Morphogenesis of rat myotendinous junction

Myotendinous junction (MTJ) is the highly specialized complex which connects the skeletal muscle to the tendon for transmitting the contractile force between the two tissues. The purpose of this study was to investigate the MTJ development and rat EDL was chosen as a model. 1, 15, 30 day animals were considered and the junctions were analyzed by light and electron microscopy. The MTJ interface architecture increased during the development, extending the interaction between muscle and tendon. 1-day-old rats showed disorganized myofibril bundles, spread cytosol and incomplete rough endoplasmic reticulum, features partially improved in 15-day-old rats, and completely developed in 30-day-old animals. These findings indicate that muscle-tendon interface displays, during rat lifetime, numerically increased and longer tendon interdigitations, correlated with an improved organization of both tissues and with a progressive acquirement of full functionalit

Further considerations on in vitro skeletal muscle cell death

The present review discusses the apoptotic behavior induced by chemical and physical triggers in C2C12 skeletal muscle cells, comparing myoblast to myotube sensitivity, and investigating it by means of morphological, biochemical and cytofluorimetric analyses. After all treatments, myotubes, differently from myoblasts, showed a poor sensitivity to cell death. Intriguingly, in cells exposed to staurosporine, etoposide and UVB radiation, apoptotic and normal nuclei within the same fibercould be revealed. The presence of nuclear-dependent “territorial” death domains in the syncytium could explain a delayed cell death of myotubes compared to mononucleated cells. Moreover, autophagic granules abundantly appeared in myotubes after each treatment. Autophagy could protect muscle cell integrity against chemical and physical stimuli, making C2C12 myotubes, more resistant to cell death inductio

Melatonin role in skeletal muscle disorders

Abstract. – OBJECTIVE: This review discusses the impact of the neuro-hormone melatonin on skeletal muscle disorders based on recent literature data with the aim to clarify the utility of the melatonin therapy in patients affected by muscle diseases. MATERIALS AND METHODS: It has been pointed out the possible role of melatonin as a food supplement to cure muscular disorders characterized by muscle wasting. Oxidative damage has been proposed as one of the major contributors of the skeletal muscle decline occurring both in physiological and pathological conditions. It is known that excessive oxidant levels lead to mitochondrial damage, and in turn, contribute to apoptotic signaling activation and autophagic impairment. This condition is common in a variety of skeletal muscle disorders. RESULTS: The scientific evidence enhances the antioxidant effect of melatonin, that has been demonstrated by several studies both in vitro and in vivo. This effect counteracts mitochondrial impairments and reduces oxidative stress and autophagic alterations in muscle fibers. Its beneficial role in restoring muscle decline, takes place mainly in atrophic conditions correlated to muscle aging. CONCLUSIONS: The findings of the research suggest that melatonin may be considered as a valid dietary supplement, useful to prevent muscle wasting, in particular, in sarcopenia-associated diseases

Protective effect of different antioxidant agents in UVB-irradiated keratinocytes

Skin cells can respond to UVB-induced damage either by tolerating it, or restoring it through antioxidant activation and DNA repair mechanisms or, ultimately, undergoing programmed cell death, when damage is massive. Nutritional factors, in particular, food antioxidants, have attracted much interest because of their potential use in new preventive, protective, and therapeutic strategies for chronic degenerative diseases, including skin inflammation and cancer. Some polyphenols, present in virgin olive oil, well tolerated by organism after oral administration, show a variety of pharmacological and clinical benefits such as anti-oxidant, anti-cancer, anti-inflammatory, and neuro-protective activities. Here, the protective effects of antioxidant compounds against UV-induced apoptosis have been described in HaCat cell line. Human keratinocytes were pre-treated with antioxidants before UVB exposure and their effects have been evaluated by means of ultrastructural analyses. After UVB radiation, a known cell death trigger, typical apoptotic features, absent in control condition and in antioxidant alone-treated cells, appear. An evident numerical decrease of ultrastructural apoptotic patterns and TUNEL positive nuclei can be observed when natural antioxidants were supplied before cell death induction. These data have been confirmed by molecular investigation of caspase activity. In conclusion, this paper highlights antioxidant compound ability to prevent apoptotic cell death in human keratinocytes exposed to UVB, suggesting, for these molecules, a potential role in preventing skin damage

