Substrate Stiffness and Microtopography in PIP2 Regulation of the Actin Cytoskeleton in Primary Cardiac Fibroblasts

Abstract

Following tissue injury, fibroblasts are one of the primary responding cell types in the wound healing process. Upon myocardial infarction, local fibroblasts divide and increase collagen secretion, leading to cardiac fibrosis. In this study, the effect of the mechanical environment, through substrate stiffness and three‐dimensional topography, is examined in relation to the distribution and levels of phosphatidylinositol 4,5‐bisphosphate (PIP2) in primary neonatal rat ventricular fibroblasts. PIP2 is a membrane component and known regulator of the actin cytoskeleton. Substrates possessing 10 µm high microprojections are fabricated from polydimethylsiloxane to a stiffness of either 100 kPa or 400 kPa (Young's Modulus). A 3‐fold cellular decrease (P<.001) in PIP2 is found between flat‐soft substrates and flat‐polystyrene culture dishes. Additionally, microprojections are shown to blunt PIP2 by a similar amount within each stiffness condition. Fluorescence recovery after photobleaching, yielding the rate constant Kfrap, allows study of actin dynamics in the various micromechanical conditions. A 4‐fold decrease (P<.0001) is found between actin dynamics within lamellipodia to those within stress cables, correlating to a migrating or an anchored cell. This difference in dynamics coincides with an increased redistribution of PIP2 to the lamella in migrating cells. An improved understanding of the intracellular lipid mechanisms involved in wound healing could aid in prevention of maladaptive scarring following major injury. Funded by NIH HL62426, T32HL07692