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Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts
Abstract
Muscle "stem cells", or satellite cells, play an important role in the maintenance and repair of muscle tissue and have the capacity to proliferate and differentiate in response to physiological or environmental changes. Although extensively studied, the key regulatory steps and the complex temporal protein dynamics accompanying the differentiation of primary human muscle cells remain poorly understood. Here, we demonstrate the advantages of applying a MS-based quantitative approach, stable isotope labeling by amino acids in cell culture (SILAC), for studying human myogenesis in vitro and characterize the fine-tuned changes in protein expression underlying the dramatic phenotypic conversion of primary mononucleated human muscle cells during in vitro differentiation to form multinucleated myotubes. Using an exclusively optimized triple encoding SILAC procedure, we generated dynamic expression profiles during the course of myogenic differentiation and quantified 2240 proteins, 243 of which were regulated. These changes in protein expression occurred in sequential "waves" and underlined vast reprogramming in key processes governing cell fate decisions i.e. cell cycle withdrawal, RNA metabolism, cell adhesion, proteolysis and cytoskeletal organization. In silico transcription factor target analysis demonstrated that the observed dynamic changes in the proteome could be attributed to a cascade of transcriptional events involving key myogenic regulatory factors as well as additional regulators not yet known to act on muscle differentiation. In addition, we created of a dynamic map of the developing myofibril, providing valuable insights into the formation and maturation of the contractile apparatus in vitro. Finally, our SILAC-based quantitative approach offered the possibility to follow the expression profiles of several "muscle disease-associated proteins simultaneously and therefore could be a valuable resource for future studies investigating pathogenesis of degenerative muscle disorders as well as assessing new therapeutic strategies.
Supplementary resources (3)
... Immortalized C2C12 muscle cell line has been extensively used to elucidate molecular mechanisms driving myogenesis and myotube maturation [5][6][7] Valuable information on changes in protein expression were thus identified using a proteomic profiling approach during differentiation of this immortalized C2C12 cell line [5]. A recent study used triple stable isotope labeling by amino acid in cell culture (SILAC) strategy to profile the proteome of primary human myoblast during differentiation and started shedding light on the complex dynamic changes in protein expression during myogenesis [8]. The authors identified 243 regulated proteins that were clustered into proteins involved in cell differentiation, RNA metabolism, cell adhesion and cytoskeleton organization. ...
... This provided useful insights about protein expression dynamics involved in myogenesis and identified key changes in protein expression associated with myogenesis. Although C2C12 cell line has been criticized as not being the ideal cell model to study skeletal muscle biology, it provided useful insights about protein expression dynamics and changes associated with myogenesis [5] that agree with a more recent study that used triple SILAC strategy to study temporal protein expression changes associated with differentiation of primary human myoblast into myotubes [8]. In this study we used human myoblast cells lines that were derived from muscle biopsies of a healthy subject and a DMD patient and immortalized by transduction with both human telomerase reverse transcriptase and cyclin dependent kinase 4 expressing vectors [12]. ...
... Perhaps the most striking changes in temporal protein expression were observed in the first and second cluster when myoblasts started switching from proliferating to differentiating state. These two-cluster consisted mainly of proteins involved in mRNA splicing and processing, regulation of cell proliferation, and in translation initiation machinery in agreement with previous study [8]. Interestingly the largest group of proteins belonging to clusters 1 and 2 (61 proteins) were those involved in mRNA splicing and processing. ...
Background:
Myogenesis is a dynamic process involving temporal changes in the expression of many genes. Lack of dystrophin protein such as in Duchenne muscular dystrophy might alter the natural course of gene expression dynamics during myogenesis.
Objective:
To gain insight into the dynamic temporal changes in protein expression during differentiation of normal and dystrophin deficient myoblasts to myotubes.
Method:
A super SILAC spike-in strategy in combination and LC-MS/MS was used for temporal proteome profiling of normal and dystrophin deficient myoblasts during differentiation. The acquired data was analyzed using Proteome Discoverer 2.2. and data clustering using R to define significant temporal changes in protein expression.
Results:
sFour major temporal protein clusters that showed sequential dynamic expression profiles during myogenesis of normal myoblasts were identified. Clusters 1 and 2, consisting mainly of proteins involved mRNA splicing and processing expression, were elevated at days 0 and 0.5 of differentiation then gradually decreased by day 7 of differentiation, then remained lower thereafter. Cluster 3 consisted of proteins involved contractile muscle and actomyosin organization. They increased in their expression reaching maximum at day 7 of differentiation then stabilized thereafter. Cluster 4 consisting of proteins involved in skeletal muscle development glucogenesis and extracellular remodeling had a lower expression during myoblast stage then gradually increased in their expression to reach a maximum at days 11-15 of differentiation. Lack of dystrophin expression in DMD muscle myoblast caused major alteration in temporal expression of proteins involved in cell adhesion, cytoskeleton, and organelle organization as well as the ubiquitination machinery.
Conclusion:
Time series proteome profiling using super SILAC strategy is a powerful method to assess temporal changes in protein expression during myogenesis and to define the downstream consequences of lack of dystrophin on these temporal protein expressions. Key alterations were identified in dystrophin deficient myoblast differentiation compared to normal myoblasts. These alterations could be an attractive therapeutic target.
... In the present study, the effects of the steroidal hormones found in anabolic implants (E2 and/or TBA) on abundance of mRNA involved in the early stages of BSC differentiation (PAX7, MYF5, and MYOD) were investigated within the first 48 h of induction of differentiation. Investigating differentiation in the first 48 h of induction is important, as changes in myoblast differentiation have been reported to occur before 24 h [45]. A potential limitation of this study is that protein expression was not also investigated, as gene abundance can be influenced by the stability of the transcript and rate of transcription [46]. ...
... The earliest changes that occur in differentiation are Pax7 decreasing, while abundance of Myf5 and MyoD increases [6,9,10]. The increase in MyoD, is then eventually followed by an increase in myogenin, but this happens much later in differentiating satellite cells [45]. In human skeletal muscle myoblasts, changes in myogenin are not detected until 72 h post-induction to differentiate [45], in rat satellite cells, myogenin peaks 3 d after being induced to differentiate [49], and in BSCs, myogenin is barely detectable after 48 h in differentiation media [50]. ...
... The increase in MyoD, is then eventually followed by an increase in myogenin, but this happens much later in differentiating satellite cells [45]. In human skeletal muscle myoblasts, changes in myogenin are not detected until 72 h post-induction to differentiate [45], in rat satellite cells, myogenin peaks 3 d after being induced to differentiate [49], and in BSCs, myogenin is barely detectable after 48 h in differentiation media [50]. Due to the fact that myogenin is barely detectable after 48 h in BSCs induced to differentiate, when mRNA abundance of myogenin was measured in this study it was too early for it be expressed as this study was looking at the mRNA tanscripts in the first 48 h of BSCs induced to differentiate. ...
Emerging research suggests that hormones found in anabolic implants interact with polyamine biosynthesis. The objective of this study was to determine the effects of steroidal hormones, polyamines and polyamine precursors on bovine satellite cell (BSC) differentiation and polyamine biosynthesis temporally. Primary BSCs were induced to differentiate in 3% horse serum (CON) and treated with 10 nM trenbolone acetate (TBA), 10 nM estradiol (E2), 10 nM TBA and 10 nM E2, 10 mM methionine, 8 mM ornithine, 2 mM putrescine, 1.5 mM spermidine, or 0.5 mM spermine. Total mRNA was isolated 0, 2, 4, 8, 12, 24, and 48 h post-treatment. Abundance of mRNA for genes associated with induction of BSC differentiation: paired box transcription factor 7, myogenic factor 5, and myogenic differentiation factor 1 and genes in the polyamine biosynthesis pathway: ornithine decarboxylase and S-adenosylmethionine—were analyzed. Overall, steroidal hormones did not impact (p > 0.05) mRNA abundance of genes involved in BSC differentiation, but did alter (p = 0.04) abundance of genes involved in polyamine biosynthesis. Polyamine precursors influenced (p < 0.05) mRNA of genes involved in BSC differentiation. These results indicate that polyamine precursors and polyamines impact BSC differentiation and abundance of mRNA involved in polyamine biosynthesis, while steroidal hormones altered the mRNA involved in polyamine biosynthesis.
... However, it is necessary to study proteome performance through all the processes or stages in order to understand the molecular mechanisms underlying the phenotypic transition during myogenic differentiation, beyond regulatory factors. Proteomic tools have helped to determine the groups of proteins that are expressed in the development of myotubes in mammals, both in primary cultures of muscle cells 22 in established cell lines [23][24][25][26] and by complementing the profile of genes expressed during the process of myogenesis. This proteome work has provided a holistic framework to understand how different biochemical processes are coordinated at the cellular level: cell cycle exit, cell adhesion and migration, metabolism, proteolysis, extra cellular matrix (ECM) remodelling and fusion, and muscle contraction. ...
... Alpha-tubulin, expressed in most organisms as the major constituent of microtubules, is involved in the intracellular processes of transport, cytokinesis, cell shape maintenance and migration 32 . During differentiation of human primary myoblast α-tubulin remained constant, as do many other housekeeping proteins 22 . So, the downregulation of α-tubulin should be a signal that the proliferation process is ending. ...
... The fusion of post-mitotic mononucleated myoblasts to form syncytial myofibres requires coordination of two processes: first, the remodelling of cell migration, adhesion, membrane fusion and the extra-cellular matrix 22 , and second, the maturation of both the contractile apparatus and entire organelles, peroxisomes and mitochondria 25 . In Fig. 10, two functional clusters related to these processes are shown, both of them with the highest local clustering coefficient, 1. ...
Myogenesis is a complex two-phase process of proliferation and differentiation, which seems to be greatly conserved in vertebrates. For the first time in fish, we identify the changes that occur in the proteome during this process in a gilthead sea bream (Sparus aurata) myocyte primary cell culture (on days 4, 8 and 12), using 2-D gel electrophoresis and LC-MS/MS. A significant increase of myogenin expression at day 8 marked the transition from proliferation to differentiation. Of the 898 spots in the proteome analysis, the 25 protein spots overexpressed on day 4 and the 15 protein spots overexpressed on day 8 indicate the end of proliferation and the beginning of differentiation, respectively. Proliferation was characterized by enrichment of proteins involved in actin cytoskeleton remodelling and in cellular metabolic processes (transcription, ubiquitination, response to stress and glucose metabolism). During differentiation, 41 proteins were overexpressed and 51 underexpressed; many of them related to biosynthetic processes (RNA and protein synthesis and folding, and pentose pathways), terminal myotube formation and muscle contraction. The main cellular processes of both phases of muscle development in fish are similar with those observed in mammals but extended in time, allowing sequential studies of myogenesis.
