Henk ter Laak

Skeletal muscles of mice deficient in muscle creatine kinase lack burst activity

To understand the physiological role of the creatine kinase-phosphocreatine (CK-PCr) system in muscle bioenergetics, a null mutation of the muscle CK (M-CK) gene was introduced into the germline of mice. Mutant mice show no alterations in absolute muscle force, but lack the ability to perform burst activity. Their fast-twitch fibers have an increased intermyofibrillar mitochondrial volume and an increased glycogenolytic/glycolytic potential. PCr and ATP levels are normal in resting M-CK-deficient muscles, but rates of high energy phosphate exchange between PCr and ATP are at least 20-fold reduced. Strikingly, PCr levels decline normally during muscle exercise, suggesting that M-CK-mediated conversion is not the only route for PCr utilization in active muscle.

Congenital muscular dystrophy

Histomorphological and histochemical variability was studied in muscle specimens from 30 patients with congenital muscular dystrophy (CMD). We found involvement of the central nervous system in 8 patients (Fukuyama CMD, F-CMD), involvement of the brain and the eyes in 5 patients (muscle, eye and brain disease, MEB-D) and hypodense white matter on the CT scans of 2 patients with (sub)normal intelligence (occidentaltype cerebromuscular dystrophy, O-CMD). No morphological hallmarks were found to differentiate these subgroups. Only fat cell infiltration was found to be increased with increasing age in ‘pure’ CMD (pure-CMD). The morphological data did not appear to be correlated with the clinical severity or type of dystrophy (pure-CMD, F-CMD, MEB-D and O-CMD). Immunohistochemistry with dystrophin, vimentin and desmin antibodies in 14 patients (6 pure-CMD, 5 F-CMD, 2 MEB-D and 1 O-CMD) showed a normal expression pattern.

Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins

Mitochondrial dysfunction during acute metabolic crises is considered an important pathomechanism in inherited disorders of propionate metabolism, i.e. propionic and methylmalonic acidurias. Biochemically, these disorders are characterized by accumulation of propionyl-CoA and metabolites of alternative propionate oxidation. In the present study, we demonstrate uncompetitive inhibition of PDHc (pyruvate dehydrogenase complex) by propionyl-CoA in purified porcine enzyme and in submitochondrial particles from bovine heart being in the same range as the inhibition induced by acetyl-CoA, the physiological product and known inhibitor of PDHc. Evaluation of similar monocarboxylic CoA esters showed a chain-length specificity for PDHc inhibition. In contrast with CoA esters, non-esterified fatty acids did not inhibit PDHc activity. In addition to PDHc inhibition, analysis of respiratory chain and tricarboxylic acid cycle enzymes also revealed an inhibition by propionyl-CoA on respiratory chain complex III and alpha-ketoglutarate dehydrogenase complex. To test whether impairment of mitochondrial energy metabolism is involved in the pathogenesis of propionic aciduria, we performed a thorough bioenergetic analysis in muscle biopsy specimens of two patients. In line with the in vitro results, oxidative phosphorylation was severely compromised in both patients. Furthermore, expression of respiratory chain complexes I-IV and the amount of mitochondrial DNA were strongly decreased, and ultrastructural mitochondrial abnormalities were found, highlighting severe mitochondrial dysfunction. In conclusion, our results favour the hypothesis that toxic metabolites, in particular propionyl-CoA, are involved in the pathogenesis of inherited disorders of propionate metabolism, sharing mechanistic similarities with propionate toxicity in micro-organisms.

