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Aberrant chromatin reorganization in cells from diseased fibrous connective tissue in response to altered chemomechanical cues

Nature Biomedical Engineering volume 7, pages 177–191 (2023)Cite this article

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Abstract

Changes in the micro-environment of fibrous connective tissue can lead to alterations in the phenotypes of tissue-resident cells, yet the underlying mechanisms are poorly understood. Here, by visualizing the dynamics of histone spatial reorganization in tenocytes and mesenchymal stromal cells from fibrous tissue of human donors via super-resolution microscopy, we show that physiological and pathological chemomechanical cues can directly regulate the spatial nanoscale organization and density of chromatin in these tissue-resident cell populations. Specifically, changes in substrate stiffness, altered oxygen tension and the presence of inflammatory signals drive chromatin relocalization and compaction into the nuclear boundary, mediated by the activity of the histone methyltransferase EZH2 and an intact cytoskeleton. In healthy cells, chemomechanically triggered changes in the spatial organization and density of chromatin are reversible and can be attenuated by dynamically stiffening the substrate. In diseased human cells, however, the link between mechanical or chemical inputs and chromatin remodelling is abrogated. Our findings suggest that aberrant chromatin organization in fibrous connective tissue may be a hallmark of disease progression that could be leveraged for therapeutic intervention.

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Fig. 1: Altered nanoscale H2B localization in hTCs with degeneration.
Fig. 2: Substrate stiffness regulates H2B localizations in hMSCs.
Fig. 3: Substrate-stiffness-dependent histone methylation in hMSCs.
Fig. 4: A phase-field model of heterochromatin formation incorporating the kinetics of acetylation and methylation recapitulates experimental results.
Fig. 5: Effect of in situ stiffening on nanoscale chromatin organization in hMSCs.
Fig. 6: Altered nanoscale H2B localization in hTCs with presentation of degenerative chemophysical cues.
Fig. 7: Altered mechanical sensitivity of hTCs with tissue degeneration and ageing.
Fig. 8: Proposed model for how substrate stiffness alters nanoscale chromatin organization, mediated by changes in cellular contractility or Rho–ROCK pathways and mechano-activation and nuclear localization of chromatin modifiers.

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Data availability

The authors declare that the main data supporting the findings in this study are available within the paper and its Supplementary Information. The raw STORM super-resolution data files are too large to be publicly shared but are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

The custom code for the super-resolution image analysis is available at https://github.com/melikelab/Su-Chin/tree/main/New%20folder.

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Acknowledgements

The authors thank K.H. Song for assistance with the design of the microfluidic chamber, E. Sorokina for assistance with the preparation of reagents for STORM imaging and P. K. Relich for help with data visualization and analysis tools. The research was funded by the National Institutes of Health (grant no. K01 AR07787 to S-J.H.), Penn Center for Musculoskeletal Disorders (grant no. P30 AR069619 to S-J.H, M.L. and R.L.M.), Department of Veterans Affairs (grant no. IK6 RX003416 to R.L.M.), R01 GM133842 (to M.L.), U01DA052715 (to M.L.) and NSF Science and Technology Center for Engineering Mechanobiology (grant no. CMMI-1548571 to M.L., R.L.M and V.S.). The theoretical work was supported by National Cancer Institute Awards R01CA232256 and U54CA261694; National Institute of Biomedical Imaging and Bioengineering Awards R01EB017753 and R01EB030876; and NSF Grants MRSEC/DMR-1720530 and DMS-1953572.

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  1. These authors contributed equally: Robert L. Mauck, Melike Lakadamyali.

