Biomimetic poypeptides: a new strategy for muscle tissue regeneration

Despite recent progress in regenerative medicine, functional muscle tissue restoration still represent a challenge, being unable to self- restore significant tissue loss, as consequence of trauma, congenital defects, tumor ablation or denervation. The creation of new muscle through tissue engineering represents an alternative for the replacement of tissue after severe damage. Among the many materials available, those of natural origin are preferable for their biocompatibility and their capacity to resemble the native physiologic environment of cells. A new opportunity is represented by the adoption of a biomimetic approach that allows to realize compounds that are in –between natural and synthetic origin with limited variability and controlled features. Human elastin-like polypeptides that have been designed and produced in our laboratory represent an innovative and promising tool to be exploited in muscle tissue engineering

The microtransplantation technique: a simple ad useful approach to study receptors transplanted into xenopus oocytes

Neuroreceptors are involved in many neurological diseases and represent the preferential target for the pharmacological treatments. Thus functional studies of their activity, by the use of electrophysiological techniques, are a fundament approach to understand not only the pathological mechanism of many neurological diseases but also the mechanism of action of potential drugs. Unfortunately, this cannot be applied for studying the receptor activity in all the human tissues. The option is the use of animal models, however they often resemble only some of the neurological diseases in human. In addition, adult or old animals are not always suitable for electrophysiological studies of age-related diseases. Here, we propose the microtransplantation technique as a novel and useful method to study receptors in humans and, more in general, in adult animals

Improving the regeneration of skeletal muscle

Several pathological conditions of the skeletal muscle (myopathies, dystrophies and age-related atrophy) represent a burden for health care system. Satellite cells are postnatal resident myogenic precursors present in the skeletal muscles throughout the entire lifespan. The identification of novel therapeutic strategies able to enhance their regenerative capacity is one of the most promising tools to compensate muscle degeneration and to restore functional muscle performance in patients and elderly. Our research activity is aiming to contribute to this crucial topic

Epidermal Growth Factor \u2013 based adhesion substrates elicit myoblast scattering, proliferation, differentiation and promote satellite cell myogenic activation

The biochemical properties of muscle extracellular matrix are essential for stem cell adhesion, motility, proliferation and myogenic development. Recombinant elastin-like polypeptides are synthetic polypeptides that, besides maintaining some properties of the native protein, can be tailored by fusing bioactive sequences to their C-terminal. Our laboratory synthesized several Human Elastin-Like Polypeptides (HELP) derived from the sequence of human tropoelastin. Here, we developed a novel HELP family member by fusing the elastin-like backbone to the sequence of human Epidermal Growth Factor. We employed this synthetic protein, named HEGF, either alone or in combination with other proteins of the HELP family carrying RGD-integrin binding sites, as adhesion substrate for C2C12 myoblasts and satellite cells primary cultures. Adhesion of myoblasts to HEGF-based substrates induced scattering, decreased adhesion and cytoskeleton assembly; the concomitant presence of the RGD motifs potentiated all these effects. Recombinant substrates induced myoblasts proliferation, differentiation and the development of multinucleated myotubes, thus favoring myoblasts expansion and preserving their myogenic potential. The effects induced by adhesion substrates were inhibited by AG82 (Tyrphostin 25) and herbimycin A, indicating their dependence on the activation of both the EGF receptor and the tyrosine kinase c-src. Finally, HEGF increased the number of muscle stem cells (satellite cells) derived from isolated muscle fibers in culture, thus highlighting its potential as a novel substrate for skeletal muscle regeneration strategies

A preliminary study on the role of Piezo1 channels in myokine release from cultured mouse myotubes

It has long been known that regular physical exercise induces short and long term benefits reducing the risk of cardiovascular disease, diabetes, osteoporosis, cancer and improves sleep quality, cognitive level, mobility, autonomy in enderly. More recent is the evidence on the endocrine role of the contracting skeletal muscle. Exercise triggers the release of miokines, which act in autocrine, paracrine and endocrine ways controlling the activity of muscles but also of other tissues and organs such as adipose tissue, liver, pancreas, bones, and brain. The mechanism of release is still unclear. Neuromuscular electrical stimulation reproduces the beneficial effects of physical activity producing physiological metabolic, cardiovascular, aerobic responses consistent with those induced by exercise. In vitro, Electrical Pulse Stimulations (EPS) of muscle cells elicit cell contraction and mimic miokine release in the external medium. Here we show that, in cultured mouse myotubes, EPS induce contractile activity and the release of the myokine IL-6. Gadolinium highly reduces EPS-induced IL-6 release, suggesting the involvement of mechanical activated ion channels. The chemical activation of mechanosensitive Piezo1 channels with the specific agonist Yoda1 stimulates IL-6 release similarly to EPS, suggesting the involvement of Piezo1 channels in the control of the myokine release. The expression of Piezo1 protein in myotubes was confirmed by the Western blot analysis. To the best of our knowledge, this is the first evidence of a Piezo1-mediated effect in myokine release and suggests a potential translational use of specific Piezo1 agonists for innovative therapeutic treatments reproducing/enhancing the benefits of exercise mediated by myokines

