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

INTRODUCTION. Transient Receptor Potential Vanilloid 4 (TRPV4) is a mechanosensitive Ca²⁺-permeable channel, which is 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. Since the intervertebral disc is exposed to high osmotic and hydrostatic pressure, we hypothesized that TRPV4 contributes to intradiscal homeostasis. Our objective was to elucidate that TRPV4 activation by agonist promotes autophagy and extracellular matrix (ECM) synthesis in the rat intervertebral disc.

METHODS. In-vitro study: Disc NP cells harvested from 12-week-old male Sprague-Dawley rats were used. (1) Cytotoxicity of the agonist was evaluated using the Cell Counting Kit-8 (CCK-8) after 24-h treatment of 0–100-nM TRPV4 agonist (GSK1016790A, Sigma-Aldrich) in DMEM with 10% FBS. (2) Ca²⁺ imaging was performed using Fluo-4 AM to detect intracellular Ca²⁺ changes. (3) After culturing cells for 24 h in serum-free DMEM with or without 10-ng/ml interleukin-1 beta (IL-1β) and TRPV4 agonist, expression of phospho-AMPK (pAMPK), autophagy markers (mTOR, RAPTOR, p70/S6K, LC3-II, and a substrate p62/SQSTM1), ECM molecules (COL2a1 and Aggrecan), catabolic matrix metalloproteinases (MMPs), anti-catabolic tissue inhibitor of metalloproteinases (TIMPs), apoptosis markers (PARP, cleaved PARP, and cleaved Caspase-9), and senescence markers (p53, p21/CIP1, and p16/INK4a) were assessed by Western blotting.

In-vivo study: Thirty-six 12-week-old male SD rats were used, and TRPV4 agonist and control (dimethyl sulfoxide) were injected into respective discs using a 33-G needle. (4) A rat tail model of disc degeneration induced by temporary static compression was designed. Rat tails were affixed with an Ilizarov-type apparatus with springs between the 8^th and 10^th coccygeal (C) vertebrae. While DMSO was injected into C9/10 (loaded control) and C12/13 (unloaded control), TRPV4 agonist was injected into C8/9 (loaded experimental) and C11/12 (unloaded experimental). Then, axial force at 1.3 MPa was applied for 24 h and subsequently released. Radiographic degeneration was assessed at 0, 28, and 56 d after compression.

RESULTS. In-vitro study: (1) TRPV4 agonist significantly decreased cell viability at 20 nM and higher (P<0.05). Therefore, 10-nM TRPV4 agonist was used for subsequent experiments. (2) Intracellular Ca²⁺ level increased immediately after the administration of TRPV4 agonist. (3) pAMPK expression significantly increased by TRPV4 agonist in serum-free DMEM (P<0.05). In serum-free DMEM with IL-1β, TRPV4 agonist increased pAMPK, LC3-II, COL2a1, Aggrecan, and TIMPs, and decreased mTOR, RAPTOR, p70/S6K, p62/SQSTM1, and MMPs (P<0.05), indicating the promotion of autophagy and ECM synthesis through AMPK pathway. TRPV4 agonist also increased PARP, and decreased cleaved PARP, cleaved Caspase-9, p53, p21/CIP1, and p16/INK4a (P<0.05), indicating the suppression of apoptosis and senescence (Fig. 1).

In-vivo study: (4) In the loaded, TRPV4 agonist-injected discs, radiographic disc height was significantly maintained compared to the control at 56 d after compression (P<0.05) (Fig. 2).

DISCUSSION. In vitro, the TRPV4 activation by agonist promoted autophagy and ECM synthesis through AMPK pathway under pro-inflammatory IL-1β stimulation. In vivo, intradiscal injection of TRPV4 agonist suppressed radiographic disc degeneration. The TRPV4 agonist could be a new therapeutic agent for intervertebral disc diseases.