Authors and Affiliations

  1. McKay Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Su-Jin Heo, Boao Xia & Robert L. Mauck

  2. Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, USA

    Su-Jin Heo, Claudia Loebel, Boao Xia, Jason A. Burdick, Vivek B. Shenoy & Robert L. Mauck

  3. Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA, USA

    Su-Jin Heo, Xingyu Chen, Claudia Loebel, Jason A. Burdick, Vivek B. Shenoy, Robert L. Mauck & Melike Lakadamyali

  4. Translational Musculoskeletal Research Center, Corporal Michael J. Crescenz VA Medical Center, Philadelphia, PA, USA

    Su-Jin Heo & Robert L. Mauck

  5. Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Shreyasi Thakur & Melike Lakadamyali

  6. Department of Materials Science Engineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, USA

    Xingyu Chen & Vivek B. Shenoy

  7. Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA, USA

    Rowena McBeath

  8. BioFrontiers Institute and Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, USA

    Jason A. Burdick

  9. Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA

    Melike Lakadamyali

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Contributions

S.-J.H., S.T., X.C., C.L., B.X., R.M., J.A.B., V.B.S., R.L.M. and M.L. designed the studies, and analysed and interpreted the data. S.-J.H., S.T., X.C., C.L. and B.X. performed the experiments. S.-J.H., R.L.M. and M.L. drafted the manuscript, and all authors edited the final submission.

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Correspondence to Robert L. Mauck or Melike Lakadamyali.

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Extended data

Extended Data Fig. 1 Custom-PDMS microfluidic chamber, and changes in chromatin distribution.

(a) Picture and schematic of a custom-PDMS microfluidic fluid shear stress (FSS) device. (b) Representative STORM super-resolution images of H2B rendered as a density map showing redistribution of H2B in hMSCs in response to FSS of varying magnitude 1 ~ 5 dyne/cm2) and duration (0.5 ~ 2 h). (c) Quantification of changes in the ratio of the total number of H2B localizations per unit area at the nuclear border to the total number of H2B localizations at the inner part of the nucleus with/without the application of FSS (*: p < 0.05 vs. Ctrl, +: p < 0.05 vs. 1D/0.5 h, a: p < 0.05 vs. 5D/0.5 h). The values have been normalized to Ctrl cells. (d) Changes in chromatin condensation with the application of FSS in (d) sparse chromatin compartment or (e) dense chromatin compartment in hMSCs. The Voronoi polygon density of the dense or the sparse chromatin compartment in cells subjected to FSS is shown normalized to the Voronoi polygon density in the absence of FSS (n = 5 nuclei/group, *: p < 0.001 vs. Ctrl, +: p < 0.001 vs. 1D/0.5 h, a: p < 0.001 vs. 5D/0.5 h, one-way ANOVA). Experiments were carried out at least in duplicate. Error bars, means ± s.d.

Source data

Extended Data Fig. 2 Changes in chromatin condensation with substrate stiffness in sparse chromatin or dense chromatin compartments.

a,b, The Voronoi polygon density of the sparse (a) or dense (b) chromatin compartment is shown normalized to the Voronoi polygon density in human tenocytes grown on Glass, showing a decrease/increase in the condensation of dense chromatin compartment on stiff/soft substrates (n = 5 nuclei/group, *: p < 0.001 vs. glass, +: p < 0.001 vs. soft, one-way ANOVA). Changes in chromatin condensation in sparse chromatin (c) or dense chromatin (d) compartments. The Voronoi polygon density of the sparse or dense chromatin compartment is shown normalized to the Voronoi polygon density in human young tenocytes grown under normoxic (N) conditions, showing an increase in the condensation of dense chromatin under hypoxic (H) conditions (n = 5 nuclei/group, *: p < 0.001 vs. young healthy tenocyte under the normoxic conditions, +: p < 0.001 vs. tendinosis tenocyte under the normoxic conditions, one-way ANOVA). Changes in chromatin condensation with exposure to inflammatory cytokines in sparse chromatin (e) or dense chromatin (f) compartments. The Voronoi polygon density of the sparse or dense chromatin compartment is shown normalized to the Voronoi polygon density in human young tenocytes without treatment with inflammatory cytokines, showing an increase in the condensation of dense chromatin upon treatment (n = 5 nuclei/group, *: p < 0.001 vs. young human tenocyte control, +: p < 0.001 vs. tendinosis young human tenocyte, One-way ANOVA). Error bars, means ± s.d.

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Heo, SJ., Thakur, S., Chen, X. et al. Aberrant chromatin reorganization in cells from diseased fibrous connective tissue in response to altered chemomechanical cues. Nat. Biomed. Eng 7, 177–191 (2023). https://doi.org/10.1038/s41551-022-00910-5

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