Mice lacking growth-associated protein 43 develop cardiac remodeling and hypertrophy.

Growth-associated protein 43 (GAP43) is found in skeletal muscle, localized near the calcium release units. In interaction with calmodulin (CaM), it indirectly modulates the activity of dihydropyridine and ryanodine Ca2+ channels. GAP43-CaM interaction plays a key role in intracellular Ca2+ homeostasis and, consequently, in skeletal muscle activity. The control of intracellular Ca2+ signaling is also an important functional requisite in cardiac physiology. The aim of this study is to define the impact of GAP43 on cardiac tissue at macroscopic and cellular levels, using GAP43 knockout (GAP43-/-) newborn C57/BL6 mice. Hearts from newborn GAP43-/- mice were heavier than hearts from wild-type (WT) ones. In these GAP43-/- hearts, histological section analyses revealed a thicker ventricular wall and interventricular septum with a reduced ventricular chamber area. In addition, increased collagen deposits between fibers and increased expression levels of myosin were observed in hearts from GAP43-/- mice. Cardiac tropism and rhythm are controlled by multiple intrinsic and extrinsic factors, including cellular events such those linked to intracellular Ca2+ dynamics, in which GAP43 plays a role. Our data revealed that, in the absence of GAP43, there were cardiac morphological alterations and signs of hypertrophy, suggesting that GAP43 could play a role in the functional processes of the whole cardiac muscle. This paves the way for further studies investigating GAP43 involvement in signaling dynamics at the cellular level.

Antioxidant Strategy to Prevent Simulated Microgravity-Induced Effects on Bone Osteoblasts.

The effects induced by microgravity on human body functions have been widely described, in particular those on skeletal muscle and bone tissues. This study aims to implement information on the possible countermeasures necessary to neutralize the oxidative imbalance induced by microgravity on osteoblastic cells. Using the model of murine MC3T3-E1 osteoblast cells, cellular morphology, proliferation, and metabolism were investigated during exposure to simulated microgravity on a random positioning machine in the absence or presence of an antioxidant-the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Our results confirm that simulated microgravity-induced morphological and metabolic alterations characterized by increased levels of reactive oxygen species and a slowdown of the proliferative rate. Interestingly, the use of Trolox inhibited the simulated microgravity-induced effects. Indeed, the antioxidant-neutralizing oxidants preserved cell cytoskeletal architecture and restored cell proliferation rate and metabolism. The use of appropriate antioxidant countermeasures could prevent the modifications and damage induced by microgravity on osteoblastic cells and consequently on bone homeostasis.

Physiological Responses of Jurkat Lymphocytes to Simulated Microgravity Conditions.

The presence of microgravity conditions deeply affects the human body functions at the systemic, organ and cellular levels. This study aimed to investigate the effects induced by simulated-microgravity on non-stimulated Jurkat lymphocytes, an immune cell phenotype considered as a biosensor of the body responses, in order to depict at the cellular level the effects of such a peculiar condition. Jurkat cells were grown at 1 g or on random positioning machine simulating microgravity. On these cells we performed: morphological, cell cycle and proliferation analyses using cytofluorimetric and staining protocols-intracellular Ca2+, reactive oxygen species (ROS), mitochondria membrane potential and O2- measurements using fluorescent probes-aconitase and mitochondria activity, glucose and lactate content using colorimetric assays. After the first exposure days, the cells showed a more homogeneous roundish shape, an increased proliferation rate, metabolic and detoxifying activity resulted in decreased intracellular Ca2+ and ROS. In the late exposure time, the cells adapted to the new environmental condition. Our non-activated proliferating Jurkat cells, even if responsive to altered external forces, adapted to the new environmental condition showing a healthy status. In order to define the cellular mechanism(s) triggered by microgravity, developing standardized experimental approaches and controlled cell culture and simulator conditions is strongly recommended.

Calcitonin-induced effects on amniotic fluid-derived mesenchymal stem cells.

