Chaperone-mediated autophagy components are upregulated in sporadic inclusion-body myositis muscle fibres.

AIMS: Sporadic inclusion-body myositis (s-IBM) is an age-associated degenerative muscle disease. Characteristic features are muscle-fibre vacuolization and intramuscle-fibre accumulations of multiprotein aggregates, which may result from the demonstrated impairments of the 26S proteasome and autophagy. Chaperone-mediated autophagy (CMA) is a selective form of lysosomal degradation targeting proteins carrying the KFERQ motif. Lysosome-associated membrane protein type 2A (LAMP2A) and the heat-shock cognate protein 70 (Hsc70) constitute specific CMA components. Neither CMA components nor CMA activity has been studied in normal or disease human muscle, to our knowledge. METHODS: We studied CMA components by immunocytochemistry, immunoblots, real-time PCR and immunoprecipitation in: (a) 16 s-IBM, nine aged-matched normal and nine disease control muscle biopsies; and (b) cultured human muscle fibres (CHMFs) with experimentally inhibited activities of either the 26S proteasome or autophagy. RESULTS: Compared with age-matched controls, in s-IBM muscle, LAMP2A and Hsc70 were on a given transverse section accumulated as aggregates in approximately 5% of muscle fibres, where they (a) colocalized with each other and α-synuclein (α-syn), a CMA-targeted protein; and (b) were bound to each other and to α-syn by immunoprecipitation. By immunoblots, LAMP2A was increased sevenfold P < 0.001 and Hsc70 2.6-fold P < 0.05. LAMP2A mRNA was increased 4.4-fold P < 0.001 and Hsc70 mRNA 1.9-fold P < 0.05. In CHMFs inhibition of either the 26S proteasome or autophagy induced CMA, evidenced by a significant increase of both LAMP2A and Hsc70. CONCLUSIONS: Our study demonstrates, for the first time, up-regulation of CMA components in s-IBM muscle, and it provides further evidence that altered protein degradation is likely an important pathogenic aspect in s-IBM.

Expression of muscle-gene-specific isozymes of phosphorylase and creatine kinase in innervated cultured human muscle.

Isozymes of creatine kinase and glycogen phosphorylase are excellent markers of skeletal muscle maturation. In adult innervated muscle only the muscle-gene-specific isozymes are present, whereas aneurally cultured human muscle has predominantly the fetal pattern of isozymes. We have studied the isozyme pattern of human muscle cultured in monolayer and innervated by rat embryo spinal cord explants for 20-42 d. In this culture system, large groups of innervated muscle fibers close to the ventral part of the spinal cord explant continuously contracted. The contractions were reversibly blocked by 1 mM d-tubocurarine. In those innervated fibers, the total activity and the muscle-gene-specific isozymes of both enzymes increased significantly. The amount of muscle-gene-specific isozymes directly correlated with the duration of innervation. Control noninnervated muscle fibers from the same dishes as the innervated fibers remained biochemically immature. This study demonstrated that de novo innervation of human muscle cultured in monolayer exerts a time-related maturational influence that is not mediated by a diffusable neural factor.

Myostatin and its precursor protein are increased in the skeletal muscle of patients with Type-II muscle fibre atrophy.

Preferential atrophy of Type-II muscle fibres occurs in several clinical situations, including cachexia, muscle disuse, chronic glucocorticoid treatment, remote neoplasia, and sometimes as an aspect of recent-denervation. For the patient, the Type-II atrophy itself might be unfavourable (as a glucocorticoid side-effect) or favourable (survivalistic via the muscle-alanine liver-gluconeogenesis pathway in starvation). The cellular mechanisms underlying Type-II fibre atrophy are unclear. Myostatin (Mstn) is physiologically a negative regulator of muscle mass and strength. In this study we evaluated a possible role of Mstn in Type-II fibre atrophy in human muscle. Mstn and Mstn precursor protein (MstnPP) were studied in 10-muscle biopsies containing Type-II fibre atrophy and in 17 disease and normal control muscle biopsies. When comparison was made with normal control fibres, we found the following: 1) by immunocytochemistry, diffusely increased Mstn/MstnPP in the atrophic Type-II muscle fibres; 2) by immunoblots, Mstn/MstnPP increased individually; 3) by RT-PCR, no increase in MstnPP mRNA. In conclusion, our results a) suggest that Mstn/ /MstnPP might play a role in the pathogenic cascade of Type-II muscle fibre atrophy; b) broaden our previously-described associations of Mstn in human muscle pathology, and c) could possibly lead to clinical prevention when Type-II muscle fibre atrophy is unfavourable, for instance in glucocorticoid therapy.

