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The extracellular matrix (ECM) of decellularized organs possesses the characteristics of the ideal tissue-engineering scaffold (i.e., histocompatibility, porosity, degradability, non-toxicity). We previously observed that the muscle acellular scaffold (MAS) is a pro-myogenic environment in vivo. In order to determine whether MAS, which is basically muscle ECM, behaves as a myogenic environment, regardless of its location, we analyzed MAS interaction with both muscle and non-muscle cells and tissues, to assess the effects of MAS on cell differentiation. Bone morphogenetic protein treatment of C2C12 cells cultured within MAS induced osteogenic differentiation in vitro, thus suggesting that MAS does not irreversibly commit cells to myogenesis. In vivo MAS supported formation of nascent muscle fibers when replacing a muscle (orthotopic position). However, heterotopically grafted MAS did not give rise to muscle fibers when transplanted within the renal capsule. Also, no muscle formation was observed when MAS was transplanted under the xiphoid process, in spite of the abundant presence of cells migrating along the laminin-based MAS structure. Taken together, our results suggest that MAS itself is not sufficient to induce myogenic differentiation. It is likely that the pro-myogenic environment of MAS is not strictly related to the intrinsic properties of the muscle scaffold (e.g., specific muscle ECM proteins). Indeed, it is more likely that myogenic stem cells colonizing MAS recognize a muscle environment that ultimately allows terminal myogenic differentiation. In conclusion, MAS may represent a suitable environment for muscle and non-muscle 3D constructs characterized by a highly organized structure whose relative stability promotes integration with the surrounding tissues. Our work highlights the plasticity of MAS, suggesting that it may be possible to consider MAS for a wider range of tissue engineering applications than the mere replacement of volumetric muscle loss. [ABSTRACT FROM AUTHOR]

Background: The majority of cancer patients experience dramatic weight loss, due to cachexia and consisting of skeletal muscle and fat tissue wasting. Cachexia is a negative prognostic factor, interferes with therapy and worsens the patients' quality of life by affecting muscle function. Mice bearing ectopically-implanted C26 colon carcinoma are widely used as an experimental model of cancer cachexia. As part of the search for novel clinical and basic research applications for this experimental model, we characterized novel cellular and molecular features of C26-bearing mice. Methods: A fragment of C26 tumor was subcutaneously grafted in isogenic BALB/c mice. The mass growth and proliferation rate of the tumor were analyzed. Histological and cytofluorometric analyses were used to assess cell death, ploidy and differentiation of the tumor cells. The main features of skeletal muscle atrophy, which were highlighted by immunohistochemical and electron microscopy analyses, correlated with biochemical alterations. Muscle force and resistance to fatigue were measured and analyzed as major functional deficits of the cachectic musculature. Results: We found that the C26 tumor, ectopically implanted in mice, is an undifferentiated carcinoma, which should be referred to as such and not as adenocarcinoma, a common misconception. The C26 tumor displays aneuploidy and histological features typical of transformed cells, incorporates BrdU and induces severe weight loss in the host, which is largely caused by muscle wasting. The latter appears to be due to proteasome-mediated protein degradation, which disrupts the sarcomeric structure and muscle fiber-extracellular matrix interactions. A pivotal functional deficit of cachectic muscle consists in increased fatigability, while the reported loss of tetanic force is not statistically significant following normalization for decreased muscle fiber size. Conclusions: We conclude, on the basis of the definition of cachexia, that ectopically-implanted C26 carcinoma represents a well standardized experimental model for research on cancer cachexia. We wish to point out that scientists using the C26 model to study cancer and those using the same model to study cachexia may be unaware of each other's works because they use different keywords; we present strategies to eliminate this gap and discuss the benefits of such an exchange of knowledge. [ABSTRACT FROM AUTHOR]

