Diseased Human Skeletal Muscle Research Articles

Coordinated changes in mitochondrial function and biogenesis in healthy and diseased human skeletal muscle

We examined the transcriptional signaling cascade involved in the changes of mitochondrial biogenesis and mitochondrial function of skeletal muscle and of the exercise capacity of humans in response to long-term physical activity and chronic heart failure (CHF). Biopsy samples of vastus lateralis muscle were obtained from 18 healthy subjects with different fitness levels (assessed by maximal oxygen uptake, VO2 peak). We compared 9 sedentary subjects with 10 CHF patients undergoing transplantation. Muscle oxidative capacity was measured in permeabilized fibers (Vmax). Transcript levels of target genes were quantified by RT-PCR. In healthy subjects, VO2 peak was linearly related to Vmax (P<0.01) and to the gene expression of mitochondrial proteins and of the coactivator PGC-1alpha and its downstream transcription factors. A coordinate increase in PGC-1alpha and mRNA levels of proteins involved in degradation, fusion, and fission of mitochondria was observed associated with calcineurin activation. Despite decreased VO2 peak, in CHF patients skeletal muscles showed preserved Vmax in accordance with preserved markers and transcription factors of mitochondrial biogenesis and dynamics, with no calcineurin activation. The results provide strong support for a central role for PGC-1alpha and calcineurin activation in mitochondrial biogenesis in healthy and diseased human skeletal muscles.

Overexpression of semicarbazide-sensitive amine oxidase in human myopathies.

Oxidative stress has been implicated in the pathogenesis of several muscle diseases. Semicarbazide-sensitive amine oxidase (SSAO) metabolizes oxidative deamination of primary aromatic and aliphatic amines. In the oxidative reactions, amine substrates are converted into the aldehyde, followed by the production of ammonia and H(2)O(2). Although normal levels in muscle are very low, SSAO is expressed in almost all mammalian tissues. In this study, we examined the possible implication of SSAO as an additional source of oxidative stress in the pathogenesis of muscle disorders. The expression of SSAO was examined immunohistochemically in muscle biopsy specimens from patients with inclusion-body myositis (IBM; n = 5), desmin-related myopathy (DRM; n = 3), dermatomyositis (n = 3), granulomatous (sarcoid) myopathy (n = 2), muscle denervation-reinnervation (n = 3), and rhabdomyolysis (n = 2), as well as from control subjects (n = 3). Strong SSAO immunoreactivity was present in vacuolated and nonvacuolated fibers in IBM, in abnormal fibers in DRM, and in degenerating and regenerating fibers in dermatomyositis and rhabdomyolysis. In addition, SSAO overexpression was observed in muscle fibers adjacent to granulomas in sarcoid myopathy. These results suggest that SSAO is a source of oxidative stress in diseased human skeletal muscle and that it contributes to oxidative stress-induced damage in various inflammatory and other myopathies. Alternatively, the expression of SSAO in muscle fibers may be a consequence of muscle fiber injury.

Beyond LGMD1A: myotilin is a component of central core lesions and nemaline rods

Myotilin is a Z-disc protein that binds α-actinin, γ-filamin and F-actin. The essential role of myotilin in skeletal muscle is highlighted by the recent observation that autosomal dominant limb girdle muscular dystrophy type 1A is caused by mutations in the human myotilin gene. We studied the expression and subcellular distribution of myotilin in nemaline myopathy, central core disease, centronuclear myopathy, and myopathies with tubular aggregates. A prominent myotilin immunostaining of nemaline rods and core lesions was detected in all ten cases of nemaline myopathy and five cases of central core disease. This renders myotilin a sensitive, though non-specific marker for these structural lesions. Western blot analysis did not indicate an increased myotilin expression in nemaline myopathy muscle. However, the analysis indicated upregulation of a 75 kDa immunoreactive band, very weak in control muscle but previously detected in limb girdle muscular dystrophy 1A samples. Our findings indicate that myotilin is a core structural molecule in nemaline rods and central core lesions and suggest modification of myotilin in nemaline myopathy, and further support the notion that myotilin may have a key role in the dynamic molecular events mediating myofibril assembly in normal and diseased human skeletal muscle.

