(H-285) Phenotype Propagation through Cardiac Injury by Biomechanical Perturbations

Introduction:: Stiffness is a key marker of heart failure that diminishes the contractile function of the heart. The development of heart failure is an evolving sequence of events that typically stems from a localized pathologic process, the most common being myocardial infarction, that progresses into a whole organ disease. While it is widely accepted that stiffness profoundly influences cardiac remodeling, it is unknown how stiffening of the local myocardium is transduced to the remaining healthy tissue. Specifically, a cell can be triggered by its local matrix not shared with its neighbor, but in turn, provide mechanical cues to those neighbors. Therefore, we have designed a magnetically tunable platform that mimics an index event through local control of stiffness, where we can measure mechanosensitive biomarkers propagation within sub-cellular, cellular, and super-cellular networks beyond the stiffened area.


Materials and Methods:: Our novel platform uses magneto-rheological elastomers that allow for a small, localized region of a population of cells to be mechanically stressed and relaxed through a tunable and reversible change in substrate modulus. Critically, this innovative platform uniquely allows us to examine how the effects of stiffening spread both spatially and temporally. We designed our culture platform with “lego-like” alignment slots for magnets on the backside (Fig. A) as well as the sidewalls were designed for easy insertion of immersion objectives. MREs consist of Sylgard 527 elastomers (Dow Corning) added with carbonyl iron particles.1 The presence of an applied magnetic field can control the stiffness of the MRE. We used an axially magnetized N42 neodymium rare earth magnet, with a magnetic flux density of 440 mT at the surface of the magnet in the center. We measured the elastic modulus of the MRE at different locations using micro indenter and compared the values with a magnetic model on COMSOL. Normal Human Cardiac Fibroblasts (NHCF-V) were cultured on fibronectin coated MREs at ~450 cells/mm² (85-90% confluency) for 24 hours and the cells were fixed, stained, and imaged (Zeiss AxioImager) at different time intervals (10mins, 30 mins, 1h, 3h, 6h, and 24h) for investigating time-sensitive biomarkers. We focused on studying the mechanosensitive proteins RhoA, Vinculin, and YAP, which have shown control over contractility, proliferation, and cellular behavior by cytoskeleton remodeling.


Results, Conclusions, and Discussions:: Results and Discussion: We both modeled and experimentally verified (Fig. A) the elastic modulus of the MRE using microindentation and found that the local stiffened region could be stimulated from physiologic, 15kPa, to pathologic, 96kPa, stiffness of the human heart2 while the rest of the substrate remained at physiologic stiffness. To explore the influence of localized stiffness, we first looked at YAP translocation with position. We observed a statistical difference of YAP translocation at both 1 and 3 hrs post stiffening Fig. B, however the difference in magnitude between time points compelled efforts to extend our time course. We constructed heat-maps that illustrate the spatio-temporal fold-changes of each mechanosensitive protein upstream from YAP (Fig. C). This side-by-side portrayal accentuates the ability of our tool to drive a wave propagation of a mechanosensitive pathway from a local biomechanical cue originating at the extracellular matrix. Overall, we see RhoA activating quickly < 10mins and then showing a spike in the propagation with Vinculin at 1hr and YAP at 3hr. From this wave of propagation in time, we can determine the magnitude, distance, and speed at which these mechanosensitive proteins activate.

Conclusion: Our unique model establishes that local biomechanical signals originating from the extracellular matrix can be propagated within a cellular network of cardiac tissue. Our preliminary results show that within the cellular network, the mechanosensitive pathways get activated with time and propagate in space beyond the originated area allowing us to understand how fast and far cardiac tissue remodels post-myocardial infarction. Ultimately, this will allow us to better understand cellular crosstalk providing a mechanistic insight to better model cardiac tissue function and diseases. This can also be a path forward to novel and targeted pharmacological therapies for cardiac injury.


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References (Optional): : [1] Corbin, E.A., et al. ACS (2019).
[2] Janmey, P. A., et.al. Physiol. Rev. (2020).