Diseased Human Muscle Research Articles

FiNuTyper: Design and validation of an automated deep learning-based platform for simultaneous fiber and nucleus type analysis in human skeletal muscle.

While manual quantification is still considered the gold standard for skeletal muscle histological analysis, it is time-consuming and prone to investigator bias. To address this challenge, we assembled an automated image analysis pipeline, FiNuTyper (Fiber and Nucleus Typer). We integrated recently developed deep learning-based image segmentation methods, optimized for unbiased evaluation of fresh and postmortem human skeletal muscle, and utilized SERCA1 and SERCA2 as type-specific myonucleus and myofiber markers after validating them against the traditional use of MyHC isoforms. Parameters including cross-sectional area, myonuclei per fiber, myonuclear domain, central myonuclei per fiber, and grouped myofiber ratio were determined in a fiber-type-specific manner, revealing that a large degree of sex- and muscle-related heterogeneity could be detected using the pipeline. Our platform was also tested on pathological muscle tissue (ALS and IBM) and adapted for the detection of other resident cell types (leucocytes, satellite cells, capillary endothelium). In summary, we present an automated image analysis tool for the simultaneous quantification of myofiber and myonuclear types, to characterize the composition and structure of healthy and diseased human skeletal muscle.

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Single Stem Cell Imaging and Analysis Reveals Telomere Length Differences inDiseased Human and Mouse Skeletal Muscles.

SummaryMuscle stem cells (MuSCs) contribute to muscle regeneration following injury. In many muscle disorders, the repeated cycles of damage and repair lead to stem cell dysfunction. While telomere attrition may contribute to aberrant stem cell functions, methods to accurately measure telomere length in stem cells from skeletal muscles have not been demonstrated. Here, we have optimized and validated such a method, named MuQ-FISH, for analyzing telomere length in MuSCs from either mice or humans. Our analysis showed no differences in telomere length between young and aged MuSCs from uninjured wild-type mice, but MuSCs isolated from young dystrophic mice exhibited significantly shortened telomeres. In corroboration, we demonstrated that telomere attrition is present in human dystrophic MuSCs, which underscores its importance in diseased regenerative failure. The robust technique described herein provides analysis at a single-cell resolution and may be utilized for other cell types, especially rare populations of cells.

Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

In myocytes, small heat shock proteins (sHSPs) are preferentially translocated under stress to the sarcomeres. The functional implications of this translocation are poorly understood. We show here that HSP27 and αB-crystallin associated with immunoglobulin-like (Ig) domain-containing regions, but not the disordered PEVK domain (titin region rich in proline, glutamate, valine, and lysine), of the titin springs. In sarcomeres, sHSP binding to titin was actin filament independent and promoted by factors that increased titin Ig unfolding, including sarcomere stretch and the expression of stiff titin isoforms. Titin spring elements behaved predominantly as monomers in vitro. However, unfolded Ig segments aggregated, preferentially under acidic conditions, and αB-crystallin prevented this aggregation. Disordered regions did not aggregate. Promoting titin Ig unfolding in cardiomyocytes caused elevated stiffness under acidic stress, but HSP27 or αB-crystallin suppressed this stiffening. In diseased human muscle and heart, both sHSPs associated with the titin springs, in contrast to the cytosolic/Z-disk localization seen in healthy muscle/heart. We conclude that aggregation of unfolded titin Ig domains stiffens myocytes and that sHSPs translocate to these domains to prevent this aggregation.

DNAJB2 Expression in Normal and Diseased Human and Mouse Skeletal Muscle

DNAJB2, a co-chaperone regulator of Hsp70 that is expressed principally in the nervous system, has been recently reported to be up-regulated in human skeletal muscle during its recovery from damage. Here we identified DNAJB2 expression in regenerating fibers in skeletal muscles of the dystrophic mdx mouse and patients with Duchenne muscular dystrophy. Surprisingly, in both dystrophic and control mice and patients, DNAJB2 was also expressed in non-regenerating fibers at the postsynaptic side of the neuromuscular junction. DNAJB2 functions as an adaptor molecule for the evacuation and degradation of proteins through the ubiquitin-proteasome system, and overexpression of DNAJB2 in models of the neurodegenerative disease spinobulbar muscular atrophy was shown to result in the reduction of protein inclusions. We therefore studied the possible relation of DNAJB2 expression to protein inclusion formation in skeletal muscle in biopsies of several muscle pathologies associated with protein aggregation and found in all of them a strong immunoreactivity with anti-DNAJB2 in aggregates and vacuoles. We conclude that DNAJB2 is expressed in mouse and human skeletal muscle at the neuromuscular junction of normal fibers, in the cytoplasm and membrane of regenerating fibers, and in protein aggregates and vacuoles in protein aggregate myopathies. Therefore, we propose a role for DNAJB2 in protein turnover processes in skeletal muscle.

