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
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
