Ultrastructural mitochondrial alterations in Equine myopathies of unknown origin.

Background: Very few mitochondrial myopathies have been described in horses. Objective: To examine the ultrastructure of muscle mitochondria in equine cases of myopathy of unknown origin. Materials & methods: Biopsies of vastus lateralis of the Musculus quadriceps femoris were taken predominantly immediately post mortem and processed for transmission electron microscopy. As a result, electron micrographs of 90 horses in total were available for analysis comprising 4 control horses, 16 horses suffering from myopathy and 70 otherwise diseased horses. Results: Following a thorough clinical and laboratory work-up, four out of five patients that did not fit into the usual algorithm to detect known causes of myopathy showed ultrastructural mitochondrial alterations. Small mitochondria with zones with complete disruption of cristae associated with lactic acidemia were detected in a 17-year-old pony mare, extremely long and slender mitochondria with longitudinal cristae in a 5-year-old Quarter horse stallion, a mixture of irregular extremely large mitochondria (measuring 2500 by 800 nm) next to smaller ones in an 8-year-old Hanoverian mare and round mitochondria with only few cristae in a 11-year-old pony gelding. It remains uncertain whether the subsarcolemmal mitochondrial accumulations observed in the fifth patient have any pathological significance. Conclusions: Ultrastructural alterations in mitochondria were detected in at least four horses. To conclude that these are due to mitochondrial dysfuntions, biochemical tests should be performed. Practical applications: The possibility of a mitochondrial myopathy should be included in the differential diagnosis of muscle weakness.

The role of autophagy in anticancer therapy: promises and uncertainties

Autophagy (literally self-eating) is a catabolic mechanism involved in the recycling and turnover of cytoplasmic constituents. Although often referred to as type II programmed cell death, autophagy is primarily a survival rather than a cell death mechanism in response to different stress stimuli. Autophagy is a process in which part of the cytoplasm or entire organelles are sequestered into double-membrane vesicles, called autophagosomes, which ultimately fuse with lysosomes to degrade their contents. Studies show that autophagy is associated with a number of pathological conditions, including cancer, infectious diseases, myopathies and neurodegenerative disorders. With respect to cancer, it has been suggested that the early stages of tumourigenesis are associated with downregulation of autophagy-related (ATG) genes. Indeed, several ATG genes display tumour suppressor function, including Beclin1, which is frequently hemizygously deleted in breast cancer cells. Conversely, in advanced stages of tumourigenesis or during anticancer therapy, autophagy may promote survival of tumour cells in adverse environmental conditions. Therefore, a thorough understanding of autophagy in different cancer types and stages is a prerequisite to determine an autophagy-activating or autophagy-inhibiting treatment strategy.

Lethal toxin of Clostridium sordellii is associated with fatal equine atypical myopathy

The lethal toxin of Clostridium sordellii (TcsL) evokes severe, mostly fatal disease patterns like toxic shock syndrome in humans and animals. Since this large clostridial toxin-induced severe muscle damaging when injected intramuscularly into mice, we hypothesized that TcsL is also associated with equine atypical myopathy (EAM), a fatal myodystrophy of hitherto unknown etiology. Transmission electron microscopy revealed skeletal and heart muscles of EAM-affected horses to undergo degeneration ultrastructurally similar to the damage found in TcsL-treated mice. Performing immunohistochemistry, myofibers of EAM-affected horses specifically reacted with sera derived from horses with EAM as well as an antibody specific for the N-terminal part of TcsL, while both antibodies failed to bind to the myofibers of either healthy horses or those with other myopathies. The presence of TcsL in myofibers of horses with EAM suggests that it plays a role as trigger or even as lethal factor in this disease.

Velocity recovery cycles of human muscle action potentials: Repeatability and variability

Velocity recovery cycles (VRCs) of human muscle action potentials have been proposed as a new technique for assessing muscle membrane function in myopathies. This study was undertaken to determine the variability and repeatability of VRC measures such as supernormality, to help guide future clinical use of the method.