BACKGROUND/AIMS: Mesenchymal stem cells from human amniotic fluid (huAFMSCs) can differentiate into multiple lineages and are not tumorigenic after transplantation, making them good candidates for therapeutic purposes. The aim was to determine the effects of calcitonin on these huAFMSCs during osteogenic differentiation, in terms of the physiological role of calcitonin in bone homeostasis. METHODS: For huAFMSCs cultured under different conditions, we assayed: expression of the calcitonin receptor, using immunolabelling techniques; proliferation and osteogenesis, using colorimetric and enzymatic assays; intracellular Ca(2+) and cAMP levels, using videomicroscopy and spectrophotometry. RESULTS: The calcitonin receptor was expressed in proliferating and osteo-differentiated huAFMSCs. Calcitonin triggered intracellular Ca(2+) increases and cAMP production. Its presence in cell medium also induced dose-dependent inhibitory effects on proliferation and increased osteogenic differentiation of huAFMSCs, as also indicated by enhancement of specific markers and alkaline phosphatase activity. CONCLUSIONS: These data show that huAFMSCs represent a potential osteogenic model to study in-vitro cell responses to calcitonin (and other members of the calcitonin family). This leads the way to the opening of new lines of research that will add new insight both in cell therapies and in the pharmacological use of these molecules.

Grape seed extract triggers apoptosis in Caco-2 human colon cancer cells through reactive oxygen species and calcium increase: extracellular signal-regulated kinase involvement.

Grape seed extract (GSE) from Italia, Palieri and Red Globe cultivars inhibits cell growth and induces apoptosis in Caco-2 human colon cancer cells in a dose-dependent manner. In order to investigate the mechanism(s) supporting the apoptotic process, we analysed reactive oxygen species (ROS) production, intracellular Ca2+ handling and extracellular signal-regulated kinase (ERK) activation. Upon exposure to GSE, ROS and intracellular Ca2+ levels increased in Caco-2 cells, concomitantly with ERK inactivation. As ERK activity is thought to be essential for promoting survival pathways, inhibition of this kinase is likely to play a relevant role in GSE-mediated anticancer effects. Indeed, pretreatment with N-acetyl cysteine, a ROS scavenger, reversed GSE-induced apoptosis, and promoted ERK phosphorylation. This effect was strengthened by ethylene glycol tetraacetic acid-mediated inhibition of extracellular Ca2+ influx. ROS and Ca2+ influx inhibition, in turn, increased ERK phosphorylation, and hence almost entirely suppressed GSE-mediated apoptosis. These data suggested that GSE triggers a previously unrecognised ERK-based mechanism, involving both ROS production and intracellular Ca2+ increase, eventually leading to apoptosis in cancer cells.

Simulated Microgravity Exposure Induces Antioxidant Barrier Deregulation and Mitochondria Enlargement in TCam-2 Cell Spheroids.

One of the hallmarks of microgravity-induced effects in several cellular models is represented by the alteration of oxidative balance with the consequent accumulation of reactive oxygen species (ROS). It is well known that male germ cells are sensitive to oxidative stress and to changes in gravitational force, even though published data on germ cell models are scarce. We previously studied the effects of simulated microgravity (s-microgravity) on a 2D cultured TCam-2 seminoma-derived cell line, considered the only human cell line available to study in vitro mitotically active human male germ cells. In this study, we used a corresponding TCam-2 3D cell culture model that mimics cell-cell contacts in organ tissue to test the possible effects induced by s-microgravity exposure. TCam-2 cell spheroids were cultured for 24 h under unitary gravity (Ctr) or s-microgravity conditions, the latter obtained using a random positioning machine (RPM). A significant increase in intracellular ROS and mitochondria superoxide anion levels was observed after RPM exposure. In line with these results, a trend of protein and lipid oxidation increase and increased pCAMKII expression levels were observed after RPM exposure. The ultrastructural analysis via transmission electron microscopy revealed that RPM-exposed mitochondria appeared enlarged and, even if seldom, disrupted. Notably, even the expression of the main enzymes involved in the redox homeostasis appears modulated by RPM exposure in a compensatory way, with GPX1, NCF1, and CYBB being downregulated, whereas NOX4 and HMOX1 are upregulated. Interestingly, HMOX1 is involved in the heme catabolism of mitochondria cytochromes, and therefore the positive modulation of this marker can be associated with the observed mitochondria alteration. Altogether, these data demonstrate TCam-2 spheroid sensitivity to acute s-microgravity exposure and indicate the capability of these cells to trigger compensatory mechanisms that allow them to overcome the exposure to altered gravitational force.

