Measuring and Modelling Nonlinear Elasticity of Ex Vivo Mouse Muscles.

Elastography is a noninvasive imaging technique that provides information on soft tissue stiffness. Young's modulus is typically used to characterize soft tissues' response to the applied force, as soft tissues are often considered linear elastic, isotropic, and quasi-incompressible materials. This approximation is reasonable for small strains, but soft tissues undergo large deformations also for small values of force and exhibit nonlinear elastic behavior. Outside the linear regime, the elastic modulus is dependent on the strain level and is different for any kind of tissue. The aim of this study was to characterize, ex vivo, the mechanical response of two different mice muscles to an external force. A system for transverse force-controlled uniaxial compression enabled obtaining the stress-strain (σ-ε) curve of the samples. The strain-dependent Young's modulus (SYM) model was adopted to reproduce muscle compression behavior and to predict the elastic modulus for large deformations. After that, a recursive linear model was employed to identify the initial linear region of the σ-ε curve. Results showed that both muscle types exhibited a strain hardening effect and that the SYM model provided good fitting of the entire σ-ε curves. The application of the recursive linear model allowed capturing the initial linear region in which the approximation of these tissues as linear elastic materials is reasonable. The residual analysis displayed that even if the SYM model better summarizes the muscle behavior on the entire region, the linear model is more precise when considering only the initial part of the σ-ε curve.

Pharmacological Inhibition of PKCθ Counteracts Muscle Disease in a Mouse Model of Duchenne Muscular Dystrophy.

Inflammation plays a considerable role in the progression of Duchenne Muscular Dystrophy (DMD), a severe muscle disease caused by a mutation in the dystrophin gene. We previously showed that genetic ablation of Protein Kinase C θ (PKCθ) in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease. RESEARCH IN CONTEXT: Duchenne muscular dystrophy (DMD) is a severe muscle disease affecting 1:3500 male births. DMD is caused by a mutation in dystrophin gene, coding for a protein required for skeletal and cardiac muscle integrity. Lack of a functional dystrophin is primarily responsible for the muscle eccentric contraction-induced muscle damage, observed in dystrophic muscle. However, inflammation plays a considerable role in the progression of DMD. Glucocorticoids, which have anti-inflammatory properties, are being used to treat DMD with some success; however, long term treatment with these drugs induces muscle atrophy and wasting, outweighing their benefit. The identification of specific targets for anti-inflammatory therapies is one of the ongoing therapeutic options. Although blunting inflammation would not be a "cure" for the disease, the emerging clue is that multiple strategies, addressing different aspects of the pathology, which may eventually converge, may be successful. In this context, we previously showed that genetic ablation of Protein Kinase C θ (PKCθ), an enzyme known to be involved in immune response, in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease.

TAp63gamma is required for the late stages of myogenesis.

p53 family members, p63 and p73, play a role in controlling early stage of myogenic differentiation. We demonstrated that TAp63gamma, unlike the other p53 family members, is markedly up-regulated during myogenic differentiation in murine C2C7 cell line. We also found that myotubes formation was inhibited upon TAp63gamma knock-down, as also indicated by atrophyic myotubes and reduction of myoblasts fusion index. Analysis of TAp63gamma-dependend transcripts identified several target genes involved in skeletal muscle contractility energy metabolism, myogenesis and skeletal muscle autocrine signaling. These results indicate that TAp63gamma is a late marker of myogenic differentiation and, by controlling different sub-sets of target genes, it possibly contributes to muscle growth, remodeling, functional differentiation and tissue homeostasis.

Lifelong physical exercise delays age-associated skeletal muscle decline.

Aging is usually accompanied by a significant reduction in muscle mass and force. To determine the relative contribution of inactivity and aging per se to this decay, we compared muscle function and structure in (a) male participants belonging to a group of well-trained seniors (average of 70 years) who exercised regularly in their previous 30 years and (b) age-matched healthy sedentary seniors with (c) active young men (average of 27 years). The results collected show that relative to their sedentary cohorts, muscle from senior sportsmen have: (a) greater maximal isometric force and function, (b) better preserved fiber morphology and ultrastructure of intracellular organelles involved in Ca(2+) handling and ATP production, (c) preserved muscle fibers size resulting from fiber rescue by reinnervation, and (d) lowered expression of genes related to autophagy and reactive oxygen species detoxification. All together, our results indicate that: (a) skeletal muscle of senior sportsmen is actually more similar to that of adults than to that of age-matched sedentaries and (b) signaling pathways controlling muscle mass and metabolism are differently modulated in senior sportsmen to guarantee maintenance of skeletal muscle structure, function, bioenergetic characteristics, and phenotype. Thus, regular physical activity is a good strategy to attenuate age-related general decay of muscle structure and function (ClinicalTrials.gov: NCT01679977).

