Figure S4. Immunolocalization of filamin C and myotilin in a longitudinal muscle section. (TIF 1576 kb)

Introduction Myofibrillar myopathies are characterized by progressive muscle weakness and impressive abnormal protein aggregation in muscle fibers. In about 10 % of patients, the disease is caused by mutations in the MYOT gene encoding myotilin. The aim of our study was to decipher the composition of protein deposits in myotilinopathy to get new information about aggregate pathology. Results Skeletal muscle samples from 15 myotilinopathy patients were included in the study. Aggregate and control samples were collected from muscle sections by laser microdissection and subsequently analyzed by a highly sensitive proteomic approach that enables a relative protein quantification. In total 1002 different proteins were detected. Seventy-six proteins showed a significant over-representation in aggregate samples including 66 newly identified aggregate proteins. Z-disc-associated proteins were the most abundant aggregate components, followed by sarcolemmal and extracellular matrix proteins, proteins involved in protein quality control and degradation, and proteins with a function in actin dynamics or cytoskeletal transport. Forty over-represented proteins were evaluated by immunolocalization studies. These analyses validated our mass spectrometric data and revealed different regions of protein accumulation in abnormal muscle fibers. Comparison of data from our proteomic analysis in myotilinopathy with findings in other myofibrillar myopathy subtypes indicates a characteristic basic pattern of aggregate composition and resulted in identification of a highly sensitive and specific diagnostic marker for myotilinopathy. Conclusions Our findings i) indicate that main protein components of aggregates belong to a network of interacting proteins, ii) provide new insights into the complex regulation of protein degradation in myotilinopathy that may be relevant for new treatment strategies, iii) imply a combination of a toxic gain-of-function leading to myotilin-positive protein aggregates and a loss-of-function caused by a shift in subcellular distribution with a deficiency of myotilin at Z-discs that impairs the integrity of myofibrils, and iv) demonstrate that proteomic analysis can be helpful in differential diagnosis of protein aggregate myopathies.

Sporadic inclusion body myositis (sIBM) is the most common acquired myopathy in patients >50 years and characterized by both degenerative and autoimmune features. Rimmed vacuoles (RV) are a typical histological finding in sIBM. The aim of our study was to identify proteins that accumulate in RV by using a proteomic approach. Skeletal muscle samples of 18 sIBM patients were included in this study. RV samples and intraindividual control samples (CS; from muscle fibers without vacuolar changes) were collected from 10 μm H&E stained muscle sections by laser microdissection and analyzed by a highly sensitive mass spectrometry approach. A total of 3875 different proteins were identified and 215 of these showed a statistically significant accumulation in RV samples compared to CS. Proteins of the extracellular matrix and of the basal lamina, sarcolemmal proteins and intermediate filaments were the most abundant over-represented proteins in RV. In addition, we found an accumulation of proteins that play a role in protein degradation and of several chaperones including BiP and calreticulin, two proteins that are involved in unfolded protein response and SR stress, and components of a chaperone complex that was not described in the context of sIBM so far. Our proteomic approach also revealed an over-representation of proteins that play a role in inflammatory pathways, e.g. interferon-induced proteins and proteins involved in T-cell activation. In conclusion, our proteomic data provide essential new insights into the composition of rimmed vacuoles in sIBM. The application of a combined laser microdissection and mass spectrometry approach enabled the identification of more than 200 proteins that accumulate in RV areas. The results not only confirm previous findings but expand our knowledge about proteins and pathways that seem to be relevant in pathogenesis of sIBM.

