Area Of Myofibers Research Articles

PECTORALIS THORACICUS MUSCLE PERFORMANCE OF HYBRID CHICKEN (F1) DERIVED FROM CROSSBREED BETWEEN BROILER AND PELUNG (Gallus gallus gallus)

This research purpose was to study the phenotype characters of body weight and pectoralis thoracicus muscle performance of hybrid chicken (F1) derived from crossbreed between parent stock broiler strain Cobb-500 and Pelung (Gallus gallus gallus). The chicks were divided into 3 groups and each group consists of 5 days-old chicks (DOC). Group 1 (T1) was used as control involving DOC derived from crossbreed between female and male of broiler Cobb-500 strain. Group 2 (T2) involving DOC derived from crossbreed between female and male Pelung, while Group 3 (T3) was DOC derived from crossbreed between female broiler strain Cobb-500 and male Pelung. The chicken was grown up to 49 days to evaluate the body weight, the pectoralis thoracicus weight, the pectoralis thoracicus area, and myofiber area. Data were statistically analyzed using one way Anova. The results showed that the average of body weight in T1; T2; and T3 were 2,170±86.02; 506.2±20.02; and 1,238±68.25 g, respectively (P<0.05). The average of pectoralis thoracicus muscle weight in T1; T2; and T3 were 165.04±11.97; 21.26±2.44; and 68.74±1.94 g, respectively (P<0.05). The average of pectoralis thoracicus muscle area in T1; T2; and T3 were 97.0±9,7; 61±6,4; and 63,4±1,8 cm2 , respectively (P<0,05). The average of pectoralis thoracicus myofiber area in T1; T2; and T3 were 14.72±1.04; 4.66±2.1; and 13.13±1.3 µm2 , respectively (P<0.05). It was concluded that crossbreed between broiler strain Cobb-500 and Pelung improved the body weight and pectoralis thoracicus muscle performance of hybrid chicken (F1).

Delayed Effect of Blood Flow-restricted Resistance Training on Rapid Force Capacity.

The aim of the present study was to investigate the effect and time course of high-frequent low-load blood flow-restricted (BFR) resistance training on rapid force capacity (i.e., rate of torque development [RTD]). Ten male subjects (22.8 ± 2.3 yr) performed four sets of knee extensor exercise (20% one-repetition maximum) to concentric failure during concurrent BFR of the thigh (100 mm Hg), and eight work-matched controls (21.9 ± 3.0 yr) trained without BFR (CON). Twenty-three training sessions were performed within 19 d. Maximal slow and fast knee joint velocity muscle strength and rapid force capacity (e.g., RTD) and evoked twitch contractile parameters were assessed before (Pre) and 5 and 12 d after (Post5 and Post12) training. Muscle biopsies were obtained Pre, after 8 d (Mid8), and 3 and 10 d after (Post3 and Post10) training to examine changes in myofiber area and expression of myocellular proteins known to be modified by cellular stress (CaMKII, annexin A6, SNO-CYS). RTD remained unchanged after BFR training at Post5, while increasing 15%-20% Post12 (P < 0.01). Evoked muscle twitch parameters showed a general decline Post5 (P < 0.01) while returning to baseline levels at Post12. All contractile parameters essentially remained unchanged in CON. Elevated CaMKII was observed with BFR training at Post3 (57%) and Post10 (71%) (P < 0.05), whereas SNO-CYS increased in CON at Mid8 (P < 0.05). This study is the first to show that low-load resistance exercise performed with BFR leads to marked increases in rapid force capacity (RTD). However, a general delayed adaptive response was observed for voluntary contractile parameters (including RTD) in parallel with a decline and subsequent recovery in evoked contractile properties, suggesting the delayed gain in rapid force capacity mainly have a peripheral origin.