TDP-43 accumulation is common in myopathies with rimmed vacuoles

TAR-DNA-binding protein-43 (TDP-43) is a nuclear protein that is thought to play a regulatory role in gene expression. Ubiquitinated TDP-43 is found in cytoplasmic inclusions in motor neuron disease (MND), frontotemporal lobar degeneration (FTLD-TDP), and MND with FTLD [4]. This shared histopathological hallmark led to the classiWcation of a new class of diseases, the TDP-43-proteinopathies [3]. Recently, TDP-43-positive inclusions have been described in skeletal muscle in sporadic inclusion body myositis (sIBM) and in IBM due to mutations in the valosin-containing protein (VCP) [6]. These myopathies are accompanied by vacuolar changes commonly known as rimmed vacuoles (although these vacuoles are morphologically not always rimmed). These characteristic morphological changes are not exclusive to sIBM and IBM with VCP-mutations but are also found in oculopharyngeal muscular dystrophy (OPMD) [2] and distal myopathies with rimmed vacuoles (DMRV), both of which are hereditary myopathies with inclusion bodies. Although DMRV are a genetically heterogeneous group of diseases, the term is often used in the context of the Nonaka myopathy with mutations of the UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase (GNE) gene [5]. We studied whether TDP-43 aggregates were present in muscle biopsies of sIBM, OPMD and DMRV or in myopathies without the mentioned vacuolar changes. TDP-43 immunostaining was present in some, but not all, rimmed vacuoles and/or basophilic Wbers in most biopsies from patients with sIBM (77.8%) and OPMD (83.3%) and in all biopsies from patients with DMRV (one with conWrmed GNE mutation) (Table 1). TDP-43 immunostaining was not detected in muscle biopsies from patients with myopathies without vacuolar changes. The pattern of TDP-43 staining was variable: in some biopsies only a single TDP-43 positive inclusion was visible even though more than one rimmed vacuole was present, and in other biopsies multiple TDP-43-positive aggregates were visualized. Both granular and more dot-like aggregates in rimmed vacuoles and basophilic Wbers were detected. The morphology of the aggregates was similar in sIBM, OPMD, and DMRV (Fig. 1a–c). Many aggregates also stained positive for ubiquitin (Fig. 1d–f). Occasionally subsarcolemmal TDP-43 staining was present in Wbers that did not exhibit rimmed vacuoles or subsarcolemmal basophilia (Fig. 1g). In conclusion, we conWrm the recent data of Weihl et al. [6], showing the presence of TDP-43-positive inclusions in the majority of muscle biopsies from patients with sIBM. However, we found TDP-43-positive aggregates not only in sIBM but also in other vacuolar myopathies. Thus TDP-43positive aggregates would seem to be a general phenomenon among the myopathies associated with rimmed vacuoles. This suggests that abnormal TDP-43 accumulation is more likely to be a common endpoint of (muscle) cell degeneration rather than a primary pathological mechanism underlying these myopathies. These Wndings may provide insight into the pathobiological relevance of TDP-43 B. Kusters and B. J. A. van Hoeve have contributed equally to this work.

Effects of the creatine analogue β-guanidinopropionic acid on skeletal muscles of mice deficient in muscle creatine kinase

To evaluate the effects of phosphocreatine (PCr) and creatine (Cr) depletion on skeletal muscles of mice deficient in muscle creatine kinase (M-CK), we have fed mutant mice a diet containing the creatine analogue beta-guanidinopropionic acid (beta GPA). After 8-10 weeks of feeding, accumulation of the creatine analogue in M-CK-deficient muscles was comparable to that observed in muscles of wild-type mice. Strikingly, and unlike wild types, mutants did not accumulate phosphorylated beta GPA, indicating that MM-CK is the only muscle CK isoform which can phosphorylate beta GPA. In M-CK-deficient muscles there was respective depletion of PCr, Cr and ATP levels to 31, 41 and 83% of normal. The average cross-sectional area of type 2B fibres in gastrocnemius muscles was very much reduced and was similar to type 1 and type 2A fibres which maintained their normal size. The maximal isometric twitch force developed by gastrocnemius-plantaris-soleus (GPS) muscle complexes of beta GPA-treated mutants was reduced by about 30%, but these muscles showed an increased fatigue resistance during 1 and 5 Hz contraction. Mitochondrial enzyme activities in the upper hind limb musculature of null mutants were 20-35% increased by the beta GPA diet. Altogether, these results provide evidence that certain functions of the creatine kinase/phosphocreatine (CK/PCr) system are not eliminated solely by the loss of M-CK.

