Muscle Protein Levels Research Articles

Lipocalin 2 Does Not Play A Role in Celastrol-Mediated Reduction in Food Intake and Body Weight

Celastrol is a leptin-sensitizing agent with profound anti-obesity effects in diet-induced obese (DIO) mice. However, the genes and pathways that mediate celastrol-induced leptin sensitization have not been fully understood. By comparing the hypothalamic transcriptomes of celastrol and vehicle-treated DIO mice, we identified lipocalin-2 (Lcn2) as the gene most strongly upregulated by celastrol. LCN2 was previously suggested as an anorexigenic and anti-obesity agent. Celastrol increased LCN2 protein levels in hypothalamus, liver, fat, muscle, and bone marrow, as well as in the plasma. However, genetic deficiency of LCN2 altered neither the development of diet-induced obesity, nor the ability of celastrol to promote weight loss and improve obesity-associated dyshomeostasis. We conclude that LCN2 is dispensable for both high fat diet-induced obesity and its therapeutic reduction by celastrol.

Deficiency of fibroblast growth factor 21 aggravates obesity-induced atrophic responses in skeletal muscle

BackgroundObesity-induced skeletal muscle inflammation is a major contributor of skeletal muscle loss/atrophy and is implicated in metabolic complications such as insulin resistance. Fibroblast growth factor 21 (FGF21) is known to be an important metabolic regulator with anti-inflammatory properties. However, the effect of FGF21 on skeletal muscle atrophy is unclear. In this study, we investigated the effect of FGF21 deficiency on obesity-induced skeletal muscle inflammation and atrophy in mice.ResultsThe expression of atrophic factors (MuRF1 and Atrogin-1) was upregulated at the mRNA and/or protein levels in the skeletal muscle of FGF21-deficient obese mice compared with wild type obese control mice. This was accompanied by an increase in levels of inflammatory cytokines (TNFα and MCP-1) and a reduction in AMPK phosphorylation. FGF21 treatment markedly suppressed TNFα-mediated inflammatory and atrophic responses in cultured myotubes, and the actions of FGF21 were blunted by the AMPK inhibitor compound C.ConclusionThese findings suggest that FGF21 deficiency aggravates obesity-induced inflammation and atrophic responses in the skeletal muscle of obese mice, and FGF21 may protect inflammation-mediated atrophy through the AMPK pathway.

Effects of vitamin D on primary human skeletal muscle cell proliferation, differentiation, protein synthesis and bioenergetics

The active form of Vitamin D (1,25(OH)2D), has been suggested to have a regulatory role in skeletal muscle function and metabolism, however, the effects and mechanisms of vitamin D (VitD) action in this tissue remain to be fully established. In this study, we have used primary human skeletal muscle myoblast (HSMM) cells that display typical characteristics of human skeletal muscle function and protein levels, to investigate the effects of the active form of VitD on proliferation, differentiation, protein synthesis and bioenergetics. Myoblast cells were treated with 100 nM of VitD for 24 h, 48 h, 72 h and five days (cells were differentiated into myotubes) and then analyses were performed. We report that VitD inhibits myoblast proliferation and enhances differentiation by altering the expression of myogenic regulatory factors. In addition, we found that protein synthesis signaling improved in myotubes after VitD treatment in the presence of insulin. We also report an increase in oxygen consumption rate after 24 h of treatment in myoblasts and after 5 days of treatment in myotubes after VitD exposure. VitD significantly impacted HSMM myogenesis, as well as protein synthesis in the presence of insulin.

