Histological analysis of the gastrocnemius muscle in preschool and school age children with cerebral palsy compared with age-matched typically developing children.

Inconsistent alterations in skeletal muscle histology have been reported in adolescents with cerebral palsy (CP) and whether alterations are present in young children and differ from older children is not yet known. This study aimed to define histological alterations in the medial gastrocnemius (MG) of ambulant CP (gross-motor classification system, GMFCS I-III) stratified in two age groups (preschool children, PS: 2-5 and school age children, SA: 6-9-yr old) compared with age-matched typically developing (TD) children. We hypothesized that alterations in muscle microscopic properties are already present in PS-CP and are GMFCS level specific. Ultrasound guided percutaneous microbiopsies were collected in 46 CP (24-PS) and 45 TD (13-PS) children. Sections were stained to determine fiber cross-sectional area (fCSA) and proportion, capillary, and satellite cell amount. Average absolute and normalized fCSA were similar in CP and TD, but a greater percentage of smaller fibers was found in CP. Coefficient of variation (CV) was significantly larger in PS-CP-GMFCS I-II and for type I fiber. In SA-CP, all fiber types contributed to the higher CV. Type IIx proportion was higher and type I was lower in PS-CP-GMFCS-III and for all SA-CP. Reduced capillary-to-fiber ratio was present in PS-CP-GMFCS II-III and in all SA-CP. Capillary fiber density was lower in SA-CP. Capillary domain was enhanced in all CP, but capillary spatial distribution was maintained as was satellite cell content. We concluded that MG histological alterations are already present in very young CP but are only partly specific for GMFCS level and age.NEW & NOTEWORTHY Inconsistent histological alterations have been reported in children with cerebral palsy (CP) but whether they are present in very young and ambulant CP children and differ from those reported in old CP children is not known. This study highlighted for the first time that enhanced muscle fiber size variability and loss of capillaries are already present in very young CP children, even in the most ambulant ones, and these alterations seem to extend with age.

Longitudinal trajectory of medial gastrocnemius muscle growth in the first years of life.

AIM: To define the longitudinal trajectory of gastrocnemius muscle growth in 6- to 36-month-old children with and without spastic cerebral palsy (SCP) and to compare trajectories by levels of gross motor function (Gross Motor Function Classification System, GMFCS) and presumed brain-lesion timing. METHOD: Twenty typically developing children and 24 children with SCP (GMFCS levels I-II/III-IV = 15/9), were included (28/16 females/males; mean age at first scan 15.4 months [standard deviation 4.93, range 6.24-23.8]). Three-dimensional freehand ultrasound was used to repeatedly assess muscle volume, length, and cross-sectional area (CSA), resulting in 138 assessments (mean interval 7.9 months). Brain lesion timing was evaluated with magnetic resonance imaging classification. Linear mixed-effects models defined growth rates, adjusted for GMFCS levels and presumed brain-lesion timing. RESULTS: At age 12 months, children with SCP showed smaller morphological muscle size than typically developing children (5.8 mL vs 9.8 mL, p < 0.001), while subsequently no differences in muscle growth were found between children with and without SCP (muscle volume: 0.65 mL/month vs 0.74 mL/month). However, muscle volume and CSA growth rates were lower in children classified in GMFCS levels III and IV than typically developing children and those classified in GMFCS levels I and II, with differences ranging from -56% to -70% (p < 0.001). INTERPRETATION: Muscle growth is already hampered during infancy in SCP. Muscle size growth further reduces with decreasing functional levels, independently from the brain lesion. Early monitoring of muscle growth combined with early intervention is needed.

Zeb2 Regulates Myogenic Differentiation in Pluripotent Stem Cells.

Skeletal muscle differentiation is triggered by a unique family of myogenic basic helix-loop-helix transcription factors, including MyoD, MRF-4, Myf-5, and Myogenin. These transcription factors bind promoters and distant regulatory regions, including E-box elements, of genes whose expression is restricted to muscle cells. Other E-box binding zinc finger proteins target the same DNA response elements, however, their function in muscle development and regeneration is still unknown. Here, we show that the transcription factor zinc finger E-box-binding homeobox 2 (Zeb2, Sip-1, Zfhx1b) is present in skeletal muscle tissues. We investigate the role of Zeb2 in skeletal muscle differentiation using genetic tools and transgenic mouse embryonic stem cells, together with single-cell RNA-sequencing and in vivo muscle engraftment capability. We show that Zeb2 over-expression has a positive impact on skeletal muscle differentiation in pluripotent stem cells and adult myogenic progenitors. We therefore propose that Zeb2 is a novel myogenic regulator and a possible target for improving skeletal muscle regeneration. The non-neural roles of Zeb2 are poorly understood.

