TGFβ /Smad2/3‐Mediated Crosstalk Between Vocal Fold Fibroblasts and Myoblasts In Vitro

ABSTRACT Objectives Traditionally, disorders of the vocal fold (VF) mucosa and underlying musculature have been regarded as mutually exclusive entities. However, emerging evidence from other organ systems suggests mucosal and muscle compartments engage in reciprocal interactions with functional consequences. We hypothesized that similar crosstalk exists in the VF, whereby fibrotic mucosa influences adjacent muscle. To model this process, we stimulated human VF fibroblasts (HVOX) with TGF‐β1, a central mediator of fibrosis, and examined the effects on rat VF myoblasts (rVF‐Mbs), as well as reciprocal influences of rVF‐Mbs on fibroblasts. Methods HVOX fibroblasts were stimulated with 10 ng/mL TGF‐β1, and the effects on rVF‐Mbs were assessed using conditioned media and co‐culture. Myotube formation was evaluated by immunofluorescence, and nuclear localization of Smad2/3 was examined in conditioned media experiments. qRT‐PCR quantified transcripts related to myogenic differentiation and Smad2/3 signaling. ALK4/5 inhibition was performed in co‐culture to test TGF‐β/Smad2/3‐signaling pathway involvement. Reciprocal effects were examined by changes in fibrogenic gene expression in HVOX fibroblasts. Results Both conditioned media and co‐culture suppressed myogenic differentiation in rVF‐Mbs; increased inhibition was observed in co‐culture, as indicated by reduced myotube formation, decreased Myh2 expression, and activation of Smad2/3 signaling. ALK4/5 inhibition abrogated these effects. Differentiating rVF‐Mbs attenuated the fibrogenic phenotype of HVOX fibroblasts. Conclusions Fibrotic VF mucosal cells can impair myogenic differentiation through TGF‐β/Smad2/3‐mediated fibroblast–myoblast crosstalk, and myogenic cells may exert reciprocal anti‐fibrotic effects. These findings suggest mucosa–muscle interactions may contribute to VF pathology and highlight Smad2/3 as a potential therapeutic target. Level of Evidence NA Study Design In vitro.