Deletion of Kvβ2 Alters Murine Skeletal Muscle Morphology and Phenotype

IntroductionMammalian skeletal muscle expresses various potassium channels that regulate myogenesis. Recent evidence points to the role of Kv1.5 in cell cycle progression in myoblasts as pharmacological inhibition of this channel in L6E9 cells inhibited G0/G1 transition. Additionally, skeletal muscle also expresses voltage‐gated potassium channel beta subunits 1–3 (Kvβ, 2, 3) of aldo‐keto reductases (AK6) family, although their role in skeletal myogenesis is not yet known. Given that Kvβ subunits binds to and regulate members of Kv1 family including Kv1.5, it is plausible that Kvβ subunits play a significant role in skeletal myogenesis.Hypothesiswe hypothesized that Kvβ2 modulates skeletal myogenesis.MethodsAge matched, 8–12 week old, wild type (Wt) and Kvβ2 knockout mice (KO) mice were used in this study. Transcript expression analysis of key transcription factors, muscle growth regulators and contractile genes was conducted utilizing real time quantitative PCR (qPCR). Myofiber size was estimated in biceps femoris muscle cross‐sections using Hematoxylin and eosin staining. Protein expression was assessed by Western blots in lysates isolated from Biceps femoris muscle.ResultsBody weight of KO mice recorded at 6, 11 or 16 weeks of age, was significantly (p &lt; 0.05) less than that of age matched Wt mice. Body weight normalized to tibia length in 16 week old KO mice was nearly 30% less than that of age matched Wt mice. Biceps femoris muscle cross‐sections in KO mice revealed significantly smaller myofiber area when compared to that of Wt mice (p &lt; 0.05). Myosin heavy chain mRNA expression profiles were also significantly different between the KO and Wt mice. when compared with that of Wt mice, gastrocnemius and biceps femoris of KO mice showed marginal (p &lt; 0.1) to significantly (p &lt; 0.05) higher expression of myosin heavy chain IIA and IIX isoforms, while myosin heavy chain I mRNA expression was not different between the groups. Soleus muscle of KO mice showed significantly higher myosin heavy chain I expression, while the expression of myosin heavy chain 2B was profoundly (p &lt; 0.05) decreased. Interestingly, the expression of major myogenic regulatory factors was unchanged between the two genotypes, with the exception that MyoD expression showed a consistently decreasing trend in all three muscles of KO mice when compared with that of Wt. In C2C12 cells, Kvβ2 mRNA expression declined steadily during the course of differentiation reaching to approximately 10% of that observed before differentiation (p &lt; 0.05).ConclusionOur data suggest that Kvβ2 subunit is essential for skeletal myogenesis. Kvβ2 appear to effect muscle fitness by altering myosin heavy chain composition, suggesting a significant role of Kvβ subunits in skeletal muscle size and phenotype determination.

Impact of Amyotrophic Lateral Sclerosis on Slow Tonic Myofiber Composition in Human Extraocular Muscles.

To analyze the proportion and cross-sectional area of myofibers containing myosin heavy chain slow-twitch (MyHCI) and myosin heavy chain slow tonic (MyHCsto) in extraocular muscles of autopsied amyotrophic lateral sclerosis (ALS) patients with either spinal or bulbar site of disease onset. Whole-muscle cross sections from the middle portion of the medial rectus were labeled with antibodies against MyHCI or MyHCsto and laminin. Myofibers labeled with the MyHC antibodies (MyHCI+sto) and the total number of myofibers were quantified in the orbital and global layer of 6 control individuals and 18 ALS patients. The cross-sectional area of myofibers labeled for either MyHC was quantified in 130 to 472 fibers/individual in the orbital and in 180 to 573 fibers/individual in the global layer of each specimen. The proportion of MyHCI+sto myofibers was significantly smaller in the orbital and global layer of ALS compared to control individuals. MyHCI+sto myofibers were significantly smaller in the global layer than in the orbital layer of ALS, whereas they were of similar size in control subjects. The decreased proportion of MyHCI+sto fibers correlated significantly with the age of death, but not disease duration, in patients who had the bulbar-onset variant of ALS but not in patients with spinal variant. ALS, regardless of site of onset, involves a loss of myofibers containing MyHCI+sto. Only in bulbar-onset cases did aging seem to play a role in the pathophysiological processes underlying the loss of MyHCI+sto fibers.

Diminished force production and mitochondrial respiratory deficits are strain-dependent myopathies of subacute limb ischemia