G.P.9.07 DNAJB2 gene expression in normal and diseased human and mouse skeletal muscle

G.P.9.07 DNAJB2 gene expression in normal and diseased human and mouse skeletal muscle

Taurine in normal and diseased human skeletal muscle

Taurine content of 199 clinical muscle biopsies was determined and correlated to histometric data of 121 cases. Taurine concentration in muscles was markedly dependent on fiber type distribution, taurine being more abundant in the slow, oxidative type 1 fibers than in the type 2 fibers. Taurine concentration rose slightly with age and tended to be higher in denervations, muscular dystrophies and myotonias, but the differences from the control values were non-significant.

The cardiac isoform of α-actin in regenerating and atrophic skeletal muscle, myopathies and rhabdomyomatous tumors: an immunohistochemical study using monoclonal antibodies

The two sarcomeric isoforms of actins, cardiac and skeletal muscle alpha-actin, are highly homologous so that their immunohistochemical distinction is extremely difficult. Taking advantage of monoclonal antibodies distinguishing the two conservative amino acid exchanges near the aminoterminus, we have performed an extended immunohistochemical analysis of the cardiac alpha-actin (CAA) isoform in normal, regenerating, diseased and neoplastic human muscle tissues. Intense and uniform CAA staining is seen in fetal and adult myocardium and in fetal skeletal muscle while adult skeletal muscle is essentially negative, except for muscle spindle myocytes and a few scattered muscle fibres with overall reduced diameter. By contrast, CAA synthesis is markedly induced in regenerating skeletal muscle cells, in Duchenne muscular dystrophy and upon degenerative atrophy. CAA has also been detected in certain vascular and visceral smooth muscle cells. Among tumors, CAA has consistently been seen in rhabdomyosarcomas and rhabdomyomatous cells of nephroblastomas, whereas, smooth muscle tumors have shown only occasional staining. While the synthesis of this actin isoform is less restricted than previously thought, monoclonal antibodies against CAA provide a well-defined, reliable and sensitive diagnostic tool for the definition and detection of aberrant differentiation in diseased skeletal muscle and of striated muscle differentiation in rhabdomyosarcomas.

Localization of coxsackie virus and adenovirus receptor (CAR) in normal and regenerating human muscle

The primary receptor for Adenovirus and Coxsackie virus (CAR) serves as main port of entry of the adenovirus vector mediating gene transfer into skeletal muscle. Information about CAR expression in normal and diseased human skeletal muscle is lacking. C′- or N′-terminally directed polyclonal antibodies against CAR were generated and immunohistochemical analysis of CAR on morphologically normal and regenerating human skeletal muscle of children and adults was performed. In morphologically normal human muscle fibers, CAR immunoreactivity was limited to the neuromuscular junction. In regenerating muscle fibers, CAR was abundantly co-expressed with markers of regeneration. The function of CAR at the neuromuscular junction is currently unknown. Co-expression of CAR with markers of regeneration suggests that CAR is developmentally regulated, and may serve as a marker of skeletal muscle fiber regeneration.

Hsp27-2D-gel electrophoresis is a diagnostic tool to differentiate primary desminopathies from myofibrillar myopathies

Small heat shock proteins prevent abnormal protein folding and accumulation. We analyzed the expression of hsp27 and αB-crystallin in skeletal muscle specimens of patients with desminopathies, plectinopathies, myotilinopathy, and other myofibrillar myopathies by means of differential centrifugation, 2D-gel electrophoresis, Western blotting, and mass spectrometry. Hsp27-P82 and -P15 as well as αB-crystallin-P59 and -P45 are the major serine phosphorylation isoforms in normal and diseased human skeletal muscle. 2D-gel-electrophoresis revealed spots of hsp27 in a range of pH 5.3–6.4 in samples of all skeletal muscle specimens, except for the seven desminopathies. They indicated a shift of the main hsp27-spot to alkaline pH degrees, which may help to differentiate primary desminopathies from other myopathies with structural pathology of the desmin cytoskeleton.

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.

Reduced expression of aquaporin 4 in human muscles with amyotrophic lateral sclerosis and other neurogenic atrophies

Aquaporin 4 (AQP4) is a water channel protein that is widely distributed in human tissues. However, the precise functional role of AQP4 in skeletal muscle tissue has not yet been determined. Expression of AQP4 was reported to be reduced in muscle tissue from Duchenne muscular dystrophy patients. In the regenerating phase of skeletal muscle, AQP4 expression was reduced when nerve supply was not present. However, in diseased human muscles with neurogenic atrophy including amyotrophic lateral sclerosis, there has been no data on the changes in AQP4 expression. In the present study, we investigated the expression of AQP4 at mRNA and protein levels in human muscles with neurogenic atrophy. The mean level of AQP4 mRNA was significantly lower in muscles with neurogenic atrophy than that in muscles from normal controls. The myofiber surface immunostaining with anti-AQP4 antibody in muscles with neurogenic atrophy was reduced on the surface of scattered myofibers, small angulated myofibers, and myofibers in small- and large-group atrophy despite the presence of dystrophin. Based on the present findings, we conclude that the expression of AQP4 is affected by nerve supply and is down-regulated in human muscles with neurogenic atrophy.

