Hindlimb Skeletal Muscle Research Articles

Genome-wide Associations Reveal Human-Mouse Genetic Convergence and Modifiers of Myogenesis, CPNE1 and STC2

Muscle bulk in adult healthy humans is highly variable even after height, age, and sex are accounted for. Low muscle mass, due to fewer and/or smaller constituent muscle fibers, would exacerbate the impact of muscle loss occurring in aging or disease. Genetic variability substantially influences muscle mass differences, but causative genes remain largely unknown. In a genome-wide association study (GWAS) on appendicular lean mass (ALM) in a population of 85,750 middle-aged (aged 38-49 years) individuals from theUKBiobank(UKB), we found 182 loci associated with ALM (p < 5× 10-8). We replicated associations for 78% of these loci (p<5× 10-8) with ALM in a population of 181,862 elderly (aged 60-74 years) individuals from UKB. We also conducted a GWAS on hindlimb skeletal muscle mass of 1,867 mice from an advanced intercross between two inbred strains (LG/J and SM/J); this GWAS identified 23 quantitative trait loci. Thirty-eight positional candidates distributed across five loci overlapped between the two species. Invitro studies of positional candidates confirmed CPNE1 and STC2 as modifiers of myogenesis. Collectively, these findings shed light on the genetics of muscle mass variability in humans and identify targets for the development of interventions for treatment of muscle loss. The overlapping results between humans and the mouse model GWAS point to shared genetic mechanisms across species.

Antioxidant Effect of Sechium edule (Chayote) Supplement Intake in Older Adults with Metabolic Syndrome: An Exploratory Study

Chlorpyrifos (CPF) is a toxic organophosphate commonly used worldwide. Its residues are being detected in different environmental matrixes and hence in the food chain. Repeated CPF exposure might pose health risk for the general population on long term. This data article contains the data of contractility impairment further to dietary exposure to CPF on a hind limb skeletal muscle; soleus, a typical slow twitch skeletal muscle. Thirty adult male rats Sprague Dawley are divided into three groups receiving the following daily diet for 6 weeks: Group 1 (vehicle), Group 2: CPF1 (CPF 1mg/kg/day) and Group 3: CPF5 (CPF 5 mg/kg/day). Soleus twitch tension and fatigability index are determined at the end of the treatment. The activity of acteylcholinesterase enzyme is assessed in the tissues homogenate. Additionally, we examined the expression levels of ryanodine type 1 receptor (RyR1), ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 1 (Atp2a1), ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 2 (Atp2a2) and nicotinic acetylcholine receptor (nAChR) in CPF-exposed skeletal muscle tissue using quantitative real time polymerase chain reaction.CPF exposure at two different doses induced an increase in twitch contraction in soleus muscle along with an increase in fatigability index. These increases are accompanied by low level of acetylcholinesterase enzyme activity as well as modification in genes level expression of nAChR, RyR1, Atp2a1 and Atp2a2 involved in contractility.

FDG uptake tracks the oxidative damage in diabetic skeletal muscle: An experimental study

ObjectivesThe present study aims to verify the relationship between glucose consumption and uptake of 18F-2-deoxy-glucose (FDG) in the skeletal muscle (SM) of experimental models of streptozotocin-induced diabetes mellitus (STZ-DM). MethodsThe study included 36 Balb/c mice. Two weeks after intraperitoneal administration of saline (control group, n = 18) or 150 mg streptozotocin (STZ-DM group, n = 18), the two cohorts were submitted to an oral glucose tolerance test and were further subdivided into three groups (n = 6 each): untreated and treated with metformin (MTF) at low or high doses (10 or 750 mg/kg daily, respectively). Two weeks thereafter, all mice were submitted to dynamic micro–positron emission tomography (PET) imaging after prolonged fasting. After sacrifice, enzymatic pathways and response to oxidative stress were evaluated in harvested SM. ResultsOn PET imaging, the FDG uptake rate in hindlimb SM was significantly lower in nondiabetic mice as compared with STZ-DM–untreated mice. MTF had no significant effect on SM FDG uptake in untreated mice; however, its high dose induced a significant decrease in STZ-DM animals. Upon conventional analysis, the SM standard uptake value was higher in STZ-DM mice, while MTF was virtually ineffective in either control or STZ-DM models. This metabolic reprogramming was not explained by any change in cytosolic glucose metabolism. By contrast, it closely agreed with the catalytic function of hexose-6P-dehydrogenase (H6PD; i.e., the trigger of a specific pentose phosphate pathway selectively located within the endoplasmic reticulum). In agreement with this role, the H6PD enzymatic response to both STZ-DM and MTF matched the activation of the NADPH-dependent antioxidant responses to the increased generation of reactive oxygen species caused by chronic hyperglycemia. Ex vivo analysis of tracer kinetics confirmed that the enhanced SM avidity for FDG occurred despite a significant reduction in glucose consumption, while it was associated with increased radioactivity transfer to the endoplasmic reticulum. ConclusionsThese data challenge the current dogma linking FDG uptake to the glycolytic rate. They instead introduce a new model considering a strict link between the uptake of this glucose analog, H6PD reticular activity, and oxidative damage in diabetes, at least under fasting condition.

