(E-199) Characterizing valve interstitial cell stress fiber formation in valve interstitial cells: A computational-experimental approach

Introduction:: Aortic valve stenosis (AVS) is a leading cause of regurgitation and can lead to death if not treated by replacement or repair. Currently there is no pharmacological therapy for this disease. A hallmark of AVS is activation of the aortic valve interstitial cells (AVICs) into myofibroblasts, where the AVICs secrete copious extracellular matrix and produce robust, highly-contractile actin stress fibers (SFs). Additionally, we have recently reported that human AVICs from highly stenotic areas of bicuspid aortic valves have a higher basal contractility than those of the surrounding healthier tissue. To better understand the link between the underlying biochemistry and mechanics of AVIC contraction, aiding in identifying druggable targets, we have developed an integrated cell signaling/mechanics computational model of endothelin-1-driven contraction, allowing for the separation of effects due to SF mass, spatial distribution, and unit contractility.


Materials and Methods:: A preliminary reaction diagram facilitated inhibitor target selection to alter actin polymerization and/or MLC2 phosphorylation, manipulating SF mass and/or unit contractility, respectively, resulting in the selection of GRK2/3, ROCK1/2, and GEFH1/LARG. Endothelin-1 time courses included ultracentrifugation followed by Western Blot to assess G/F-actin ratios and quantitative ICC of F-actin and MLC2 phosphorylation. These data, along with literature values, were used to parameterize a mechanistic model of endothelin-1-induced contractility.

The resultant cell signaling simulations were then used to predict the local SF mass fraction and intrinsic SF contraction levels. These outputs were subsequently integrated into a recently developed AVIC mechanics model of AVICs in traction force microscopy (TFM) studies to predict changes in endothelin-1-driven AVIC contraction.


Results, Conclusions, and Discussions:: Cell signaling results indicate that MLC2 phosphorylation and actin polymerization follow disparate time-dependent responses, with MLC2 activity dynamics occurring more quickly. VIC myosin phosphorylation and actin polymerization changes upon activation with endothelin-1 are GRK2/3, ROCK1/2, and RhoA GEF-dependent. The resultant AVIC mechanics model was able to successfully utilize the AVIC signaling model to accurately predict changes in AVIC shape and TFM forces.

This mechanistic model is now allowing us to delineate contributions to endothelin-1 contractility observed in 3D poly(ethylene)glycol gels in 3D traction force microscopy (TFM) due to SF mass and SF unit contractility. Novel to our approach is the ability to directly link AVIC signaling and mechanics modeling into a single integrated simulation. Future work will add uncertainty quantification to the cell signaling model, facilitating the design of new experiments.


Acknowledgements (Optional): :

References (Optional): :