Browsing by Subject "Muscle Proteins"
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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 Myokine Musclin Expression Is Elevated in Rats after Burn(2017-01-17) Maredia, Navin; Song, Juquan; Sehat, Alvand; Maxwell, Christian; Panwar, Kunal; Kulangara, Rohan; Carlson, Deborah; Huebinger, Ryan; Wolf, StevenINTRODUCTION: Annually, over 2 million people in the US experience severe burns, a condition marked by a hypercatabolic state with significant muscle loss. Muscle is necessary for glucose and lipid metabolism. Previous studies have shown detrimental effects of insulin resistance and hyperglycemia associated with muscle loss due to burns. Recently, the novel skeletal muscle myokine musclin has been found to regulate glucose in vitro. Thus, we attempted to better understand the effects of burns on musclin levels. We aimed to investigate the effects of burns on 1.) musclin levels systemically and 2.) musclin mRNA expression in vitro. METHODS: Thirty-one adult male Sprague-Dawley rats received 40% total body surface area (TBSA) burns. Rat serum was collected from 6 hours to 14 days after burn. Nine animals without injury served as control. Musclin levels in serum were measured by ELISA. Mouse C2C12 myoblasts were stimulated with 10% rat burn serum for 24 hours. Cells were stimulated with non-burn serum, 6-hour post burn serum, 72-hour post burn serum, and 14-day post burn serum. Following stimulation for 24 hours, C2C12 cells were collected and musclin expression was quantified by real time PCR analysis. RESULTS: Circulating musclin levels were 59.3 ± 3.3 ng/mL in non burned control rats. Musclin levels in the serum significantly increased to 76.7 ± 6.0 ng/mL at 6 hours and 76.7 ± 1.9 ng/mL at 24 hours after burn (p<0.05). Musclin levels in the serum returned to baseline until 14 days. Normalized to GAPDH mRNA level, musclin mRNA expression was 7.87 ± 0.56 fold in C2C12 myoblasts with 10% non-burn serum stimulation. Musclin mRNA expression significantly increased with the addition of the following burn rat serums: 22.66 ± 5.18 fold with 6-hour post-burn serum, 15.00 ± 1.93 fold with 72-hour post-burn serum, and 12.17 ± 0.82 fold with 14-day post-burn serum (p<0.05). CONCLUSIONS: Musclin levels increase in rat serum following 40% TBSA burn injury. In vitro stimulation of muscle cells with burn serum increases musclin expression.Item [News](1978-04-27) Land, ChrisItem Slow Inactivation of Sodium Channels: Structural Clues and Disease Associations(2009-06-19) Webb, Jadon Ray; Cannon, Stephen C.Voltage gated sodium channels underlie the rapid upstroke of action potentials in electrically excitable mammalian tissues. A cardinal feature of Na+ channels is their ability to rapidly inactivate to a refractory state during membrane depolarization, in a process known as 'fast inactivation'. During sustained membrane depolarization or prolonged busts of discharges, channels can further inactivate to non-conducting states collectively referred to as 'slow inactivation'. Fast inactivation occurs by occlusion of the inner pore by the intracellular III-IV Loop, and defects in fast inactivation gating are known to underlie certain forms of myotonia, periodic paralysis, epilepsy, and cardiac arrhythmias. The mechanism of slow inactivation and its relevance to human disease, on the other hand, are much less understood. The primary aim of this thesis was to characterize the mechanism of sodium channel slow inactivation, and also to further define its role in disease. In Chapter 1, an overview of sodium channel structure and gating is provided as background for understanding the rational and interpretation of the experimental studies. The experiments in Chapter 2 characterized the gating of a sodium channel mutation (P1158S) associated with temperature-sensitive periodic paralysis. This disease mutation caused a robust defect in slow inactivation, in accordance with an emerging model that associates defective slow inactivation with increased susceptibility to paralytic attacks. Additionally, the slow inactivation gating defects were elicited by cold temperature, analogous to the temperature-dependent provocation of paralysis. This finding further strengthens the association between defective slow inactivation gating and a specific disease phenotype. Chapter 3 explores the interaction of the sodium channel Beta-1 subunit and slow inactivation, which is incompletely characterized especially in mammalian cell expression systems. I found that co-expression of wild-type Beta-1 significantly depolarized the voltage-dependence of steady-state slow inactivation and also reduced the number of channels occupying the slow state (IS) after a long depolarizing conditioning pulse, but did not affect the kinetics of slow inactivation.. To understand which region(s) of Beta-1 are important for modulation of slow inactivation, two mutant constructs were tested. A point mutation in the extracellular N-terminus associated with epilepsy (C121W) disrupts a critical disulfide bond in an Ig-like fold and abolished the ability of Beta-1 to modulate slow inactivation. Conversely, truncation of the short cytoplasmic C-terminus did not alter the effects of Beta-1 on slow inactivation. These observations parallel the structure-function relations that have been established for Beta-1 modulation of fast inactivation. Interestingly, however, I used a mutant fast-inactivation deficient alpha-subunit to show that the Beta-1 effect on slow inactivation was independent of coupling to fast inactivation. In Chapter 4, the interaction of slow inactivation and alkali metal cations is explored. External cations have been shown to influence slow inactivation, but little is known about the location and mechanism of this interaction. To address this, I examined the interaction of Group IA alkali metal cations with slow inactivation in rat Nav1.4 channels expressed in HEK293t cells. Slow inactivation was significantly impeded by external, but not internal Na+ and Li+ cations in the buffer solutions. External K+, Rb+, and Cs+, on the other hand, caused little effect compared to sucrose (cation-free) buffer. Cation effects on slow inactivation were found to be very low affinity and were not dependent on the ability of cations to permeate deep into the channel. Indeed, Na+ interaction occurred at a shallow apparent electrical distance of 0.15 relative to the outside of the channel, and was affected by mutagenesis in the outer