Mechanotransduction in Cardiac Fibroblasts on a Novel Device Mimicking Myocardial Infarction

Introduction: : Stiffening of cardiac tissue is a hallmark of heart failure and significantly impairs the heart’s contractile function. Heart failure often originates from a localized injury, most commonly a myocardial infarction (MI), that progresses into a systemic condition affecting the entire organ. Following MI, the myocardium undergoes dynamic spatiotemporal remodeling, particularly in the infarct border zone and extending into remote, initially unaffected regions. We aim to understand how localized mechanical changes in stiffness communicate with the extracellular matrix (ECM) and neighboring cells to drive remodeling or scar expansion beyond the original site of injury. To replicate this evolving mechanical environment in vitro, we use magnetorheological elastomers (MREs), which allow reversible, bidirectional modulation of substrate stiffness by applying an external magnetic field. Here, we examined normal human cardiac fibroblasts (NHCF-Vs), focusing on phenotypic changes and mechanotransduction dynamics—specifically YAP nuclear translocation, actin stress fiber formation, vinculin phosphorylation, and RhoA activation—to investigate how mechanical cues are propagated through fibroblast networks. To quantitatively interpret the kinetics underlying the qualitatively observed changes, we propose an initial modeling framework to elucidate the mechanistic basis of the mechanosensitive signaling cascade implicated in the mechanobiology of heart failure progression.

Materials and

Methods: : We developed a custom culture platform to locally stiffen a small culture area. Designed in SolidWorks, the 60mm × 13mm platform was 3D-printed in PLA. Sylgard 527 was prepared at a 1:1 ratio (Part A:B) with 50 wt% carbonyl iron particles, mixed at 2500 rpm for 1 minute, and 5g was poured into each device. After degassing for 5–7 minutes, the polymer was pre-cured at 60 °C for 15 minutes and then cured overnight at 45 °C to form a magneto-rheological elastomer (MRE). Devices were sterilized, washed, and coated with 10 µg/mL fibronectin for 2 hours.
NHCF-Vs were seeded at ~450 cells/mm² (85–90% confluency). After overnight culture without a magnet, cells were cultured for 24 hours with either a magnet at position 1 or no magnet (control) to assess stiffness-dependent mechanosensitive responses. Responses were measured at four locations (13 mm apart) and tracked over time intervals (10 min, 30 min, 1 h, 3 h, 6 h, 24 h) to capture the dynamics of mechanosensitive pathways. Cells were stained for YAP, RhoA, activated RhoA, vinculin, phosphorylated vinculin, and F-actin, then imaged using a 20× dip-in lens on an upright microscope. Images were analyzed using FIJI and MATLAB.
We created a mathematical model to better understand the kinetics of the mechanobiological response to a point-wise mechanical stimulation. To model how mechanical dosing influences fibroblast crosstalk through mechanobiological pathways, we use coupled ordinary differential equations combined with a non-linear optimizer to model the fibroblast network and responses in RhoA, Vinculin, Actomyosin, and YAP.

Results, Conclusions, and Discussions:: This study simulates an acute injury environment with our dynamic MRE platform, whereby we can spatially capture mechanosensitive changes. Specifically, we focused on YAP translocation through the Rho signaling pathway. We observed spatiotemporal changes in RhoA activity, vinculin phosphorylation, and YAP nuclear localization extending outward from the stiffened region over time. These responses diminished in intensity while spreading spatially, suggesting the establishment of a spatiotemporal equilibrium (Figure 1). Notably, we identified distinct peak activation times for key mechanotransduction markers: RhoA at 10 minutes, vinculin at 1 hour, and both Actin and YAP at 3 hours post-stimulation. These findings demonstrate that cardiac fibroblasts rapidly respond to localized mechanical stimuli, with activation kinetics specific to each pathway. These initial results validate our ability to spatiotemporally tune stiffness using MREs and to direct cell responses accordingly, providing a powerful tool for dissecting mechanotransduction mechanisms in complex tissue environments.
To characterize signaling kinetics, we developed a mathematical model incorporating a forcing function, u(t), to simulate stiffness perturbations at the pathway's entry point. Time-delay elements captured the temporal progression between signaling nodes. The model closely aligns with experimental data across all pathway components (Figure 2). Estimated transfer rates (e.g kρ=1.509, kρV=3.4964, kV=2.0728, kVA=-0.9432, kA=0.27, kAY=1.6467, kY=1.6545; units: 1/hr) indicate transition speeds and directionality—where large positive values reflect slower transitions and negative values imply reverse flow. Notably, the model reveals a “leap-frogging” effect, suggesting RhoA may influence actin directly, partially bypassing vinculin.
To further dissect this phenomenon, we plan to prevent vinculin phosphorylation by using an adenoviral construct with silencing shRNA targeting human VCL. This approach will help determine whether mechanosensitive signaling pathways operate cooperatively or competitively to propagate mechanical cues across space and time. Additionally, the resulting data will support the refinement of our computational model, enhancing its ability to predict how fibroblast networks transmit mechanical signals.

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