Browsing by Author "Grange, Robert W."
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- The activity and content of calpains in maturing dystrophic muscle membranesWang, Qiong (Virginia Tech, 2005-05-11)Increased calcium-activated calpain proteolysis in the sarcolemma membrane is thought to be a primary mechanism in the pathophysiology of Duchenne Muscular Dystrophy (DMD). However, few studies have tested this possibility prior to the overt signs of the dystrophy. The purpose of this study was to test the hypothesis that there is greater calpain content and total relative calpain activity in membranes obtained from dystrophic (mdx; mdx:utrophin-deficient (mdx:utrn-/-)) compared to wildtype (wt) mouse skeletal muscles during maturation at ages 7- and 21-d,and at a post-maturation age of 35-d. Calpain activity was determined as the calcium-dependent cleavage of the flurogenic substrate SLY-AMC, and content was determined by Western analysis with an anti-calpain antibody. There were several intriguing findings: 1. There was an inverse relationship between calpain content and relative activity in the whole muscle in both wt and mdx mice from age 7- to 35-d: calpain content decreased, and relative calpain activity increased as the mice aged. This suggests a similar role for calpain in both genotypes, which might relate to specific maturation processes, possibly up to age 21-d. Although the inverse relation was evident at 35-d, the targets for calpain in mdx compared to wt likely differed. 2. The increased relative calpain activity in the membrane fraction of mdx mice at age 35-d (26.73 Arbitrary Units, (AU)) compared to that of age 7- (4.9AU; p<0.05) and 21-d (8.74AU; p<0.05) is temporally related to degeneration and regeneration processes, and may also indicate activation of apoptosis, in mdx muscles at this age. 3. At age 7-d, there were no significant differences in either calpain content or relative calpain activity in all subcellular fractions for wt and mdx mice. This result might suggest similar calpain distribution and activities that are related to the regulation of muscle maturation and differentiation in both genotypes. (Note:data were not obtained for the mdx:utrn-/- mice at age 7-d because of insufficient animals). 4. At age 21-d, there was greater relative calpain activity in the myofibrillar supernatant fraction in mdx (15.13AU) than wt mice (1.18AU; p<0.05). This could indicate calpain's role in the initiation of myofibrillar protein turnover and the proteolysis of submembranous networks in the mdx muscles. 5. At age 21-d, greater calpain content in the mdx (1.40ìg) compared to wt (0.23 ìg; p<0.05) membrane fraction might suggest a broader distribution of calpain along membranes that contributes to the onset of dystrophy in the mdx muscles. 6. At age 35-d, there was greater calpain content in the mdx:utrn-/- compared to the wt membrane (0.48ìg vs 0.13 ìg), cytosolic (0.88ìg vs 0.30ìg), and myofibrillar supernatant (0.49ìg vs 0.17ìg; p<0.05 ) fractions This increased content and broad distribution across several subcellular fractions may reflect degeneration and regeneration processes, and potentially activation of apoptosis, in the mdx:utrn-/- muscles. These data suggest that calpain activity contributes to dystrophic pathophysiology mainly in the membrane fraction of mdx skeletal muscles at age ~21-d, but appears to contribute later at 35-d and in more subcellular fractions in mdx:utrn-/- skeletal muscles.
- Blastocyst Injection of Wild Type Embryonic Stem Cells Induces Global Corrections in Mdx MiceStillwell, Elizabeth; Vitale, Joseph; Zhao, Qingshi; Beck, Amanda; Schneider, Joel S.; Khadim, Farah; Elson, Genie; Altaf, Aneela; Yehia, Ghassan; Dong, Jia-hui; Liu, Jing; Mark, Willie; Bhaumik, Mantu; Grange, Robert W.; Fraidenraich, Diego (PLOS, 2009-03-11)Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology and function. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection increases the amount of fat in the chimeras to reach WT levels. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally in a dose-dependent manner.
- Characterization of an in vitro exercise model and the effects of a metabolic endotoxemia on skeletal muscle adaptation to electric pulse stimulationHarvey, Mordecai Micah (Virginia Tech, 2017-06-20)The prevalence of obesity and type II diabetes is increasing. Although exercise is widely accepted for prevention and treatment, evidence of resistance to exercise in patients with these diseases is also mounting. Muscle contraction during exercise stimulate cellular responses important for adaptation. These responses include the release of myokines and the subsequent increase in substrate metabolism. This study aimed to define a culture model for simulating exercise in human primary skeletal muscle cells. We hypothesized that chronic electric pulse stimulation (EPS) of human myotubes in vitro would emulate cellular and molecular responses to exercise observed in vivo. To define this model, we applied EPS to human myotubes for varied lengths of time and measured interleukin-6 (Il-6), peroxisome proliferator-activated receptor gamma coactivator 1- (PGC1-), superoxide dismutase 2 (SOD2), substrate metabolism, metabolic enzyme activity, heat stress markers, and pH. To recreate the inflammatory milieu observed in metabolic disease states we treated the myotubes with a low dose of 20 EU lipopolysaccharide (LPS). Following the 24-hour stimulation we observed significant increases in transcription of Il-6, PGC1-, and SOD2. Basal glucose and fatty acid oxidation were also markedly increased in the cells after EPS. Cells treated with LPS elicited a blunted transcriptional, metabolic, and enzymatic response to EPS. These findings suggest that EPS is a viable model for simulating the effects of exercise. Our observations also indicate that an inflammatory environment could play a role in interfering with the adaptations to exercise.