Expression of muscle-type phosphorylase in innervated and aneural cultured muscle of patients with myophosphorylase deficiency.

Patients with McArdle's myopathy lack muscle glycogen phosphorylase (M-GP) activity. Regenerating and cultured muscle of patients with McArdle's myopathy presents a glycogen phosphorylase (GP) activity, but it is not firmly established whether M-GP or non-M-GP isoforms are expressed. We have cultured myoblasts from biopsy specimen of five patients with McArdle's myopathy. Skeletal muscle was cultured aneurally or was innervated by coculture with fetal rat spinal cord explants. In the patients' muscle biopsies and in their cultured innervated and aneural muscle we studied total GP activity, isoenzymatic pattern, reactivity with anti-M-GP antiserum, and presence of M-GP mRNA. There was no detectable enzymatic activity, no immunoreactivity with anti-M-GP antiserum, and no M-GP mRNA in the muscle biopsy of all patients. GP activity, M-GP isozyme, and anti-M-GP antiserum reactivity were present in patients' aneural cultures, increased after innervation, and were undistinguishable from control. M-GP mRNA was demonstrated in both aneural and innervated cultures of patients and control by primer extension and PCR amplification of total RNA. Our studies indicate that the M-GP gene is normally transcribed and translated in cultured muscle of patients with myophosphorylase deficiency.

Increased expression of Noga-A in ALS muscle biopsies is not unique for this disease.

Nogo (RTN4) belongs to the reticulon (RTN) family of integral membrane proteins. RTN4A (Nogo-A), RTN4B (Nogo-B) and RTN4C (Nogo-C) are isoforms of RTN4. In the gastrocnemius muscle of transgenic mice bearing an SOD1 mutation ("ALS model"), increased Nogo-A mRNA and protein was reported, and similar changes were reported in muscle biopsies of patients with amyotrophic lateral sclerosis (ALS) but not with peripheral neuropathy or primary muscle diseases, leading to the proposal that Nogo-A in skeletal muscle is a new specific molecular marker of ALS. Here we report, based on studies of muscle biopsies from patients with ALS, peripheral neuropathies, polymyositis, dermatomyositis and morphologically nonspecific myopathies that, in addition of strong Nogo-A immunoreactivity within apparently-denervated small angular fibers in ALS and peripheral neuropathies, Nogo-A was strongly immunoreactive within desmin-positive regenerating muscle fibers in various myopathies, and its expression on immunoblots was increased in all those neuromuscular diseases. In conclusion, we have found that the presence of Nogo-A in diseased human muscle biopsies is not limited to ALS.

Parkin and its association with alpha-synuclein and AbetaPP in inclusion-body myositis and AbetaPP-overexpressing cultured human muscle fibers.

UNLABELLED: Parkin, an E3-ubiquitin ligase in the ubiquitin-proteasome system, facilitates degradation of alpha-synuclein and other proteins. Since ubiquitinated multiprotein-aggregates containing amyloid-beta (Abeta), alpha-synuclein, and other proteins, are characteristic of sporadic inclusion-body myositis (s-IBM) muscle fibers, we asked whether parkin might have a role in s-IBM pathogenesis. We studied the association of parkin with alpha-synuclein and Abeta-precursor protein (AbetaPP) in s-IBM muscle biopsies and in our IBM model based on overexpression of AbetaPP into cultured human muscle fibers. We report the following in s-IBM muscle fibers: a) parkin was increased 2.7 fold and accumulated in aggregates also containing Abeta and alpha-synuclein; b) alpha-synuclein was increased 6.3 fold; c) parkin physically associated with alpha-synuclein and AbetaPP; d) alpha-synuclein and AbetaPP were ubiquitinated. In the IBM model: a) parkin was increased 2.7 fold, b) it associated with alpha-synuclein and AbetaPP. CONCLUSION: 1. This is the first demonstration that in a human muscle disease alpha-synuclein associates with parkin, and might be ubiquitinated by it. 2. The small increase of parkin relative to the much larger increase of alpha-synuclein might be insufficient to secure complete ubiquitination and consequent degradation of alpha-syn. 3. AbetaPP might be a novel substrate of parkin.