Cytoplasmic Ca[sup 2+] concentration ([Ca[sup 2+]][sub i]) variation is a key event in myoblast differentiation, but the mechanism by which it occurs is still debated. Here we show that increases of extracellular Ca[sup 2+] concentration ([Ca[sup 2+]][sub o]) produced membrane hyperpolarization and a concentrationdependent increase of [Ca[sup 2+]][sub i] due to Ca[sup 2+] influx across the plasma membrane. Responses were not related to inositol phosphate turnover and Ca[sup 2+]-sensing receptor. [Ca[sup 2+]][sub o]-induced [Ca[sup 2+]][sub i] increase was inhibited by Ca[sup 2+] channel inhibitors and appeared to be modulated by several kinase activities. [Ca[sup 2+]][sub i] increase was potentiated by depletion of intracellular Ca[sup 2+] stores and depressed by inactivation of the Na[sup +]/Ca[sup 2+] exchanger. The response to arginine vasopressin (AVP), which induces inositol 1,4,5-trisphosphate-dependent [Ca[sup 2+]][sub i] increase in L6-C5 cells, was not modified by high [Ca[sup 2+]][sub o]. On the contrary, AVP potentiated the [Ca[sup 2+]][sub i] increase in the presence of elevated [Ca[sup 2+]][sub o]. Other clones of the L6 line as well as the rhabdomyosarcoma RD cell line and the satellite cell-derived C2-C12 line expressed similar responses to high [Ca[sup 2+]][sub o], and the amplitude of the responses was correlated with the myogenic potential of the cells. [ABSTRACT FROM AUTHOR]

Cachexia is associated with poor prognosis in patients with chronic disease. Tumor necrosis factor-alpha (TNFα) plays a pivotal role in mediating cachexia and has been demonstrated to inhibit skeletal muscle differentiation in vitro. It has been proposed that TNFα- mediated activation of NEκ leads to down regulation of MyoD, however the mechanisms underlying TNFα effects on skeletal muscle remain poorly understood. We report here a novel pathway by which TNEα inhibits muscle differentiation through activation of caspases in the absence of apoptosis. TNFα-mediated caspase activation and block of differentiation are dependent upon the expression of PW1, but occur independently of NEκB activation. PW1 has been implicated previously in p53-mediated cell death and can induce bax transtocation to the mitochondria. We show that bax-deficient myoblasts do not activate caspases and differentiate in the presence of TNFα, high- lighting a rote for bax-dependent caspase activation in mediating TNFα effects. Taken together, our data reveal that TNFα inhibits myogenesis by recruiting components of apoptotic pathways through PW1. [ABSTRACT FROM AUTHOR]

Cachexia, sarcopenia and anorexia are characterised by muscle wasting. This condition is a weakening, shrinking, and loss of muscle caused by a disease or lack of use. The loss of muscle causes a decrease in strength and inability to move compromising the quality of life. Recently we demonstrated that the skeletal muscle of endurance trained Balb/c mice release IL-6 and Hsp60 (inside exosomes) in the blood stream. We studied the expression of Hsp60 in the muscles of trained and untrained C26-bearing mice, to understand if Hsp60 was over-expression may improve muscle performance and reduce cachexia. Four different interleukins have been also studied in cachectic mice, to understand which was their effect on Hsp60 expression both in the tumor mass and the trained muscle. In the present study we demonstrated that: 1) IL-6 is released by the trained muscle; 2) IL-6 is release also by the tumor mass, 3) in animals inoculated with the C26 tumor and trained after inoculation, IL-6 is synthesized mainly by the skeletal muscle and the tumor mass undergo apoptosis. This work was funded by PRIN2012 - Prof. Farina. [ABSTRACT FROM AUTHOR]

Recent studies strengthen the belief that physical activity as a behavior has a genetic basis. Screening wheel-running behavior in inbred mouse strains highlighted differences among strains, showing that even very limited genetic differences deeply affect mouse behavior. We extended this observation to substrains of the same inbred mouse strain, that is, BALB/c mice. We found that only a minority of the population of one of these substrains, the BALB/c J, performs spontaneous physical activity. In addition, the runners of this substrain cover a significantly smaller distance than the average runners of two other substrains, namely, the BALB/c ByJ and the BALB/c AnNCrl. The latter shows a striking level of voluntary activity, with the average distance run/day reaching up to about 12 kilometers. These runners are not outstanders, but they represent the majority of the population, with important scientific and economic fallouts to be taken into account during experimental planning. Spontaneous activity persists in pathological conditions, such as cancer-associated cachexia. This important amount of physical activity results in a minor muscle adaptation to endurance exercise over a three-week period; indeed, only a nonsignificant increase in NADH transferase+ fibers occurs in this time frame.