Ether-Oxygen Containing Electrospun Microfibrous and Sub-Microfibrous Scaffolds Based on Poly(butylene 1,4-cyclohexanedicarboxylate) for Skeletal Muscle Tissue Engineering.

We report the study of novel biodegradable electrospun scaffolds from poly(butylene 1,4-cyclohexandicarboxylate-co-triethylene cyclohexanedicarboxylate) (P(BCE-co-TECE)) as support for in vitro and in vivo muscle tissue regeneration. We demonstrate that chemical composition, i.e., the amount of TECE co-units (constituted of polyethylene glycol-like moieties), and fibre morphology, i.e., aligned microfibrous or sub-microfibrous scaffolds, are crucial in determining the material biocompatibility. Indeed, the presence of ether linkages influences surface wettability, mechanical properties, hydrolytic degradation rate, and density of cell anchoring points of the studied materials. On the other hand, electrospun scaffolds improve cell adhesion, proliferation, and differentiation by favouring cell alignment along fibre direction (fibre morphology), also allowing for better cell infiltration and oxygen and nutrient diffusion (fibre size). Overall, C2C12 myogenic cells highly differentiated into mature myotubes when cultured on microfibres realised with the copolymer richest in TECE co-units (micro-P73 mat). Lastly, when transplanted in the tibialis anterior muscles of healthy, injured, or dystrophic mice, micro-P73 mat appeared highly vascularised, colonised by murine cells and perfectly integrated with host muscles, thus confirming the suitability of P(BCE-co-TECE) scaffolds as substrates for skeletal muscle tissue engineering.

Fate choice of post-natal mesoderm progenitors: skeletal versus cardiac muscle plasticity.

Regenerative medicine for skeletal and cardiac muscles still constitutes a fascinating and ambitious frontier. In this perspective, understanding the possibilities of intrinsic cell plasticity, present in post-natal muscles, is vital to define and improve novel therapeutic strategies for acute and chronic diseases. In addition, many somatic stem cells are now crossing the boundaries of basic/translational research to enter the first clinical trials. However, it is still an open question whether a lineage switch between skeletal and cardiac adult myogenesis is possible. Therefore, this review focuses on resident somatic stem cells of post-natal skeletal and cardiac muscles and their plastic potential toward the two lineages. Furthermore, examples of myogenic lineage switch in adult stem cells are also reported and discussed.

Role of Inflammation in Muscle Homeostasis and Myogenesis.

Skeletal muscle mass is subject to rapid changes according to growth stimuli inducing both hypertrophy, through increased protein synthesis, and hyperplasia, activating the myogenic program. Muscle wasting, characteristic of several pathological states associated with local or systemic inflammation, has been for long considered to rely on the alteration of myofiber intracellular pathways regulated by both hormones and cytokines, eventually leading to impaired anabolism and increased protein breakdown. However, there are increasing evidences that even alterations of the myogenic/regenerative program play a role in the onset of muscle wasting, even though the precise mechanisms involved are far from being fully elucidated. The comprehension of the links potentially occurring between impaired myogenesis and increased catabolism would allow the definition of effective strategies aimed at counteracting muscle wasting. The first part of this review gives an overview of skeletal muscle intracellular pathways determining fiber size, while the second part considers the cells and the regulatory pathways involved in the myogenic program. In both parts are discussed the evidences supporting the role of inflammation in impairing muscle homeostasis and myogenesis, potentially determining muscle atrophy.

Autophagic degradation contributes to muscle wasting in cancer cachexia.

