Melatonin role in skeletal muscle disorders.

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.

Diet Modulation Restores Autophagic Flux in Damaged Skeletal Muscle Cells.

OBJECTIVES: Autophagy is a physiological and highly regulated mechanism, crucial for cell homeostasis maintenance. Its impairment seems to be involved in the onset of several diseases, including muscular dystrophies, myopathies and sarcopenia. According to few papers, chemotherapeutic drug treatment is able to trigger side effects on skeletal muscle tissue and, among these, a defective autophagic activation, which leads to the persistence of abnormal organelles within cells and, finally, to myofiber degeneration. The aim of this work is to find a strategy, based on diet modulation, to prevent etoposide-induced damage, in a model of in vitro skeletal muscle cells. METHODS: Glutamine supplementation and nutrient deprivation have been chosen as pre-treatments to counteract etoposide effect, a chemotherapeutic drug known to induce oxidative stress and cell death. Cell response has been evaluated by means of morpho-functional, cytofluorimetric and molecular analyses. RESULTS: Etoposide treated cells, if compared to control, showed dysfunctional mitochondria presence, ER stress and lysosomal compartment damage, confirmed by molecular investigations. CONCLUSIONS: Interestingly, both dietary approaches were able to rescue myofiber from etoposide-induced damage. Glutamine supplementation, in particular, seemed to be a good strategy to preserve cell ultrastructure and functionality, by preventing the autophagic impairment and partially restoring the normal lysosomal activity, thus maintaining skeletal muscle homeostasis.

Melatonin action in tumor skeletal muscle cells: an ultrastructural study.

Melatonin (Mel), or N-acetyl-5-methoxytryptamine, is a circadian hormone that can diffuse through all the biological membranes thanks to its amphiphilic structure, also overcoming the blood-brain barrier and placenta. Although Mel has been reported to exhibit strong antioxidant properties in healthy tissues, studies carried out on tumor cultures gave a different picture of its action, often describing Mel as effective to trigger the cell death of tumor cells by enhancing oxidative stress. Based on this premise, here Mel effect was investigated using a tumor cell line representative of the human alveolar rhabdomyosarcoma (ARMS), the most frequent soft tissue sarcoma affecting childhood. For this purpose, Mel was given either dissolved in ethanol (EtOH) or dimethyl sulfoxide (DMSO) at different concentrations and time exposures. Cell viability assays and ultrastructural observations demonstrated that Mel was able to induce a dose- and time-dependent cell death independently on the dissolution solvent. Microscopy analyses highlighted the presence of various apoptotic and necrotic patterns correlating with the increasing Mel dose and time of exposure. These findings suggest that Mel, triggering apoptosis in ARMS cells, could be considered as a promising drug for future multitargeted therapies.

Three-dimensional apoptotic nuclear behavior analyzed by means of Field Emission in Lens Scanning Electron Microscope.

Apoptosis is an essential biological function required during embryogenesis, tissue homeostasis, organ development and immune system regulation. It is an active cell death pathway involved in a variety of pathological conditions. During this process cytoskeletal proteins appear damaged and undergo an enzymatic disassembling, leading to formation of apoptotic features. This study was designed to examine the three-dimensional chromatin behavior and cytoskeleton involvement, in particular actin re-modeling. HL-60 cells, exposed to hyperthermia, a known apoptotic trigger, were examined by means of a Field Emission in Lens Scanning Electron Microscope (FEISEM). Ultrastructural observations revealed in treated cells the presence of apoptotic patterns after hyperthermia trigger. In particular, three-dimensional apoptotic chromatin rearrangements appeared involving the translocation of filamentous actin from cytoplasm to the nucleus. FEISEM immunogold techniques showed actin labeling and its precise three-dimensional localization in the diffuse chromatin, well separated from the condensed one. The actin presence in dispersed chromatin inside the apoptotic nucleus can be considered an important feature, indispensable to permit the apoptotic machinery evolution.

α-Actinin involvement in Z-disk assembly during skeletal muscle C2C12 cells in vitro differentiation.