- Chronic administration of a leupeptin-derived calpain inhibitor fails to ameliorate severe muscle pathology in a canine model of Duchenne muscular dystrophyChilders, Martin K.; Bogan, Janet R.; Bogan, Daniel J.; Greiner, Hansel; Holder, Melanie; Grange, Robert W.; Kornegay, Joe N. (Frontiers, 2012-01-09)Calpains likely play a role in the pathogenesis of Duchenne muscular dystrophy (DMD). Accordingly, calpain inhibition may provide therapeutic benefit to DMD patients. In the present study, we sought to measure benefit from administration of a novel calpain inhibitor, C101, in a canine muscular dystrophy model. Specifically, we tested the hypothesis that treatment with C101 mitigates progressive weakness and severe muscle pathology observed in young dogs with golden retriever muscular dystrophy (GRMD). Young (6-week-old) GRMD dogs were treated daily with either C101 (17 mg/kg twice daily oral dose, n = 9) or placebo (vehicle only, n = 7) for 8 weeks. A battery of functional tests, including tibiotarsal joint angle, muscle/fat composition, and pelvic limb muscle strength were performed at baseline and every 2 weeks during the 8-week study. Results indicate that C101-treated GRMD dogs maintained strength in their cranial pelvic limb muscles (tibiotarsal flexors) while placebo-treated dogs progressively lost strength. However, concomitant improvement was not observed in posterior pelvic limb muscles (tibiotarsal extensors). C101 treatment did not mitigate force drop following repeated eccentric contractions and no improvement was seen in the development of joint contractures, lean muscle mass, or muscle histopathology. Taken together, these data do not support the hypothesis that treatment with C101 mitigates progressive weakness or ameliorates severe muscle pathology observed in young dogs with GRMD.
- Chronic Dietary Supplementation of Branched-Chain Amino Acids Does Not Attenuate Muscle Torque Loss in a Mouse Model of Duchenne Muscular DystrophySperringer, Justin Edward (Virginia Tech, 2019-09-12)Duchenne Muscular Dystrophy (DMD) is an X-linked recessive, progressive muscle-wasting disease characterized by mutations in the dystrophin gene. Duchenne muscular dystrophy is the most common and most severe form of inherited muscle diseases, with an incidence of 1 in 3,500 male births1,2. Mutations in the dystrophin gene result in non-functional dystrophin or the complete absence of the protein dystrophin, resulting in necrosis and fibrosis in the muscle, loss of ambulation, cardiomyopathies, inadequate or failure of respiratory function, and decreased lifespan. Although there has been little research for effective nutritional strategies, dietary intervention may be effective as an adjuvant treatment. In this study, wild type (WT) and mdx animals were provided either a control or elevated branched chain amino acid (BCAA) diet nocturnally for 25 weeks to determine if the elevated BCAAs would attenuate muscle torque loss. Twenty-five weeks of chronic, elevated BCAA supplementation had no impact on muscle function measures. Interestingly, mdx and WT animals had the same torque responses in the low stimulation frequencies (1 Hz – 30 Hz) compared to higher stimulation frequencies. Tetanus was reached at a much lower stimulation frequency in mdx animals compared to WT animals (100 Hz vs +150 Hz). The mdx mouse consistently had more cage activity in the light cycle X- and Y-planes. Interestingly, animals on the BCAA diet increased X-, Y-, and Z-plane activity in the dark cycles at four weeks while animals on the control diet more Z-plane activity at 25 weeks, although not significant. All three BCAAs were elevated in the plasma at 25 weeks, although only Leu was significantly elevated. The BCAAs had no effect on. The diaphragm and skeletal muscle masses were larger in mdx animals, and WT animals had a significantly larger epididymal fat pad. The active state of BCKDC determined by phosphorylation of the E1α enzyme was greater in WT animals in white skeletal muscle, but not red skeletal muscle. Protein synthesis effectors of the mTORC1 signaling pathway and autophagy markers were similar among groups. Wild type animals had increased mTORC1 effectors and animals on the BCAA diet had decreased autophagy markers, although not significant. Although BCAAs did not affect muscle function, fibrosis, or protein synthesis effectors, this study illustrates the functionality of mdx muscles over time. It would be interesting to see how the different muscle fiber types are affected by DMD, noting the differences between the diaphragm, heart, red muscle, and white muscle fibrosis markers. Although there was no increase in mTORC1 effectors with an elevated BCAA diet, it would be interesting to determine muscle protein synthesis, myofibrillar protein synthesis, and total protein turnover in the mdx mouse with an elevated BCAA diet, although the dietary intervention started when mice arrived at 4 weeks of age, earlier intervention may be beneficial early in the disease process.
