RFLNA mitigates heat stress-impaired chondrocyte proliferation and vertebral development through cytoskeletal regulation in pigs

BACKGROUND: Global climate change has brought severe challenges to the livestock industry, among which heat stress (HS) acts as a key factor impairing the growth and development of pigs. It has been established that ambient temperature influences body size traits mainly by directly affecting skeletal development, yet the molecular mechanisms by which HS inhibits this process remain largely unclear. The cytoskeleton is critical for mediating cellular morphological adaptation to environmental stimuli, and HS can disturb cellular development by destroying cytoskeletal homeostasis. Previous studies have demonstrated that RFLNA regulates bone development through the cytoskeleton, but whether it alleviates HS-induced chondrocyte damage by modulating the cytoskeleton has not been reported. Therefore, this study was conducted to explore the effects of HS on the proliferation and differentiation of pig thoracic vertebral growth plate chondrocytes (PTVCs), and to clarify the regulatory mechanism by which the regulatory factor RFLNA endows PTVCs with HS resistance. RESULTS: Transcriptomic profiling of PTVCs cultured under control (37 °C) and HS (41 °C) conditions at multiple differentiation time points revealed that HS suppressed cell proliferation and extracellular matrix synthesis. Differentially expressed genes (DEGs) in the early HS response (6 h and 24 h) were enriched in inflammation and stress-response pathways. In contrast, DEGs from later-phases (48, 96, and 144 h) were linked to cytoskeletal reorganization. Further analysis revealed that RFLNA expression was upregulated both by HS and during chondrocyte differentiation. RFLNA expression was higher in the thoracic vertebrae tissues from large-sized Yorkshire pigs than in those from small-sized Wuzhishan pigs. Spatial expression analysis indicated that RFLNA was predominantly expressed in thoracic and lumbar vertebrae, with subcellular localization to the cytoskeleton. Functional assays demonstrated that RFLNA overexpression under HS conditions promoted PTVC proliferation and adhesion while inhibiting migration, thereby mitigating HS-induced growth suppression. Conversely, RFLNA knockdown exacerbated the detrimental effects of HS. CONCLUSIONS: Our findings show RFLNA might be a critical mediator of thermal adaptation and vertebral development, which links cytoskeletal regulation to temperature-related variations in body size. This work provides a theoretical foundation for strategies aimed at enhancing climate resilience in swine.