α-Actinin is involved in the assembly and maintenance of muscle fibers. α-Actinin is required to cross-link actin filaments and to connect the actin cytoskeleton to the cell membrane and it is necessary for the attachment of actin filaments to Z-disks in skeletal muscle fibers and to dense bodies in smooth muscle ones. In addition to its mechanical role, sarcomeric α-actinin interacts with proteins involved in a variety of signaling and metabolic pathways. The aim of this work is to monitor Z-disk formation, in order to clear up the role of sarcomeric α-actinin in undifferentiated stage, after 4 days of differentiation (intermediate differentiation stage) and after 7 days of differentiation (fully differentiated stage). For this purpose, C2C12 murine skeletal muscle cells, grown in vitro, were analyzed at three time points of differentiation. Confocal laser scanner microscopy and transmission electron microscopy have been utilized for α-actinin immunolocalization. Both techniques reveal that in undifferentiated cells labeling appears uniformly distributed in the cytoplasm with punctate α-actinin Z-bodies. Moreover, we found that when differentiation is induced, α-actinin links at first membrane-associated proteins, then it aligns longitudinally across the cytoplasm and finally binds actin, giving rise to Z-disks. These findings evidence α-actinin involvement in sarcomeric development, suggesting for this protein an important role in stabilizing the muscle contractile apparatus.

Analysis of cell death by electron microscopy.

Cell death is said to occur mostly by two alternative, opposite modes: apoptosis, which involves a highly genetically regulated and elaborate network of biochemical events and cascades, and necrosis, considered a passive cell death without underlying regulatory mechanisms. Here, we describe the different morphological features of cells undergoing apoptotic and necrotic cell death, through the analysis of transmission (TEM) and scanning (SEM) electron microscopy. TEM allows detailed studies of ultrastructural changes, within the cell, such as the nuclear alteration, the cytoplasmic reorganization, and the loss of membrane integrity. The cell-surface changes, including membrane blebbing and loss of features, such as microvilli, can be assessed by SEM.

Hemidesmus indicus induces apoptosis as well as differentiation in a human promyelocytic leukemic cell line.

ETHNOPHARMACOLOGICAL RELEVANCE: The decoction of the roots of Hemidesmus indicus is widely used in the Indian traditional medicine for the treatment of blood diseases, dyspepsia, loss of taste, dyspnea, cough, poison, menorrhagia, fever, and diarrhea. Poly-herbal preparations containing Hemidesmus are often used by traditional medical practitioners for the treatment of cancer. The aim of this study was to investigate the cytodifferentiative, cytostatic and cytotoxic potential of a decoction of Hemidesmus indicus's roots (0.31-3 mg/mL) on a human promyelocytic leukemia cell line (HL-60). MATERIALS AND METHODS: The decoction of Hemidesmus indicus was characterized by HPLC to quantify its main phytomarkers. Induction of apoptosis, cell-cycle analysis, levels of specific membrane differentiation markers were evaluated by flow cytometry. The analysis of cell differentiation by nitroblue tetrazolium (NBT) reducing activity, adherence to the plastic substrate, α-napthyl acetate esterase activity and morphological analysis was performed through light microscopy (LM) and transmission electron microscopy (TEM). RESULTS: Starting from the concentration of 0.31 mg/ml, Hemidesmus indicus induced cytotoxicity and altered cell-cycle progression, through a block in the G0/G1 phase. The decoction caused differentiation of HL-60 cells as shown by NBT reducing activity, adherence to the plastic substrate, α-naphtyl acetate esterase activity, and increasing expression of CD14 and CD15. The morphological analysis by LM and TEM clearly showed the presence of granulocytes and macrophages after Hemidesmus indicus treatment. CONCLUSIONS: The cytodifferentiating, cytotoxic and cytostatic activities of Hemidesmus indicus offers a scientific basis for its use in traditional medicine. Its potent antileukemic activity provides a pre-clinical evidence for its traditional use in anticancer pharmacology. Further experiments are worthwhile to determine the in vivo anticancer potential of this plant decoction and its components.

Growth hormone plus resistance exercise attenuate structural changes in rat myotendinous junctions resulting from chronic unloading.

Myotendinous junctions (MTJs) are specialized sites on the muscle surface where forces generated by myofibrils are transmitted across the sarcolemma to the extracellular matrix. At the ultrastructural level, the interface between the sarcolemma and extracellular matrix is highly folded and interdigitated at these junctions. In this study, the effect of exercise and growth hormone (GH) treatments on the changes in MTJ structure that occur during muscle unloading, has been analyzed. Twenty hypophysectomized rats were assigned randomly to one of five groups: ambulatory control, hindlimb unloaded, hindlimb unloaded plus exercise (3 daily bouts of 10 climbs up a ladder with 50% body wt attached to the tail), hindlimb unloaded plus GH (2 daily injections of 1 mg/kg body wt, i.p.), and hindlimb unloaded plus exercise plus GH. MTJs of the plantaris muscle were analyzed by electron microscopy and the contact between muscle and tendon was evaluated using an IL/B ratio, where B is the base and IL is the interface length of MTJ's digit-like processes. After 10 days of unloading, the mean IL/B ratio was significantly lower in unloaded (3.92), unloaded plus exercise (4.18), and unloaded plus GH (5.25) groups than in the ambulatory control (6.39) group. On the opposite, the mean IL/B ratio in the group treated with both exercise and GH (7.3) was similar to control. These findings indicate that the interaction between exercise and GH treatments attenuates the changes in MTJ structure that result from chronic unloading and thus can be used as a countermeasure to these adaptations.

