I13 - Increased Nuclear Reorganization Following Repetitive Stiffening in NIH-3T3 Fibroblasts

Introduction: : Fibroblasts continuously sense and transfer mechanical signals from the extracellular environment to their nucleus. Nuclear mechanosensing is critical for quiescence, but sustained mechanical imbalances can trigger an irreversible phenotype that negatively impacts tissue composition [1]. Fibroblast mechanosensitivity is responsible for fibrotic transitions that occur following acute conditions such as myocardial infarction [2]. Mechanistically, nuclear deformation alters transcriptional activity by stimulating chromatin reorganization [3]. Prior work has revealed that chromatin reorganization occurs within minutes of a mechanical stimulus and can permanently impact cell fate depending on the intensity and duration [4,5]. Specifically, methylation of Histone 3 Lysine 27 (H3K27me3) has been shown to compact chromatin and consequently reduce its accessibility following nuclear deformation [1]. Monitoring H3K27me3 activity in response to repeated mechanical stresses, such as cyclic stiffening, can reveal the extent of plasticity the nucleus has before it becomes unresponsive to restored mechanical homeostasis. Clinically, fibroblast nuclear plasticity may provide insight into timeframes for disease intervention and therapeutic delivery. Unfortunately, the impact of repetitive mechanical stress on nuclear plasticity is not completely understood. Experimental models that instantaneously deliver biophysical stimulation are advantageous because they provide controlled starting points to investigate nuclear mechanobiology. Magnetorheological elastomers (MREs) are functional substrates that grant instantaneous stiffening due to their reactivity to applied magnetic fields [6]. Ferrous particles embedded within a viscoelastic polymer provide a dynamic cytocompatible substrate to study nuclear mechanobiology. Herein, we deliver precise stiffening cycles to NIH-3T3 fibroblasts using an MRE substrate and quantify nuclear reorganization with each cycle.

Materials and

Methods: : MRE Preparation: Sylgard 527 was prepared according to the manufacturer’s directions, mixed with carbonyl iron powder at varying PDMS:iron powder mass ratios for stiffness characterization and a 1:1 PDMS:iron powder mass ratio for proceeding experiments. The mixture was poured into custom dishes, degassed for 10 min, and cured overnight at 60°C.
Mechanical characterization: Bulk MRE dynamic stiffness was measured (Fig. 1B). Compressive moduli were obtained using a DMA (10-lbf load cell/4mm spherical probe). A quasistatic load was applied up to 30% strain in three distinct areas per sample, with and without a magnet. Force and position data were converted and plotted as stress and strain. The compressive modulus was determined to be the linear slope for each curve.
The strain generated on MRE surfaces was quantified. MREs were treated with 50g/ml fibronectin overnight, incubated with fluorescent beads and imaged on a confocal microscope before and after magnet application. Image stacks were processed through the Particle Image Velocimetry plugin on ImageJ to obtain position vectors and then ran through a custom MATLAB code to calculate strain (Fig. 1D).
Experimental

Design: NIH-3T3 fibroblasts were seeded for 48hrs on stiff (magnet) or soft (no magnet) MREs. The experimental group then underwent four 1hr stiffening cycles with 1hr softening between each cycle (Fig. 1C). Samples were fixed according to the timepoints in Fig. 1C, stained with DAPI, Phalloidin, and H3K27me3, and imaged on a confocal microscope (40x objective). A custom MATLAB code quantified foci count, nuclear aspect ratio, and nuclear area (Fig. 1E).

Results, Conclusions, and Discussions:: We utilized a dynamic in vitro substrate to examine the impacts of repetitive stiffening on NIH-3T3 nuclear reorganization (Fig. 1A). We confirmed a 1:1 PDMS:iron powder mass ratio with a 5mm depth provided the greatest MRE stiffness range and closely resembled native heart stiffness (Fig. 1B) [6]. Bead displacement on coated MRE surfaces revealed an average strain of 2.23% when stiffened (Fig. 1D). This strain is hypothesized to be induced by the alignment of iron particles within the elastomer. We quantified nuclear area, aspect ratio, and H3K27me3 foci in the mechanically treated NIH-3T3s (Fig. 1E). In these preliminary studies, we found that the stiff control and all experimental groups displayed increased nuclear area relative to the soft control. The aspect ratio following one round of stiffening more closely resembled the soft control, while the subsequent rounds experienced a slight increase and became more indicative of the stiff control. Finally, an increase in H3K27me3 foci was observed as stiffening cycles progressed relative to the controls. Interestingly, a jump in foci was observed in the later cycles (cycles 3-4) compared to the earlier cycles (cycles 1-2).
Here, we show that modulating stiffness induces concomitant strain on MRE surfaces. We use the reversible stiffening capability of the MRE to monitor nuclear reorganization over four stiffening cycles in NIH-3T3 fibroblasts. Trending increases in H3K27me3 foci, nuclear area, and aspect ratio with increasing cycles suggest that repeated rounds of stiffening may alter cell fate in a manner that reduces their cellular plasticity. Incorporating more stiffening cycles with increased recovery time may elucidate the ability of the cell to revert to baseline nuclear organization. Future work will extend the sample size to verify trends and expand into the role of H3K27me3 in mechanical memory. Complementing changes observed in nuclear morphology with cytoskeletal organization and nuclear mechanics may add context to the potency of nuclear reorganization. Finally, extending this approach to human-based cardiac cells will improve translational relevance in modeling fibrotic epigenetic mechanisms. This study opens the door for future work surrounding mechanical memory or the effects of cell therapeutics on nuclear mechanobiology.

Acknowledgements and/or References (Optional):: This work was supported by funding from NIH R01 AR083379 and T32 EB034215.
[1] Felisbino, M. B. et al., American Journal of Physiology-Heart and Circulatory Physiology, H61–H73, 2023. [2] Braidotti, N. et al., Sci Rep, 10365, 2024. [3] Seelbinder, B., et al., Nat Biomed Eng, 1500–1516, 2021. [4] Walker Cierra J. et al., Nature Biomed Eng, 1485-1499, 2021. [5] Heo, SJ. et al., Sci Rep, 16895, 2015. [6] Corbin Elise A. et al., ACS Applied Materials & Interfaces, 20603-20614, 2019. [7] Berry Mark F. et al., American Journal of Physiology-Heart and Circulatory Physiology, H2196-H2203, 2006.