H.R. Scholte

AN X-LINKED MITOCHONDRIAL DISEASE AFFECTING CARDIAC MUSCLE, SKELETAL MUSCLE AND NEUTROPHIL LEUCOCYTES

An X-linked recessive disease is reported in a large pedigree. The disease is characterised by a triad of dilated cardiomyopathy, neutropenia and skeletal myopathy. The untreated patients, all boys, died in infancy or early childhood from septicemia or cardiac decompensation. Ultrastructural abnormalities were observed in mitochondria in cardiac muscle cells, neutrophil bone marrow cells and to a lesser extent (0-9%) in skeletal muscle cells. Membrane-bound vacuoles were seen in neutrophil bone marrow cells. Intramuscular fat droplets were increased in type I skeletal muscle fibres. An affected patient had intermittent lactic acidemia, borderline low plasma carnitine, the latter decreasing during periods of illness, and low muscle carnitine (27% pretreatment; 35-40% posttreatment). While on treatment with oral carnitine he had less weakness and no cardiac complaints, but his neutropenia was not affected. Respiratory chain abnormalities were observed in this patients isolated skeletal muscle mitochondria. These were: (1) diminished concentrations of cytochromes c1 + c, b and aa3 to 29, 47 and 64% of the averaged controls, and (2) a lowered P:0 ratio for oxidation of ascorbate + TMPD, with diminished uncoupler stimulated Mg2+-ATPase activity. Muscle AMP deaminase was deficient (5 resp. 17%). Only one previous report (Neustein et al. 1979) on X-linked mitochondrial cardiomyopathy exists, which probably refers to the same entity. Biochemical studies and haematological abnormalities (neutropenia) are reported for the first time.

Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting. To examine whether these different phenotypes are due to differences in the VLCAD genotype, we investigated 58 different mutations in 55 unrelated patients representing all known clinical phenotypes and correlated the mutation type with the clinical phenotype. Our results show a clear relationship between the nature of the mutation and the severity of disease. Patients with the severe childhood phenotype have mutations that result in no residual enzyme activity, whereas patients with the milder childhood and adult phenotypes have mutations that may result in residual enzyme activity. This clear genotype-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established.

Fatty Acid Activation: Specificity, Localization, and Function

Publisher Summary This chapter discusses fatty acid activation. Most cells can metabolize fatty acids. The most important pathways are β-oxidation to acetyl-CoA; esterification with glycerol phosphate, glycerol derivatives, sphingosine, glycol, and long-chain alcohols; chain elongation; desaturation; and α and ω oxidation. Fatty acid molecules are completely ionized at physiological pH values. The carboxylate ion is chemically unreactive toward nucleophilic substitution. Esters, thioesters, and acid anhydrides, in which both the carboxylate resonance and the negative charge on the carboxyl group disappear, are more reactive. Both microsomal and mitochondrial long-chain fatty acid activation is stimulated by the presence of high concentrations of several salts. Both activities are also strongly stimulated by the addition of the particle-free supernatant from several rat tissues. Purified mammalian acyl-CoA synthetases are very specific in their nucleotide requirement.

Riboflavin-responsive oxidative phosphorylation complex I deficiency caused by defective ACAD9: new function for an old gene

Mitochondrial complex I deficiency is the most common oxidative phosphorylation defect. Mutations have been detected in mitochondrial and nuclear genes, but the genetics of many patients remain unresolved and new genes are probably involved. In a consanguineous family, patients presented easy fatigability, exercise intolerance and lactic acidosis in blood from early childhood. In muscle, subsarcolemmal mitochondrial proliferation and a severe complex I deficiency were observed. Exercise intolerance and complex I activity was improved by a supplement of riboflavin at high dosage. Homozygosity mapping revealed a candidate region on chromosome three containing six mitochondria-related genes. Four genes were screened for mutations and a homozygous substitution was identified in ACAD9 (c.1594 C>T), changing the highly conserved arginine-532 into tryptophan. This mutation was absent in 188 ethnically matched controls. Protein modelling suggested a functional effect due to the loss of a stabilizing hydrogen bond in an α-helix and a local flexibility change. To test whether the ACAD9 mutation caused the complex I deficiency, we transduced fibroblasts of patients with wild-type and mutant ACAD9. Wild-type, but not mutant, ACAD9 restored complex I activity. An unrelated patient with the same phenotype was compound heterozygous for c.380 G>A and c.1405 C>T, changing arginine-127 into glutamine and arginine-469 into tryptophan, respectively. These amino acids were highly conserved and the substitutions were not present in controls, making them very probably pathogenic. Our data support a new function for ACAD9 in complex I function, making this gene an important new candidate for patients with complex I deficiency, which could be improved by riboflavin treatment.