How physical exercise changes rat myotendinous junctions: an ultrastructural study.

Myotendinous junctions can be easily injured by overloading or trauma, and exercise training may be a way of increasing their resistance to mechanical stress. To this end, we examined herein the morphological changes induced by moderate exercise training in the myotendinous junctions of extensor digitorum longus and gastrocnemius muscles in rats. Twelve Sprague-Dawley rats were used in this investigation. Six of them were trained to run on a treadmill for 1 h/day, 3 days/week over 10 weeks in order for them to achieve a running rate of 25 m/min at the end of the training period. Six age-matched sedentary rats were used as controls. The rats were sacrificed 24 h after the final training session, and the extensor digitorum longum (EDL) and the gastrocnemium were excised; the myotendinous junctions (MTJ) were then prepared and observed with electron microscopy. Digitation branching was evaluated by counting the bifurcations in the MTJ protrusions. Our observations indicate that exercise does indeed induce changes in MTJ morphology. In both muscles the number of bifurcated interdigitations increased significantly, as well as, in gastrocnemius, the branching of the finger-like processes. It was demonstrated that the MTJ is able to adapt to an increase in tensile force by enlarging the muscle-tendon contact area and, consequently, mechanical resistance.

Morphological and biochemical patterns in skeletal muscle apoptosis.

Some neuromuscular disorders, such as Duchenne muscular dystrophy, hereditary inclusion body myopathy, malignant hyperthermia, alcoholic myopathy and mitochondrial myopathies are characterized by oxidative stress and loss of muscle fibres due to apoptosis. In this study we have analyzed muscle cell death in vitro utilizing C2C12 myoblasts and myotubes, inducing apoptosis by means of UVB irradiation. C2C12 cells were analysed by scanning and transmission electron microscopy (SEM, TEM) as well as by TUNEL reaction. DNA analysis was performed by gel electrophoresis and flow cytometry. MitoTracker red CMXRos and JC-1 fluorescent probes were also used to study mitochondrial behavior. Finally, caspase activity was investigated by means of Western blot, while caspase-9 and -3 inhibitor effects by means of SEM. SEM showed the typical membrane blebbing while TEM revealed the characteristic chromatin condensation. The TUNEL reaction presented a certain positivity too. Apoptotic and non-apoptotic nuclei in the same myotube were identified both by TUNEL and TEM. Gel electrophoresis never showed oligonucleosomal DNA fragmentation, in agreement with the cell cycle analysis performed by flow cytometry which did not reveal a sharp subdiploid peak. Mitochondrial response to UVB was later investigated and a decrease in mitochondrial functionality appeared. Caspase-9 and -3 cleavage, and, consequently, the activation of the caspase cascade, was also demonstrated by Western blot. Moreover a decrease in apoptotic cell number was noted after caspase-9 and-3 inhibitor treatment. All these results indicated that UVB irradiation induces apoptosis, both in myoblasts and in myotubes, the second being more resistant. DNA fragmentation, at least the nucleosomic type, does not occur. A certain double-strand cleavage appears in TUNEL analysis, as well as characteristic ultrastructural changes in chromatin.

Proteomics-based investigation in C2C12 myoblast differentiation.

Skeletal muscle cell differentiation is a multistage process extensively studied over the years. Even if great improvements have been achieved in defining biological process underlying myogenesis, many molecular mechanisms need still to be clarified.To further highlight this process, we studied cells at undifferentiated, intermediate and highly differentiated stages, and we analyzed, for each condition, morphological and proteomic changes. We also identified the proteins that showed statistical significant changes by a ESI-Q-TOF mass spectrometer. This work provides further evidence of the involvement of particular proteins in skeletal muscle development. Furthermore, the high level of expression of many heat shock proteins, suggests a relationship between differentiation and cellular stress. Intriguingly, the discovery of myogenesis-correlated proteins, known to play a role in apoptosis, suggests a link between differentiation and this type of cell death.

