Preclinical Multi-Omic Assessment of Pioglitazone in Skeletal Muscles of Mice Implanted with Human HER2/neu Overexpressing Breast Cancer Xenografts.

Breast cancer (BC) is the most prevalent cancer worldwide and is accompanied by fatigue during both active disease and remission in the majority of cases. Our lab has measured fatigue in isolated muscles from treatment-naive BC patient-derived orthotopic xenograft (BC-PDOX) mice. Here, we conducted a preclinical trial of pioglitazone in BC-PDOX mice to determine its efficacy in ameliorating BC-induced muscle fatigue, as well as its effects on transcriptomic, metabolomic, and lipidomic profiles in skeletal muscle. Methods: The pioglitazone and vehicle groups were treated orally for 4 weeks upon reaching a tumor volume of 600 mm3. Whole-animal indirect calorimetry was used to evaluate systemic metabolic states. The transcriptome was profiled using short-read bulk RNA sequencing (RNA-seq). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to profile the metabolome and lipidome. Fast and slow skeletal muscle function were evaluated using isolated ex vivo testing. Results: Pioglitazone was associated with a significant overall decrease in metabolic rate, with no changes in substrate utilization. RNA-seq supported the downstream effects of pioglitazone on target genes and displayed considerable upregulation of mitochondrial bioenergetic pathways. Skeletal muscle metabolomic and lipidomic profiles exhibited dysregulation in response to BC, which was partially restored in pioglitazone-treated mice compared to vehicle-treated BC-PDOX mice. Despite molecular support for pioglitazone's efficacy, isolated muscle function was not affected by pioglitazone treatment. Conclusions: BC induces multi-omic dysregulation in skeletal muscle, which pioglitazone partially ameliorates. Future research should focus on profiling systemic metabolic dysfunction, identifying molecular biomarkers of fatigue, and testing alternative pioglitazone treatment regimens.

Sexual Dimorphism of Skeletal Muscle in a Mouse Model of Breast Cancer: A Functional and Molecular Analysis.

Breast cancer incidence in men is statistically rare; however, given the lack of screening in males, more advanced stages at initial diagnosis result in lower 5-year survival rates for men with breast cancer compared to women. A sexual dimorphism, with respect to the effect of tumor growth on cachexia incidence and severity, has also been reported across cancer types. The purpose of this study was to examine the sexual dimorphism of breast cancer as it pertains to skeletal muscle function and molecular composition. Using female and male transgenic PyMT mice, we tested the hypothesis that the isometric contractile properties and molecular composition of skeletal muscle would be differentially affected by breast tumors. PyMT tumor-bearing mice of each sex, corresponding to maximal tumor burden, were compared to their respective controls. RNA sequencing of skeletal muscle revealed different pathway alterations that were exclusive to each sex. Further, differentially expressed genes and pathways were substantially more abundant in female tumor mice, with only minimal dysregulation in male tumor mice, each compared to their respective controls. These differences in the transcriptome were mirrored in isometric contractile properties, with greater tumor-induced dysfunction in females than male mice, as well as muscle wasting. Collectively, these data support the concept of sexually dimorphic responses to cancer in skeletal muscle and suggest that these responses may be associated with the clinical differences in breast cancer between the sexes. The identified sex-dependent pathways within the muscle of male and female mice provide a framework to evaluate therapeutic strategies targeting tumor-associated skeletal muscle alterations.

Sexual Dimorphism of Skeletal Muscle in a Mouse Model of Breast Cancer: A Functional and Molecular Analysis.