Reduced skeletal muscle mitochondrial function might be a contributing mechanism to the myopathy and activity based limitations that typically plague patients with peripheral arterial disease (PAD). We hypothesized that mitochondrial dysfunction, myofiber atrophy, and muscle contractile deficits are inherently determined by the genetic background of regenerating ischemic mouse skeletal muscle, similar to how patient genetics affect the distribution of disease severity with clinical PAD. Genetically ischemia protected (C57BL/6) and susceptible (BALB/c) mice underwent either unilateral subacute hind limb ischemia (SLI) or myotoxic injury (cardiotoxin) for 28days. Limbs were monitored for blood flow and tissue oxygen saturation and tissue was collected for the assessment of histology, muscle contractile force, gene expression, mitochondrial content, and respiratory function. Despite similar tissue O2 saturation and mitochondrial content between strains, BALB/c mice suffered persistent ischemic myofiber atrophy (55.3% of C57BL/6) and muscle contractile deficits (approximately 25% of C57BL/6 across multiple stimulation frequencies). SLI also reduced BALB/c mitochondrial respiratory capacity, assessed in either isolated mitochondria (58.3% of C57BL/6 at SLI on day (d)7, 59.1% of C57BL/6 at SLI d28 across multiple conditions) or permeabilized myofibers (38.9% of C57BL/6 at SLI d7; 76.2% of C57BL/6 at SLI d28 across multiple conditions). SLI also resulted in decreased calcium retention capacity (56.0% of C57BL/6) in BALB/c mitochondria. Nonischemic cardiotoxin injury revealed similar recovery of myofiber area, contractile force, mitochondrial respiratory capacity, and calcium retention between strains. Ischemia-susceptible BALB/c mice suffered persistent muscle atrophy, impaired muscle function, and mitochondrial respiratory deficits during SLI. Interestingly, parental strain susceptibility to myopathy appears specific toregenerative insults including an ischemic component. Our findings indicate that the functional deficits that plague PAD patients could include mitochondrial respiratory deficits genetically inherent to the regenerating muscle myofibers.

Prolongation of Relaxation Time in Extraocular Muscles With Brain Derived Neurotrophic Factor in Adult Rabbit.

PurposeWe tested the hypothesis that short-term treatment with brain derived neurotrophic factor (BDNF) would alter the contractile characteristics of rabbit extraocular muscle (EOM).MethodsOne week after injections of BDNF in adult rabbit superior rectus muscles, twitch properties were determined in treated and control muscles in vitro. Muscles were also examined for changes in mean cross-sectional areas, neuromuscular junction size, and percent of myofibers expressing specific myosin heavy chain isoforms, and sarcoendoplasmic reticulum calcium ATPases (SERCA) 1 and 2.ResultsBrain derived neurotrophic factor–treated muscles had prolonged relaxation times compared with control muscles. Time to 50% relaxation, time to 100% relaxation, and maximum rate of relaxation were increased by 24%, 27%, and 25%, respectively. No significant differences were seen in time to peak force, twitch force, or maximum rate of contraction. Brain derived neurotrophic factor treatment significantly increased mean cross-sectional areas of slow twitch and tonic myofibers, with increased areas ranging from 54% to 146%. Brain derived neurotrophic factor also resulted in an increased percentage of slow twitch myofibers in the orbital layers, ranging from 54% to 77%, and slow-tonic myofibers, ranging from 44% to 62%. No significant changes were seen SERCA1 or 2 expression or in neuromuscular junction size.ConclusionsShort-term treatment with BDNF significantly prolonged the duration and rate of relaxation time and increased expression of both slow-twitch and slow-tonic myosin-expressing myofibers without changes in neuromuscular junctions or SERCA expression. The changes induced by BDNF treatment might have potential therapeutic value in dampening/reducing uncontrolled eye oscillations in nystagmus.

Skeletal muscle fiber-type specific succinate dehydrogenase activity in cerebral palsy.

Children with cerebral palsy (CP) exhibit increased energy expenditure during movement, but whether this is due in part to decrements in skeletal muscle mitochondrial oxidative capacity is unknown. Accordingly, we compared fiber-type specific succinate dehydrogenase (SDH) activity in children with CP with typically developing (TD) children. SDH activity and myofiber areas of type 1 and 2A fibers were measured in semitendinosus biopsies of both groups (n = 5/group). SDH activity was ∼35% higher in type 1 compared with type 2A fibers, but there were no differences between groups. Average myofiber area was 45% smaller in CP versus TD (P < 0.05), and type 2A fibers were 32% larger than type 1 fibers (P < 0.05) only in TD children. Fiber-type specific SDH activity is similar between TD children and children with CP. This suggests that increased energy expenditure in children with CP is not related to impaired mitochondrial oxidative capacity. Muscle Nerve, 2016 Muscle Nerve 55: 122-124, 2017.

Activation of Wnt3a signaling promotes myogenic differentiation of mesenchymal stem cells in mdx mice.