- Chronic Hypoxia and Cardiovascular Dysfunction in Sleep Apnea SyndromeChittenden, Thomas William (Virginia Tech, 2002-07-24)The purpose of the current study was to test the hypothesis that chronic hypoxia associated with sleep-disordered breathing relates to abnormal Nitric Oxide (NO) production and vascular endothelial growth factor (VEGF) expression patterns that contribute to aberrancy of specific determinates of cardiovascular and cardiopulmonary function before, during, and after graded exercise. These patterns may further reflect pathologic alteration of signaling within the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt-1) transduction network. To this end, 7 medically diagnosed OSA patients (3 male, 4 female), mean age 48 years and 7 apparently healthy control subjects (3 male, 4 female), mean age 42 years, underwent baseline venous blood draws and maximal bicycle ergometry. Mononuclear cells isolated from peripheral blood were utilized as reporter cells for measurement of VEGF, Akt-1, hypoxia inducible factor-1 alpha (HIF-1 alpha), and vascular endothelial growth factor receptor-2 (VEGFR2) gene expression by redundant oligonucleotide DNA microarray and real-time PCR technologies. Circulating angiogenic progenitor cells expressing VEGFR2 were profiled by flow cytometry. Plasma and serum concentrations of VEGF, nitrates/nitrites, catecholamines, and dopamine were measured by enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC). Arterial blood pressure, cardiac output, oxygen consumption and total peripheral resistance were determined at Baseline, 100W, and peak ergometric stress by standard techniques. There were no apparent differences (p < .05) observed in biochemical markers relating to vascular function and adaptation including, serum nitrates/nitrites, norepinephrine, dopamine, and plasma VEGF. No differences were found relative to cardiac output, stroke volume, cardiopulmonary or myocardial oxygen consumption, expired ventilation, heart rate, arteriovenous oxygen difference, total peripheral resistance, and mean arterial pressure. Due to methodological issues related to the redundant oligonucleotide DNA microarray and real-time PCR gene expression analyses, results of these experiments were uninterpretable. Thus, the research hypothesis was rejected. Conversely, significant (p < .05) differences were observed in waist: hip ratios, recovery: peak systolic blood pressure ratio at 1 minute post-exercise and %VEGFR2 expression. OSA was associated with elevations in both waist: hip ratios and recovery: peak systolic blood pressure ratio at 1 minute post-exercise as well as significant depression of %VEGFR2 profiles. Moreover, significant negative correlations were found regarding waist: hip ratios and %VEGFR2 expression (r = -.69;p =.005) and recovery: peak systolic blood pressure ratio at 1 minute post-exercise and %VEGFR2 expression (r = -.65;p =.01). These findings did not provide evidence that NO-dependent vasoactive mechanisms are suppressed nor did they support the supposition that angiogenic mechanisms are pathologically activated in sleep-disordered breathing.
- Classical and adaptive control of ex vivo skeletal muscle contractions using Functional Electrical Stimulation (FES)Cienfuegos, Paola Jaramillo; Shoemaker, Adam; Grange, Robert W.; Abaid, Nicole; Leonessa, Alexander (PLOS, 2017-03-08)Functional Electrical Stimulation is a promising approach to treat patients by stimulating the peripheral nerves and their corresponding motor neurons using electrical current. This technique helps maintain muscle mass and promote blood flow in the absence of a functioning nervous system. The goal of this work is to control muscle contractions from FES via three different algorithms and assess the most appropriate controller providing effective stimulation of the muscle. An open-loop system and a closed-loop system with three types of model-free feedback controllers were assessed for tracking control of skeletal muscle contractions: a Proportional-Integral (PI) controller, a Model Reference Adaptive Control algorithm, and an Adaptive Augmented PI system. Furthermore, a mathematical model of a muscle-mass-spring system was implemented in simulation to test the open-loop case and closed-loop controllers. These simulations were carried out and then validated through experiments ex vivo. The experiments included muscle contractions following four distinct trajectories: a step, sine, ramp, and square wave. Overall, the closed-loop controllers followed the stimulation trajectories set for all the simulated and tested muscles. When comparing the experimental outcomes of each controller, we concluded that the Adaptive Augmented PI algorithm provided the best closed-loop performance for speed of convergence and disturbance rejection.
- Closed Loop Control of Muscle Contraction using Functional Electrical StimulationJaramillo Cienfuegos, Paola (Virginia Tech, 2016-02-05)A promising approach to treat patients with vocal fold paralysis using electrical stimulation is investigated throughout this research work. Functional Electrical Stimulation works by stimulating the atrophied muscle or group of muscles directly by current when the transmission lines between the central nervous system are disrupted. This technique helps maintain muscle mass and promote blood flow in the absence of a functioning nervous system. The goal of this work is two-fold: develop control techniques for muscle contraction to optimize muscle stimulation and develop a small-scale electromagnetic system to provide stimulation to the laryngeal muscles for patients with vocal fold paralysis. These studies; therefore, focus on assessing a linear Proportional-Integral (PI) controller and two nonlinear controllers: Model Reference Adaptive Controller (MRAC) and an Adaptive Augmented PI (ADP-PI) system to identify the most appropriate controller providing effective stimulation of the muscle. Direct stimulation is applied to mouse skeletal muscle in vitro to test the controllers along with numerical simulations for validation of these experimental tests. The experiments included muscle contractions following four distinct trajectories: a step, sine, ramp, and square wave. Overall, the closed-loop controllers followed the stimulation trajectories set for all the simulated and tested muscles. When comparing the experimental outcomes of each controller, we concluded that the ADP-PI algorithm provided the best closed-loop performance for speed of convergence and disturbance rejection. Next, the focus of the research work was on the implementation of an electromagnetic system to generate appropriate currents of stimulation using the aforementioned controllers. For this study, Nickel-Titanium shape memory alloys were used to assess activation (contraction) through a two-coil system guided by the controllers. The application of the two-coil system demonstrated the effectiveness of the approach and a main effect was observed between the PI, MRAC, and ADP-PI controllers when following the trajectories. Lastly, a small scale two-coil system is developed for animal testing in the muscle-mass-spring setup. Experiments were successful in generating the appropriate stimulation controlled by the output-based algorithms for muscle contraction. Trials conducted for this study were compared to the muscle contractions observed in the first study. The controllers were able to provide appropriate stimulation to the muscle system to follow the set trajectories: a step, ramp, and sinusoidal input. More trials are required to draw statistical conclusions about the performance of each controller. Regardless, the small-scale two-coil system along with the applied controllers can be reconfigured to be an implantable system and tested for appropriate stimulation of the laryngeal muscles.
