Resistance‐exercise‐induced stress intervenes in TGF ‐β signaling by cooperatively downregulating nuclear αB ‐crystallin and SMAD4 in human skeletal muscle fibers

Alpha-crystallin B chain (αB-crystallin; CRYAB) helps to maintain proteostasis following cellular stress. Additionally, recent studies in fibroblasts have shown its function in the transforming growth factor beta (TGF-β) pathway, where it stabilizes mothers against decapentaplegic homolog 4 (SMAD4) in the nucleus, enabling target gene transcription. Specifically in skeletal muscle fibers (SkMFs), TGF-β/SMAD4 signaling via myostatin regulates fiber size by inducing growth inhibitors and suppressing the serine/threonine-protein kinase (mTOR) pathway. Conversely, resistance exercise (RE) promotes muscle growth and should therefore affect CRYAB/SMAD4 interaction. However, in human SkMFs, CRYAB's nuclear localization and role in TGF-β/SMAD4 signaling, at rest or after RE, remain undescribed. Therefore, we first validated CRYAB's nuclear localization by small interfering RNA (siRNA)-mediated CRYAB silencing in C2C12 cells (mouse skeletal muscle myoblast cell line) and in human SkMFs via subcellular fractionation and confocal microscopy. To determine the effect of RE on this pathway, skeletal muscle biopsies were taken at rest and 60 min post-RE and analyzed for nuclear localization and phosphorylation of CRYAB and SMAD4, CRYAB/SMAD4 colocalization, and anabolic signaling. CRYAB was localized to C2C12 myoblast nuclei during proliferation but disappeared early during differentiation and was absent in nuclei of myotubes. In contrast, CRYAB was present in nuclei of resting human SkMFs. However, acute RE reduced total CRYAB in SkMF nuclei and increased its phosphorylation at serine 59, which likely promotes its export. Acute RE also reduced both nuclear and cytoplasmic SMAD4 while enhancing anabolic signaling by mTOR and small ribosomal subunit protein eS6 (rpS6) phosphorylation. We conclude that CRYAB and SMAD4 interact in nuclei of human SkMFs and have a coordinated regulation in response to RE-induced mechanical stress, revealing a new perspective on a yet-undescribed mechanism contributing to skeletal muscle adaptation.