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Phospho-tyrosine phosphatase inhibitor Bpv(Hopic) enhances C2C12 myoblast migration in vitro. Requirement of PI3K/AKT and MAPK/ERK pathways

  • Georgi A. Dimchev
  • Nasser Al-Shanti
  • Claire E. Stewart
Original Paper

Abstract

Muscle progenitor cell migration is an important step in skeletal muscle myogenesis and regeneration. Migration is required for muscle precursors to reach the site of damage and for the alignment of myoblasts prior to their fusion, which ultimately contributes to muscle regeneration. Limited spreading and migration of donor myoblasts are reported problems of myoblast transfer therapy, a proposed therapeutic strategy for Duchenne Muscular Dystrophy, warranting further investigation into different approaches for improving the motility and homing of these cells. In this article, the effect of protein phospho-tyrosine phosphatase and PTEN inhibitor BpV(Hopic) on C2C12 myoblast migration and differentiation was investigated. Applying a wound healing migration model, it is reported that 1 μM BpV(Hopic) is capable of enhancing the migration of C2C12 myoblasts by approximately 40 % in the presence of myotube conditioned media, without significantly affecting their capacity to differentiate and fuse into multinucleated myotubes. Improved migration of myoblasts treated with 1 μM BpV(Hopic) was associated with activation of PI3K/AKT and MAPK/ERK pathways, while their inhibition with either LY294002 or UO126, respectively, resulted in a reduction of C2C12 migration back to control levels. These results propose that bisperoxovanadium compounds may be considered as potential tools for enhancing the migration of myoblasts, while not reducing their differentiation capacity and underpin the importance of PI3K/AKT and MAPK/ERK signalling for the process of myogenic progenitor migration.

Keywords

Skeletal muscle Myoblast Migration Bisperoxovanadium Differentiation 

Abbreviations

Bpv

Bisperoxovanadium

CM

Conditioned media

DMD

Duchenne muscular dystrophy

Notes

Acknowledgments

Work was sponsored by Institute for Biomedical Research into Human Movement and Health (IRM), Manchester Metropolitan University, Faculty of Science and Engineering, John Dalton Building, Oxford Road, Manchester, UK.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10974_2013_9340_MOESM1_ESM.png (6 mb)
Supplemental figure S1. Myoblasts in serum free media were seeded in Corning® Costar® 6.5mm (8.0μm pore size) permeable polycarbonate transwell membrane supports and following 4 hours of incubation at 37ºC with either serum free media (A) or myotube conditioned media (B) in the bottom chamber, cells were fixed with 2.5% gluteraldehyde, permeabilized for 3min in 0.5% Triton X-100 (BDH Labs, Poole, England), membranes were stained in Gill’s no.1 haematoxylin and non-migrated cells on the upper side of the microporous membrane were gently removed by a cotton swab. Images are representative of the cells adherent on the bottom side of the microporous membrane, i.e. cells that have migrated through the pores of the membrane. Images demonstrate that, as expected, cells do not migrate to the bottom side of the membrane in response to serum-free media present in the bottom chamber, while when myotube conditioned media is present in the bottom chamber, cells potently migrate to and form a confluent layer of cells on the bottom part of the microporous membrane. Data are representative of 3 experiments performed in duplicate. (PNG 6162 kb)
10974_2013_9340_MOESM2_ESM.png (3.7 mb)
Supplemental figure S2. SDS-PAGE reveals that 1µM BpV(Hopic) treatment maintains pAKT and pERK1/2 levels above control CM levels for the investigated time course to 60min. LY294002 (A) and UO126 (B) inhibitors were applied to inhibit, respectively, PI3K/AKT and MAPK/ERK signaling pathways. (PNG 3835 kb)
10974_2013_9340_MOESM3_ESM.png (2.6 mb)
Supplemental figure S3. Analysis of directionality of cell movement of myoblasts treated with CM indicates an increased directionality at 10-20hrs vs 0-10hrs (0.81+/-0.006 vs 0.78+/-0.007, p<0.0001, represented by *) (A). Analysis of directionality of cell movement of myoblasts treated with BpV(Hopic) indicates an increased directionality at 10-20hrs vs 0-10hrs (0.8+/-0.006 vs 0.73+/-0.008, p<0.0001, represented by *) (B). (PNG 2688 kb)
10974_2013_9340_MOESM4_ESM.png (1.6 mb)
Supplemental figure S4. The number of dying cells were manually counted frame by frame in each area for the duration of the movie (20hours). Dying cells were clearly obvious as floating cell bodies in the supernatant. The numbers of dying cells from the 6 random fields were summed and represented as percentage of the total number of cells in each field (alive or dead). Cell death quantifications and analyses found no significant difference between CM, CM+1μM Bpv(Hopic) and CM+1μM Bpv(Hopic)+10μM LY294002. (A). Cell death quantifications indicated decreased cell death with CM+1μM Bpv(Hopic)+5μM UO126 vs CM (0.46 +/-0.29% vs 2.07 +/-0.28%, p<0.05, represented by *) (B) Data are representative of minimum 4 experiments performed in duplicate. (PNG 1670 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Georgi A. Dimchev
    • 1
    • 2
  • Nasser Al-Shanti
    • 1
  • Claire E. Stewart
    • 1
    • 3
  1. 1.Institute for Biomedical Research into Human Movement and Health (IRM), Faculty of Science and Engineering, Manchester Metropolitan UniversityManchesterUK
  2. 2.Institute of Genetics, University of BonnBonnGermany
  3. 3.Research Institute for Sport and Exercise Sciences, School of Sport and Exercise Sciences, Liverpool John Moores UniversityLiverpoolUK

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