Search

BiP (binding immunoglobulin protein) is a key chaperone mostly residing in the Endoplasmic / Sarcoplasmic Reticulum (ER/SR). It plays an important role in maintaining protein homeostasis by facilitating correct folding, assembly, and clearance of proteins across diverse cellular compartments. Despite its implication in diverse (neuro-)muscular disorders associated with ER stress, the precise involvement of BiP in muscle physiology and pathology, as in the context of IIM, remains poorly understood. To study the role of BiP in IIM, quadriceps muscle biopsy specimens obtained from IIM patients were subjected to unbiased proteomic profiling, and results were validated by immunostaining. Hereby, following diseases were studied: anti-synthetase syndrome (ASS), dermatomyositis (DM), inclusion body myositis (IBM), immune-mediated necrotizing myopathy (IMNM), and systemic sclerosis associated with myositis and vasculopathy (SSc/MMCP). In patients with idiopathic inflammatory myopathies (IIM), proteomics revealed the most substantial elevation in BiP levels in muscle biopsies derived from dermatomyositis (DM) patients, with a median increase of 3.49-fold. This was followed by patients with immune-mediated necrotizing myopathy (IMNM), showing a mean increase of 2.62-fold in SRP54-seropositive individuals and a mean increase of 2.23-fold in HMGCR-seropositive individuals. In patients with inclusion body myositis (IBM), a mean increase of 2.33-fold in BiP levels was identified. Among anti-synthetase syndrome patients, PL-7-seropositive individuals exhibited a median increase of 1.56-fold, while PL-12- and JO-1-seropositive patients showed elevations, with mean increases of 1.31-fold and 1.51-fold, respectively. Additionally, muscle protein extracts from patients with systemic sclerosis/mixed connective tissue disease (SSc/MMCP) showed increased BiP levels, with a mean increase of 1.40-fold. Immunostaining confirmed the elevation of BiP in the sarcoplasm of patients with IMNM, IBM, ASS and DM. Interestingly, muscle biopsy samples from systemic sclerosis (MMCP) patients showed an extracellular increase in BiP levels indicative for a function in immunomodulation. In conclusion, BiP is upregulated throughout all IIM subtypes, with DM showing the highest intracellular abundance suggesting a role in proteostasis. However, BiP was also found in the extracellular space in SSc patients, hinting at additional biological roles in the molecular aetiology of muscular diseases with an autoimmune background. [ABSTRACT FROM AUTHOR]

Systemic sclerosis (SSc) is a rare autoimmune disease belonging to the group of connective tissue disorders. It is characterized by obliterative vasculopathy, accumulation of extracellular matrix in terms of fibrosis and autoimmune-mediated inflammation; all of these three features can affect almost any organ, including skeletal muscle, leading to potentially life-threatening complications such as interstitial lung disease or pulmonary-arterial hypertension. There is therefore a great medical need for a better understanding of the pathophysiology and thus improved therapy. Recent studies unveiled involvement of ER-stress in white blood cells and muscle tissue, which lead to activation of the unfolded protein response (UPR). BiP, a chaperone, is a major player that binds nascent polypeptides as they translocate into the ER and misfolded/aberrant proteins destined for degradation. Since therapy in SSc is currently oriented towards individual symptoms rather than the causes of the disease, a deeper understanding of the pathophysiology is invaluable to provide improved therapies. Therefore, UPR modulation represents an attractive target, as it is therapeutically addressable by e.g. small molecules. Hence, we investigated the potential involvement of BiP in the etiology of SSc. For this we analyzed muscle tissue from 15 patients with SSc in comparison to five non-disease controls by immune histochemistry, unbiased proteomic profiling, and sera by ELISA. Proteomic profiling revealed clear increase of BiP in skeletal muscle from SSc patients. Immunostaining also revealed an increase of BiP. Interestingly, BiP-deposits were not identified within the muscle fibers, but rather within the extracellular space. Notably, previous studies have shown that extracellular BiP-localization impacts immune cell function and thus immune response. Especially T cells are attracted by high extracellular BiP concentrations. Our ELISA-based quantification showed lower serum concentrations. In contrast, anti-BiP in serum showed a significant increase, supporting the concept of cellular BiP-release and thus supports the interesting finding of BiP in the extracellular space seen in skeletal muscle. This migth hint to an inflammatory role of BiP in SSc. Our combined results unravel BiP as a tissue and blood biomarker with potential impact on immune responses in SSc. Further functional studies are crucial to address a potential therapeutic impact of this finding. [ABSTRACT FROM AUTHOR]