PP-115 - Extended half-life product (rFVIIIFC) for treatment of children with hemophilia A: preliminary post-switch experience from a tertiary care center

Chlorpyrifos (CPF) is a toxic organophosphate commonly used worldwide. Its residues are being detected in different environmental matrixes and hence in the food chain. Repeated CPF exposure might pose health risk for the general population on long term. This data article contains the data of contractility impairment further to dietary exposure to CPF on a hind limb skeletal muscle; soleus, a typical slow twitch skeletal muscle. Thirty adult male rats Sprague Dawley are divided into three groups receiving the following daily diet for 6 weeks: Group 1 (vehicle), Group 2: CPF1 (CPF 1mg/kg/day) and Group 3: CPF5 (CPF 5 mg/kg/day). Soleus twitch tension and fatigability index are determined at the end of the treatment. The activity of acteylcholinesterase enzyme is assessed in the tissues homogenate. Additionally, we examined the expression levels of ryanodine type 1 receptor (RyR1), ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 1 (Atp2a1), ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 2 (Atp2a2) and nicotinic acetylcholine receptor (nAChR) in CPF-exposed skeletal muscle tissue using quantitative real time polymerase chain reaction.CPF exposure at two different doses induced an increase in twitch contraction in soleus muscle along with an increase in fatigability index. These increases are accompanied by low level of acetylcholinesterase enzyme activity as well as modification in genes level expression of nAChR, RyR1, Atp2a1 and Atp2a2 involved in contractility.

Investigation of the mechanisms of cyclooxygenase-mediated mechanoreflex sensitization in a rat model of simulated peripheral artery disease.

Mechanical and metabolic stimuli within contracting skeletal muscles reflexly increase sympathetic nervous system activity and blood pressure. That reflex, termed the exercise pressor reflex, is exaggerated in patients with peripheral artery disease (PAD) and in a rat PAD model with a chronically ligated femoral artery. The cyclooxygenase (COX) pathway contributes to the exaggerated pressor response during rhythmic skeletal muscle contractions in patients with PAD, but the specific mechanism(s) of the COX-mediated exaggeration are not known. In decerebrate, unanesthetized rats with a chronically ligated femoral artery ("ligated" rats), we hypothesized that hindlimb arterial injection of the COX inhibitor indomethacin would reduce the pressor response during 1-Hz dynamic hindlimb skeletal muscle stretch; a model of the activation of the mechanical component of the exercise pressor reflex (i.e., the mechanoreflex). In ligated rats (n = 7), indomethacin reduced the pressor response during stretch (control: 30 ± 4; indomethacin: 12 ± 3 mmHg; P < 0.01), whereas there was no effect in rats with "freely perfused" femoral arteries (n = 6, control: 18 ± 5; indomethacin: 17 ± 5 mmHg; P = 0.87). In ligated rats (n = 4), systemic indomethacin injection had no effect on the pressor response during stretch. Femoral artery ligation had no effect on skeletal muscle COX protein expression or activity or concentration of the COX metabolite prostaglandin E2. Conversely, femoral artery ligation increased expression of the COX metabolite receptors endoperoxide 4 and thromboxane A2-R in dorsal root ganglia tissue. We conclude that, in ligated rats, the COX pathway sensitizes the peripheral endings of mechanoreflex afferents, which occurs principally as a result of increased expression of COX metabolite receptors.NEW & NOTEWORTHY We demonstrate that the mechanoreflex is sensitized by the cyclooxygenase (COX) pathway within hindlimb skeletal muscles in the rat chronic femoral artery ligation model of simulated peripheral artery disease (PAD). The mechanism of sensitization appears attributable to increased receptors for COX metabolites on sensory neurons and not increased concentration of COX metabolites. Our data may carry important clinical implications for patients with PAD who demonstrate exaggerated increases in blood pressure during exercise compared with healthy counterparts.

Placental growth factor levels in quadriceps muscle are reduced by a Western diet in association with advanced glycation end products.