Ultrastructural modifications of myotendinous junction with disuse

The skeletal muscle contraction is transmitted to the tendon through the myotendinous junction (MTJ), where the proximal extremity of tendon forms characteristic finger-like processes, penetrating into the muscle mass (Ciena et al., 2010). MTJ represents an intriguing and complex structure from both biological and functional point of view. We recently demonstrated that changes at MTJ level occur as an adaptation to exercise-induced tension increase. In particular, branching of finger-like processes increases, so enlarging the whole tendon-muscle surface area and, consequently, allowing a better tension resistance (Curzi et al., 2012). The aim of this study is to analyse MTJ behavior in disuse condition, furtherly investigating the strict morphofunctional correlation. Therefore, 4 hind-limb suspended and 4 control rats were studied. After sacrifice, MTJs of plantaris muscle were withdrawn, fixed in 1.4% glutaraldehyde in 0.2M sodium cacodylate and processed as previously described (Tidball et al., 1992; D’Emilio et al., 2010). After 5 days of suspension, skeletal muscle highlights signs of atrophy in response to decreased mechanical loading (Linderman et al., 1994). In fact, close to muscle-tendon interface, irregular misaligned sarcomeres, with absent Z-lines, were observed. Muscle intermyofibrillar component spreads, especially between terminal filaments and tendon finger-like processes. A lower number of muscle-tendon interdigitations, characteristically not bifurcated, also appears. In conclusion, along with muscle atrophy features, ultrastructural changes occur at the MTJ organization level, as an adaptation to muscle unloading

Melatonin effects in normal and tumoral skeletal muscle cells: a preliminary study

Melatonin (MEL), also chemically known as N-acetyl-5-methoxytryptamine, is a hormone found in animals, plants, and microbes. It exhibits strong antioxidant effects and thanks to its structure it is able to diffuse through all the biological membranes, also overcoming the blood-brain barrier and the placenta (Salucci et al., 2014). Numerous in vitro and in vivo studies have documented Mel ability to induce apoptosis in tumor cells while inhibiting it in the normal ones (Cristofanos et al, 2009; Lanoix et al., 2011). In this study MEL activity has been investigated in vitro both in murine skeletal muscle (C2C12) and in alveolar rhabdomyosarcoma (RH30) cell lines by means of morpho-functional approaches. If MEL low concentrations are well tolerated by normal skeletal muscle cells, its effect appears completely different in tumor cells, where MEL can be considered a powerful apoptotic trigger. In RH30 cells, blebbing, chromatin condensation and margination, apoptotic bodies occur as well as necrotic cell death features. The latter appeared after prolonged exposure to MEL. In conclusion, the neuro-hormone shows a strong dose and time dependent pro-apoptotic activity and it could represent a potential tool in association with the current chemotherapeutic compounds to resolve alveolar rhabdomyosarcoma, the most common pediatric skeletal muscle tissue malignancy

Cytoprotective effects of melatonin in C2C12 skeletal muscle cells: a multiple technical approach

Melatonin has a wide range of physiological functions including protection against oxidative stress, which is carried out through its ability to act as a free radical scavenger and to stimulate antioxidant enzyme production (Allegra et al., 2003). Oxidative stress is a major player in initiating apoptosis in skeletal muscle, as well as in other tissues. Apoptosis is essential for skeletal muscle development and homeostasis; nevertheless, its misregulation has been frequently observed in various myopathies (Loro et al., 2010). Several authors demonstrated that melatonin exerts antiapoptotic actions in various cell models (Hibaoui et al., 2009) and our previous studies evidenced that it prevents apoptosis induced by UV-B and H2O2 in U937 cells (Luchetti et al., 2006; Salucci et al., 2010). In this work, melatonin activity has been investigated in C2C12 cells, after apoptotic chemical treatments. Myoblasts and myotubes were pre-treated with melatonin and then exposed to H2O2, cisplatin, etoposide and staurosporine. Data, obtained by means of TEM and TUNEL-CLSM, show that melatonin prevents apoptosis induced by H2O2, cisplatin and etoposide. Differently, staurosporine-induced apoptosis is not inhibited, probably because this trigger has a mechanism of action different from free radical increase. These results confirm melatonin ability to act as an antioxidant and anti-apoptotic molecule, thus suggesting a possible therapeutic strategy for myophaties involving apoptosis misregulation