The role of titin in muscular disorders

Titin, the biggest single (poly) peptide found in humans, and throughout nature so far, was long considered as a good candidate for inherited muscle diseases. However, disease-causing defects were not known until recently, when this central sarcomeric protein was associated with human skeletal tibial muscular dystrophy (TMD/LGMD2J), dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). Several mutations in different parts of titin have now been identified and more are expected. Spontaneous mouse and zebrafish mutants have also been reported. Experimental knock-outs are not viable, even in cases where just a c-terminal part of the gene was silenced, telling something of the basic importance of titin for life. In this article we review the current known structure and functions of this elementary molecule with some emphasis on the only defects so far known to cause human skeletal muscle disease, mutations in the c-terminal M-line part of titin.

Tibial Muscular Dystrophy Is a Titinopathy Caused by Mutations in TTN, the Gene Encoding the Giant Skeletal-Muscle Protein Titin

Tibial muscular dystrophy (TMD) is an autosomal dominant late-onset distal myopathy linked to chromosome 2q31. The linked region includes the giant TTN gene, which encodes the central sarcomeric protein, titin. We have previously shown a secondary calpain-3 defect to be associated with TMD, which further underscored that titin is the candidate. We now report the first mutations in TTN to cause a human skeletal-muscle disease, TMD. In Mex6, the last exon of TTN, a unique 11-bp deletion/insertion mutation, changing four amino acid residues, completely cosegregated with all tested 81 Finnish patients with TMD in 12 unrelated families. The mutation was not found in 216 Finnish control samples. In a French family with TMD, a Leu-->Pro mutation at position 293,357 in Mex6 was discovered. Mex6 is adjacent to the known calpain-3 binding site Mex5 of M-line titin. Immunohistochemical analysis using two exon-specific antibodies directed to the M-line region of titin demonstrated the specific loss of carboxy-terminal titin epitopes in the TMD muscle samples that we studied, thus implicating a functional defect of the M-line titin in the genesis of the TMD disease phenotype.

P0071, a member of the armadillo multigene family, is a constituent of sarcomeric I-bands in human skeletal muscle.

p0071 is a member of the armadillo gene family that is expressed in a wide variety of mammalian tissues and cell types with a prominent cell-cell contact association in epithelial cells. Here, we report the expression and localization patterns of p0071 in differentiating human skeletal muscle cells and in normal and diseased human skeletal muscle tissues. Northern blots revealed expression of p0071 mRNA in adult skeletal muscle tissue. RT-PCR analysis and Western blotting experiments identified two differentially spliced isoforms of p0071. The balance between these isoforms shifted during in vitro differentiation of isolated muscle cells from predominant expression of the short variant to a preponderance of the larger variant from day 6 onwards. Immunolocalization studies in mature skeletal muscle tissue revealed that p0071 is a constituent of myofibrils with a distinct localization at the level of sarcomeric N2-lines. During myofibrillogenesis, p0071 was not detected in non-striated nascent myofibrils, but became apparent shortly after the development of compact Z-discs in early myotubes. Furthermore, we studied the expression of p0071 in a wide variety of neuromuscular disorders by indirect immunofluorescence. Here, the myofibrillar staining of p0071 was preserved in all the disease entities included in our study. Our results provide the first evidence that a member of the armadillo multigene family is a constituent of the contractile apparatus in human skeletal muscle. The localization of p0071 at the level of I-bands and the timepoint of its integration into developing myofibrils suggest a possible role in the organization of thin filaments.