Sporadic inclusion-body myositis: a proposed key pathogenetic role of the abnormalities of the ubiquitin-proteasome system, and protein misfolding and aggregation.

Sporadic inclusion-body myositis (s-IBM), the most common muscle disease of older persons, is of unknown cause and there is no successful treatment. We summarize our most recent findings in s-IBM muscle fibers, which demonstrate abnormalities of the ubiquitin-proteasome system, and abnormal accumulation, misfolding and aggregation of proteins. We propose that these changes, possibly provoked by the aging intra-muscle fiber cellular milieu, and aggravated by the oxidative stress, play a key pathogenic role in s-IBM. This evidence strongly suggests that mechanisms other than the immune/inflammatory response play the important role in s-IBM muscle fiber degeneration.

Inclusion-body myositis: newest concepts of pathogenesis and relation to aging and Alzheimer disease.

We review the newest advances related to seeking the pathogenic mechanism(s) of sporadic inclusion-body myositis (s-IBM) and present the pathologic diagnostic criteria of s-IBM. We discuss the possible pathogenic role of several themes, such as 1) increased amyloid-beta precursor protein (AbetaPP) and of its fragment Abeta; 2) phosphorylation of tau protein; 3) oxidative stress; 4) abnormal a) signal-transduction, b) transcription, and c) RNA accumulation; 5) "junctionalization" and myogenous" denervation; and 6) lymphocytic inflammation. Evidence is provided supporting our hypothesis that overexpression of AbetaPP within the aging muscle fibers is an early upstream event causing the subsequent pathogenic cascade. The remarkable pathologic similarities between s-IBM muscle and Alzheimer disease (AD) brain are discussed, and the possible cause and significance are addressed.

Novel immunolocalization of alpha-synuclein in human muscle of inclusion-body myositis, regenerating and necrotic muscle fibers, and at neuromuscular junctions.

Alpha-synuclein (alpha-syn) is an important component of neuronal and glial inclusions in brains of patients with several neurodegenerative disorders. Sporadic inclusion-body myositis (s-IBM) is the most common progressive muscle disease of older patients. Its muscle phenotype shows several similarities with Alzheimer disease brain. A distinct feature of s-IBM pathology is specific vacuolar degeneration of muscle fibers characterized by intracellular amyloid inclusions formed by both amyloid-beta (Abeta) and paired-helical filaments composed of phosphorylated tau. We immunostained alpha-syn in muscle biopsies of s-IBM, disease-control, and normal patients. Approximately 60% of Abeta-positive vacuolated muscle fibers (VMF) contained well-defined inclusions immunoreactive with antibodies against alpha-syn. In those fibers. alpha-syn co-localized with Abeta, both by light microscopy, and ultrastructurally. Paired-helical filaments did not contain alpha-syn immunoreactivity. In all muscle biopsies, alpha-syn was strongly immunoreactive at the postsynaptic region of the neuromuscular junctions. alpha-syn immunoreactivity also occurred diffusely in regenerating and necrotic muscle fibers. In cultured human muscle fibers, alpha-syn and its mRNA were expressed by immunocytochemistry, immunoblots, and Northern blots. Our study provides the first demonstration that alpha-syn participates in normal and pathologic processes of human muscle. Therefore. its function is not exclusive to the brain and neurodegenerative diseases.

Difference in expression of phosphorylated tau epitopes between sporadic inclusion-body myositis and hereditary inclusion-body myopathies.

Sporadic inclusion-body myositis (s-IBM) and the hereditary inclusion-body myopathies (h-IBMs) are severe and progressive muscle diseases, characterized pathologically by vacuolated muscle fibers containing paired-helical filaments (PHFs). An interesting feature of the s- and h-IBM muscle phenotype is its striking similarity to Alzheimer-disease (AD) brain. We immunostained muscle biopsies of 9 s-IBM patients, 9 autosomal-recessive h-IBM patients, 1 autosomal-dominant h-IBM patients, and 18 normal and disease-controls with several antibodies known to react with the hyperphosphorylated tau of AD-PHFs. Those included SMI-31, SMI-310, PHF-1, and AT8. In both s- and h-IBM, virtually all vacuolated muscle fibers had strongly immunoreactive inclusions with SMI-31, and by immuno-electronmicroscopy SMI-31 was exclusively localized to PHFs. Approximately 40 to 50% of both s- and h-IBM vacuolated muscle fibers were also immunoreactive with AT8 antibody. To the contrary, in h-IBM, there was no immunoreactivity with SMI-310 and PHF-1 antibodies, whereas in s-IBM the vacuolated muscle fibers had strong immunoreactivity with those two antibodies. By immunoelectronmicorscopy, SMI-310 and PHF-1 also were localized to PHFs. Within s-IBM muscle fibers, the structures immunoreactive with SMI-310 were congophilic, whereas h-IBM muscle fibers did not have congophilia. Our studies: (a) demonstrate a distinct difference between s-IBM and the h-IBMs in regard to expression of immunoreactive phosphorylated tau and congophilia; (b) demonstrate a new "diagnostic duo" combination of SMI-31 and SMI-310 antibodies for identifying and distinguishing s-IBM and the h-IBMs; and (c) provide another close similarity of pathologic phenotypes between s-IBM muscle and AD brain, suggesting that similar cellular pathogenic mechanisms may be active in both diseases.