Surface and inner cell behaviour along skeletal muscle cell in vitro differentiation.

During muscle tissue differentiation, in particular in the formation of myotubes from the myoblasts, plasma membrane changes its morpho-functional characteristics. In this study, muscle cell membrane behaviour has been studied along the differentiation of C2C12, a mouse myoblastic adherent cell line. Flat undifferentiated cells, cultured for 3-4 days in the differentiation medium, progressively become thick, long and multinucleated myotubes covered with microvilli. They lose stress fibers and adhesion to the underlying substrate evidentiating an actin redistribution, followed by the spatial organization of thick and thin myofilaments. Sarcomeres and myofibrils occasionally appear, even if a certain percentage of "myosacs" containing randomly oriented filaments can be identified all along the differentiation. M-cadherin, a molecule involved in cell-cell adhesion, also appears in the early differentiation stage, during myoblast fusion. Occasional focal contractions can also be observed in myotubes, which prompt an electrophysiological membrane analysis. When studied by means of patch clamp technique, resting membrane potential appears to undergo a transient depolarization, while input resistance increases until day 5 after differentiation induction, then successively decreases. Capacitance declines until day 5, later appearing enhanced. Moreover, with the induction of differentiation, the pattern of functional voltage-dependent ion channels changes. Therefore, during myogenesis, cell maturation is coupled with changes in cell membrane morphological features and functional characteristics.

Effects of hyperthermia in human neuroblastoma cells.

Neuroblastoma is a childhood malignancy derived from the developing sympathetic nervous system (SNS) and often diagnosed during early infancy. To investigate its metastatic properties, also in response to anti-cancer treatment, we have studied hyperthermia (HT) effects on the ultrastructure of SK-N-MC human neuroblastoma tumor cells. Cells undergoing HT showed a significant increase in apoptotic and necrotic events, but also a rearrangement of the cellular shape, appearing as cell detachment and rounding. The most striking effects of HT can be so correlated to primary tumor mass decrease and to a certain impairment of cell adhesion properties and consequently metastatic diffusion potential.

Flow cytometric evaluation of Vibrio parahaemolyticus adhesion inhibition to human epithelial cells.

BACKGROUND: The present report demonstrates the usefulness of flow cytometry for a quantitative assessment of adhesion inhibition of a Vibrio parahaemolyticus strain to human epithelial cells to acquire more information about the nature of its adhesins. METHODS: The inhibition of the adhesive process to Hep-2 was assayed by adding several monosaccharides to infected cells monolayers. The quantification of the adherent bacteria, labeled with a specific primary antibody plus a secondary fluorescein isothiocyanate-conjugated antibody, was performed by flow cytometry in comparison with light microscopy. The adherence was quantified in terms of the proportion of cells with adherent V. parahaemolyticus and as the mean of adherent bacteria per cell. RESULTS: The adhesion showed a percentage of 98% with a mean fluorescence channel of 331 comparable to those obtained by light microscopy. The addition of monosaccharides resulted in a D-mannose and N-acetyl-galactosamine sensitive adherence. Even if this environmental strain also showed a mannose-sensitive cell-associated hemoagglutination that could mediate V. parahaemolyticus adherence, our results suggest that different sites for an irreversible adherence to host cell are involved. CONCLUSIONS: Flow cytometry in combination with indirect immunofluorescence is an effective tool to investigate the adhesive process of bacteria to epithelial cells because it is more sensitive and reproducible than visual counting of bacteria performed in light microscopy.

C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization.

In this study, the differentiation of C2C12 cells, a primary line of murine myoblasts, was investigated by a multiple technical approach. Undifferentiated cells, and those at intermediate and final differentiation times, were studied at the reverted microscope, by conventional and confocal immunofluorescence, and by transmission and scanning electron microscopy. The general monolayer architecture changed during differentiation from fusiform or star-shaped cells to elongated confluent cells, finally originating long, multinucleated myotubes. Sarcomeric actin and myosin are present also in undifferentiated myoblasts, but progressively acquire a structured pattern up to the appearance of sarcomeres and myofibrils at about 5 days after differentiation induction. Myotubes show a particular positivity for actin and myosin, and M-cadherin, an adhesion molecule characteristic, as known, of satellite cells, also seems to be involved in their assembling. Rare apoptotic patterns, as evidenced by the TUNEL technique, appear during myoblast maturation.

