Skeletal muscles are highly resilient due to the capacity to regenerate damaged muscle tissue. Despite the regenerative capacity, pathological conditions can lead to reduced muscle mass and... Show moreSkeletal muscles are highly resilient due to the capacity to regenerate damaged muscle tissue. Despite the regenerative capacity, pathological conditions can lead to reduced muscle mass and strength, also known as muscle wasting. Muscle wasting, seen in pathological conditions, is often progressive and concomitant with excessive or prolonged inflammation, fibrosis and fat infiltration. Aging is a risk factor for a large number of diseases that can result in muscle wasting. The prevalence of sarcopenia, an age-associated decline in skeletal muscle mass and strength in elderly, surges as life expectancy increases. In combination with muscle wasting caused by neuromuscular disorders, the burden of muscle wasting on society is significant. Gaining a better understanding of the cellular and molecular processes underlying muscle wasting conditions is critical for creating better treatments. Aging and thereby age-associated muscle wasting are multifactorial involving changes in different tissues and multiple cell systems. This thesis demonstrates how different omics approaches, like transcriptomics, proteomics and metabolomics, can elucidate the complexity of those molecular mechanisms and cellular processes involved in muscle wasting conditions. Employing a combination of different omics-techniques (multi-omics) specifically proved powerful and will continue to illuminate impaired mechanisms and consequently help to battle muscle wasting. Show less
The ubiquitin proteasomal system is a critical regulator of muscle physiology, and impaired UPS is key in many muscle pathologies. Yet, little is known about the function of deubiquitinating... Show moreThe ubiquitin proteasomal system is a critical regulator of muscle physiology, and impaired UPS is key in many muscle pathologies. Yet, little is known about the function of deubiquitinating enzymes (DUBs) in the muscle cell context. We performed a genetic screen to identify DUBs as potential regulators of muscle cell differentiation. Surprisingly, we observed that the depletion of ubiquitin-specific protease 18 (USP18) affected the differentiation of muscle cells. USP18 depletion first stimulated differentiation initiation. Later, during differentiation, the absence of USP18 expression abrogated myotube maintenance. USP18 enzymatic function typically attenuates the immune response by removing interferon-stimulated gene 15 (ISG15) from protein substrates. However, in muscle cells, we found that USP18, predominantly nuclear, regulates differentiation independent of ISG15 and the ISG response. Exploring the pattern of RNA expression profiles and protein networks whose levels depend on USP18 expression, we found that differentiation initiation was concomitant with reduced expression of the cell-cycle gene network and altered expression of myogenic transcription (co) factors. We show that USP18 depletion altered the calcium channel gene network, resulting in reduced calcium flux in myotubes. Additionally, we show that reduced expression of sarcomeric proteins in the USP18 proteome was consistent with reduced contractile force in an engineered muscle model. Our results revealed nuclear USP18 as a critical regulator of differentiation initiation and maintenance, independent of ISG15 and its role in the ISG response. Show less
Cancer risk after ionizing radiation (IR) is assumed to be linear with the dose; however, for low doses, definite evidence is lacking. Here, using temporal multi-omic systems analyses after a low ... Show moreCancer risk after ionizing radiation (IR) is assumed to be linear with the dose; however, for low doses, definite evidence is lacking. Here, using temporal multi-omic systems analyses after a low (LD; 0.1 Gy) or a high (HD; 1 Gy) dose of X-rays, we show that, although the DNA damage response (DDR) displayed dose proportionality, many other molecular and cellular responses did not. Phosphoproteomics uncovered a novel mode of phospho-signaling via S12-PPP1R7, and large-scale dephosphorylation events that regulate mitotic exit control in undamaged cells and the G2/M checkpoint upon IR in a dose-dependent manner. The phosphoproteomics of irradiated DNA double-strand breaks (DSBs) repair-deficient cells unveiled extended phospho-signaling duration in either a dose-dependent (DDR signaling) or independent (mTOR-ERK-MAPK signaling) manner without affecting signal magnitude. Nascent transcriptomics revealed the transcriptional activation of genes involved in NRF2-regulated antioxidant defense, redox-sensitive ERK-MAPK signaling, glycolysis and mitochondrial function after LD, suggesting a prominent role for reactive oxygen species (ROS) in molecular and cellular responses to LD exposure, whereas DDR genes were prominently activated after HD. However, how and to what extent the observed dose-dependent differences in molecular and cellular responses may impact cancer development remain unclear, as the induction of chromosomal damage was found to be dose-proportional (10-200 mGy). Show less
Background Degeneration of shoulder muscle tissues often result in tearing, causing pain, disability and loss of independence. Differential muscle involvement patterns have been reported in tears... Show moreBackground Degeneration of shoulder muscle tissues often result in tearing, causing pain, disability and loss of independence. Differential muscle involvement patterns have been reported in tears of shoulder muscles, yet the molecules involved in this pathology are poorly understood. The spatial distribution of biomolecules across the affected tissue can be accurately obtained with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The goal of this pilot study was to decipher the metabolic landscape across shoulder muscle tissues and to identify signatures of degenerated muscles in chronic conditions. Methods Paired biopsies of two rotator cuff muscles, torn infraspinatus and intact teres minor, together with an intact shoulder muscle, the deltoid, were collected during an open tendon transfer surgery. Five patients, average age 65.2 +/- 3.8 years, were selected for spatial metabolic profiling using high-spatial resolution (MALDI-TOF) and high-mass resolution (MALDI-FTICR) MSI in negative or positive ion mode. Metabolic signatures were identified using data-driven analysis. Verifications of spatial localization for selected metabolic signatures were carried out using antibody immunohistology. Results Data-driven analysis revealed major metabolic differences between intact and degenerated regions across all muscles. The area of degenerated regions, encompassed of fat, inflammation and fibrosis, significantly increased in both rotator cuff muscles, teres minor (27.9%) and infraspinatus (22.8%), compared with the deltoid (8.7%). The intact regions were characterized by 49 features, among which lipids were recognized. Several of the identified lipids were specifically enriched in certain myofiber types. Degenerated regions were specifically marked by the presence of 37 features. Heme was the most abundant metabolite in degenerated regions, whereas Heme oxygenase-1 (HO-1), which catabolizes heme, was found in intact regions. Higher HO-1 levels correlated with lower heme accumulation. Conclusions Degenerated regions are distinguished from intact regions by their metabolome profile. A muscle-specific metabolome profile was not identified. The area of tissue degeneration significantly differs between the three examined muscles. Higher HO-1 levels in intact regions concurred with lower heme levels in degenerated regions. Moreover, HO-1 levels discriminated between dysfunctional and functional rotator cuff muscles. Additionally, the enrichment of specific lipids in certain myofiber types suggests that lipid metabolism differs between myofiber types. The signature metabolites can open options to develop personalized treatments for chronic shoulder muscles degeneration. Show less
Bindellini, D.; Voortman, L.M.; Olie, C.S.; Putten, M. van; Akker, E. van den; Raz, V. 2021
Skeletal muscle function is inferred from the spatial arrangement of muscle fiber architecture, which corresponds to myofiber molecular and metabolic features. Myofiber features are often... Show moreSkeletal muscle function is inferred from the spatial arrangement of muscle fiber architecture, which corresponds to myofiber molecular and metabolic features. Myofiber features are often determined using immunofluorescence on a local sampling, typically obtained from a median region. This median region is assumed to represent the entire muscle. However, it remains largely unknown to what extent this local sampling represents the entire muscle. We present a pipeline to study the architecture of muscle fiber features over the entire muscle, including sectioning, staining, imaging to image quantification and data-driven analysis with Myofiber type were identified by the expression of myosin heavy chain (MyHC) isoforms, representing contraction properties. We reconstructed muscle architecture from consecutive cross-sections stained for laminin and MyHC isoforms. Examining the entire muscle using consecutive cross-sections is extremely laborious, we provide consideration to reduce the dataset without loosing spatial information. Data-driven analysis with over 150,000 myofibers showed spatial variations in myofiber geometric features, myofiber type, and the distribution of neuromuscular junctions over the entire muscle. We present a workflow to study histological changes over the entire muscle using high-throughput imaging, image quantification, and data-driven analysis. Our results suggest that asymmetric spatial distribution of these features over the entire muscle could impact muscle function. Therefore, instead of a single sampling from a median region, representative regions covering the entire muscle should be investigated in future studies. Show less
Background Interferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often... Show moreBackground Interferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often deregulated in cancer. The deubiquitylating enzyme USP18 is a major negative regulator of the IFN signalling cascade and is the predominant human protease that cleaves ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. Methods In this study, using advanced proteomic techniques, we have significantly expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line. USP18-dependent effects were explored further in CML and colorectal carcinoma cellular models. Results Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. Conclusions Our results provide strong evidence for USP18 in regulating antigenicity and radiosensitivity, highlighting its potential as a cancer target. Show less
Olie, C.S.; Wal, E. van der; Domagoj, C.; Maton, L.; Greef, J.C. de; Lin, I.H.; ... ; Raz, V. 2020
Muscle wasting and atrophy are regulated by multiple molecular processes, including mRNA processing. Reduced levels of the polyadenylation binding protein nucleus 1 (PABPN1), a multifactorial... Show moreMuscle wasting and atrophy are regulated by multiple molecular processes, including mRNA processing. Reduced levels of the polyadenylation binding protein nucleus 1 (PABPN1), a multifactorial regulator of mRNA processing, cause muscle atrophy. A proteomic study in muscles with reduced PABPN1 levels suggested dysregulation of sarcomeric and cytoskeletal proteins. Here we investigated the hypothesis that reduced PABPN1 levels lead to an aberrant organization of the cytoskeleton. MURC, a plasma membrane-associated protein, was found to be more abundant in muscles with reduced PABPN1 levels, and it was found to be expressed at regions showing regeneration. A polarized cytoskeletal organization is typical for muscle cells, but muscle cells with reduced PABPN1 levels (named as shPAB) were characterized by a disorganized cytoskeleton that lacked polarization. Moreover, cell mechanical features and myogenic differentiation were significantly reduced in shPAB cells. Importantly, restoring cytoskeletal stability, by actin overexpression, was beneficial for myogenesis, expression of sarcomeric proteins and proper localization of MURC in shPAB cell cultures and in shPAB muscle bundle. We suggest that poor cytoskeletal mechanical features are caused by altered expression levels of cytoskeletal proteins and contribute to muscle wasting and atrophy. Show less
Olie, C.S.; Riaz, M.; Konietzny, R.; Charles, P.D.; Pinto-Fernandez, A.; Kielbasa, S.M.; ... ; Raz, V. 2019
