M4 - Senescent cardiac fibroblasts alter cardiomyocyte nuclear mechanics

Introduction: : Cellular senescence, initially defined as an arrest in cell cycle, is now understood to be induced by factors such as oncogene disruption, irradiation, chemotherapy, developmental cues, and tissue damage [1,2]. These factors give rise to subpopulations of senescence cells with unique biophysical features, gene expression profiles, and secretory phenotypes [3]. The contribution of senescent cells in the progression of disease is being examined and targeted in aging and disease states such as osteoarthritis, cancers, and cardiac disease, the leading cause of mortality in the United States [3,4]. With cardiovascular disease and aging, tissue stiffening coupled with altered contraction mechanics (e.g., increased contraction frequency, decreased strain magnitude), and heterogeneity of the cellular populations including increased prevalence of senescent cells. While senolytics have shown improved cardiac function [3], the impact of senescent cells on tissue, cellular, and nuclear mechanics remains under investigation and is a target for potential therapeutics to improve functional and structural integrity of the tissue.
In cardiovascular mechanobiology, the cardiac tissue adapts rapidly to changing biomechanical parameters, and understanding how mechanical forces influence cellular fate is essential [5]. Nuclear mechanics, including chromatin architecture, and intranuclear strain, contribute to cellular responses [6]. Our laboratory has previously shown in cardiomyocytes (CMs) that increased substrate stiffness influences contraction dynamics, nuclear architecture, and intranuclear strains which guide cell differentiation [6,7]. Using this established in vitro model, we combined premature senescent cardiac fibroblast (SntCFs) with primary murine CMs co-cultures to examine how SntCFs influence cellular structural integrity, specifically within the cardiomyocyte nucleus.

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. Embryonic hearts were isolated, digested, and cells were plated on TCP at 37°C. After 1 hr, non-adherent (CM-enriched) cells were collected and plated at a concentration of 1x106 cells/mL on Geltrex-coated PDMS dishes. Adherent cardiac fibroblasts (CFs) were cultured on TCP for 7 days after CM removal. Treatments: On day 7, CFs were treated with 1 µM Doxorubicin for 24 hrs [8], labeled with Cell Tracker CMRA (5µM), and transferred to the CM cultures on Stiff PDMS at a 10% (i.e., 1:10 CF:CM) ratio (based on initial cell plating concentration). CM cultures on Soft and Stiff PDMS without SntCFs served at culture-time controls. CM/CFs ± SntCFs were co-cultured for an additional 4 days before live imaging and fixing (Fig. 1A). Doxorubicin-treated CMRA-labeled CFs were cultured as confirmation of a senescent phenotype. Microscopy & Analysis: Nuclei of CMs were imaged on DIV12 in an Oko incubation chamber (5% CO2, 37°C) with a 60× objective. Fixed cells were stained with Phalloidin AF405, gH2A.X (633 nm), and imaged with a 60× objective. Morphological analysis and ɣH2A.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 [7]. Statistical analysis and graphs were made in OriginPro.

Results, Conclusions, and Discussions:: The study explored how a pathologically stiff environment, mimicking disease, affects cardiomyocyte mechanics during culture with SntCFs (Fig. 1A). Live imaging of nuclear deformation during the contraction cycle on different substrates and with the addition of SntCFs revealed alterations in 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. 1B). A significant difference in the absolute hydrostatic strain was observed for CM nuclei plated on Soft PDMS compared to those on Stiff PDMS or on Stiff PDMS plus the addition of SntCFs (Fig. 1C) indicating both substrate stiffness and SntCFs alter CM nuclear mechanics. Dividing hydrostatic strain into its tensile and compressive components, we observed differing trends based on substrate stiffness and SntCFs. Interestingly, the decreased absolute hydrostatic strain observed in the Stiff PDMS and Stiff PDMS with SntCFs was mainly driven by significant decreases in the tensile component (Fig. 1B). However, no difference in the compressive component was found between the three groups. We analyzed the 3 groups for morphological differences and stained for ɣH2A.X, a marker of double stranded breaks and DNA damage. CMs on Stiff PDMS with SntCFs had increased nuclear area compared to CMs on Soft or Stiff PDMS (Fig. 1D). Interestingly, we did not observe an increase in ɣH2A.X foci in the CMs on Stiff PDMS with SntCFs (Fig. 1D). However, while no difference in foci per nucleus was observed in the Stiff PDMS with SntCF group, we did observe the relative size of the foci in the nuclei were larger than CM on Soft or Stiff PDMS. Additional work is expected to quantify this finding.
In this study, we show how the addition of SntCFs alters cellular mechanics and morphology. Specifically, we found significant decreases in absolute hydrostatic strain, driven mainly through decrease tensile strain, and increased nuclear area in co-cultured groups. Since many senescence markers are linked to changes in nuclear structure and integrity, these findings, through examination of the CM nucleus, demonstrate the importance of understanding how senescence influences cellular and nuclear mechanics.

Acknowledgements and/or References (Optional):: Acknowledgements: This work was supported by funding from NIH R01 AR063712 and NSF 2212121.

References: [1] Rhinn, M et al., Development. 146(20), 2019. [2] Rodier, F et al., J Cell Biol, 192(4):547-556, 2011. [3] Chaib, S et al., Nat Med., 28(8):1556-1568, 2022. [4] Roger, L et al., Int J Mol Sci., 22(23):13173., 2021. [5] Münch and Abdelilah-Seyfried, Front Cell Dev Biol., 26:9:642840, 2021. [6] Seelbinder, B et al., Nat Biomed Eng., 12:1500-1516, 2021. [7] Ghosh, S et al., Cell Rep., 27(5):1607-1620, 2019. [8] Skaggs, C et al., BMC Res Notes. 16:340, 2023.