Browsing by Subject "Muscle, Skeletal"

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Control of Skeletal Muscle Fiber Types by Calcium Signaling Pathways
(2002-08-01) Wu, Hai; William, R. Sanders
Different patterns of motor nerve activity drive distinctive programs of gene expression in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Previous studies have demonstrated that calcineurin activity is required to maintain slow myofiber identity. I am interested in determining the transcription factors downstream of calcineurin and other calcium-regulated signaling pathways in the control of myofiber specialization. By analyzing two fiber type-specific enhancers, I was able to demonstrate that there are functional NFAT (nuclear factor of activated T cells) and MEF2 (myocyte-specific enhancer factor 2) binding sites within the enhancer of troponin I slow, and both sites are required for slow fiber specific activity of this enhancer. Next, I identified MEF2 as a target of calcineurin in cultured myogenic cells. Calcineurin physically interacts with MEF2 and dephosphorylates MEF2. C-terminal transactivation domain, but not N-terminal DNA binding domain of MEF2, responds to calcineurin activation. The use of "MEF2 indicator" transgenic mice that harbor a MEF2-dependent lacZ transgene enabled us to monitor the endogenous activities of MEF2 transcription factors. MEF2 is selectively active in slow and oxidative myofibers. Calcineurin is both necessary and sufficient for MEF2 activation in skeletal muscles. I also found a dose-response relationship between calcineurin activity and expression level of slow, oxidative fiber-specific and MEF2 target genes. Furthermore, I observed that functional activity of MEF2 transcription factors was stimulated by sustained periods of endurance exercise or low-frequency motor nerve pacing in a calcineurin-dependent manner. In addition to calcineurin, CaMKs (calcium, calmodulin-dependent kinases) also transduce their signaling through MEF2. CaMKIV synergistically activates MEF2-dependent gene expression together with calcineurin. Transgenic mice expressing constitutively active CaMKIV in their skeletal muscles showed increased percentage of slow and oxidative myofibers, which was accompanied by increased mitochondrial biogenesis mediated through the upregulation of PGC-1 (PPARg co-activator). Taken together, these findings delineate a molecular pathway in which MEF2 and NFAT integrate signaling inputs from multiple calcium-regulated pathways in the control of skeletal muscle fiber types.
Discovery and Characterization of Genes Involved in Muscle Calcium Handling
(2015-08-10) Nelson, Benjamin Rhett; Wilkie, Thomas; Olson, Eric N.; Mendell, Joshua T.; Stull, James
Muscle tissue requires continuous cycling of calcium release and clearance to generate and sustain contraction. When the plasma membrane of a muscle fiber becomes electrically excited, a voltage sensor in the membrane, called the dihydropyridine receptor (DHPR), becomes activated and signals to the calcium release channel located in the membrane of the sarcoplasmic reticulum (SR), called the ryanodine receptor (RyR). When the RyR channel is activated, calcium is released into the cytoplasm from the SR, the primary calcium storage compartment of muscle fibers. Calcium then binds to the sarcomere, activating the motor activity of the myosin filaments, and causes the fiber to contract. Following contraction, most of the cytosolic and sarcomeric calcium is recycled back to the SR by the sarco/endoplasmic reticulum ATPase (SERCA). Although most components of the calcium handling pathway are thought to be already known, the large number muscle-specific genes with unknown functions would suggest that additional components may yet be undiscovered. The first goal of this study was to carry out initial functional characterization of the Stac3 knockout mouse, in which muscle contraction is severely defective. We found that mice lacking Stac3, a gene with skeletal muscle-specific expression, lack muscle contraction because of a defect in excitation-contraction coupling, that is the link between membrane excitation and SR calcium release. The second goal was to identify small peptides that may play a role in muscle function. We examined codon conservation in transcripts annotated as long non-coding RNAs and discovered a transcript that encodes a 34-amino-acid transmembrane peptide with cardiac and slow-twitch muscle expression that we have named Dwarf Open Reading Frame or DWORF. Overexpression of this peptide in cardiac myocytes increases the peak calcium release during contraction while also increasing the rate of calcium clearance. We conclude that DWORF likely serves to increase the apparent enzymatic activity of SERCA. Together the discoveries of Stac3 and Dworf suggest that many important genes in muscle function may be awaiting a closer look or have not yet been discovered at all.
Durability and Longevity of Gene Edited DMD Skeletal Muscle
(2022-08) Karri, Dileep Reddy; Sadek, Hesham A.; Olson, Eric N.; Chen, Elizabeth; Mendell, Joshua T.
