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Hypotension reduces cerebral O2supply, which may result in brain cell damage and loss of brain cell function in the near-term neonate. The aim is to elucidate 1) to what extent the functional disturbance of the cerebral cortex, as measured with electrocortical brain activity (ECBA), is related to cerebral cortical tissue damage, as estimated by MAP2; and 2) whether there is a relationship between the glutamate, nitric oxide (NO), cGMP pathway and the development of cerebral cortical tissue damage after hemorrhagic hypotension. Seven lambs were delivered at 131 d of gestation. Hypotension was induced by withdrawal of blood until mean arterial blood pressure was reduced to 30% of normotension. Cerebral O2supply, consumption, and ECBA were calculated in normotensive conditions and after 2.5 h of hypotension. Concentrations of glutamate and aspartate in cerebrospinal fluid (CSF), NO2and NO3(NOx) in plasma, and cGMP in cortical brain tissue were determined in both conditions. CSF and brain tissue from siblings were used to determine normotensive values. Cortical neuronal damage was detected after 2.5 h of hypotension. ECBA was negatively related to the severity of the cortical damage. ECBA was related to respectively glutamate, NOx, and cGMP concentrations. In conclusion, cortical neuronal damage is detected after 2.5 h of hemorrhagic hypotension in the near-term born lamb. The damage is reflected by a reduction of ECBA. The glutamate, NOx, cGMP pathway is likely to be involved in the pathogenesis of cerebral cortical damage.

Hypotension reduces cerebral O2supply, which may result in brain cell damage and loss of brain cell function in the near-term neonate. The aim is to elucidate 1) to what extent the functional disturbance of the cerebral cortex, as measured with electrocortical brain activity (ECBA), is related to cerebral cortical tissue damage, as estimated by MAP2; and 2) whether there is a relationship between the glutamate, nitric oxide (NO), cGMP pathway and the development of cerebral cortical tissue damage after hemorrhagic hypotension. Seven lambs were delivered at 131 d of gestation. Hypotension was induced by withdrawal of blood until mean arterial blood pressure was reduced to 30% of normotension. Cerebral O2supply, consumption, and ECBA were calculated in normotensive conditions and after 2.5 h of hypotension. Concentrations of glutamate and aspartate in cerebrospinal fluid (CSF), NO2and NO3(NOx) in plasma, and cGMP in cortical brain tissue were determined in both conditions. CSF and brain tissue from siblings were used to determine normotensive values. Cortical neuronal damage was detected after 2.5 h of hypotension. ECBA was negatively related to the severity of the cortical damage. ECBA was related to respectively glutamate, NOx, and cGMP concentrations. In conclusion, cortical neuronal damage is detected after 2.5 h of hemorrhagic hypotension in the near-term born lamb. The damage is reflected by a reduction of ECBA. The glutamate, NOx, cGMP pathway is likely to be involved in the pathogenesis of cerebral cortical damage.

Sengers syndrome is characterized by congenital cataracts, hypertrophic cardiomyopathy, mitochondrial myopathy, and lactic acidosis, but no abnormalities have been found with routine mitochondrial biochemical diagnostics (the determination of pyruvate oxidation rates and enzyme measurements). In immunoblot analysis, the protein content of the mitochondrial adenine nucleotide translocator 1 (ANT1) was found to be strongly reduced in the muscle tissues of two unrelated patients with Sengers syndrome. In addition, low residual adenine nucleotide translocator activity was detected upon the reconstitution of detergent‐solubilized mitochondrial extracts from the patients' skeletal or heart muscle into liposomes. Sequence analysis and linkage analysis showed that ANT1 was not the primary genetic cause of Sengers syndrome. We propose that transcriptional, translational, or posttranslational events are responsible for the ANT1 deficiency associated with the syndrome.