Myofibrillar myopathies (MFM) are a group of usually autosomal dominant inherited muscle disorders characterized by focal disintegration of myofibrils and by the formation of intramyoplasmic protein aggregates. Known diseases genes encode proteins that are located at or associated with the Z-disc. We extended our previous proteomic analysis in MFM to identify novel disease-relevant proteins that accumulate in aggregate areas and to search for subtype-specific proteomic profiles. We analyzed skeletal muscle samples from 72 MFM patients. Aggregate samples and intraindividual control samples (from normally looking muscle fibers) were collected from 10 μ m muscle sections by laser microdissection and analyzed by a label-free mass spectrometric approach for identification and relative quantification of proteins. We detected 4716 different proteins in the samples and 291 of these showed a statistically significant accumulation in aggregate samples with a ratio >1.5 compared to controls (mean ratio 4.5). Z-disc and Z-disc-associated proteins, especially desmin, filamin C and their binding partners, constituted the most abundant group of over-represented aggregate proteins followed by proteins involved in protein quality control and protein degradation, extracellular and sarcolemmal proteins, components of signaling pathways and proteins involved in actin dynamics and myofibrillar organization. Subgroup analysis revealed a characteristic basic pattern of aggregate composition but also significant differences regarding the accumulation ratio, order and proportion of individual proteins that enabled the definition of subtype-specific proteomic profiles. Our proteomic findings expand the knowledge about proteins and pathways that seem to be involved in the pathogenesis of MFM. The identification of specific proteomic profiles in different MFM subtypes can be useful in differential diagnosis of protein aggregation myopathies.

Hereditary myopathy with early respiratory failure (HMERF) caused by A-band TTN mutations shows a clinical and histopathological overlap with myofibrillar myopathies (MFM) characterized by disintegration of myofibrils and protein aggregation in muscle fibers. We applied a proteomic approach to decipher the aggregate composition in HMERF and compared the proteomic profile with findings in MFM subtypes. Nine skeletal muscle samples from HMERF patients with three different mutations in A-band titin and histopathological findings consistent with MFM were analyzed. Protein aggregates and intraindividual control samples (from aggregate-free muscle fibers) were collected by laser microdissection and analysed by a label-free mass spectrometric approach for identification and relative quantification of proteins. Fifty-eight proteins showed a statistically significant accumulation in aggregates with a ratio > 1.8 compared to control samples. The over-represented proteins desmin, filamin C, Xirp2, N-RAP, alphaB-crystallin, nestin, myotilin, Xin and Hsp27 were highly abundant in aggregates. The same was found in different MFM subtypes. The detection of further Z-disc proteins, chaperones, proteins involved in protein degradation and sarcolemmal proteins was also typical of MFM. In addition, we identified an over-representation of proteins that are involved in signaling pathways (e.g. inhibitors of the Rho/ROCK signaling pathway and ankyrin repeat domain-containing proteins). Titin was slightly but significant under-represented in aggregates (ratio 0.83). In conclusion, the proteomic profile of aggregates in HMERF associated with mutations in A-band titin is typical of MFM and confirms that this type of titinopathy is a new MFM subtype. The finding that titin is under-represented in aggregates indicates that aggregate formation is mediated by secondary effects. The detected over-representation of proteins involved in signaling pathways may play a role in pathogenesis. Hereditary myopathy with early respiratory failure (HMERF) caused by A-band TTN mutations shows a clinical and histopathological overlap with myofibrillar myopathies (MFM) characterized by disintegration of myofibrils and protein aggregation in muscle fibers. We applied a proteomic approach to decipher the aggregate composition in HMERF and compared the proteomic profile with findings in MFM subtypes. Nine skeletal muscle samples from HMERF patients with three different mutations in A-band titin and histopathological findings consistent with MFM were analyzed. Protein aggregates and intraindividual control samples (from aggregate-free muscle fibers) were collected by laser microdissection and analysed by a label-free mass spectrometric approach for identification and relative quantification of proteins. Fifty-eight proteins showed a statistically significant accumulation in aggregates with a ratio > 1.8 compared to control samples. The over-represented proteins desmin, filamin C, Xirp2, N-RAP, alphaB-crystallin, nestin, myotilin, Xin and Hsp27 were highly abundant in aggregates. The same was found in different MFM subtypes. The detection of further Z-disc proteins, chaperones, proteins involved in protein degradation and sarcolemmal proteins was also typical of MFM. In addition, we identified an over-representation of proteins that are involved in signaling pathways (e.g. inhibitors of the Rho/ROCK signaling pathway and ankyrin repeat domain-containing proteins). Titin was slightly but significant under-represented in aggregates (ratio 0.83). In conclusion, the proteomic profile of aggregates in HMERF associated with mutations in A-band titin is typical of MFM and confirms that this type of titinopathy is a new MFM subtype. The finding that titin is under-represented in aggregates indicates that aggregate formation is mediated by secondary effects. The detected over-representation of proteins involved in signaling pathways may play a role in pathogenesis.