... More indepth investigations are required to understand the differentiation-dependent susceptibility to ZIKV infection. In this way, proteomic studies illustrating the different changes that occur during human muscle cell differentiation, as already performed [44] could constitute the first step to assess the potential virus receptors or host cell restriction factors that govern muscle cells susceptibility to ZIKV infection during differentiation. According to Le Bihan et al., at least 243 proteins are modulated during myoblasts differentiation into myotubes [44]. ...
... In this way, proteomic studies illustrating the different changes that occur during human muscle cell differentiation, as already performed [44] could constitute the first step to assess the potential virus receptors or host cell restriction factors that govern muscle cells susceptibility to ZIKV infection during differentiation. According to Le Bihan et al., at least 243 proteins are modulated during myoblasts differentiation into myotubes [44]. ...
Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, that may be involved in the physiopathological course of the infection. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human primary myoblasts that can be differentiated into myotubes, we found that myoblasts can be productively infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection, suggesting a differentiation-dependent susceptibility. Infection was accompanied by a caspase-independent cytopathic effect, associated with paraptosis-like cytoplasmic vacuolization. Proteomic profiling was performed 24h and 48h post-infection in cells infected with two different isolates. Proteome changes indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, and differentiation-dependent restriction of infection from myoblasts to myotubes. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences in ZIKV infection pathogenesis.
... The systematic profiling of myogenesis using mass spectrometry-based proteomics has covered postnatal growth and development [63,64], but focused mostly on studying cell culture models [65][66][67] and specifically the skeletal muscle secretome during myoblast differentiation and myotube formation [68][69][70][71][72]. The concept that skeletal muscle cells act as secretory tissues has recently been reviewed by Pedersen [73]. ...
... The temporal profiling of the human myoblast proteome during in vitro differentiation highlighted the importance of ECM rearrangement during early myogenesis and showed a drastic increase in key ECM components, including several α-isoforms of collagen COL VI and COL XVIII, as well as the heparan sulfate proteoglycan HSPG2 of the basement membrane, the elastin-microfibril interface-located ECM glycoprotein EMILIN2 and nidogen isoform NID2 [76]. These findings confirm the developmental concept that enhanced synthesis of ECM proteins occurs during the transition from myoblasts to syncytial myotubes [72] and that complex interactions at the cell-ECM interface facilitate the fusion of myoblasts [35]. ...
... Thus, in addition to their contractile properties, myofibers have also a central role in cell-cell communication since they are able to secrete the so-called myokines, especially during regeneration and differentiation, including dedicated vesicles acting on muscle adaptation to damage or exercise (26). This myofibers secretome plays important roles in intercellular communication of the muscle resident cells, i.e., MuSCs, myofibers themselves, the fibro-adipogenic progenitors (FAPs), and macrophages (27). ...
Overactivation of the transforming growth factor-β (TGFβ) signaling in Duchenne muscular dystrophy (DMD) is a major hallmark of disease progression, leading to fibrosis and muscle dysfunction. Here, we investigated the role of SETDB1 (SET domain, bifurcated 1), a histone lysine methyltransferase involved in muscle differentiation. Our data show that, following TGFβ induction, SETDB1 accumulates in the nuclei of healthy myotubes while being already present in the nuclei of DMD myotubes where TGFβ signaling is constitutively activated. Transcriptomics revealed that depletion of SETDB1 in DMD myotubes leads to down-regulation of TGFβ target genes coding for secreted factors involved in extracellular matrix remodeling and inflammation. Consequently, SETDB1 silencing in DMD myotubes abrogates the deleterious effect of their secretome on myoblast differentiation by impairing myoblast pro-fibrotic response. Our findings indicate that SETDB1 potentiates the TGFβ–driven fibrotic response in DMD muscles, providing an additional axis for therapeutic intervention.
... The dynamic proteome of voluntary striated muscles is majorly influenced by alterations in an individual's lifestyle that affects the nutritional status [14] and physical activity levels [15]. Drastic and highly varied changes/adaptations in the skeletal muscle proteome have been described to occur during myogenesis [16] and in response to exercise training [17], muscular atrophy [18], traumatic injury [19], weight loss [20], hypoxia [21] and the natural aging process [22]. Physiological or pathophysiological alterations within the skeletal musculature also strongly influence the composition of the muscle secretome [23]. ...
Introduction:
Skeletal muscles contain large numbers of high-molecular-mass protein complexes in elaborate membrane systems. Integral membrane proteins are involved in diverse cellular functions including the regulation of ion handling, membrane homeostasis, energy metabolism and force transmission.
Areas covered:
The proteomic profiling of membrane proteins and large protein assemblies in skeletal muscles are outlined in this article. This includes a critical overview of the main biochemical separation techniques and the mass spectrometric approaches taken to study membrane proteins. As an illustrative example of an analytically challenging large protein complex, the proteomic detection and characterization of the Ca2+-ATPase of the sarcoplasmic reticulum is discussed. The biological role of this large protein complex during normal muscle functioning, in the context of fiber type diversity and in relation to mechanisms of physiological adaptations and pathophysiological abnormalities is evaluated from a proteomics perspective.
Expert opinion:
Mass spectrometry-based muscle proteomics has decisively advanced the field of basic and applied myology. Although it is technically challenging to study membrane proteins, innovations in protein separation methodology in combination with sensitive mass spectrometry and improved systems bioinformatics has allowed the detailed proteomic detection and characterization of skeletal muscle membrane protein complexes, such as Ca2+-pump proteins of the sarcoplasmic reticulum.
... Cellular differentiation in multicellular organisms is often accompanied by programmed proteome reshaping and cellular reorganization to accomplish cell-type-specific functions. For instance, during myogenesis proliferative myoblasts undergo a differentiation programme with induction of specialized cytoskeletal proteins to form myofibrils in terminally differentiated myofibers (Chal and Pourquie, 2017;Le Bihan et al., 2015), whereas adipose stem cells induce differentiation cues controlling expression of proteins involved in lipid storage and lipid synthesis (Tsuji et al., 2014). ...
The development of haematopoietic stem cells into mature erythrocytes – erythropoiesis – is a controlled process characterized by cellular reorganization and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the ubiquitin system is poorly understood. By measuring the proteomic landscape of in vitro human erythropoiesis models, we found dynamic differential expression of subunits of the CTLH E3 ubiquitin ligase complex that formed maturation stage-dependent assemblies of topologically homologous RANBP9- and RANBP10-CTLH complexes. Moreover, protein abundance of CTLH’s cognate E2 ubiquitin conjugating enzyme UBE2H increased during terminal differentiation, and UBE2H expression depended on catalytically active CTLH E3 complexes. CRISPR-Cas9-mediated inactivation of CTLH E3 assemblies or UBE2H in erythroid progenitors revealed defects, including spontaneous and accelerated erythroid maturation as well as inefficient enucleation. Thus, we propose that dynamic maturation stage-specific changes of UBE2H-CTLH E2-E3 modules control the orderly progression of human erythropoiesis.
... Replicatively aged C2C12 myoblasts exhibit impaired differentiation and fusion [11,12] similar to human replicatively-aged primary myoblasts [13] and cells and tissue isolated from older rodents and humans [13-18]. Myoblast differentiation is orchestrated by complex programmes of cell signalling [19] and altered gene expression [17] that result in dynamic changes to the cell proteome during differentiation [20,21]. Accordingly, the reductions in differentiation and fusion capacity of replicatively aged cells are accompanied by impaired cell signalling and gene expression [22,23]. ...
Age-related impairments in myoblast differentiation may contribute to reductions in muscle function in older adults, however, the underlying proteostasis processes are not well understood. Young (P6-10) and replicatively aged (P48-50) C2C12 myoblast cultures were investigated during early (0h-24h) and late (72h-96h) stages of differentiation using deuterium oxide (D2O) labelling and mass spectrometry. The absolute dynamic profiling technique for proteomics (Proteo-ADPT) was applied to quantify the absolute rates of abundance change, synthesis and degradation of individual proteins. Proteo-ADPT encompassed 116 proteins and 74 proteins exhibited significantly (P<0.05, FDR <5 %) different changes in abundance between young and aged cells at early and later periods of differentiation. Young cells exhibited a steady pattern of growth, protein accretion and fusion, whereas aged cells failed to gain protein mass or undergo fusion during later differentiation. Maturation of the proteome was retarded in aged myoblasts at the onset of differentiation, but their proteome appeared to "catch up" with the young cells during the early phase of the differentiation period. However, this "catch up" process in aged cells was not accomplished by higher levels of protein synthesis. Instead, a lower level of protein degradation in aged cells was responsible for the elevated gains in protein abundance. Our novel data point to a loss of proteome quality as a precursor to the lack of fusion of aged myoblasts and highlights dysregulation of protein degradation, particularly of ribosomal and chaperone proteins, as a key mechanism that may contribute to age-related declines in the capacity of myoblasts to undergo differentiation.
... A previous report showed that MyoD-hiPSCs showed high similarity with primary myotubes except for MYF5 expression 52 . Moreover, another report has shown that primary myotubes express sarcomere isoforms such as MYH3, MYH7, MYH8, and MYH13, but not adult fast skeletal muscle isoforms (MYH1, MYH2, and MYH4) 53 . This discrepancy could be a strength of MyoD-hiPSCs. ...
MYOD-induced microRNA-494-3p expression inhibits fast oxidative myotube formation by downregulating myosin heavy chain 2 (MYH2) in human induced pluripotent stem cells (hiPSCs) during skeletal myogenesis. However, the molecular mechanisms regulating MYH2 expression via miR-494-3p remain unknown. Here, using bioinformatic analyses, we show that miR-494-3p potentially targets the transcript of the E1A-binding protein p300 at its 3′-untranslated region (UTR). Myogenesis in hiPSCs with the Tet/ON-myogenic differentiation 1 ( MYOD1 ) gene (MyoD-hiPSCs) was induced by culturing them in doxycycline-supplemented differentiation medium for 7 days. p300 protein expression decreased after transient induction of miR-494-3p during myogenesis. miR-494-3p mimics decreased the levels of p300 and its downstream targets MYOD and MYH2 and myotube formation efficiency. p300 knockdown decreased myotube formation efficiency, MYH2 expression, and basal oxygen consumption rate. The binding of miR-494-3p to the wild type p300 3′-UTR, but not the mutated site, was confirmed using luciferase assay. Overexpression of p300 rescued the miR-494-3p mimic-induced phenotype in MyoD-hiPSCs. Moreover, miR-494-3p mimic reduced the levels of p300, MYOD, and MYH2 in skeletal muscles in mice. Thus, miR-494-3p might modulate MYH2 expression and fast oxidative myotube formation by directly regulating p300 levels during skeletal myogenesis in MyoD-hiPSCs and murine skeletal muscle tissues.