Muscle-fiber conduction velocity and electromyography as diagnostic tools in patients with suspected inflammatory myopathy: A prospective study

Combinations of different techniques can increase the diagnostic yield from neurophysiological examination of muscle. In 25 patients with suspected inflammatory myopathy, we prospectively performed needle electromyography (EMG) and measured muscle‐fiber conduction velocity (MFCV) in a single muscle, using a technique with direct muscle‐fiber stimulation and recording. Results of MFCV were compared with final diagnosis, EMG, and needle muscle biopsy. Diagnostic accuracy of combined MFCV and EMG studies was 72%, compared to 60% for EMG alone. This improvement was due to a gain in specificity. The MFCV did not prove useful in discriminating inflammatory myopathy from other myopathies. Furthermore, we found a correlation of 92% between variability of MFCV and myopathic changes in muscle biopsy. We conclude that the utility of electrodiagnostic examination can be increased if EMG examination is combined with MFCV studies. Muscle Nerve 29: 46–50, 2004

Congenital hypertrophic cardiomyopathy, cataract, mitochondrial myopathy and defective oxidative phosphorylation in two siblings with Sengers-like syndrome

We describe two siblings with a Sengers-like syndrome, who presented with congenital hypertrophic cardiomyopathy, infantile cataract, mitochondrial myopathy, lactic acidosis and normal mental development. A mitochondrial adenine nucleotide translocator 1 (ANT1) defect was detected since the ANT1 protein was not detectable by immmunoblotting in muscle samples of the patients. Additionally to these features of classical Sengers syndrome (OMIM 212350), we found that the mitochondrial oxidative phosphorylation, measured by biochemical analysis, was severely compromised in skeletal muscle in both children. Biochemical and morphological analysis of the fibroblasts revealed normal results. The association of significantly decreased pyruvate oxidation rates, deficient energy production and decreased multiple mitochondrial enzyme-complex activities in the muscle samples of our patients is a new finding which differs from previous results in patients with Sengers syndrome. Conclusion:we recommend a muscle biopsy and the biochemical analysis of the oxidative phosphorylation system in patients with muscle hypotonia, cardiomyopathy and congenital or infantile cataract.

Muscle 3243A-->G mutation load and capacity of the mitochondrial energy-generating system.

The mitochondrial energy‐generating system (MEGS) encompasses the mitochondrial enzymatic reactions from oxidation of pyruvate to the export of adenosine triphosphate. It is investigated in intact muscle mitochondria by measuring the pyruvate oxidation and adenosine triphosphate production rates, which we refer to as the “MEGS capacity.” Currently, little is known about MEGS pathology in patients with mutations in the mitochondrial DNA. Because MEGS capacity is an indicator for the overall mitochondrial function related to energy production, we searched for a correlation between MEGS capacity and 3243A→G mutation load in muscle of patients with the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) syndrome.

Postnatal centralization of muscle fibre nuclei in centronuclear myopathy

Postnatal centralization of muscle fibre nuclei, which were previously located subsarcolemmally, is described in a case of centronuclear myopathy (CNM) in a male patient with generalized muscle weakness since birth. A muscle biopsy was taken at the age of 11 months; no particular abnormalities were observed at this stage apart from an unusual variation in fibre size. A distinctly below average muscle fibre diameter, increased endomysial connective tissue, and features typical for CNM were found in a biopsy taken 9 yr later. Immunohistochemical studies using antibodies to desmin, vimentin, laminin and type IV collagen revealed altered staining patterns compared with normal fibres. The abnormalities in the patterns of cytoskeletal proteins point to a defective regulation of the composition and organization of the cytoskeletal network during development, paralleled by abnormalities in the extracellular matrix.

Concomitant dermatomyositis and myasthenia gravis presenting with respiratory insufficiency.

We report a 28‐year‐old woman with a history of chronic immune‐mediated hepatitis, in whom the simultaneous manifestation of dermatomyositis and myasthenia gravis resulted in severe neck extensor weakness and subacute respiratory insufficiency, followed by proximal muscle weakness and external ophthalmoplegia. Radiological signs of a thymoma were absent. The distinguishing clinical, electrophysiological, and biopsy findings are discussed. We suggest that an underlying immunoregulatory disorder was present, explaining the occurrence of three rare immune‐mediated diseases in one patient.