In peripheral artery disease (PAD), atherosclerotic occlusion chronically impairs limb blood flow. Arteriogenesis (collateral artery remodeling) is a vital adaptive response to PAD that protects tissue from ischemia. People with type II diabetes have a high risk of developing PAD and would benefit from arteriogenesis. However, arteriogenesis is suppressed in people with diabetes by a multifaceted mechanism which remains incompletely defined. Upregulation of placental growth factor (PLGF) is a key early step in arteriogenesis. Therefore, we hypothesized that metabolic dysfunction would impair PLGF expression in skeletal muscle. We tested this hypothesis in C57BL/6J and ApoE-/- mice of both sexes fed a Western diet (WD) for 24 wk. We first assessed baseline levels of PLGF, vascular endothelial growth factor (VEGF-A), and VEGF receptor 1 (VEGFR1) protein in hindlimb skeletal muscle. Only PLGF was consistently decreased by the WD. We next investigated the effect of 24 wk of the WD on the response of PLGF, VEGF-A, VEGFR1, and monocyte chemoattractant protein-1 (MCP-1) to the physiological stimulus of vascular occlusion. Hindlimb ischemia was induced in mice by gradual femoral artery occlusion using an ameroid constrictor. Growth factor levels were measured 3-28 days postsurgery. In C57BL/6J mice, the WD decreased and delayed upregulation of PLGF and abolished upregulation of VEGF-A and VEGFR1 but had no effect on MCP-1. In ApoE-/- mice fed either diet, all factors tested failed to respond to occlusion. Metabolic phenotyping of mice and in vitro studies suggest that an advanced glycation end product/TNFα-mediated mechanism could contribute to the effects observed in vivo.NEW & NOTEWORTHY In this study, we tested the effect of a Western diet on expression of the arteriogenic growth factor placental growth factor (PLGF) in mouse skeletal muscle. We provide the first demonstration that a Western diet interferes with both baseline expression and hindlimb ischemia-induced upregulation of PLGF. We further identify a potential role for advanced glycation end product/TNFα signaling as a negative regulator of PLGF. These studies provide insight into one possible mechanism by which type II diabetes may limit collateral growth.

Assessment of Age-related Oxygenation Changes in Calf Skeletal Muscle by Photoacoustic Imaging: A Potential Tool for Peripheral Arterial Disease.

Peripheral artery disease is often asymptomatic, and various imaging and nonimaging techniques have been used for assessment and monitoring treatments. This study is designed to demonstrate the ability of photoacoustic imaging to noninvasively determine changes in tissue oxygenation that occur in mice's hind limb skeletal muscle as they age. Mice from two age cohorts were scanned bilaterally with a pulsed laser. The photoacoustic signal was unmixed to generate a parametric map of estimated oxygen saturation and then overlaid on grayscale ultrasound images. Tissue oxygenation measured in young and old mice was compared. Photoacoustic imaging visually and quantitatively showed the decrease in skeletal muscle oxygenation that occurs with age. Percent tissue oxygenation decreased from 30.2% to 3.5% (p < 0.05). This reduction corresponded to reduced fractional area of oxygenation, which decreased from 60.6% to 6.0% (p < 0.05). The change in oxygenation capacity of the still active vascular regions was insignificant (p > 0.05). Intrasubject, intra-, and interobserver comparisons showed low variability in measurements, exhibited by high regression and intraclass correlations exceeding 0.81 for all ages. The decrease in oxygenation detected by photoacoustic imaging paralleled the known oxygenation decrease observed in aging tissues, demonstrating that photoacoustic imaging can assess age-related changes in a mouse calf muscle. These intramuscular changes could potentially act as a strong diagnostic marker for peripheral artery disease. This study thus opens the doors for a novel, affordable, noninvasive method of evaluation free of radiation or exogenous material.

Cyclooxygenase inhibition does not impact the pressor response during static or dynamic mechanoreflex activation in healthy decerebrate rats.

Passive limb movement and limb muscle stretch in humans and animals are common experimental strategies used to investigate activation of the muscle mechanoreflex independent of contraction-induced metabolite production. Cyclooxygenase (COX) metabolites, however, are produced by skeletal muscle stretch in vitro and have been found to impact various models of mechanoreflex activation. Whether COX metabolites influence the decerebrate rat triceps surae muscle stretch mechanoreflex model remains unknown. We examined the effect of rat triceps surae muscle stretch on the interstitial concentration of the COX metabolite prostaglandin E2 (PGE2). Interstitial PGE2 concentration was increased above baseline values by 4 min of both static (38% increase, P = 0.01) and dynamic (56% increase, P < 0.01) triceps surae muscle stretch (n = 10). The 4-min protocol was required to collect enough microdialysis fluid for PGE2 detection. The finding that skeletal muscle stretch in vivo was capable of producing COX metabolites prompted the hypothesis that intra-arterial administration of the COX inhibitor indomethacin (1 mg/kg) would reduce the pressor and cardioaccelerator responses evoked during 30 s (the duration most commonly used in the rat mechanoreflex model) of static and dynamic rat triceps surae muscle stretch. We found that indomethacin had no effect (P > 0.05, n = 9) on the pressor or cardioaccelerator response during 30 s of either static or dynamic stretch. We conclude that, despite the possibility of increased COX metabolite concentration, COX metabolites do not activate or sensitize thin-fiber muscle afferents stimulated during 30 s of static or dynamic hindlimb skeletal muscle stretch in healthy rats.