Breast cancer incidence in men is statistically rare; however, given the lack of screening in males, more advanced stages at initial diagnosis results in lower 5-year survival rates for men with breast cancer compared to women. A sexual dimorphism, with respect to the effect of tumor growth on cachexia incidence and severity, has also been reported across cancer types. The purpose of this study was to examine the sexual dimorphism of breast cancer as it pertains to skeletal muscle function and molecular composition. Using female and male transgenic PyMT mice, we tested the hypothesis that isometric contractile properties and molecular composition of skeletal muscle would be differentially affected by breast tumors. PyMT tumor-bearing mice of each sex, corresponding to maximal tumor burden, were compared to their respective controls. RNA-sequencing of skeletal muscle revealed different pathway alterations that were exclusive to each sex. Further, differentially expressed genes and pathways were substantially more abundant in female tumor mice, with only minimal dysregulation in male tumor mice, each compared to their respective controls. These differences in the transcriptome were mirrored in isometric contractile properties, with greater tumor-induced dysfunction in females than male mice, as well as muscle wasting. Collectively, these data support the concept of sexually dimorphic responses to cancer in skeletal muscle and suggest these responses may be associated with the clinical differences in breast cancer between the sexes. The identified sex-dependent pathways within muscle of male and female mice provide a framework to evaluate therapeutic strategies targeting tumor-associated skeletal muscle alterations.

Chronic stress induced perivascular adipose tissue impairment of aortic function and the therapeutic effect of exercise.

NEW FINDINGS: What is the central question of this study? Thoracic perivascular adipose tissue (tPVAT) is known to, in part, regulate aortic function: what are the effects of unpredictable chronic mild stress (UCMS) on the tPVAT regulation of aortic function and what is the role of exercise training in alleviating the potential negative actions of UCMS on tPVAT? What is the main finding and its importance? UCMS causes tPVAT to disrupt endothelium-dependent dilatation, increases inflammatory cytokine production and diminishes tPVAT-adiponectin. Exercise training proved efficacious in preventing tPVAT-mediated disruption of aortic function. The data support a tPVAT mechanism through which chronic stress negatively impacts vascular health, which adds to our knowledge of how psychological disorders might increase the risk of cardiovascular disease. ABSTRACT: Chronic stress is a major risk for cardiovascular disease. Perivascular adipose tissue (PVAT) has been shown to regulate vascular function; however, the impact of chronic stress and the comorbidity of metabolic syndrome (MetS) on thoracic (t)PVAT is unknown. Additionally, aerobic exercise training (AET) is known to combat the pathology of MetS and chronic stress, but the role of tPVAT in these actions is also unknown. Therefore, the purpose of this study was to examine the effects of unpredictable chronic mild stress (UCMS) on the tPVAT regulation of aortic function and the preventative effect of AET. Lean (LZR) and obese (OZR) Zucker rats (16-17 weeks old) were exposed to 8 weeks of UCMS with and without treadmill exercise (AET). In LZR, UCMS impaired aortic endothelium-dependent dilatation (EDD) (assessed ex vivo by wire myography) and aortic stiffness (assessed by elastic modulus) with no change in OZR subject to UCMS. However, both LZR and OZR UCMS tPVAT impaired EDD compared to respective controls. LZR and OZR subject to UCMS had higher oxidative stress production, diminished adiponectin and impaired aortic nitric oxide levels. Divergently, UCMS induced greater inflammatory cytokine production in LZR UCMS tPVAT, but not in OZR UCMS tPVAT. AET prevented the tPVAT impairment of aortic relaxation with UCMS in LZR and OZR. Additionally, AET reduced aortic stiffness in both LZR and OZR. These beneficial effects on tPVAT regulation of the aorta are likely due to AET preservation of adiponectin, reduced oxidative stress and inflammation, and enhanced nitric oxide. UCMS impaired tPVAT-regulated aortic function in LZR, and augmented MetS-induced EDD in OZR. Conversely, AET in combination with UCMS largely preserved aortic function and the tPVAT environment, in both groups.

Breast cancer-associated skeletal muscle mitochondrial dysfunction and lipid accumulation is reversed by PPARG.

The peroxisome proliferator-activated receptors (PPARs) have been previously implicated in the pathophysiology of skeletal muscle dysfunction in women with breast cancer (BC) and animal models of BC. This study investigated alterations induced in skeletal muscle by BC-derived factors in an in vitro conditioned media (CM) system and tested the hypothesis that BC cells secrete a factor that represses PPAR-γ (PPARG) expression and its transcriptional activity, leading to downregulation of PPARG target genes involved in mitochondrial function and other metabolic pathways. We found that BC-derived factors repress PPAR-mediated transcriptional activity without altering protein expression of PPARG. Furthermore, we show that BC-derived factors induce significant alterations in skeletal muscle mitochondrial function and lipid accumulation, which are rescued with exogenous expression of PPARG. The PPARG agonist drug rosiglitazone was able to rescue BC-induced lipid accumulation but did not rescue effects of BC-derived factors on PPAR-mediated transcription or mitochondrial function. These data suggest that BC-derived factors alter lipid accumulation and mitochondrial function via different mechanisms that are both related to PPARG signaling, with mitochondrial dysfunction likely being altered via repression of PPAR-mediated transcription, and lipid accumulation being altered via transcription-independent functions of PPARG.

