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

TRPC6 regulates inflammation and catabolism in intervertebral disc degeneration (#MP-12a)

Janitri Venkatachala Babu 1 , Alexandra Sadowska 2 , Addisu Mesfin 3 , Varun Puvanesarajah 3 , Karin Wuertz 1 4 5
  1. Rochester Institute of Technology, Rochester, NEW YORK, United States
  2. Institute for Biomechanics, ETH Zurich, Zurich, Switzerland
  3. Department of Orthopedics and Rehabilitation, University of Rochester Medical Centre, Rochester, New York, United States
  4. Spine Research Institute of the Paracelsus Medical University, Salzburg, Austria
  5. Schon Clinic Munich Harlaching, Munich, Germany

Introduction

Low back pain is the leading cause of global disability, with discogenic etiology being the prime contributor1,2. The intervertebral disc degeneration exhibits increased levels of pro-inflammatory cytokines, catabolic enzymes, and nerve ingrowth, causing nociception, for which no treatment targeting the pathophysiology exists3-5. Transient receptor potential channels are cation-selective transmembrane receptors activated by a diverse set of stimulants and are particularly promising as drug targets due to their involvement in many pathologies6,7. In discogenic chronic back pain (DCBP), the canonical subtype 6, TRPC6, is noteworthy for regulating inflammation and pain in various tissues8,9. Therefore, we hypothesize that TRPC6 activation in IVD cells pharmacologically will increase catabolism and inflammation, key characteristics of DCBP. Conversely, pharmacological inhibition of TRPC6 will mitigate inflammation and catabolism in vitro and alleviate pain in vivo, presenting a novel non-opioid pharmacological therapeutic for DCBP.

Methods

Human IVD samples of degenerated (n=22) and non-degenerated (n=12) discs were analyzed using RT-qPCR for TRPC6 expression. TRPC6 channel activity in isolated human IVD cells (n=3) was assessed through calcium flux measurement (Flex Station-3, Fura-2 QBT™ Calcium kit) following treatment with different concentrations of pharmacological activator (HYP9) or inhibitor (larixyl acetate). The effects of TRPC6 modulation on gene expression were analyzed through mRNA isolation and qPCR analysis in human IVD cells following exposure to HYP9 (1 μM) for 18 hours for activation (n=7) and larixyl acetate (1 μM or 10 μM for 18 hours) after 2 hours of pre-treatment with HYP9 (1 μM) for inhibition (n=3) studies. Untreated cells and vehicle controls were included. Statistical tests (normality test, t-tests) conducted using GraphPad Prism 10.0.2 for Windows.

Results

TRPC6 exhibits significantly elevated gene and protein expression in degenerated human IVDs compared to healthy controls, with particularly pronounced upregulation in IVDs associated with pain10,11 (Figure 1). Pharmacological activation of TRPC6 using HYP9 elicited dose-dependent influx of calcium ions in human IVD cells, while inhibition with larixyl acetate reduced the influx (Figure 2A & 3A). The pharmacological activation with HYP9 in human IVDs (Figure 2B-D) significantly increased gene expression levels of inflammatory cytokines like interleukins (IL 6, IL 8 – p<0.05) and prostaglandin (PTGS2 – p=0.053), neurotropic factors (NGF, BDNF – p<0.05), and catabolic enzymes (MMP1, 2 and 3 – p<0.05). The targeted inhibition of TRPC6 (Figure 3B-D) reversed these effects significantly with decreased mRNA expression of matrix-degrading enzymes (MMP1 – p<0.01 and MMP3 – p<0.05), neurotrophic factors (NGF p<0.05) and inflammatory cytokines, including PTGS2 (p<0.01).

Discussion

The increased TRPC6 expression in degeneration supports targeting this channel as a potential therapeutic for DCBP. Our in vitro results of pharmacological TRPC6 activation show an inflammatory-catabolic response that is associated with increased expression of neurotrophins that promote IVD innervation. Importantly application of the pharmacological inhibitor reversed the catabolic-inflammatory responses. These findings highlight the potential of modulating TRPC6 as a molecularly targeted therapy to mitigate degenerative processes in IVDs. Further experimentation in vitro will validate the role of TRPC6 in IVD degeneration and pathophysiology. Additionally, in vivo animal studies will be performed to assess changes in pain.

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