Subphysiological loading initiates a mechanically-induced premature senescent state in cardiac fibroblast cultures

Introduction:: While originally characterized as an arrest in cell cycle, cellular senescence is now understood to be induced by extrinsic factors such as oncogene disruption, irradiation, chemotherapy, developmental cues, and tissue damage. These extrinsic factors have been shown to give rise to a new form of senescence, stress-induced senescence [1]. The contribution of senescent cells in the progression of diseases is now being examined and targeted in disease states such as osteoarthritis, solid tumor cancers, and cardiac disease [1-3]. Further exploration defining the biomarkers of the senescent phenotype are necessary as a combination of biophysical and biochemical is required to identify senescent cells. Interestingly, alterations in the nuclear area, chromatin organization and disruption of nuclear envelope proteins are all established markers of senescence. While chemical treatments have been shown to induce senescence, mimicking those cells found in several disease states, it is still unclear how mechanical perturbations initiate cellular senescence.

Using a simplified in vitro model of cardiovascular disease, we designed our loading scheme to mimic the alterations of contractile dysfunction which manifest as decreased strain and increased frequency [4,5]. During cardiovascular disease, remodeling of the extracellular matrix leading to this contractile dysfunction is mainly attributed to the cardiac fibroblasts [6]. Our objective is to study how mechanical perturbations may initiate a premature senescent state, and more specifically, alterations in the nucleus. We compared our findings with a common method to induce senescence through ROS (reactive oxygen species, H₂0₂) treatment.


Materials and Methods:: Cell culture: Primary murine cardiac fibroblast (CF) cultures were derived from Lmna wild type (WT) mice (Jackson Labs, Stock No.: 009125) between 3-9 weeks of age with no exclusion of gender. Minced cardiac tissue was digested with 0.2% Collagenase P for 35mins at 37°C, filtered, and plated in petri dishes for 2hr at 37°C. After 2hrs, non-adherent cells were removed and remaining cells received D10 media (DMEM/F12, 10% FBS, 1X Pen/Strep, 1µM ascorbic acid). CFs were cultured to confluency with passage 1 and 2 cultures being used for all experiments. Mechanical Stimulation: CFs were cultured on Geltrex coated membranes and uniaxially stretched by a MCFX device (CellScale) for two 4-day cyclic routines: Control and Perturbed. For the Control routine, 8.5% strain at 1 Hz was applied for 4 days. For the Perturbed routine, cells experienced 1 day at 8.5% strain (1 Hz) and 3 days at 2.5% strain (2 Hz). Cells were fixed in 4% PFA on day 4. Chemical Stimulation: CFs were treated 3 times with 300µM H₂0₂ for 2hrs over 6 days and then cultured for an additional 8 days before being fixed with 4% PFA. Microscopy & Analysis: Fixed cells were stained to visualize Lamin B, Emerin, DAPI, and Phalloidin Texas-Red and imaged with a 60X objective on a Nikon AIR confocal. A custom MATLAB code calculated the Lamin B or Emerin thickness. Statistics: A linear mixed effects model with Type II ANOVA was used to examine interactions and Tukey post-hoc tested for means comparisons between groups.


Results, Conclusions, and Discussions:: Examining the nuclear area revealed that CFs exposed to perturbed loading had a slightly increased nuclear area compared to Control CFs (Fig 1A). In contrast, senescent cells induced through chemical treatment (ROS) had a significant increase in nuclear area (Fig 1B). Analyzing a second marker of cellular senescence, Lamin B, we found a significant decrease in the Lamin B ring thickness of the Perturbed CFs compared to Control CFs (Fig 1A). This was similarly observed in ROS-induced senescent CFs, suggesting an induction of a senescent-like state in the Perturbed CFs (Fig 1B). Given that many alterations and markers found in senescent cells also overlap with modifications in the nucleus and nuclear envelope, we examined mechanically-sensitive proteins in the nuclear envelope. Using the same mechanical loading scheme, we next looked for the presence of Emerin. Perturbed CFs showed a significant decrease in Emerin ring thickness compared to Control CFs (Fig 1C). However, ROS-induced senescent CFs did not show any alteration in Emerin ring thickness compared to CFs plated on TCP (Fig 1D).

In this study, we show how subphysiologic cyclic loading of CFs can induce a premature senescent-like state. Increases in cellular and nuclear area along with decreases in Lamin B are established markers of cellular senescence [1]. Perturbed loading conditions resulted in decreased Lamin B ring thickness similar to ROS-induced senescent CFs. With further examination of nuclear envelope proteins which may be involved in the induction of senescence, we found a decrease in Emerin ring thickness which was not observed with ROS-induced senescent CFs. This may indicate separate and unique pathways of initiation of a senescent state. However, it should be noted that ROS-induced senescent cells were in culture an additional 12 days which could explain the increased nuclear area. Future work will further define the premature mechanically-induced senescent-like state of the CFs through established markers of senescence including decreases in proliferation. Additionally, examination of other nuclear envelope proteins will help to establish the role of nuclear alterations in the cellular senescent phenotype.


Acknowledgements (Optional): : This work was supported by funding from NIH R01 AR063712, NSF 2212121, and Biophysics Training Grant T32 GM-065103

References (Optional): : [1] Rodier, F et al., J Cell Biol, 192(4):547-556, 2011. [2] Gualda-González, E et al., FEBS J, 288(1):56-80, 2021. [3] Childs, B et al., J Clin Invest, 128(4):1217-1228, 2018. [4] Tang, X et al. Front Endocrinol, 11: 280, 2020. [5] Ugolini, G et al., eLife, 6, 2017. [6] van Putten, S et al., J Mol Cell Cardiol, 93:133-142, 2016.