Poster Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Introduction. Transient Receptor Potential Vanilloid 4 (TRPV4) has been identified as a Ca²⁺-permeable channel and reported to be activated under a physiological mechanical stimulation in disc nucleus pulposus (NP) cells in vitro. Autophagy via Ca²⁺-dependent AMPK/mTOR pathway is activated under hyperosmotic stress in notochordal cells. Our objective was to elucidate the role of TRPV4 in rat intervertebral disc autophagy and extracellular matrix metabolism through loss-of-function study with the RNA interference (RNAi) technique.

Methods. In-vitro study: (1) Disc NP cells harvested from 12-week-old male Sprague-Dawley (SD) rats were used. Small interfering RNA (siRNA) was applied to knockdown TRPV4 by the reverse transfection method. Expression of AMPK, mTOR, LC3-II, and a substrate p62/SQSTM1 as well as TRPV4 was measured by Western blotting (WB). (2) Next, cells after the transfection were cultured in serum-free DMEM with 10-ng/ml interleukin-1 beta (IL-1β) for 24 h. Autophagy markers and extracellular matrix molecules (COL2a1 and Aggrecan), catabolic matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) were assessed by WB.

In-vivo study: Thirty-six 12-week-old male SD rats were used, and TRPV4 and control siRNAs were injected into respective discs using a 33-G needle. (3) To confirm in-vivo transfection, WB for TRPV4 and α-tubulin was conducted in rat disc NP-tissue protein extracts 2 and 28 d after injection. (4) A rat tail model of disc degeneration induced by temporary static compression was designed. Radiographic and histological (Safranin-O) degeneration was assessed at 0, 7, 28, and 56 d after compression. (5) Extracellular matrix metabolism (COL2a1 and Aggrecan) and the notochordal Brachyury were assessed by immunofluorescence at 0, 28, and 56 d after compression.

Results. In-vitro study: (1) In rat disc NP cells, TRPV4 expression significantly decreased by TRPV4 RNAi (70% or more). The LC3-II decreased and p70/S6K and p62/SQSTM1 increased, indicating autophagy suppression. In addition, AMPK decreased and mTOR increased, suggesting a possible pathway between TRPV4 and autophagy (Figure. 1). (2) Pro-inflammatory IL-1β stimulation with TRPV4 RNAi further decreased AMPK, LC3-II, COL2a1, Aggrecan, and TIMPs and increased mTOR, p70/S6K, p62/SQSTM1, and MMPs, indicating enhancement of the effect of TRPV4 knockdown (Figure. 2).

In-vivo study: (3) WB displayed sustained decreases in TRPV4 protein expression 2, 28, and 56 d after injection (P<0.05). (4) In the loaded, TRPV4 siRNA-injected discs, radiographic disc height significantly decreased compared to the other conditions at 28 and 56 d after compression (P<0.05) (Figure. 3). Safranin-O staining also showed progressive degenerative findings in the loaded, TRPV4 siRNA-injected discs relative to the other conditions at 28 and 56 d after compression (P<0.05). (5) Immunofluorescence showed that signals of COL2a1, Aggrecan, and Brachyury were significantly decreased in the loaded, TRPV4 siRNA-injected discs at 28 and 56 d after compression (P<0.05).

Conclusions. In vitro, TRPV4 knockdown suppressed autophagy with AMPK inhibition and suppressed extracellular matrix metabolism under pro-inflammatory IL-1β stimulation. In vivo, intradiscal injection of TRPV4 siRNA was effective as long as 56 d, resulting in radiographic and histomorphological disc degeneration. The TRPV4 could be a therapeutic target for intervertebral disc diseases via modulating autophagy.