In the pursuit of a transplantable construct for the replacement of large skeletal muscle defects arising from traumatic or pathological conditions, several attempts have been made to obtain a highly oriented, vascularized and functional skeletal muscle. Acellular scaffolds derived from organ decellularization are promising, widely used biomaterials for tissue engineering. However, the acellular skeletal muscle extra cellular matrix (ECM) has been poorly characterized in terms of production, storage and host-donor interactions. We have produced acellular scaffolds at the whole organ scale from various skeletal muscles explanted from mice. The acellular scaffolds conserve chemical and architectural features of the tissue of origin, including the vascular bed. Scaffolds can be sterilely stored for weeks at +4°C or +37°C in tissue culture grade conditions. When transplanted in wt mice, the grafts are stable for several weeks, whilst being colonized by inflammatory and stem cells. We demonstrate that the acellular scaffold per se represents a pro-myogenic environment supporting de novo formation of muscle fibers, likely derived from host cells with myogenic potential. Myogenesis within the implant is enhanced by immunosuppressive treatment. Our work highlights the fundamental role of this niche in tissue engineering application and unveils the clinical potential of allografts based on decellularized tissue for regenerative medicine., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Skeletal muscle has a remarkable capacity to regenerate after mechanical or pathological injury. We show that the V1a receptor (V1aR) for vasopressin, a potent myogenic-promoting factor that stimulates differentiation and hypertrophy in vitro, is expressed in mouse skeletal muscle and modulated during regeneration after experimental injury. We used gene delivery by electroporation to overexpress the myc-tagged vasopressin V1aR in specific muscles, thus sensitizing them to circulating vasopressin. The correct localization on the surface of the fibers of the recombinant product was demonstrated by confocal immunofluorescence directed against the myc tag. V1aR overexpression dramatically enhanced regeneration. When compared with mock-transfected controls, V1aR overexpressing muscles exhibited significantly accelerated activation of satellite cells and increased expression of differentiation markers. Downstream of V1aR activation, calcineurin was strongly up-regulated and stimulated the expression of IL-4, a potent mediator of myogenic cell fusion. The central role of calcineurin in mediating V1aR-dependent myogenesis was also demonstrated by using its specific inhibitor, cyclosporine A. This study identifies skeletal muscle as a physiological target of hormones of the vasopressin family and reveals a novel in vivo role for vasopressin-dependent pathways. These findings unveil several steps, along a complex signaling pathway, that may be exploited as potential targets for the therapy of diseases characterized by altered muscle homeostasis and regeneration.

Culturing of skeletal muscle cells on conductive surfaces is required to develop electronic device-muscle junctions for tissue engineering and medical applications. We characterized from a molecular and morphological point of view myogenic cells cultured on gold and on cysteamine-coated gold, as compared to the standard plastic for cell culture. Our results show that cell proliferation and survival are comparable between cells grown on either of the gold surface or plastic. The majority of the cells cultured on gold surfaces retain the ability to respond to differentiation cues, as shown by nuclear translocation of myogenin. Following terminal differentiation, the myotubes cultured on cysteamine-coated gold resemble myotube cultures obtained on plastic for the size and orientation of the myotube bundles retaining most of myosin expression; on the contrary, the myotube cultures on gold show a clumped morphology, likely due to repulsive cell-substratum interaction resulting in aberrant differentiation. On the basis of the aforementioned evidences, the culture of muscle cells on cysteamine-coated gold represents an advance with respect to previously reported substrata. The cysteamine self-assembled monolayer coating is a simple approach to accomplish cultures of myotubes in unprecedented tight proximity to conductive surfaces., ((c) 2008 Wiley Periodicals, Inc.)