... Over the last decade, mass spectrometry (MS)-based proteomics has evolved as a powerful platform for studying complex biological processes at a systemwide level. We and others have previously used MS-based quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC) to provide a global overview of human embryonic stem cell proteomes and phosphoproteomes during differentiation (Prokhorova et al. 2009;Van Hoof et al. 2009;Rigbolt et al. 2011a;Rigbolt and Blagoev 2012), to monitor receptor tyrosine kinase signaling in MSCs differentiation to osteoblasts (Kratchmarova et al. 2005), and to determine secreted factors during OB differentiation (Kristensen et al. 2012), as well as proteome and secretome variations during muscle cell differentiation (Henningsen et al. 2010;Le Bihan et al. 2015). Here, we combined SILAC-based quantitative proteomic and phosphoproteomic analyses to elucidate the signaling events occurring during the first 24 h of OB differentiation of human MSCs, identifying more than 6000 proteins and 15,000 phosphorylation sites. ...
Bone marrow-derived mesenchymal stem cells (MSCs) differentiate into osteoblasts upon stimulation by signals present in their niche. Because the global signaling cascades involved in the early phases of MSCs osteoblast (OB) differentiation are not well-defined, we used quantitative mass spectrometry to delineate changes in human MSCs proteome and phosphoproteome during the first 24 h of their OB lineage commitment. The temporal profiles of 6252 proteins and 15,059 phosphorylation sites suggested at least two distinct signaling waves: one peaking within 30 to 60 min after stimulation and a second upsurge after 24 h. In addition to providing a comprehensive view of the proteome and phosphoproteome dynamics during early MSCs differentiation, our analyses identified a key role of serine/threonine protein kinase D1 (PRKD1) in OB commitment. At the onset of OB differentiation, PRKD1 initiates activation of the pro-osteogenic transcription factor RUNX2 by triggering phosphorylation and nuclear exclusion of the histone deacetylase HDAC7.
... Protein concentration was determined using BCA assay. A total of 50 μg aliquots from each extract were spiked with 12.5 μg of stable isotope labeled protein extract prepared from stable isotope labeling with amino acids in human myotube (SILAC), leading to 13 C and 15 N labeling at lysine and arginine in all proteins [14]. The myotubes were labelled for 12-15 days before they were detached from the flask using Trypsin-EDTA (Millipore, MA, USA). ...
Aim: Detection of drug-induced dystrophin in patient muscle biopsy is a surrogate outcome measure for Duchenne muscular dystrophy. We sought to establish and validate an orthogonal approach to measurement of dystrophin protein and RNA in muscle biopsies. Materials & methods: Validated methods were developed for dystrophin Western blotting, mass spectrometry, immunostaining and reverse transcriptase PCR of biopsy mRNA using muscle biopsy standards. Results: Both Western blotting and mass spectrometry validated methods demonstrated good linearity, and acceptable precision and accuracy with a lower limit of quantitation at 1%. Immunostaining and reverse transcriptase PCR methods were shown to be reliable. Conclusion: The described orthogonal approach is sufficient to support measures of dystrophin as a surrogate outcome in a clinical trial.
... Primary cells are collected directly from mature tissue and cultured to obtain the desired cell number and form tissue constructs. However, quick phenotypic changes, limited proliferation numbers, and aging of primary cells inhibit their use once the cells are transferred from their natural living conditions to artificial ones [132,136]. While CMs can be taken from specific tissue sources for targeted applications, robust scaffolds and engineered biological tissues are needed to improve to CM characteristics in new implanting environments. ...
The purpose of this review is to assess the state-of-the-art fabrication methods, advances in genome editing, and the use of machine learning to shape the prospective growth in cardiac tissue engineering. Those interdisciplinary emerging innovations would move forward basic research in this field and their clinical applications. The long-entrenched challenges in this field could be addressed by novel 3-dimensional (3D) scaffold substrates for cardiomyocyte (CM) growth and maturation. Stem cell-based therapy through genome editing techniques can repair gene mutation, control better maturation of CMs or even reveal its molecular clock. Finally, machine learning and precision control for improvements of the construct fabrication process and optimization in tissue-specific clonal selections with an outlook of cardiac tissue engineering are also presented.
... The column showed excellent run-to-run reproducibility (repeatability) and durability during a proteomics study of muscle cell (myoblast) differentiation (Supplementary Fig. S6). The quantitative proteomics data obtained using FlashPack columns was in accordance with previously published results [22]. Our results reconfirmed that column packing from high sorbent slurry concentrations did not negatively affect chromatographic resolution [12,21,23]. ...
Capillary ultrahigh-pressure liquid chromatography (cUHPLC) is essential for in-depth characterization of complex biomolecule mixtures by LC-MS. We developed a simple and fast method called FlashPack for custom packing of capillary columns of 50-100 cm length with sub- 2 m sorbent particles. FlashPack uses high sorbent concentrations of 500-1,000 mg/ml for packing at relatively low pressure of 100 bar. Column blocking by sorbent aggregation is avoided during the packing by gentle mechanical tapping of the capillary proximal end by a slowly rotating magnet bar. Utilizing a standard 100-bar pressure bomb, Flashpack allows for production of 15-25 cm cUHPLC columns within a few minutes and of 50 cm cUHPLC columns in less than an hour. Columns exhibit excellent reproducibility of back-pressure, retention time, and resolution (CV 8.7%). FlashPack cUHPLC columns are inexpensive, robust and deliver performance comparable to commercially available cUHPLC columns. The FlashPack method is versatile and enables production of cUHPLC columns using a variety of sorbent materials. © 2019 Kovalchuk et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
... The column showed excellent run-to-run reproducibility (repeatability) and durability during a proteomics study of muscle cell (myoblast) differentiation (Supplementary Fig. S6). The quantitative proteomics data obtained using FlashPack columns was in accordance with previously published results [22]. Our results reconfirmed that column packing from high sorbent slurry concentrations did not negatively affect chromatographic resolution [12,21,23]. ...
Capillary ultra-high-pressure liquid chromatography (cUHPLC) is essential for in-depth characterization of complex biomolecule mixtures by LC-MS. We developed a simple and fast method called FlashPack for custom packing of capillary columns of 50-100 cm length with sub-2-μm sorbent particles. FlashPack uses high sorbent concentrations of 500-1000 mg/ml for packing at relatively low pressure of 100 bar. Column blocking by sorbent aggregation is avoided during the packing of sorbent particles by gentle mechanical tapping of the capillary proximal end by a slowly rotating magnet bar. Utilizing a standard 100 bar pressure bomb, Flashpack allows for production of 15-25 cm cUHPLC columns within a few minutes and of 50 cm cUHPLC columns in less than an hour. Columns exhibit excellent reproducibility of back-pressure, retention time and resolution (CV 8.7 %). FlashPack cUHPLC columns are inexpensive, robust and deliver performance comparable to commercially available cUHPLC columns. The FlashPack method is versatile and enables production of cUHPLC columns using a variety of sorbent materials.
... These data are compiled from proteins identified in previous comparative proteome profiling experiments. [58][59][60][61][62][63] In the context of the laboratory standard cell lines, it is worth noting that RCMH cells have already been extensively characterized on the morphological and biochemical level. In contrast to C2C12 mouse muscle cells, RCMH cells present with so-called myopodia, which are important cellular structures for the formation of NMJs. ...
Congenital myasthenic syndromes (CMS) are a group of rare disorders that cause fatigable muscle weakness due to defective signal transmission at the neuromuscular junction, a specialized synapse between peripheral motor neurons and their target muscle fibers. There are now over 30 causative genes that have been reported for CMS. Of these, there are 10 that are associated with a limb-girdle pattern of muscle weakness and are thus classed as LG-CMS. Next-generation sequencing and advanced methods of data sharing are likely to uncover further genes that are associated with similar clinical phenotypes, contributing to better diagnosis and effective treatment of LG-CMS patients. This review highlights clinical and pathological hallmarks of LG-CMS in relation to the underlying genetic defects and pathways. Tailored animal and cell models are essential to elucidate the exact function and pathomechanisms at the neuromuscular synapse that underlie LG-CMS. The integration of genomics and proteomics data derived from these models and patients reveals new and often unexpected insights that are relevant beyond the rare genetic disorder of LG-CMS and may extend to the functioning of mammalian synapses in health and disease more generally.
... The cellular proteomic data of myogenesis. This cellular proteomic data [32] monitor the changes in protein expression underlying the phenotypic conversion of human primary myoblasts; from primary mononucleated muscle cells to multinucleated myotubes, using the SILAC method. The authors used human satellite cells isolated from a quadriceps muscle biopsy of a 5-day old infant. ...
Approaches to identify significant pathways from high-throughput quantitative data have been developed in recent years. Still, the analysis of proteomic data stays difficult because of limited sample size. This limitation also leads to the practice of using a competitive null as common approach; which fundamentally implies genes or proteins as independent units. The independent assumption ignores the associations among biomolecules with similar functions or cellular localization, as well as the interactions among them manifested as changes in expression ratios. Consequently, these methods often underestimate the associations among biomolecules and cause false positives in practice. Some studies incorporate the sample covariance matrix into the calculation to address this issue. However, sample covariance may not be a precise estimation if the sample size is very limited, which is usually the case for the data produced by mass spectrometry. In this study, we introduce a multivariate test under a self-contained null to perform pathway analysis for quantitative proteomic data. The covariance matrix used in the test statistic is constructed by the confidence scores retrieved from the STRING database or the HitPredict database. We also design an integrating procedure to retain pathways of sufficient evidence as a pathway group. The performance of the proposed T2-statistic is demonstrated using five published experimental datasets: the T-cell activation, the cAMP/PKA signaling, the myoblast differentiation, and the effect of dasatinib on the BCR-ABL pathway are proteomic datasets produced by mass spectrometry; and the protective effect of myocilin via the MAPK signaling pathway is a gene expression dataset of limited sample size. Compared with other popular statistics, the proposed T2-statistic yields more accurate descriptions in agreement with the discussion of the original publication. We implemented the T2-statistic into an R package T2GA, which is available at https://github.com/roqe/T2GA.
... To identify the myogenic factors of the conditioned media, proteomic analyses of the secretome of the adipogenic lineage were carried out. Typically for such analyses, cells are grown in the absence of serum 15,16 . We therefore verified that the cells of the adipogenic lineage proliferated and secreted the myogenic factors in the absence of serum. ...
Myogenic differentiation results in different cell type cooperation, but the molecules involved in the myogenic cell activation remain elusive. Here, we show that muscle-resident pre-adipocytes promote myogenic differentiation through the secretion of factors. Using proteomic and transcriptomic analyses, we identified that proliferative adipogenic lineage cells produce and secrete a key factor of the innate immune system, the complement C3. Cell culture experiments revealed that C3 promotes the differentiation of myogenic progenitors following internalisation of the immune molecule. These data demonstrate that the third component of the complement system, which is a pivotal factor in the immune response to pathogens, is also involved in the differentiation of myogenic progenitor cells.
... Skeletal muscle development is a highly complex cell biological process that is associated with considerable alterations in protein expression patterns. Substantial proteome-wide changes during the phenotypic conversions of myoblasts into post-mitotic myotubes were confirmed by MS studies [151]. Embryonic and adult myogenesis is regulated by a variety of factors, including the transcription factors PAX3 and PAX7 during the initial induction of mesodermal precursor cells and during regenerative processes following muscle injury. ...