Muscle protein wasting in cancer cachexia is a critical problem. The underlying mechanisms are still unclear, although the ubiquitin-proteasome system has been involved in the degradation of bulk myofibrillar proteins. The present work has been aimed to investigate whether autophagic degradation also plays a role in the onset of muscle depletion in cancer-bearing animals and in glucocorticoid-induced atrophy and sarcopenia of aging. The results show that autophagy is induced in muscle in three different models of cancer cachexia and in glucocorticoid-treated mice. In contrast, autophagic degradation in the muscle of sarcopenic animals is impaired but can be reactivated by calorie restriction. These results further demonstrate that different mechanisms are involved in pathologic muscle wasting and that autophagy, either excessive or defective, contributes to the complicated network that leads to muscle atrophy. In this regard, particularly intriguing is the observation that in cancer hosts and tumor necrosis factor α-treated C2C12 myotubes, insulin can only partially blunt autophagy induction. This finding suggests that autophagy is triggered through mechanisms that cannot be circumvented by using classic upstream modulators, prompting us to identify more effective approaches to target this proteolytic system.

Short-Term Effects of Botulinum Toxin-A Injection on the Medial Gastrocnemius Histological Features in Ambulant Children with Cerebral Palsy: A Longitudinal Pilot Study.

Botulinum toxin-A (BoNT-A) injection is known to exert beneficial effects on muscle tone, joint mobility and gait in children with cerebral palsy (CP). However, recent animal and human studies have raised the concern that BoNT-A might be harmful to muscle integrity. In CP-children, the impact of BoNT-A on muscle structure has been poorly studied, and inconsistent results have been reported. This study was aimed at determining the time course effect of a single BoNT-A administration on medial gastrocnemius (MG) morphology in CP-children. MG microbiopsies from 12 ambulant and BoNT-A-naïve CP-children (age, 3.4 (2.3) years, ranging from 2.5 to 7.8 years; seven boys and five girls; GMFCS I = 5, II = 4 and III = 3) were collected before and 3 and 6 months after BoNT-A treatment to analyze the fiber cross-sectional area (fCSA) and proportion; capillarization; and satellite cell (SC) content. Compared with the baseline, the fCSA decreased at 3 months (-14%, NS) and increased at 6 months (+13%, NS). Fiber size variability was significantly higher at 3 months (type I: +56%, p = 0.032; type IIa: +37%, p = 0.032) and 6 months (type I: +69%, p = 0.04; type IIa: +121%, p = 0.032) compared with the baseline. The higher type I proportion seen at 3 months was still present and more pronounced at 6 months (type I: +17%, p = 0.04; type IIx: -65%, p = 0.032). The capillary fiber density was reduced at 3 months (type I: -43%, NS; type II: -44%, p = 0.0320) but normalized at 6 months. There was a non-significant increase in SC/100 fibers at 3 months (+75%, NS) and 6 months (+40%, NS) compared with the baseline. These preliminary data suggest that BoNT-A induced alterations in the MG of children with CP, which were still present 6 months after BoNT-A injection but with signs of muscle recovery.

Histological analysis of the medial gastrocnemius muscle in young healthy children.

Introduction: Histological data on muscle fiber size and proportion in (very) young typically developing (TD) children is not well documented and data on capillarization and satellite cell content are also lacking. Aims: This study investigated the microscopic properties of the medial gastrocnemius muscle in growing TD children, grouped according to age and gender to provide normal reference values in healthy children. Methods: Microbiopsies of the medial gastrocnemius (MG) muscle were collected in 46 TD boys and girls aged 2-10 years subdivided into 4 age groups (2-4, 4-6, 6-8 and 8-10 years). Sections were immunostained to assess fiber type cross-sectional area (fCSA) and proportion, the number of satellite cells (SC), capillary to fiber ratio (C/F), capillary density for type I and II fiber (CFD), capillary domain, capillary-to-fiber perimeter exchange index (CFPE) and heterogeneity index. fCSA was normalized to fibula length2 and the coefficient of variation (CV) was calculated to reflect fCSA intrasubject variability. Results: Absolute fCSA of all fibers increased with age (r = 0.72, p < 0.001) but more in boys (+112%, p < 0.05) than in girls (+48%, p > 0.05) Normalized fCSA, CV and fiber proportion did not differ between age groups and gender. C/F was strongly correlated with age in boys (r = 0.83, p < 0.001), and to a lesser extent in girls (r = 0.37, p = 0.115), while other capillary parameters as well as the number of SC remained stable with increasing age in boys and girls. Discussion: This study provides reference values of histological measures in MG according to age in normally growing boys and girls. These data may be used as a reference to determine disease impact and efficacy of therapeutic approach on the muscle.