Duchenne muscular dystrophy (DMD) is an X-linked genetic muscular disorder with no effective treatment at present. Mesenchymal stem cell (MSC) transplantation has been used to treat DMD, but the efficiency is low. Our previous studies show that activation of Wnt3a signaling promotes myogenic differentiation of MSCs in vitro. Here we report an effective MSC transplantation therapy in mdx mice by activation of Wnt3a signaling. MSCs were isolated from mouse bone marrow, and pretreated with Wnt3a-conditioned medium (Wnt3a-CM), then transplanted into mdx mice. The recipient mice were euthanized at 4, 8, 12, 16 weeks after the transplantation, and muscle pathological changes were examined. The expression of dystrophin in muscle was detected using immunofluorescence staining, RT-PCR and Western blotting. Sixteen weeks later, transplantation of Wnt3a-pretreated MSCs in mdx mice improved the characteristics of dystrophic muscles evidenced by significant reductions in centrally nucleated myofibers, the variability range of cross-sectional area (CSA) and the connective tissue area of myofibers. Furthermore, transplantation of Wnt3a-pretreated MSCs in mdx mice gradually and markedly increased the expression of dystrophin in muscle, and improved the efficiency of myogenic differentiation. Transplantation of Wnt3a-pretreated MSCs in mdx mice results in long-term amelioration of the dystrophic phenotype and restores dystrophin expression in muscle. The results suggest that Wnt3a may be a promising candidate for the treatment of DMD.

AMP-activated kinase α2 deficiency protects mice from denervation-induced skeletal muscle atrophy

AMP-activated protein kinase (AMPK) is a master regulator of skeletal muscle metabolic pathways. Recently, AMPK activation by AICAR has been shown to increase myofibrillar protein degradation in C2C12 myotubes via stimulating autophagy and ubiquitin proteasome system. However, the impact of AMPKα on denervation induced muscle atrophy has not been tested. In this study, we performed sciatic denervation on hind limb muscles in both wild type (WT) and AMPKα2−/− mice. We found that AMPKα was phosphorylated in atrophic muscles following denervation. In addition, deletion of AMPKα2 significantly attenuated denervation induced skeletal muscle wasting and protein degradation, as evidenced by preserved muscle mass and myofiber area, as well as lower levels of ubiquitinated protein, Atrogin-1 and MuRF-1 expression, and LC3-II/I ratio in tibial anterior (TA) muscles. Interestingly, the phosphorylated FoxO3a at Ser253 was significantly decreased in atrophic TA muscles, which was preserved in AMPKα2−/− mice. Collectively, our data support the notion that the activation of AMPKα2 contributes to the atrophic effects of denervation.

Abnormally Small Neuromuscular Junctions in the Extraocular Muscles From Subjects With Idiopathic Nystagmus and Nystagmus Associated With Albinism.

PurposeInfantile nystagmus syndrome (INS) is often associated with abnormalities of axonal outgrowth and connectivity. To determine if this manifests in extraocular muscle innervation, specimens from children with idiopathic INS or INS and albinism were examined and compared to normal age-matched control extraocular muscles.MethodsExtraocular muscles removed during normal surgery on children with idiopathic INS or INS and albinism were immunostained for neuromuscular junctions, myofiber type, the immature form of the acetylcholine receptor, and brain-derived neurotrophic factor (BDNF) and compared to age-matched controls.ResultsMuscles from both the idiopathic INS and INS and albinism groups had neuromuscular junctions that were 35% to 71% smaller based on myofiber area and myofiber perimeter than found in age-matched controls, and this was seen on both fast and slow myosin heavy chain isoform–expressing myofibers (all P < 0.015). Muscles from subjects with INS and albinism showed a 7-fold increase in neuromuscular junction numbers on fast myofibers expressing the immature gamma subunit of the acetylcholine receptor. The extraocular muscles from both INS subgroups showed a significant increase in the number and size of slow myofibers compared to age-matched controls. Brain-derived neurotrophic factor was expressed in control muscle but was virtually absent in the INS muscles.ConclusionsThese studies suggest that, relative to the final common pathway, INS is not the same between different patient etiologies. It should be possible to modulate these final common pathway abnormalities, via exogenous application of appropriate drugs, with the hope that this type of treatment may reduce the involuntary oscillatory movements in these children.

Effects of GHRP-2 and Cysteamine Administration on Growth Performance, Somatotropic Axis Hormone and Muscle Protein Deposition in Yaks (Bos grunniens) with Growth Retardation.