Skeletal muscle is susceptible to injury following trauma, neurological dysfunction, and genetic diseases. Skeletal muscle homeostasis is maintained by a pronounced regenerative capacity, which includes the recruitment of stem cells. Chronic exposure to tumor necrosis factor-alpha (TNF) triggers a muscle wasting reminiscent of cachexia. To better understand the effects of TNF upon muscle homeostasis and stem cells, we exposed injured muscle to TNF at specific time points during regeneration. TNF exposure delayed the appearance of regenerating fibers, without exacerbating fiber death following the initial trauma. We observed modest cellular caspase activation during regeneration, which was markedly increased in response to TNF exposure concomitant with an inhibition in regeneration. Caspase activation did not lead to apoptosis and did not involve caspase-3. Inhibition of caspase activity improved muscle regeneration in either the absence or the presence of TNF, revealing a nonapoptotic role for this pathway in the myogenic program. Caspase activity was localized to the interstitial cells, which also express Sca-1, CD34, and PW1. Perturbation of PW1 activity blocked caspase activation and improved regeneration. The restricted localization of Sca-1+, CD34+, PW1+ cells to a subset of interstitial cells with caspase activity reveals a critical regulatory role for this population during myogenesis, which may directly contribute to resident muscle stem cells or indirectly regulate stem cells through cell-cell interactions.

Muscle wasting (cachexia) is an incurable complication associated with chronic infection and cancers that leads to an overall poor prognosis for recovery. Tumor necrosis factor-alpha (TNFalpha) is a key inflammatory cytokine associated with cachexia. TNFalpha inhibits myogenic differentiation and skeletal muscle regeneration through downstream effectors of the p53 cell death pathway including PW1/Peg3, bax, and caspases. We report that p53 is required for the TNFalpha-mediated inhibition of myogenesis in vitro and contributes to muscle wasting in response to tumor load in vivo. We further demonstrate that PW1 and p53 participate in a positive feedback regulatory loop in vitro. Consistent with this observation, we find that the number of PW1-expressing stem cells in skeletal muscle declines significantly in p53 nullizygous mice. Furthermore, gene transfer of a dominant-negative form of PW1 into muscle tissue in vivo blocks myofiber atrophy in response to tumor load. Taken together, these results show a novel role for p53 in mediating muscle stem cell behavior and muscle atrophy, and point to new targets for the therapeutic treatment of muscle wasting.

Chronic disease states are associated with elevated levels of inflammatory cytokines that have been demonstrated to lead to severe muscle wasting. A mechanistic understanding of muscle wasting is hampered by limited in vivo cytokine models which can be applied to emerging mouse mutants as they are generated. We developed a simple and novel approach to induce adult mouse skeletal muscle wasting based on direct gene transfer of an expression vector encoding the secreted form of the murine tumor necrosis factor-alpha (mTNFalpha). This procedure results in the production of elevated levels of circulating mTNFalpha followed by body weight loss, upregulation of Atrogin1, and muscle atrophy, including muscles distant from the site of gene transfer. We also found that mTNFalpha gene transfer resulted in a significant inhibition of regeneration following muscle injury. We conclude that in addition to being a potent inducer of cachexia, TNFalpha is a potent inhibitor of myogenesis in vivo.

Cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) variation is a key event in myoblast differentiation, but the mechanism by which it occurs is still debated. Here we show that increases of extracellular Ca(2+) concentration ([Ca(2+)](o)) produced membrane hyperpolarization and a concentration-dependent increase of [Ca(2+)](i) due to Ca(2+) influx across the plasma membrane. Responses were not related to inositol phosphate turnover and Ca(2+)-sensing receptor. [Ca(2+)](o)-induced [Ca(2+)](i) increase was inhibited by Ca(2+) channel inhibitors and appeared to be modulated by several kinase activities. [Ca(2+)](i) increase was potentiated by depletion of intracellular Ca(2+) stores and depressed by inactivation of the Na(+)/Ca(2+) exchanger. The response to arginine vasopressin (AVP), which induces inositol 1,4,5-trisphosphate-dependent [Ca(2+)](i) increase in L6-C5 cells, was not modified by high [Ca(2+)](o). On the contrary, AVP potentiated the [Ca(2+)](i) increase in the presence of elevated [Ca(2+)](o). Other clones of the L6 line as well as the rhabdomyosarcoma RD cell line and the satellite cell-derived C2-C12 line expressed similar responses to high [Ca(2+)](o), and the amplitude of the responses was correlated with the myogenic potential of the cells.