DNA damage-activated ABL-MyoD signaling contributes to DNA repair in skeletal myoblasts.

Previous works have established a unique function of MyoD in the control of muscle gene expression during DNA damage response in myoblasts. Phosphorylation by DNA damage-activated ABL tyrosine kinase transiently inhibits MyoD-dependent activation of transcription in response to genotoxic stress. We show here that ABL-MyoD signaling is also an essential component of the DNA repair machinery in myoblasts exposed to genotoxic stress. DNA damage promoted the recruitment of MyoD to phosphorylated Nbs1 (pNbs1)-containing repair foci, and this effect was abrogated by either ABL knockdown or the ABL kinase inhibitor imatinib. Upon DNA damage, MyoD and pNbs1 were detected on the chromatin to MyoD target genes without activating transcription. DNA damage-mediated tyrosine phosphorylation was required for MyoD recruitment to target genes, as the ABL phosphorylation-resistant MyoD mutant (MyoD Y30F) failed to bind the chromatin following DNA damage, while retaining the ability to activate transcription in response to differentiation signals. Moreover, MyoD Y30F exhibited an impaired ability to promote repair in a heterologous system, as compared with MyoD wild type (WT). Consistently, MyoD-null satellite cells (SCs) displayed impaired DNA repair that was rescued by reintroduction of MyoD WT but not by MyoD Y30F. In addition, inhibition of ABL kinase prevented MyoD WT-mediated rescue of DNA repair in MyoD-null SCs. These results identify an unprecedented contribution of MyoD to DNA repair and suggest that ABL-MyoD signaling coordinates DNA repair and transcription in myoblasts.

Signalling pathways regulating muscle mass in ageing skeletal muscle: the role of the IGF1-Akt-mTOR-FoxO pathway.

During ageing skeletal muscles undergo a process of structural and functional remodelling that leads to sarcopenia, a syndrome characterized by loss of muscle mass and force and a major cause of physical frailty. To determine the causes of sarcopenia and identify potential targets for interventions aimed at mitigating ageing-dependent muscle wasting, we focussed on the main signalling pathway known to control protein turnover in skeletal muscle, consisting of the insulin-like growth factor 1 (IGF1), the kinase Akt and its downstream effectors, the mammalian target of rapamycin (mTOR) and the transcription factor FoxO. Expression analyses at the transcript and protein level, carried out on well-characterized cohorts of young, old sedentary and old active individuals and on mice aged 200, 500 and 800 days, revealed only modest age-related differences in this pathway. Our findings suggest that during ageing there is no downregulation of IGF1/Akt pathway and that sarcopenia is not due to FoxO activation and upregulation of the proteolytic systems. A potentially interesting result was the increased phosphorylation of the ribosomal protein S6, indicative of increased activation of mTOR complex1 (mTORC1), in aged mice. This result may provide the rationale why rapamycin treatment and caloric restriction promote longevity, since both interventions blunt activation of mTORC1; however, this change was not statistically significant in humans. Finally, genetic perturbation of these pathways in old mice aimed at promoting muscle hypertrophy via Akt overexpression or preventing muscle loss through inactivation of the ubiquitin ligase atrogin1 were found to paradoxically cause muscle pathology and reduce lifespan, suggesting that drastic activation of the IGF1-Akt pathway may be counterproductive, and that sarcopenia is accelerated, not delayed, when protein degradation pathways are impaired.

Dystrophic tendon functionality is recovered by muscle-specific expression of insulin-like growth factor in mdx mice.