The objective of this study was to investigate the effects of growth hormone-releasing peptide-2 (GHRP-2) and cysteamine (CS) administration on growth performance in yaks with growth retardation and try to elucidate its regulatory mechanisms. Trial 1, thirty-six 1-year-old Qinghai high plateau yaks (body weight 38–83.2 kg) were randomly chosen for body weight and jugular blood samples collection. The relationship between body weight and serum GHRH (P < 0.05, R = 0.45), GH (P < 0.05, R = 0.47), IGF-1 (P < 0.05, R = 0.62) was significantly correlated in yaks colonies with lighter body weights. Trial 2, fifteen 1-year-old Qinghai high plateau yaks with growth retardation (average body weight 54.8 ± 8.24 kg) were randomly selected and assigned to negative control group (NG), GHRP-2 injection group (GG) and cysteamine feeding group (CG), with 5 yaks per group. Another five 1-year-old Qinghai high plateau yaks with normal growth performance (average body weight 75.3 ± 2.43 kg) were selected as positive control group (PG). The average daily gain (ADG) of the GG and CG were significantly higher than those in the PG and NG (P < 0.05). Both GHRP-2 and CS administration significantly enhanced the myofiber diameter and area of skeletal muscle (P<0.05). GHRP-2 significantly enhanced the serum GH and IGF-1 levels (P < 0.05), and up-regulated GHR, IGF-1 and IGF-1R mRNA expression in the liver and skeletal muscle (P < 0.05), enhanced the mRNA expression of PI3K, AKt and mTOR in the skeletal muscle (P<0.05). CS significantly reduced the serum SS levels and the hypothalamus SS mRNA expression (P < 0.05), and enhanced GHR and IGF-1 mRNA expression in the liver (P < 0.05), decreased the mRNA expression of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) mRNA (P < 0.05). Conclusions: Growth retardation in yaks was primarily due to somatotropic axis hormones secretion deficiency. Both GHRP-2 and CS administration can accelerate growth performance and GH, IGF-1 secretion in yaks with growth retardation. GHRP-2 enhanced muscle protein deposition mainly by up-regulated the protein synthesis pathways, whereas CS worked mainly by down-regulated the ubiquitin-proteasome pathway.

Glycoprotein nonmetastatic melanoma protein B ameliorates skeletal muscle lesions in a SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons and subsequent muscular atrophy. The quality of life of patients with ALS is significantly improved by ameliorating muscular symptoms. We previously reported that glycoprotein nonmetastatic melanoma protein B (GPNMB; osteoactivin) might serve as a target for ALS therapy. In the present study, superoxide dismutase 1/glycine residue 93 changed to alanine (SOD1(G93A) ) transgenic mice were used as a model of ALS. Expression of the C-terminal fragment of GPNMB was increased in the skeletal muscles of SOD1(G93A) mice and patients with sporadic ALS. SOD1(G93A) /GPNMB transgenic mice were generated to determine whether GPNMB expression ameliorates muscular symptoms. The weight and cross-sectional area of the gastrocnemius muscle, number and cross-sectional area of myofibers, and denervation of neuromuscular junctions were ameliorated in SOD1(G93A) /GPNMB vs. SOD1(G93A) mice. Furthermore, direct injection of a GPNMB expression plasmid into the gastrocnemius muscle of SOD1(G93A) mice increased the numbers of myofibers and prevented myofiber atrophy. These findings suggest that GPNMB directly affects skeletal muscle and prevents muscular pathology in SOD1(G93A) mice and may therefore serve as a target for therapy of ALS.

Myostatin Neutralization Results in Preservation of Muscle Mass and Strength in Preclinical Models of Tumor-Induced Muscle Wasting.

Skeletal muscle wasting occurs in a great majority of cancer patients with advanced disease and is associated with a poor prognosis and decreased survival. Myostatin functions as a negative regulator of skeletal muscle mass and has recently become a therapeutic target for reducing the loss of skeletal muscle and strength associated with clinical myopathies. We generated neutralizing antibodies to myostatin to test their potential use as therapeutic agents to attenuate the skeletal muscle wasting due to cancer. We show that our neutralizing antimyostatin antibodies significantly increase body weight, skeletal muscle mass, and strength in non-tumor-bearing mice with a concomitant increase in mean myofiber area. The administration of these neutralizing antibodies in two preclinical models of cancer-induced muscle wasting (C26 colon adenocarcinoma and PC3 prostate carcinoma) resulted in a significant attenuation of the loss of muscle mass and strength with no effect on tumor growth. We also show that the skeletal muscle mass- and strength-preserving effect of the antibodies is not affected by the coadministration of gemcitabine, a common chemotherapeutic agent, in both non-tumor-bearing mice and mice bearing C26 tumors. In addition, we show that myostatin neutralization with these antibodies results in the preservation of skeletal muscle mass following reduced caloric intake, a common comorbidity associated with advanced cancer. Our findings support the use of neutralizing antimyostatin antibodies as potential therapeutics for cancer-induced muscle wasting.