Imbalances in the male reproductive function of the estuarine crab Neohelice granulata, caused by glyphosate

The effect of glyphosate, both pure and formulated (Roundup Ultramax®), was evaluated on males of the estuarine crab Neohelice granulata, by means of both in vivo and in vitro assays. The in vivo assays comprised the exposure for 30 d to 1 mg/L of the herbicide, until finally assessing weight gain, levels of energy reserves, sperm number per spermatophore, proportion of abnormal spermatophores, and sperm viability. At the end of this assay, significant (p < 0.05) decrease in weight gain and muscle protein levels was detected by effect of both pure and formulated glyphosate. In spermatophores from the vas deferens, a significant (p < 0.05) decrease of the sperm count was observed by effect of Roundup, while a significant incidence (p < 0.05) of abnormal spermatophores was observed either with glyphosate or with Roundup treatment. No changes were seen in the spermatophore area or in vas deferens secretions. Since no sperm mortality was induced by the formulated herbicide, we propose a probable inhibiting effect on spermatogenesis might explain the observed sperm count decrease. In this sense, an in vitro assay was designed by incubating testes and vasa deferentia with Roundup, in order to corroborate the possible interference of glyphosate with the secretion of the androgenic gland hormone that controls the spermatogenesis, in the presence or absence of the androgenic gland. Although the herbicide per se was able to reduce the sperm count to some extent, the increase in the number of spermatozoa/spermatophore produced by the co-incubation with the androgenic gland was completely reverted by the addition of Roundup (1 mg/L of glyphosate a.e.), suggesting that an inhibition on the secretion and/or transduction of the androgenic gland hormone could be taking place.

Peritubular cells of the human testis: prostaglandin E2 and more.

Several layers of slender, smooth muscle-like, peritubular cells and extracellular matrix (ECM) form the peritubular compartment of the human testis. Peritubular cells are the least explored testicular cells. Human testicular peritubular cells (HTPCs) can be isolated from small testicular fragments of patients and studied in vitro. We have used this cellular model, in combination with human testicular samples, to examine how peritubular cells may contribute to male (in)fertility. Human testicular peritubular cells(HTPCs) retain contractile abilities in vitro and secrete many proteins. Among them are factors, which serve intra-testicular roles, for example, glial cell line-derived neurotrophic factor (GDNF), thought to be important for the renewal of spermatogonial stem cells (SSCs). Studies in mutant mice indicated that peritubular cell-derived GDNF is crucial for lifelong spermatogenesis. Thus, peritubular cells are a functional part of the SSC niche. Peritubular cells of mice and men express androgen receptors (AR). In mouse peritubular cells, androgens enhanced GDNF production, but not in HTPCs. Rather, AR activation increased the levels of AR and smooth muscle proteins and thereby enhanced the smooth muscle-like phenotype. Following the lead of a proteomic analysis, which identified the key prostaglandin (PG)-synthesizing enzyme (PTGS1=COX1), we found that HTPCs secrete PGE2 . COX1, and PGE2 receptors (EP1, 2, and 4) were identified in peritubular cells in situ, supporting in vivo relevance. In HTPCs, activation of EP1/4 increased GDNF and a smooth muscle protein. Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID), which blocks PG synthesis. Added to HTPCs it reduced PGE2 and GDNF production and lowered smooth muscle protein levels. If applicable to the in vivo situation, the results suggest that ibuprofen and possibly other NSAIDs may impair important peritubular cell functions and consequently testicular functions. The few examples highlighted, together with others not mentioned here, indicate that HTPCs provide an experimental window into the human testis.

Paraspinal muscle ladybird homeobox 1 (LBX1) in adolescent idiopathic scoliosis: a cross-sectional study