Phosphocreatine interacts with phospholipids, affects membrane properties and exerts membrane-protective effects

A broad spectrum of beneficial effects has been ascribed to creatine (Cr), phosphocreatine (PCr) and their cyclic analogues cyclo-(cCr) and phospho-cyclocreatine (PcCr). Cr is widely used as nutritional supplement in sports and increasingly also as adjuvant treatment for pathologies such as myopathies and a plethora of neurodegenerative diseases. Additionally, Cr and its cyclic analogues have been proposed for anti-cancer treatment. The mechanisms involved in these pleiotropic effects are still controversial and far from being understood. The reversible conversion of Cr and ATP into PCr and ADP by creatine kinase, generating highly diffusible PCr energy reserves, is certainly an important element. However, some protective effects of Cr and analogues cannot be satisfactorily explained solely by effects on the cellular energy state. Here we used mainly liposome model systems to provide evidence for interaction of PCr and PcCr with different zwitterionic phospholipids by applying four independent, complementary biochemical and biophysical assays: (i) chemical binding assay, (ii) surface plasmon resonance spectroscopy (SPR), (iii) solid-state (31)P-NMR, and (iv) differential scanning calorimetry (DSC). SPR revealed low affinity PCr/phospholipid interaction that additionally induced changes in liposome shape as indicated by NMR and SPR. Additionally, DSC revealed evidence for membrane packing effects by PCr, as seen by altered lipid phase transition. Finally, PCr efficiently protected against membrane permeabilization in two different model systems: liposome-permeabilization by the membrane-active peptide melittin, and erythrocyte hemolysis by the oxidative drug doxorubicin, hypoosmotic stress or the mild detergent saponin. These findings suggest a new molecular basis for non-energy related functions of PCr and its cyclic analogue. PCr/phospholipid interaction and alteration of membrane structure may not only protect cellular membranes against various insults, but could have more general implications for many physiological membrane-related functions that are relevant for health and disease.

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Elevated transaminases in asymptomatic patients can be detected in more than 5 % of the investigations. If there are no obvious reasons, the finding should be confirmed within the next 3 months. Frequent causes are non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcohol, hepatitis B or C, hemochromatosis and drugs or toxins. Rarer causes are autoimmune hepatitis, M. Wilson and α1-antitrypsine deficiency. There are also non-hepatic causes such as celiac disease or hemolysis and myopathies in the case of an exclusive increase of ASAT. I recommend a two-step investigational procedure; the more frequent causes are examined first before the rare causes are studied. The value of the proposed investigations is discussed.

Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways

AIMS: Cardiac myopathies are the second leading cause of death in patients with Duchenne and Becker muscular dystrophy, the two most common and severe forms of a disabling striated muscle disease. Although the genetic defect has been identified as mutations of the dystrophin gene, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Here, we examined cellular mechanisms linking the initial membrane damage to the dysfunction of dystrophic heart. METHODS AND RESULTS: Cardiac ventricular myocytes were enzymatically isolated from 5- to 9-month-old dystrophic mdx and wild-type (WT) mice. Cells were exposed to mechanical stress, applied as osmotic shock. Stress-induced cytosolic and mitochondrial Ca(2+) signals, production of reactive oxygen species (ROS), and mitochondrial membrane potential were monitored with confocal microscopy and fluorescent indicators. Pharmacological tools were used to scavenge ROS and to identify their possible sources. Osmotic shock triggered excessive cytosolic Ca(2+) signals, often lasting for several minutes, in 82% of mdx cells. In contrast, only 47% of the WT cardiomyocytes responded with transient and moderate intracellular Ca(2+) signals. On average, the reaction was 6-fold larger in mdx cells. Removal of extracellular Ca(2+) abolished these responses, implicating Ca(2+) influx as a trigger for abnormal Ca(2+) signalling. Our further experiments revealed that osmotic stress in mdx cells produced an increase in ROS production and mitochondrial Ca(2+) overload. The latter was followed by collapse of the mitochondrial membrane potential, an early sign of cell death. CONCLUSION: Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.