Skeletal muscle reprogramming by breast cancer regardless of treatment history or tumor molecular subtype.

Increased susceptibility to fatigue is a negative predictor of survival commonly experienced by women with breast cancer (BC). Here, we sought to identify molecular changes induced in human skeletal muscle by BC regardless of treatment history or tumor molecular subtype using RNA-sequencing (RNA-seq) and proteomic analyses. Mitochondrial dysfunction was apparent across all molecular subtypes, with the greatest degree of transcriptomic changes occurring in women with HER2/neu-overexpressing tumors, though muscle from patients of all subtypes exhibited similar pathway-level dysregulation. Interestingly, we found no relationship between anticancer treatments and muscle gene expression, suggesting that fatigue is a product of BC per se rather than clinical history. In vitro and in vivo experimentation confirmed the ability of BC cells to alter mitochondrial function and ATP content in muscle. These data suggest that interventions supporting muscle in the presence of BC-induced mitochondrial dysfunction may alleviate fatigue and improve the lives of women with BC.

Human Breast Cancer Xenograft Model Implicates Peroxisome Proliferator-activated Receptor Signaling as Driver of Cancer-induced Muscle Fatigue.

PURPOSE: This study tested the hypothesis that a patient-derived orthotopic xenograft (PDOX) model would recapitulate the common clinical phenomenon of breast cancer-induced skeletal muscle (SkM) fatigue in the absence of muscle wasting. This study additionally sought to identify drivers of this condition to facilitate the development of therapeutic agents for patients with breast cancer experiencing muscle fatigue. EXPERIMENTAL DESIGN: Eight female BC-PDOX-bearing mice were produced via transplantation of tumor tissue from 8 female patients with breast cancer. Individual hind limb muscles from BC-PDOX mice were isolated at euthanasia for RNA-sequencing, gene and protein analyses, and an ex vivo muscle contraction protocol to quantify tumor-induced aberrations in SkM function. Differentially expressed genes (DEG) in the BC-PDOX mice relative to control mice were identified using DESeq2, and multiple bioinformatics platforms were employed to contextualize the DEGs. RESULTS: We found that SkM from BC-PDOX-bearing mice showed greater fatigability than control mice, despite no differences in absolute muscle mass. PPAR, mTOR, IL6, IL1, and several other signaling pathways were implicated in the transcriptional changes observed in the BC-PDOX SkM. Moreover, 3 independent in silico analyses identified PPAR signaling as highly dysregulated in the SkM of both BC-PDOX-bearing mice and human patients with early-stage nonmetastatic breast cancer. CONCLUSIONS: Collectively, these data demonstrate that the BC-PDOX model recapitulates the expected breast cancer-induced SkM fatigue and further identify aberrant PPAR signaling as an integral factor in the pathology of this condition.

Dysregulation of metabolic-associated pathways in muscle of breast cancer patients: preclinical evaluation of interleukin-15 targeting fatigue.

