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
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
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
Nonsense mutations in the gene encoding dystrophin cause Duchenne muscular dystrophy (DMD), a lethal and debilitating neuromuscular disorder. Dystrophin is an important muscle structural protein... Show moreNonsense mutations in the gene encoding dystrophin cause Duchenne muscular dystrophy (DMD), a lethal and debilitating neuromuscular disorder. Dystrophin is an important muscle structural protein that protects muscle membrane from contraction-induced damage. Therefore, in the absence of dystrophin, the integrity of muscle fibers will be compromised and severe degeneration will take place. When the regeneration process mediated by satellite cells can no longer compensate, muscle fibers is eventually replaced by connective or fibrotic tissue, leading to the loss of muscle function. Multiple stages in DMD pathology are associated with the Transforming Growth Factor (TGF)-_ signaling pathway (Chapter 1). The TGF-_ superfamily consists of more than 30 secreted proteins including TGF-_, bone morphogenetic protein (BMP), activin/inhibins and growth and differentiation factor (GDF). These proteins regulate many biological processes, such as cell growth and differentiation, and maintain homeostasis during development and in multiple adult tissues. To elicit these diverse physiological responses, a fairly simple and yet powerful signaling pathway is utilized by the TGF-_ family members. The basic signaling engine consists of two receptor serine/threonine kinases, termed receptor types I and II, and intracellular Smad proteins. The ligand assembles a receptor complex that activates Smad proteins, which will assemble multisubunit complexes that regulate transcription. Two general steps thus actually suffice to carry the TGF-_ stimuli to target genes. How can such a simple system mediate a variety of cell-specific gene response? It is now apparent that TGF-_ signaling pathways have equally important extracellular and intracellular control mechanism. This includes myostatin (GDF-8), one of the members that is highly expressed in skeletal muscle. In addition to being a negative regulator of myoblast differentiation, myostatin also plays role in adipogenesis, skeletal muscle fibrosis and myometrial cell proliferation. Genetic mutation of myostatin leads to a remarkable increase of muscle mass, but, as myostatin is found in the circulation, effects on other tissues are somehow expected. We hypothesized that such remarkable effects of myostatin in the muscle are controlled by a unique modulatory mechanism. Indeed, we found that myostatin signaling in myogenic and non myogenic cells are conferred by different utilization of type I receptors, which are also termed activin receptor-like kinases (ALKs), and co-receptor (Chapter 2). In myogenic cells, myostatin signaling is dependent on activin receptor-like kinase-4 (ALK4), whereas ALK5 is utilized in non myogenic cells. Furthermore, we found that the ALK4-dependent myostatin signaling in muscle is largely conferred by a membrane-associated co-receptor Cripto, which is predominantly expressed in myogenic cells but absent in non muscle cells. Moreover, Cripto has different influences on TGF-_ family members that play a role in muscle, i.e. myostatin, activin and TGF-_. As such, Cripto may also be an interesting therapeutic target to follow up in the future. As DMD is caused by the lack of dystrophin, one strategy is to bring back dystrophin in the dystrophic muscle. Antisense oligonucleotide (AON)-mediated exon skipping has been used to reframe the mutated DMD gene and restore dystrophin protein synthesis. It will, however, be less effective in the later stage of the disease where fibrosis is already extensive. This thesis explores the possibility of using exon skipping AONs to inhibit several components of the TGF-_ family signaling and blunt their inhibitory effects on muscle regeneration and fibrosis. In Chapter 3, we first used AONs to functionally knockdown myostatin expression. They efficiently downregulate myostatin in vitro, but induce only subtle exon skipping in vivo. Nevertheless, in a relatively straightforward manner, we were able to combine myostatin and dystrophin AONs and induce exon skipping of both genes without functional interference. This provides a conceptual foundation for a combinatorial therapeutic approach, which targets the primary genetic defect and attempts to improve muscle quality. We further sought to use AON to functionally knockdown myostatin and/or TGF-_ receptors ALK4 and/or ALK5 (Chapter 4). This strategy allowed us to target the activity of a broader spectrum of TGF-_ members, including but not limited to myostatin. Administration in dystrophic mice reduces fibrosis in the diaphragm, which is known to be the most affected muscle. Interestingly, combination of both ALK4 and ALK5 inhibition induces most pronounced effects. The beneficial response after targeting ALK4 or ALK5 separately demonstrates the involvement of TGF-_ and activin in DMD pathology. Overall, in addition to its therapeutic potential, the AON-mediated exon skipping approach also enables the dissection of the roles of TGF-_ family members in muscle regeneration and fibrosis, and potentially other aspects of DMD pathology. In summary, this thesis discusses how the inhibition of several members of the TGF-_ signaling pathway has been implicated in ameliorating DMD pathology. Furthermore, it also increases the awareness that more knowledge on how these family members actually play role in (dystrophic) muscle may still be needed. Finally, alteration of TGF-_ signaling components is involved in various diseases with multilayered pathophysiology, including but not limited to other neuromuscular disorders. Thus, the use of AONs has potential therapeutic value for other TGF-_-related disorders and is also important research tools to study the effect of modulation of TGF-_ receptor family members in the different facets of these diseases. 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