RNA Expression of Cardiac Troponin T Isoforms in Diseased Human Skeletal Muscle

The expression of multiple cardiac troponin T (cTnT) isoforms has been demonstrated in diseased human skeletal muscle. However, cardiac troponin I (cTnI) expression has been described only in heart muscle. The goal of this study was to determine whether mRNA for cTnT, slow skeletal troponin T (sTnT), or cTnI was expressed in skeletal muscle biopsies obtained from patients with end-stage renal disease (ESRD) and Duchenne muscular dystrophy (DMD). Total mRNA was extracted from healthy human heart (n = 4), healthy human skeletal muscle (n = 5), and skeletal muscle from patients with ESRD (n = 7) and DMD (n = 5). Total RNA (1 microg) was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase. The reverse-transcribed cDNAs were amplified by PCR using oligonucleotide primers specific for cTnT, sTnT, and cTnI sequences (GenBank accession numbers X74819, m19308, and X54163, respectively). In all heart specimens, a 150-bp cTnT amplicon was detected. Skeletal muscle from four of seven patients with ESRD and two of five patients with DMD showed expression of a 150-bp amplicon. Using DNA sequencing and a comparison program, the 150-bp amplicons found in heart and diseased skeletal muscle specimens were 100% identical and specific to the cTnT mRNA sequence. No cTnT mRNA expression was found in healthy skeletal muscle. No evidence of sTnT mRNA was found in heart muscle. A 200-bp sTnT amplicon specific to a human sTnT sequence was detected in all skeletal muscle specimens. A 250-bp cTnI amplicon specific to the cTnI sequence was detected in all heart specimens. However, no cTnI mRNA expression was found in healthy or diseased skeletal muscle specimens. cTnT mRNA expression in both heart and diseased skeletal muscles corresponded with cTnT isoform expression, respectively, as determined by Western blot analysis. Our findings demonstrate cTnT mRNA expression, but no cTnI mRNA expression, by reverse transcription-PCR in diseased human skeletal muscle that expresses cTnT isoforms.

Cardiac troponin-I is not expressed in fetal and healthy or diseased adult human skeletal muscle tissue

Cardiac troponin-I (cTnI) is not found in sera of patients with skeletal muscle disease in the absence of myocardial injury. It is not known, however, whether trace amounts of cTnI are expressed in regenerating human skeletal muscle, as has been observed with creatine kinase MB. Using immunohistochemical and biochemical techniques, we investigated cTnI expression in various human muscle tissues: human heart tissue (n = 5), normal adult skeletal muscle (n = 3), and fetal heart (n = 3) and skeletal muscle (n = 3) obtained, respectively, during heart transplant, from autopsy, or from a tissue bank. Specimens from diagnostic tissue biopsies were used as diseased skeletal muscle: polymyositis (PM), n = 13; Duchenne muscular dystrophy (DMD), n = 6. Frozen sections 8 microns thick were stained immunohistochemically for either cTnI or TnI (cardiac or skeletal) by using monoclonal antibodies (MAb) 2B1.9 (cTnI specific) or 3C5.10 (reactive with all TnI isoforms), respectively. cTnI was measured in tissue homogenates by an immunofluorometric assay. Cardiac muscle was stained by both MAbs. Normal fetal and adult skeletal muscle, and samples from all of the PM and DMD patients, stained only with the nonspecific MAb (3C5.10), confirming the sole presence of skeletal TnI. No cTnI was detectable by immunoassay in any skeletal muscle sample. We conclude that cTnI is not expressed in human skeletal muscle during development or during regenerative muscle disease processes such as PM or DMD.

Localized in vivo 1H spectroscopy of human skeletal muscle: Normal and pathologic findings

To obtain high signal to noise ratio in small volume elements (8 cm 3), in vivo 1H NMR spectroscopy of normal and diseased human skeletal muscle was performed using a double spin-echo localization method on a 1.5-T whole body system. High resolved spectra of normal calf muscle show the well known resonances of lipids (methyl, methylene, olefinic, and other fatty acid resonances), creatine/phosphocreatine, choline/carnitine, taurine, and histidine with good intraindividual reproducibility. Pronounced intraindividual differences in the lipid range were found between different upper thigh muscle groups. On pathologic conditions like myopathia, myositis or irradiation damage the spectral lipid content was increased. Three months after local irradiation of the medial vastus muscle (50 Gy), the localized 1H NMR spectrum showed a complete loss of the choline and creatine signals. In a case of M. Behçet with muscular involvement the relative reduction of the choline signal may provide an insight in the pathobiochemistry. The results of our investigations in nine healthy volunteers and three patients are presented in detail including relaxation times of the metabolites.