Duchenne Muscular Dystrophy (DMD) is a lethal muscle disease caused by mutations in the dystrophin gene. CRISPR/Cas9 genome editing has been used to correct DMD mutations in animal models at young ages. However, the longevity and durability of CRISPR/Cas9 editing remained to be determined. To address these issues, I subjected ΔEx44 DMD mice to systemic delivery of AAV9 expressing CRISPR/Cas9 gene editing components to reframe exon 45 of the dystrophin gene, allowing robust dystrophin expression and maintenance of muscle structure and function. I found that genome correction by CRISPR/Cas9 confers lifelong expression of dystrophin in mice and that corrected skeletal muscle is highly durable and resistant to myofiber necrosis and fibrosis, even in response to chronic injury. In contrast, when muscle fibers were ablated by barium chloride injection I observed a loss of gene edited dystrophin expression. Analysis of on-target and off-target editing in aged mice confirmed the stability of gene correction and the lack of significant off-target editing at 18 months of age. These findings demonstrate the long-term durability of CRISPR/Cas9 genome editing as a therapy for maintaining the integrity and function of DMD muscle, even under conditions of stress.
Fiber Orientation Modeling: a Method to Improve Quantitation of Intramyocellular Lipids in Human Subjects at 7 Tesla
(2011-10-03) Khuu, Anthony N.; Malloy, Craig R.
BACKGROUND: Increased intramyocellular lipid (IMCL) content in skeletal muscle has been suggested to be a biomarker for insulin resistance. As a noninvasive method of estimating IMCL, 1H MR spectroscopy of muscle fat has been a popular method for measuring the concentration of IMCLs, a goal highly desirable for research in the pathogenesis of type 2 diabetes. Extramyocellular lipids (EMCL) are often considered to be deposited along strands that are parallel to Bo (the applied field) whereas IMCL are assumed to be spherical droplets in the muscle cells’ cytoplasm. Resolution between IMCL and EMCL signals mainly results from the angle-dependant bulk susceptibility of the 2 geometric structures. However, IMCL signal is usually contaminated by a broad and asymmetrical EMCL . Conventional fitting methods usually assume that both the IMCL and EMCL signals to be symmetrical, represented by a single Lorentzian, Gaussian or Voigt (hybrid lineshape between Gaussian and Lorentzian) lineshapes. However, significant asymmetry in the resonance assigned to the methylene protons (-CH2-)n in extramyocellular lipids (EMCL) interfered with fitting the spectra. In this work, we explore another approach, named Fiber Orientation Modeling (FOM) by using the bulk susceptibility effect theory to accurately assess the lineshape of EMCL. METHODS: The distribution of EMCL strand orientation at any angle from 0 degrees to 90 degrees relative to Bo was described by a Gaussian function, centered at a specific angle and a width representing a dispersion of EMCL strands. The chemical shift OMEGA from each strand was translated by the well-known orientation dependence interaction OMEGA = 3cos2THETA-1, where THETA is the angle between EMCL and the applied field. As the result, the location and amplitude of individual curves representing each strand could be derived. Depend on the aforementioned Gaussian distribution, the combination of these individual curves generated a unique EMCL shape. In this work, spectral simulations were generated using muscle fiber orientation reported previously. The phantom experiment with a fat cylinder (representing EMCL) submerged in Intralipid(TM) solution (representing IMCL) was also performed to determine the maximal shift at 0 degrees and 90 degrees. Under IRB approval, single voxel and chemical shift images were acquired from soleus and gastrocnemius muscle of healthy human subjects at 7T (Phillips Medical system, Cleveland, Ohio). All the spectra were fitted with the hybrid Voigt lineshape and the experimental lineshape. RESULTS: In simulated spectra with dominant angle of 00 to the applied field and little dispersion, fitting with the Voigt lineshape accurately determined IMCL/EMCL ratio over a range of different linewidths. Increasing dispersion and central angle caused overestimation of IMCL/EMCL ratios, up to three-fold when fitted with the Voigt lineshape. The error was substantially reduced using our method. The improvement is also observed in phantom spectra and human spectra. Estimates of [IMCL]/[EMCL] were significantly improved by including variations in fiber orientation in the lineshape analysis (fiber orientation modeling, FOM). Calculated soleus [IMCL] using FOM, 4.43 ± 2.32 mmol/kg wet weight, was lower compared to most previous reports in soleus. The average orientation of EMCL was calculated to be 35 degrees relative to Bo with a dispersion width of 24 degrees CONCLUSION: Since prominent asymmetrical EMCL signal tends to contaminate into IMCL region, this interaction results in the amplitude-dependence of IMCL signal on the average orientation and dispersion of EMCL. As the result, the use of symmetrical lineshape tends to overestimate the IMCL signal if all strands of EMCL are not parallel to Bo and one another. By accounting for the angular dispersion& orientation, the fit would improve both the residual and the IMCL estimate.