Cardioprotective effect of virgin coconut oil in heated palm oil diet-induced hypertensive rats

Context: Virgin coconut oil (VCO) contains high antioxidant activity which may have protective effects on the heart in hypertensive rats.Objectives: The study investigated the effects of VCO on blood pressure and cardiac tissue by measuring angiotensin-converting enzyme (ACE) activity and its histomorphometry in rats fed with a heated palm oil (HPO) diet.Materials and methods: Thirty-two male Sprague–Dawley rats were randomly divided into four groups: (i) control, (ii) orally given VCO (1.42 ml/kg), (iii) fed with a HPO (15%) diet, and (iv) fed with a HPO diet and supplemented with VCO (1.42 ml/kg, po) (HPO+VCO) for 16 weeks. Blood pressure was measured monthly. After 16 weeks, rat hearts were dissected for lipid peroxidation (TBARS) and ACE activity measurement and histomorphometric study.Results: Systolic blood pressure was significantly increased in the HPO group compared with the control starting at week eight (112.91 ± 1.32 versus 98.08 ± 3.61 mmHg, p < 0.05) which was prevented by VCO supplementation (91.73 ± 3.42 mmHg). The consumption of HPO increased TBARS and ACE activity in heart, which were inhibited by VCO supplementation. The increases in the myofiber width and area as well as nuclear size reduction in the HPO group were significantly prevented by VCO supplementation.Conclusion: These results suggested that VCO supplementation possesses a cardioprotective effect by preventing the increase in blood pressure via an antioxidant mechanism and remodeling in rats fed repeatedly with a HPO diet.

Disease course in mdx:utrophin+/- mice: comparison of three mouse models of Duchenne muscular dystrophy.

The mdx mouse model of Duchenne muscular dystrophy (DMD) is used to study disease mechanisms and potential treatments, but its pathology is less severe than DMD patients. Other mouse models were developed to more closely mimic the human disease based on knowledge that upregulation of utrophin has a protective effect in mdx muscle. An mdx:utrophin−/− (dko) mouse was created, which had a severe disease phenotype and a shortened life span. An mdx:utrophin+/− mouse was also created, which had an intermediate disease phenotype compared to the mdx and dko mice. To determine the usefulness of mdx:utrophin+/− mice for long-term DMD studies, limb muscle pathology and function were assessed across the life span of wild-type, mdx, mdx:utrophin+/−, and dko mice. Muscle function assessment, specifically grip duration and rotarod performance, demonstrated that mdx:utrophin+/− mice were weaker for a longer time than mdx mice. Mean myofiber area was smaller in mdx:utrophin+/− mice compared to mdx mice at 12 months. Mdx:utrophin+/− mice had a higher percentage of centrally nucleated myofibers compared to mdx mice at 6 and 12 months. Collagen I and IV density was significantly higher in mdx:utrophin+/− muscle compared to mdx at most ages examined. Generally, mdx:utrophin+/− mice showed an intermediate disease phenotype over a longer time course compared to the mdx and dko mice. While they do not genetically mirror human DMD, mdx:utrophin+/− mice may be a more useful animal model than mdx or dko mice for investigating long-term efficacy of potential treatments when fibrosis or muscle function is the focus.

Effects of chronic high-fat feeding on skeletal muscle mass and function in middle-aged mice.

Increased adipose tissue may promote catabolic events in skeletal muscle. The aim of this study was to test whether high-fat diet (HFD)-induced obesity would accelerate the onset of muscle wasting in middle-aged mice. Muscle was collected from C57BL/6 mice at 9months of age (baseline) and 14months of age after consuming a control (C) or HFD. Mice in C and HFD were also subjected to evaluations of body composition and function before and after their respective diets. HFD demonstrated significant (p<0.05) losses of grip strength (-15%) and sensorimotor coordination (-11%), whereas C did not. Lean mass decreased to a greater degree in HFD although not significantly (C: -20.69±7.94 vs. HFD: -31.14±5.49%, p>0.05). Gastrocnemius, quadriceps, and hamstrings mass in C and HFD were significantly reduced from baseline (-27 to 43 and -39 to 47%, respectively, p<0.05) with no differences between the two; however, soleus mass was lower only in HFD (-24%, p=0.03). Myofiber area, satellite cells, and myonuclei of the gastrocnemius were lower only in HFD (-23, -19, and -16%, respectively, p<0.05) compared to baseline. HFD-induced obesity adversely affected function in middle-aged mice. Atrophy of the soleus in HFD but not C suggests sensitivity of oxidative muscle to HFD-dependent catabolism more so than aging. In the muscles containing fast/mixed fibers, aging effects may have concealed the catabolic nature of HFD; however, morphological changes in the gastrocnemius including decreased fiber area, satellite cells, and myonuclei are consistent with an atrophic phenotype related to HFD.