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.

BACE1 and BACE2 in pathologic and normal human muscle

BACE1 and BACE2 are recently discovered enzymes participating in processing of amyloid β precursor protein (AβPP). Their discovery is contributing importantly to understanding the mechanism of amyloid-β generation, and hence the pathogenesis of Alzheimer’s disease (AD). Sporadic inclusion-body myositis (s-IBM) and hereditary inclusion-body myopathy (h-IBM) are progressive muscle diseases in which overproduction of AβPP and accumulation of its presumably toxic proteolytic product amyloid-β (Aβ) in abnormal muscle fibers appear to play an important upstream role in the pathogenic cascade. In normal human muscle AβPP was also shown to be present and presumably playing a role (a) at neuromuscular junctions and (b) during muscle development. To investigate whether BACE1 and BACE2 play a role in normal and diseased human muscle, we have now studied them by immunocytochemistry and immunoblotting in 35 human muscle biopsies, including: 5 s-IBM; 5 chromosome-9p1-linked quadriceps-sparing h-IBM; and 25 control muscle biopsies. In addition, expression of BACE1 and BACE2 was studied in normal cultured human muscle. Our studies demonstrate that BACE1 and BACE2 (a) are expressed in normal adult muscle at the postsynaptic domain of neuromuscular junctions, and in cultured human muscle; (b) are accumulated in the form of plaque-like inclusions in both s-IBM and h-IBM vacuolated muscle fibers; and (c) are immunoreactive in necrotizing muscle fibers. Accordingly, BACE1 and BACE2 participate in normal and abnormal processes of human muscle, suggesting that their functions are broader than previously thought.

Ultrastructural findings at the satellite cell-myofiber border in normal and diseased human muscle biopsy specimens.

Satellite cells (SC) are mononuclear myoblasts located between the plasma membrane and the basement membrane of a myofiber. In normal adult muscle, SC are quiescent in the G0 phase of the cell cycle. The contact of the SC with the myofiber plasma membrane imposes a mitotic inhibition on the SC. Sarcolemmal molecules which might explain this membrane-imparted mitotic inhibition have not yet been identified. In this study we examined the border of the SC and the adjacent myofiber electron microscopically, assessing the number of SC showing encroachments of basement membrane (BM) material, secretion of cellular degradation products into the intercellular space, and caveolae. We studied normal and diseased muscle including Duchenne muscular dystrophy, Becker muscular dystrophy, idiopathic inflammatory myopathies, and neurogenic atrophies. Caveolae were present in SC from normal muscle, but they were more abundant in SC from diseased muscle, and they significantly prevailed at the outer surface of SC in all of the diseased muscle groups. Encroachments of BM material was only present in SC from diseased muscle, and mostly so in neurogenic atrophies. Secretion of cellular degradation products into the intercellular cleft occurred in normal and diseased muscle. We conclude that degradation products in the intercellular cleft do not disturb SC adhesion and that there is a neural influence on SC adhesion. The significance of the abundance of caveolae at the outer surface of the SC when compared with the inner surface in diseased muscle remains at present unknown.

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.

Transforming growth factor-beta2 is elevated in skeletal muscle disorders.

The transforming growth factor betas (TGF-betas) are multifunctional growth factors that act on both fibroblasts and myosatellite cells. In rodent models of muscle diseases, high levels of TGF-beta2 are expressed by myogenic cells. We have examined whether the expression of TGF-beta2 is also elevated in diseased human muscles. The disorders examined were Duchenne muscular dystrophy, myotonic dystrophy, myotubular myopathy, spinal muscular atrophy, and amyotrophic lateral sclerosis. The levels of TGF-beta2 immunoreactivity were elevated in atrophic, necrotic, and regenerating fibers and in fibers with central nuclei or cytoplasmic masses, irrespective of whether fibrosis was present. We therefore suggest that TGF-beta2 is important for muscle repair and that the presence of a TGF-beta within a muscle only leads to fibrosis if certain other factors are present.

Automated segmentation of muscle fiber images using active contour models.

The cross-sectional area of different fiber types is an important anatomic feature in studying the structure and function of healthy and diseased human skeletal muscles. However, such studies are hampered by the thousands of fibers involved when manual segmentation has to be used. We have developed a semiautomatic segmentation method that uses computational geometry and recent computer vision techniques to significantly reduce the time required to accurately segment the fibers in a sample. The segmentation is achieved by simply pointing to the approximate centroid of each fiber. The set of centroids is then used to automatically construct the Voronoi polygons, which correspond to individual fibers. Each Voronoi polygon represents the initial shape of one active contour model, called a snake. In the energy minimization process, which is executed in several stages, different external forces and problem-specific knowledge are used to guide the snakes to converge to fiber boundaries. Our results indicate that this approach for segmenting muscle fiber images is fast, accurate, and reproducible compared with manual segmentation performed by experts.