The Oxidative Balance Orchestrates the Main Keystones of the Functional Activity of Cardiomyocytes.

This review is aimed at providing an overview of the key hallmarks of cardiomyocytes in physiological and pathological conditions. The main feature of cardiac tissue is the force generation through contraction. This process requires a conspicuous energy demand and therefore an active metabolism. The cardiac tissue is rich of mitochondria, the powerhouses in cells. These organelles, producing ATP, are also the main sources of ROS whose altered handling can cause their accumulation and therefore triggers detrimental effects on mitochondria themselves and other cell components thus leading to apoptosis and cardiac diseases. This review highlights the metabolic aspects of cardiomyocytes and wanders through the main systems of these cells: (a) the unique structural organization (such as different protein complexes represented by contractile, regulatory, and structural proteins); (b) the homeostasis of intracellular Ca2+ that represents a crucial ion for cardiac functions and E-C coupling; and (c) the balance of Zn2+, an ion with a crucial impact on the cardiovascular system. Although each system seems to be independent and finely controlled, the contractile proteins, intracellular Ca2+ homeostasis, and intracellular Zn2+ signals are strongly linked to each other by the intracellular ROS management in a fascinating way to form a "functional tetrad" which ensures the proper functioning of the myocardium. Nevertheless, if ROS balance is not properly handled, one or more of these components could be altered resulting in deleterious effects leading to an unbalance of this "tetrad" and promoting cardiovascular diseases. In conclusion, this "functional tetrad" is proposed as a complex network that communicates continuously in the cardiomyocytes and can drive the switch from physiological to pathological conditions in the heart.

A Protective Strategy to Counteract the Oxidative Stress Induced by Simulated Microgravity on H9C2 Cardiomyocytes.

Microgravity affects human cardiovascular function inducing heart rhythm disturbances and even cardiac atrophy. The mechanisms triggered by microgravity and the search for protection strategies are difficult to be investigated in vivo. This study is aimed at investigating the effects induced by simulated microgravity on a cardiomyocyte-like phenotype. The Random Positioning Machine (RPM), set in a CO2 incubator, was used to simulate microgravity, and H9C2 cell line was used as the cardiomyocyte-like model. H9C2 cells were exposed to simulated microgravity up to 96 h, showing a slower cell proliferation rate and lower metabolic activity in comparison to cell grown at earth gravity. In exposed cells, these effects were accompanied by increased levels of intracellular reactive oxygen species (ROS), cytosolic Ca2+, and mitochondrial superoxide anion. Protein carbonyls, markers of protein oxidation, were significantly increased after the first 48 h of exposition in the RPM. In these conditions, the presence of an antioxidant, the N-acetylcysteine (NAC), counteracted the effects induced by the simulated microgravity. In conclusion, these data suggest that simulated microgravity triggers a concomitant increase of intracellular ROS and Ca2+ levels and affects cell metabolic activity which in turn could be responsible for the slower proliferative rate. Nevertheless, the very low number of detectable dead cells and, more interestingly, the protective effect of NA, demonstrate that simulated microgravity does not have "an irreversible toxic effect" but, affecting the oxidative balance, results in a transient slowdown of proliferation.

Effects of acute and chronic low frequency electromagnetic field exposure on PC12 cells during neuronal differentiation.