The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins.
Poly(ADP-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.
Objectives
Idiopathic inflammatory myopathies (IIMs) are a group of heterogeneous autoimmune diseases. Intron retention (IR) serves as an important post-transcriptional and translational regulatory mechanism. This study aims to identify changes in IR profiles in IIM subtypes, investigating their influence on proteins and their correlations with clinical features.
Methods
RNA sequencing and liquid chromatography-tandem mass spectrometry were performed on muscle tissues obtained from 174 patients with IIM and 19 controls, following QC procedures. GTFtools and iREAD software were used for IR identification. An analysis of differentially expressed IRs (DEIs), exons and proteins was carried out using edgeR or DEP. Functional analysis was performed with clusterProfiler, and SPIRON was used to assess splicing factors.
Results
A total of 6783 IRs located in 3111 unique genes were identified in all IIM subtypes compared with controls. IIM subtype-specific DEIs were associated with the pathogenesis of respective IIM subtypes. Splicing factors YBX1 and HSPA2 exhibited the most changes in dermatomyositis and immune-mediated necrotising myopathy. Increased IR was associated with reduced protein expression. Some of the IIM-specific DEIs were correlated with clinical parameters (skin rash, MMT-8 scores and muscle enzymes) and muscle histopathological features (myofiber necrosis, regeneration and inflammation). IRs in IFIH1 and TRIM21 were strongly correlated with anti-MDA5+ antibody, while IRs in SRP14 were associated with anti-SRP+ antibody.
Conclusion
This study revealed distinct IRs and specific splicing factors associated with IIM subtypes, which might be contributing to the pathogenesis of IIM. We also emphasised the potential impact of IR on protein expression in IIM muscles.
Tamoxifen (TMX), a selective estrogen receptor modulator, is commonly used in the treatment of hormone-responsive cancers. However, the effects of TMX in anabolic tissues harboring estrogen receptors, such as skeletal muscle, are poorly understood. We report a tandem mass-tag approach to TMX-treated myogenesis in C2C12 cells, a well-characterized model of in vitro murine skeletal muscle differentiation. A longitudinal analysis of >10,000 proteins identified in untreated C2C12 myogenesis revealed a novel subset of 1,062 myogenically regulated proteins. These proteins clustered into five distinct longitudinal expression trends which significantly overlap those obtained in similar analyses performed in human myocytes. We document a specific functional enrichment for adiponectin-signaling unique to TMX-treated myogenesis, as well as a subset of 198 proteins that are differentially expressed in TMX-treated cells relative to controls at one or more stages of myogenesis, the majority of which were involved in steroid and lipid metabolism. Further analysis highlights metallothionein-1 as a novel target of TMX treatment at each stage of C2C12 myogenesis. Finally, we present a powerful, self-validating pipeline for analyzing the total proteomic response to in vitro treatment across every stage of muscle cell development which can be easily adapted to study the effects of other drugs on myogenesis.
Sensitivity and plasticity of insulin signaling and glucose uptake in skeletal muscle depends on determinants such as genetic variation and obesity. We collected muscle biopsies and isolated myoblasts from a multi-ethnic cohort of lean South Asians (N=10), lean Chinese (N=10), and obese Chinese (N=10), and analysed the proteome and phosphoproteome dynamics in terminally differentiated myotubes after a low-dose insulin stimulation (10nM at 0, 5, 30 min). The myotubes initially responded with increased abundance and phosphorylation level changes along the PI3K/AKT/mTOR axis, decreased abundance of translation initiation factors, and increased phosphorylation levels on proteins involved in mRNA processing at 5 min. After the acute response, protein abundance returned to baseline at 30 min, while phosphorylation changes persisted in proteins including AKT, RPS6 and AS160 (TBC1D4). A joint kinase-substrate statistical analysis revealed that protein abundance changes of AKT, PAK1 and CDK1 showed concordant phosphorylation changes in their respective substrates upon insulin stimulation. We also observed increased phosphorylation of some substrates uniquely in each group, particularly the substrates of CDKs showing stronger changes in South Asians than in Chinese. Pharmacological inhibition and siRNA knockdown of CDK1, a non-myogenic kinase, in terminally differentiated myotubes reduced glucose uptake and desensitized several phosphorylation-mediated signaling on protein translation initiation factors, IRS1, and AS160. Our data suggest that basal extramitotic activity of CDK1 is required for PI3K/AKT/mTORC1 signaling cascade and glucose uptake in insulin-stimulated myotubes. The data also provide a rich resource for studying the role of other kinases in the mechanism of insulin resistance in human myotubes.
The development of haematopoietic stem cells into mature erythrocytes – erythropoiesis – is a controlled process characterized by cellular reorganisation and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin (UB) system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the UB system is poorly understood. By measuring the proteomic landscape of in vitro human erythropoiesis models, we found dynamic differential expression of subunits of the CTLH E3 ubiquitin ligase complex that formed distinct maturation stage-dependent assemblies of structurally homologous RANBP9-and RANBP10-CTLH complexes. Moreover, protein abundance of CTLH’s cognate E2-conjugating enzyme UBE2H increased during terminal differentiation, which depended on catalytically active CTLH E3 complexes. CRISPR-Cas9 mediated inactivation of all CTLH E3 assemblies by targeting the catalytic subunit MAEA, or UBE2H , triggered spontaneous and accelerated maturation of erythroid progenitor cells including increased heme and haemoglobin synthesis. Thus, the orderly progression of human erythropoiesis is controlled by the assembly of distinct UBE2H-CTLH modules functioning at different developmental stages.
New findings:
What is the central question of this study? What is the impact of obesity-independent hyperlipidaemia on skeletal muscle stem cell function of ApoE-deficient (ApoE-/- ) mice? What is the main finding and its importance? Compromised muscle stem cell function accounts for the impaired muscle regeneration in hyperlipidaemic ApoE-/- mice. Importantly, impaired muscle regeneration is normalised by administration of platelet releasate.
Abstract:
Muscle satellite cells are important stem cells for skeletal muscle regeneration and repair after injury. ApoE-deficient mice, an established mouse model of hyperlipidaemia and atherosclerosis, show evidence of oxidative stress-induced lesions and fat infiltration in skeletal muscle followed by impaired repair after injury. However, the mechanisms underpinning attenuated muscle regeneration remain to be fully defined. Key to addressing the latter is to understand the properties of muscle stem cells from ApoE-deficient mice and their myogenic potential. Muscle stem cells from ApoE-deficient mice were cultured both ex vivo (on single fibres) and in vitro (primary myoblasts) and their myogenic capacity was determined. Skeletal muscle regeneration was studied on days 5 and 10 after cardiotoxin injury. ApoE-deficient muscle stem cells showed delayed activation and differentiation on single muscle fibres ex vivo. Impaired proliferation and differentiation profiles were also evident on isolated primary muscle stem cells in culture. ApoE-deficient mice displayed impaired skeletal muscle regeneration after acute injury in vivo. Administration of platelet releasate in ApoE-deficient mice reversed the deficits of muscle regeneration after acute injury to wild-type levels. These findings indicate that muscle stem cell myogenic potential is perturbed in skeletal muscle of a mouse model of hyperlipidaemia. We propose that platelet releasate could be a therapeutic intervention for conditions with associated myopathy such as peripheral arterial disease.
The C2C12 myoblast is a model that has been used extensively to study the process of skeletal muscle differentiation. Proteomics has advanced our understanding of skeletal muscle biology and also the differentiation process of skeletal muscle cells. However, there is still no comprehensive analysis of C2C12 myoblast proteomes, which is important for the understanding of key drivers for the differentiation of skeletal muscle cells. Here, we conducted multidimensional proteome profiling to get a comprehensive analysis of proteomes and PTMomes of C2C12 myoblasts with a TiSH strategy. A total of 8313 protein groups were identified, including 7827 protein groups from nonmodified peptides, 3803 phosphoproteins, and 977 formerly sialylated N-linked glycoproteins. Integrated analysis of proteomic and PTMomic data showed that almost all of the kinases and transcription factors in the muscle cell differentiation pathway were phosphorylated. Further analysis indicated that sialylation might play a role in the differentiation of C2C12 myoblasts. Further functional analysis demonstrated that C2C12 myoblasts showed a decreased level of sialylation during skeletal muscle cell differentiation. Inhibition of sialylation with the sialyltransferase inhibitor 3Fax-Neu5Ac resulted in the lower expression of MHC and suppression of myoblast fusion. In all, these results indicate that sialylation has an effect on the differentiation of skeletal muscle cells.
Background
Proteomic studies of the secretome of skeletal muscle cells can help us understand the processes that govern the synthesis, systemic interactions and organization of skeletal muscle and identify proteins that are involved in muscular adaptations to exercise, ageing and degeneration. In this systematic review, we aimed to summarize recent mass-spectrometry based proteomics discoveries on the secretome of skeletal muscle cells in response to disease, exercise or metabolic stress.
Methods
A literature search was performed in the Medline/Ovid and Scopus electronic bibliographic databases. Only papers reporting the analysis of the secretome by mass spectrometry were included.
Results
A total of 19 papers met the inclusion criteria for this systematic review. These papers included comparative analysis of differentially expressed proteins between healthy and unhealthy muscle cells and comparison of the secretome of skeletal muscle cells during myogenesis and after insulin stimulation or exercising. The proteins were separated into several categories and their differential secretion was compared. In total, 654 proteins were listed as being present in the secretome of muscle cells. Among them, 30 proteins were differentially regulated by physical exercise, 130 during myogenesis, 114 by dystrophin deficiency, 26 by muscle atrophy, 27 by insulin stimulation and finally 176 proteins secreted by insulin-resistant muscle cells.
Conclusions
This systematic review of the secretome of skeletal muscle cell in health and disease provides a comprehensive overview of the most regulated proteins in pathological or physiological conditions. These proteins might be therapeutic targets or biochemical markers of muscle diseases.
Comprehensive and accurate proteome profiling of skeletal muscle remains challenging owing to the large proteome dynamic range in this tissue and the increased sensitivity needed to detect low-abundant proteins. Sarcomeric and glycolytic enzymes are by far the most abundant proteins in muscle, masking detection and quantification of low-abundant proteins such as dystrophin, dystrophin-associated protein complex, cell signaling proteins, and transcription factors. About 5400 unique proteins have been identified so far in skeletal muscle using extensive pre-fractionation methods and mass spectrometry [1]. While this is good for cataloging the muscle proteome, pre-fractionation methods are often not compatible with quantification or comparative proteomics because of inherent technical variability from experiment to experiment leading to false and inaccurate quantification. To overcome these challenges, several stable isotope labeling strategies have been developed and tested in the past in different cell culture models and tissues. Typically, the two samples to be compared are tagged with heavy and light stable isotope-labeled moieties, respectively, either at the peptide level, after digestion of proteins with a protease (e.g., trypsin or endoproteinase Lys-C), or even at the cellular level before protein extraction and processing. This book chapter, we will focus on stable isotope labeling by amino acid in cell culture (SILAC), which is by far the most accurate method for quantitative proteomics in cell culture systems.