Gene Expression Changes in Response to Severe Burn Injury in Rat Achilles Tendon
(2017-01-17) Buller, Dustin; Mitchell, Wes; Manchanda, Kshitij; Song, Juquan; Hernandez, Paula
IMPORTANCE: Severe burn injury, occurring at a global rate of 5/100,000 per year, can result in serious sequelae such as infection, shock, and direct organ damage. It has been shown that severe burn also results in both systemic inflammation and distant inflammatory effects, specifically in skeletal muscle and bone. However, we are unaware of any work that investigates similar effects in the tendon, which connects muscle and bone. OBJECTIVE: To determine whether, after distant severe burn injury, changes occur in gene expression, protein synthesis, and biomechanical properties in rat Achilles tendon. METHODS: Rats were subjected to thoracolumbar full-thickness severe burn injury under anesthesia before harvesting the Achilles tendon at time points of 6h, 1d, 3d, 7d, and 14d. Unburned rats were used for control. RNA expression of collagen I (Col1), collagen III (Col3), MMP9, MMP13, IL-1β, IL-6, TNF-α, and tenomodulin was measured using qPCR. Quantified Western blots were performed to assess relative protein content for Col1, Col3, and MMP9. Finally, biomechanical testing was performed on a separate group of rats to assess biomechanical changes at 14d versus control (n = 8). RESULTS: The MMP9 gene was upregulated on the order of 20-fold at 14d (p = 0.0016) and 10-fold at 3d (p = 0.0255) and MMP13 showed a trend toward 12-fold increase at 3d (p = 0.079). A significant increase in expression of IL-1β and a trend toward significance IL-6 were also observed at 3d (p < 0.01, and p = 0.059, respectively). Differences in expression of Col1, Col3, TNF-α, and tenomodulin were not significant compared to control. Preliminary data from Western blots showed 5-fold decrease of collagen 1 at 7d (p < 0.01) and 2-fold increase of collagen 3 at 14d (p = 0.016). Significant changes were not found in MMP9. Preliminary biomechanical data shows a trend toward a 4-fold decrease in stiffness in the burn group. All p values are by t-test. CONCLUSIONS: These results newly confirm the existence of inflammation in tendon distant from the burn site after severe burn injury. Specifically, these changes could indicate initial matrix remodeling as carried out by the MMPs beginning at 3d, followed by collagen deposition with a decreased Col1:Col3 ratio, resulting in decreased tissue stiffness. These findings are consistent with the repair processes known to occur in other tissues after inflammation. Additionally, IL-6 and IL-1β may have a more significant role in post-burn acute-phase inflammation than other acute-phase reactants like TNF-α in the Achilles tendon.
Mitochondrial Fission with Function Impairment in Burn Serum Treated C2C12 Cells
(2016-01-19) Sehat, Alvand; Song, Juquan; Kumar, Puneet; Cai, Anthony; Huebinger, Ryan M.; Carlson, Deborah, L.; Zang, Qun S.; Wolf, Steven E.
BACKGROUND: Burn patients suffer muscle mass loss associated with a hypercatabolic status. Mitochondria dynamics cycle is affected by metabolic status, and mitochondrial fission mediated high glucose induced cell death. Mitochondria function impairment associated with muscle mass loss has been observed in severe burn patients. We hypothesize that severe burn impaired muscle mass loss is associated with increased mitochondria fission with function impairment. The study was to investigate mitochondrial dynamics in response to burn serum stimulation. METHODS: Murine myoblast C2C12 cells were treated with DMEM media containing 10% rat serum isolated either from 40% TBSA scald burn rats, or control rats. Mitochondria was labeled with 3nM of MitoTracker Green FM dye, and live cell images were taken sequentially under a Nikon Ti Eclipse Confocal microscope. Cell lysates were collected for molecular biological analysis. Mitochondrial function was evaluated with Enzo Mito-ID membrane potential cytotoxicity kit. Target protein signals from cell lysate were detected by SDS-PAGE and western blot analysis. RESULTS: Mitochondrial morphology maintained the elongated linear shape in C2C12 cells when treated with 10% control rat serum. In contrast, when cells were treated with 10% burn serum, mitochondria reduced the elongated linear shape at 24 to 48 hours, and the florescent dye diffused at 72 hours. The cell florescent images showed an increase in circularity and fragmentation of mitochondria in C2C12 cells with burn serum stimulation. Meanwhile mitochondrial membrane potential decreased with 6hr post-burn serum stimulation. Western blot data showed that mitofusion-1 (Mfn1) significantly decreased in C2C12 cells with burn serum stimulation, confirming the observation of mitochondrial fission in response to burn serum. Cell death marker caspase 3 increased its expression in C2C12 cells with burn serum stimulation, suggesting a superfluous cell death in skeletal muscle after burn. CONCLUSION: Our results show an increase in the mitochondria fission/fusion ratio in C2C12 cells stimulated with burn serum isolated 6 hours after burn. The mechanism of mitochondrial fission with function impairment leading to muscle death is under investigation.