BACKGROUND CONTEXTAdolescent idiopathic scoliosis (AIS) is the leading cause of spinal deformity in adolescents globally. Recent evidence from genome-wide association studies has implicated variants in or near the ladybird homeobox 1 (LBX1) gene, encoding the ladybird homeobox 1 transcription factor, in AIS development. This gene plays a critical role in guiding embryonic neurogenesis and myogenesis and is vital in muscle mass determination. Despite the confirmation of the role for LBX1 gene variants in the development of AIS, the biological basis of LBX1 contribution to AIS remains mostly unknown. PURPOSETo investigate the potential role of LBX1 in driving spinal curving, curve laterality, and progression through muscle-based mechanisms in AIS patients by analyzing its gene and protein expression. STUDY DESIGNThis is a cross-sectional study using clinical data and biological samples from the Immunometabolic CONnections to Scoliosis study (ICONS study). PATIENT SAMPLETwenty-five patients with AIS provided informed consent. Paraspinal muscle biopsies from the maximal points of concavity and convexity for gene expression and protein analysis were obtained at the start of corrective spinal surgery. OUTCOME MEASURESThe outcome measures included the detection of paraspinal muscle LBX1 mRNA abundance and LBX1 protein expression and the correlation of the latter with age, sex, and curve severity. METHODSThe measurement of mRNA abundance was done using quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, protein lysates from the biopsied muscle samples were probed with a monoclonal LBX1 antibody to compare the muscle protein levels on either side of the scoliotic curve by western blot. This study received funding from the Division of Orthopedics, Department of Surgery, McMaster University, Hamilton, Ontario, Canada ($39,900 CAN for 2 years). The authors have no conflicts of interest to disclose. RESULTSLBX1 mRNA abundance (concave 2.98±0.87, convex 3.40±1.10, p value 0.73) and protein expression (concave 1.20±0.13, convex 1.21±0.10, p value 0.43) were detected on both sides of the scoliotic curve at equivalent levels. The expression of LBX1 protein did not correlate with age (concave: correlation coefficient 0.32, p value 0.12; convex: correlation coefficient 0.08, p value 0.69), sex (concave: correlation coefficient −0.03, p value 0.08; convex: correlation coefficient 0.07, p value 0.72), or the severity of spinal curving measured using the Cobb angle (concave: correlation coefficient −0.16, p value 0.45; convex: correlation coefficient −0.08, p value 0.69). CONCLUSIONSLBX1 is expressed in erector spinae muscles, and its levels are equal in muscles on both sides of the scoliotic curve in AIS. The expression of LBX1 on the convex and concave sides of the scoliotic curve did not correlate with age, sex, or the severity of spinal curving. The molecular mechanisms by which LBX1contributes to the development and propagation of AIS need to be explored further in muscle and other tissues.

Golden sea cucumber extract revives glucose transporter-4 and interleukin-6 protein level in diabetic mouse muscle.

Background:Streptozotocin (STZ)-induced free radical oxidant activity resulted in muscle wasting due to protein carbonyl (PC), glucose transporter-4 (Glut-4), and interleukin-6 (IL-6) protein alteration. Antioxidant ingredient in the golden sea cucumber extract was found in promising level to inhibit free radical activity.Aim:This study was aimed to investigate the effect of golden sea cucumber extract on PC, IL-6, and Glut-4 level of STZ-induced diabetes mouse.Materials and Methods:This study was performed using mice, which were grouped into non-diabetes, diabetes, and diabetes-treated extract groups. The golden sea cucumber was extracted using 70% ethanol, which was administered by oral gavage twice a day for 5 consecutive days.Results:The extract reduced PC level and improved muscle Glut-4 and IL-6 protein level of diabetic mouse.Conclusion:The extract of golden sea cucumber revived muscle Glut-4 and IL-6 protein level in protection against muscle wasting.

Micro(RNA)-managing muscle wasting.

Progressive skeletal muscle wasting is a natural consequence of aging and is common in chronic and acute diseases. Loss of skeletal muscle mass and function (strength) often leads to frailty, decreased independence, and increased risk of hospitalization. Despite progress made in our understanding of the mechanisms underlying muscle wasting, there is still no treatment available, with exercise training and dietary supplementation improving, but not restoring, muscle mass and/or function. There has been slow progress in developing novel therapies for muscle wasting, either during aging or disease, partially due to the complex nature of processes underlying muscle loss. The mechanisms of muscle wasting are multifactorial, with a combination of factors underlying age- and disease-related functional muscle decline. These factors include well-characterized changes in muscle such as changes in protein turnover and more recently described mechanisms such as autophagy or satellite cell senescence. Advances in transcriptomics and other high-throughput approaches have highlighted significant deregulation of skeletal muscle gene and protein levels during aging and disease. These changes are regulated at different levels, including posttranscriptional gene expression regulation by microRNAs. microRNAs, potent regulators of gene expression, modulate many processes in muscle, and microRNA-based interventions have been recently suggested as a promising new therapeutic strategy against alterations in muscle homeostasis. Here, we review recent developments in understanding the aging-associated mechanisms of muscle wasting and explore potential microRNA-based therapeutic avenues.