- Creating Human-Like Facial Expressions Utilizing Artificial Muscles and SkinTadesse, Yonas Tegegn (Virginia Tech, 2009-11-18)Mimicking facial structures for a robotic head requires integration of multiple structural and mechanical parameters, design, synthesis and control of muscle actuation, architecture of the linkages between actuation points within skin, and implementation of the deformation matrix with respect to global skull coordinates. In this dissertation, humanoid faces were designed and fabricated to investigate all the parameters mentioned above. A prototype face and neck was developed using servo motors and extensively characterized. In this prototype, a neck mechanism was designed using a four bar mechanism to achieve nodding and turning motions. The modular neck prototype simplifies the assembly and statically in equilibrium and hence demands less torque from the cost-effective RC servo motor. The mechanism was critically investigated for dynamic performance and it was found out that RC servo based robotic head requires a PD external controller to overcome inherent overshoot. The servo based robotic head was analyzed for design and control of anchor, architecture of linkages between actuation points within skin, and deformation matrix with respect to global coordinate for creating specific expressions. A functional relationship between deformation vector of facial control points and actuator parameter, skin elasticity and angular position of actuator was derived. The developed analysis method is applicable to any rotary actuator technology utilized for facial expressions and takes into account the skin stiffness. The artificial skin materials for facial expression were synthesized using platinum-cured silicone elastomeric material (Reynolds Advanced Materials Inc.) with base consisting of mainly polyorganosiloxanes, amorphous silica and platinum-siloxane complex compounds. Systematic incorporation of porosity in this material was found to lower the force required to deform the skin in the axial direction. The performance of the servo motor based face was quite realistic but it suffers from the drawback of large power consumption, bulky, heavy, and limited functionality. Thus, significant effort was made in developing a Biometal fiber and Flexinol shape memory alloy actuator (SMA) based biped mountable baby head facial structure which resembles the form and functionality of a human being. SMAs were embedded inside a skull and connected to elastomeric skin at control points. An engineered architecture of skull was fabricated that incorporates all the muscles with their 35 routine pulleys, two fire wire CMOS cameras that serve as eyes, and a battery powered microcontroller base driving circuit within the total dimensions of 140 mm x 90 mm x 110 mm. The driving circuit was designed such that it can be easily integrated with biped and processed in real-time. The humanoid face with 12DOF was mounted on the body of DARwIn (Dynamic Anthropomorphic Robot with Intelligence) robot which has 21 DOF resulting in a total of 33 DOF system. Characterization results on the face and associated design issues are described that provide pathways for developing human-like facial anatomy. Numerical simulation using Simulink was conducted to assess the performance of a prototypic robotic face mainly focusing on jaw movement. A graphical method “Graphical Facial Expression Analysis and Design (GFEAD)” was developed that can be used to allocate the sinking points on robotic head. The method assumes that the origin of the action units are known prior and the underlying criterion in the design of faces being deformation of a soft elastomeric skin through tension in anchoring wires attached on one end to the sinking point and on the other to the actuator. Experimental characterization on a prototyping humanoid face was performed to validate the model and demonstrate the applicability on a generic platform. During characterization of the SMA based face, it was found that the currently available artificial muscle technologies do not meet the entire requirement for being embedded in the skin and provide the required strain rate, maximum strain, blocking force, response time and energy density. Thus an effort was made to develop conducting polymer based artificial muscles which can meet the metrics of human muscle. Composite stripe and zigzag actuators consisting of a sandwich structure polypyrrole /poly(vinylidene difluoride) (PPy/PVDF) were synthesized using potentiodynamic film growth on gold electrodes. The synthesis was done from an aqueous solution containing tetrabutylammonium Perchlorate (TBAP) and pyrrole by polymerization at room temperature. For depositing thin PPy films and thereby minimizing the response time, an experimental optimization of the deposition conditions was performed. The number of current-potential (potentiodynamic) growth cycles and the thickness of the deposited PPy film were highly correlated in the initial stages of polymer film growth. Strip actuator of size 11 x 5 mm2 with 63μM exhibited a deflection of 3mm under 1V DC voltage and 2mm deflection under 8V AC voltage at 0.5 Hz. It was found that three-segment zigzag actuator of segment length 15x2.5mm and thickness 63μM amplifies the displacement by 1.5 times. A study was also conducted on the synthesis and characterization of thick and thin film polypyrrole (PPy) – metal composite actuators. The fabrication method consisted of three steps based upon the approach proposed by Ding et al.: (i) winding the conductive spiral structure around the platinum (Pt)-wire core, (ii) deposition of PPy film on the Pt-wire core, and (iii) removal of the Pt-wire core. This approach yielded good performance from the synthesized actuators, but was complex to implement due to the difficulty in implementing the third step. To overcome the problem of mechanical damage occurring during withdrawal of Pt-wire, the core was replaced with a dispensable gold coated polylactide fiber that could be dissolved at the end of deposition step. Experimental results indicate that thin film actuators perform better in terms of response time and blocking force. A unique muscle-like structure with smoothly varying cross-section was grown by combining layer by layer deposition with changes in position and orientation of the counter electrode in reference to the working electrode. Synthesis of polypyrrole–metal coil was conducted in aqueous solution containing 0.25 M Pyrrole, 0.10 M TBAP and 0.50 M KCl. The actuator consisted of a single layer of platinum winding on a core substrate. Electrochemical characterization for free strain and blocking stress was conducted 0.1 M TBAP solution and a 6% free strain was obtained at an applied potential of 6V DC after 80 s stimulation time. The blocking stress 18 kPa was estimated by extrapolating the strain magnitude on stress-strain diagram. For axial type actuator with coil winding, a generalized governing equation for the electrochemical stress generated from polypyrrole–metal coil which accommodates the effect of magnetic field due to winding was proposed and numerically studied. It was considered as insightful modeling.
