Abstract
Follistatin (FS) is a high affinity activin-binding protein, neutralizing the effects of the Transforming Growth Factor-beta (TGF-β) superfamily members, as myostatin (MSTN). Since MSTN emerged as a negative regulator, FS has been considered as a stimulator of skeletal muscle growth and differentiation. Here, we studied the effect of FS administration on the Ca2+-homeostasis of differentiating C2C12 skeletal muscle cells. FS-treatment increased the fusion index, the size of terminally differentiated myotubes, and transiently elevated the expression of the calcium-dependent protein phosphatase, calcineurin, at the beginning of differentiation. Functional experiments did not detect any alterations in the Ca2+ transients following the stimulation by KCl or caffeine in myotubes. On the other hand, decreased Ca2+-uptake capability was determined by calculating the maximal pump rate (332 ± 17 vs. 279 ± 11 µM/s, in control and FS-treated myotubes, respectively; p < 0.05). In the same way, the expression and ATPase activity of the neonatal sarcoplasmic/endoplasmic reticulum Ca2+ATPase (SERCA1b) were decreased (0.59 ± 0.01 vs. 0.19 ± 0.01 mM ATP/min, in control and FS-treated myotubes, respectively; p < 0.05). However, the expression level of other proteins involved in Ca2+-homeostasis and differentiation (calsequestrin, STIM1, MyoD) were not affected. Our results suggest that the FS controlled myotube growth is paralleled with the tight regulation of cytosolic calcium concentration, and the decline of SERCA1b appears to be one of the key components in this process.
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Abbreviations
- BCA:
-
Bicinchoninic acid
- BSA:
-
Bovine serum albumin
- CaN:
-
Calcineurin
- CPA:
-
Cyclopiazonic acid
- CSQ:
-
Calsequestrin
- ECL:
-
Enhanced chemiluminescence
- FBS:
-
Foetal bovine serum
- FITC:
-
Fluoresceinisothiocyanate
- FS:
-
Follistatin
- HS:
-
Horse serum
- MSTN:
-
Myostatin
- PBS:
-
Phosphate-buffered saline
- RyR:
-
Ryanodine receptor
- SDS–PAGE:
-
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
- SERCA:
-
Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase
- SOCE:
-
Store-operated Ca2+ entry
- SR:
-
Sarcoplasmic reticulum
- STIM:
-
Stromal interaction molecule
- TGF-β:
-
Transforming Growth Factor-beta
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Acknowledgements
JA and JF were the recipients of the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences and JA the Lajos Szodoray Scholarship of the University of Debrecen, Faculty of Medicine. The work was supported by the National Research, Development and Innovation Office (NKFIH K115461, and PD 112199). We are grateful to Luca Mendler (Department of Biochemistry, Faculty of Medicine, University of Szeged) for support. The publication is supported by the GINOP-2.3.2-15-2016-00040 project. The project is co-financed by the European Union and the European Regonal Development Fund.
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Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work: JF, AG, TO, JA, EZ, LC. Drafting the work or revising it critically for important intellectual content: JF, AG, TO, JA, EZ, LC. Final approval of the version to be published: JF, AG, TO, JA, EZ, LC. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: JF, AG, TO, JA, EZ, LC.
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Fodor, J., Gomba-Tóth, A., Oláh, T. et al. Follistatin treatment suppresses SERCA1b levels independently of other players of calcium homeostasis in C2C12 myotubes. J Muscle Res Cell Motil 38, 215–229 (2017). https://doi.org/10.1007/s10974-017-9474-8
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DOI: https://doi.org/10.1007/s10974-017-9474-8