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are small channel proteins involved in the translocation of metabolites across the mitochondrial outer membrane. A single channel-forming protein is found in yeast, whereas higher eukaryotes express multiple VDACs, with humans and mice each harboring three distinct channels (VDAC1–3) encoded by separate genes. To begin to assess the functions of each of the three isoforms, the VDAC3 gene was inactivated by targeted disruption in embryonic stem cells. Here we show that mice lacking VDAC3 are healthy, but males are infertile. Although there are normal sperm numbers, the sperm exhibit markedly reduced motility. Structural defects were found in two-thirds of epididymal axonemes, with the most common abnormality being loss of a single microtubule doublet at a conserved position within the axoneme. In testicular sperm, the defect was only rarely observed, suggesting that instability of a normally formed axoneme occurs with sperm maturation. In contrast, tracheal epithelial cilia showed no structural abnormalities. In addition, skeletal muscle mitochondria were abnormally shaped, and activities of the respiratory chain complexes were reduced. These results demonstrate that axonemal defects may be caused by associated nonaxonemal components such as mitochondrial channels and illustrate that normal mitochondrial function is required for stability of the axoneme.

A patient with a deficient voltage-dependent anion channel (VDAC) is reported, presenting clinically with psychomotor retardation and minor dysmorphic features. Biochemical studies on muscle mitochondria showed impaired rates of pyruvate oxidation and ATP production; however, no specific deficient activity of one of the mitochondrial enzymes was involved. Western blotting experiments indicated an almost complete VDAC deficiency in skeletal muscle. The only moderately decreased VDAC content in the patient's fibroblasts might indicate that VDAC is expressed in a tissue-specific manner. The deficiency is likely caused by a mutation in the HVDAC1 gene or by a disturbed posttranslational modification. This is the first described deficiency of a component of the outer mitochondrial membrane associated with the pyruvate oxidation pathway. Defects in this membrane should be considered as a possible cause of otherwise unexplained mitochondrial disorders.

• We describe a 6-year-old boy who presented with progressive muscle weakness. Additional investigations revealed the existence of a myopathy and a pure motor neuropathy. Biochemical studies in muscle tissue showed a defect of NADH dehydrogenase (complex I). The patient dramatically improved on treatment with riboflavin and l-carnitine. Seven months after the start of the treatment, complex I activity was determined again and appeared to be normalized. Normalization of the enzymatic defect at this level has not been reported before. We provide a survey of nine patients with pure myopathy, associated with complex I deficiency and onset of symptoms in childhood.

Soluble enzymes were immobilized and visualized by polyacrylamide gel slabs, impregnated with the incubation medium including auxiliairy enzymes. The method has several advantages over existing techniques which make use of gel films or a semipermeable membrane. The diffusion of tissue compounds is effectively limited, while auxiliary enzymes may be operative. Moreover the viscosity of the medium is temperature-independent so that the incubation temperature can be varied.

In a substantial group of subjects suspected to have a mitochondriopathy no defect in the mitochondrial energy metabolism (pyruvate dehydrogenase complex or respiratory chain complexes) can be demonstrated. At least in some of these subjects it seems justified to consider a defect in one of the proteins which mediate the transport of several ions and substrates across the mitochondrial membranes. Of particular interest are proteins which are directly involved in the process of oxidative phosphorylation, such as the adenine nucleotide translocator (ANT) and the phosphate carrier (PiC). However, defects in transmembrane ion transporters also may induce impaired energy metabolism probably as a result of osmotic disturbances within the mitochondrial matrix. In this respect, the voltage-dependent anion channel (VDAC) and other ion channels have to be taken into consideration. Here we review the still incomplete knowledge of the occurrence of ANT, PiC, VDAC, cation channels, and a few substrate carriers in human tissues, as well as their possible role in pathology.

ABSTRACT: In a patient with a mitochondrial myopathy, presenting with lactic acidosis, 31P-nuclear magnetic resonance spectroscopy in resting muscle showed half the creatine phosphate level of controls. The creatine phosphate resynthesis rate after aerobic exercise was only 18% of that in controls. However, the activities of complexes I to V catalyzing oxidative phosphorylation and the pyruvate and the 2-oxoglutarate dehydrogenase complexes showed a 2- to 20-fold increase. In line with this, the uncoupled mitochondrial respiration rate was significantly higher than in controls. In contrast, the respiration of the mitochondria from the patient was less stimulated by ADP than that of control mitochondria. This finding could point to a defect in complex V, the enzyme directly involved in ATP synthesis. The activity of complex V, measured as the mitochondrial ATPase activity, and its concentration, as judged from Western blots using antisera against the F1part of complex V, were, however, also greatly increased in the patient. Alternatively, the transport system, importing ADP into and exporting ATP out of the mitochondrial matrix, the ADP/ATP or adenine nucleotide translocator, could be affected. Immunostaining of Western blots revealed a 4-fold decrease in the concentration of the adenine nucleotide translocator in the patient. Because oxidative phosphorylation was not disturbed in fibroblasts and lymphocytes, we conclude that this patient suffers from a muscle-specific deficiency of his mitochondrial adenine nucleotide translocator, a defect unknown so far.