Duchenne muscular dystrophy (DMD) is a severe genetic disorder of skeletal muscle, characterized by a steady muscle weakness. By using the animal model for DMD, the mdx mice, we have previously demonstrated that biomechanical properties of tendinous tissue are also significantly affected in this muscle pathology. Muscle specific over-expression of insulin like growth factor-1 (mIgf-1) is known to induce a partial recovery in muscle functionality, in particular increasing the muscle absolute force, but not the specific force. To test whether Igf-1 muscle specific over-expression helps the recovery also in tendinous tissue, mechanical and cellular evaluation of mdx and mdx:MLC/mIgf-1 mice tendons has been performed. Mechanical properties were investigated by measuring the viscoelastic response of the tissue, while cell viability was evaluated by molecular assays. An absolute recovery in the mechanical properties of EDL and TA tendons was observed through the measurement of tissue viscoelasticity for several different frequencies of interest. Moreover, when compared with tendons from dystrophic mdx animals, mdx:MLC/mIgf-1 specimens showed an almost complete recovery in the number of viable cells for both extensor digitorum longus (EDL) and tibialis anterior (TA) tendons. Of note, the partial recovery in muscle functionality and the full recovery in tendons response, suggests that mIgf-1 muscle specific over-expression exerts its effect on tendons either indirectly, improving the tendon viability and its functional properties as a consequence of the reduction of the hostile muscle dystrophic environment, or acting directly on the tendon tissue, as a paracrine trophic factor.

Injected matrix stimulates myogenesis and regeneration of mouse skeletal muscle after ischaemic injury.

Biomaterial-guided regeneration represents a novel approach for the treatment of myopathies. Revascularisation and the intramuscular extracellular matrix are important factors in stimulating myogenesis and regenerating muscle damaged by ischaemia. In this study, we used an injectable collagen matrix, enhanced with sialyl LewisX (sLeX), to guide skeletal muscle differentiation and regeneration. The elastic properties of collagen and sLeX-collagen matrices were similar to those of skeletal muscle, and culture of pluripotent mESCs on the matrices promoted their differentiation into myocyte-like cells expressing Pax3, MHC3, myogenin and Myf5. The regenerative properties of matrices were evaluated in ischaemic mouse hind-limbs. Treatment with the sLeX-matrix augmented the production of myogenic-mediated factors insulin-like growth factor (IGF)-1, and IGF binding protein-2 and -5 after 3 days. This was followed by muscle regeneration, including a greater number of regenerating myofibres and increased transcription of Six1, M-cadherin, myogenin and Myf5 after 10 days. Simultaneously, the sLeX-matrix promoted increased mobilisation and engraftment of bone marrow-derived progenitor cells, the development of larger arterioles and the restoration of tissue perfusion. Both matrix treatments tended to reduce maximal forces of ischaemic solei muscles, but sLeX-matrix lessened this loss of force and also prevented muscle fatigue. Only sLeX-matrix treatment improved mobility of mice on a treadmill. Together, these results suggest a novel approach for regenerative myogenesis, whereby treatment only with a matrix, which possesses an inherent ability to guide myogenic differentiation of pluripotent stem cells, can enhance the endogenous vascular and myogenic regeneration of skeletal muscle, thus holding promise for future clinical use.

AvidinOX™ for tissue targeted delivery of biotinylated cells.

AvidinOX™, a product containing aldehyde groups, generated by ligand-assisted sugar oxidation of avidin by sodium periodate, maintains the capacity to bind biotin with very high affinity and exhibits the property to chemically link cellular and tissue proteins through Schiff's base formation thus residing in tissues for weeks. In recent studies, we have shown that AvidinOX exhibits much higher persistency in the skeletal muscle than native avidin. The aim of the present study is to evaluate whether AvidinOX-biotin interaction might be exploited to target biotinylated cells to an AvidinOX pre-treated muscle. To accomplish this we performed the following experiments: 1) The proliferation and differentiation properties of biotinylated C2C12 myoblasts were tested in vitro upon linkage to AvidinOX; 2) Bone marrow-derived cells (BMDC) were isolated from GFP positive transgenic mice [strain C57 BL/6-tg (UBC-GFP)] and after biotinylation (bBMDC) were intravenously administered to naive and MAVA+ (Mouse anti Avidin Antibody) C57/B6 mice previously injected with AvidinOX in a tibial muscle (TM). Localization efficiency of GFP+ bBMDC was evaluated on serial sections of the AvidinOX- and vehicle-treated (contra lateral limb) TM, 5 days after transplantation. Results show that biotinylated C2C12 cells, once linked to AvidinOX, maintain their proliferation and differentiation capacity, in vitro. Intravenous injection of biotinylated GFP+ bone marrow-derived cells leads to their specific and efficient localization in the AvidinOX-pre-treated, but not contra lateral muscle of both naive and MAVA+ mice. The present data suggest a potential use of AvidinOX to improve tissue targeted delivery of biotinylated cells.

A stromal cell-derived factor-1 releasing matrix enhances the progenitor cell response and blood vessel growth in ischaemic skeletal muscle.

