Poster P15 - Divergent responses in decoupling the role of stiffness and contraction dynamics in cardiomyocyte behavior

Introduction: In cardiovascular mechanobiology, the cardiac tissue adapts rapidly to changing biomechanical parameters, and understanding how mechanical forces influence cellular function is essential [1]. Nuclear mechanics, including nuclear shape, chromatin architecture, and intranuclear strain, contribute to cellular responses [2]. Specifically, within cardiac tissue, cardiomyocytes and cardiac fibroblasts utilize mechanotransduction pathways to detect alterations in tissue stiffness and modify gene expression [3]. Mechanosensing systems within cells directly or indirectly measure mechanical parameters, such as changes in environmental stiffness which impact processes such as DNA damage response [4]. Our laboratory has previously shown in cardiomyocytes that increased substrate stiffness influences contraction dynamics, nuclear architecture, and intranuclear strains which guide cell differentiation [2,5]. With the adaptation of multiple mechanical stresses, it is still unclear if changes in the biomechanical properties of the cardiomyocyte (e.g., contraction) or mechanical environment (e.g., stiffness) are driving cell fate.

To study this, we modulated the cardiomyocyte contraction cycle through two treatments, increased substrate stiffness and calcium signaling. We examine intranuclear mechanics on soft (i.e., healthy) and stiff (i.e., diseased) substrates, and with altered contraction dynamics using an L-type calcium channel blocker, verapamil, on soft substrates. Studies using verapamil have shown decreased contraction frequency and contraction velocity [6] which is a similar behavior to cardiomyocytes exposed to stiff substrates, representing that of a diseased environment [5,7]. The objective of this study is to decouple the role of stiffness and altered contraction dynamics for an improved understanding of how mechanical stresses influence cardiomyocyte nuclear mechanics, and in turn, cellular differentiation.

Materials and

Methods: Substrate Preparation: 3.5cm2 ibidi dishes were coated with 100µm layer of Sylgard 527 PDMS at two ratios (A:B), 1:1 (Soft, healthy) and 1:4 (Stiff, diseased). Cell Culture: Primary cardiac cultures were derived from embryonic (E18.5) H2b-eGFP mice (Jax# 00609) for live imaging or C57BL/6J (Jax# 00064) for RNA sequencing. Embryonic hearts were isolated, digested, and enriched for cardiomyocytes by plating on TCP for 1 hr at 37°C prior and then plated at a concentration of 1x106 cells/mL on Geltrex-coated PDMS dishes. On day 4, cultures were live imaged on a Nikon AIR confocal in a temperature-controlled incubation chamber and then fixed with 4% PFA or lysed in Qiazol. Chemical Treatments: Cultures on Soft PDMS were treated with 814nM Verapamil or DMSO (0.025%, vehicle control). Cultures on Stiff PDMS received DMSO (0.025%) only. Microscopy & Analysis: Fixed cells were stained with Phalloidin AF405, gH2A.X (561nm), and imaged with a 60X objective. Max intensity z-projected stacks from were created in ImageJ and gH2A.X foci were counted using a custom MATLAB code. Strains were calculated from undeformed and maximum deformed template images from the live imaging run through deformation microscopy. RNA sequencing: RNA was isolated, and poly A-selected RNA was sequenced at the UC Anschutz. Data were processed using the Nextflow nf-core/rnaseq (version 3.11.2) with alignment to the mm10 genome. The resulting counts matrix was normalized using DESeq2’s rlog method. A heatmap of the top 50 genes with the highest variance across treatments was generated with pheatmap.

Results, Conclusions, and Discussions: Live imaging of nuclear deformation during the contraction cycle on different substrates and altered calcium dynamics revealed changes in frequency (data not shown) and intranuclear strains. Using an imaged-based displacement tracking tool developed in our laboratory, deformation microscopy, we calculated intranuclear strains from undeformed and deformed images for each treatment (Fig 1A). A decreasing trend in absolute hydrostatic strain was observed for nuclei in which contraction dynamics were changed or were plated on stiffer substrates (Fig 1B). Dividing hydrostatic strain into its tensile and compressive components, we observed differing trends based on substrate stiffness or contraction dynamics. For hydrostatic tensile strain, cardiomyocytes treated with Verapamil had similar intranuclear strains as cardiomyocytes on stiff substrates (Fig 1B). However, for compressive strain, we observed cardiomyocytes treated with Verapamil had similar strains to cardiomyocytes plated on soft substrates. Given the diffuse signal of the inherent GFP fluorescence in the nucleus, future processing will include binning the GFP intensity to determine if these trends map to heterochromatin or euchromatin regions of the nucleus. As contraction dynamics are modified by either substrate or chemical treatment, we examined how this might affect the DNA damage response. Staining for ɣH2A.X, a marker of double stranded breaks, we quantified the number ɣH2A.X foci per nucleus in all treatments. Interestingly, we found that stiffness had a significant effect on the DNA damage response represented by increased foci per nucleus (Fig 1C). Finally, we performed RNA sequencing and examined the variance between the three treatments. We found a greater effect in gene expression with the Verapamil treatment than by substrate alone (Fig 1D).

Decoupling the influence of stiffness and contraction dynamics in cardiomyocyte mechanics has potential to improve understanding of cardiomyocyte differentiation through alteration in nuclear mechanics. Subsequent studies will merge strain maps from deformation microscopy to map nuclear architecture and ɣH2A.X foci with intranuclear strains. Differences in the DNA damage response and gene expression between the three treatments might indicate unique pathways of differentiation through alterations in nuclear mechanics. Future work will continue to distill mechanical mechanisms influencing cardiomyocyte mechanobiology and differentiation.

Acknowledgements (Optional): Acknowledgements: This work was supported by funding from NIH R01 AR063712, NSF 2212121.

References: [1] Münch and Abdelilah-Seyfried, Front Cell Dev Biol., 26:9:642840., 2021. [2] Seelbinder, B et al., Nat Biomed Eng., 12:1500-1516. 2021. [3] Powers and McCulloch, Curr Opin Physiol., 25:100482, 2022. [4] Dos Santos and Toseland, Int J Mol Sci., 22(6):3178, 2021. [4] Ghosh, S et al., Cell Rep., 27(5):1607-1620. 2019. [6] Lam, CK et al., Circ Res.,125(2):212-222, 2019. [7] Schuftan, D et al., J Biomed Mater Res.,112:534–548., 2024