BACKGROUND/AIMS: The purpose of this study was to provide information about the in vitro neuritogenesis during cell exposure to extremely low frequency electromagnetic fields (ELF-EMFs) of different intensities and durations using pheochromocytoma-derived cell line (PC12 cells) as neuronal model. METHODS: Proliferative rates and neuritogenesis were tested by colorimetric assay and morphological analysis, respectively; reactive oxygen species (ROS) levels and intracellular Ca(2+) variations monitored using single cell videomicroscopy. RESULTS: The long-lasting ELF-EMF exposure (0.1-1.0 mT) did not appear to significantly affect the biological response (proliferation and neuritogenesis). However, during the acute ELF-EMF exposure (30 min), in undifferentiated PC12 cells, there were increased ROS levels and decreased catalase activity, that, conversely, resulted increased after chronic exposure (7 days) at 1.0 mT. Acute exposure (0.1-1.0 mT) affected the spontaneous intracellular Ca(2+) variations in undifferentiated cells, in which basal intracellular Ca(2+) resulted increased after chronic exposure. In addition acute exposure affected cell response to a depolarizing agent, while basal membrane potential was not changed. CONCLUSION: Even if further studies remain necessary to identify the ROS/intracellular Ca(2+)cross-talking pathway activated by ELF-EMF exposure, we support the hypothesis that ROS and Ca(2+) could be the cellular "primum movens" of the ELF-EMF induced effects on biological systems.

Involvement of P2X7 Receptors in the Osteogenic Differentiation of Mesenchymal Stromal/Stem Cells Derived from Human Subcutaneous Adipose Tissue.

The ionotropic P2X7 receptor (P2X7R) is involved in bone homeostasis but its role in osteogenesis is controversial. Thus, we investigated the expression of P2X7R and the effects exerted by its modulation in mesenchymal stromal cells from human subcutaneous adipose tissue (S-ASCs), which have potential therapeutic application in bone regenerative medicine. We found that undifferentiated S-ASCs expressed P2X7R and its functional splice variants P2X7AR and P2X7BR. Cell stimulation by P2X7R agonist BzATP (100 µM) neither modified proliferation nor caused membrane pore opening while increasing intracellular Ca2+ levels and migration. The P2X7R antagonist A438079 reversed these effects. However, 25-100 µM BzATP, administered to S-ASCs undergoing osteogenic differentiation, dose-dependently decreased extracellular matrix mineralization and expression of osteogenic transcription factors Runx2, alkaline phosphatase and osteopontin. These effects were not coupled to cell proliferation reduction or to cell death increase, but were associated to decrease in P2X7AR and P2X7BR expression. In contrast, expression of P2X7R, especially P2X7BR isoform, significantly increased during the osteogenic process. Noteworthy, the antagonist A438079, administered alone, at first restrained cell differentiation, enhancing it later. Accordingly, A438079 reversed BzATP effects only in the second phase of S-ASCs osteogenic differentiation. Apyrase, a diphosphohydrolase converting ATP/ADP into AMP, showed a similar behavior. Altogether, findings related to A438079 or apyrase effects suggest an earlier and prevailing pro-osteogenic activity by endogenous ATP and a later one by adenosine derived from endogenous ATP metabolism. Conversely, P2X7R pharmacological stimulation by BzATP, mimicking the effects of high ATP levels occurring during tissue injuries, depressed receptor expression/activity impairing MSC osteogenic differentiation.

IgIII (270-280)-fragment-like H2N-DDSDEEN-COOH peptide modulates N-CAM expression via Ca2+-dependent ERK signaling during "in vitro neurogenesis".

The two major isoforms (180 kDa and 140 kDa) of the neural cell adhesion molecule (N-CAM) are crucially involved in neurogenesis and brain repair via activation of the mitogen-activated protein kinase (MAPK) cascade. Modification by glycosylation, and homophilic and heterophilic interactions regulate the function of N-CAM, but little is known about the interplay of these processes. In the neuron-like PC12 cell line, extracellular small acidic peptides have been shown to modulate the expression of N-CAM mRNA and protein and regulate its translocation to the plasma membrane. Among these peptides, a synthetic Ig-III-like short sequence (H2N-DDSDEEN-COOH), designated sSP, was particularly potent. In this study, we analyzed the cross-talk between nerve growth factor (NGF) and extracellular sSP in native and N-CAM-transfected PC12 cells to determine if these systems interact to modulate transduction pathways and regulate early steps of neurogenesis in vitro. Our results indicate that sSP accelerated the phosphorylation of extracellular regulated kinase-1 (ERK1) and -2 (ERK2) and promoted plasma membrane translocation of 180 kDa N-CAM. By stabilizing cell-cell contacts and promoting cell cluster formation, these events, which were mediated via a significant increase in intracellular Ca2+, regulated some of the early stages of the NGF-induced differentiation process.