Skeletal muscle is the largest organ in the body by mass [1] and as such can affect many processes in addition to its obvious roles in locomotion and breathing. In these roles, diseases of skeletal muscle can have profound effects on human health. Because it also is sensitive to the effect of insulin to enhance glucose uptake, storage, and metabolism, it also is a key organ in insulin resistance and deranged glucose metabolism in obesity and type 2 diabetes mellitus [2], one of the most common chronic diseases in the world. The complex polygenic nature of this disease, reaching the limits of predictive power of knowledge gained by genome-wide association studies [3], and the profound genotype/environment interaction required to develop type 2 diabetes mellitus compel additional approaches to understanding pathogenesis of the disease. These approaches by necessity, therefore, must be able to deal with complexities of biological changes in tissues. Proteomics offers investigators such an approach. Skeletal muscle is the largest single contributor when insulin stimulates clearance of glucose from the blood in humans [4] and is one of the major regulators of body amino acid metabolism [5]. Because of these and other vitally important functions, human skeletal muscle has been a widely studied organ, with nearly 20,000 citations in PubMed as of June 2018. Moreover, although not as accessible as blood, skeletal muscle can be sampled readily by the use of percutaneous needle biopsies taken under local anesthesia. Because of the low rate of adverse events during these procedures and the lack of lasting effects [6], muscle biopsies can readily and ethically be performed not only in patients in need of diagnosis of disease but also in healthy control volunteers. This chapter will focus on studies of human skeletal muscle using mass spectrometry-based proteomics technologies. The primary focus will be on studies using biopsies to obtain skeletal muscle, but with selected references to studies using in vitro primary cultures of human myoblasts or differentiated myotubes. This chapter is not intended to be exhaustive, but rather to represent the array of areas which have proven to be amenable to mass spectrometry-based proteomics analysis in humans and for which such analyses have contributed in a major way to our base of knowledge.
Proteomic quantification at very high sensitivity is not achieved yet, even if they are a need to realize this quantification for the analysis of uncommon samples at a single cell level, or for the detection of low abundance protein. To improve this sensitivity, one way is to use a microscope able to detect single-molecule. In this optic, the first step to enable precise quantification is to measure the proportion of the proteome that is labeled by a fluorescent probe. When using strong denaturant conditions combined with a probe able to detect the amine of the protein, we are able to label up to 75% of the proteome from a cell lysate, with an increase in the labeling efficiency when the protein is bigger. The second step necessitates the protein separation by size in order to realize a proteome profile. Two technics were used for that, the microfluidic chip and the micro SDS-PAGE. The second one enables the possibility to scan the profile by microscopy, allowing the detection of up 10 ng of protein and then permits the analysis of only 100 cells. This sensitivity enables the differentiation of 4 different proteome profiles from cell lines originated from breast cancer, with a potential in the diagnostic of cancer cell from a smaller biopsy, allowing a less painful experience for the patient.
The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast which were differentiated into myotubes followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we analyzed changes in the relative abundance of 2,588 proteins, of which 387 were significantly regulated in myoblasts versus myotubes. Changes in protein expression generally reflected the drastic phenotypic conversion of the cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Since an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an exemplary fashion, we analyzed the actin-binding multi-adaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our dataset provides an invaluable resource for further identification of kinase-mediated changes in myofibrillar protein interactions, kinetics and mobility that will greatly advance our understanding of Z disc dynamics and signaling.
Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high-sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity.
© 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.
Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging due to highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art mass spectrometric (MS) workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compare to tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
Defining the transcriptional dynamics of a temporal process such as cell differentiation is challenging owing to the high variability in gene expression between individual cells. Time-series gene expression analyses of bulk cells have difficulty distinguishing early and late phases of a transcriptional cascade or identifying rare subpopulations of cells, and single-cell proteomic methods rely on a priori knowledge of key distinguishing markers. Here we describe Monocle, an unsupervised algorithm that increases the temporal resolution of transcriptome dynamics using single-cell RNA-Seq data collected at multiple time points. Applied to the differentiation of primary human myoblasts, Monocle revealed switch-like changes in expression of key regulatory factors, sequential waves of gene regulation, and expression of regulators that were not known to act in differentiation. We validated some of these predicted regulators in a loss-of function screen. Monocle can in principle be used to recover single-cell gene expression kinetics from a wide array of cellular processes, including differentiation, proliferation and oncogenic transformation.
CD13 is a multifunctional cell surface molecule that regulates inflammatory and angiogenic mechanisms in vitro, but its contribution to these processes in vivo or potential roles in stem cell biology remains unexplored. We investigated the impact of loss of CD13 on a model of ischemic skeletal muscle injury that involves angiogenesis, inflammation and stem cell mobilization. Consistent with its role as an inflammatory adhesion molecule, lack of CD13 altered myeloid trafficking in the injured muscle, resulting in cytokine profiles skewed toward a pro-healing environment. Despite this healing-favorable context, CD13(KO) animals showed significantly impaired limb perfusion with increased necrosis, fibrosis and lipid accumulation. Capillary density was correspondingly decreased, implicating CD13 in skeletal muscle angiogenesis. The number of CD45-/Sca1-/α7-integrin+/β1-integrin+ satellite cells was markedly diminished in injured CD13(KO) muscles and adhesion of isolated CD13(KO) satellite cells was impaired while their differentiation was accelerated. Bone marrow transplantation studies showed contributions from both host and donor cells to wound healing. Importantly, CD13 was co-expressed with Pax7 on isolated muscle-resident satellite cells. Finally, phosphorylated-FAK and ERK levels were reduced in injured CD13(KO) muscles, consistent with CD13 regulating satellite cell adhesion, potentially contributing to the maintenance and renewal of the satellite stem cell pool and facilitating skeletal muscle regeneration. Stem Cells 2013.
In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the
analysis of scientific images. We discuss the origins, challenges and solutions of these two programs,
and how their history can serve to advise and inform other software projects.
The fusion of myoblasts into multinucleate syncytia plays a fundamental role in muscle function, as it supports the formation of extended sarcomeric arrays, or myofibrils, within a large volume of cytoplasm. Principles learned from the study of myoblast fusion not only enhance our understanding of myogenesis, but also contribute to our perspectives on membrane fusion and cell-cell fusion in a wide array of model organisms and experimental systems. Recent studies have advanced our views of the cell biological processes and crucial proteins that drive myoblast fusion. Here, we provide an overview of myoblast fusion in three model systems that have contributed much to our understanding of these events: the Drosophila embryo; developing and regenerating mouse muscle; and cultured rodent muscle cells.
Skeletal muscle tissue contains slow as well as fast twitch muscle fibers that possess different metabolic and contractile properties. Although the distribution of individual proteins in fast and slow fibers has been investigated extensively, a comprehensive proteomic analysis, which is key for any systems biology approach to muscle tissues, is missing. Here, we compared the global protein levels and gene expression profiles of the predominantly slow soleus and fast extensor digitorum longus muscles using the principle of in vivo stable isotope labeling with amino acids based on a fully lysine-6 labeled SILAC-mouse. We identified 551 proteins with significant quantitative differences between slow soleus and fast extensor digitorum longus fibers out of >2000 quantified proteins, which greatly extends the repertoire of proteins differentially regulated between both muscle types. Most of the differentially regulated proteins mediate cellular contraction, ion homeostasis, glycolysis, and oxidation, which reflect the major functional differences between both muscle types. Comparison of proteomics and transcriptomics data uncovered the existence of fiber-type specific posttranscriptional regulatory mechanisms resulting in differential accumulation of Myosin-8 and α-protein kinase 3 proteins and mRNAs among others. Phosphoproteome analysis of soleus and extensor digitorum longus muscles identified 2573 phosphosites on 973 proteins including 1040 novel phosphosites. The in vivo stable isotope labeling with amino acids-mouse approach used in our study provides a comprehensive view into the protein networks that direct fiber-type specific functions and allows a detailed dissection of the molecular composition of slow and fast muscle tissues with unprecedented resolution.
SMART (Simple Modular Architecture Research Tool) is an online resource (http://smart.embl.de/) for the identification and annotation of protein domains and the analysis of protein domain architectures. SMART version
7 contains manually curated models for 1009 protein domains, 200 more than in the previous version. The current release introduces
several novel features and a streamlined user interface resulting in a faster and more comfortable workflow. The underlying
protein databases were greatly expanded, resulting in a 2-fold increase in number of annotated domains and features. The database
of completely sequenced genomes now includes 1133 species, compared to 630 in the previous release. Domain architecture analysis
results can now be exported and visualized through the iTOL phylogenetic tree viewer. ‘metaSMART’ was introduced as a novel
subresource dedicated to the exploration and analysis of domain architectures in various metagenomics data sets. An advanced
full text search engine was implemented, covering the complete annotations for SMART and Pfam domains, as well as the complete
set of protein descriptions, allowing users to quickly find relevant information.
Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies.
Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders.
The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice.
Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.
Protein ubiquitination is a dynamic reversible post-translational modification that plays a key role in the regulation of numerous cellular processes including signal transduction, endocytosis, cell cycle control, DNA repair and gene transcription. The conjugation of the small protein ubiquitin or chains of ubiquitin molecules of various types and lengths to targeted proteins is known to alter proteins' lifespan, localization and function and to modulate protein interactions. Despite its central importance in various aspects of cellular life and function there are only a limited number of reports investigating ubiquitination on a proteomic scale, mainly due to the inherited complexity and heterogeneity of ubiquitination. We describe here a quantitative proteomics strategy based on the specificity of ubiquitin binding domains (UBDs) and Stable Isotope Labeling by Amino acids in Cell culture (SILAC) for selectively decoding ubiquitination-driven processes involved in the regulation of cellular signaling networks. We applied this approach to characterize the temporal dynamics of ubiquitination events accompanying epidermal growth factor receptor (EGFR) signal transduction. We used recombinant UBDs derived from endocytic adaptor proteins for specific enrichment of ubiquitinated complexes from the EGFR network and subsequent quantitative analyses by high accuracy mass spectrometry. We show that the strategy is suitable for profiling the dynamics of ubiquitination occurring on individual proteins as well as ubiquitination-dependent events in signaling pathways. In addition to a detailed seven time-point profile of EGFR ubiquitination over 30 minutes of ligand stimulation, our data determined prominent involvement of Lysine-63 ubiquitin branching in EGF signaling. Furthermore, we found two centrosomal proteins, PCM1 and Azi1, to form a multi-protein complex with the ubiquitin E3 ligases MIB1 and WWP2 downstream of the EGFR, thereby revealing possible ubiquitination cross-talk between EGF signaling and centrosomal-dependent rearrangements of the microtubules. This is a general strategy that can be utilized to study the dynamics of other cellular systems and post-translational modifications.