292-LB: The Exercise-Like Impacts of Conditioned Media Harvested from Cultured Dental Pulp Stem Cells on Myokine Expressions in Skeletal Muscles

Exercise improves not only glycemic control, but also other metabolic disorders in diabetic patients. Furthermore, exercise also stimulate muscle-derived secreted factors, myokines, via muscle contraction. The receptors of myokines expressed in the whole body and myokines affect autocrine, paracrine, and systemic functions for all people and patients with diabetes. In this study, we have demonstrated that there are exercise-like impacts of conditioned media from cultured dental pulp stem cells (DPSCs) on myokine expressions in skeletal muscles. DPSCs were isolated and cultured from the mandibular incisors of 6-week-old male Sprague-Dawley (SD) rats. Conditioned media from DPSCs (DPSC-CM) was collected after 24-hour culture of DPSCs in the serum-free medium and concentrated by a factor of 10. We firstly investigated the effects of DPSC-CM on exercise-induced myokines, follistatin-like 1 (Fstl1), Fractalkine, and fibroblast growth factor 21 (FGF-21) in C2C12 myotubes. DPSC-CM significantly increased the gene expressions of Fstl1 (209±16%, P&amp;lt;0.01), Fractalkine(182±41%, P&amp;lt;0.05), and FGF-21 (1021±39%, P&amp;lt;0.01) in C2C12 myotubes. To confirm these effects in vivo, we administered DPSC-CM (1.0 ml/rat) into 10 sites of unilateral hindlimb skeletal muscles of male SD rats, and examined the effects on myokine gene and protein expression. DPSC-CM significantly increased Fstl1 gene expression in the gastrocnemius muscles (477±140%, P&amp;lt;0.05). Immunohistochemical staining revealed that the expression of Fstl1 was increased in the DPSC-CM-injected gastrocnemius muscles. These results suggest that there is a novel exercise-like impact of DPSC-CM on skeletal muscles. Disclosure S. Kanada: None. E. Makino: None. N. Nakamura: None. M. Miyabe: None. M. Ito: None. M. Hata: None. T. Saiki: None. T. Minato: None. K. Miyazawa: None. S. Goto: None. T. Matsubara: None. K. Naruse: None.

Phosphorylated ERK1/2 protein levels are closely associated with the fast fiber phenotypes in rat hindlimb skeletal muscles.

The relationship between the extracellular signal-regulated kinase 1 and 2 (ERK1/2), one of the mitogen-activated protein kinases (MAPKs), and mammalian skeletal muscle fiber phenotype is unclear. We looked at this relationship in three in vivo conditions in male Wistar rats. First, the levels of phosphorylated (active) ERK1/2 protein were closely associated with the fiber type composition of sedentary rat hindlimb muscles: highest in the superficial portion of the gastrocnemius (100% fast fibers), lower in the plantaris (~ 80% fast fibers), and lowest in the soleus (~ 15% fast fibers). Second, during growth, there was a gradual decrease in the percentage of fast fibers from 40% at 3weeks to 1.5% at 65weeks and a concomitant gradual decrease in the levels of phosphorylated ERK1/2 in the soleus muscle. Third, sciatic nerve denervation induced a significant decrease in the weight of both the soleus and plantaris, but a slow-to-fast fiber type shift and increase in phosphorylated ERK1/2 protein were observed only in the soleus. Although only a few fast and fast + slow hybrid fibers of the denervated soleus muscle reacted positively to the anti-phosphorylated ERK1/2 antibody by immuno-histochemical analysis, our results suggest that the phosphorylated form of ERK1/2 seems to be closely related to the fast fiber phenotype program. Further evidence for this relationship was provided by the observation that several slow fiber phenotype-specific proteins, i.e., Hsp72, Hsp60, and PGC-1, changed in the opposite direction of the levels of phosphorylated ERK1/2 protein.

Conditioned media from dental pulp stem cells improved diabetic polyneuropathy through anti-inflammatory, neuroprotective and angiogenic actions: Cell-free regenerative medicine for diabetic polyneuropathy.

Aims/IntroductionDental pulp stem cells (DPSCs) can be easily obtained from teeth for general orthodontic reasons. We have previously reported the therapeutic effects of DPSC transplantation for diabetic polyneuropathy. As abundant secretomes from DPSCs are considered to play a central role in the improvement of diabetic polyneuropathy, we investigated whether direct injection of DPSC‐conditioned media (DPSC‐CM) into hindlimb skeletal muscles ameliorates diabetic polyneuropathy in diabetic rats.Materials and MethodsDPSCs were isolated from the dental pulp of Sprague–Dawley rats. Eight weeks after the induction of diabetes, DPSC‐CM was injected into the unilateral hindlimb skeletal muscles in both normal and diabetic rats. The effects of DPSC‐CM on diabetic polyneuropathy were assessed 4 weeks after DPSC‐CM injection. To confirm the angiogenic effect of DPSC‐CM, the effect of DPSC‐CM on cultured human umbilical vascular endothelial cell proliferation was investigated.ResultsThe administration of DPSC‐CM into the hindlimb skeletal muscles significantly ameliorated sciatic motor/sensory nerve conduction velocity, sciatic nerve blood flow and intraepidermal nerve fiber density in the footpads of diabetic rats. We also showed that DPSC‐CM injection significantly increased the capillary density of the skeletal muscles, and suppressed pro‐inflammatory reactions in the sciatic nerves of diabetic rats. Furthermore, an in vitro study showed that DPSC‐CM significantly increased the proliferation of umbilical vascular endothelial cells.ConclusionsWe showed that DPSC‐CM injection into hindlimb skeletal muscles has a therapeutic effect on diabetic polyneuropathy through neuroprotective, angiogenic and anti‐inflammatory actions. DPSC‐CM could be a novel cell‐free regenerative medicine treatment for diabetic polyneuropathy.