Low-Intensity Exercise in Mice Is Sufficient to Protect Retinal Function During Light-Induced Retinal Degeneration.

PurposeWe previously reported that a specific treadmill running exercise regimen protects against light-induced retinal degeneration (LIRD) in mice. We hypothesized that this protective effect varies with running intensity. To test this, mice undergoing LIRD were run at different treadmill speeds and retinal function was assessed.MethodsBALB/c mice were assigned to LIRD groups at varying treadmill speeds—0, 5, 10, or 20 m/min labeled inactive, low, medium, and high, respectively—and compared with naïve mice exposed to standard lighting (50 lux; naïve). Following 2 weeks of exercise, a subset of mice were exposed to toxic light (10,000 lux; LIRD) for 4 hours. After 5 additional days of exercise, retinal function was assessed by ERG. Corticosterone levels in serum and cathepsin B (CTSB) protein levels in muscle, brain, serum, and retina were measured. The retinal gene expression of complement factor 1qa (C1qa) and CTSB were measured.ResultsThe low+LIRD and medium+LIRD exercise groups had greater a- and b-wave ERG amplitudes when compared with the inactive+LIRD group (P < 0.02). The high+LIRD mice only differed from the inactive+LIRD mice in their dark-adapted b-waves. Serum corticosterone increased in the high+LIRD mice (P < 0.006). Retinal CTSB protein levels were higher in the low+LIRD versus high+LIRD mice (P < 0.004) but were otherwise unchanged. Exercise of any intensity decreased C1qa gene expression.ConclusionsFaster running did not additionally protect against LIRD, but it did increase serum corticosterone, suggesting stress-induced limits to exercise benefits. Unexpectedly, exercise did not increase CTSB proteins levels in muscle or serum, suggesting that it may not mediate exercise effects. Our results have implications for the use of low-intensity exercise as a vision loss treatment.

Se Regulates the Contractile Ability of Uterine Smooth Musclevia Selenoprotein N, Selenoprotein T, and Selenoprotein Win Mice.

Selenium (Se) is an essential micronutrient that maintains normal physiological functions in humans and animals. Se plays a vital role in regulating smooth muscle contractions, and selenoprotein N (SelN), selenoprotein T (SelT), and selenoprotein W (SelW) are closely related to the release of Ca2+. The present study analyzed the effects and mechanisms of SelN, SelT, and SelW in uterine smooth muscle contractions in a mouse model fed Se. The mRNA and protein levels in the uterine smooth muscle of mice were detected by qPCR, Western blot, and immunohistochemical analysis. The results showed that Se played an indispensable role in uterine smooth muscle contractions. Increased Se concentration in food increased the release of Ca2+ to a certain extent, causing CaM expression, MLCK expression, and MLC phosphorylation, which can lead to uterine smooth muscle contractions. In contrast, Se deficiency reduced the release of Ca2+ to a certain degree, thereby reducing the contractile ability of uterine smooth muscle. In this study, genes related to SelN, SelT, and SelW expression in uterine smooth muscle cells were investigated. The results showed that the Se concentration had an effect on the expression of SelN, SelT, and SelW in uterine smooth muscle cells. Se influences the release of Ca2+ through SelN, SelT, and SelW, which changes the expression of MLCK and then affects uterine smooth muscle contractions. The three selenoproteins SelN, SelT, and SelW play a very important role in uterine smooth muscle contractions, and the absence of any of these proteins affects the contractility of the uterus.

The pleural mesothelium and transforming growth factor-β1 pathways in restrictive allograft syndrome: A pre-clinical investigation.