BACKGROUND: Breast cancer patients report a perception of increased muscle fatigue, which can persist following surgery and standardized therapies. In a clinical experiment, we tested the hypothesis that pathways regulating skeletal muscle fatigue are down-regulated in skeletal muscle of breast cancer patients and that different muscle gene expression patterns exist between breast tumour subtypes. In a preclinical study, we tested the hypothesis that mammary tumour growth in mice induces skeletal muscle fatigue and that overexpression of the cytokine interleukin-15 (IL-15) can attenuate mammary tumour-induced muscle fatigue. METHODS: Early stage non-metastatic female breast cancer patients (n = 14) and female non-cancer patients (n = 6) provided a muscle biopsy of the pectoralis major muscle during mastectomy, lumpectomy, or breast reconstruction surgeries. The breast cancer patients were diagnosed with either luminal (ER+ /PR+ , n = 6), triple positive (ER+ /PR+ /Her2/neu+ , n = 5), or triple negative (ER- /PR- /Her2/neu- , n = 3) breast tumours and were being treated with curative intent either with neoadjuvant chemotherapy followed by surgery or surgery followed by standard post-operative therapy. Biopsies were used for RNA-sequencing to compare the skeletal muscle gene expression patterns between breast cancer patients and non-cancer patients. The C57BL/6 mouse syngeneic mammary tumour cell line, E0771, was used to induce mammary tumours in immunocompetent mice, and isometric muscle contractile properties and fatigue properties were analysed following 4 weeks of tumour growth. RESULTS: RNA-sequencing and subsequent bioinformatics analyses revealed a dysregulation of canonical pathways involved in oxidative phosphorylation, mitochondrial dysfunction, peroxisome proliferator-activated receptor signalling and activation, and IL-15 signalling and production. In a preclinical mouse model of breast cancer, the rate of muscle fatigue was greater in mice exposed to mammary tumour growth for 4 weeks, and this greater muscle fatigue was attenuated in transgenic mice that overexpressed the cytokine IL-15. CONCLUSIONS: Our data identify novel genes and pathways dysregulated in the muscles of breast cancer patients with early stage non-metastatic disease, with particularly aberrant expression among genes that would predispose these patients to greater muscle fatigue. Furthermore, we demonstrate that IL-15 overexpression can attenuate muscle fatigue associated with mammary tumour growth in a preclinical mouse model of breast cancer. Therefore, we propose that skeletal muscle fatigue is an inherent consequence of breast tumour growth, and this greater fatigue can be targeted therapeutically.

Aortic dysfunction in metabolic syndrome mediated by perivascular adipose tissue TNFα- and NOX2-dependent pathway.

NEW FINDINGS: What is the central question of this study? Tumour necrosis factor-α (TNFα) has been shown to impair vascular function, but the impact of thoracic aorta perivascular adipose tissue (tPVAT)-derived TNFα on tPVAT and aortic function in metabolic syndrome is unknown. What is the main finding and its importance? Release of TNFα by tPVAT causes production of reactive oxygen species in tPVAT through activation of an NADPH-oxidase 2 (NOX2)-dependent pathway, activates production of aortic reactive oxygen species and mediates aortic stiffness, potentially through matrix metalloproteinase 9 activity. Neutralization of TNFα and/or inhibition of NOX2 blocks the tPVAT-induced impairment of aortic function. These data partly implicate tPVAT NOX2 and TNFα in mediating the vascular pathology of metabolic syndrome. ABSTRACT: Perivascular adipose tissue (PVAT) is recognized for its vasoactive effects, but it is unclear how metabolic syndrome impacts thoracic aorta (t)PVAT and the subsequent effect on functional and structural aortic stiffness. Thoracic aorta and tPVAT were removed from 16- to 17-week-old lean (LZR, n = 16) and obese Zucker rats (OZR, n = 16). The OZR presented with aortic endothelial dysfunction, assessed by wire myography, and increased aortic stiffness, assessed by elastic modulus. The OZR tPVAT exudate further exacerbated the endothelial dysfunction, reducing nitric oxide and endothelium-dependent relaxation (P < 0.05). Additionally, OZR tPVAT exudate had increased MMP9 activity (P < 0.05) and further increased the elastic modulus of the aorta after 72 h of co-culture (P < 0.05). We found that the observed aortic dysfunction caused by OZR tPVAT was mediated through increased production and release of tumour necrosis factor-α (TNFα; P < 0.01), which was dependent on tPVAT NADPH-oxidase 2 (NOX2) activity. The OZR tPVAT release of reactive oxygen species and subsequent aortic dysfunction were inhibited by TNFα neutralization and/or inhibition of NOX2. Additionally, we found that OZR tPVAT had reduced activity of the active sites of the 20S proteasome (P < 0.05) and reduced superoxide dismutase activity (P < 0.01). In conclusion, metabolic syndrome causes tPVAT dysfunction through an interplay between TNFα and NOX2 that leads to tPVAT-mediated aortic stiffness by activation of aortic reactive oxygen species and increased MMP9 activity.