Immune-mediated necrotizing myopathy (IMNM) is a form of idiopathic inflammatory myopathy (IIM) characterized by severe, rapidly progressing muscle weakness, creatine kinase elevation and muscle fibre necrosis. IMNMs are divided into three subgroups depending on the presence of muscle specific autoantibodies: anti-SRP-positive, anti-HMGCR-positive and seronegative IMNM. Vascular abnormalities, particularly alterations in capillaries, have been mentioned in IMNM. However, the link between autoimmune muscle damage and abnormal capillary morphology has remained unclear. The type of capillary pathology is a relevant feature, since vascular damage is key e.g. in dermatomyositis and systemic sclerosis as highlighted recently. In this project, we aimed to characterize vascular/capillary abnormalities in muscle biopsies derived from HMGCR-Ab, SRP-Ab, and seronegative IMNM patients. Light and electron microscopy were used to assess structural changes in capillaries, while quantitative real-time PCR was used to study the expression of genes related to vascular function. Additionally, we analyzed the proteomic signature of IMNM in relation to vascularization. Distinct patterns of capillary abnormalities were identified in all IMNM subgroups. Capillaries showed a spectrum of alterations, including a broad range of basement membrane duplications, activation of endothelial subcellular organelles featuring vesicle formation, proliferation of pericytes and degeneration to string vessels, but they do not show endothelial tubuloreticular inclusions. By integrating these methods, this study sought to provide a comprehensive understanding of the vascular abnormalities observed in IMNM, shedding light on a previously neglected aspect of the disease's pathophysiology. [ABSTRACT FROM AUTHOR]

Anti-synthetase syndrome (ASyS) is an autoimmune condition, characterized by the presence of autoantibodies directed against an aminoacyl-tRNA synthetase (anti-ARS). Patients present clinical symptoms such as myositis, interstitial lung disease, Raynaud's phenomenon, and arthritis. Anti-Jo-1, anti-PL-7 and anti-PL-12 are the most frequent anti-ARS. However, their role in ASyS pathogenesis remains incompletely understood. Therefore, robust animal models are essential to gain a detailed insight into the underlying pathophysiology. Aiming to characterize these pathophysiological features, we established and studied a mouse model for Jo-1, PL-7 and PL-12 associated ASyS. ASyS was induced in NOD.Idd 3/5 mice by injection of 200 µg Jo-1, PL-7 or PL-12 recombinant protein emulsified in Complete Freund's Adjuvant (CFA) in combination with OX86. Controls received CFA and Phosphate Buffered Saline only. Muscle strength was assessed by rotarod tests and the effects on the peripheral immune system were investigated by flow cytometry in spleen and lymph nodes. Morphological characteristics of skeletal muscle and lung tissue of immunized mice and the tissue infiltrating immune cells were validated using histology and immunohistochemistry. Immunization of mice led to clinical symptoms including muscle weakness and demonstrated variations in the immune cell response between the ARS subtypes. Histological analysis of skeletal muscle tissues showed infiltration by immune cells in the epimysium, spreading into the adjacent perifascicular area with progressing disease. Analysis of lung specimens by immunohistological staining demonstrated peribronchial accentuated accumulation of lymphocyte aggregates. We present a mouse model, which recapitulates features of the human phenotype of ASyS, to study the molecular pathogenesis and provide new insights into the pathomechanisms. [ABSTRACT FROM AUTHOR]