Chronic lung allograft dysfunction (CLAD) hampers long-term survival after lung transplantation. Common fibrosis-related mechanisms in idiopathic pulmonary fibrosis and CLAD instigated the consideration of investigating the differential regulation of pleural mesothelium and transforming growth factor-β1 (TGF-β1) in restrictive allograft syndrome (RAS). TGF-β1 was assessed in bronchoalveolar lavage (BAL) fluid using enzyme-linked immunoassay and via immune staining of explant biopsies. To assess the role of the pleura, explanted bronchiolitis obliterans syndrome (BOS) and RAS lungs were compared using computed tomography scans, calretinin stainings, Western blot, and quantititative real-time PCR. Last, a pleural mesothelial cell line was used to assess mesothelial-to-mesenchymal transition and its inhibition. TGF-β1 was increased in BAL of RAS patients (p = 0.035), and was present in the (sub)pleural area of biopsies. Explanted RAS lungs demonstrated an increased volume fraction of pleura (p = 0.0004), a higher proportion of calretinin-positive stainings (p = 0.0032), and decreased E-cadherin (p = 0.019) and increased α-smooth muscle actin (p = 0.0089) mRNA expression and protein levels in isolated pleural tissue. Moreover, TGF-β1 stimulation of pleural mesothelial cells led to a phenotypical switch to mesenchymal cells, accompanied with an increased migratory capacity. Interleukin-1α was able to accentuate TGF-β1‒induced mesothelial-to-mesenchymal transition. None of the tested drugs could inhibit mesothelial-to-mesenchymal transition at the used concentrations. Our results support an interplay between TGF-β1 and the pleural mesothelium in the pathophysiology of RAS.

Fasting in the ureotelic Lake Magadi tilapia, Alcolapia grahami, does not reduce its high metabolic demand, increasing its vulnerability to siltation events.

Lake Magadi, Kenya, is one of the most extreme aquatic environments on Earth (pH~10, anoxic to hyperoxic, high temperatures). Recently, increased water demand and siltation have threatened the viable hot springs near the margins of the lake where Alcolapia grahami, the only fish surviving in the lake, live. These Lake Magadi tilapia largely depend on nitrogen-rich cyanobacteria for food and are 100% ureotelic. Their exceptionally high aerobic metabolic rate, together with their emaciated appearance, suggests that they are energy-limited. Therefore, we hypothesized that during food deprivation, Magadi tilapia would economize their energy expenditure and reduce metabolic rate, aerobic performance and urea-N excretion. Surprisingly, during a 5-day fasting period, routine metabolic rates increased and swimming performance (critical swimming speed) was not affected. Urea-N excretion remained stable despite the lack of their N-rich food source. Their nitrogen use switched to endogenous sources as liver and muscle protein levels decreased after a 5-day fast, indicating proteolysis. Additionally, fish relied on carbohydrates with lowered muscle glycogen levels, but there were no signs indicating use of lipid stores. Gene expression of gill and gut urea transporters were transiently reduced as were gill rhesus glycoprotein Rhbg and Rhcg-2. The reduction in gill glutamine synthetase expression concomitant with the reduction in Rh glycoprotein gene expression indicates reduced nitrogen/ammonia metabolism, most likely decreased protein synthesis. Additionally, fish showed reduced plasma total CO2, osmolality and Na+ (but not Cl−) levels, possibly related to reduced drinking rates and metabolic acidosis. Our work shows that Lake Magadi tilapia have the capacity to survive short periods of starvation which could occur when siltation linked to flash floods covers their main food source, but their seemingly hardwired high metabolic rates would compromise long-term survival.

High-intensity exercise training ameliorates aberrant expression of markers of mitochondrial turnover but not oxidative damage in skeletal muscle of men with essential hypertension.