Hippocampus-specific deficiency of IL-15Rα contributes to greater anxiety-like behaviors in mice.

A hippocampus-specific IL15RαKO mouse (hipIl15ra fl/fl /Cre+) was generated to test the hypothesis that the targeted deletion of interleukin-15 receptor alpha (IL-15Rα) in the hippocampus contributes to altered behavior, including greater levels of anxiety and ambulatory activity. Using Cre-loxP, exons 2 and 3 of the IL-15Rα gene were excised within the hippocampus, while normal expression was maintained within the rest of the brain. In the open field test (OFT), hipIl15ra fl/fl /Cre+ spent a greater amount of time in the periphery and less time in the central portions of the chamber, and there was also a noticeable trend for decreased rearing activity; these behaviors are consistent with greater levels of anxiety-like behavior in these mice. However, there were no differences in the overall locomotor counts in the OFT when comparing hipIl15ra fl/fl /Cre+ mice to their littermate controls. These data implicate IL-15-related signaling within the hippocampus has a role in anxiety-like behavior.

IL-15Rα deficiency in skeletal muscle alters respiratory function and the proteome of mitochondrial subpopulations independent of changes to the mitochondrial genome.

Interleukin-15 receptor alpha knockout (IL15RαKO) mice exhibit a greater skeletal muscle mitochondrial density with an altered mitochondrial morphology. However, the mechanism and functional impact of these changes have not been determined. In this study, we characterized the functional, proteomic, and genomic alterations in mitochondrial subpopulations isolated from the skeletal muscles of IL15RαKO mice and B6129 background control mice. State 3 respiration was greater in interfibrillar mitochondria and whole muscle ATP levels were greater in IL15RαKO mice supporting the increases in respiration rate. However, the state 3/state 4 ratio was lower, suggesting some degree of respiratory uncoupling. Proteomic analyses identified several markers independently in mitochondrial subpopulations that are associated with these functional alterations. Next Generation Sequencing of mtDNA revealed a high degree of similarity between the mitochondrial genomes of IL15RαKO mice and controls in terms of copy number, consensus coding and the presence of minor alleles, suggesting that the functional and proteomic alterations we observed occurred independent of alterations to the mitochondrial genome. These data provide additional evidence to implicate IL-15Rα as a regulator of skeletal muscle phenotypes through effects on the mitochondrion, and suggest these effects are driven by alterations to the mitochondrial proteome.

Interleukin-15 directly stimulates pro-oxidative gene expression in skeletal muscle in-vitro via a mechanism that requires interleukin-15 receptor alpha.

Interleukin-15 (IL-15) signaling is heavily regulated by a high specificity IL-15 binding protein known as interleukin-15 receptor alpha (IL-15Rα). In-vivo disruption of IL-15Rα in the constitutive IL-15Rα knock-out (IL-15RαKO) mouse results in a shift towards an oxidative muscle phenotype characterized by dramatic increases in mitochondrial density. The IL-15RαKO mouse displays elevated levels of IL-15 transcript in muscle tissue, along with increased circulating levels of IL-15. As a result, it has been suggested that loss of IL-15Rα from skeletal muscle enhances muscle IL-15 secretion, and that muscle-derived IL-15 acts in an autocrine fashion to elicit pro-oxidative effects. However, this proposed mechanism of IL-15/IL-15Rα action in skeletal muscle is based primarily on in-vivo associative observations, and has yet to be explored in a direct manner. Thus, our purpose was to assess the immediate influence of IL-15Rα on the capacity of skeletal muscle to secrete and respond to IL-15, and also to determine whether IL-15 has the ability to act directly on skeletal muscle to induce pro-oxidative changes. These aims were addressed in-vitro using primary myogenic cultures derived from IL-15RαKO mice and B6129 controls, as well as cultures of the C2C12 immortalized myogenic cell line. Cultures obtained from IL-15RαKO mice displayed a diminished capacity to secrete IL-15 in relation to B6129 controls. Acute treatment of B6129-derived cultures with recombinant IL-15 increased transcriptional expression of the pro-oxidative genes PGC1α and PPARδ. IL-15 treatment failed to elicit a similar response in cultures generated from IL-15RαKO mice. Chronic treatment of C2C12 cultures with IL-15 during myogenic differentiation resulted in mature myocytes with greater mitochondrial density in relation to vehicle treated controls. Collectively, these results provide evidence that IL-15 has the capacity to act directly on skeletal muscle in a pro-oxidative manner, and that disruption of IL-15Rα ablates the ability of skeletal muscle to secrete and respond to IL-15.