- Defective excitation-contraction coupling is partially responsible for impaired contractility in hindlimb muscles of Stac3 knockout miceCong, Xiaofei; Doering, Jonathan; Grange, Robert W.; Jiang, Honglin (Nature, 2016-05-17)The Stac3 gene is exclusively expressed in skeletal muscle, and Stac3 knockout is perinatal lethal in mice. Previous data from Stac3-deleted diaphragms indicated that Stac3-deleted skeletal muscle could not contract because of defective excitation-contraction (EC) coupling. In this study, we determined the contractility of Stac3-deleted hindlimb muscle. In response to frequent electrostimulation, Stac3- deleted hindlimb muscle contracted but the maximal tension generated was only 20% of that in control (wild type or heterozygous) muscle (P < 0.05). In response to high [K⁺], caffeine, and 4-chloro-m-cresol (4-CMC), the maximal tensions generated in Stac3-deleted muscle were 29% (P < 0.05), 58% (P = 0.08), and 55% (P < 0.05) of those in control muscle, respectively. In response to 4-CMC or caffeine, over 90% of myotubes formed from control myoblasts contracted, but only 60% of myotubes formed from Stac3- deleted myoblasts contracted (P = 0.05). However, in response to 4-CMC or caffeine, similar increases in intracellular calcium concentration were observed in Stac3-deleted and control myotubes. Gene expression and histological analyses revealed that Stac3-deleted hindlimb muscle contained more slow type-like fibers than control muscle. These data together confirm a critical role of STAC3 in EC coupling but also suggest that STAC3 may have additional functions in skeletal muscle, at least in the hindlimb muscle.
- Development of a SNP Assay for the Differentiation of Allelic Variations in the mdx Dystrophic Mouse ModelMisyak, Sarah A. (Virginia Tech, 2008-04-21)The purpose of this study was to develop a SNaPshot® assay to simultaneously discriminate between the dystrophic and wild type (wt) alleles in mdx mice. The mdx mouse is an animal model for Duchenne muscular dystrophy (DMD), a severe and fatal muscle wasting disease. To evaluate possible treatments and to carry out genetic studies, it is essential to distinguish between mice that carry the mutant dystrophic or wt allele(s). The current Amplification-Resistant Mutation System (ARMS) assay used to genotype mdx mice is labor intensive and sometimes fails to yield typing results, which reduce its efficiency as a screening tool. An alternative assay based on single nucleotide polymorphism (SNP) extension technology (i.e., SNaPshot®) would be advantageous because its specificity and capability to be automated would reduce the labor involved and increase the fidelity of each assay. A SNaPshot® assay has been developed that provides a robust and potentially automatable assay that discriminates between the wt and dystrophic alleles. The assay has been optimized to use: an undiluted DNA in the PCR, a 0.1 µM PCR primer concentration, a full PCR product for the SNP extension reaction, a 50ºC annealing temperature for the SNP extension in accordance with standard SNaPshot® conditions, and a 0.4 µM concentration of the SNP extension primer. The advantages of the resultant SNaPshot® assay over the ARMS assay include higher fidelity, robustness, and more consistent performance within and among laboratories, and reduced risk of human error.
- Dietary Conjugated Linoleic Acid Reduces Body Weight and Fat in Snord116m+/p- and Snord116m-/p- Mouse Models of Prader-Willi SyndromeKnott, Brittney; Kocher, Matthew A.; Paz, Henry A.; Hamm, Shelby E.; Fink, William; Mason, Jordan; Grange, Robert W.; Wankhade, Umesh D.; Good, Deborah J. (MDPI, 2022-02-18)Prader–Willi Syndrome (PWS) is a human genetic condition that affects up to 1 in 10,000 live births. Affected infants present with hypotonia and developmental delay. Hyperphagia and increasing body weight follow unless drastic calorie restriction is initiated. Recently, our laboratory showed that one of the genes in the deleted locus causative for PWS, Snord116, maintains increased expression of hypothalamic Nhlh2, a basic helix–loop–helix transcription factor. We have previously also shown that obese mice with a deletion of Nhlh2 respond to a conjugated linoleic acid (CLA) diet with weight and fat loss. In this study, we investigated whether mice with a paternal deletion of Snord116 (Snord116m+/p−) would respond similarly. We found that while Snord116m+/p− mice and mice with a deletion of both Snord116 alleles were not significantly obese on a high-fat diet, they did lose body weight and fat on a high-fat/CLA diet, suggesting that the genotype did not interfere with CLA actions. There were no changes in food intake or metabolic rate, and only moderate differences in exercise performance. RNA-seq and microbiome analyses identified hypothalamic mRNAs, and differentially populated gut bacteria, that support future mechanistic analyses. CLA may be useful as a food additive to reduce obesity in humans with PWS.