Myotonic dystrophy type 2 (DM2) is an autosomal-dominant disorder characterized by a spectrum of systemic manifestations, primarily presenting with proximal muscle weakness, myalgia, myotonia, and muscle atrophy. The underlying pathology involves a CCTG tetranucleotide expansion within the CNBP gene, resulting in an RNA-dominated spliceopathy. DM2 lacks effective treatment options, necessitating a deeper understanding of its pathophysiological mechanisms. In the context of Myotonic dystrophy type 1 (DM1), promising treatment targets, including disturbed mitochondrial metabolism, have emerged. Of note, diverse biomarkers have been identified in DM1, while there is currently no biomarker for DM2. Thus, there are considerable and unaddressed scientific questions in DM2 compared to DM1. We have previously identified mitochondrial abnormalities on both, biochemical and morphological level in muscle biopsy specimens derived from DM2 patients. In this follow-up study, using blood samples obtained from DM2 patients, we aimed to correlate muscle- and blood-derived molecular signatures to (1) investigate multi-systemic aspects of the previously identified mitochondrial alterations and (2) to screen for circulating disease biomarkers of pathophysiological relevance. From EDTA-blood samples obtained from DM2 patients, we isolated white blood cells (WBC) and serum. Both WBC and serum underwent unbiased proteomic analysis by LC-MS/MS, and data was intersected with muscle tissue-derived transcriptomic and proteomic signatures. Using this approach, we identified alterations of several mitochondrial proteins in WBC derived from DM2 patients, thus confirming our results obtained from skeletal muscle specimens and pointing at (multi-)systemic mitochondrial alterations in DM2. Dysregulated proteins were linked to respiratory chain complexes I and III. Other prominently downregulated proteins in WBC were involved in DNA and RNA binding. Prominently upregulated proteins in WBC were linked to protein quality control and splicing, pointing toward compensatory mechanisms in the context of wide-spread, disease-associated mis-splicing events. In addition, we identified several new biomarker candidates in serum samples derived from DM2 patients. In conclusion, DM2 emerges as a disease characterized by systemic mitochondrial dysfunction. This disease-specific molecular signature might serve as a promising treatment target and, in addition, provide new biomarker candidates. [ABSTRACT FROM AUTHOR]

Autoimmune necrotizing myopathies (ANM) represent a well-recognized entity among the so-called idiopathic inflammatory myopathies (IIMs), which also comprise dermatomyositis (DM), polymyositis (PM), sporadic inclusion body myositis (sIBM) and non-specific myositis. The diagnosis of these IIMs is established on the basis of specific clinical symptoms, results derived from muscle biopsy and ancillary data, such as creatine kinase (CK) levels, electromyography (EMG) and modern imaging techniques. Myositis-associated and myositis-specific autoantibodies play an important role and may prove to be clues for understanding details of the pathogenesis of these conditions, which to date remain largely unknown. ANMs probably represent a heterogeneous group of diseases all of which are characterized by the striking presence of necrotic fibres on muscle biopsy. However, additional morphological features as well as certain autoantibodies (e.g. SRP or HMGCR auto-antibodies) may be helpful to define subgroups of ANM. Subclassification of ANMs and definition of specific syndromes with precise and characteristic immunological features is a subject of intense research, especially since clinical trials with anti-inflammatory agents or so called ’biologicals’ should follow universally defined and accepted criteria. We present molecular and morphological data of subgroups within the spectrum of autoimmune necrotizing myopathies. [Copyright &y& Elsevier]

Involvement of the complement system in the pathogenesis of myasthenia gravis (MG) depends on the IgG-subtype. Treatment with complement inhibitors is currently restricted to AChR-ab+MG, mainly belonging to the IgG1-subtype with strong capacity for complement activation. The main pathomechanism of LRP4-ab is thought to be disruption of AChR-clustering at the postsynaptic membrane. However, LRP4-ab have found to be of IgG1 or IgG2-subtypes with different capacities to activate complement. This study aimed to investigate the role of complement as well as IgG-subtypes in LRP4-ab+MG. In this study we analyzed 7 patients with LRP4-ab+MG who underwent external intercostal muscle (ICM) biopsy. 2/7 patients were double-positive for LRP4- and AChR-ab. Specimens were analyzed by stainings, electron microscopy (EM) and gene transcript analyses. All results were compared to 'diseased controls' (AChR-ab+MG) as well as NDC (patients that underwent ICM biopsy due to e.g. scoliosis). In all biopsies endplates were identified by histology. In 6/7 patients C5b-9 (MAC) was clearly identified at a variable percentage of NMJs (range 6-75%). In gene transcript analyses, complement factors C1QC and C3 were increased in LRP4-ab+ as well as AChR-ab+MG compared to NDC, without statistical significance. IgG subtyping via immunofluorescence was performed in 4 LRP4-ab+MG patients. All samples evidenced co-localization of MAC with IgG1. One of the double-positive patients showed additional positivity for IgG3 on single endplates. There was no IgG2 or IgG4 staining. All NDC were negative for IgG1-4 and C5b-9. On electron microscopy LRP4-ab+MG showed on the postsynaptic side that clefts are scarce in number and some of them are short or plump. Our data suggest a crucial role of the complement system in LRP4-ab+MG. Targeted complement inhibition might therefore be a therapeutic option in LRP4-ab+MG. [ABSTRACT FROM AUTHOR]