The dystrophin-related protein, utrophin, is expressed on the sarcolemma of regenerating human skeletal muscle fibres in dystrophies and inflammatory myopathies

Utrophin is the 400 kDa protein product of an autosomal homologue (DMDL) of the dystrophin gene. In normal skeletal muscle, utrophin is expressed in vascular smooth muscle, endothelium and nerves but not in mature muscle fibres except at the neuromuscular junction. We have examined the expression of utrophin in a wide range of human skeletal muscle diseases using monoclonal antibodies against three C-terminal epitopes. Utrophin is consistently expressed in all basophilic, regenerating fibres irrespective of the underlying disease or expression of dystrophin. It is also found in regenerating fibres from a normal volunteer. In Duchenne and Becker dystrophies, as well as in dermatomyositis, sarcolemmal staining for utrophin is also seen in larger fibres which are not obviously regenerating. These studies do not support the idea that utrophin occupies membrane attachment sites only when dystrophin is absent or reduced, but would be consistent with utrophin expression as part of an activated foetal programme during regeneration.

Enhancement of K+ conductance improves in vitro the contraction force of skeletal muscle in hypokalemic periodic paralysis

An abnormal ratio between Na+ and K+ conductances seems to be the cause for the depolarization and paralysis of skeletal muscle in primary hypokalemic periodic paralysis. Recently we have shown that the "K+ channel opener" cromakalim hyperpolarizes mammalian skeletal muscle fibers. Now we have studied the effects of this drug on the twitch force of muscle biopsies from normal and diseased human skeletal muscle. Cromakalim had little effect on the twitch force of normal muscle whereas it strongly improved the contraction force of fibers from patients suffering from hypokalemic periodic paralysis. Recordings of intracellular K+ and Cl- activities in human muscle and isolated rat soleus muscle support the view that cromakalim enhances the membrane K+ conductance (gK+). These data indicate that "K+ channel openers" may have a beneficial effect in primary hypokalemic periodic paralysis.

(Immuno) electron microscopy of mitochondrial crystals in diseased human skeletal muscle fibres

(Immuno) electron microscopy of mitochondrial crystals in diseased human skeletal muscle fibres

Histochemical features of tubular aggregates in diseased human skeletal muscle fibres

This communication presents the results obtained in tubular aggregates of 24 enzyme histochemical techniques for demonstrating activity of oxidoreductases, transferases, hydrolases and isomerases. The activity characteristics of the tubular aggregates in m. gluteus medius of 18 patients with diseases of the neuromuscular system were almost identical. A high activity of the mitochondrial enzymes, NADPH: tetrazolium oxidoreductase, NADH:tetrazolium oxidoreductase and cytochrome c oxidase, could be shown in the pathological structures, whereas the activity of the mitochondrial enzymes, glycerol-3-phosphate:menadione oxidoreductase, succinate:PMS oxidoreductase, malate:NAD + oxidoreductase and isocitrate:NAD + oxidoreductase, and the partial mitochondrial enzymes, malate:NADP + oxidoreductase and isocitrate:NADP + oxidoreductase, was very slight or even absent. There was a moderate to strong activity of the glycolytic enzymes lactate : NAD + oxidoreductase, glyceraldehyde-3-phosphate : NAD + oxidoreductase, phosphofructokinase, phosphoglucomutase and glucose phosphate isomerase. In contrast, the activity of α-glucan phosphorylase was slight. The activity of phosphogluconate : NADP + oxidoreductase, glucose-6-phosphate : NADP + oxidoreductase and 5′-nucleotidase was slight, whereas there was no activity of myosine ATPase and mitochondrial ATPase, acid phosphatase or alkaline phosphatase. The high activity of AMP-deaminase was very striking. The activity of peroxidase was moderate. Results obtained with adsorption studies point to adsorption of some of the enzymes studied to the tubular aggregates in vivo and this phenomenon very probably determined the histochemical characteristics of these structures.

Two types of mitochondrial crystals in diseased human skeletal muscle fibers.