Although many regenerative cell therapies are being developed to replace or regenerate ischaemic muscle, the lack of vasculature and poor persistence of the therapeutic cells represent major limiting factors to successful tissue restoration. In response to ischaemia, stromal cell-derived factor-1 (SDF-1) is up-regulated by the affected tissue to stimulate stem cell-mediated regenerative responses. Therefore, we encapsulated SDF-1 into alginate microspheres and further incorporated these into an injectable collagen-based matrix in order to improve local delivery. Microsphere-matrix impregnation reduced the time for matrix thermogelation, and also increased the viscosity reached. This double-incorporation prolonged the release of SDF-1, which maintained adhesive and migratory bioactivity, attributed to chemotaxis in response to SDF-1. In vivo, treatment of ischaemic hindlimb muscle with microsphere-matrix led to increased mobilisation of bone marrow-derived progenitor cells, and also improved recruitment of angiogenic cells expressing the SDF-1 receptor (CXCR4) from bone marrow and local tissues. Both matrix and SDF-1-releasing matrix were successful at restoring perfusion, but SDF-1 treatment appeared to play an earlier role, as evidenced by arterioles that are phenotypically older and by increased angiogenic cytokine production, stimulating the generation of a qualitative microenvironment for a rapid and therefore more efficient regeneration. These results support the release of implanted SDF-1 as a promising method for enhancing progenitor cell responses and restoring perfusion to ischaemic tissues via neovascularisation.

Measuring tendon properties in mdx mice: cell viability and viscoelastic characteristics.

Muscular dystrophy is a genetic disorder of skeletal muscle characterized by progressive muscle weakness. Here we assessed whether muscle wasting affects cell viability and mechanical properties of extensor digitorum longus (EDL) and of tibialis anterior (TA) tendons from mdx dystrophic mice compared to wild type (WT) mice. mdx mice represent the classical animal model for human Duchenne muscular dystrophy, and show several signs of the pathology, including a decrease in specific force and an increase of fibrotic index. Cell viability of tendons was evaluated by histological analysis, and viscoelastic properties have been assessed by a rapid measurement protocol that allowed us to compute, at the same time, tissue complex compliance for all the frequencies of interest. Confocal microscopy and mechanical properties measurements revealed that mdx tendons, compared to WT ones, have an increase in the number of dead cells and a significant reduction in tissue elasticity for all the frequencies that were tested. These findings indicate a reduced quality of the tissue. Moreover, mdx tendons have an increase in the viscous response, indicating that during dynamic loading, they dissipate more energy compared to WT. Our results demonstrate that muscular dystrophy involves not only muscle wasting, but also alteration in the viscoelastic properties of tendons, suggesting a paracrine effect of altered skeletal muscle on tendinous tissue.

Stem cell-mediated muscle regeneration and repair in aging and neuromuscular diseases.

One of the most exciting aspirations of current medical science is the regeneration of damaged body parts. The capacity of adult tissues to regenerate in response to injury stimuli represents an important homeostatic process that until recently was thought to be limited in mammals to tissues with high turnover such as blood and skin. However, it is now generally accepted that each tissue type, even those considered post-mitotic, such as nerve or muscle, contains a reserve of undifferentiated progenitor cells, loosely termed stem cells, participating in tissue regeneration and repair. Skeletal muscle regeneration is a coordinate process in which several factors are sequentially activated to maintain and preserve muscle structure and function upon injury stimuli. In this review, we will discuss the role of stem cells in muscle regeneration and repair and the critical role of specific factors, such as IGF-1, vasopressin and TNF-alpha, in the modulation of the myogenic program and in the regulation of muscle regeneration and homeostasis.

Growth factor enhancement of muscle regeneration: a central role of IGF-1.

The prolongation of skeletal muscle strength in aging and neuromuscular disease has been the objective of numerous studies employing a variety of approaches. In the last decade, dramatic progress has been made in elucidating the molecular defects underlying a number of muscle diseases. With the characterization of mutations responsible for muscle dysfunction in several inherited pathologies, and the identification of novel signaling pathways, subtle alterations in which can lead to significant defects in muscle metabolism, the field is poised to devise successful strategies for treatment of this debilitating and often fatal group of human ailments. Yet progress has been slow in therapeutic applications of our newly gained knowledge. The complexity of muscle types, the intimate relationship between structural integrity and mechanical function, and the sensitivity of skeletal muscle to metabolic perturbations have impeded rapid progress in successful clinical intervention. The relatively poor regenerative properties of striated muscle compound also the devastating effects of muscle degeneration.

Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle.