Botulinum Toxin Treatment of Adult Muscle Stem Cells from Children with Cerebral Palsy and hiPSC-Derived Neuromuscular Junctions.

Botulinum neurotoxin type-A (BoNT) injections are commonly used as spasticity treatment in cerebral palsy (CP). Despite improved clinical outcomes, concerns regarding harmful effects on muscle morphology have been raised, and the BoNT effect on muscle stem cells remains not well defined. This study aims at clarifying the impact of BoNT on growing muscles (1) by analyzing the in vitro effect of BoNT on satellite cell (SC)-derived myoblasts and fibroblasts obtained from medial gastrocnemius microbiopsies collected in young BoNT-naïve children (t0) compared to age ranged typically developing children; (2) by following the effect of in vivo BoNT administration on these cells obtained from the same children with CP at 3 (t1) and 6 (t2) months post BoNT; (3) by determining the direct effect of a single and repeated in vitro BoNT treatment on neuromuscular junctions (NMJs) differentiated from hiPSCs. In vitro BoNT did not affect myogenic differentiation or collagen production. The fusion index significantly decreased in CP at t2 compared to t0. In NMJ cocultures, BoNT treatment caused axonal swelling and fragmentation. Repeated treatments impaired the autophagic-lysosomal system. Further studies are warranted to understand the long-term and collateral effects of BoNT in the muscles of children with CP.

Fine-needle percutaneous muscle microbiopsy technique as a feasible tool to address histological analysis in young children with cerebral palsy and age-matched typically developing children.

Cerebral palsy (CP) is a heterogeneous group of motor disorders attributed to a non-progressive lesion in the developing brain. Knowledge on skeletal muscle properties is important to understand the impact of CP and treatment but data at the microscopic levels are limited and inconsistent. Currently, muscle biopsies are collected during surgery and are restricted to CP eligible for such treatment or they may refer to another muscle or older children in typically developing (TD) biopsies. A minimally invasive technique to collect (repeated) muscle biopsies in young CP and TD children is needed to provide insights into the early muscle microscopic alterations and their evolution in CP. This paper describes the protocol used to 1) collect microbiopsies of the medial gastrocnemius (MG) and semitendinosus (ST) in CP children and age-matched TD children, 2) handle the biopsies for histology, 3) stain the biopsies to address muscle structure (Hematoxylin & Eosin), fiber size and proportion (myosin heavy chain), counting of the satellite cells (Pax7) and capillaries (CD31). Technique feasibility and safety as well as staining feasibility and measure accuracy were evaluated. Two microbiopsies per muscle were collected in 56 CP (5.8±1.1 yr) and 32 TD (6±1.1 yr) children using ultrasound-guided percutaneous microbiopsy technique. The biopsy procedure was safe (absence of complications) and well tolerated (Score pain using Wong-Baker faces). Cross-sectionally orientated fibers were found in 86% (CP) and 92% (TD) of the biopsies with 60% (CP) and 85% (TD) containing more than 150 fibers. Fiber staining was successful in all MG biopsies but failed in 30% (CP) and 16% (TD) of the ST biopsies. Satellite cell staining was successful in 89% (CP) and 85% (TD) for MG and in 70% (CP) and 90% (TD) for ST biopsies, while capillary staining was successful in 88% (CP) and 100% (TD) of the MG and in 86% (CP) and 90% (TD) for the ST biopsies. Intraclass coefficient correlation showed reliable and reproducible measures of all outcomes. This study shows that the percutaneous microbiopsy technique is a safe and feasible tool to collect (repeated) muscle biopsies in young CP and TD children for histological analysis and it provides sufficient muscle tissue of good quality for reliable quantification.

The Myotube Analyzer: how to assess myogenic features in muscle stem cells.

