Three-dimensional analysis of hydrogel-imbedded aortic valve interstitial cell shape and its relation to contractile behavior

Abstract

Cell-shape is a conglomerate of mechanical, chemical, and biological mechanisms that reflects the cell biophysical state. In a specific application, we consider aortic valve interstitial cells (AVICs), which maintain the structure and function of aortic heart valve leaflets. Actomyosin stress fibers help determine AVIC shape and facilitate processes such as adhesion, contraction, and mechanosensing. However, detailed 3D assessment of stress fiber architecture and function is currently impractical. Herein, we assessed AVIC shape and contractile behaviors using hydrogel-based 3D traction force microscopy to intuit the orientation and behavior of AVIC stress fibers. We utilized spherical harmonics (SPHARM) to quantify AVIC geometries through three days of incubation, which demonstrated a shift from a spherical shape to forming substantial protrusions. Furthermore, we assessed changes in post-three day AVIC shape and contractile function within two testing regimes: (1) normal contractile level to relaxation (cytochalasin D), and (2) normal contractile level to hyper-contraction (endothelin-1). In both scenarios, AVICs underwent isovolumic shape changes and produced complex displacement fields within the hydrogel. AVICs were more elongated when relaxed and more spherical in hyper-contraction. Locally, AVIC protrusions contracted along their long axis and expanded in their circumferential direction, indicating predominately axially aligned stress fibers. Furthermore, the magnitude of protrusion displacements was correlated with protrusion length and approached a consistent displacement plateau at a similar critical length across all AVICs. This implied that stress fiber behavior is conserved, despite great variations in AVIC shapes. We anticipate our findings will bolster future investigations into AVIC stress fiber architecture and function. STATEMENT OF SIGNIFICANCE: Within the aortic valve there exists a population of aortic valve interstitial cells, which orchestrate the turnover, secretion, and remodeling of its extracellular matrix, maintaining tissue integrity and ultimately sustaining the proper mechanical function. Alterations in these processes are thought to underlie diseases of the aortic valve, which affect hundreds of thousands domestically and world-wide. Yet, to date, there are no non-surgical treatments for aortic heart valve disease, in part due to our limited understanding of the underlying disease processes. In the present study, we built upon our previous study to include a full 3D analysis of aortic valve interstitial cell shapes at differing contractile levels. The resulting detailed shape and deformation analysis provided insight into the underlying stress-fiber structures and mechanical behaviors.

Keywords: Aortic valve interstitial cell; Contraction; Imaging; Kinematics; Protrusion; Shape; Size; Stress fibers; Three dimensional traction force microscopy; Volume.