• A 17-year-old patient had a progressive hypokinetic-rigid syndrome and several other signs and symptoms that indicated central nervous system involvement. Biochemical studies revealed a reduced form of nicotinamide-adenine dinucleotide dehydrogenase deficiency in skeletal muscle. Clinical signs and symptoms, and their association with an established defect of energy metabolism, led us to classify this disorder as a mitochondrial encephalomyopathy of Leigh's type.

Eukaryotic porins or VDACs (Voltage-Dependent Anion-selective Channels) are integral membrane proteins forming large hydrophilic pores. Three functioning genes for VDAC isoforms have been detected in mouse and the corresponding cDNAs are known also in humans. Tissue-specific VDAC isoform 1 (HVDAC1) deficiency in human skeletal muscle is responsible of a rare mitochondrial encephalomyopathy, fatal in childhood. Since coding sequences are not affected in the patient, we focused our interest in the gene structure. HVDAC1 and 2 have been previously mapped at chromosomes Xq13-21 and 21, respectively. Screening of an human chromosome X cosmid library resulted only in the isolation of processed pseudogenes, finely mapped at Xq22 and Xp11.2. Here, we report the mapping of HVDAC1 to chromosome 5q31 and HVDAC2 to chromosome 10q22 by FISH. Exon/intron probes, designed on the basis of the mouse gene structures, were obtained by long extension PCR amplification using the whole genomic DNA as a template. The sequence of the probe extremities clearly pointed to a genuine VDAC genomic sequence. Human and mouse regions where VDAC 1 and 2 genes were mapped are known to be synthetic, thus reinforcing the mapping of the human homologues.

Disturbances in substrate oxidations in muscle mitochondria from patients with a suspicion of a mitochondrial myopathy may arise from a deficiency of one or more of the complexes of the respiratory chain or of the pyruvate dehydrogenase complex. However, we found no clear-cut defect in a substantial part of such patients. In this report we discuss some of the other possibilities which could account for the disturbed substrate oxidation rates. Special attention will be paid to defects which are localized outside the respiratory chain, such as defects in post-respiratory chain enzymes, defects in transport mechanisms of the mitochondrial inner or outer membrane, deficiency of cofactors and deficiency of heat-shock protein. (Mol Cell Biochem 174: 243–247, 1997)

We have blocked creatine kinase (CK) mediated phosphocreatine (PCr) ⇄ ATP transphosphorylation in mitochondria and cytosol of skeletal muscle by knocking out the genes for the mitochondrial (ScCKmit) and the cytosolic (M-CK) CK isoforms in mice. Animals which carry single or double mutations, if kept and tested under standard laboratory conditions, have surprisingly mild changes in muscle physiology. Strenuous ex vivo conditions were necessary to reveal that MM-CK absence in single and double mutants leads to a partial loss of tetanic force output. Single ScCKmit deficiency has no noticeable effects but in combination the mutations cause slowing of the relaxation rate. Importantly, our studies revealed that there is metabolic and cytoarchitectural adaptation to CK defects in energy metabolism. The effects involve mutation type-dependent alterations in the levels of AMP, IMP, glycogen and phosphomonoesters, changes in activity of metabolic enzymes like AMP-deaminase, alterations in mitochondrial volume and contractile protein (MHC isoform) profiles, and a hyperproliferation of the terminal cysternae of the SR (in tubular aggregates). This suggests that there is a compensatory resiliency of loss-of-function and redirection of flux distributions in the metabolic network for cellular energy in our mutants.