BACKGROUND: The analysis of in vitro cultures of human adult muscle stem cells obtained from biopsies delineates the potential of skeletal muscles and may help to understand altered muscle morphology in patients. In these analyses, the fusion index is a commonly used quantitative metric to assess the myogenic potency of the muscle stem cells. Since the fusion index only partly describes myogenic potency, we developed the Myotube Analyzer tool, which combines the definition of the fusion index with extra features of myonuclei and myotubes obtained from satellite cell cultures. RESULTS: The software contains image adjustment and mask editing functions for preprocessing and semi-automatic segmentation, while other functions can be used to determine the features of nuclei and myotubes. The fusion index and a set of five novel parameters were tested for reliability and validity in a comparison between satellite cell cultures from children with cerebral palsy and typically developing children. These novel parameters quantified extra nucleus and myotube properties and can be used to describe nucleus clustering and myotube shape. Two analyzers who were trained in cell culture defined all parameters using the Myotube Analyzer app. Out of the six parameters, five had good reliability reflected by good intra-class correlation coefficients (> 0.75). Children with cerebral palsy were significantly different from the typically developing children (p < 0.05) for five parameters, and for three of the six parameters, these differences exceeded the minimal detectable differences. CONCLUSIONS: The Myotube Analyzer can be used for the analysis of fixed differentiated myoblast cultures with nuclear and MyHC staining. The app can calculate the fusion index, an already existing parameter, but also provides multiple new parameters to comprehensively describe myogenic potential in its output. The raw data used to determine these parameters are also available in the output. The parameters calculated by the tool can be used to detect differences between cultures from children with cerebral palsy and typically developing children. Since the program is open source, users can customize it to fit their own analysis requirements.

Autologous iPSC-Derived Human Neuromuscular Junction to Model the Pathophysiology of Hereditary Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) is a heterogeneous group of genetic neurodegenerative disorders, characterized by progressive lower limb spasticity and weakness resulting from retrograde axonal degeneration of motor neurons (MNs). Here, we generated in vitro human neuromuscular junctions (NMJs) from five HSP patient-specific induced pluripotent stem cell (hiPSC) lines, by means of microfluidic strategy, to model disease-relevant neuropathologic processes. The strength of our NMJ model lies in the generation of lower MNs and myotubes from autologous hiPSC origin, maintaining the genetic background of the HSP patient donors in both cell types and in the cellular organization due to the microfluidic devices. Three patients characterized by a mutation in the SPG3a gene, encoding the ATLASTIN GTPase 1 protein, and two patients with a mutation in the SPG4 gene, encoding the SPASTIN protein, were included in this study. Differentiation of the HSP-derived lines gave rise to lower MNs that could recapitulate pathological hallmarks, such as axonal swellings with accumulation of Acetyl-α-TUBULIN and reduction of SPASTIN levels. Furthermore, NMJs from HSP-derived lines were lower in number and in contact point complexity, denoting an impaired NMJ profile, also confirmed by some alterations in genes encoding for proteins associated with microtubules and responsible for axonal transport. Considering the complexity of HSP, these patient-derived neuronal and skeletal muscle cell co-cultures offer unique tools to study the pathologic mechanisms and explore novel treatment options for rescuing axonal defects and diverse cellular processes, including membrane trafficking, intracellular motility and protein degradation in HSP.

Reduced Cross-Sectional Muscle Growth Six Months after Botulinum Toxin Type-A Injection in Children with Spastic Cerebral Palsy.

Botulinum Neurotoxin type-A (BoNT-A) injections are widely used as first-line spasticity treatment in spastic cerebral palsy (SCP). Despite improved clinical outcomes, concerns regarding harmful effects on muscle morphology have been raised. Yet, the risk of initiating BoNT-A to reduce muscle growth remains unclear. This study investigated medial gastrocnemius (MG) morphological muscle growth in children with SCP (n = 26, median age of 5.2 years (3.5)), assessed by 3D-freehand ultrasound prior to and six months post-BoNT-A injections. Post-BoNT-A MG muscle growth of BoNT-A naive children (n = 11) was compared to (a) muscle growth of children who remained BoNT-A naive after six months (n = 11) and (b) post-BoNT-A follow-up data of children with a history of BoNT-A treatment (n = 15). Six months after initiating BoNT-A injection, 17% decrease in mid-belly cross-sectional area normalized to skeletal growth and 5% increase in echo-intensity were illustrated. These muscle outcomes were only significantly altered when compared with children who remained BoNT-A naive (+4% and -3%, respectively, p < 0.01). Muscle length growth persevered over time. This study showed reduced cross-sectional growth post-BoNT-A treatment suggesting that re-injections should be postponed at least beyond six months. Future research should extend follow-up periods investigating muscle recovery in the long-term and should include microscopic analysis.