Contributions of biarticular myogenic components to the limitation of the range of motion after immobilization of rat knee joint.

BackgroundMuscle atrophy caused by immobilization in the shortened position is characterized by a decrease in the size or cross-sectional area (CSA) of myofibers and decreased muscle length. Few studies have addressed the relationship between limitation of the range of motion (ROM) and the changes in CSA specifically in biarticular muscles after atrophy because of immobilization. We aimed to determine the contribution of 2 distinct muscle groups, the biarticular muscles of the post thigh (PT) and those of the post leg (PL), to the limitation of ROM as well as changes in the myofiber CSAs after joint immobilization surgery.MethodsMale Wistar rats (n = 40) were randomly divided into experimental and control groups. In the experimental group, the left knee was surgically immobilized by external fixation for 1, 2, 4, 8, or 16 weeks (n = 5 each) and sham surgery was performed on the right knee. The rats in the control groups (n = 3 per time point) did not undergo surgery. After the indicated immobilization periods, myotomy of the PT or PL biarticular muscles was performed and the ROM was measured. The hamstrings and gastrocnemius muscles from the animals operated for 1 or 16 weeks were subjected to morphological analysis.ResultsIn immobilized knees, the relative contribution of the PT biarticular myogenic components to the total restriction reached 80% throughout the first 4 weeks and decreased thereafter. The relative contribution of the PL biarticular myogenic components remained <20% throughout the immobilization period. The ratio of the myofiber CSA of the immobilized to that of the sham-operated knees was significantly lower at 16 weeks after surgery than at 1 week after surgery only in the hamstrings.ConclusionsThe relative contribution of the PT and PL components to myogenic contracture did not significantly change during the experimental period. However, the ratio of hamstrings CSAs to the sham side was larger than the ratio of medial gastrocnemius CSAs to the sham side after complete atrophy because of immobilization.

Molecular and metabolomic effects of voluntary running wheel activity on skeletal muscle in late middle‐aged rats (1040.7)

We examined the molecular and metabolomic effects of low‐intensity exercise in late middle‐aged male Sprague Dawley rats (16‐17 months). Animals were assigned either continuous running wheel access for 8 weeks (RW+) or cage‐matched without running wheels (RW‐). The 9 RW+ rats averaged just 83 m/day (range: 8‐163 m), yet exhibited 6% reduced individual average weekly food intake (145 g vs 154 g, p&lt;0.01) and 84% reduced individual body weight gain (4.3 g vs 26.3 g, p=0.02) over the 8 weeks. Muscle weights and myofiber area showed no difference between groups. Western blots of gastrocnemius lysates with a panel of antibodies suggest that running wheel access improved oxidative metabolism (53% increase in PGC1‐α, p=0.02), increased autophagy (36% increase in LC3B‐II:‐I ratio; p=0.03), and modulated growth signaling (26% increase in myostatin, p=0.04). RW+ gastrocnemius lysates also showed 45% increased glycogen content (p=0.04) and 43% increased glycogen phosphorylase expression (p=0.04). Metabolomic profiling of plantaris and soleus muscles indicated that even low‐volume voluntary wheel running is associated with decreases in many long chain fatty acids (e.g., palmitoleate, myristoleate) relative to RW‐ rats. Increases in acylcarnitines and glycerophosphocholines were more specific to plantaris. These data further establish extensive metabolic and behavioral remodeling due to small amounts of exercise.

Muscle‐specific AMPK β1β2‐null mice display a myopathy due to loss of capillary density in nonpostural muscles