Skeletal muscle fibres represent one of the most abundant cell types in mammals. Their highly specialised contractile and metabolic functions depend on a large number of membrane-associated proteins with very high molecular masses, proteins with extensive posttranslational modifications and components that exist in highly complex supramolecular structures. This makes it extremely difficult to perform conventional biochemical studies of potential changes in protein clusters during physiological adaptations or pathological processes.
Skeletal muscle proteomics attempts to establish the global identification and biochemical characterisation of all members of the muscle-associated protein complement. A considerable number of proteomic studies have employed large-scale separation techniques, such as high-resolution two-dimensional gel electrophoresis or liquid chromatography, and combined them with mass spectrometry as the method of choice for high-throughput protein identification. Muscle proteomics has been applied to the comprehensive biochemical profiling of developing, maturing and aging muscle, as well as the analysis of contractile tissues undergoing physiological adaptations seen in disuse atrophy, physical exercise and chronic muscle transformation. Biomedical investigations into proteome-wide alterations in skeletal muscle tissues were also used to establish novel biomarker signatures of neuromuscular disorders. Importantly, mass spectrometric studies have confirmed the enormous complexity of posttranslational modifications in skeletal muscle proteins.
This review critically examines the scientific impact of modern muscle proteomics and discusses its successful application for a better understanding of muscle biology, but also outlines its technical limitations and emerging techniques to establish new biomarker candidates.
The global analysis of protein composition, modifications, and dynamics are important goals in cell biology. Mass spectrometry (MS)-based proteomics has matured into an attractive technology for this purpose. Particularly, high resolution MS methods have been extremely successful for quantitative analysis of cellular and organellar proteomes. Rapid advances in all areas of the proteomic workflow, including sample preparation, MS, and computational analysis, should make the technology more easily available to a broad community and turn it into a staple methodology for cell biologists.
Satellite cells are quiescent cells located under the basal lamina of skeletal muscle fibers that contribute to muscle growth, maintenance, repair, and regeneration. Mouse satellite cells have been shown to be muscle stem cells that are able to regenerate muscle fibers and self-renew. As human skeletal muscle is also able to regenerate following injury, we assume that the human satellite cell is, like its murine equivalent, a muscle stem cell. In this review, we compare human and mouse satellite cells and highlight their similarities and differences. We discuss gaps in our knowledge of human satellite cells, compared with that of mouse satellite cells, and suggest ways in which we may advance studies on human satellite cells, particularly by finding new markers and attempting to re-create the human satellite cell niche in vitro.
During recent years, increased efforts have focused on elucidating the secretory function of skeletal muscle. Through secreted molecules, skeletal muscle affects local muscle biology in an auto/paracrine manner as well as having systemic effects on other tissues. Here we used a quantitative proteomics platform to investigate the factors secreted during the differentiation of murine C2C12 skeletal muscle cells. Using triple encoding stable isotope labeling by amino acids in cell culture, we compared the secretomes at three different time points of muscle differentiation and followed the dynamics of protein secretion. We identified and quantitatively analyzed 635 secreted proteins, including 35 growth factors, 40 cytokines, and 36 metallopeptidases. The extensive presence of these proteins that can act as potent signaling mediators to other cells and tissues strongly highlights the important role of the skeletal muscle as a prominent secretory organ. In addition to previously reported molecules, we identified many secreted proteins that have not previously been shown to be released from skeletal muscle cells nor shown to be differentially released during the process of myogenesis. We found 188 of these secreted proteins to be significantly regulated during the process of myogenesis. Comparative analyses of selected secreted proteins revealed little correlation between their mRNA and protein levels, indicating pronounced regulation by posttranscriptional mechanisms. Furthermore, analyses of the intracellular levels of members of the semaphorin family and their corresponding secretion dynamics demonstrated that the release of secreted proteins is tightly regulated by the secretory pathway, the stability of the protein, and/or the processing of secreted proteins. Finally, we provide 299 unique hydroxyproline sites mapping to 48 distinct secreted proteins and have discovered a novel hydroxyproline motif.
To investigate the temporal regulation of the DNA damage response, we applied quantitative mass spectrometry-based proteomics to measure site-specific phosphorylation changes of nuclear proteins after ionizing radiation. We profiled 5204 phosphorylation sites at five time points following DNA damage of which 594 sites on 209 proteins were observed to be regulated more than 2-fold. Of the 594 sites, 372 are novel phosphorylation sites primarily of nuclear origin. The 594 sites could be classified to distinct temporal profiles. Sites regulated shortly after radiation were enriched in the ataxia telangiectasia mutated (ATM) kinase SQ consensus sequence motif and a novel SXXQ motif. Importantly, in addition to induced phosphorylation, we identified a considerable group of sites that undergo DNA damage-induced dephosphorylation. Together, our data extend the number of known phosphorylation sites regulated by DNA damage, provides so far unprecedented temporal dissection of DNA damage-modified phosphorylation events, and elucidate the cross-talk between different types of post-translational modifications in the dynamic regulation of a multifaceted DNA damage response.
Defining the mechanisms governing myogenesis has advanced in recent years. Skeletal-muscle differentiation is a multi-step process controlled spatially and temporally by various factors at the transcription level. To explore those factors involved in myogenesis, stable isotope labeling with amino acids in cell culture (SILAC), coupled with high-accuracy mass spectrometry (LTQ-Orbitrap), was applied successfully. Rat L6 cell line is an excellent model system for studying muscle myogenesis in vitro. When mononucleate L6 myoblast cells reach confluence in culture plate, they could transform into multinucleate myotubes by serum starvation. By comparing protein expression of L6 myoblasts and terminally differentiated multinucleated myotubes, 1170 proteins were quantified and 379 proteins changed significantly in fully differentiated myotubes in contrast to myoblasts. These differentially expressed proteins are mainly involved in inter-or intracellular signaling, protein synthesis and degradation, protein folding, cell adhesion and extracellular matrix, cell structure and motility, metabolism, substance transportation, etc. These findings were supported by many previous studies on myogenic differentiation, of which many up-regulated proteins were found to be involved in promoting skeletal muscle differentiation for the first time in our study. In summary, our results provide new clues for understanding the mechanism of myogenesis.
Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations.
Biological experiments are most often performed with immortalized cell lines because they are readily available and can be expanded without limitation. However, cell lines may differ from the in vivo situation in important aspects. Here we introduce a straightforward methodology to compare cell lines to their cognate primary cells and to derive a comparative functional phenotype. We used SILAC (stable isotope labeling by amino acids in cell culture) for quantitative, mass spectrometry-based comparison of the hepatoma cell line Hepa1-6 with primary hepatocytes. The resulting quantitative proteome of 4,063 proteins had an asymmetric distribution, with many proteins down-regulated in the cell line. Bioinformatic analysis of the quantitative proteomics phenotypes revealed that Hepa1-6 cells were deficient in mitochondria, reflecting re-arrangement of metabolic pathways, drastically up-regulate cell cycle-associated functions and largely shut down drug metabolizing enzymes characteristic for the liver. This quantitative knowledge of changes provides an important basis to adapt cell lines to more closely resemble physiological conditions.
In many genomics projects, numerous lists containing biological identifiers are produced. Often it is useful to see the overlap between different lists, enabling researchers to quickly observe similarities and differences between the data sets they are analyzing. One of the most popular methods to visualize the overlap and differences between data sets is the Venn diagram: a diagram consisting of two or more circles in which each circle corresponds to a data set, and the overlap between the circles corresponds to the overlap between the data sets. Venn diagrams are especially useful when they are 'area-proportional' i.e. the sizes of the circles and the overlaps correspond to the sizes of the data sets. Currently there are no programs available that can create area-proportional Venn diagrams connected to a wide range of biological databases.
We designed a web application named BioVenn to summarize the overlap between two or three lists of identifiers, using area-proportional Venn diagrams. The user only needs to input these lists of identifiers in the textboxes and push the submit button. Parameters like colors and text size can be adjusted easily through the web interface. The position of the text can be adjusted by 'drag-and-drop' principle. The output Venn diagram can be shown as an SVG or PNG image embedded in the web application, or as a standalone SVG or PNG image. The latter option is useful for batch queries. Besides the Venn diagram, BioVenn outputs lists of identifiers for each of the resulting subsets. If an identifier is recognized as belonging to one of the supported biological databases, the output is linked to that database. Finally, BioVenn can map Affymetrix and EntrezGene identifiers to Ensembl genes.
BioVenn is an easy-to-use web application to generate area-proportional Venn diagrams from lists of biological identifiers. It supports a wide range of identifiers from the most used biological databases currently available. Its implementation on the World Wide Web makes it available for use on any computer with internet connection, independent of operating system and without the need to install programs locally. BioVenn is freely accessible at http://www.cmbi.ru.nl/cdd/biovenn/.
Extracellular matrix proteins produced by a mouse skeletal muscle cell line, G8-1, were isolated and characterized. Cultures were incubated with [35S]methionine or [3H]glycine and [3H]proline, and the labeled, substrate-attached proteins were obtained after cellular proteins were extracted by deoxycholate in neutral salt. The labeled matrix was analyzed by gel electrophoresis and fluorography before and after enzymatic digestion. Of the nine major bands present in the matrix, four were identified. Fibronectin and collagen were detected on the bases of their relative mobilities, differential labeling with 3H-versus 35S-labeled amino acids and their solubilization by protease free collagenase. High molecular weight material which was present in the matrix was also sensitive to collagenase and probably included cross-linked collagen and laminin. Proteins co-migrating with actin and myosin were also present in the extracellular matrix. These results are novel in that they demonstrate that the skeletal muscle phenotype, not contaminated with fibroblastic elements, is able to synthesize basal lamina-type macromolecules and incorporate them into an insoluble, extracellular matrix. Since this cell line is able to form functional synaptic contacts with neuronal cells, the influence of nerve on basal lamina production by muscle in vitro is possible.
Cadherins are a gene family encoding calcium-dependent cell adhesion proteins which are thought to act in the establishment and maintenance of tissue organization. M-cadherin, one member of the family, has been found in myogenic cells of somitic origin during embryogenesis and in the adult. These findings have suggested that M-cadherin is involved in the regulation of morphogenesis of skeletal muscle cells. Therefore, we investigated the function of M-cadherin in the fusion of myoblasts into myotubes (terminal differentiation) in cell culture. Furthermore, we tested whether M-cadherin might influence (a) the expression of troponin T, a typical marker of biochemical differentiation of skeletal muscle cells, and (b) withdrawal of myoblasts from the cell cycle (called terminal commitment). The studies were performed by using antagonistic peptides which correspond to sequences of the putative M-cadherin binding domain. Analogous peptides of N-cadherin have previously been shown to interfere functionally with the N-cadherin-mediated cell adhesion. In the presence of antagonistic M-cadherin peptides, the fusion of myoblasts into myotubes was inhibited. Analysis of troponin T revealed that it was downregulated at the protein level although its mRNA was still detectable. In addition, withdrawal from the cell cycle typical for terminal commitment of muscle cells was not complete in fusion-blocked myogenic cells. Finally, expression of M-cadherin antisense RNA reducing the expression of the endogenous M-cadherin protein interfered with the fusion process of myoblasts. Our data imply that M-cadherin-mediated myoblast interaction plays an important role in terminal differentiation of skeletal muscle cells.
Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
We discuss the upstream regulators of myogenesis that lead to the activation of myogenic determination genes and subsequent differentiation, focusing on the mouse model. Key upstream genes, such as Pax3 and Pax7, Six1 and Six4, or Pitx2, participate in gene regulatory networks at different sites of skeletal muscle formation. MicroRNAs also intervene, with emerging evidence for the role of other noncoding RNAs. Myogenic determination and subsequent differentiation depend on members of the MyoD family. We discuss new insights into mechanisms underlying the transcriptional activity of these factors.
The discovery of the transcription factor MyoD and its ability to induce muscle differentiation was the first demonstration of genetically programmed cell transdifferentiation. MyoD functions by activating a feed-forward circuit to regulate muscle gene expression. This requires binding to specific E-boxes throughout the genome, followed by recruitment of chromatin modifying complexes and transcription machinery. MyoD binding can be modified by both cooperative factors and inhibitors, including microRNAs that may serve as important developmental switches. Recent studies indicate that epigenetic regulation of MyoD binding sites is another important mechanism for controlling MyoD activity, which may ultimately limit its ability to induce transdifferentiation to cells with permissive epigenetic 'landscapes.'
Efficient muscle regeneration requires cross talk between multiple cell types via secreted signaling molecules. However, as yet there has been no comprehensive analysis of this secreted signaling network in order to understand how it regulates myogenesis in humans. Using integrated proteomic and genomic strategies, we show that human muscle cells release not only soluble secreted proteins through conventional secretory mechanisms but also complex protein and nucleic acid cargos via membrane microvesicle shedding. The soluble secretome of muscle cells contains 253 conventionally secreted signaling proteins, including 43 previously implicated in myogenesis, while others are known to modulate various cell types thus implying a much broader role for myoblasts in muscle remodeling. We also isolated and characterized two types of secreted membrane-derived vesicles: nanovesicles harboring typical exosomal features and larger, morphologically distinct, microvesicles. While they share some common features, their distinct protein and RNA cargos suggest independent functions in myogenesis. We further demonstrate that both types of microvesicles can dock and fuse with adjacent muscle cells but also deliver functional protein cargo. Thus, the intercellular signaling networks invoked during muscle differentiation and regeneration may employ conventional soluble signaling molecules acting in concert with muscle derived microvesicles delivering their cargos directly into target cells.
Regeneration of adult tissues depends on stem cells that are primed to enter a differentiation program, while remaining quiescent. How these two characteristics can be reconciled is exemplified by skeletal muscle in which the majority of quiescent satellite cells transcribe the myogenic determination gene Myf5, without activating the myogenic program. We show that Myf5 mRNA, together with microRNA-31, which regulates its translation, is sequestered in mRNP granules present in the quiescent satellite cell. In activated satellite cells, mRNP granules are dissociated, relative levels of miR-31 are reduced, and Myf5 protein accumulates, which initially requires translation, but not transcription. Conditions that promote the continued presence of mRNP granules delay the onset of myogenesis. Manipulation of miR-31 levels affects satellite cell differentiation ex vivo and muscle regeneration in vivo. We therefore propose a model in which posttranscriptional mechanisms hold quiescent stem cells poised to enter a tissue-specific differentiation program.
The nucleobindins, nucleobindin 1 (NUCB1) and nucleobindin 2 (NUCB2), are homologous multidomain calcium and DNA binding proteins. NUCB1 is a well-characterized Golgi protein found within the rat pituitary, liver and kidney with functions related to immunity, calcium homeostasis and G protein signaling. NUCB2 is found both in the hypothalamus and brain stem centers, as well as peripherally in the digestive tract. Renewed interest in the nucleobindins has been sparked by the recent discovery of nesfatin-1, an endocrine factor post-translationally processed from the N-terminal of NUCB2. Nesfatin-1 has quickly established itself as a novel regulator of appetite, insulin secretion, energy homeostasis and reproduction with important consequences to the etiology of metabolic diseases including diabetes and obesity. The discovery of nesfatin-1 and it endocrine functions attracted more attention to the nucleobindins that are already known to have important intracellular functions. From the sequence information available, it is possible that nucelobindins itself or nesfatin-1 like peptides within the NUCB1 could also elicit nesfatin-1-like biological functions. The research on nesfatin-1 in last 5years further adds to the importance of nucleobindins as potential endocrine precursors. This review aims to summarize some of the most recent findings on the functional significance of NUCB1, NUCB2, as well as encoded proteins and highlights the questions that remain unanswered.
Mammalian skeletal muscle comprises different fiber types, whose identity is first established during embryonic development by intrinsic myogenic control mechanisms and is later modulated by neural and hormonal factors. The relative proportion of the different fiber types varies strikingly between species, and in humans shows significant variability between individuals. Myosin heavy chain isoforms, whose complete inventory and expression pattern are now available, provide a useful marker for fiber types, both for the four major forms present in trunk and limb muscles and the minor forms present in head and neck muscles. However, muscle fiber diversity involves all functional muscle cell compartments, including membrane excitation, excitation-contraction coupling, contractile machinery, cytoskeleton scaffold, and energy supply systems. Variations within each compartment are limited by the need of matching fiber type properties between different compartments. Nerve activity is a major control mechanism of the fiber type profile, and multiple signaling pathways are implicated in activity-dependent changes of muscle fibers. The characterization of these pathways is raising increasing interest in clinical medicine, given the potentially beneficial effects of muscle fiber type switching in the prevention and treatment of metabolic diseases.
Skeletal muscle development, repair and function are dependent on highly coordinated expression of many genes. RNA-binding proteins are crucial determinants of gene expression in the health and disease of various tissues, including skeletal muscle. A variety of RNA-binding proteins are associated with a transcript during its life cycle and define the lifetime, cellular localization, processing and rate at which that transcript is translated and ultimately degraded. The focus of this review is to highlight the roles of the best-characterized RNA-binding proteins in muscle, including HuR, KSRP, CUGBP1, PABPN1, Lin-28 and TTP. Recent studies indicate key functions for these RNA-binding proteins in different aspects of muscle physiology. Understanding the role of specific RNA-binding proteins in skeletal muscle will provide insights not only into basic mechanisms regulating gene expression in muscle, but also into the etiology and pathology of muscle disease.
Cells anchor to substrates by binding to extracellular matrix (ECM). In addition to this anchoring function however, cell-ECM binding is a mechanism for cells to sense their surroundings and to communicate and coordinate behaviour amongst themselves. Several ECM molecules and their receptors play essential roles in muscle development and maintenance. Defects in these proteins are responsible for some of the most severe muscle dystrophies at every stage of life from neonates to adults. However, recent studies have also revealed a role of cell-ECM interactions at much earlier stages of development as skeletal muscle forms. Here we review which ECM molecules are present during the early phases of myogenesis, how myogenic cells interact with the ECM that surrounds them and the potential consequences of those interactions. We conclude that cell-ECM interactions play significant roles during all stages of skeletal muscle development in the embryo and suggest that this "extracellular matrix dimension" should be added to our conceptual network of factors contributing to skeletal myogenesis.
Muscle represents a large fraction of the human body mass. It is an extremely heterogeneous tissue featuring in its contractile structure various proportions of heavy- and light-chain slow type 1 and fast types 2A and 2X myosins, actins, tropomyosins, and troponin complexes as well as metabolic proteins (enzymes and most of the players of the so-called excitation-transcription coupling). Muscle is characterized by wide plasticity, i.e. capacity to adjust size and functional properties in response to endogenous and exogenous influences. Over the last decade, proteomics has become a crucial technique for the assessment of muscle at the molecular level and the investigation of its functional changes. Advantages and shortcomings of recent techniques for muscle proteome analysis are discussed. Data from differential proteomics applied to healthy individuals in normal and unusual environments (hypoxia and cold), in exercise, immobilization, aging and to patients with neuromuscular hereditary disorders (NMDs), inclusion body myositis and insulin resistance are summarized, critically discussed and, when required, compared with homologous data from pertinent animal models. The advantages as well as the limits of proteomics in view of the identification of new biomarkers are evaluated.
A key step in mass spectrometry (MS)-based proteomics is the identification of peptides in sequence databases by their fragmentation spectra. Here we describe Andromeda, a novel peptide search engine using a probabilistic scoring model. On proteome data, Andromeda performs as well as Mascot, a widely used commercial search engine, as judged by sensitivity and specificity analysis based on target decoy searches. Furthermore, it can handle data with arbitrarily high fragment mass accuracy, is able to assign and score complex patterns of post-translational modifications, such as highly phosphorylated peptides, and accommodates extremely large databases. The algorithms of Andromeda are provided. Andromeda can function independently or as an integrated search engine of the widely used MaxQuant computational proteomics platform and both are freely available at www.maxquant.org. The combination enables analysis of large data sets in a simple analysis workflow on a desktop computer. For searching individual spectra Andromeda is also accessible via a web server. We demonstrate the flexibility of the system by implementing the capability to identify cofragmented peptides, significantly improving the total number of identified peptides.
The plasticity of skeletal muscle allows the body to adapt to various physiological demands such as growth, exercise and tissue regeneration and repair. The secreted factors from muscle exert their action via auto-, para-, and endocrine mechanisms, thereby influencing the maintenance of total body homeostasis. In addition, the regulation of muscle proliferation, differentiation, and regeneration is often perturbed by inflammatory processes and is dependent on the pattern of expression of pro-inflammatory stimuli. Studies examining the cross talk between factors released by muscle and cytokines secreted by other tissues are still very limited. In order to comprehensively characterize the low abundant low molecular weight secreted proteins during the course of muscle differentiation we used a mass spectrometry-based proteomics strategy. The application of the triple encoding Stable Isotope Labeling by Amino acids in Cell culture (SILAC) method for quantitative analysis resulted in the identification and generation of quantitative profiles of 59 growth factors and cytokines, including 9 classical chemokines. The members of the CC chemokine family of proteins such as monocyte chemotactic proteins 1, 2, and 3 (MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7) showed a distinct pattern of secretion during differentiation. Further analysis using combinatorial RNA and protein approaches demonstrated that the MCPs are regulated via both post-transcriptional and post-translational mechanisms. Analyses of the autocrine function of all three MCPs reveal similar activation of downstream effectors in muscle cells. Finally, we show that the expression of the MCPs in skeletal muscle is also regulated by pro-inflammatory stimuli, indicating a much broader cross talk and interaction between inflammatory-dependent systems than previously anticipated.