Extremely Low-Frequency Electromagnetic Fields Affect Myogenic Processes in C2C12 Myoblasts: Role of Gap-Junction-Mediated Intercellular Communication.

Extremely low-frequency electromagnetic fields (ELF-EMFs) can interact with biological systems. Although they are successfully used as therapeutic agents in physiatrics and rehabilitative practice, they might represent environmental pollutants and pose a risk to human health. Due to the lack of evidence of their mechanism of action, the effects of ELF-EMFs on differentiation processes in skeletal muscle were investigated. C2C12 myoblasts were exposed to ELF-EMFs generated by a solenoid. The effects of ELF-EMFs on cell viability and on growth and differentiation rates were studied using colorimetric and vital dye assays, cytomorphology, and molecular analysis of MyoD and myogenin expression, respectively. The establishment of functional gap junctions was investigated analyzing connexin 43 expression levels and measuring cell permeability, using microinjection/dye-transfer assays. The ELF-EMFs did not affect C2C12 myoblast viability or proliferation rate. Conversely, at ELF-EMF intensity in the mT range, the myogenic process was accelerated, through increased expression of MyoD, myogenin, and connexin 43. The increase in gap-junction function suggests promoting cell fusion and myotube differentiation. These data provide the first evidence of the mechanism through which ELF-EMFs may provide therapeutic benefits and can resolve, at least in part, some conditions of muscle dysfunction.

Evidence for Altered Ca2+ Handling in Growth Associated Protein 43-Knockout Skeletal Muscle.

Neuronal growth-associated protein 43 (GAP43) has crucial roles in the nervous system, and during development, regeneration after injury, and learning and memory. GAP43 is expressed in mouse skeletal muscle fibers and satellite cells, with suggested its involvement in intracellular Ca2+ handling. However, the physiological role of GAP43 in muscle remains unknown. Using a GAP43-knockout (GAP43-/-) mouse, we have defined the role of GAP43 in skeletal muscle. GAP43-/- mice showed low survival beyond weaning, reduced adult body weight, decreased muscle strength, and changed myofiber ultrastructure, with no significant differences in the expression of markers of satellite cell and myotube progression through the myogenic program. Thus, GAP43 expression is involved in timing of muscle maturation in-vivo. Intracellular Ca2+ measurements in-vitro in myotubes revealed GAP43 involvement in Ca2+ handling. In the absence of GAP43 expression, the spontaneous Ca2+ variations had greater amplitudes and higher frequency. In GAP43-/- myotubes, also the intracellular Ca2+ variations induced by the activation of dihydropyridine and ryanodine Ca2+ channels, resulted modified. These evidences suggested dysregulation of Ca2+ homeostasis. The emerging hypothesis indicates that GAP43 interacts with calmodulin to indirectly modulate the activities of dihydropyridine and ryanodine Ca2+ channels. This thus influences intracellular Ca2+ dynamics and its related intracellular patterns, from functional excitation-contraction coupling, to cell metabolism, and gene expression.

Nuclear translocation of PKCα isoenzyme is involved in neurogenic commitment of human neural crest-derived periodontal ligament stem cells.