Human iPSC model reveals a central role for NOX4 and oxidative stress in Duchenne cardiomyopathy.

Duchenne muscular dystrophy (DMD) is a progressive muscle disorder caused by mutations in the Dystrophin gene. Cardiomyopathy is a major cause of early death. We used DMD-patient-specific human induced pluripotent stem cells (hiPSCs) to model cardiomyopathic features and unravel novel pathologic insights. Cardiomyocytes (CMs) differentiated from DMD hiPSCs showed enhanced premature cell death due to significantly elevated intracellular reactive oxygen species (ROS) resulting from depolarized mitochondria and increased NADPH oxidase 4 (NOX4). CRISPR-Cas9 correction of Dystrophin restored normal ROS levels. ROS reduction by N-acetyl-L-cysteine (NAC), ataluren (PTC124), and idebenone improved hiPSC-CM survival. We show that oxidative stress in DMD hiPSC-CMs was counteracted by stimulating adenosine triphosphate (ATP) production. ATP can bind to NOX4 and partially inhibit the ROS production. Considering the complexity and the early cellular stress responses in DMD cardiomyopathy, we propose targeting ROS production and preventing detrimental effects of NOX4 on DMD CMs as promising therapeutic strategy.

PGC-1α in the myofibers regulates the balance between myogenic and adipogenic progenitors affecting muscle regeneration.

Skeletal muscle repair is accomplished by satellite cells (MuSCs) in cooperation with interstitial stromal cells (ISCs), but the relationship between the function of these cells and the metabolic state of myofibers remains unclear. This study reports an altered proportion of MuSCs and ISCs (including adipogenesis-regulatory cells; Aregs) induced by the transgenic overexpression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in the myofibers (MCK-PGC-1α mice). Although PGC-1α-driven increase of MuSCs does not accelerate muscle regeneration, myogenic progenitors isolated from MCK-PGC-1α mice and transplanted into intact and regenerating muscles are more prone to fuse with recipient myofibers than those derived from wild-type donors. Moreover, both young and aged MCK-PGC-1α animals exhibit reduced perilipin-positive areas when challenged with an adipogenic stimulus, demonstrating low propensity to accumulate adipocytes within the muscle. Overall, these results unveil that increased PGC-1α expression in the myofibers favors pro-myogenic and anti-adipogenic cell populations in the skeletal muscle.

Glutamine prevents myostatin hyperexpression and protein hypercatabolism induced in C2C12 myotubes by tumor necrosis factor-α.

Depletion of skeletal muscle protein mainly results from enhanced protein breakdown, caused by activation of proteolytic systems such as the Ca2+-dependent and the ATP-ubiquitin-dependent ones. In the last few years, enhanced expression and bioactivity of myostatin have been reported in several pathologies characterized by marked skeletal muscle depletion. More recently, high myostatin levels have been associated with glucocorticoid-induced hypercatabolism. The search for therapeutical strategies aimed at preventing/correcting protein hypercatabolism has been directed to inhibit humoral mediators known for their pro-catabolic action, such as TNFα. The present study has been aimed to investigate the involvement of TNFα in the regulation of both myostatin expression and intracellular protein catabolism, and the possibility to interfere with such modulations by means of amino acid supplementation. For this purpose, C2C12 myotubes exposed to TNFα in the presence or in the absence of amino acid (glutamine or leucine) supplementation have been used. Myotube treatment with TNFα leads to both hyperexpression of the muscle-specific ubiquitin ligase atrogin-1, and enhanced activity of the Ca(2+)-dependent proteolytic system. These changes are associated with increased myostatin expression. Glutamine supplementation effectively prevents TNFα-induced muscle protein loss and restores normal myostatin levels. The results shown in the present study indicate a direct involvement of TNFα in the onset of myotube protein loss and in the perturbation of myostatin-dependent signaling. In addition, the protective effect exerted by glutamine suggests that amino acid supplementation could represent a possible strategy to improve muscle mass.

Isolation of Mammalian Mesoangioblasts: A Subset of Pericytes with Myogenic Potential.