To examine whether hypertensive individuals exhibit altered muscle mitochondrial turnover and redox homeostasis compared with healthy normotensive counterparts, and whether the antihypertensive effect of high-intensity exercise training is associated with improved mitochondrial quality and enhanced anti-oxidant defence. In a cross-sectional and longitudinal parallel design, 24 essential hypertensive (HYP) and 13 healthy normotensive (NORM) men completed 6weeks of high-intensity interval training (HIIT). Twenty four-hour ambulatory blood pressure, body composition, cardiorespiratory fitness, exercise capacity and skeletal muscle characteristics were examined before and after HIIT. Expression of markers of mitochondrial turnover, anti-oxidant protection and oxidative damage was determined in vastus lateralis muscle biopsies. Muscle protein levels of eNOS and VEGF, and muscle capillarity were also evaluated. At baseline, HYP exhibited lower expression of markers of mitochondrial volume/biogenesis, mitochondrial fusion/fission and autophagy along with depressed eNOS expression compared with NORM. Expression of markers of anti-oxidant protection was similar in HYP and NORM, whereas oxidative damage was higher in HYP than in NORM. In HYP, HIIT lowered blood pressure, improved body composition, cardiorespiratory fitness and exercise capacity, up-regulated markers of mitochondrial volume/biogenesis and autophagy and increased eNOS and VEGF protein content. Furthermore, in HYP, HIIT induced divergent responses in markers of mitochondrial fusion and anti-oxidant protection, did not affect markers of mitochondrial fission, and increased apoptotic susceptibility and oxidative damage. The present results indicate aberrant muscle mitochondrial turnover and augmented oxidative damage in hypertensive individuals. High-intensity exercise training can partly reverse hypertension-related impairments in muscle mitochondrial turnover, but not redox imbalance.

Gene editing and muscular dystrophy

Muscle Disease Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness and a shortened life span. The disease is caused by mutations that reduce or prevent expression of dystrophin, an essential structural protein in skeletal and heart muscle. The gene editing technology CRISPR-Cas9 can correct disease-causing mutations and has yielded promising results in mouse models of DMD. In a small, short-term study, Amoasii et al. tested this strategy in a dog model of DMD that exhibits many features of the human disease. Intramuscular or systemic delivery of the gene editing components resulted in a substantial increase in dystrophin protein levels in skeletal and heart muscle. Restoration of dystrophin expression was accompanied by improved muscle histology. Science , this issue p. [86][1] [1]: /lookup/doi/10.1126/science.aau1549

PL - 041 Effects of power resistance exercise and feeding on the expression of putative mechanosensing proteins in skeletal muscle of resistance-trained men

Objective Power resistance exercise involves high intensity (load and velocity) dynamic muscular contractions and is frequently performed by athletes to enhance performance via improved muscle function. To investigate the remodelling processes that contribute to improved muscle function, we investigated the expression of putative mechanosensing genes implicated in this process (Kojic et al., 2011): titin-linked Muscle Ankyrin Repeat Protein (MARPs) family CARP, Ankrd 2 and DARP, and the Z-disc associated muscle-LIM protein (MLP) in healthy, resistance-trained men (n = 7) following 90 min of rest (Rest) or power resistance exercise, with (Ex + Meal) or without (Ex only) feeding during recovery.&#x0D; Methods Percutaneous needle biopsy samples were obtained from the vastus lateralis of resistance-trained males using local anesthetic (2% Xylocaine), 3 h after performing each of the three experimental trials on separate days.&#x0D; Previously, we presented results from this study showing that the mRNA levels of CARP (~15-fold) and MLP (~2.5-fold) were upregulated in human skeletal muscle 3 h post power resistance exercise (Wette et al., 2012). Based on these results, we performed protein analyses on the same muscle samples to determine the protein levels of all MARPs and MLP in whole muscle homogenates after Rest, Ex only and Ex + Meal. To assess whether the exercise elicited a stress response in these resistance-trained individuals, the level of phosphorylated heat shock protein 27 at serine 15 (pHSP27-Ser15) was measured at Rest and 3 h after Ex only and Ex + Meal. The levels of pHSP27-Ser15 are typically upregulated 3 h after eccentric exercise in human skeletal muscle (Frankenberg et al., 2014).&#x0D; Results The 90 min exercise session consisted of 180 intermittent muscular contractions at high intensity (70-96% maximal strength). Compared to Rest, there were ~5.8- and 12.6-fold increases in pHSP27-Ser15 levels at 3 h post Ex only and Ex + Meal (both P = 0.049, one-way ANOVA) respectively. CARP protein levels were elevated ~2.7-fold after Ex only (P = 0.049, one-way ANOVA) and ~7.6-fold after Ex + Meal (P = 0.326), due to markedly higher levels (6-40-fold) in three of the seven participants. Pearson correlation analysis revealed a significant positive correlation between the levels of pHSP27-Ser-15 and CARP protein (r = 0.56, P = 0.008). Ankrd 2, DARP and MLP protein levels were unchanged (all P &gt; 0.05) following Ex only and Ex + Meal.&#x0D; Conclusions These findings indicate that CARP is highly responsive to increased mechanical loading because the protein levels in skeletal muscle can be substantially increased as early as 3 h after stressful resistance exercise. This suggests a specialised role for CARP protein during the early phases of muscle remodelling that occur as a consequence of performing high intensity resistance exercise.