- Differential Requirement for Utrophin in the Induced Pluripotent Stem Cell Correction of Muscle versus Fat in Muscular Dystrophy MiceBeck, Amanda J.; Vitale, Joseph M.; Zhao, Qingshi; Schneider, Joel S.; Chang, Corey; Altaf, Aneela; Michaels, Jennifer; Bhaumik, Mantu; Grange, Robert W.; Fraidenraich, Diego (PLOS, 2011-05-16)Duchenne muscular dystrophy (DMD) is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs) into mdx and mdx∶utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx <<< mdx∶utrophin). In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdx∶utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdx∶utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdx∶utrophin chimeras, despite the fact that utrophin was compromised in the mdx∶utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent) non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle.
- Dystrophin-deficient dogs with reduced myostatin have unequal muscle growth and greater joint contracturesKornegay, Joe N.; Bogan, Daniel J.; Bogan, Janet R.; Dow, Jennifer L.; Wang, Jiahui; Fan, Zheng; Liu, Naili; Warsing, Leigh C.; Grange, Robert W.; Ahn, Mihye; Balog-Alvarez, Cynthia J.; Cotten, Steven W.; Willis, Monte S.; Brinkmeyer-Langford, Candice; Zhu, Hongtu; Palandra, Joe; Morris, Carl A.; Styner, Martin A.; Wagner, Kathryn R. (2016-04-04)Background Myostatin (Mstn) is a negative regulator of muscle growth whose inhibition promotes muscle growth and regeneration. Dystrophin-deficient mdx mice in which myostatin is knocked out or inhibited postnatally have a less severe phenotype with greater total mass and strength and less fibrosis and fatty replacement of muscles than mdx mice with wild-type myostatin expression. Dogs with golden retriever muscular dystrophy (GRMD) have previously been noted to have increased muscle mass and reduced fibrosis after systemic postnatal myostatin inhibition. Based partly on these results, myostatin inhibitors are in development for use in human muscular dystrophies. However, persisting concerns regarding the effects of long-term and profound myostatin inhibition will not be easily or imminently answered in clinical trials. Methods To address these concerns, we developed a canine (GRippet) model by crossbreeding dystrophin-deficient GRMD dogs with Mstn-heterozygous (Mstn +/−) whippets. A total of four GRippets (dystrophic and Mstn +/−), three GRMD (dystrophic and Mstn wild-type) dogs, and three non-dystrophic controls from two litters were evaluated. Results Myostatin messenger ribonucleic acid (mRNA) and protein levels were downregulated in both GRMD and GRippet dogs. GRippets had more severe postural changes and larger (more restricted) maximal joint flexion angles, apparently due to further exaggeration of disproportionate effects on muscle size. Flexors such as the cranial sartorius were more hypertrophied on magnetic resonance imaging (MRI) in the GRippets, while extensors, including the quadriceps femoris, underwent greater atrophy. Myostatin protein levels negatively correlated with relative cranial sartorius muscle cross-sectional area on MRI, supporting a role in disproportionate muscle size. Activin receptor type IIB (ActRIIB) expression was higher in dystrophic versus control dogs, consistent with physiologic feedback between myostatin and ActRIIB. However, there was no differential expression between GRMD and GRippet dogs. Satellite cell exhaustion was not observed in GRippets up to 3 years of age. Conclusions Partial myostatin loss may exaggerate selective muscle hypertrophy or atrophy/hypoplasia in GRMD dogs and worsen contractures. While muscle imbalance is not a feature of myostatin inhibition in mdx mice, findings in a larger animal model could translate to human experience with myostatin inhibitors.
