Dynamic Tensile Loading Modulates Gene Expression Through Epigenetic Regulation in Tenocytes

Introduction: Tendinopathy, particularly prevalent in the Achilles tendon, presents a significant clinical challenge due to its chronic degeneration nature. Despite its widespread occurrence, the precise molecular mechanisms driving tendinopathy progression remain unclear. Overuse injury, characterized by repetitive tendon strain leading to micro-tears, is a primary cause of tendinopathy [1]. Pathologically, cyclic tensile loading profoundly influences tendon mechanical properties, including collagen organization, tissue composition, and gene expression [2, 3], thereby influencing their phenotype and contributing to pathology. Epigenetic modifications play a crucial role in regulating chromatin organization, influencing gene expression and cell differentiation [4]. However, the specific impact of altered biophysical environments on the epigenetic status in tenocytes within tendon degeneration remains unknown. Thus, this study investigates how repetitive tensile loading, mimicking an overuse tendon model in vitro, influences gene expression and histone modifications in bovine Achilles tenocytes, aiming to enhance understanding of the molecular basis of tendinopathy and contribute to the development of precise therapeutic strategies.

Materials and

Methods: Tenocytes from juvenile bovine Achilles tendons were expanded on tissue culture plastic up to passage 1 (P1). To investigate the effects of dynamic tensile loading, tenocytes were seeded onto aligned poly(ε−caprolactone) nanofibrous scaffolds followed by 2 days of culture in basal growth media. A custom tensile loading bioreactor applied cyclic uniaxial force at 3% to 8% strain amplitudes (at 1 Hz) to the tenocyte-seeded scaffolds for 1 to 5 hours over 1 or 3 days. Following loading, samples were processed for RNA or histone extraction. Relative mRNA expression changes for type-I collagen (COL-I), type-III (COL-III), and tenascin-C (TNC) were determined by RT-qPCR, using GAPDH as the reference gene. Immunoblotting assessed protein expression changes of H3K9ac and H3K4me3 (epigenetic activators), or H3K27me3 and H3K9me3 (epigenetic repressors), normalized to H3 levels and further normalized to non-loading free-swelling conditions (Ctrl).

Results, Conclusions, and Discussions: The RT-qPCR analysis showed a significant decrease in the ratio between COL-I and COL-III in tenocytes subjected to 8% strain for 5 hours loading for 1 day, compared to non-loading and 3% strain conditions (Fig. 1A). However, no significant changes were observed in TNC expression between the two loading conditions (Fig. 1A). Application of 3% strain loading for 3 hours for 1 day resulted in approximately a two-fold decrease in H3K9ac and H3K4me3 expression, associated with gene activation, as well as a reduction in markers associated with gene repression (H3K27me3 and H3K9me3) (Fig. 1B, C). For the 3-day loading duration (Fig. 2A), the RT-qPCR analysis indicated an increase in the COL-I/COL-III ratio with 3% strain for 1-hour loading compared to control, while a general reduction in the ratio was observed with 3% strain for 4 hours and 8% strain for 4 hours (Fig. 2B). There was a significant decrease in H3K9ac expression across all conditions and an overall decrease in H3K9me3 expression for all loading conditions (Fig. 2C). These findings suggest that prolonged overloading leads to heightened structural protein remodeling, and both types of histone modifications are responsible for the response to repetitive tensile overloading. Overall, these findings suggest that extended periods of cyclic loading trigger a turnover of structural proteins in parallel to the response observed in tendon repair and regeneration. Prolonged tensile loading reduces the COL-I/COL-III ratio, indicating increased remodeling activity in tenocytes, characteristic of overuse injuries. The differential expression of histone modifications associated with gene activation and repression following repetitive tensile loading emphasizes the regulatory role of epigenetic modifications in tenocytes. Ongoing studies are focused on defining parameters for physiological and pathological in vitro loading in conjunction with identifying how mechanically induced changes in histone modifications correlate with altered gene expression by profiling accessible chromatin through ATAC-seq and by understanding the impact of the epigenetic landscape through ChIP-seq. This study reveals molecular responses in tenocytes to various dynamic tensile loading conditions, providing insights into tendon degeneration and facilitating the development of targeted strategies for tendon regeneration and repair.

Acknowledgements (Optional): This work was supported by the NIH (K01 AR077087, P50 AR080581, R01 HL163168).

References: [1] Roberts+ 2003, Arthritis Res Ther; [2] Ma+ 2013, Osteoarthritis Cartilage; [3] Heo+ 2020, Sci Adv; [4] Sung + 2008, Blood.