A “noisy” electrical stimulation protocol favors muscle regeneration in vitro through release of endogenous ATP. [Reserch paper]
Alessandra Bosutti1, Annalisa Bernareggi1, Gabriele Massaria1,2, Paola D'Andrea1, Giuliano Taccola3,4, Paola Lorenzon1, Marina Sciancalepore1
1. Department of Life Sciences and Centre for Neuroscience B.R.A.I.N., University of Trieste, Trieste, Italy
2. Area Science Park, Padriciano, Trieste, Italy
3. Department of Neuroscience, SISSA, Trieste, Italy
4. SPINAL (Spinal Person Injury Neurorehabilitation Applied Laboratory), Istituto di Medicina Fisica e Riabilitazione (IMFR), Udine, Italy
Abstract. An in vitro system of electrical stimulation was used to explore whether an innovative “noisy” stimulation protocol derived from human electromyographic recordings (EMGstim) could promote muscle regeneration. EMGstim was delivered to cultured mouse myofibers isolated from Flexor Digitorum Brevis, preserving their satellite cells. In response to EMGstim, immunostaining for the myogenic regulatory factor myogenin, revealed an increased percentage of elongated myogenin-positive cells surrounding the myofibers. Conditioned medium collected from EMGstim-treated cell cultures, promoted satellite cells differentiation in unstimulated myofiber cell cultures, suggesting that extracellular soluble factors could mediate the process. Interestingly, the myogenic effect of EMGstim was mimicked by exogenously applied ATP (0.1 μM), reduced by the ATP diphosphohydrolase apyrase and prevented by blocking endogenous ATP release with carbenoxolone.
In conclusion, our results show that “noisy” electrical stimulations favor muscle progenitor cell differentiation most likely via the release of endogenous ATP from contracting myofibres. Our data also suggest that “noisy” stimulation protocols could be potentially more efficient than regular stimulations to promote in vivo muscle regeneration after traumatic injury or in neuropathological diseases.
Keywords: Differentiation, Electrical stimulation, Skeletal muscle, Myofibers, ATP, Satellite cells
Figure: EMGstim effect on myogenin-positive cells.
a) Representative immunofluorescence-labeled myogenin-positive cells (green), DAPI counterstained nuclei (blue) and corresponding merged micrographs, at 72 h of seeding, in control conditions and in EMGstim treated cultures. Scale bars, 100 μm.
b) Percentage of myogenin-positive cells in control and EMGstim treated cultures.
c) Representative immunofluorescence-labeled myogenin-positive cells (green) merged with the same images captured in bright field microscopy in control and EMGstim treated cultures. Scale bars, 20 μm.
d) The aspect ratio of myogenin positive-cells in control and in EMGstim treated cultures.
***p < 0.001 vs controls.
Acknowledgments: We are grateful to Dr. Andrew Constanti (UCL, School of Pharmacy, London, UK) for critically reading the manuscript and to Gianluigi Nocera for doing preliminary experiments. This work was supported by Fondazione Beneficentia Stiftung (Liechtenstein), by MEMORI-net project, Interreg V-A Italia-Slovenija 2014–2020 Programme. G.M. is working within MADE in TRIESTE, an Area Science Park initiative financed by Commissariato del Governo nella Regione Friuli Venezia Giulia – “Fondo Trieste”.
Published in: Experimental Cell Research
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Cite this paper as: Bosutti A., Bernareggi A., Massaria G., D'Andrea P., Taccola G., Lorenzon P., Sciancalepore M. A “noisy” electrical stimulation protocol favors muscle regeneration in vitro through release of endogenous ATP. Experimental Cell Research, Elsevier, 2019; DOI: 10.1016/j.yexcr.2019.05.012.
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Moreover, on 27th May 2019 in Lubiana, at the Symposium & Workshop "Skeletal Muscle Research - from cell to human 2019", Alessandra Bosutti will present the abstract titled: "NOISY ELECTRICAL STIMULATION TO COUNTERACT SKELETAL MUSCLE ATROPHY" (authors: A. Bosutti, A. Bernareggi, G. Massaria, P. D’Andrea, G. Taccola, P. Lorenzon and M. Sciancalepore)
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