Stem cells isolated from human adult tissue niche represent a promising source for neural differentiation. Human Periodontal Ligament Stem Cells (hPDLSCs) originating from the neural crest are particularly suitable for induction of neural commitment. In this study, under xeno-free culture conditions, in undifferentiated hPDLSCs and in hPDLSCs induced to neuronal differentiation by basic Fibroblast Growth Factor, the level of some neural markers have been analyzed. The hPDLSCs spontaneously express Nestin, a neural progenitor marker. In these cells, the neurogenic process induced to rearrange the cytoskeleton, form neurospheres and express higher levels of Nestin and Tyrosine Hydroxylase, indicating neural induction. Protein Kinase C (PKC) is highly expressed in neural tissue and has a key role in neuronal functions. In particular the Ca(2+) and diacylglycerol-dependent activation of PKCα isozyme is involved in the regulation of neuronal differentiation. Another main component of the pathways controlling neuronal differentiation is the Growth Associated Protein-43 (GAP-43), whose activity is strictly regulated by PKC. The aim of this study is to investigate the role of PKCα/GAP-43 nuclear signal transduction pathway during neuronal commitment of hPDLSCs. During hPDLSCs neurogenic commitment the levels of p-PKC and p-GAP-43 increased both in cytoplasmic and nuclear compartment. PKCα nuclear translocation induced GAP-43 movement to the cytoplasm, where it is known to regulate growth cone dynamics and neuronal differentiation. Moreover, the degree of cytosolic Ca(2+) mobilization appeared to be more pronounced in differentiated hPDLSCs than in undifferentiated cells. This study provides evidences of a new PKCα/GAP-43 nuclear signalling pathway that controls neuronal differentiation in hPDLSCs, leading the way to a potential use of these cells in cell-based therapy in neurodegenerative diseases.

RCCS bioreactor-based modelled microgravity induces significant changes on in vitro 3D neuroglial cell cultures.

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions.

Molecular and phenotypic characterization of human amniotic fluid-derived cells: a morphological and proteomic approach.

Mesenchymal Stem Cells derived from Amniotic Fluid (AFMSCs) are multipotent cells of great interest for regenerative medicine. Two predominant cell types, that is, Epithelial-like (E-like) and Fibroblast-like (F-like), have been previously detected in the amniotic fluid (AF). In this study, we examined the AF from 12 donors and observed the prevalence of the E-like phenotype in 5, whereas the F-like morphology was predominant in 7 samples. These phenotypes showed slight differences in membrane markers, with higher CD90 and lower Sox2 and SSEA-4 expression in F-like than in E-like cells; whereas CD326 was expressed only in the E-like phenotype. They did not show any significant differences in osteogenic, adipogenic or chondrogenic differentiation. Proteomic analysis revealed that samples with a predominant E-like phenotype (HC1) showed a different profile than those with a predominant F-like phenotype (HC2). Twenty-five and eighteen protein spots were differentially expressed in HC1 and HC2 classes, respectively. Of these, 17 from HC1 and 4 from HC2 were identified by mass spectrometry. Protein-interaction networks for both phenotypes showed strong interactions between specific AFMSC proteins and molecular chaperones, such as preproteasomes and mature proteasomes, both of which are important for cell cycle regulation and apoptosis. Collectively, our results provide evidence that, regardless of differences in protein profiling, the prevalence of E-like or F-like cells in AF does not affect the differentiation capacity of AFMSC preparations. This may be valuable information with a view to the therapeutic use of AFMSCs.

Modification of the functional capacity of sarcoplasmic reticulum membranes in patients suffering from chronic fatigue syndrome.

In chronic fatigue syndrome, several reported alterations may be related to specific oxidative modifications in muscle. Since sarcoplasmic reticulum membranes are the basic structures involved in excitation-contraction coupling and the thiol groups of Ca(2+) channels of SR terminal cisternae are specific targets for reactive oxygen species, it is possible that excitation-contraction coupling is involved in this pathology. We investigated the possibility that abnormalities in this compartment are involved in the pathogenesis of chronic fatigue syndrome and consequently responsible for characteristic fatigue. The data presented here support this hypothesis and indicate that the sarcolemmal conduction system and some aspects of Ca(2+) transport are negatively influenced in chronic fatigue syndrome. In fact, both deregulation of pump activities (Na(+)/K(+) and Ca(2+)-ATPase) and alteration in the opening status of ryanodine channels may result from increased membrane fluidity involving sarcoplasmic reticulum membranes.