Mitochondrial transmembrane carrier deficiencies are a recently discovered group of disorders, belonging to the so-called mitochondriocytopathies. We examined the human tissue distribution of carriers which are involved in the process of oxidative phosphorylation (adenine nucleotide translocator, phosphate carrier, and voltage-dependent anion channel) and some mitochondrial substrate carriers (2-oxoglutarate carrier, carnitine-acylcarnitine carrier, and citrate carrier). The tissue distribution on mRNA level of mitochondrial transport proteins appears to be roughly in correlation with the dependence of these tissues on mitochondrial energy production capacity. In general the main mRNA expression of carriers involved in mitochondrial energy metabolism occurs in skeletal muscle and heart. Expression in liver and pancreas differs between carriers. Expression in brain, placenta, lung, and kidney is lower than in the other tissues. Western and Northern blotting experiments show a comparable HVDAC1 protein and mRNA distribution for the tested tissues. Patient's studies showed that cultured skin fibroblasts may not be a reliable alternative for skeletal muscle in screening for human mitochondrial carrier defects.

In a patient with a mitochondrial myopathy, presenting with lactic acidosis, 31Pnuclear magnetic resonance spectroscopy in resting muscle showed half the creatine phosphate level of controls. The creatine phosphate resynthesis rate after aerobic exercise was only 18 of that in controls. However, the activities of complexes I to V catalyzing oxidative phosphorylation and the pyruvate and the 2oxoglutarate dehydrogenase complexes showed a 2 to 20fold increase. In line with this, the uncoupled mitochondrial respiration rate was significantly higher than in controls. In contrast, the respiration of the mitochondria from the patient was less stimulated by ADP than that of control mitochondria. This finding could point to a defect in complex V, the enzyme directly involved in ATP synthesis. The activity of complex V, measured as the mitochondrial ATPase activity, and its concentration, as judged from Western blots using antisera against the F1part of complex V, were, however, also greatly increased in the patient. Alternatively, the transport system, importing ADP into and exporting ATP out of the mitochondrial matrix, the ADPATP or adenine nucleotide translocator, could be affected. Immunostaining of Western blots revealed a 4fold decrease in the concentration of the adenine nucleotide translocator in the patient. Because oxidative phosphorylation was not disturbed in fibroblasts and lymphocytes, we conclude that this patient suffers from a musclespecific deficiency of his mitochondrial adenine nucleotide translocator, a defect unknown so far. Pediatr Res33 412417, 1993

A patient with a deficient voltage-dependent anion channel (VDAC) is reported, presenting clinically with psychomotor retardation and minor dysmorphic features. Biochemical studies on muscle mitochondria showed impaired rates of pyruvate oxidation and ATP production; however, no specific deficient activity of one of the mitochondrial enzymes was involved. Western blotting experiments indicated an almost complete VDAC deficiency in skeletal muscle. The only moderately decreased VDAC content in the patient's fibroblasts might indicate that VDAC is expressed in a tissue-specific manner. The deficiency is likely caused by a mutation in the HVDAC1 gene or by a disturbed posttranslational modification. This is the first described deficiency of a component of the outer mitochondrial membrane associated with the pyruvate oxidation pathway. Defects in this membrane should be considered as a possible cause of otherwise unexplained mitochondrial disorders.

Age dependency of enzymes involved in the energy generating system was investigated in 600 g supernatants of quadriceps muscle from rats of different ages. (14-C) pyruvate oxidation rates (± ADP) increased a significantly from low early values in the neonatal period (321 ± 113 nmol/h/mg prot.) to nearly adult values at the end of the suckling period (859 ± 199 nmol/h/mg prot.) (p < 0.01). Pyruvate dehydrogenase complex (PDHC), citrate synthase and cytochrome c oxidase showed similar patterns of development. Immunoblot studies of PDHC indicated that E1a, the regulatory subunit of the multi-enzyme complex, is ihe most rapidly increasing protein with age. This study demonstrates a clear increase in mitochondrial oxidative capacity in rat muscle up until weaning which is in accordance with the known differententiation pattern of skeletal muscle fibers.