Substrate Stiffness, Topography, and TRPV4 in AF Mechanotransduction

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SUMMARY Over the past decade(s), research has highlighted that substrate stiffness and architecture/topography can be recognized by cells and serve as mechanical and topographical cues that ultimately drive cell behavior through mechanoreceptors. Substrate changes can also affect the mechanical stimulation of cells and thus their response to loading. These cell responses are largely governed through mechanosensitive ion channels, such as the transient receptor potential (TRP) channels. TRPV4 is of specific interest as its activation and expression can be affected by matrix stiffness and topography. Furthermore, its activation controls extracellular matrix (ECM) synthesis, matrix-degrading enzyme expression, and ECM remodeling in various cell types. The annulus fibrosus (AF), the outer area of the intervertebral disc (IVD), is a mechanosensitive tissue in which topographical and mechanical cues change during degeneration, thus likely affecting cell fate, cellular activity, and disease progression. The AF plays a crucial role in the development of low back pain as its structural failure can lead to IVD herniation. Surprisingly, only very few studies have thus far investigated cell-substrate interactions in AF cells and no data exists on the relevance of substrate stiffness/topography on TRPV4 activation in AF cells. It is also unknown whether TRPV4 regulates ECM synthesis/remodeling in the AF, which would, in turn, affect its activation and hence create a crucial feedback loop. Our long-term goal is to reveal the relevance of cell-substrate processes in IVD health and disease and to use this knowledge in the development of regenerative approaches. Specifically, this project aims to: (1) Determine the relevance of substrate stiffness on TRPV4 activation in AF cells in response to (a) a pharmacological TRPV4 agonist and (b) cyclic stretching. (2) Determine the relevance of substrate topography on TRPV4 activation in AF cells in response to (a) a pharmacological TRPV4 agonist and (b) cyclic stretching. (3) Determine the importance of TRPV4 activation in AF cells in regulating ECM synthesis and remodeling The proposed project will use an innovative design of stretching chambers that allows investigating the integrative role of substrate cues (stiffness, topography) and mechanical stimulation in modulating cell function and fate. TRPV4 will be activated by specific agonists or stretching upon seeding in these chambers and cell responses will be determined by qPCR, ELISA, and Western Blot for targets selected based on RNA-seq data. Furthermore, ECM synthesis and remodeling following TRPV4 activation will be evaluated. This will be the first study to investigate TRPV4 in the context of substrate stiffness and topography in AF cells. As the developed tools will also apply to other research areas, I can help advance the fundamental understanding of mechanotransduction processes in health and disease. The gained knowledge will be applicable in tissue engineering and support the identification of new drug targets related to dysregulated mechanotransduction.