The feasibility of non-viral gene transfer to the diaphragm in vivo

Gene transfer using electroporation is an essential method for the study of developmental biology, especially to understand the internal control of degeneration and apoptosis of the muscle cells that occurs earlier and quicker than the usual degeneration process occurring by aging. Such experimental studies may have a role in developing new strategies for treating patients suffering from inherited primary myopathies such as Duchenne muscular dystrophy (DMD). The present study was designed to evaluate the feasibility of electroporation mediated transfer of reporter genes to the diaphragm in vivo. This is the first report of gene transfer of naked plasmid DNA into the diaphragm muscle in vivo using electroporation. Our results showed that in vivo gene transfer of naked plasmid DNA into the diaphragm muscle using electroporation is feasible.

Teaching video neuroimages: Beevor sign: when the umbilicus is pointing to neurologic disease

A 57-year-old man with genetically proven facioscapulohumeral muscular dystrophy (FSHMD 1A) demonstrated Beevor sign (video on the Neurology Web site at www.neurology.org). The upward movement of the umbilicus in a supine patient flexing the neck or sitting up is named after the British neurologist Charles Edward Beevor (1854-1908). He described a "marked elevation of the umbilicus in the act of sitting up" due to a paralyzed infraumbilical part of the rectus abdominis muscle, indicating a lesion of the spinal cord between the segments T10 and T12 or its nerve roots.(1) Beevor sign may also be present, as in our patient, in myopathies affecting the abdominal muscles, particularly in FSHMD, in which predominant involvement of the lower part of the rectus abdominis muscle is typical.(2).

Myositides : What is the current situation?

This article gives a review of the classification, diagnostic procedures and treatment of idiopathic inflammatory myopathies from a neurological point of view. The myositis syndromes can be subdivided into four groups, polymyositis (PM), dermatomyositis (DM), inclusion body myositis (IBM) and necrotizing myopathy (NM), which substantially differ clinically and pathophysiologically. Myositis may also occur in association with cancer or autoimmune systemic diseases (overlap syndrome). Diagnosis of inflammatory myopathies is based on clinical symptoms, determination of creatine phosphokinase and acute phase parameters in blood (e.g. C-reactive protein and erythrocyte sedimentation rate), electromyography results and findings of magnetic resonance imaging (MRI) in muscle. A muscle biopsy is mandatory to confirm the diagnosis. High quality randomized controlled trials of treatment regimens for inflammatory myopathies are sparse; however, empirical experience indicates a clear effectiveness of immunosuppressive treatment of PM, DM and NM.

Central and peripheral defects in motor units of the diaphragm of spinal muscular atrophy mice.

Spinal muscular atrophy (SMA) is characterized by motoneuron loss and muscle weakness. However, the structural and functional deficits that lead to the impairment of the neuromuscular system remain poorly defined. By electron microscopy, we previously found that neuromuscular junctions (NMJs) and muscle fibres of the diaphragm are among the earliest affected structures in the severe mouse SMA model. Because of certain anatomical features, i.e. its thinness and its innervation from the cervical segments of the spinal cord, the diaphragm is particularly suitable to characterize both central and peripheral events. Here we show by immunohistochemistry that, at postnatal day 3, the cervical motoneurons of SMA mice receive less stimulatory synaptic inputs. Moreover, their mitochondria become less elongated which might represent an early stage of degeneration. The NMJs of the diaphragm of SMA mice show a loss of synaptic vesicles and active zones. Moreover, the partly innervated endplates lack S100 positive perisynaptic Schwann cells (PSCs). We also demonstrate the feasibility of comparing the proteomic composition between diaphragm regions enriched and poor in NMJs. By this approach we have identified two proteins that are significantly upregulated only in the NMJ-specific regions of SMA mice. These are apoptosis inducing factor 1 (AIFM1), a mitochondrial flavoprotein that initiates apoptosis in a caspase-independent pathway, and four and a half Lim domain protein 1 (FHL1), a regulator of skeletal muscle mass that has been implicated in several myopathies.