Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Collaborative Specialization

Musculoskeletal Health Research

Supervisor

Séguin, Cheryle A.

Abstract

IVD degeneration is a multifactorial pathological process associated with back pain. While biomechanical factors are important regulators of IVD homeostasis, mechanical loading also contribute to the onset of IVD degeneration. Importantly, the mechanotransduction pathways that mediate cell type-specific responses to mechanical loading are not well understood. Transient receptor potential vanilloid 4 (TRPV4) is a multimodally activated cell surface cation channel implicated as a mechanoreceptor regulating the mechano-response in other musculoskeletal cell types. Using both in vitro and in vivo models, the current study aimed to characterize the role of TRPV4 in annulus fibrosus (AF) cell mechanotransduction. Using a mechanically dynamic bioreactor system, AF cells were exposed to cyclic tensile strain (CTS) to assess mechanically-induced changes in gene expression and mitogen activated protein kinase (MAPK) pathway activation. Next, a novel transgenic Trpv4-reporter mouse model was used to determine the expression pattern of Trpv4 during mouse spine development and aging. TRPV4 function in AF cells was then characterized using live cell calcium imaging and treatment with pharmacological modulators of TRPV4 during CTS. Lastly, conditional Trpv4 knockout mice (Col2-Cre;Trpv4fl/fl) were used to determine the role of TRPV4 signalling in IVD health and injury-induced degeneration. These studies demonstrated that the mechano-response of AF cells was frequency-dependent, showing increased stress fibre formation, ERK1/2 pathway activation, and gene expression changes (i.e. Extracellular matrix (ECM) genes, matrix remodelling genes, mechano-sensitive genes, inflammatory cytokine genes, and mechanoreceptor genes). Trpv4 expression was first detected during spine development, was maintained in NP and inner AF tissues and subsequently decreased with age. Activation of TRPV4 elicited intracellular calcium response in AF cells that was shown to regulate cytoskeletal remodelling and CTS-induced changes in Acan, Col1a1, and Prg4 expression. Furthermore, loss of Trpv4 led to decreased proteoglycan staining and the attenuation of degenerative changes in IVDs experiencing aberrant load following injury. Taken together, our findings suggest the existence of mechanical threshold that regulates IVD health and degeneration through TRPV4-mediated mechanotransduction pathways.

Summary for Lay Audience

Back pain is a major global health challenge and one of the most common causes of disability worldwide, imposing high socioeconomic burden on individuals and healthcare systems. In North America, four out of five adults will experience back pain at some point in their lives. Currently, back pain is treated mostly with pain management strategies and disc surgery, but the outcomes are disappointing, as the causes of back pain are rarely addressed. Back pain is a complex condition with multiple causes but is typically associated with intervertebral disc (IVD) degeneration. The IVD is a joint found between the spinal column that acts as a shock absorber during movement. Similar to muscles and bones, the force experienced by IVD is important for maintaining joint health, but abnormal levels can cause damage. This adaptive response to mechanical load is regulated at the cellular level by a process called mechanotransduction, in which cells sense and change external mechanical force into biological chemical signals. However, this cellular mechanism is not well understood. Using different experimental approaches, we aimed to determine how IVD cells adapt and respond to mechanical stimulation. Our studies showed that IVD cells respond to mechanical stimulation by changing their stiffness, activating specific biochemical signals, and regulating gene expression. Interestingly, the cellular responses differed depending on the frequency of the mechanical stimulation applied to the cells. These responses were found to be mediated by a receptor protein found on the cell surface that is activated by normal and abnormal levels of mechanical loading, serving to regulate IVD health and degeneration. Our findings contribute to an important biological question in the field of spine and IVD research regarding how mechanical forces regulate joint health.

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