Distal myopathies are a group of rare progressive genetic muscle disorders typically causing weakness and atrophy in the feet and/or hand muscles. The family under study is affected by an adult-onset dominant distal myopathy and the objective was to identify the pathogenic variant causing the disease. After negative gene panel and exome data, linkage analysis was conducted with genotyped single nucleotide polymorphisms to identify loci that co-segregated with the symptoms. Rare small-scale variants were analyzed with short-read whole exome and genome sequencing. Structural variants (SV) and repeat expansions were detected from whole genome sequenced samples with bioinformatic tools. SVs were further revealed with optical genome mapping. A muscle biopsy of a patient was used for untargeted proteomic profiling to recognize dysregulated proteins, and RNA sequencing was performed to identify splicing alterations and altered transcriptional regulation. Genetic regions identified by linkage analysis did not contain rare segregating variants likely to be pathogenic. Linkage analysis was then repeated several times with different parameters, to account for possible presymptomatic carriers, and a patient with uncertain disease status. But none of the potential new linked regions did contain any potentially promising variants. Proteomic profiling identified over 200 dysregulated proteins, many of which were encoded by muscle-associated genes but were not located in any of the linked regions and did not contain rare segregating variants. RNA sequencing detected splicing alterations in five genes and expression changes in two genes, which were all seemingly unrelated to muscle disorders. To date we haven't been able to identify the causative variant, but further analyses are ongoing. The cause may turn out to be a complex mechanisms such as digenic/oligenic inheritance or limitations of the current next-generation sequencing. [ABSTRACT FROM AUTHOR]

Inclusion body myositis (IBM) is a unique entity in the group of idiopathic inflammatory myopathies (IIM). The underlying pathomechanisms remain unclear although both autoimmunity as well as myodegenerative processes seem to play a role. Another feature of IBM skeletal muscle are dysfunctional mitochondria as evidenced by pathological findings such as cytochrome c oxidase negative (COX-)fibers and mitochondrial DNA (mtDNA) alterations. Providing energy for the cell, mitochondria are essential organelles for muscle cell function and survival given their high energy demand. However, mitochondria also contribute to apoptosis through a process termed mitochondrial outer membrane permeabilization (MOMP). Interestingly, in the event of sub-lethal cellular stress, mtDNA entering the cytosol does not cause apoptosis but engages a variety of downstream effects including the activation of inflammatory pathways. This condition is termed minority MOMP (miMOMP). We hypothesize that miMOMP might provide a pathophysiological link between impaired mitochondrial function and the inflammatory response in IBM. To test this hypothesis, we aimed to visualize and quantify mitochondrial leakage. We used skeletal muscle biopsies obtained from clinically and histologically diagnosed IBM patients. The muscle specimens were sliced into 4 and 8μm sections and cryopreserved at -80°C prior to immunostaining. We performed immunofluorescence stains with the following antibodies: TOMM20 (translocase of outer mitochondrial membrane 20), cytochrome C and anti-DNA. Specimen were analyzed using wide-field fluorescence microscopy (ObserverZ.1, Zeiss, 25x and 63x objectives) and superresolution microscopy (ELYRA PS.1, Zeiss, 100x/1.46NA) in cooperation with the Core Facility for Advanced Light Microscopy, Medical Faculty of the Heinrich Heine University, Düsseldorf. Depicting relative intensity of fluorescence signals over distance, our aim was to quantify the localization of cytochrome C and mtDNA within the boundaries of intact mitochondrial membranes marked by TOMM20. Intensity values were generated by ZEISS Zen 3.7. We were able to visualize both mitochondrial networks in the sarcoplasma of skeletal muscle cells as well as single mitochondria at a larger magnification. Testing different pre-processing approaches in our staining protocol, cell permeabilization with 1% saponin proved most effective for high-resolution depiction of mitochondria. We observed differences in mtDNA localization between IBM and non-diseased controls with an increase of mitochondrial leakage into the cytosol of IBM skeletal muscle cells as compared to healthy controls. Mitochondrial integrity may be objectively examined by fluorescence intensity measurements. We plan to use this approach staining for mitochondrial membranes and DNA both in skeletal muscle tissue as well as in cell culture of myoblasts to identify "leaky" mitochondria and investigate their pathophysiological implications in IBM. Given that the release of mtDNA into the cytosol of cells is considered a strong inflammatory trigger, it is our objective to examine whether miMOMP and its downstream effects are engaged in IBM muscle cells, too. Confirmation of the role of mitochondria as promoters of inflammation in the muscle of IBM could introduce new therapeutic targets in this so far treatment-refractory disease. [ABSTRACT FROM AUTHOR]