Muscle-specific deletion of exons 2 and 3 of the IL15RA gene in mice: effects on contractile properties of fast and slow muscles.

Interleukin-15 (IL-15) is a putative myokine hypothesized to induce an oxidative skeletal muscle phenotype. The specific IL-15 receptor alpha subunit (IL-15Rα) has also been implicated in specifying this contractile phenotype. The purposes of this study were to determine the muscle-specific effects of IL-15Rα functional deficiency on skeletal muscle isometric contractile properties, fatigue characteristics, spontaneous cage activity, and circulating IL-15 levels in male and female mice. Muscle creatine kinase (MCK)-driven IL-15Rα knockout mice (mIl15ra(fl/fl)/Cre(+)) were generated using the Cre-loxP system. We tested the hypothesis that IL-15Rα functional deficiency in skeletal muscle would increase resistance to contraction-induced fatigue, cage activity, and circulating IL-15 levels. There was a significant effect of genotype on the fatigue curves obtained in extensor digitorum longus (EDL) muscles from female mIl15ra(fl/fl)/Cre(+) mice, such that force output was greater during the repeated contraction protocol compared with mIl15ra(fl/fl)/Cre(-) control mice. Muscles from female mIl15ra(fl/fl)/Cre(+) mice also had a twofold greater amount of the mitochondrial genome-specific COXII gene compared with muscles from mIl15ra(fl/fl)/Cre(-) control mice, indicating a greater mitochondrial density in these skeletal muscles. There was a significant effect of genotype on the twitch:tetanus ratio in EDL and soleus muscles from mIl15ra(fl/fl)/Cre(+) mice, such that the ratio was lower in these muscles compared with mIl15ra(fl/fl)/Cre(-) control mice, indicating a pro-oxidative shift in muscle phenotype. However, spontaneous cage activity was not different and IL-15 protein levels were lower in male and female mIl15ra(fl/fl)/Cre(+) mice compared with control. Collectively, these data support a direct effect of muscle IL-15Rα deficiency in altering contractile properties and fatigue characteristics in skeletal muscles.

Aging alters contractile properties and fiber morphology in pigeon skeletal muscle.

In this study, we tested the hypothesis that skeletal muscle from pigeons would display age-related alterations in isometric force and contractile parameters as well as a shift of the single muscle fiber cross-sectional area (CSA) distribution toward smaller fiber sizes. Maximal force output, twitch contraction durations and the force-frequency relationship were determined in tensor propatagialis pars biceps muscle from young 3-year-old pigeons, middle-aged 18-year-old pigeons, and aged 30-year-old pigeons. The fiber CSA distribution was determined by planimetry from muscle sections stained with hematoxylin and eosin. Maximal force output of twitch and tetanic contractions was greatest in muscles from young pigeons, while the time to peak force of twitch contractions was longest in muscles from aged pigeons. There were no changes in the force-frequency relationship between the age groups. Interestingly, the fiber CSA distribution in aged muscles revealed a greater number of larger sized muscle fibers, which was verified visually in histological images. Middle-aged and aged muscles also displayed a greater amount of slow myosin containing muscle fibers. These data demonstrate that muscles from middle-aged and aged pigeons are susceptible to alterations in contractile properties that are consistent with aging, including lower force production and longer contraction durations. These functional changes were supported by the appearance of slow myosin containing muscle fibers in muscles from middle-aged and aged pigeons. Therefore, the pigeon may represent an appropriate animal model for the study of aging-related alterations in skeletal muscle function and structure.

Non-invasive serial casting to treat idiopathic toe walking in an 18-month old child.