MON-LB038 Reduced Exercise Endurance And Skeletal Muscle Alterations In A Prenatally Androgenized Rat Model For Polycystic Ovary Syndrome

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women, impacting female reproductive, cardiometabolic, and psychological health. Studies of metabolic dysfunction in women with PCOS have primarily focused on adipose tissue and obesity-related changes. Some studies have reported changes in lean body mass and muscle function in women with PCOS. OBJECTIVE: To determine if prenatally androgenized (PNA) female rats exhibit altered exercise endurance or differences in skeletal muscle mass or morphology compared to control rats. METHODS: Sprague-Dawley rats were prenatally exposed to 5 mg testosterone or vehicle alone from gestational days 16-19. At 15 weeks of age, females were subjected to rotarod time trials as a measure of exercise endurance. At 16 weeks of age, hindlimb skeletal muscle mass, fiber cross-sectional area (CSA), and fiber composition were determined. Expression of ATP synthase was also investigated. RESULTS: Compared with controls, PNA female rats demonstrated greater total body and hindlimb muscle weights, and trends toward diminished time on the rotarod, as well as greater type II muscle fiber composition and levels of ATP synthase in the gastrocnemius. No changes were observed in gastrocnemius or soleus muscle fiber CSA, or in soleus fiber type composition or ATP synthase expression. CONCLUSIONS: PNA female rats exhibit changes in gastrocnemius muscle that are consistent with a change in muscle fiber composition, which may affect exercise endurance. Findings in this rat model for PCOS are consistent with findings in women with the disorder. Sources of Research Support: LECOM institutional funding to DS. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Immune Challenge During Reproduction Results in Tissue Specific and Intensity‐Dependent Responses for Mitochondrial Stress

Life‐history theory suggests that tradeoffs can occur between self‐maintance and an animal's capacity to allocate resources to reproduction. This effect is thought to be exacerbated when a female is faced with an immune challenge during a reproductive event. Ultimately, such tradeoffs are thought to curtail lifespan, but the bioenergetic underpinnings of this effect are not well understood. We determined the extent of any effects in mitochondrial function and oxidative damage in female mice when exposed to an immune challenge (keyhole limpet haemocyanin) during reproduction. Female ICR mice were paired with a male until just before giving birth and randomly assigned to one of three groups. Female mice in the control group (LC) were injected with saline 2 days after parturition, and then sacrificed 1 week after weaning. In the lactating immune challenge group (LI), females were treated similarly except they were injected with KLH. Finally, in the pregnant‐lactating immune challenge group (PLI), females were treated the same as LI group, except the male was left in the box with the female until 3 days post‐partum, so the female became pregnant again while lactating. The second litter was born within 3 days of weaning, immediately removed, and females were sacrificed 1 week later. Mitochondrial function and reactive oxygen species (ROS) emission were determined in the liver and hindlimb skeletal muscle. Antioxidant protein levels and oxidative damage markers were also analyzed in these tissues. A one‐way ANOVA was performed on all measures, with a Tukey's post hoc test to determine group differences. No significant differences were observed in liver or hindlimb skeletal muscle mitochondrial coupling as measured by the respiratory control ratio (RCR) for both complex I and complex II (p&gt;0.05). Isolated mitochondrial ROS emission in the hindlimb skeletal muscle was significantly lower in LI compared to LC (p=0.041), but no differences were observed in the liver. Additionally, skeletal muscle PGC1‐α was significantly reduced in LI mice compared to LC (p=0.01). In skeletal muscle the antioxidant GPX was significantly increased in the PLI animals compared to LI animals (p=0.022). Lastly, liver SOD1 was significantly higher in LC compared to PLI (p=0.039). The opposite response was observed in HSP70, in which PLI had significantly increased expression compared to LC (p=0.018). In conclusion, females that experienced an immune challenge during reproduction did not appear to experience a decline in mitochondrial respiratory performance but a drop in PGC1‐a in skeletal muscle that could contribute to a reduction in future performance associated with a reduction in mitochondrial density.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic Femoral Artery Ligation Increases Skeletal Muscle Interstitial Prostaglandin E2 in the Rat: Implications for the Exercise Pressor Reflex