- The Effect of AICAR Treatment on Sarcoplasmic Reticulum Function and Possible Links to Skeletal Muscle FatigueVidt, Stacey Elizabeth (Virginia Tech, 2007-05-21)A compelling mystery in the study of exercise is mechanisms of skeletal muscle fatigue. Broadly described, muscle fatigue is the uncomfortable sensation that particular muscle groups are shutting down and muscle force production cannot continue. More specifically, muscle fatigue is defined as an activity-induced inability to continue to produce a desired level of force. Several groups suggest that a major cause of force loss during fatigue is reductions in the rates of sarcoplasmic reticulum (SR) calcium (Ca2+) release and uptake. These changes result in diminished contractile machinery activation, reduced force production and slowed relaxation. During exercise, adenosine 5'-triphosphate (ATP) is the energy currency that is used to support force production. As a result of ATP hydrolysis and re-synthesis, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) levels rise. AMP kinase (AMPK) is an enzyme that becomes activated as a result of increased AMP levels. It is thought to function as a metabolic "master switch" within the muscle and plays a major role in carbohydrate and fat metabolism. Once AMPK is activated it regulates several ATP consuming and producing pathways. The overall objective of this project was to determine if increased metabolism during exercise contributes to SR Ca2+ dysfunction during fatigue. If this is true, artificial activation of AMPK via 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) should induce changes in SR function that are qualitatively similar to those caused by fatigue. In study 1, mice were injected with 0.85 mg/kg AICAR (or saline solution) and both gastrocnemius muscles were removed one hour later. In study 2, EDL muscles were placed in a muscle bath and incubated in AICAR (4mM) or stimulated to fatigue. Glycogen, glucose-6-phosphate (G-6-P), ATP, ADP, and phosphocreatine (PCr) were examined in all groups of muscles. Alterations in SR calcium uptake and release rates due to the presence of AICAR were also studied as a likely cause of muscle fatigue. AICAR treatment in vivo did not alter muscle glycogen, glucose, ATP, ADP or PCr concentrations. However, G-6-P levels were increased by 137%. This was accompanied by a 36% reduction in the SR Ca2+ uptake rate and a 42% reduction in Ca2+-stimulated Ca2+ ATPase activity as well as 13-15% reductions in the rates of Ca2+ release. These changes were not associated with SR Ca2+ pump content. Administration of AICAR in vitro also increased G-6-P content (200%) without altering the concentrations of glycogen, glucose, G-6-P, ATP, ADP or PCr. AICAR decreased SR Ca2+ uptake rate by 28% and the rate of Ca2+ release by 16%. For comparison, fatiguing stimulation reduced the rates of SR Ca2+ uptake and release by 31 and 41%, respectively. Taken together, these results indicate that when administered to skeletal muscle both in vivo and in vitro, AICAR evokes metabolic stress. More importantly, activation of AMPK alters skeletal muscle SR function to an extent that is qualitatively similar to that caused by fatiguing activity. At present, it is not clear how AMPK activation causes changes in SR function. However, the present finding is consistent with the notion that metabolic stress caused by exercise, affects SR function. This, in turn, leads to force loss but reduces energy demand and protects the cell from ATP depletion during maximal contractile activity.
- The Effects of Dha Supplementation on Markers of Inflammation and Muscle Damage Following an Acute Eccentric Exercise BoutDiLorenzo, Frank Michael (Virginia Tech, 2012-07-09)Aim: The purpose of this study was to investigate the influence of docosahexaenoic acid (DHA) on muscle damage and inflammation following an acute eccentric exercise bout. Methods: A double-blind placebo-controlled, study was performed using 41 healthy, untrained males aged 18-28 y who consumed either 2 g/d DHA or placebo (PL, corn oil) for 32 days. Supplements were consumed for 28 days prior to exercise. Participants completed an eccentric exercise procedure of the elbow flexors at 140% of 1-RM (6 sets x 10 repetitions). The time under tension (TUT) for each set of eccentric contractions was recorded manually from the investigators voice commands. Fasted blood samples for prostaglandin E2 (PGE2), interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL1-ra), C-reactive protein and creatine kinase (CK) were assessed on days 1, 2 and 4. Fasted serum DHA was measured at baseline (day -28) and on day 1. Peak isometric strength of the elbow flexors, delayed-onset muscle soreness, and range of motion were measured on day 1 prior to exercise and days 2, 3, and 4 following exercise. Results: DHA significantly reduced natural log of CK (p<0.05) response over 4 d. Additionally, IL-6 area under the curve (AUC) was reduced for DHA compared to PL (3.6 ± 2.5 pg/mL vs. 5.3 ± 2.7 pg/mL) (p<0.05). TUT/set was higher in the DHA group compared to placebo (p<0.05). There were no other significant differences between treatments. Conclusion: DHA supplementation produced lower indicators of muscle damage (CK) and inflammation (IL-6 AUC). DHA supplementation resulted in greater TUT/set.
- The Effects of Job Rotation Parameters on Localized Muscle Fatigue and Performance: An Investigation of Rotation Frequency and Task OrderHorton, Leanna Marie (Virginia Tech, 2012-04-17)Work-related musculoskeletal disorders (WMSDs) remain a substantial problem in the workplace. Rotation, in which workers are rotated between tasks, is widely used as an administrative control, as it is considered to reduce WMSD risk through reducing physical exposures and increasing exposure variation. However, despite its widespread use, there is limited evidence that rotating between tasks is effective in reducing the risk of WMSDs. Inconsistencies in measured outcomes of rotation may be attributed to the variety of parameters involved in determining rotation schedules, including which tasks to include in a schedule, the rate at which workers rotate, and the order in which tasks are performed. This research assessed the effects of rotation, specifically focusing on rotation frequency and task order, on muscle fatigue and performance when included tasks loaded the same muscle group. Twelve participants completed six experimental sessions in each of three studies, during which repetitive tasks were performed for one hour either with or without rotation. Each study simulated a different task, including static shoulder abduction, box lifting, and a light assembly task. Rotation occurred between lower and higher exertion levels, and each rotation schedule varied in both rotation frequency (rotating every 15 minutes vs. 30 minutes) and task order (starting with the lower vs. higher intensity task). Muscle fatigue was assessed through several measures, including electromyography, and ratings of perceived discomfort. Performance was assessed through the accuracy of shoulder moment output, the accuracy of box placement, or the speed of assembly completion. As expected, rotation was effective in reducing fatigue compared to higher intensity tasks with no rotation, although it increased fatigue compared to the lower intensity with no rotation. While effects of rotation frequency and task order were seen on some measures, results across all three studies did not indicate consistent effects of either rotation frequency or task order on fatigue or performance. As such, the practical relevance of these rotation parameters and the likely impacts of rotation are not yet clear, and further assessments are needed. Such assessments should ideally involve longer durations, field studies, and/or more direct measures of injury or injury risk.