On the triple localization of creatine kinase in heart and skeletal muscle cells of the rat: Evidence for the existence of myofibrillar and mitochondrial isoenzymes

Abstract 1. 1. In order to assess the intracellular localization of creatine kinase (EC 2.7.3.2) in muscle, a fractionation prodecure was developed in which (a) 1 mM EDTA was used to prevent the Ca2+-dependent binding of cytosolic creatine kinase to myosin A; (b) 0.2 mM dithiothreitol was used to prevent binding of the cytosolic marker enzyme pyruvate, kinase (EC 2.7.1.40) to the heavy particulate fraction; (c) the myofibrils were solubilized by ultrasonic treatment in 1 M KCI; (d) the washing of the minced tissue was omitted since it was found that this gave rise to the loss of up to 40% of pyruvate kinase. The tissues were fractionated in three fractions enriched in the myofibrillar marker enzyme myosin ATPase (EC 3.6.1.3.), the mitochondrial marker enzyme cytochrome c oxidase (EC 1.9.3.1) or the cytosolic marker enzyme pyruvate kinase. 2. 2. Creatine kinase was found to be located in the myofibrils, the mitochondria and the cytosol of both rat heart and rat masseter muscle. With the aid of the marker enzymes mentioned above, it was calculated that in heart 33% of the total cellular creatine kinase activity (measured with 25 mM creatine at 25°C) resides in the myofibrils, 19% in the mitochondria and 48% in the cytosol. For rat masseter muscle these values were 4.4, 2.3 and 93% respectively. The total creatine kinase activity was in heart 145 units/g wet weight, and in masseter muscle 432 units/g wet weight. 3. 3. The electrophoretic mobility at pH 9 on cellulose acetate was different for the three isoenzymes. The mobility towards the cathode increased in the following order:cytosolic (MM-type), myofibrillar and mitochondrial isoenzyme, extracted from mitochondria. Mitochondrial creatine kinase, when bound to the outside of the inner membrane, where it resides in situ, was not mobile. 4. 4. The apparent Km values for creatine for reactions catalyzed by rat-heart myofibrillar, mitochondrial and cytosolic subcellular fractions were 4.3, 13 and 20 mM, respectively. In the corresponding fractions of masseter muscle these values were 18, 13 and 28 mM, respectively. For creatine phosphate as the substrate the values were for heart 0.9, 1.0 and 1.8 mM, and for masseter muscle 3.0, 1.6 and 2.6 mM, respectively. 5. 5. It is proposed that the physiological function of the mitochondrial and cytosolic creatine kinases is the synthesis of creatine phosphate, when fatty acids or carbohydrates, respectively, are the main source of energy. As the function for myofibrillar creatine kinase is proposed the degradation of creatine phosphate to yield ATP for muscular contraction.

X-linked cardioskeletal myopathy and neutropenia (Barth syndrome): respiratory-chain abnormalities in cultured fibroblasts

ConclusionsThe ATP production from glutamate + malate, pyruvate + malate, or succinate (+ rotenone was lower than the mean values found in control fibroblasts in patient 1 and in the low-normal range in patient 2. Decreased activities of complex III (±30% of controls) and complex IV (±60% of controls) are in agreement with the previous studies in muscle. These findings provide evidence that respiratory-chain dysfunction is an essential component of Barth syndrome. Results further indicate that the gene product is essential for the function, formation or stability of both complex III and complex IV. Both complex III and complex IV contain subunits that are encoded by mtDNA. The results of emetine labelling make it unlikely that the gene is involved in the expression of mtDNA. We furthermore suspect that 3-methylglutaconic aciduria in this syndrome represents an epiphenomenon which is as yet unexplained but has been described so far in patients with separate mitochondrial disorders (Gibson et al 1995).

Carnitine palmitoyltransferase II deficiency with normal carnitine palmitoyltransferase I in skeletal muscle and leucocytes

Deficiency of carnitine palmitoyltransferase II (CPT II), was found to be the cause of the syndrome of muscle pain and myoglobinuria following strenuous exercise in an otherwise healthy young man. During fasting, serum creatine kinase remained low and ketogenesis was normal. The clearance of a fat emulsion and the activity of extrahepatic lipoprotein lipase was lowered, while the hepatic lipoprotein lipase was normal. A skeletal muscle biopsy did not show abnormal lipid storage. CPT II was deficient in skeletal muscle and leucocytes, while CPT I activity was normal and exhibited normal kinetic properties. CPT I has a higher affinity for palmitoylcarnitine than CPT II, and is more inhibited at increasing palmitoylcarnitine concentrations. In erythrocytes only CPT I is present.