Dihydropyridine-sensitive Ca2+ channel in aneurally cultured human muscles. Relationship between high-affinity binding site and inhibition of calcium uptake.

Dihydropyridine-sensitive Ca2+ channels and the relationship between binding of dihydropyridine derivatives and depolarization-induced Ca2+ uptake have been studied in aneurally cultured human muscle. Analysis of the equilibrium binding of the 1,4-dihydropyridine derivative (+)-PN200-110 revealed a single high-affinity binding site with a Kd of 0.15 +/- 0.05 nM and a Bmax of 87 +/- 12 fmol/mg protein. Inhibition of (+)-[3H]PN200-110 binding by nitrendipine revealed a Ki of 0.8 nM for the nitrendipine-receptor complex. Depolarization of cultured human muscle achieved by elevating the K+ concentration increased the uptake 45Ca2+ which was inhibited by nitrendipine with an IC50 of 1.1 nM. This study demonstrates that aneurally cultured human muscle has dihydropyridine-sensitive voltage-dependent Ca2+ channels which are functional when the fibers are depolarized.

Synergistic influence of polypeptide growth factors on cultured human muscle.

In two- to five-week tissue cultures of biopsied adult human skeletal muscle, combined addition to the culture medium of insulin, fibroblast growth factor, and epidermal growth factor synergistically increased creatine kinase activity 17-fold, increased acetylcholine receptors tenfold, and accelerated muscle differentiation. This study provides the first demonstration of the beneficial influence of these peptides on human muscle. It also establishes a new culture medium, resulting in the following: (1) much better long-term growth and differentiation of biopsied adult human muscle; and (2) by allowing elimination of embryo extract and reduction of serum, an important step toward developing a fully defined medium for culturing biopsied adult human normal and pathologic muscle tissue.

Abnormality of cultured muscle and Schwann's cells in familial lipid neuromyopathy. Muscle corrected by neural influence.

Muscle and Schwann's cell cultures were established from a family (mother and two sons) with non-carnitine deficient neuromyopathy. Electron microscopy of noninnervated cultured muscle showed poorly matured muscle fibers containing large "mushy" mitochondria, lipid droplets, abundant multilaminated inclusions, and dense-core dark osmiophilic bodies. Parallel innervated muscle fibers (cocultured with normal-rat motor neurons for two to three weeks) were well cross-striated, had well-developed T tubules and sarcoplasmic reticulum, and none of the abnormalities of aneural muscle cultures. Cultured Schwann's cells were normal by light microscopy, but had ultrastructurally abnormal mitochondria, lipid droplets, dark osmiophilic granular inclusions, and numerous "foamy" vacuoles. Those studies demonstrated (1) intrinsic muscle and Schwann's cell defects because of reproduction of abnormalities in culture, (2) normal muscle cell responsiveness to innervation reflected by development of contractions and longer survival in culture, and (3) beneficial influence of a neural factor(s) on the endogenous muscle cell abnormality.

Adult-onset acid maltase deficiency. Electrophysiological properties of aneurally cultured muscle.

Electrophysiological studies were performed on aneurally cultured muscle cells from one patient with adult-onset acid maltase deficiency (AAMD) and from controls. The cells from the patient with AAMD had a higher mean resting membrane potential, a lower input resistance, and a higher incidence of action potentials at resting membrane potential than the control cells. Therefore, sarcolemma maturation was not adversely affected. The AAMD cells had membrane thresholds and action potential amplitudes similar to those of the control cells, and rarely produced repetitive action potentials. Therefore, the membrane instability noted in adult muscle fibers from the patient with AAMD was not present in cultured cells. This study does not support the suggestion that the biochemical and morphological abnormalities present in muscle fibers of patients with AAMD are sufficient to cause the electrical abnormalities.