- The Effects of Low Dose Endotoxin on Glucose HomeostasisStevens, Joseph R. (Virginia Tech, 2014-08-28)Obese individuals present with an increased inflammatory tone as compared to healthy, normal-weight individuals, which is associated with insulin resistance. One factor hypothesized to contribute to increased inflammation in obese and diabetic states is elevated blood endotoxin levels, also known as metabolic endotoxemia. In healthy rodents (non-obese and insulin sensitive), there is evidence that blood endotoxin levels fluctuate over the course of the day with elevations in the post-prandial state that return to baseline levels in the post-absorptive state. High-fat feeding in these animals altered these fluctuations causing endotoxin levels to remain high throughout the day. The effects of alterations in endotoxin levels on glucose metabolism are not understood. The goal of this study was to determine the effects of short-term and long-term increases in endotoxin of a low magnitude on insulin signaling in a human primary cell line as well as the effects of short-term endotoxin treatments on glucose homeostasis in a C57/Bl6 mouse model. First, we tested the hypothesis in cell culture that short-term low-dose endotoxin treatments would enhance insulin-signaling and glycogen synthesis while long-term treatments would have inhibitory effects. Under our second hypothesis, we examined whether short-term low-dose treatments of endotoxin would contribute to improvements in glucose tolerance in a mouse model. In contrast to our first hypothesis, short-term endotoxin treatments did not improve insulin signaling or glycogen synthesis although long-term treatments did contribute to decreases in glycogen synthesis. Interestingly, short-term endotoxin treatments resulted in significant improvements in glucose clearance in the mouse model; this is believed to be partly attributed to LPS inhibiting gluconeogenesis. Future studies are necessary to understand the mechanisms responsible for altered glucose metabolism in response to low magnitude changes in LPS levels.
- Effects of Perfusate Composition (Na+ and Ca2+) on Cardiac Electrical and Mechanical Function in the Isolated Langendorff-Perfused HeartKing, David Ryan (Virginia Tech, 2021-02-11)Following the landmark studies on scientific reproducibility, or the lack thereof, by Bayer and Amgen in the past decade, there has been a renewed interest in scientific rigor and reproducibility in both the scientific and public media. In several recent reports, the high attrition rate observed in clinical trials has been attributed to irreproducibility at the preclinical level. Cardiology is no exception to this rule. In our systematic review of the ex vivo Langendorff-perfused heart, we found methods reporting to be sparse at best, specifically as it pertains to documenting the ex vivo perfusate compositions employed in the Langendorff heart. Our lab has demonstrated that variation in perfusate compositions can unmask disease states in genetically modified animals. In this dissertation, we exploit this concept with a therapeutic end-point in mind. We show that perfusate variation, specifically sodium and calcium elevations, can attenuate conduction slowing associated with severe hyperkalemia. Likewise, elevating sodium is capable of sustaining intrinsic rhythm where hearts would otherwise go asystolic. In doing so, elevated sodium prevents repolarization prolongation in these hearts. Together, these studies would suggest that elevating extracellular sodium, and calcium, should be considered as therapeutic targets in the context of conduction defects. However, when considering the heart's primary role as a pump, we found that elevating sodium actually depresses cardiac mechanical function. This is both in a pre- and post-ischemic setting. In short, we show that electrolyte variation may influence both cardiac electrophysiology and contraction; however, an improvement in one does not guarantee an improvement in both. Maintaining proper cardiac physiological function is a complex process that is tightly regulated by the ionic makeup of the extracellular environment. To improve insights from preclinical studies at the clinical level it is paramount that researchers properly document methods so that any significant results may be properly interpreted in clinical trial design.
- The Effects of Resistance Wheel Running on Skeletal Muscle Function and Adaptation in C57BL/10SnJ MiceRodden, Gregory Robert (Virginia Tech, 2015-07-21)Background: Resistance wheel running (RWR) can promote resistance-like training adaptations in mouse skeletal muscle (SkM), but its endurance-training effects are lesser known. Methods: Voluntary RWR was modulated as an exercise model to increase mouse hind-limb plantar-flexor torque and to promote endurance-training adaptations. Thirty male mice (cohort 1, n= 16; cohort 2, n= 14), were trained on a prototype RWR system that applied resistance relative to body mass (BM). Mice were sequentially, (1) screened for running ability (screening; 3-days); (2) trained with incremental adjustments to wheel loads (pre-training; 8-weeks); (3) grouped into cage-activity only (CA), and constant Low-0%, Med-15%, or High-25% BM resistance conditions (static training; 5-weeks); (4) trained with resistance adjusted in real-time (dynamic training; cohort 1, 7-weeks; cohort 2, 10-weeks); and (5) sacrificed for various assays. Plantar-flexor torque was determined during each training phase. After dynamic training, resistance runners in each cohort were sub-grouped post-hoc by work tertiles. Results: Wheel running distance varied between cohorts (cohort 2 > 1). During dynamic training, wheel running (±added-resistance) improved plantar flexor torque normalized to BM by 19% only in cohort 2 (p= 0.007). Muscle mass and cross-sectional area were unchanged. Runners in both cohorts (±added-resistance) improved maximal running capacity vs. CA-controls (+69% and +115%; both p < 0.05), but metabolic training adaptations were less evident. Conclusions: Wheel running promoted SkM strength and endurance, but there was a greater increase in endurance capacity than strength. This outcome may be due to adaptive signaling interference.
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