Protein aggregation in skeletal muscle fibers is a hallmark of myofibrillar myopathies and relevant in pathogenesis. Our previous proteomic studies deciphered details of aggregate composition and revealed specific proteomic profiles in different MFM subtypes. The aim of this study was to validate if these profiles are helpful in differential diagnosis in a patient with new mutations in two different MFM genes. The index patient presented with adult-onset weakness affecting limb and respiratory muscles. Two novel and heterozygous nucleotide exchanges in exon 1 of DES and exon 2 of MYOT, both predicted missense mutations, were identified and MFM was proven on the muscle biopsy. Protein aggregates from abnormal fibers and control samples from normally looking muscle fibers were collected by laser microdissection and analyzed by a combination of mass spectrometry (LC-MS/MS) and spectral index calculation. Our proteomic approach detected 113 proteins that were over-represented in intramyoplasmic aggregates of the index patient with a ratio >1.8 compared to control sample. The proteomic profile was consistent with desminopathy: desmin, filamin C, XIRP2, N-Rap and αB-crystallin showed the highest spectral indices of over-represented proteins. Accumulation of the desmin binding partner desmuslin also pointed to a pathogenic desmin mutation. The myotilinopathy markers plectin and obscurin were not over-represented in aggregates of the index patient and spectral index and ratio of myotilin also argued against a MFM-causing MYOT mutation. The results of our study indicate that the index patient harbors a disease-causing desmin mutation and demonstrate that our combined laser microdissection and mass spectrometric approach is a helpful new tool in differential diagnostics of MFM patients. Subtype-specific proteomic profiles can contribute to evaluate the pathogenicity of new mutations in MFM genes. Protein aggregation in skeletal muscle fibers is a hallmark of myofibrillar myopathies and relevant in pathogenesis. Our previous proteomic studies deciphered details of aggregate composition and revealed specific proteomic profiles in different MFM subtypes. The aim of this study was to validate if these profiles are helpful in differential diagnosis in a patient with new mutations in two different MFM genes. The index patient presented with adult-onset weakness affecting limb and respiratory muscles. Two novel and heterozygous nucleotide exchanges in exon 1 of DES and exon 2 of MYOT, both predicted missense mutations, were identified and MFM was proven on the muscle biopsy. Protein aggregates from abnormal fibers and control samples from normally looking muscle fibers were collected by laser microdissection and analyzed by a combination of mass spectrometry (LC-MS/MS) and spectral index calculation. Our proteomic approach detected 113 proteins that were over-represented in intramyoplasmic aggregates of the index patient with a ratio >1.8 compared to control sample. The proteomic profile was consistent with desminopathy: desmin, filamin C, XIRP2, N-Rap and αB-crystallin showed the highest spectral indices of over-represented proteins. Accumulation of the desmin binding partner desmuslin also pointed to a pathogenic desmin mutation. The myotilinopathy markers plectin and obscurin were not over-represented in aggregates of the index patient and spectral index and ratio of myotilin also argued against a MFM-causing MYOT mutation. The results of our study indicate that the index patient harbors a disease-causing desmin mutation and demonstrate that our combined laser microdissection and mass spectrometric approach is a helpful new tool in differential diagnostics of MFM patients. Subtype-specific proteomic profiles can contribute to evaluate the pathogenicity of new mutations in MFM genes.