Muscle Function Improvement in Injured Mice with Combination Treatment
(2017-01-17) Kulangara, Rohan G.; Sehat, Alvand, J.; Maredia, Navin; Maxwell, Christian; Liu, Ming-Mei; DeSpain, Kevin; Wolf, Steven E.; Song, Juquan
INTRODUCTION: Loss of skeletal muscle from direct injury presents debilitating effects to an individual. Current treatments addressing muscle loss are limited by insufficient reconstitution of functioning muscle. Novel regenerative medicine technologies include the application of Urinary Bladder Matrix (UBM) and mesenchymal stem cells (MSCs) to restore functional muscle tissue. In our previous studies, we found that UBM increased muscle myoblast cell proliferation. Therefore, we examined whether co-treatment with MSCs would further augment regeneration as compared to individual treatments. METHODS: Twenty C57BL/6 male adult mice received bilateral laceration injuries on the gastrocnemius muscle under anesthesia, and were randomly grouped to a designed treatment applied 14 days after injury. Treatment groups were 1) DMEM culture medium, 2) UBM only (150μg), 3) MSCs only (1 million mouse derived cells), and 4) UBM+MSCs. 4 additional mice served as a control baseline not receiving injury. Efficacy of treatment was analyzed through isometric muscle force testing as well as histomorphologic examination at 50 days after injury. Two-way ANOVA was applied for statistical analysis. RESULTS: Isometric muscle force was measured, including twitch (Pt), tetanic (Po), and fatigue isometric functions with the muscle stretched to optimal length (Lo). Muscle twitch (Pt) significantly decreased in the DMEM group compared to the non-injured group at day 50 (p < 0.05). Furthermore, twitch significantly increased with UBM treatment, but not with MSC treatment. Regenerating myofiber nuclei were counted and myofiber cross sectional area was measured with histology. New myotubes were identified as having centrally located nuclei. Further, Ki-67 nuclear immunofluorescence staining was performed to demonstrate proliferating satellite cells. The myofiber cross sectional area and the number of Ki-67/DAPI overlapping stained nuclei significantly increased in the DMEM group compared to the non-injured group (p < 0.05). No differences were observed with other treatments in injured mice at day 50. CONCLUSION: We observed a significant improvement in muscle function with combination treatment and single UBM treatment applied 50 days post-injury. The current animal model provides a tool to study muscle regeneration, and is feasible for clinical translation to address impairment in skeletal muscle function after burn injury.
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.
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, Steven
INTRODUCTION: 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.
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(1984-02-17) Rutherford, Susan
[News]
(1978-04-27) Land, Chris
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.
Regulation of Skeletal Muscle Innervation and ALS Pathogenesis by MicroRNA 206
(2010-01-12) Williams, Andrew H.; Olson, Eric N.
Motor neurons and the skeletal muscle fibers they innervate maintain an intimate relationship that requires bidirectional signaling for the establishment and maintenance of neuromuscular synapses and muscle function. Abnormalities in the regulation of neuromuscular gene expression often result in neuropathies and myopathies, reflecting the intimate communication between muscle and motor nerve. In this thesis, I present my studies on the function of microRNAs in neuromuscular synapse regeneration and neurodegenerative disease. First, I show that the expression of a muscle-specific microRNA (miRNA), miR-206, is dramatically upregulated following surgical denervation of skeletal muscle and in a mouse model of amyotrophic lateral sclerosis (ALS). The responsiveness of the miR-206 gene to the state of motor innervation is dependent on binding sites for MyoD in an upstream enhancer. Based on the upregulation of miR-206 following denervation and its synapse-enriched expression pattern, I hypothesized that miR-206 is an important regulator of neuromuscular junction (NMJ) physiology and I generated miR-206 mutant mice. Using these mice, I demonstrated that miR-206 is an essential regulator of neuromuscular synapse reinnervation following nerve injury. The requirement of miR-206 for efficient reinnervation reflects, at least in part, its repressive influence on histone deacetylase 4 (HDAC4). I also explored another function of miR-206, as an essential modulator of retrograde growth factor signaling during the progression of neurodegenerative disease. By crossing miR-206 mutant mice to G93A-SOD1 transgenic mice, which express a mutant form of superoxide dismutase (SOD), I determined that the loss of miR-206 accelerates the pathogenesis of ALS due to the loss of functional NMJs. Thus, the results of my thesis research demonstrate that miR-206 functions as a sensor of motor innervation and regulates a retrograde signaling pathway required for nerve-muscle interactions during stress and disease.