Asbestos Fibers Enhance the TMEM16A Channel Activity in Xenopus Oocytes

Background: The interaction of asbestos fibers with target cell membranes is still poorly investigated. Here, we detected and characterized an enhancement of chloride conductance in Xenopus oocyte cell membranes induced by exposure to crocidolite (Croc) asbestos fibers. Methods: A two-microelectrode voltage clamp technique was used to test the effect of Croc fiber suspensions on outward chloride currents evoked by step membrane depolarization. Calcium imaging experiments were also performed to investigate the variation of ‘resting’ oocyte [Ca2+]i following asbestos exposure. Results: The increase in chloride current after asbestos treatment, was sensitive to [Ca2+]e, and to specific blockers of TMEM16A Ca2+-activated chloride channels, MONNA and Ani9. Furthermore, asbestos treatment elevated the ‘resting’ [Ca2+]i likelihood by increasing the cell membrane permeability to Ca2 in favor of a tonic activation of TMEME16A channels. Western blot analysis confirmed that TMEME16A protein was endogenously present in the oocyte cell membrane and absorbed by Croc. Conclusion: the TMEM16A channels endogenously expressed by Xenopus oocytes are targets for asbestos fibers and represent a powerful tool for asbestos–membrane interaction studies. Interestingly, TMEM16A channels are highly expressed in many types of tumors, including some asbestos-related cancers, suggesting them, for the first time, as a possible early target of crocidolite-mediated tumorigenic effects on target cell membranes

Intrinsic ionic conductances mediate the spontaneous electrical activity of cultured mouse myotubes

AbstractMouse skeletal myotubes differentiated in vitro exhibited spontaneous contractions associated with electrical activity. The ionic conductances responsible for the origin and modulation of the spontaneous activity were examined using the whole-cell patch-clamp technique and measuring [Ca2+]i transients with the Ca2+ indicator, fura 2-AM. Regular spontaneous activity was characterized by single TTX-sensitive action potentials, followed by transient increases in [Ca2+]i. Since the bath-application of Cd2+ (300 μM) or Ni2+ (50 μM) abolished the cell firing, T-type (ICa,T) and L-type (ICa,L) Ca2+ currents were investigated in spontaneously contracting myotubes. The low activation threshold (around −60 mV) and the high density of ICa,T observed in contracting myotubes suggested that ICa,T initiated action potential firing, by bringing cells to the firing threshold. The results also suggested that the activity of ICa,L could sustain the [Ca2+]i transients associated with the action potential, leading to the activation of apamin-sensitive SK-type Ca2+-activated K+ channels and the afterhyperpolarization (AHP) following single spikes. In conclusion, an interplay between voltage-dependent inward (Na+ and Ca2+) and outward (SK) conductances is proposed to mediate the spontaneous pacemaker activity in cultured muscle myotubes during the process of myogenesis

The State of the Art of Piezo1 Channels in Skeletal Muscle Regeneration

Piezo1 channels are highly mechanically-activated cation channels that can sense and transduce the mechanical stimuli into physiological signals in different tissues including skeletal muscle. In this focused review, we summarize the emerging evidence of Piezo1 channel-mediated effects in the physiology of skeletal muscle, with a particular focus on the role of Piezo1 in controlling myogenic precursor activity and skeletal muscle regeneration and vascularization. The disclosed effects reported by pharmacological activation of Piezo1 channels with the selective agonist Yoda1 indicate a potential impact of Piezo1 channel activity in skeletal muscle regeneration, which is disrupted in various muscular pathological states. All findings reported so far agree with the idea that Piezo1 channels represent a novel, powerful molecular target to develop new therapeutic strategies for preventing or ameliorating skeletal muscle disorders characterized by an impairment of tissue regenerative potential

Sanitary problems related to the presence of Ostreopsis spp. in the Mediterranean Sea: a multidisciplinary scientific approach

The increased presence of potentially toxic microalgae in the Mediterranean area is a matter of great concern. Since the end of the last century, microalgae of the genus Ostreopsis have been detected more and more frequently in the Italian coastal waters. The presence of Ostreopsis spp. has been accompanied by the presence of previously undetected marine biotoxins (palytoxins) into the ecosystem with the increased possibility of human exposure. In response to the urgent need for toxicity characterization of palytoxin and its congeners, an integrated study encompassing both in vitro and in vivo methods was performed

Effect of Synthetic Vitreous Fiber Exposure on TMEM16A Channels in a Xenopus laevis Oocyte Model

Many years ago, asbestos fibers were banned and replaced by synthetic vitreous fibers because of their carcinogenicity. However, the toxicity of the latter fibers is still under debate, especially when it concerns the early fiber interactions with biological cell membranes. Here, we aimed to investigate the effects of a synthetic vitreous fiber named FAV173 on the Xenopus laevis oocyte membrane, the cell model we have already used to characterize the effect of crocidolite asbestos fiber exposure. Using an electrophysiological approach, we found that, similarly to crocidolite asbestos, FAV173 was able to stimulate a chloride outward current evoked by step membrane depolarizations, that was blocked by the potent and specific TMEM16A channel antagonist Ani9. Exposure to FAV173 fibers also altered the oocyte cell membrane microvilli morphology similarly to crocidolite fibers, most likely as a consequence of the TMEM16A protein interaction with actin. However, FAV173 only partially mimicked the crocidolite fibers effects, even at higher fiber suspension concentrations. As expected, the crocidolite fibers’ effect was more similar to that induced by the co-treatment with (Fe3+ + H2O2), since the iron content of asbestos fibers is known to trigger reactive oxygen species (ROS) production. Taken together, our findings suggest that FAV173 may be less harmful that crocidolite but not ineffective in altering cell membrane properties