BACKGROUND: Idiopathic toe walking is characterized by persistent toe walking in the absence of clinically diagnosed neuromuscular disease. Treatment options in children diagnosed with idiopathic toe walking include: observation, physical therapy, serial casting, or Achilles tendon (heel cord) lengthening surgery. OBJECTIVE: In this case report, we present a non-invasive serial casting protocol to treat severe and persistent toe walking in an 18-month old child, diagnosed as an idiopathic toe walker following neurological examination. METHODS: A series of below knee casts was used to provide a consistent stretch to the plantar flexor muscles. Upon removal of each set of casts, passive range of motion at the ankles was measured with a goniometer. RESULTS: Four sets of casts, each lasting approximately one week, increased passive ankle dorsiflexion to 10° of neutral and established a heel-toe walking gait. Improvements in ankle range of motion and gait were maintained upon repeated examinations at 3, 7, and 12 months post-casting. CONCLUSIONS: These results demonstrate that non-invasive procedures, such as serial casting, can be successful in very young children diagnosed as idiopathic toe walkers. Early identification and intervention for this diagnosis may eliminate the need for invasive surgeries and associated risks in this population.

FER-1/Dysferlin promotes cholinergic signaling at the neuromuscular junction in C. elegans and mice.

Dysferlin is a member of the evolutionarily conserved ferlin gene family. Mutations in Dysferlin lead to Limb Girdle Muscular Dystrophy 2B (LGMD2B), an inherited, progressive and incurable muscle disorder. However, the molecular mechanisms underlying disease pathogenesis are not fully understood. We found that both loss-of-function mutations and muscle-specific overexpression of C. elegans fer-1, the founding member of the Dysferlin gene family, caused defects in muscle cholinergic signaling. To determine if Dysferlin-dependent regulation of cholinergic signaling is evolutionarily conserved, we examined the in vivo physiological properties of skeletal muscle synaptic signaling in a mouse model of Dysferlin-deficiency. In addition to a loss in muscle strength, Dysferlin -/- mice also exhibited a cholinergic deficit manifested by a progressive, frequency-dependent decrement in their compound muscle action potentials following repetitive nerve stimulation, which was observed in another Dysferlin mouse model but not in a Dysferlin-independent mouse model of muscular dystrophy. Oral administration of Pyridostigmine bromide, a clinically used acetylcholinesterase inhibitor (AchE.I) known to increase synaptic efficacy, reversed the action potential defect and restored in vivo muscle strength to Dysferlin -/- mice without altering muscle pathophysiology. Our data demonstrate a previously unappreciated role for Dysferlin in the regulation of cholinergic signaling and suggest that such regulation may play a significant pathophysiological role in LGMD2B disease.

Microarray analysis reveals novel features of the muscle aging process in men and women.

To develop a global view of muscle transcriptional differences between older men and women and sex-specific aging, we obtained muscle biopsies from the biceps brachii of young and older men and women and profiled the whole-genome gene expression using microarray. A logistic regression-based method in combination with an intensity-based Bayesian moderated t test was used to identify significant sex- and aging-related gene functional groups. Our analysis revealed extensive sex differences in the muscle transcriptome of older individuals and different patterns of transcriptional changes with aging in men and women. In older women, we observed a coordinated transcriptional upregulation of immune activation, extracellular matrix remodeling, and lipids storage; and a downregulation of mitochondrial biogenesis and function and muscle regeneration. The effect of aging results in sexual dimorphic alterations in the skeletal muscle transcriptome, which may modify the risk for developing musculoskeletal and metabolic diseases in men and women.

IL-15Rα deficiency leads to mitochondrial and myofiber differences in fast mouse muscles.

The purpose of this study was to determine mitochondrial changes in fast muscles from interleukin-15 receptor alpha knockout (IL-15RαKO) mice. We tested the hypothesis that fast muscles from IL-15RαKO mice would have a greater mitochondrial density and altered internal structure compared to muscles from control mice. In fast muscles from IL-15RαKO mice, mitochondrial density was 48% greater with a corresponding increase in mitochondrial DNA content. Although there were no differences in the relative size of isolated mitochondria, internal complexity was lower in mitochondria from IL-15RαKO mice. These data support an increase in mitochondrial biogenesis and provide direct evidence for a greater density and altered internal structure of mitochondria in EDL muscles deficient in IL-15Rα.