Desminopathy is a subtype of myofibrillar myopathy caused by desmin mutations and characterized by protein aggregates accumulating in muscle fibers. The aim of this study was to assess the protein composition of these aggregates. Aggregates and intact myofiber sections were obtained from skeletal muscle biopsies of five desminopathy patients by laser microdissection and analyzed by a label-free spectral count-based proteomic approach. We identified 397 proteins with 22 showing significantly higher spectral indices in aggregates (ratio >1.8, p <0.05). Fifteen of these proteins not previously reported as specific aggregate components provide new insights regarding pathomechanisms of desminopathy. Results of proteomic analysis were supported by immunolocalization studies and parallel reaction monitoring. Three mutant desmin variants were detected directly on the protein level as components of the aggregates, suggesting their direct involvement in aggregate-formation and demonstrating for the first time that proteomic analysis can be used for direct identification of a disease-causing mutation in myofibrillar myopathy. Comparison of the proteomic results in desminopathy with our previous analysis of aggregate composition in filaminopathy, another myofibrillar myopathy subtype, allows to determine subtype-specific proteomic profile that facilitates identification of the specific disorder.Biological significanceOur proteomic analysis provides essential new insights in the composition of pathological protein aggregates in skeletal muscle fibers of desminopathy patients. The results contribute to a better understanding of pathomechanisms in myofibrillar myopathies and provide the basis for hypothesis-driven studies. The detection of specific proteomic profiles in different myofibrillar myopathy subtypes indicates that proteomic analysis may become a useful tool in differential diagnosis of protein aggregate myopathies. This article is part of a Special Issue entitled: From Genome to Proteome: Open Innovations. (C) 2013 Elsevier B.V. All rights reserved.

Desminopathy is a subtype of myofibrillar myopathies (MFM) caused by mutations in DES, the gene encoding desmin. A histopathologic hallmark of the disease is a massive protein aggregation within skeletal muscle fibers. The aim of our study was to elucidate the composition of aggregates in MFM patients with different desmin mutations by using a label-free mass spectrometry approach. Aggregates and control tissue from muscle biopsies of MFM patients with four different mutations in DES were collected by laser microdissection and analyzed by a combination of mass-spectrometry and spectral index calculation. Proteins with a ratio >1.8 (sum of peptides identified in aggregates compared to intraindividual controls) were accepted as accumulated in aggregates. Results of selected proteins were validated by immunofluorescence studies. Mass spectrometric data were searched against an extended human protein database to detect desmin mutations at the protein level. Three hundred and seventeen different proteins were identified and 98 of them showed an accumulation in aggregates. Aggregate compositions were more heterogenous than in other MFM subtypes, depending on individual mutations, but desmin was on top of the list of abundant proteins in all cases except of one. Immunolocalization findings were consistent with proteomic data. Three out of four desmin mutations were identified at the protein level. Our proteomic approach enabled the identification of many novel components of pathologic protein aggregates within skeletal muscle fibers of desminopathy patients. This provides new insights in the pathogenesis of the disease. Differences in aggregate composition in patients with different desmin mutations indicate diverse pathomechanisms, consistent with data of previous functional studies.

Myofibrillar myopathies (MFM) encompass a genetic heterogenous group of muscle disorders characterized by formation of intracellular protein aggregates in skeletal muscle fibers. We applied a proteomic approach to decipher the aggregate composition in MFM subtypes with the aim to identify novel disease-relevant proteins, disease-specific proteomic profiles and new candidates for MFM-causing proteins. Muscle biopsies of 21 genetically clarified MFM patients (filaminopathy, myotilinopathy, desminopathy, ZASPopathy) were analyzed. Aggregates and intraindividual controls (muscle fibers without aggregates) were collected by laser microdissection. A label-free mass spectrometric approach was used for identification and relative quantification of proteins. A total of 588 different proteins were identified. Up to 193 proteins showed an accumulation in protein aggregates in the different MFM subtypes (ratio >1.8 compared to intraindividual controls). Many of these proteins have never been described in the context of MFM so far. A subset of proteins including desmin, filamin C, Xin, Xirp2 and many other proteins was detected in aggregates in all patients. The comparison of MFM subtypes revealed disease-specific patterns of aggregate compositions with clear differences in ratios of most abundant proteins. Filamin C, desmin, myotilin and ZASP showed the highest accumulation in aggregates of related MFM subtypes. Our proteomic data provide essential new insights in the composition of pathological protein aggregates in MFM. Proteomic profiles of aggregates seem to be specific for the different MFM subtypes and expand our knowledge about proteins involved in pathogenesis of filaminopathy, desminopathy, myotilinopathy and ZASPopathy. The list of abundant proteins in aggregates also include potential new MFM proteins. These should to be considered in future genetic studies in MFM patients with so far unknown mutation.