Cardiomyopathy and altered integrin-actin signaling in Fhl1 mutant female mice.

X-linked scapuloperoneal myopathy (X-SM), one of Four-and-a-half LIM 1 (FHL1) related diseases, is an adult-onset slowly progressive myopathy, often associated with cardiomyopathy. We previously generated a knock-in mouse model that has the same mutation (c.365 G>C, p.W122S) as human X-SM patients. The mutant male mouse developed late-onset slowly progressive myopathy without cardiomyopathy. In this study, we observed that heterozygous (Het) and homozygous (Homo) female mice did not show alterations of skeletal muscle function or histology. In contrast, 20-month-old mutant female mice showed signs of cardiomyopathy on echocardiograms with increased systolic diameter [wild-type (WT): 2.74±0.22mm, mean±standard deviation (SD); Het: 3.13±0.11mm, P<0.01; Homo: 3.08±0.37mm, P<0.05) and lower fractional shortening (WT: 31.1±4.4%, mean±SD; Het: 22.7±2.5%, P<0.01; Homo: 22.4±6.9%, P<0.01]. Histological analysis of cardiac muscle revealed frequent extraordinarily large rectangular nuclei in mutant female mice that were also observed in human cardiac muscle from X-SM patients. Western blot demonstrated decreased Fhl1 protein levels in cardiac muscle, but not in skeletal muscle, of Homo mutant female mice. Proteomic analysis of cardiac muscle from 20-month-old Homo mutant female mice indicated abnormalities of the integrin signaling pathway (ISP) in association with cardiac dysfunction. The ISP dysregulation was further supported by altered levels of a subunit of the ISP downstream effectors Arpc1a in Fhl1 mutant mice and ARPC1A in X-SM patient muscles. This study reveals the first mouse model of FHL1-related cardiomyopathy and implicates ISP dysregulation in the pathogenesis of FHL1 myopathy.

CONGENITAL MYOPATHIES (CNM): P.143Myostatin as a novel blood based biomarker for antisense oligonucleotide-mediated Dnm2 knockdown to treat myotubular myopathy in mice

Centronuclear myopathies (CNM) are non-dystrophic muscle diseases for which no effective therapy is currently available. The most severe form, X-linked CNM, is caused by myotubularin 1 (MTM1) loss-of-function mutations, while the main autosomal dominant form is due to dynamin2 (DNM2) mutations. We previously showed that genetic reduction of DNM2 expression in Mtm1 knockout (Mtm1KO) mice prevents development of muscle pathology. Most recently we have shown that weekly systemic delivery of Dnm2 antisense oligonucleotides (ASOs) into Mtm1KO mice efficiently reduces DNM2 protein level in muscle and prevents the myopathy from developing. In addition, systemic weekly ASO injection into severely affected mice leads to reversal of muscle pathology within two weeks. Here we perform a single injection of ASO into 3-week-old Mtm1KO mice, and complete clinical, histological and molecular analysis of the duration of the effects after a single injection. A single injection of 25mg/kg of ASO targeting DNM2 increased the lifespan, whole body strength, and reduced disease severity in Mtm1KO mice compared to untreated controls. Despite these results, a single injection alone was not sufficient to rescue the disease for the lifespan of the mice, suggesting repeated treatments will be required. Thus, ASO-mediated DNM2 knockdown can efficiently correct muscle defects due to loss of MTM1, providing an attractive therapeutic strategy for this disease. We next analyzed myostatin levels in muscles and blood of Mtm1KO mice. We show myostatin levels in plasma are significantly reduced in Mtm1KO mice, and that treatment by ASOs targeting DNM2 significantly improves myostatin levels in plasma. In addition plasma myostatin levels inversely correlated with the level of DNM2 mRNA in muscles after injection with ASOs. This provides the first evidence of a blood based biomarker that can be used to monitor disease state and rescue in myotubular myopathy mice. With clinical trials for myotubular myopathy currently in progress, these results are of high relevance to the research field.