α-Actinin behavior during C2C12 along differentiation

α-Actinin is a cytoskeletal actin-binding protein (Ogura et al., 2009) that provides structural integrity of the sarcomeres and is located in the skeletal muscle Z-lines. It creates cross-links between actin filaments and, besides, it contributes to cytoskeleton organization and muscle contraction (Sjöblom et al., 2008). The aim of this work was to clear up the behavior of sarcomeric α-actinin in Z-lines formation during myogenic differentiation. For this purpose, C2C12 cells were analyzed at 0, 3, 7 days of differentiation, monitoring cell maturation and viability by means of inverted microscopy. Immuno-labeling of sarcomeric α-actinin was investigated both at CLSM and at TEM, using a mouse anti-α-actinin antibody followed by a FITC-conjugated goat antimouse or a 10nm colloidal gold conjugated anti-mouse antibody (Ferri et al., 2009), respectively. Immunofluorescence analysis reveals that, when differentiation is induced, initially α-actinin colocalizes with membrane-associated proteins, then it aligns longitudinally across the cytoplasm and, finally, it binds actin, giving rise to Z-lines. Immunogold study generally evidences a cytoplasmic and nuclear positivity, indicating a role for α-actinin in signaling, chromatin remodeling and in shuttle between these compartments (Dingovà et al., 2009; Lin et al., 2010). This study shows an α-actinin specific distribution and dynamic organization along the differentiation process

Skeletal muscle cell death induced by physical agents

Apoptosis plays a pivotal role in the deletion of unwanted, damaged, or infected cells in multicellular organisms, as well as in development and tissue homeostasis, cell differentiation, and proliferation. In skeletal muscle cells it is unique for several reasons. First, skeletal muscle fibre is multinucleated. So muscle cell death is correlated to a loss of gene expression within the local myonuclear domain, potentially leading to muscle atrophy. In addition, skeletal muscle is a plastic tissue capable of changing its mitochondrial content and/or composition in response to chronic alterations in muscle use or disuse (Siu et al., 2009). Most of the research evidenced that many of the external apoptotic stimuli activate signaling pathways that converge on the mitochondria, determining cell death (Adhietty et al., 2008). Physical triggers such as UVB (D’Emilio et al., 2010), hyperthermia (Lee et al., 2011) and hypothermia (Pizanis et al., 2011) induced cell death by mitochondrial pathways in various cell types. In addition also low pH usually induced DNA damage in other cell lines (Xiao et al., 2003). The aim of this work is to investigate in vitro skeletal muscle cell death appearing after exposure to physical triggers, by means of TUNEL reaction, analysed at confocal microscope, and of electron microscopy. C2C12 myoblasts and myotubes, grown as previously reported (D’Emilio et al., 2010), were exposed to UV-B (312nm) for 30 min, hyperthermia 45°C for 1h and hypothermia (2-6°C) and low pH (5) for 4h. All treatments were followed by 2h recovery. Control cell evidentiated a good morphology and appeared negative to TUNEL reaction. UVB - treated sample presented nuclear features suggest apoptosis both at electron and confocal microscopy and in undifferentiated and differentiated conditions. Hyperthermia induced both apoptosis and necrosis with cell rounding and a certain positivity to TUNEL reaction both in myoblasts and myotubes. After hypothermia apoptosis was observed in some cells, but the majority appeared similar to the control, so evidentiating a scarce response. Cells treated with low had swollen nuclei, sometimes showing a thin film of condensed chromatin, and occasionally TUNEL-positive. In all conditions cytoplasm vacuolisaton and autophagic vacuole increase appeared. These findings suggest that skeletal muscle cells seem to be sensitive to physical agents induced cell death