The stimulation of primarily mechanically‐sensitive group III and primarily metabolically‐sensitive group IV muscle afferents during skeletal muscle contraction evokes reflex increases in blood pressure (i.e., the exercise pressor reflex). The exercise pressor reflex is exaggerated in many forms of cardiovascular disease when compared to the reflex found in healthy counterparts. In rats in which a femoral artery is ligated for 72 hours (a model of simulated peripheral artery disease), the mechanically‐sensitive component of the reflex (i.e., the mechanoreflex) contributes importantly to the overall exercise pressor reflex exaggeration. Using a dynamic hindlimb muscle stretch protocol to study the mechanoreflex, we found recently that the increase in blood pressure was greater during stretch of a ligated versus a freely perfused hindlimb. This suggested that a chronic sensitization of mechanically‐activated channels contributed to the exaggerated mechanoreflex in ligated hindlimbs. The identity of the sensitizing chemical, however, was unknown. Experimental evidence has established that cyclooxygenase (COX) products, especially prostaglandins, sensitize mechanically‐activated channels. Accordingly, the purpose of this investigation was to determine whether 72 hour femoral artery ligation increased resting skeletal muscle interstitial prostaglandin E2 (PGE2) concentration in decerebrate, unanesthetized rats. The microdialysis technique was used to collect hindlimb skeletal muscle interstitial fluid in eight rats with a previously ligated femoral artery and a freely perfused femoral artery. We found that resting PGE2 concentration was greater in the ligated (224±44 pg/ml) versus the freely perfused (180±33 pg/ml, p&lt;0.01) hindlimb. To investigate the potential underlying mechanisms of the PGE2 elevation, we quantified both COX expression and COX activity in the soleus muscles (highly oxidative) and white portion of the gastrocnemius muscles (highly glycolytic). Western blot analyses revealed that femoral artery ligation had no effect on COX‐1 or COX‐2 protein expression in the soleus or white gastrocnemius muscles (p‐value range: 0.44 to 0.78). Total COX activity, however, was higher in the white gastrocnemius muscle of ligated hindlimbs compared to freely perfused hindlimbs (p&lt;0.04). There was no difference in total COX activity in the soleus muscle between hindlimbs (p=0.21). We conclude that 72 hours of femoral artery ligation increased resting PGE2 concentration in rat hindlimb skeletal muscles. The higher PGE2 concentration in ligated hindlimb muscles appears attributable to increased COX activity in white, glycolytic muscle portions. Our findings suggest that chronically elevated skeletal muscle interstitial PGE2 concentration may contribute to the exaggerated mechanically‐sensitive component of the exercise pressor reflex found in ligated hindlimb versus a freely perfused hindlimb.Support or Funding Information1R01HL142877 to SWCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Skeletal Muscle Stretch‐Induced PGE2 Formation does not Impact the Muscle Mechanoreflex in Decerebrate Rats

Mechanical and metabolic signals associated with skeletal muscle contraction stimulate thin fiber muscle afferents and produce reflex increases in blood pressure (i.e., the exercise pressor reflex). There is an interplay between those signals as metabolites may sensitize afferent responsiveness to mechanical signals. A common strategy used to study the mechanical component of the exercise pressor reflex (i.e., the mechanoreflex) in isolation from the metabolic component is to measure the reflex increase in blood pressure during passive hindlimb muscle stretch in an experimental animal preparation. Passive muscle stretch has been presumed to impart a purely mechanical stimulus. However, intermittent but not static mechanical stimulation of cultured skeletal muscle cells was found to increase PGE2 production and PGE2 has been shown to sensitize the muscle mechanoreflex. Based on those findings, the purpose of this investigation was to test the hypothesis that in decerebrate, unanesthetized rats, dynamic but not static hindlimb skeletal muscle stretch would increase interstitial PGE2 concentration. Moreover, we tested the hypothesis that COX inhibition with indomethacin would attenuate the stretch‐induced increase in PGE2 concentration and the increase in blood pressure during mechanoreflex activation. Experiments were performed on 28 young adult male Sprague‐Dawley rats. The microdialysis technique was used to collect triceps surae muscle interstitial fluid at rest and during 4 minutes of 1 Hz dynamic muscle stretch and 4 minutes of static muscle stretch (performed in random order and separated by 10 minutes). Muscle stretch was performed by manually turning the rack and pinion. We found that triceps surae muscle interstitial PGE2 concentration was significantly increased during both static (↑38%, p=0.01) and dynamic (↑56%, p&lt;0.01) stretch above their respective baseline concentrations (n=9). Indomethacin (1μg/kg) significantly reduced baseline PGE2 concentration and prevented the increase in PGE2 during static (n=4) and dynamic (n=5) stretch. Importantly, indomethacin had no effect on the peak pressor response during static (control: 15±4, indomethacin: 16±4 mmHg; p=0.82) and dynamic (control: 17±2, indomethacin: 18±4 mmHg; p=0.68) stretch (n=7). Collectively, these data indicate that static and dynamic hindlimb muscle stretch in decerebrate, unanaesthetized rats increased muscle interstitial PGE2 concentration. The magnitude of increase in PGE2, however, was not sufficient to impact the muscle mechanoreflex.Support or Funding Information1R01HL142877 to SWCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SELENON (SEPN1) protects skeletal muscle from saturated fatty acid-induced ER stress and insulin resistance

Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to a congenital myopathy associated with insulin resistance (SEPN1-related myopathy). The exact cause of the insulin resistance in patients with SELENON loss of function is not known. Skeletal muscle is the main contributor to insulin-mediated glucose uptake, and a defect in this muscle-related mechanism triggers insulin resistance and glucose intolerance. We have studied the chain of events that connect the loss of SELENON with defects in insulin-mediated glucose uptake in muscle cells and the effects of this on muscle performance. Here, we show that saturated fatty acids are more lipotoxic in SELENON-devoid cells, and blunt the insulin-mediated glucose uptake of SELENON-devoid myotubes by increasing ER stress and mounting a maladaptive ER stress response. Furthermore, the hind limb skeletal muscles of SELENON KO mice fed a high-fat diet mirrors the features of saturated fatty acid-treated myotubes, and show signs of myopathy with a compromised force production. These findings suggest that the absence of SELENON together with a high-fat dietary regimen increases susceptibility to insulin resistance by triggering a chronic ER stress in skeletal muscle and muscle weakness.Importantly, our findings suggest that environmental cues eliciting ER stress in skeletal muscle (such as a high-fat diet) affect the pathological phenotype of SEPN1-related myopathy and can therefore contribute to the assessment of prognosis beyond simple genotype-phenotype correlations.

Carboxyl-terminal modulator protein regulates Akt signaling during skeletal muscle atrophy in vitro and a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease involving motor neuron death, paralysis and, ultimately, respiratory failure. Motor neuron dysfunction leads to target skeletal muscle atrophy involving dysregulation of downstream cell survival, growth and metabolic signaling. Decreased Akt activity is linked to muscle atrophy in ALS and is associated with increased atrophy gene expression. Unfortunately, the regulating mechanism of Akt activity in atrophic muscle remains unclear. Recent research indicates a role of carboxyl-terminal modulator protein (CTMP) in Akt-signaling related neurologic dysfunction and skeletal muscle metabolism. CTMP is known to bind and reduce Akt phosphorylation and activation. We hypothesized that CTMP expression might progressively increase in ALS skeletal muscle as the disease progresses, downregulating Akt activity. We found that CTMP protein expression significantly increased in hindlimb skeletal muscle in the mSOD1G93A mouse model of ALS in late stages of the disease (P < 0.05), which negatively correlated with Akt phosphorylation over this period (R2 = −0.77). Co-immunoprecipitation of Akt revealed CTMP binding in pre-symptomatic and end-stage skeletal muscle, suggesting a possible direct role in reduced Akt signaling during disease progression. Inflammatory TNFα and downstream cellular degradation process markers for autophagy, lysosome production, and atrophy significantly increased in a pattern corresponding to increased CTMP expression and reduced Akt phosphorylation. In an in vitro model of skeletal muscle atrophy, differentiated C2C12 cells exhibited reduced Akt activity and decreased FOXO1 phosphorylation, a process known to promote transcription of atrophy genes in skeletal muscle. These results corresponded with increased Atrogin-1 expression compared to healthy control cells (P < 0.05). Transfection with CTMP siRNA significantly increased Akt phosphorylation in atrophic C2C12 cells, corresponding to significantly decreased CTMP expression. In conclusion, this is the first study to provide evidence for a link between elevated CTMP expression, downregulated Akt phosphorylation and muscle atrophy in ALS and clearly demonstrates a direct influence of CTMP on Akt phosphorylation in an in vitro muscle cell atrophy model.

Maternal inflammation at midgestation impairs subsequent fetal myoblast function and skeletal muscle growth in rats, resulting in intrauterine growth restriction at term.