N-CAM expression and localization in PC12 cells modulated by extracellular peptides.

The neural cell adhesion molecules (N-CAMs) play an important role in mediating cell-cell interactions in the nervous system. Different isoforms of these membrane proteins are involved in the formation of the neuronal network and in the dynamic phases of neuronal plasticity. We studied the early stages of the pseudo neuronal differentiation of PC12 cells induced by a class of small acidic peptides capable of modulating gene expression in these cells. The data presented here indicate that peptides with specific sequences induce an increase in N-CAM mRNA expression and protein translocation to the plasma membrane to a comparable degree as NGF.

New insights into the relationship between mIGF-1-induced hypertrophy and Ca2+ handling in differentiated satellite cells.

Muscle regeneration involves the activation of satellite cells, is regulated at the genetic and epigenetic levels, and is strongly influenced by gene activation and environmental conditions. The aim of this study was to determine whether the overexpression of mIGF-1 can modify functional features of satellite cells during the differentiation process, particularly in relation to modifications of intracellular Ca2+ handling. Satellite cells were isolated from wild-type and MLC/mIGF-1 transgenic mice. The cells were differentiated in vitro, and morphological analyses, intracellular Ca2+ measurements, and ionic current recordings were performed. mIGF-1 overexpression accelerates satellite cell differentiation and promotes myotube hypertrophy. In addition, mIGF-1 overexpression-induced potentiation of myogenesis triggers both quantitative and qualitative changes to the control of intracellular Ca2+ handling. In particular, the differentiated MLC/mIGF-1 transgenic myotubes have reduced velocity and amplitude of intracellular Ca2+ increases after stimulation with caffeine, KCl and acetylcholine. This appears to be due, at least in part, to changes in the physico-chemical state of the sarcolemma (increased membrane lipid oxidation, increased output currents) and to increased expression of dihydropyridine voltage-operated Ca2+ channels. Interestingly, extracellular ATP and GTP evoke intracellular Ca2+ mobilization to greater extents in the MLC/mIGF-1 transgenic satellite cells, compared to the wild-type cells. These data suggest that these MLC/mIGF-1 transgenic satellite cells are more sensitive to trophic stimuli, which can potentiate the effects of mIGF-1 on the myogenic programme.

Growth associated protein 43 is expressed in skeletal muscle fibers and is localized in proximity of mitochondria and calcium release units.

The neuronal Growth Associated Protein 43 (GAP43), also known as B-50 or neuromodulin, is involved in mechanisms controlling pathfinding and branching of neurons during development and regeneration. For many years this protein was classified as neuron-specific, but recent evidences suggest that a) GAP43 is expressed in the nervous system not only in neurons, but also in glial cells, and b) probably it is present also in other tissues. In particular, its expression was revealed in muscles from patients affected by various myopathies, indicating that GAP43 can no-longer considered only as a neuron-specific molecule. We have investigated the expression and subcellular localization of GAP43 in mouse satellite cells, myotubes, and adult muscle (extensor digitorum longus or EDL) using Western blotting, immuno-fluorescence combined to confocal microscopy and electron microscopy. Our in vitro results indicated that GAP43 is indeed expressed in both myoblasts and differentiating myotubes, and its cellular localization changes dramatically during maturation: in myoblasts the localization appeared to be mostly nuclear, whereas with differentiation the protein started to display a sarcomeric-like pattern. In adult fibers, GAP43 expression was evident with the protein labeling forming (in longitudinal views) a double cross striation reminiscent of the staining pattern of other organelles, such as calcium release units (CRUs) and mitochondria. Double immuno-staining and experiments done in EDL muscles fixed at different sarcomere lengths, allowed us to determine the localization, from the sarcomere Z-line, of GAP43 positive foci, falling between that of CRUs and of mitochondria. Staining of cross sections added a detail to the puzzle: GAP43 labeling formed a reticular pattern surrounding individual myofibrils, but excluding contractile elements. This work leads the way to further investigation about the possible physiological and structural role of GAP43 protein in adult fiber function and disease.