Riboflavin-responsive complex I deficiency

Three patients from a large consanguineous family, and one unrelated patient had exercise intolerance since early childhood and improved by supplementation with a high dosage of riboflavin. This was confirmed by higher endurance power in exercise testing. Riboflavin had been given because complex I, which contains riboflavin in FMN, one of its prosthetic groups, had a very low activity in muscle. Histochemistry showed an increase of subsarcolemmal mitochondria. The low complex I activity contrasted with an increase of the activities of succinate dehydrogenase, succinate-cytochrome c oxidoreductase and cytochrome c oxidase. Isolated mitochondria from these muscle specimens proved deficient in oxidizing pyruvate plus malate and other NAD(+)-linked substrates, but oxidized succinate and ascorbate at equal or higher levels than controls. Two years later a second biopsy was taken in one of the patients, and the activity of complex I had increased from 16% to 47% of the average activity in controls. In the four biopsies, cytochrome c oxidase activity correlated negatively with age. We suspect that this is due to reactive oxygen species generated by the proliferating mitochondria and peroxidizing unsaturated fatty acids of cardiolipin. Three of the four patients had low blood carnitine, and all were found to have hypocarnitinemic family members.

Patients with chronic fatigue syndrome performed worse than controls in a controlled repeated exercise study despite a normal oxidative phosphorylation capacity

BackgroundThe aim of this study was to investigate the possibility that a decreased mitochondrial ATP synthesis causes muscular and mental fatigue and plays a role in the pathophysiology of the chronic fatigue syndrome (CFS/ME).MethodsFemale patients (n = 15) and controls (n = 15) performed a cardiopulmonary exercise test (CPET) by cycling at a continuously increased work rate till maximal exertion. The CPET was repeated 24 h later. Before the tests, blood was taken for the isolation of peripheral blood mononuclear cells (PBMC), which were processed in a special way to preserve their oxidative phosphorylation, which was tested later in the presence of ADP and phosphate in permeabilized cells with glutamate, malate and malonate plus or minus the complex I inhibitor rotenone, and succinate with rotenone plus or minus the complex II inhibitor malonate in order to measure the ATP production via Complex I and II, respectively. Plasma CK was determined as a surrogate measure of a decreased oxidative phosphorylation in muscle, since the previous finding that in a group of patients with external ophthalmoplegia the oxygen consumption by isolated muscle mitochondria correlated negatively with plasma creatine kinase, 24 h after exercise.ResultsAt both exercise tests the patients reached the anaerobic threshold and the maximal exercise at a much lower oxygen consumption than the controls and this worsened in the second test. This implies an increase of lactate, the product of anaerobic glycolysis, and a decrease of the mitochondrial ATP production in the patients. In the past this was also found in patients with defects in the mitochondrial oxidative phosphorylation. However the oxidative phosphorylation in PBMC was similar in CFS/ME patients and controls. The plasma creatine kinase levels before and 24 h after exercise were low in patients and controls, suggesting normality of the muscular mitochondrial oxidative phosphorylation.ConclusionThe decrease in mitochondrial ATP synthesis in the CFS/ME patients is not caused by a defect in the enzyme complexes catalyzing oxidative phosphorylation, but in another factor.Trial registrationClinical trials registration number: NL16031.040.07

Defects in oxidative phosphorylation. Biochemical investigations in skeletal muscle and expression of the lesion in other cells

Mitochondria are very vulnerable to genetic and environmental damage. If a patient is suspected of having a mitochondrial disease, elevated blood lactate, lowered blood free carnitine, abnormal urinary organic acids and carnitine esters and tissue histopathology may help with the diagnosis. For biochemical assessment of the defect, muscle is the tissue of choice even when involvement of other organs like heart or brain is more prominent.We have studied isolated muscle mitochondria and homogenates from muscle biopsies in 250 patients, and have detected in more than one third mitochondrial defects in oxidative phosphorylation, dehydrogenases, non-redox enzymes catalyzing synthesis of fuel molecules and in the carnitine system. Several patients showed more than one defect.We have selected eight patients to illustrate how a relatively simple series of investigations in both isolated mitochondria and homogenate can be used for the identification of defects in oxidative phosphorylation in a small amount of muscle (200 mg or more). Identification of the defect(s) is important since it may provide the basis for rational treatment. A minority of the patients recovered partly or completely, which is unique in treatment of inborn errors of subcellular organelles.An important aspect of mitochondrial dysfunction is the tissue specificity. The defect may be systemic but is often clinically expressed in only one or a few tissues. Rarely, tissue-specific defects can be understood on the basis of tissue-specificity of mitochondrial (iso-)enzymes. Mitochondrial deficiencies of all biotin enzymes and most CoA-linked enzymes are expressed in fibroblasts; most respiratory chain defects are not.When mitochondrial ATP synthesis has been compromised by a mitochondrial defect, secondary lesions may be generated by changes in mitochondrial protein synthesis, activated proteases and phospholipases, increased matrix CoA and resulting carnitine deficiency, decrease in Krebs cycle intermediates and increased free radical formation and lipid peroxidation.