Helicases are enzymes that can separate duplex oligonucleotides in a NTP-dependent fashion and are essential in all aspects of DNA and RNA metabolism. Amino acid sequence analysis identified several conserved sequence motifs in DNA and RNA helicases allowing their classification into 5 major groups (Super families SF1–SF5) [1]. DExD/H helicases share eight conserved sequence motifs, whereas the DEAD box helicase subgroup has an additional ninth conserved sequence motif [2]. These sequence motifs encompass an approximately 300–400 amino acid core region involved in ATP-binding/hydrolysis and RNA binding (Part 2: Figure 1A). Structural analyses of several DEAD-box proteins show this core region forms two RecA-like globular domains [2].
Figure 1
Human DEAD-box protein (DDX) posttranslational modifications and protein-protein interactions
Work in a variety of eukaryotes has identified the biological functions of many DEAD-box helicases. The genome of the yeast Saccharomyces cerevisiae encodes 25 DEAD-box proteins. Counterparts for each of these, along with 11 additional DEAD-box genes, are found in the human genome. Although some of the shared DEAD-box genes have similar functions in both humans and yeast, it is clear that several human DEAD-box proteins have acquired additional functions [3, 4]. How these functions are regulated within cellular or developmental contexts is less understood. The N-terminal and C-terminal sequences flanking the DEAD-box core regions are considerably more divergent among DEAD-box proteins and are thought to interact with RNA substrates or cofactors. Such interactions can thereby target and regulate their helicase activity or perform completely independent functions [3]. Although the structures of these divergent flanking sequences are largely unknown, a growing body of evidence suggests they are regulatory hot-spots for posttranslational modifications and protein-protein interactions (Figure 1). Despite DEAD-box helicase conservation throughout the animal kingdom, the most comprehensive data on their posttranslational regulation comes from the human DEAD-box helicase family (DDX proteins). For the purposes of this review, we shall focus primarily on the data concerning human DDX proteins and look at our current understanding on how posttranslational modifications and protein-protein interactions regulate DEAD-box protein functions.
Cold shock domain (CSD)-containing proteins have been found in all three domains of life and function in a variety of processes that are related, for the most part, to post-transcriptional gene regulation. The CSD is an ancient beta-barrel fold that serves to bind nucleic acids. The CSD is structurally and functionally similar to the S1 domain, a fold with otherwise unrelated primary sequence. The flexibility of the CSD/S1 domain for RNA recognition confers an enormous functional versatility to the proteins that contain them. This review summarizes the current knowledge on eukaryotic CSD/S1 domain-containing proteins with a special emphasis on UNR (upstream of N-ras), a member of this family with multiple copies of the CSD.
The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes for protein and organelle clearance. In skeletal muscle, both systems are under FoxO regulation and their excessive activation induces severe muscle loss. Although altered autophagy has been observed in various myopathies, the specific role of autophagy in skeletal muscle has not been determined by loss-of-function approaches. Here, we report that muscle-specific deletion of a crucial autophagy gene, Atg7, resulted in profound muscle atrophy and age-dependent decrease in force. Atg7 null muscles showed accumulation of abnormal mitochondria, sarcoplasmic reticulum distension, disorganization of sarcomere, and formation of aberrant concentric membranous structures. Autophagy inhibition exacerbated muscle loss during denervation and fasting. Thus, autophagy flux is important to preserve muscle mass and to maintain myofiber integrity. Our results suggest that inhibition/alteration of autophagy can contribute to myofiber degeneration and weakness in muscle disorders characterized by accumulation of abnormal mitochondria and inclusions.
RNA interference is a powerful way to study gene function and is frequently combined with microarray analysis. Here we introduce a similar technology at the protein level by simultaneously applying Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and RNA interference (RNAi) to Drosophila SL2 cells. After knockdown of ISWI, an ATP-hydrolyzing motor of different chromatin remodeling complexes, we obtained a quantitative proteome of more than 4,000 proteins. ISWI itself was reduced 10-fold as quantified by SILAC. Several hundred proteins were significantly regulated and clustered into distinct functional categories. Acf-1, a direct interaction partner of ISWI, is severely depleted at the protein, but not the transcript, level; this most likely results from reduced protein stability. We found little overall correlation between changes in the transcriptome and proteome with many protein changes unaccompanied by message changes. However, correlation was high for those mRNAs that changed significantly by microarray.
In order to test the diversification among satellite cells in man, satellite cells were isolated from human quadriceps and masseter muscles. The growth kinetics and morphological features of these cells were determined in vitro and the expression of the different myosin heavy (embryonic, fetal, fast, and slow) and light chain isoforms was analyzed. In all satellite cell cultures, only the four fast-type light chains (MLC1emb, MLC1F, MLC2F, and MLC3F) were synthesized and no slow myosin light chains were ever detected. However, we found that fused cultures of human satellite cells express both adult fast and slow myosin heavy chains (MHCs), in addition to embryonic and fetal isoforms. In order to determine if distinct fast and slow cell lineages could be detected among the satellite cells, a clonal analysis was carried out on both cell populations. This analysis was first carried out on clonal populations and was confirmed by the analysis of isolated clones. Double-labeling experiments confirmed that all myogenic clones which expressed fast MHC also coexpressed slow MHC. Therefore, we found no evidence for the existence of different fast and slow satellite cell lineages in human postnatal skeletal muscle.
Satellite cells were isolated from human quadriceps and masseter muscles and the phenotype of these cells examined in vitro. The expression of the different isoforms of the myosin heavy chains (embryonic, fetal, fast and slow) and light chain isoforms was used to assay myotube diversification. We found that fused cultures of human satellite cells express adult fast and slow MHCs in addition to the embryonic and fetal isoforms. Only the four fast light chains (MLC1emb, MLC1F, MLC2F and MLC3F) were synthesized. No slow MLCs were ever detected in these cultures. In order to determine if the human satellite cells were committed to distinct fast and slow myogenic lineages, a clonal analysis was carried out on both cell populations. All myogenic clones expressed fast and slow MHCs, suggesting that there is no evidence for different fast and slow satellite cell lineages in human skeletal muscle.
C2C12 myoblasts fuse to form multinucleated myotubes and express muscle specific proteins during differentiation. To elucidate developmental regulation of intracellular proteolytic systems, enzymatic activities, protein and mRNA levels of proteasomes (20S and 26S) and lysosomal cathepsins (B, L, and H) were examined. Myoblasts were differentiated fully to myotubes 6 days after starting differentiation. In this developmental process, the 26S proteasome activity decreased, while the 20S proteasome activity increased. Expression of proteasome subunits of 20S (RC2, RC8) and regulatory components of 26S (S4, S7) was down-regulated, though total protein levels of proteasomes showed no remarkable changes. On the other hand, enzymatic activities of cathepsins B and B + L increased in association with an increase of their transcriptional and translational levels. Expression of their specific endogenous inhibitor, cystatin beta, also increased. Maturation of the lysosomal proteolytic system was tightly linked to the differentiation process. These results suggested that signals for differentiation of myoblasts mediate a change of intracellular proteolytic systems, involving up-regulation of lysosomal cathepsins and down-regulation of proteasomes.
Myofibrillar proteins exist as multiple isoforms that derive from multigene (isogene) families. Additional isoforms, including products of tropomyosin, myosin light chain 1 fast, troponin T, titin, and nebulin genes, can be generated from the same gene through alternative splicing or use of alternative promoters. Myofibrillar protein isogenes are differentially expressed in various muscle types and fiber types but can be coexpressed within the same fiber. Isogenes are regulated by transcriptional and posttranscriptional mechanisms; however, specific regulatory sequences and transcriptional factors have not yet been identified. The pattern of isogene expression varies during muscle development in relation to the different origin of myogenic cells and primary/secondary fiber generations and is affected by neural and hormonal influences. The variable expression of myofibrillar protein isoforms is a major determinant of the contractile properties of skeletal muscle fibers. The diversity among isomyosins is related to the differences in the parameters of chemomechanical transduction as ATP hydrolysis rate and shortening velocity. Troponin and tropomyosin isoforms determine the variable sensitivity to calcium, whereas titin isoforms dictate the elastic properties of muscle fibers at rest. Both myosin and troponin isoforms contribute to the differences in the resistance to fatigue of muscle fibers.
In this study, we have evaluated the ability of human satellite cells isolated from subjects aged from 5 days to 86 years to proliferate in culture. Cells were cultivated until they became senescent. The number of cell divisions was calculated by counting the number of cells plated in culture compared to the number of cells removed following proliferation. Telomere length, which is known to decrease during each round of cell division, has been used to analyze the in vitro replicative capacity and in vivo replicative history of human satellite cells at isolation. The rate of telomere shortening in myonuclei of these muscle biopsies was also examined. Our results show that both proliferative capacity and telomere length of satellite cells decreases with age during the first two decades but that the myonuclei of human skeletal muscle are remarkably stable because telomere length in these myonuclei remains constant from birth to 86 years. The lack of shortening of mean terminal restriction fragments (TRF) in vivo confirms that skeletal muscle is a stable tissue with little nuclear turnover and therefore an ideal target for cell-mediated gene therapy. Moreover, our results show that it is important to consider donor age as a limiting factor to obtain an optimal number of cells.
Adult skeletal muscle has a remarkable ability to regenerate following myotrauma. Because adult myofibers are terminally differentiated, the regeneration of skeletal muscle is largely dependent on a small population of resident cells termed satellite cells. Although this population of cells was identified 40 years ago, little is known regarding the molecular phenotype or regulation of the satellite cell. The use of cell culture techniques and transgenic animal models has improved our understanding of this unique cell population; however, the capacity and potential of these cells remain ill-defined. This review will highlight the origin and unique markers of the satellite cell population, the regulation by growth factors, and the response to physiological and pathological stimuli. We conclude by highlighting the potential therapeutic uses of satellite cells and identifying future research goals for the study of satellite cell biology.
The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized.
We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar proteins were encoded by novel or uncharacterized genes, while the known proteins included several unexpected factors with no previously known nucleolar functions. MS analysis of nucleoli isolated from HeLa cells in which transcription had been inhibited showed that a subset of proteins was enriched. These data highlight the dynamic nature of the nucleolar proteome and show that proteins can either associate with nucleoli transiently or accumulate only under specific metabolic conditions.
This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.
We used expression arrays and chromatin immunoprecipitation assays to demonstrate that myogenesis consists of discrete subprograms of gene expression regulated by MyoD. Approximately 5% of assayed genes alter expression in a specific temporal sequence, and more than 1% are regulated by MyoD without the synthesis of additional transcription factors. MyoD regulates genes expressed at different times during myogenesis, and promoter-specific regulation of MyoD binding is a major mechanism of patterning gene expression. In addition, p38 kinase activity is necessary for the expression of a restricted subset of genes regulated by MyoD, but not for MyoD binding. The identification of distinct molecular mechanisms that regulate discrete subprograms of myogenesis should facilitate analyses of differentiation in normal development and disease.