Mesoangioblasts (MABs) are vessel-associated stem cells that express pericyte markers and are originally isolated from the embryonic dorsal aorta. From postnatal small vessels of skeletal muscle and heart, it is possible to isolate cells with similar characteristics to embryonic MABs. Adult MABs have the capacity to self-renew and to differentiate into cell types of mesodermal lineages upon proper culture conditions. To date, the origin of MABs and the relationship with other muscle stem cells are still debated. Recently, in a phase I-II clinical trial, intra-arterial HLA-matched MABs were proved to be relatively safe. Novel information on MAB pure populations is desirable, and implementation of their therapeutic potential is mandatory to approach efficacy in MAB-based treatments. This chapter provides an overview of the current techniques for isolation and characterization of rodent, canine, human, and equine adult MABs.

Muscle atrophy in experimental cancer cachexia: is the IGF-1 signaling pathway involved?

Skeletal muscle wasting, one of the main features of cancer cachexia, is associated with marked protein hypercatabolism, and has suggested to depend also on impaired IGF-1 signal transduction pathway. To investigate this point, the state of activation of the IGF-1 system has been evaluated both in rats bearing the AH-130 hepatoma and in mice transplanted with the C26 colon adenocarcinoma. In the skeletal muscle of tumor hosts, the levels of phosphorylated (active) Akt, one of the most relevant kinases involved in the IGF-1 signaling pathway, were comparable to controls, or even increased. Accordingly, downstream targets such as GSK3beta, p70(S6K) and FoxO1 were hyperphosphorylated, while the levels of phosphorylated eIF2alpha were markedly reduced with respect to controls. In the attempt to force the metabolic balance toward anabolism, IGF-1 was hyperexpressed by gene transfer in the tibialis muscle of the C26 hosts. In healthy animals, IGF-1 overexpression markedly increased both fiber and muscle size. As a positive control, IGF-1 was also overexpressed in the muscle of aged mice. In IGF-1 hyperexpressing muscles the fiber cross-sectional area definitely increased in both young and aged animals, while, by contrast, loss of muscle mass or reduction of fiber size in mice bearing the C26 tumor were not modified. These results demonstrate that muscle wasting in tumor-bearing animals is not associated with downregulation of molecules involved in the anabolic response, and appears inconsistent, at least, with reduced activity of the IGF-1 signaling pathway.

Interleukin-4 administration improves muscle function, adult myogenesis, and lifespan of colon carcinoma-bearing mice.

BACKGROUND: Anorexia, body wasting, inflammation, muscle, and adipose tissue loss are hallmarks of cancer cachexia, a syndrome that affects the majority of cancer patients, impairing their ability to endure chemotherapeutic therapies and reducing their lifespan. In the last 10 years, alterations of protein turnover and impairment of adult myogenesis have been proposed as major contributing factors. METHODS: Muscle stem cells, including satellite cells, mesoangioblasts, and fibroadipogenic progenitors, were isolated and characterized from C26 colon carcinoma-bearing (C26) mice. Circulating levels of interleukin-4/13 (IL4/IL13) were analysed by ELISA, and the effects of IL4 on muscle mass and function, protein synthesis, muscle regeneration, and myogenic progenitor cell number were analysed at both functional (treadmill and grip test) and molecular levels (qRT-PCR, immunofluorescence analysis, surface sensing of translation, and western blot). The Kaplan-Meier test was used to analyse the survival curve of IL4-treated and IL4-untreated C26 mice. RESULTS: The administration of IL4 to C26 mice rescued muscle mass by increasing protein synthesis. The IL4 treatment improved performances and prolonged survival of C26 mice. IL4 administration re-established both number and function of satellite cells and fibroadipogenic progenitors without affecting mesoangioblasts in C26 mice, rescuing myogenesis. Upon IL4 treatment, a high number of cytotoxic lymphocytes and type II macrophages were observed with a subsequent increase in necrotic areas of C26 tumours. CONCLUSIONS: The results here presented shed new light on IL4 signalling during muscle wasting and early stages of muscle regeneration that explain the beneficial effect observed in IL4-treated C26 mice. These findings might aid to develop therapeutic approaches to improve mobility and quality of life in cachectic patients.