Duchenne and Becker muscular dystrophy (DMD and BMD, respectively) are characterized by progressive loss of muscle function combined with an increase in fat tissue in muscle. In some muscles this... Show moreDuchenne and Becker muscular dystrophy (DMD and BMD, respectively) are characterized by progressive loss of muscle function combined with an increase in fat tissue in muscle. In some muscles this process of ‘fat replacement’ starts earlier or progresses faster than in others and this occurs in a consistent temporal pattern. In addition, even within muscles fat replacement seems to progress heterogeneously. Evidently there are factors that vary between and within muscles which cause differential fat replacement of muscle tissue, but these are currently unknown. The identification of factors that influence this process of muscle degeneration could support the selection of current, and the development of future, therapies.The aim of part 1 of this thesis was to identify differences between muscles that are related to muscle fat replacement over time. These can provide therapeutical targets for, and support the design of, future clinical trials in DMD and BMD. Part 2 aimed to develop new approaches to study intramuscular differences in muscle physiology and mechanics in healthy muscle. These can be applied in neuromuscular disease in the future, and can be related to intramuscular differences in disease progression. Show less
Rios-Morales, M.; Vieira-Lara, M.A.; Homan, E.; Langelaar-Makkinje, M.; Gerding, A.; Li, Z.; ... ; Bakker, B.M. 2022
Skeletal muscle insulin resistance is a key pathophysiological process that precedes the development of type 2 diabetes. Whereas an overload of long-chain fatty acids can induce muscle insulin... Show moreSkeletal muscle insulin resistance is a key pathophysiological process that precedes the development of type 2 diabetes. Whereas an overload of long-chain fatty acids can induce muscle insulin resistance, butyrate, a short -chain fatty acid (SCFA) produced from dietary fibre fermentation, prevents it. This preventive role of butyrate has been attributed to histone deacetylase (HDAC)-mediated transcription regulation and activation of mito-chondrial fatty-acid oxidation. Here we address the interplay between butyrate and the long-chain fatty acid palmitate and investigate how transcription, signalling and metabolism are integrated to result in the butyrate -induced skeletal muscle metabolism remodelling. Butyrate enhanced insulin sensitivity in palmitate-treated, insulin-resistant C2C12 cells, as shown by elevated insulin receptor 1 (IRS1) and pAKT protein levels and Slc2a4 (GLUT4) mRNA, which led to a higher glycolytic capacity. Long-chain fatty-acid oxidation capacity and other functional respiration parameters were not affected. Butyrate did upregulate mitochondrial proteins involved in its own oxidation, as well as concentrations of butyrylcarnitine and hydroyxybutyrylcarnitine. By knocking down the gene encoding medium-chain 3-ketoacyl-CoA thiolase (MCKAT, Acaa2), butyrate oxidation was inhibited, which amplified the effects of the SCFA on insulin sensitivity and glycolysis. This response was associated with enhanced HDAC inhibition, based on histone 3 acetylation levels. Butyrate enhances insulin sensitivity and induces glycolysis, without the requirement of upregulated long-chain fatty acid oxidation. Butyrate catabolism functions as an escape valve that attenuates HDAC inhibition. Thus, inhibition of butyrate oxidation indirectly prevents insulin resistance and stimulates glycolytic flux in myotubes treated with butyrate, most likely via an HDAC-dependent mechanism. Show less
Eggelbusch, M.; Shi, A.D.; Broeksma, B.C.; Vazquez-Cruz, M.; Soares, M.N.; Wit, G.M.J. de; ... ; Wust, R.C.I. 2022
Background Systemic inflammation is associated with skeletal muscle atrophy and metabolic dysfunction. Although the nucleotide-binding oligomerization domain-like receptor family pyrin domain... Show moreBackground Systemic inflammation is associated with skeletal muscle atrophy and metabolic dysfunction. Although the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cytokine production in immune cells, its role in skeletal muscle is poorly understood. Here, we studied the link between inflammation, NLRP3, muscle morphology, and metabolism in in vitro cultured C2C12 myotubes, independent of immune cell involvement.Methods Differentiated C2C12 myotubes were treated with lipopolysaccharide (LPS; 0, 10, and 100-200 ng/mL) to induce activation of the NLRP3 inflammasome with and without MCC950, a pharmacological inhibitor of NLRP3-induced IL-1 ss production. We assessed markers of the NLRP3 inflammasome, cell diameter, reactive oxygen species, and mitochondrial function.Results NLRP3 gene expression and protein concentrations increased in a time-dependent and dose-dependent manner. Intracellular IL-1 ss concentration significantly increased (P < 0.0001), but significantly less with MCC950 (P = 0.03), suggestive of moderate activation of the NLRP3 inflammasome in cultured myotubes upon LPS stimulation. LPS suppressed myotube growth after 24 h (P = 0.03), and myotubes remained smaller up to 72 h (P = 0.0009). Exposure of myotubes to IL-1 ss caused similar alterations in cell morphology, and MCC950 mitigated these LPS-induced differences in cell diameter. NLRP3 appeared to co-localize with mitochondria, more so upon exposure to LPS. Mitochondrial reactive oxygen species were higher after LPS (P = 0.03), but not after addition of MCC950. Myotubes had higher glycolytic rates, and mitochondria were more fragmented upon LPS exposure, which was not altered by MCC950 supplementation.Conclusions LPS- induced activation of the NLRP3 inflammasome in cultured myotubes contributes to morphological and metabolic alterations, likely due to its mitochondrial association. Show less