Role of KIRREL in Mammalian Myogenesis and Rhabdomyosarcoma
(2018-01-23) Reinert, Shannon; Avirneni, Usha; Galindo, Rene
Rhabdomyosarcoma is an aggressive soft-tissue malignancy comprised microscopically of neoplastic skeletal muscle-lineage precursors that fail to exit the cell-cycle and fuse into syncytial muscle - the underlying pathogenetic mechanisms for which remain unclear. We previously identified that misregulated myoblast fusion signaling via the TANC1 adaptor molecule promotes neoplastic transformation in RMS cells driven by the PAX-FOXO1 oncogenic transcription factor. Unknown from these studies are the upstream elements that participate with TANC1 in wild-type myoblasts to orchestrate myoblast fusion, and how these elements likewise participate in RMS. We are now interrogating the Immunoglobulin Superfamily Receptor Kirrel receptors in these processes. Three Kirrel orthologs are present in mammals, two of which, Kirrel-1 and -3, are expressed normally in myogenesis. Using loss-of-function (shRNA) and gain-of-function (misexpression) strategies, our preliminary data are showing a requirement for Kirrel-3 during myoblast fusion, as tube formation is significantly altered in these studies. We are now extending these for Kirrel-3 activity in RMS using cultured neoplastic rhabdomyoblast. Our data and most recent findings will be presented and discussed.
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(1985-10-21) Waggoner, Lori
Voltage-Gated Sodium Channel Activity in Mouse Skeletal Muscle Fibers: Normal Gating and Defects Associated with Periodic Paralysis Mutants
(2010-11-02) Fu, Yu; Cannon, Stephen C.
Mutations in SCN4A, the gene encoding the skeletal muscle Na+ channel (NaV1.4) α-subunit, cause several disorders related to skeletal muscle excitability. The functional consequences of these NaV1.4 mutations have been extensively characterized in heterologous expression systems. These studies have significantly advanced our understanding of the pathophysiology of these disorders. The in vivo functional consequences on channel activity, however, have yet to be defined. Animal models are now available in genetically engineered mice, which provide an opportunity to examine channel function in mature skeletal muscle. We optimized a two-electrode voltage clamp protocol to improve the fidelity of Na+ current recording from acutely dissociated intact muscle fibers. Computer simulation, incorporating measured capacitance and ionic current densities, was used to confirm sufficient voltage control and distortion-free Na+ currents. The gating properties of endogenous Na+ currents were measured and compared between two mouse strains, C57BL/6 and 129-E. The most dramatic finding was a hyperpolarized shift in the voltage dependence of activation (-25 mV) and fast inactivation (-18 mV) as compared to the studies in HEK293 cells expressing NaV1.4 plus the accessory β1-subunit. A possible contribution from NaV1.5 channels in the mouse muscle preparation was excluded by RT-PCR and TTX sensitivity. There was no significant difference in voltage dependence of fast gating between C57BL/6 and 129-E. The entry rate into slow inactivation was slower for Na+ channel in 129-E fibers; while the recovery from slow inactivation was similar between two mouse stains. Two NaV1.4 missense mutations associated with divergent clinical phenotypes - NaV1.4-M1592V in hyperkalemic periodic paralysis (HyperPP) and NaV1.4-R663H (homolog of human R669H) in hypokalemic periodic paralysis (HypoPP) - were characterized with voltage-clamp recordings in fully differentiated fibers from knock-in mutant mice. The NaV1.4-M1592V mutation produced gain-of-function defects, with the major changes being a slightly increased persistent current and moderately disrupted slow inactivation. In contrast, the HypoPP knock-in mutant R663H resulted in loss-of-function changes, due to an enhancement of inactivation, both fast and slow, and impaired activation. These observations provide important validation of prior findings using heterologous expression systems and yield quantitative information on the severity of the gating defects in mammalian skeletal muscles.