Proteolytic systems' expression during myogenesis and transcriptional regulation by amino acids in gilthead sea bream cultured muscle cells

PLoS One. 2017 Dec 20;12(12):e0187339. doi: 10.1371/journal.pone.0187339. eCollection 2017.

Abstract

Proteolytic systems exert an important role in vertebrate muscle controlling protein turnover, recycling of amino acids (AA) or its use for energy production, as well as other functions like myogenesis. In fish, proteolytic systems are crucial for the relatively high muscle somatic index they possess, and because protein is the most important dietary component. Thus in this study, the molecular profile of proteolytic markers (calpains, cathepsins and ubiquitin-proteasome system (UbP) members) were analyzed during gilthead sea bream (Sparus aurata) myogenesis in vitro and under different AA treatments. The gene expression of calpains (capn1, capn3 and capns1b) decreased progressively during myogenesis together with the proteasome member n3; whereas capn2, capns1a, capns1b and ubiquitin (ub) remained stable. Contrarily, the cathepsin D (ctsd) paralogs and E3 ubiquitin ligases mafbx and murf1, showed a significant peak in gene expression at day 8 of culture that slightly decreased afterwards. Moreover, the protein expression analyzed for selected molecules presented in general the same profile of the mRNA levels, which was confirmed by correlation analysis. These data suggest that calpains seem to be more important during proliferation, while cathepsins and the UbP system appear to be required for myogenic differentiation. Concerning the transcriptional regulation by AA, the recovery of their levels after a short starvation period did not show effects on cathepsins expression, whereas it down-regulated the expression of capn3, capns1b, mafbx, murf1 and up-regulated n3. With regards to AA deficiencies, the major changes occurred at day 2, when leucine limitation suppressed ctsb and ctsl expression. Besides at the same time, both leucine and lysine deficiencies increased the expression of mafbx and murf1 and decreased that of n3. Overall, the opposite nutritional regulation observed, especially for the UbP members, points out an efficient and complementary role of these factors that could be useful in gilthead sea bream diets optimization.

MeSH terms

  • Amino Acids / pharmacology*
  • Animals
  • Cells, Cultured
  • Gene Expression Regulation*
  • Muscle Development*
  • Muscle Proteins / metabolism
  • Muscles / cytology
  • Muscles / metabolism*
  • Proteolysis
  • Real-Time Polymerase Chain Reaction
  • Sea Bream
  • Transcription, Genetic*

Substances

  • Amino Acids
  • Muscle Proteins

Grants and funding

E.J.V. and E.L. are supported by predoctoral fellowships (BES-2013-062949 and BES-2012-061867) from the “Ministerio de Economía y Competitividad” (MINECO). This study was supported by the projects from the MINECO AGL2014-57974-R to I.N. and E.C., AGL2012-39768 and AGL2015-70679-R to J.G., the “Xarxa de Refèrencia d’R+D+I en Aqüicultura” and 2014SGR-01371 from the “Generalitat de Catalunya”, and by funds from the European Union through the project LIFECYCLE (EU-FP7 222719). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.