Reduced myotube diameter induced by combined inhibition of transforming growth factor-β type I receptors Acvr1b and Tgfbr1 is associated with enhanced β1-syntrophin expression

J Cell Physiol. 2024 Dec;239(12):e31418. doi: 10.1002/jcp.31418. Epub 2024 Aug 20.

Abstract

Simultaneous inhibition of transforming growth factor-β (TGF-β) type I receptors Acvr1b and Tgfbr1 signalling has been associated with excessive skeletal muscle hypertrophy in vivo. However, it remains unclear whether the increased muscle mass in vivo is a direct result of inhibition of intracellular TGF-β signalling or whether this is an indirect effect of an altered extracellular anabolic environment. Here, we tested whether individual or simultaneous knockdown of TGF-β type I receptors in C2C12 myotubes was sufficient to induce muscle hypertrophy. The expression levels of TGF-β type I receptors Acvr1b and Tgfbr1 in myotubes were knocked down individually or in combination in the absence or presence of TGF-β1 and myostatin. Knocking down either Acvr1b or Tgfbr1 did not significantly change cell phenotype. Unexpectedly, simultaneous knockdown of both receptors reduced C2C12 myotube diameter, mRNA expression levels of Hgf, Ccn2 and Mymx with or without TGF-β1 and myostatin administration. In spite of decreased phosphorylation of Smad2/3, phosphorylation of P70S6K was reduced. In addition, the gene expression level of β1-syntrophin (Sntb1), which encodes a protein associated with the dystrophin-glycoprotein complex, was increased. Parallel experiments where Sntb1 gene expression was reduced showed an increase in myotube diameter and fusion of C2C12 myoblasts. Together, these results indicate that the knockdown of both TGF-β type I receptors reduced myotube diameter. This atrophic effect was attributed to reduced protein synthesis signalling and an increased expression of β1-syntrophin. These results have implications for our fundamental understanding of how TGF-β signalling regulates skeletal muscle size.

Keywords: Acvr1b; TGF‐β type I receptor; Tgfbr1; myotubes; β1‐syntrophin.

MeSH terms

  • Activin Receptors, Type I* / genetics
  • Activin Receptors, Type I* / metabolism
  • Animals
  • Cell Line
  • Connective Tissue Growth Factor
  • Gene Knockdown Techniques
  • Hypertrophy / metabolism
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mice
  • Muscle Fibers, Skeletal* / drug effects
  • Muscle Fibers, Skeletal* / metabolism
  • Muscle Proteins* / genetics
  • Muscle Proteins* / metabolism
  • Myostatin* / genetics
  • Myostatin* / metabolism
  • Phosphorylation
  • Protein Serine-Threonine Kinases* / genetics
  • Protein Serine-Threonine Kinases* / metabolism
  • Receptor, Transforming Growth Factor-beta Type I* / genetics
  • Receptor, Transforming Growth Factor-beta Type I* / metabolism
  • Receptors, Transforming Growth Factor beta* / genetics
  • Receptors, Transforming Growth Factor beta* / metabolism
  • Signal Transduction*
  • Smad2 Protein / genetics
  • Smad2 Protein / metabolism
  • Smad3 Protein / genetics
  • Smad3 Protein / metabolism
  • Transforming Growth Factor beta1* / metabolism

Substances

  • Receptor, Transforming Growth Factor-beta Type I
  • Tgfbr1 protein, mouse
  • Activin Receptors, Type I
  • Myostatin
  • Protein Serine-Threonine Kinases
  • Acvr1b protein, mouse
  • Muscle Proteins
  • Transforming Growth Factor beta1
  • Receptors, Transforming Growth Factor beta
  • Membrane Proteins
  • CCN2 protein, mouse
  • Smad2 protein, mouse
  • Mstn protein, mouse
  • Smad2 Protein
  • Smad3 Protein
  • Smad3 protein, mouse
  • Connective Tissue Growth Factor