Identifying genes involved in functional differences between similar tissues from expression profiles is challenging, because the expected differences in expression levels are small. To exemplify... Show moreIdentifying genes involved in functional differences between similar tissues from expression profiles is challenging, because the expected differences in expression levels are small. To exemplify this challenge, we studied the expression profiles of two skeletal muscles, deltoid and biceps, in healthy individuals. We provide a series of guides and recommendations for the analysis of this type of studies. These include how to account for batch effects and inter-individual differences to optimize the detection of gene signatures associated with tissue function. We provide guidance on the selection of optimal settings for constructing gene co-expression networks through parameter sweeps of settings and calculation of the overlap with an established knowledge network. Our main recommendation is to use a combination of the data-driven approaches, such as differential gene expression analysis and gene co-expression network analysis, and hypothesis-driven approaches, such as gene set connectivity analysis. Accordingly, we detected differences in metabolic gene expression between deltoid and biceps that were supported by both data- and hypothesis-driven approaches. Finally, we provide a bioinformatic framework that support the biological interpretation of expression profiles from related tissues from this combination of approaches, which is available at github.com/tabbassidaloii/AnalysisFrameworkSimilarTissues. Show less
Hiller, M.; Geissler, M.; Janssen, G.; Veelen, P. van; Aartsma-Rus, A.; Spitali, P. 2020
Muscle formation is a coordinated process driven by extensive gene expression changes where single cells fuse together to form multinucleated muscle fibers. Newly synthesized mRNAs are then... Show moreMuscle formation is a coordinated process driven by extensive gene expression changes where single cells fuse together to form multinucleated muscle fibers. Newly synthesized mRNAs are then regulated by RNA binding proteins (RBPs), affecting post-transcriptional transcript metabolism. Here, we determined how large-scale gene expression changes affect the catalog of RBPs by studying proliferating and differentiated muscle cells in healthy and dystrophic conditions. Transcriptomic analysis showed that the expression of more than 7000 genes was affected during myogenesis. We identified 769 RBPs, of which 294 were muscle-specific and 49 were uniquely shared with cardiomyocytes. A subset of 32 RBPs (half of which were muscle-specific) was found to be preferentially associated with target mRNAs in either myoblasts (MBs) or myotubes (MTs). A large proportion of catalytic proteins were bound to mRNAs even though they lack classical RNA binding domains. Finally, we showed how the identification of cell-specific RBPs enabled the identification of biomarkers that can separate healthy individuals from dystrophic patients. Our data show how interactome data can shed light on new basic RNA biology as well as provide cell-specific data that can be used for diagnostic purposes. Show less
In this thesis we examined several effects of fasting in lean and obese individuals. As expected, both the hormonal response as well as the metabolic shift from glucose towards lipid oxidation was... Show moreIn this thesis we examined several effects of fasting in lean and obese individuals. As expected, both the hormonal response as well as the metabolic shift from glucose towards lipid oxidation was impaired in obese individuals. At baseline, mitochondrial protein content in skeletal muscle of obese subjects was significantly reduced compared to lean individuals. We assessed the neuronal response to fasting by fMRI scanning. We found a different neuronal response to fasting between lean and obese individuals in terms of functional connectivity between the hypothalamus and respectively the dACC and insula. Since these regions are part of the saliency network, these differences may reflect distinct perception of calorie imbalance between lean and obese subjects. The effects of fasting on sympathetic tone (estimated by heart rate variability) were studied. Our data suggests that fasting decreases sympathetic tone in lean subjects, whereas it increases sympathetic activity in obese individuals. Weight loss in obese individuals increased HRV parameters that reflect the postprandial sympathetic tone. Finally, we studied the effects of fasting in the presence and absence of food-odors since this has been shown to reduce the fasting-induced increase in life span in fruit flies __ no differences were found in our human subjects. Show less
Pedro, G.S.; Muhlmeister, M.; Seip, R.; Kaijzel, E.; Lowik, C.; Bohmer, M.; ... ; Grull, H. 2014
Sarcopenia in old age has been associated with a higher mortality, poor physical functioning, poor outcome of surgery and higher drug toxicity. There is no general consensus on the definition of... Show moreSarcopenia in old age has been associated with a higher mortality, poor physical functioning, poor outcome of surgery and higher drug toxicity. There is no general consensus on the definition of sarcopenia. The aim of the research presented in this thesis was to assess the implications of the use of different diagnostic criteria for sarcopenia, and to define the most accurate criteria for sarcopenia. Currently used diagnostic criteria for sarcopenia can be divided into criteria based on (1) low muscle mass, (2) low muscle strength, and (3) low walking speed. This thesis describes how muscle mass can be further divided into relative muscle mass and absolute muscle mass. A higher body or fat mass is associated with a lower relative muscle mass and with a higher absolute muscle mass. Higher relative muscle mass at old age is associated with better physical performance and with less insulin resistance. It is suggested to reserve the term sarcopenia to describe a low muscle mass and dynapenia to describe a low muscle strength. Most importantly, this research illustrates that it is impossible to compare studies about sarcopenia in scientific literature due to the use of different diagnostic criteria for sarcopenia. Show less
Limb Girdle Muscular Dystrophy (LGMD) is a rare progressive heterogeneous disorder that can be caused by mutations in at least 21 different genes. These genes are often widely expressed and encode... Show moreLimb Girdle Muscular Dystrophy (LGMD) is a rare progressive heterogeneous disorder that can be caused by mutations in at least 21 different genes. These genes are often widely expressed and encode proteins with highly differing functions. And yet mutations in all of them give rise to a similar clinical presentation: adult onset muscle weakness, with muscles of the pelvic and shoulder girdle as predominantly affected muscle groups. This thesis explores a potential molecular mechanism that unifies the different genetic defects, which individually can cause a limb girdle muscular dystrophy, with a focus on LGMD2A and 2B. Show less