Integrin and cytoskeleton behaviour in human neuroblastoma cells during hyperthermia-related apoptosis.

Hyperthermia induces several cellular responses leading to morphological changes, cell detachment and death. Loss of integrins from the cell surface after acute heat-treatment may block several physiological signalling pathways, but whether the assembly network between integrin and cytoskeletal actin is perturbed during hyperthermic treatment is unknown. In this study we tested this hypothesis by evaluating cell morphology, protein cytoskeletal profile and integrin CD11a content in both adherent and floating SK-N-MC human neuroblastoma cells. Morphological and cytometric analyses confirmed that hyperthermia is an effective apoptotic trigger, revealing the typical chromatin margination, cell shape changes and 7-AAD incorporation. After hyperthermia, cytoskeletal proteins showed an increase of high-molecular-weight aggregates and a significant decrease of both actin and CD11a content with respect to control cells. The integrin CD11a and membrane-bound actin alterations found in detached floating neuroblastoma cells recovered after heat-shock may cause the cytoskeletal abnormalities related to the observed surface cell rounding/blebbing and anoikis, early events of hyperthermia-induced programmed cell death.

In vitro apoptotic cell death during erythroid differentiation.

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.

Evaluation of leukocyte stabilisation in TransFix-treated blood samples by flow cytometry and transmission electron microscopy.

In this report, we have evaluated the effects of a TransFix-based stabilisation technique on leukocyte scatter characteristics, immunophenotyping, membrane permeability, absolute cell counting and morphology to extend previously reported flow cytometric data focused on the lymphocyte population. We show that scatter characteristics, immunophenotyping and absolute cell counting are well preserved, particularly in the lymphocyte population. Nevertheless, a general increase in membrane permeability, evaluated by propidium iodide (PI) uptake, was observed in TransFix-treated leukocyte subsets. Ultrastructural observations show selective morphological preservation (up to 10 days of storage) of lymphocytes and, to a lesser extent, of monocytes. In contrast, granulocytes have necrosis-like features, although the plasma membrane seems well preserved. Therefore, electron microscopy observations reflect modifications induced in different cell populations as evidenced by flow cytometry (FC). The data indicate that this short-term stabilisation method is particularly suitable for the analysis of human lymphocytes and it is a good procedure for quality control programmes for inter- and intra-laboratory performance evaluation; good results are obtained with respect to antigen definition and absolute cell counting procedures. Any apoptotic pathways in leukocyte subsets are blocked for at least 10 days.

Hyperthermia triggers apoptosis and affects cell adhesiveness in human neuroblastoma cells.

Hyperthermia is a known apoptotic inducer and has been recently utilized in combination with chemo-and/or radiotherapy in cancer treatment. In this study we have described its effect on SK-N-MC human neuroblastoma tumor cells, a line which grows as a double adherent and floating population. Considering this particular culture behavior, we also investigated the relationship between hyperthermia and cell adhesiveness by evaluating integrin expression, namely CD11a, which is, as known, closely correlated to cell adhesion properties. By a multiple, ultrastructural and flow cytometrical approach, we have demonstrated that hyperthermia, while triggering apoptosis, also determines a CD11a surface expression decrease in apoptotic and living cells. We thus suggest a further role for this treatment, which, affecting adhesion mechanisms, could down-regulate metastatic diffusion.

5-(2-Ethyl-phenyl)-3-(3-methoxy-phenyl)-1H-[1,2,4]triazole (DL-111-IT) and related compounds induce apoptotic patterns in cultures of human tumor cell lines.

5-(2-Ethyl-phenyl)-3-(3-methoxy-phenyl)-1H-[1,2,4]triazole (DL-111-IT) and related compounds were extensively studied as anti-gestational agents and some of these molecules were also described as inhibitors of ornithine decarboxylase. Polyamine depletion has been frequently related to the induction of apoptosis and consequently we investigated DL-111-IT and analogs for this effect in myeloid (HL60), neuroblastic (SK-N-MC) and epithelial (BeWo) human tumor cell lines, by means of electron microscopy and DNA electrophoresis. HL60 and SK-N-MC appeared notably sensitive to apoptosis, whereas BeWo responsiveness was variable and frequently associated with necrosis. Our results indicate that the contragestational effect of DL-111-IT and analogs is associated with apoptotic deletion of chorionic tissue and that these molecules, due to their effect on human tumor cell lines, can be considered as antiblastic lead compounds.