Aging skeletal muscles suffer a steady decline in mass and functional performance, and compromised muscle integrity as fibrotic invasions replace contractile tissue, accompanied by a characteristic loss in the fastest, most powerful muscle fibers. The same programmed deficits in muscle structure and function are found in numerous neurodegenerative syndromes and disease-related cachexia. We have generated a model of persistent, functional myocyte hypertrophy using a tissue-restricted transgene encoding a locally acting isoform of insulin-like growth factor-1 that is expressed in skeletal muscle (mIgf-1). Transgenic embryos developed normally, and postnatal increases in muscle mass and strength were not accompanied by the additional pathological changes seen in other Igf-1 transgenic models. Expression of GATA-2, a transcription factor normally undetected in skeletal muscle, marked hypertrophic myocytes that escaped age-related muscle atrophy and retained the proliferative response to muscle injury characteristic of younger animals. The preservation of muscle architecture and age-independent regenerative capacity through localized mIgf-1 transgene expression suggests clinical strategies for the treatment of age or disease-related muscle frailty.

IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1.

Localized synthesis of insulin-like growth factors (IGFs) has been broadly implicated in skeletal muscle growth, hypertrophy and regeneration. Virally delivered IGF-1 genes induce local skeletal muscle hypertrophy and attenuate age-related skeletal muscle atrophy, restoring and improving muscle mass and strength in mice. Here we show that the molecular pathways underlying the hypertrophic action of IGF-1 in skeletal muscle are similar to those responsible for cardiac hypertrophy. Transfected IGF-1 gene expression in postmitotic skeletal myocytes activates calcineurin-mediated calcium signalling by inducing calcineurin transcripts and nuclear localization of calcineurin protein. Expression of activated calcineurin mimics the effects of IGF-1, whereas expression of a dominant-negative calcineurin mutant or addition of cyclosporin, a calcineurin inhibitor, represses myocyte differentiation and hypertrophy. Either IGF-1 or activated calcineurin induces expression of the transcription factor GATA-2, which accumulates in a subset of myocyte nuclei, where it associates with calcineurin and a specific dephosphorylated isoform of the transcription factor NF-ATc1. Thus, IGF-1 induces calcineurin-mediated signalling and activation of GATA-2, a marker of skeletal muscle hypertrophy, which cooperates with selected NF-ATc isoforms to activate gene expression programs.

Transgenic mouse models of muscle aging.

In the last decade transgenic animals have been become a powerful and exciting research model to study the molecular mechanisms underlying the cellular and physiological processes such as cell growth, differentiation, apoptosis, and the regulation of specific gene expression. In the context of skeletal muscle development, transgenic mice and gene-targeting approaches have led to the definition of specific roles for Muscle Regulatory Factors (MRFs) during embryogenesis, although less is known about the molecular mechanism underlying skeletal muscle aging. Recent studies using specific models of transgenic mice have added new insights into the muscle aging process, providing a baseline for designing appropriate strategies to attenuate or to reverse the cumulative effects of aging. In this review we discuss some of the transgenic models currently available to address the molecular mechanisms of skeletal muscle senescence. Given the complexity of the aging process, this review should be regarded as a presentation of works in progress rather than a comprehensive description of muscle aging.

Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I.

The molecular mechanisms underlying myogenic induction by insulin-like growth factor I (IGF-I) are distinct from its proliferative effects on myoblasts. To determine the postmitotic role of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell lines carrying a stably transfected rat IGF-I gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC-IGF-I exclusively in differentiated L6E9 myotubes, which express the embryonic form of myosin heavy chain (MyHC) and no endogenous IGF-I, resulted in pronounced myotube hypertrophy, accompanied by activation of the neonatal MyHC isoform. The hypertrophic myotubes dramatically increased expression of myogenin, muscle creatine kinase, beta-enolase, and IGF binding protein 5 and activated the myocyte enhancer factor 2C gene which is normally silent in this cell line. MLC-IGF-I induction in differentiated L6E9 cells also increased the expression of a transiently transfected LacZ reporter driven by the myogenin promoter, demonstrating activation of the differentiation program at the transcriptional level. Nuclear reorganization, accumulation of skeletal actin protein, and an increased expression of beta1D integrin were also observed. Inhibition of the phosphatidyl inositol (PI) 3-kinase intermediate in IGF-I-mediated signal transduction confirmed that the PI 3-kinase pathway is required only at early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction in these cells. Expression of IGF-I in postmitotic muscle may therefore play an important role in the maturation of the myogenic program.