Inclusion body myositis (IBM) is a chronic inflammatory disease affecting skeletal muscles. IBM is characterized by muscle weakness mostly starting with muscle weakness in the finger flexors and the quadriceps muscle. This disease is refractory to contemporary therapies such as immunosuppressive approaches. In physiology, fibro-adipogenic progenitors (FAPs) are necessary for maintaining skeletal muscle homeostasis. However, recent data suggests that FAPs may contribute to the pathophysiology of IBM. Here, FAPs engage a pro-inflammatory and pro-fibrogenic phenotype associated with features of senescence. Besides secretion of pro-inflammatory signaling molecules, FAPs may affect the collagen homeostasis in muscles prioritizing type I collagen. To enable the study of FAPs in muscle diseases, such as IBM, reliable surface markers are required to effectively identify these cells. Previous works suggested platelet-derived growth factor receptor A (PDGFRa) as a marker for mesenchymal muscle cells, such as FAPs. In this study, we observed that PDGFRa seems to mark FAPs in the peri- and endomysium of skeletal muscle. In line with previous reports, these cells were found throughout the muscle assuming a flat and elongated shape surrounding muscle fibers. We tested the protein decorin based on transcriptomic data, however, without observing sufficient level in FAPs to serve as reliable marker. Based on these preliminary findings, we plan to perform further stainings to establish a FAP marker and therefore, investigating the role of FAPs in IBM. For this, we will employ PDGFRa to characterize the phenotype of FAPs in IBM. A better understanding of the role of FAPs in the pathophysiology of IBM patients may provide new potential targets for developing therapeutic strategies for this treatment-refractory disease. [ABSTRACT FROM AUTHOR]

Polymyositis with mitochondrial pathology (PM-Mito) was initially recognized in 1997 as a subtype of idiopathic inflammatory myopathy (IIM). Recent evidence indicates substantial clinical, histological, and molecular similarities between PM-Mito and Inclusion Body Myositis (IBM), leading to the inclusion of PM-Mito as an early form of IBM (eIBM) within the IBM spectrum. While inflammation and fatty-fibrotic tissue remodeling are less pronounced in early IBM compared to full-blown IBM, both demonstrate similar amount of mitochondrial abnormalities on skeletal muscle biopsies, suggesting mitochondrial dysfunction as an early IBM feature. This study aimed to elucidate the specificities of mitochondrial dysfunction as an early event in Inclusion Body Myositis pathophysiology, possibly linked to autoimmunity and inflammation. We analyzed 30 skeletal muscle biopsies obtained from early IBM/PM-Mito patients focusing on mitochondrial abnormalities and compared them with biopsies derived from full-blown IBM patients, and non-disease controls (NDC). Extensive light and electron microscopy were performed, and protein expression was investigated by unbiased proteomic profiling. Additionally, we conducted immunoblotting, focusing on respiratory chain subunits and inflammatory pathways associated with the release of mitochondrial damage-associated molecular patterns (DAMPs). Quantitative PCR (qPCR) and long-range PCR (LR-PCR) were utilized to evaluate mitochondrial DNA copy numbers and deletions, respectively. Our investigation revealed widespread mitochondrial abnormalities in both early and late-stage IBM patients, evidenced by histological and ultrastructural analyses. Notably, abnormal cristae architecture and inner mitochondrial membrane irregularities emerged as prominent features. Furthermore, both early and late IBM patients exhibited significantly reduced mitochondrial DNA copy numbers and age-inappropriate mtDNA deletions compared to healthy controls, without notable difference between IBM patient groups. Analysis of protein expression highlighted differential regulation of proteins associated with the inner mitochondrial membrane. Immunoblotting revealed defects in multiple respiratory chain subunits and activation of inflammatory pathways like the cGAS/STING pathway, associated with the release of mitochondrial DNA into the cytosol. Overall, our findings underscore mitochondrial dysfunction, characterized by disrupted mitochondrial ultrastructure and inner membrane defects, as an early feature of IBM. Additionally, the activation of specific inflammatory pathways suggests a significant role for mitochondria-associated inflammation in the early pathogenesis of IBM, occurring before tissue remodeling and vacuole formation. [ABSTRACT FROM AUTHOR]