Browsing by Subject "Myopathies, Nemaline"
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Item Discovery of New Regulatory Proteins and Mechanisms in Muscle Biology and Disease(2014-06-09) Garg, Ankit; Hill, Joseph A.; Olson, Eric N.; MacDonald, Raymond J.; Stull, JamesIn an effort to discover new regulators of muscle function, we identified a novel muscle-specific protein, Klhl40. Genetic deletion of Klhl40 in mice results in a nemaline myopathy-like phenotype with disruption of sarcomere function causing neonatal lethality. Nemaline myopathy (NM) typically results from sarcomere thin filament dysfunction, but the molecular function of Klhl40 is not known. We found that Klhl40 binds to two proteins: (1) nebulin (Neb), a sarcomere thin filament protein that is frequently mutated in NM; and (2) leiomodin 3 (Lmod3), a novel muscle-specific protein with putative thin filament actin polymerization activity. Klhl40 belongs to the BTB-BACK-Kelch (BBK) protein family, which typically promote protein ubiquitination and degradation, but we find that Klhl40 stabilizes its substrates. Thus, Neb and Lmod3 protein levels are diminished in Klhl40 deficient mice independent of any changes in their respective mRNA transcripts. Loss of KLHL40 in humans was recently reported to cause NM, and we find that NEB and LMOD3 are decreased in some KLHL40 mutant patients. However, the function of LMOD3 is also not known. To establish the role of LMOD3 in NM, we generated Lmod3 knockout mice by TALEN-mediated mutagenesis. Preliminary data shows that loss of Lmod3 results in a degenerative skeletal muscle myopathy. Thus, we propose that loss of Klhl40 directly results in decreased Neb and Lmod3 causing thin filament disruption and subsequent NM. In addition, we uncover the first BBK protein with a pro-stability function which has broad implications for future study of this protein family. In conjunction to our studies with Klh40, we found a closely neighboring gene in the antisense direction, Hhatl. Similar to Klhl40, we found that Hhatl expression is highly enriched in the heart and skeletal muscle although with notable expression in the central nervous system. Hhatl encodes for a putative membrane bound O-acyltransferase protein. Global deletion, but not heart or skeletal muscle-specific deletion, of Hhatl results in a failure to thrive phenotype with mid to late neonatal lethality. We outline future experiments to determine the nature and mechanism of the Hhatl knockout phenotype as well as possible means to delineate its function in striated muscles.Item Myogenic Effectors and Disease(December 2021) Ramirez Martinez, Andres; Sadek, Hesham A.; Mendell, Joshua T.; De Martino, George; Olson, Eric N.Skeletal muscle is essential for life. Inside muscle fibers, filaments of actin and myosin slide on each other to generate the mechanical forces that drive muscle contraction, movement, and breathing. Mutations in muscle-related genes can cause severe diseases in humans. Here we characterize the role of three understudied muscle-specific genes and their potential contribution to human disease. We show that constitutive and juvenile loss of the nuclear envelope protein Net39 in mice recapitulates different manifestations of Emery-Dreifuss muscular dystrophy. Deletion of Net39 caused disruption of nuclear envelope integrity and associated genomic, transcriptional, and metabolic changes that compromised muscle function. Mechanistically, Net39 regulates nuclear organization by associating with LEM proteins, and gene expression by controlling the transcription factor Mef2c. In contrast, global deletion of the Kelch protein Klhl41 in mice causes severe nemaline myopathy, including neonatal lethality and aggregation of contractile proteins in muscle, particularly Nebulin. Molecularly, Klhl41 acts as a chaperone for Nebulin, and N-terminal poly-ubiquitination of Klhl41 acts as a signal to regulate its activity. Finally, we identify a novel pathogenic mutation in the cell fusogen Myomixer. We show that patients with Carey-Fineman-Ziter syndrome lose a region of Myomixer required to destabilize opposing cell membranes during myoblast fusion. Overall, our findings here highlight the contribution of understudied genes to muscle biology and the molecular etiology of muscle disorders.Item Regulation of Skeletal Muscle Development and Disease by an Actin-Dependent Transcriptional Circuit(2018-05-17) Kutluk Cenik, Bercin; Cleaver, Ondine; Olson, Eric N.; MacDonald, Raymond J.; Mangelsdorf, David J.Congenital myopathies are a group of diseases that primarily affect skeletal muscle and cause muscle weakness that manifests at birth. With an incidence of 6 in 100,000 live births every year, myopathies are considered to be one of the top neuromuscular diseases in the world. Among congenital myopathies, nemaline myopathy (NM) is the most common variant. NM patients have generalized muscle weakness and lifelong disability, and its severest forms are neonatal lethal due to respiratory failure. Currently, there is no cure for NM, underscoring the necessity for new insights into the mechanisms of this severe disease. NM results from mutations in the actin thin filament proteins, and is associated with disorganization of myofibrils, reduced contractile force, and consequent failure to thrive. The main goal of my research has been to expand our knowledge of transcriptional networks that regulate sarcomeric actin, and to investigate how perturbations of these networks can lead to muscle disease. The expression of the actin gene, and the stability of the actin protein are tightly regulated during muscle development and maintenance. Myocardin-related transcription factors (MRTFs) play a central role in actin dynamics, by functioning as coactivators of the serum response factor (SRF), a master regulator of actin and other cytoskeletal genes. MRTFs additionally serve as sensors of actin polymerization and are sequestered in the cytoplasm by actin monomers. We explored the role of MRTFs in muscle development in vivo by generating mutant mice harboring a skeletal muscle-specific deletion of MRTF-B and a global deletion of MRTF-A and showed that the absence of MRTF-A and MRTF-B in the skeletal muscle leads to sarcomeric disarray and dramatic dysregulation of cytoskeletal genes. These findings highlight the importance of MRTFs in actin cycling and myofibrillogenesis. One of the cytoskeletal genes dysregulated in the MRTF dKO was Leiomodin-3 (Lmod3). This striated muscle-specific gene encodes for a putative actin nucleation factor and is downregulated in the MRTF dKO. Lmod3 is a component of the sarcomere thin filament, and its loss leads to compromised sarcomere integrity and nemaline myopathy (NM), a severe congenital muscle disease. We demonstrated an actin-dependent transcriptional circuit in which SRF cooperates with the myogenic transcription factor MEF2 to sustain the expression of the Lmod3 gene and other components of contractile apparatus. In turn, Lmod3 enhances MRTF-SRF activity by promoting actin polymerization. Together, these factors establish a regulatory loop to maintain skeletal muscle function. Finally, we investigated the Kelch protein family: a group of proteins that function as substrate-specific adaptors for Cullin RING E3 ligases; and are responsible for the balance between protein stability and degradation in many tissues, including striated muscle. Previously we have shown that KLHL40 is required for the stabilization of LMOD3. We further demonstrate that KLHL21, a muscle-enriched Kelch protein, operates in numerous unique pathways that potentially govern muscle and heart development, and the cell cycle. Future studies could pave the pathway to therapeutic approaches that improve heart and muscle regeneration, through our understanding of this gene and protein. Overall, our findings not only provide insight into how actin cycling networks regulate skeletal muscle specific transcripts and/or proteins to contribute to myogenesis, but also pave the way for potential new therapeutic approaches for congenital myopathies through the identification of disease-causing mutations.