Mitochondrial crystalline inclusions, frequently found in mitochondrial myopathies, were analyzed by crystallographic techniques and computer-aided image processing. It could be shown that these structures were real crystals. There are two distinct types of crystal, which can be distinguished by shape, size, and pattern. So-called type I crystals are usually present in the intracristal space, whereas the type II crystals are preferentially located in the intermembrane space between outer and inner mitochondrial membranes. The unit cell dimensions were found to be 38 x 34 x 8 nm for the type I crystals and 20 x 17 x 8 nm for the type II crystals. These results strongly suggest that the crystals are composed of macromolecules, presumably proteins. Arguments are presented that indicate that type I crystals occur only in type 1 muscle fibers and type II crystals in type 2 muscle fibers.

Proteases in normal and diseased human skeletal muscle: a preliminary histochemical survey.

Seven proteases assumed to be aminopeptidases A, B and M, dipeptidyl peptidases II and IV, esteroproteinase and gamma-glutamyltransferase were localized histochemically, using semipermeable membrane simultaneous coupling techniques, in unfixed cryostat sections of skeletal muscle removed from one healthy volunteer, six patients with disuse muscle atrophy, and 15 patients with some form of muscle disease. Normal muscle fibres showed weak reactions for aminopeptidases A and M and for the dipeptidyl peptidases, but no reactivity for gamma-glutamyltransferase or esteroproteinase. No change was detected in diseased muscle fibres except that low gamma-glutamyltransferase and esteroproteinase activities appeared in some cases. The activities of the seven enzymes were stronger in the intermyosial connective tissue than in the muscle fibres, but were also unchanged in disease. The strongest reactions were found in some interstitial cells (mast cells and macrophages) and these were much increased in diseased muscle, particularly for dipeptidyl peptidases II and IV. The findings are interpreted in terms of the release of proteases from such cells and their subsequent involvement in the breakdown of myofibrillar proteins in muscle disease.

Improvements of isoelectric focusing in agarose for direct tissue isoelectric focusing

AbstractImprovements of isoelectric focusing (IEF) in agarose are described that result in consistently obtaining broad, linear pH gradients in the range pH 4 to 9.2 at equilibrium. Cathodic drift has been reduced by the use of a blend of commercial carrier ampholytes and by incorporating lysine and arginine in the catholyte as spacers. Resolution has also been enhanced by the use of thin gels, supplemented with sorbitol and glycerol. In addition, a comparison of two agarose preparations for isoelectric focusing was performed using two commercial carrier ampholytes. To minimise background staining, Triton complexes were removed by modification of the fixing and clearing procedures. These modifications to IEF in agarose are being applied to direct tissue isoelectric focusing (DTIF) of normal and diseased human skeletal muscle.

The distribution of intermediate filament protein (skeletin) in normal and diseased human skeletal muscle: An immunohistochemical and electron-microscopic study

The presence of intermediate filaments in various mature and immature eukaryotic cells is well documented. By using antibodies against the intermediate (skeletin) filaments of cow heart Purkinje fibres, we have in the present work analyzed the distribution of skeletin in normal and diseased skeletal muscle fibres. Antiskeletin proved to be a useful tool for the identification of regenerating fibres in Duchenne muscular dystrophy. In mature muscle fibres with well-preserved myofibrils, skeletin had an intermyofibrillar localization at the Z-disk level where intermediate filaments were demonstrated. The presence of skeletin was also demonstrated around the rods of muscle fibres in nemaline myopathy biopsies, but the rods themselves did not show any cross-reactivity. This report exemplifies the usefulness of antibodies in muscle pathology.

Muscle protein analysis. II. Two-dimensional electrophoresis of normal and diseased human skeletal muscle.

We used high-resolution two-dimensional electrophoresis to analyze the major proteins of normal and pathological human-muscle samples. The normal human-muscle pattern contains four myosin light chains: three that co-migrate with the myosin light chains from rabbit fast muscle (extensor digitorum longus), and one that co-migrates with the light chain 2 from rabbit slow muscle (soleus). Of seven Duchenne muscular dystrophy samples, four yielded patterns with decreased amounts of actin and myosin relative to normal muscle, while three samples gave patterns comparable to that for normal muscle. Six samples from patients with myotonic dystrophy also gave normal patterns. In nemaline rod myopathy, in contrast, the pattern was deficient in two of the fast-type myosin light chains.

