with glutamate or palmitoyl carnitine as substrates. Enzymatic activities of mitochondrial respiratory chain complexes were measured on tissue homogenates. Morphometric analysis of mitochondria was performed on electron micrographs. Mitochondrial DNA was quantified using Southern blot analysis. RESULTS: Whereas oxidative capacity of both ventricles was decreased following 21 days of CH, oxygen consumption and ATP synthesis was maintained with the glutamate substrate in the right ventricle following 14 days of CH. As for the oxidative capacity, enzyme activities were decreased only in the left ventricle following 14 days of CH and in both ventricles following 21 days of CH. These functional alterations were associated with an increase in numerical density and a decrease in size of mitochondria without a change in volume density in both ventricles. Finally, 21 days of CH also decreased the ratio of mitochondrial DNA to nuclear DNA in both ventricles. CONCLUSIONS: CH alters morphometry and function of mitochondria in the heart, but this effect is delayed in the right compared to the left ventricle, suggesting some adaptive processes at the onset of right ventricular hypertrophy.
Asunto(s)
Metabolismo Energético , Hipoxia/metabolismo , Mitocondrias Cardíacas/metabolismo , Adenosina Trifosfatasas/biosíntesis , Animales , Southern Blotting/métodos , Núcleo Celular/ultraestructura , Enfermedad Crónica , ADN/ultraestructura , ADN Mitocondrial/ultraestructura , Ventrículos Cardíacos , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Hipertrofia Ventricular Derecha/metabolismo , Hipertrofia Ventricular Derecha/patología , Masculino , Microscopía Electrónica , Mitocondrias Cardíacas/ultraestructura , Fosforilación Oxidativa , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
OBJECTIVE: To assess mitochondrial metabolism of bladder tissue induced by small-intestinal submucosa (SIS), by comparing the mitochondrial enzyme metabolism in this tissue with that in normal bladder tissue and thus evaluate intracellular normality. MATERIAL AND METHODS: In all, 70 rats were grouped into healthy controls (10), surgical controls with a simple bladder incision (15) and rats treated by partial cystectomy with replacement by the SIS graft (45). At 1, 3 and 6 months the rats were killed, the enzymes of mitochondrial respiratory chain complexes assayed, and the respiration of permeabilized bladder fibres assessed using polarographic analysis. RESULTS: The enzyme activities of control and treated rats at 3 months were identical. The results from the polarographic analysis of respiration were also similar to that in normal tissue apart from a decrease in the number of mitochondria. Histologically, there was complete regeneration at 6 months. CONCLUSION: After a phase of inflammation the bladder regenerates after a patch is placed. The new tissue has the same enzymatic and histological features as normal bladder tissue.
Asunto(s)
Intestino Delgado/fisiología , Mitocondrias/metabolismo , Regeneración/fisiología , Vejiga Urinaria/fisiología , Animales , Mucosa Intestinal/fisiología , Intestino Delgado/citología , Masculino , Ratas , Ratas WistarRESUMEN
The existence of a biochemical threshold effect in the metabolic expression of oxidative phosphorylation deficiencies has considerable implications for the understanding of mitochondrial bioenergetics and the study of mitochondrial diseases. However, the molecular bases of this phenomenon remain unclear. We report here a new mechanism to explain this threshold effect, based on a reserve of enzymes not initially participating in the respiratory rate that can be activated either to respond to a flux increase or to compensate for a defect induced by a mutation. We show that this mobilization occurs through 1) the assembly of inactive adenine nucleotide translocator isoform 1 subunits into oligomeric active carriers or 2) conformational changes in the adenine nucleotide translocator isoform 1 in a permeability transition pore-like structure. We discuss how these transitions are sensitive to the steady state of oxidative phosphorylation functioning or tissue and analyze their consequences on the threshold effect.
Asunto(s)
Nucleótidos de Adenina/química , Atractilósido/análogos & derivados , Enfermedades Mitocondriales/metabolismo , Translocador 1 del Nucleótido Adenina/química , Animales , Atractilósido/farmacología , Biopsia , Western Blotting , Transporte de Electrón , Electroforesis en Gel de Poliacrilamida , Humanos , Cinética , Masculino , Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Modelos Biológicos , Músculos/metabolismo , Mutación , Fosforilación Oxidativa , Oxígeno/metabolismo , Consumo de Oxígeno , Fosforilación , Conformación Proteica , Isoformas de Proteínas , Ratas , Ratas Wistar , Distribución TisularRESUMEN
The study of mitochondrial diseases has revealed dramatic variability in the phenotypic presentation of mitochondrial genetic defects. To attempt to understand this variability, different authors have studied energy metabolism in transmitochondrial cell lines carrying different proportions of various pathogenic mutations in their mitochondrial DNA. The same kinds of experiments have been performed on isolated mitochondria and on tissue biopsies taken from patients with mitochondrial diseases. The results have shown that, in most cases, phenotypic manifestation of the genetic defect occurs only when a threshold level is exceeded, and this phenomenon has been named the 'phenotypic threshold effect'. Subsequently, several authors showed that it was possible to inhibit considerably the activity of a respiratory chain complex, up to a critical value, without affecting the rate of mitochondrial respiration or ATP synthesis. This phenomenon was called the 'biochemical threshold effect'. More recently, quantitative analysis of the effects of various mutations in mitochondrial DNA on the rate of mitochondrial protein synthesis has revealed the existence of a 'translational threshold effect'. In this review these different mitochondrial threshold effects are discussed, along with their molecular bases and the roles that they play in the presentation of mitochondrial diseases.
Asunto(s)
ADN Mitocondrial , Metabolismo Energético , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Fosforilación Oxidativa , Animales , Línea Celular , Regulación de la Expresión Génica , Humanos , Enfermedades Mitocondriales/fisiopatología , Mutación , FenotipoRESUMEN
BACKGROUND: Adaptation to chronic exposure to hypoxia alters energy metabolism in the heart, particularly in the left ventricle, which undergoes a loss in oxidative capacity. Highly lipophilic local anesthetics interfere with mitochondrial energy metabolism. The purpose of this study was to compare the effects of bupivacaine on mitochondrial energy metabolism in heart of rats subjected to normoxic or hypoxic environments. METHODS: Male Wistar rats (n = 10) were subjected to hypobaric hypoxia (simulated altitude = 5,000 m, 380 mmHg) for 2 weeks. Control rats (n = 10) were maintained in an ambient normoxic environment. Mitochondrial metabolism (oxygen consumption and adenosine triphosphate synthesis) was assessed using saponin-skinned ventricular fibers. Bupivacaine (0-5 mM) was tested on both left and right ventricles of normoxic or hypoxic heart. RESULTS: In animals exposed to hypobaric hypoxia for 14 days, cardiac mass significantly increased, and the right-to-left ventricular ratio was approximately twofold (0.48 +/- 0.11 vs. 0.22 +/- 0.04, P < 0.05). Oxygen consumption and adenosine triphosphate synthesis were significantly lower in the hypoxic left ventricles but not in the right ones. The uncoupling effect of bupivacaine was more pronounced in the left ventricle from hypoxic heart than in the right ventricle; the bupivacaine-induced decrease in the adenosine triphosphate synthesis rate and in the adenosine triphosphate-to-oxygen ratio was significantly greater in the hypoxic left ventricle than in the normoxic one. CONCLUSIONS: Chronic hypoxia impairs cardiac energy metabolism in left ventricles and enhances the depressant effects of bupivacaine on mitochondrial functions.