The Role of Mechanical Loading and Tropomyosin 3.1 in Stabilizing F-actin

• Mark Arranguez, Human Physiology, University of Delaware

• Justin Parreno, Biological Sciences, University of Delaware

Osteoarthritis is an irreversible, debilitating disease caused in part by aberrant mechanical overload onto cartilage. We suspect that amplified mechanical overloading of chondrocytes has major implications in the etiology of post-traumatic osteoarthritis via activation of chondrocyte degeneration and cell death pathways. We postulate that mechanical overloading disrupts cortical filamentous (F-)actin stability of chondrocytes and destabilization is pivotal by altering chondrocyte homeostasis. Therefore, maintaining proper F-actin organization may be critical for cartilage health. In this study, we examine two critical gaps in the regulation of F-actin in PTOA. First, we explore the effects of mechanical loading on cortical F-actin organization. Second, we elucidate the regulation of F-actin stability by F-actin stabilization proteins, the Tropomyosins. We hypothesize that both mechanical overloading and Tropomyosin 3.1 are key regulators of F-actin polymerization. We determined the effect of mechanical overloading on F-actin stability by exposing primary bovine chondrocytes on silicone substrates to physiological (5%) and overload (30%) uniaxial stretch (0.5Hz up to 6 hours). We determined that 5% stretch increases cortical F-actin but 30% decreases cortical F-actin in chondrocytes. Using confocal microscopy, we examined the localization of TPM3.1 in chondrocytes, both in native mouse hip tissue sections and in-vitro in isolated primary bovine cells. We determined that TPM3.1 inhibition decreases F-actin and negatively altered chondrocyte homeostasis, respectively. Additionally, using a whole mount confocal imaging protocol, we imaged mouse hip cartilage, and similarly found that Tpm3.1 knockout led to a reduction in F-actin. Therefore, this data supports the idea that F-actin polymerization is regulated by mechanical overload as well as Tpm3.1 in both mouse and bovine models.