The histochemical characterization of the coupling state of skeletal muscle mitochondria.

Isolated mitochondria from skeletal muscles of human and animals with neuromuscular diseases may reveal a loosely coupled state of oxidative phosphorylation, which is characterized by a normal phosphorylation in the presence of a phosphate acceptor and a maximal respiration in the absence of a phosphate acceptor. Moreover in these cases activity of mitochondrial Mg2+-stimulated ATPase is strongly increased and cannot be stimulated by the uncoupler 2,4-dinitrophenol. In this communication a histochemical technique for the demonstration of activity of mitochondrial Mg2+-stimulated ATPase to characterize the coupling state of muscle mitochondria in tissue sections, is described. This tissue-saving technique is especially suitable for the study of human skeletal muscle diseases.

DISEASED MUSCLE CELLS IN CULTURE

Summary(1) Cultures of differentiated muscle cells have been grown from diseased human, mouse and chick skeletal muscle, and from cardiac muscle of the myopathic hamster.(2) Methods of culture established for normal embryonic and adult skeletal muscle cells have proved suitable for cultures of diseased muscle cells.(3) Myoblasts obtained from dy2J mouse muscle crushed in vivo before explanting fuse in culture and form morphologically normal myotubes. Studies of the effects of innervation by dy2J spinal cord neurones on the differentiation of normal, dy2J and dy myotubes have been inconclusive but it is probable that innervation does not play a part in the pathogenesis of this disorder.(4) Myoblasts prepared by trypsinization of embryonic dy muscle behave normally in culture and fuse to form myotubes that appear normal. It is not clear if myoblasts that migrate from explants of adult muscle in vitro fuse. Aggregates of non‐fusing cells have been described, but under other culture conditions normal and abnormal forms of myotube have been observed. dy muscle fibres fail to regenerate even when cultured with normal spinal cord explants and dy nerves are without effect on regenerating normal muscle fibres. These tissue‐culture studies suggest that the dy mouse mutation is a myopathic disorder.(5) Embryonic mdg myoblasts have a normal cell cycle in vitro and fuse to form well‐differentiated myotubes with cross‐striations. mdg myotubes have normal electro‐physiological properties but do not contract spontaneously or on depolarization. The defect in the muscle of the mdg mutant appears to be a failure of excitation‐contraction coupling.(6) Cells migrate earlier from explants of adult dystrophic chick muscle than from normal muscle but dystrophic chick myotubes appear morphologically normal. Myotubes prepared from embryonic dystrophic chick muscle become vacuolated and degenerate, changes that can be prevented by anti‐proteases such as antipain. Lactic dehydrogenase isozyme subunit M4 is absent from dystrophic muscle in vivo but reappears in cultured myotubes. Dystrophic myotubes innervated in culture by either normal or dystrophic neurones exhibit bi‐directional lcoupling and multiple innervation. These results suggest that there are changes in dystrophic myotubes and that chick muscular dystrophy is a myopathy.(7) Cardiac muscle cells from the cardiomyopathic hamster synthesize less actin and myosin than normal cells, and Z lines in dystrophic cells are irregularly arranged. The beat frequency of myopathic cardiac cells is lower than that of normal cells and declines more rapidly. Tissue‐culture studies have not been made of hamster skeletal muscle.(8) Human dystrophic myotubes do not show degenerative changes in culture and have normal histochemical reactions. RNA synthesis appears normal in dystrophic myotubes but there may be changes in adenyl‐cyclase activity and protein synthesis in dystrophic cells. Morphological and biochemical changes have been found in muscle cells cultured from a case of acid‐maltase deficiency but phosphorylase activity re‐appeared in myotubes cultured from biopsies of phosphorylase‐deficient muscle. Innervation by normal mouse nerves does not induce degenerative changes in dystrophic myotubes.(9) Studies on the origins of myoblasts in explants of muscle fibres in culture suggest that in these conditions myoblasts are derived only from satellite cells and that this process may be the same in normal and diseased muscle.