Myosin isoenzymes in cultured human muscle.

Cultured human muscle grown aneurally and innervated by the ventral part of fetal rat spinal cord was examined using antimyosin antibodies specific for isomyosins from fast and slow mammalian skeletal muscle. Cultured muscle displayed multiple reactivity with antibodies against both types of myosins, with no evident compartmentalization of different forms of myosin into different muscle cells, such as seen in adult muscle. Innervation of cultured muscle resulted in better growth and longer survival of cultured muscle and its more advanced maturation, with a larger number of cross-striated muscle fibers. The pattern of immunofluorescence reaction, however, was the same in both innervated and noninnervated cultured muscle.

Abnormalities of aneural and innervated cultured muscle fibers from patients with myotonic atrophy (dystrophy).

Innervation of human muscle cocultured in monolayer with explants of fetal rat spinal cord plus dorsal-root ganglia produces more mature fibers, which show spontaneous, neurogenic (d-tubocurarine-blocked) contractions. On the innervated myotonic atrophy (MA) muscle fibers, 96% of acetylcholinesterase-stained patches were simple, and only 4% appeared as complicated, pretzel-like, more mature-looking structures; on control innervated fibers, 37% of the acetylcholinesterase patches had the mature appearance. The normal trend from multifocal innervation toward unifocal innervation was decreased in innervated MA muscle fibers. Microelectrode studies compared parameters of cultured aneural muscle fibers and cultured innervated-contracting muscle fibers from 7 patients with MA and 10 control patients. The mean resting membrane potentials of the two groups (aneurally cultured MA muscle fibers and innervated-contracting cultured MA muscle fibers) were 8 and 9 mV lower, respectively, than those of their counterpart controls. The mean amplitude of action potentials, the maximum rate of rise of action potentials in innervated MA muscle fibers, and the action potential amplitude in aneural MA muscle fibers were significantly smaller than in corresponding control fibers.

Human schwann cells in tissue culture: histochemical and ultrastructural studies.

Schwann cells cultures were established from 40 human peripheral nerves that underwent biopsy according to an explant-reexplantation technique that markedly reduces non-Schwann cells and achieves cultures greatly enriched in Schwann cells suitable for a wide range of studies. Described are the growth characteristics and histochemical, ultrastructural, and ultrastructural-cytochemical patterns of normal human cultured Schwann cells from 12 patients with demonstrable peripheral nerve abnormality. These findings serve as a basis of comparison when seeking abnormalities of Schwann cells grown from peripheral nerves of patients with putative dysschwannian neuropathies.

Reincarnation in cultured muscle of mitochondrial abnormalities. Two patients with epilepsy and lactic acidosis.

Two unrelated 9-year-old boys failed to thrive from ages 5 and 4 years, and had focal cerebral seizures followed by transcent hemipareses. Histochemistry of their muscle biopsies showed "ragged-red" fibers, which ultrastructurally contained clusters of mitochondria having loss of crisp delineation of crista membranes and contained amorphous inclusion material and parallel-packed cristae and sometimes paracrystalline inclusions. In the patients' cultured muscles, similar mitochondrial abnormalities were present. 2,4-Dinitrophenol, introduced to the medium of cultures of normal human muscle, produced mitochondrial abnormalities similar to those of the patients', and the medium of the patients' muscle cultures worsened the mitochondrial abnormalities. This study, in demonstrating a mitochondrial defect reproducible in the cultured muscle fibers and, therefore, intrinsic to the ragged-red muscle fibers themselves, raises the possibility of a collateral mitochondrial defect in CNS cells as part of a multicellular mitochondriopathy.

Muscle-type phosphorylase activity present in muscle cells cultured from three patients with myophosphorylase deficiency.

Skeletal muscle fibers cultured from three patients whose mature fibers are deficient in glycogen myophosphorylase (EC 2.4.1.1) were shown to become rather mature, to have no excessive glycogen accumulation, and to develop signifcant myophosphorylase activity. That activity was characterized electrophoretically and immunologically and shown to be muscle phosphorylase rather than a genetically different type, thereby demonstrating true "rejuvenation" in culture of an enzyme genetically programmed ultimately to be deficient.