Maternal inflammation induces intrauterine growth restriction (MI-IUGR) of the fetus, which compromises metabolic health in human offspring and reduces value in livestock. The objective of this study was to determine the effect of maternal inflammation at midgestation on fetal skeletal muscle growth and myoblast profiles at term. Pregnant Sprague-Dawley rats were injected daily with bacterial endotoxin (MI-IUGR) or saline (controls) from the 9th to the 11th day of gestational age (dGA; term = 21 dGA). At necropsy on dGA 20, average fetal mass and upper hindlimb cross-sectional areas were reduced (P < 0.05) in MI-IUGR fetuses compared with controls. MyoD+ and myf5+ myoblasts were less abundant (P < 0.05), and myogenin+ myoblasts were more abundant (P < 0.05) in MI-IUGR hindlimb skeletal muscle compared with controls, indicating precocious myoblast differentiation. Type I and Type II hindlimb muscle fibers were smaller (P < 0.05) in MI-IUGR fetuses than in controls, but fiber type proportions did not differ between experimental groups. Fetal blood plasma TNFα concentrations were below detectable amounts in both experimental groups, but skeletal muscle gene expression for the cytokine receptors TNFR1, IL6R, and FN14 was greater (P < 0.05) in MI-IUGR fetuses than controls, perhaps indicating enhanced sensitivity to these cytokines. Maternal blood glucose concentrations at term did not differ between experimental groups, but MI-IUGR fetal blood contained less (P < 0.05) glucose, cholesterol, and triglycerides. Fetal-to-maternal blood glucose ratios were also reduced (P < 0.05), which is indicative of placental insufficiency. Indicators of protein catabolism, including blood plasma urea nitrogen and creatine kinase, were greater (P < 0.05) in MI-IUGR fetuses than in controls. From these findings, we conclude that maternal inflammation at midgestation causes muscle-centric fetal programming that impairs myoblast function, increases protein catabolism, and reduces skeletal muscle growth near term. Fetal muscle sensitivity to inflammatory cytokines appeared to be enhanced after maternal inflammation, which may represent a mechanistic target for improving these outcomes in MI-IUGR fetuses.

Reduced Skeletal Muscle Protein Turnover and Thyroid Hormone Metabolism in Adaptive Thermogenesis That Facilitates Body Fat Recovery During Weight Regain.

Objective: The recovery of body composition after weight loss is characterized by an accelerated rate of fat recovery (preferential catch-up fat) resulting partly from an adaptive suppression of thermogenesis. Although the skeletal muscle has been implicated as an effector site for such thrifty (energy conservation) metabolism driving catch-up fat, the underlying mechanisms remain to be elucidated. We test here the hypothesis that this thrifty metabolism driving catch-up fat could reside in a reduced rate of protein turnover (an energetically costly “futile” cycle) and in altered local thyroid hormone metabolism in skeletal muscle.Methods: Using a validated rat model of semistarvation-refeeding in which catch-up fat is driven solely by suppressed thermogenesis, we measured after 1 week of refeeding in refed and control animals the following: (i) in-vivo rates of protein synthesis in hindlimb skeletal muscles using the flooding dose technique of 13C-labeled valine incorporation in muscle protein, (ii) ex-vivo muscle assay of net formation of thyroid hormone tri-iodothyronine (T3) from precursor hormone thyroxine (T4), and (iii) protein expression of skeletal muscle deiodinases (type 1, 2, and 3).Results: We show that after 1 week of calorie-controlled refeeding, the fractional protein synthesis rate was lower in skeletal muscles of refed animals than in controls (by 30–35%, p < 0.01) despite no between-group differences in the rate of skeletal muscle growth or whole-body protein deposition—thereby underscoring concomitant reductions in both protein synthesis and protein degradation rates in skeletal muscles of refed animals compared to controls. These differences in skeletal muscle protein turnover during catch-up fat were found to be independent of muscle type and fiber composition, and were associated with a slower net formation of muscle T3 from precursor hormone T4, together with increases in muscle protein expression of deiodinases which convert T4 and T3 to inactive forms.Conclusions: These results suggest that diminished skeletal muscle protein turnover, together with altered local muscle metabolism of thyroid hormones leading to diminished intracellular T3 availability, are features of the thrifty metabolism that drives the rapid restoration of the fat reserves during weight regain after caloric restriction.

GsMTx4 reduces the reflex pressor response during dynamic hindlimb skeletal muscle stretch in decerebrate rats.

Mechanical signals within contracting skeletal muscles contribute to the generation of the exercise pressor reflex; an important autonomic and cardiovascular control mechanism. In decerebrate rats, the mechanically activated channel inhibitor GsMTx4 was found to reduce the pressor response during static hindlimb muscle stretch; a maneuver used to investigate specifically the mechanical component of the exercise pressor reflex (i.e., the mechanoreflex). However, the effect was found only during the initial phase of the stretch when muscle length was changing and not during the later phase of stretch when muscle length was relatively constant. We tested the hypothesis that in decerebrate, unanesthetized rats, GsMTx4 would reduce the pressor response throughout the duration of a 30 sec, 1 Hz dynamic hindlimb muscle stretch protocol that produced repetitive changes in muscle length. We found that the injection of 10 μg of GsMTx4 into the arterial supply of a hindlimb reduced the peak pressor response (control: 15 ± 4, GsMTx4: 5 ± 2 mmHg, P < 0.05, n = 8) and the pressor response at multiple time points throughout the duration of the stretch. GsMTx4 had no effect on the pressor response to the hindlimb arterial injection of lactic acid which indicates the lack of local off‐target effects. Combined with the recent finding that GsMTx4 reduced the pressor response only initially during static stretch in decerebrate rats, the present findings suggest that GsMTx4‐sensitive channels respond primarily to mechanical signals associated with changes in muscle length. The findings add to our currently limited understanding of the channels that contribute to the activation of the mechanoreflex.