AMP-activated protein kinase (AMPK) is a master regulator of metabolism. While muscle-specific AMPK β1β2 double-knockout (β1β2M-KO) mice display alterations in metabolic and mitochondrial capacity, their severe exercise intolerance suggested a secondary contributor to the observed phenotype. We find that tibialis anterior (TA), but not soleus, muscles of sedentary β1β2M-KO mice display a significant myopathy (decreased myofiber areas, increased split and necrotic myofibers, and increased centrally nucleated myofibers. A mitochondrial- and fiber-type-specific etiology to the myopathy was ruled out. However, β1β2M-KO TA muscles displayed significant (P<0.05) increases in platelet aggregation and apoptosis within myofibers and surrounding interstitium (P<0.05). These changes correlated with a 45% decrease in capillary density (P<0.05). We hypothesized that the β1β2M-KO myopathy in resting muscle resulted from impaired AMPK-nNOSμ signaling, causing increased platelet aggregation, impaired vasodilation, and, ultimately, ischemic injury. Consistent with this hypothesis, AMPK-specific phosphorylation (Ser1446) of nNOSμ was decreased in β1β2M-KO compared to wild-type (WT) mice. The AMPK-nNOSμ relationship was further demonstrated by administration of 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) to β1β2-MKO muscles and C2C12 myotubes. AICAR significantly increased nNOSμ phosphorylation and nitric oxide production (P<0.05) within minutes of administration in WT muscles and C2C12 myotubes but not in β1β2M-KO muscles. These findings highlight the importance of the AMPK-nNOSμ pathway in resting skeletal muscle.

Abstract A6: The ES-2 ovarian cancer causes muscle wasting in vitro and in vivo: A novel experimental model of cancer cachexia

Abstract Cancer cachexia is a devastating consequence of cancer. A multifactorial syndrome characterized by skeletal muscle and adipose tissue wasting, cachexia cannot be fully reversed by nutritional support. Cachexia occurs in up to one half of all cancer patients and correlates with poor performance status, reduced quality of life and high mortality. Cachexia results both from the tumor-host interaction, as well as from the anti-cancer therapies. The only cure for cachexia is definitive removal of the tumor. In ovarian cancer, cachexia is associated with malnutrition and a general inflammatory state, characterized by high levels of pro-inflammatory cytokines (such as IL-6, TNF) in ascites and blood. Due to the absence of proper experimental models, cachexia also represents a generally understudied aspect of ovarian cancer that deserves more attention. In order to establish novel experimental models for the study of ovarian cancer cachexia, we tested several human ovarian cancer cell lines with different degrees of differentiation for their capability to induce cachexia in vivo and muscle wasting in vitro. In a xenograft model, female Nu/Nu athymic nude mice were injected i.p. with 10x106 OVCAR-3, SK-OV-3 and ES-2 human ovarian cancer cells to mimic disseminated abdominal disease. After 60 days, the OVCAR-3 and SK-OV-3 cell lines did not result in detectable tumors. In contrast, beginning 8 days after inoculation, ES-2 cells caused severe cachexia with marked loss of body weight and with massive ascites accumulation in the peritoneal cavity. Within the ascites, ES-2 cells were not shown to aggregate, but existed more as a single-cell suspension or very small cell clumps. Some ES-2 cells also disseminated and infiltrated the pancreas, diaphragm and other organs, forming multiple solid tumors of histology consistent with clear cell carcinoma, characterized by sheets of cells with irregularly shaped nuclei and abundant cytoplasm. Muscles, including tibialis, gastrocnemius, quadriceps and heart, of tumor-bearing mice appeared markedly smaller than normal controls (~-20% vs. control; p&amp;lt;0.01). Muscle loss was due to fiber atrophy, as observed by reduced tibialis myofiber area. Fat pads and spleen were reduced in size, while the liver was unchanged. Piximus imaging revealed decreased bone mineral density (-16% vs. control; p&amp;lt;0.05). Consistent with reports in human ovarian cancer cachexia, IL-6 levels were markedly elevated in both blood and ascites. In quadriceps muscle, cachexia was also associated with high levels of phosphorylated STAT3 (pSTAT3), whose causal role in tumor-associated muscle wasting was previously reported in our laboratory. In order to understand whether tumor-derived factors were directly responsible for the wasting effects observed in vivo, differentiated C2C12 murine myotubes were exposed to conditioned medium from ES-2 cell cultures and evaluated for effects on myotube size. After 48 hours, the average diameter of C2C12 myofibers was severely reduced (-28% vs. control; p&amp;lt;0.001). These effects were evident at a low concentration of supernatant and were associated with elevated pSTAT3 levels. In conclusion, here we report that ES-2 tumor cells directly induce muscle atrophy, both in vitro and in vivo. Thus this is a new experimental model for the study of ovarian cancer cachexia. This model and future similar models will aid in identifying novel molecular mediators that could be effectively targeted in order to improve muscle wasting associated with ovarian cancer. Citation Format: Andrea Bonetto, Melissa J. Puppa, James A. Carson, Teresa A. Zimmers. The ES-2 ovarian cancer causes muscle wasting in vitro and in vivo: A novel experimental model of cancer cachexia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A6.