Elucidation of the molecular alterations underlying aging-associated muscle wasting applying multi-omics

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 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.LUMC / Geneeskund

Discovering fiber type architecture over the entire muscle using data-driven analysis

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 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.Functional Genomics of Muscle, Nerve and Brain Disorder

The metabolic landscape in chronic rotator cuff tear reveals tissue-region-specific signatures

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 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.Functional Genomics of Muscle, Nerve and Brain Disorder

Elucidation of the molecular alterations underlying aging-associated muscle wasting applying multi-omics

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 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.</p

Elucidation of the molecular alterations underlying aging-associated muscle wasting applying multi-omics

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 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.</p

Divergent Molecular and Cellular Responses to Low and High-Dose Ionizing Radiation

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 (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).Cancer Signaling networks and Molecular Therapeutic

Deletion of the deISGylating enzyme USP18 enhances tumour cell antigenicity and radiosensitivity

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 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.Functional Genomics of Muscle, Nerve and Brain Disorder

Deletion of the deISGylating enzyme USP18 enhances tumour cell antigenicity and radiosensitivity

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 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

Precision medicine for more oxygen (P4O2)-study design and first results of the long COVID-19 extension

Introduction: the coronavirus disease 2019 (COVID-19) pandemic has led to the death of almost 7 million people, however, with a cumulative incidence of 0.76 billion, most people survive COVID-19. Several studies indicate that the acute phase of COVID-19 may be followed by persistent symptoms including fatigue, dyspnea, headache, musculoskeletal symptoms, and pulmonary functional-and radiological abnormalities. However, the impact of COVID-19 on long-term health outcomes remains to be elucidated. Aims: the Precision Medicine for more Oxygen (P4O2) consortium COVID-19 extension aims to identify long COVID patients that are at risk for developing chronic lung disease and furthermore, to identify treatable traits and innovative personalized therapeutic strategies for prevention and treatment. This study aims to describe the study design and first results of the P4O2 COVID-19 cohort. Methods: the P4O2 COVID-19 study is a prospective multicenter cohort study that includes nested personalized counseling intervention trial. Patients, aged 40-65 years, were recruited from outpatient post-COVID clinics from five hospitals in The Netherlands. During study visits at 3-6 and 12-18 months post-COVID-19, data from medical records, pulmonary function tests, chest computed tomography scans and biological samples were collected and questionnaires were administered. Furthermore, exposome data was collected at the patient's home and state-of-the-art imaging techniques as well as multi-omics analyses will be performed on collected data. Results: 95 long COVID patients were enrolled between May 2021 and September 2022. The current study showed persistence of clinical symptoms and signs of pulmonary function test/radiological abnormalities in post-COVID patients at 3-6 months post-COVID. The most commonly reported symptoms included respiratory symptoms (78.9%), neurological symptoms (68.4%) and fatigue (67.4%). Female sex and infection with the Delta, compared with the Beta, SARS-CoV-2 variant were significantly associated with more persisting symptom categories. Conclusions: the P4O2 COVID-19 study contributes to our understanding of the long-term health impacts of COVID-19. Furthermore, P4O2 COVID-19 can lead to the identification of different phenotypes of long COVID patients, for example those that are at risk for developing chronic lung disease. Understanding the mechanisms behind the different phenotypes and identifying these patients at an early stage can help to develop and optimize prevention and treatment strategies.</p