Estrogen replacement enhances insulin-induced AS160 activation and improves insulin sensitivity in ovariectomized rats.

Menopause predisposes women to impaired glucose metabolism, but the role of estrogen remains unclear. In this study, we examined the effects of chronic estrogen replacement on whole body insulin sensitivity and insulin signaling in ovariectomized rats. Female Wistar rats aged 9 wk were ovariectomized under anesthesia. After 4 wk, pellets containing either 17β-estradiol (E2) or placebo (Pla) were subcutaneously implanted in the rats. After 4 wk of treatment, the intra-abdominal fat accumulation was greater in the Pla group than that in the E2 group. Hyperinsulinemic-euglycemic clamp analysis and intravenous glucose tolerance test revealed that insulin sensitivity was significantly lower in the Pla group than in the E2 group. In addition, Western blotting showed that in vivo insulin stimulation increased protein kinase B (Akt) phosphorylation to a similar degree in the gastrocnemius and liver of both groups, but phosphorylated Akt2 Ser474 was enhanced in the muscle of the E2 group compared with the Pla group. Moreover, insulin-stimulated phosphorylation of Akt substrate of 160 kDa (AS160) Thr642 was observed only in the E2 group, resulting in the difference between the two groups. Additionally, AS160 protein and mRNA levels were higher in muscle of the E2 group than the Pla group. In contrast, E2 replacement had no effect on glucose transporter 4 protein levels in muscle and glycogen synthase kinase-3β in muscle and liver. These results suggest that estrogen replacement improves insulin sensitivity by activating the Akt2/AS160 pathway in the insulin-stimulated muscle of ovariectomized rats.

Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational "hotspot" in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD.

Evaluation of biological effectiveness of amino acid mixture as potential stimulator of synthetic processes in skeletal muscles

Background. The ability of certain amino acids to stimulate anabolic processes in skeletal muscles has been proved by fundamental research, which makes it important to search for effective agents based on amino acids for stimulation of synthetic processes in skeletal muscles.&#x0D; Aim. To study the effect of oral administration of the original amino acid mixture (Larginine, Lmethionine, Lleucine, Lisoleucine) on protein, lipid and carbohydrate metabolism in skeletal muscles and liver of CBA male mice.&#x0D; Material and Methods. Two series of experiments were performed. In the first series (n=36), the animals were divided into three groups. In group 1 (n = 12), the mice received a diet balanced in protein and carbohydrates for two months. The animals of group 2 (n=12) were kept on a carbohydrate, proteindepleted isocaloric diet, in which wheat gliadin served as the protein source. Mice of group 3 (n=12) were kept on a diet similar to the second group, in which deficit of protein was compensated for with the tested mixture of Lamino acids. In the animals of the second series (n=36) acute liver failure was modeled by a single intraperitoneal injection of 20% carbon tetrachloride solution (CTC) in olive oil. Three days after the injection of CTC, all animals of the second series were randomly divided into three groups, depending on the received diet.&#x0D; Results. The results of the first series of experiments showed that compensation for protein deficiency with amino acid mixture reliably prevented excessive buildup of glycogen in muscles, led to a decrease in lipids in tissue, and also prevented reduction in the level of muscle protein. The results of the second series of experiments showed that intake of the amino acid mixture prevented loss of protein in muscles and supported the proteinsynthetic function of the liver.&#x0D; Conclusion. The study demonstrated that the tested mixture, when taken orally, can prevent disorders of proteincarbohydratelipid ratio in the muscles.