Deciphering the distinct roles of extracellular matrix ligands and mechanics in cardiac aging

Introduction: : Cardiac tissue is remodeled during aging, characterized by alterations in extracellular matrix (ECM) ligand organization and composition, as well as increased tissue stiffness from ~10 kPa to ~40 kPa. Cardiac fibroblasts (CFs) are responsible for maintaining ECM homeostasis, but are affected by these age-related pro-fibrotic biochemical and mechanical stimuli, ultimately leading to age-related cardiac dysfunction. For example, both the increased collagen VI and stiffness in aging cardiac tissue have been shown to facilitate CF activation, leading to a positive feedback loop that exacerbates aberrant ECM remodeling. It is therefore important to investigate the specific roles of ECM biochemical and mechanical cues in dictating CF dysfunction in aging. However, most current biomaterial systems cannot decouple these two ECM cues in native contexts. Thus, we developed a novel biomaterial strategy termed DECIPHER (DECellularized In situ Polyacrylamide Hydrogel-ECM hybRid) that consists of a hybrid decellularized ECM-synthetic hydrogel scaffold and is capable of independently tuning native ECM ligands and mechanics. In this way, we can control ‘ligand age’ (young vs. aged ECM) and ‘mechanical age’ (soft vs. stiff), identifying the impact of these two cues on age-specific CF functions.

Materials and

Methods: : To determine native tissue properties, ECM composition and stiffness were measured in aged (~24 months) vs. young (~2 months) murine hearts using TMT-quantitative mass spectrometry and nanoindentation, respectively. To fabricate DECIPHER scaffolds, vibratome sections of young and aged heart tissue were linked to acrylamide solutions (~10 or 40 kPa, representing young and aged tissue stiffness, respectively), polymerized to stabilize tissue proteins, and decellularized in situ. This results in four scaffold combinations: young/soft, young/stiff, aged/soft, and aged/stiff. Scaffold composition and architecture pre- and post-decellularization were examined by immunohistochemistry (IHC) and quantitative assays. Nanoindentation was carried out to assess scaffold stiffness and viscoelasticity. Isolated primary young and aged CFs were seeded on the samples to investigate the influence of ECM ligands vs. mechanics on CF activation, mechanosensing, and senescence using RNA sequencing, RT-qPCR, and immunofluorescence (IF).

Results, Conclusions, and Discussions:: Compared to young tissue, aged ECM exhibits distinct compositional changes (e.g., increased collagen VI, laminin a2, and fibronectin - Figure 1B), architectural remodeling (e.g., reduced alignment and endpoints, Figure 1C), and matrix stiffening (e.g., ~40 vs. ~10 kPa). DECIPHER substrates showed the removal of cellular structures while maintaining native ECM composition and architecture (Figure 1A), while nanoindentation confirmed the tunability of substrate stiffnesses independent of the native ECM mechanics (~10 kPa to mimic young and ~40 kPa to mimic aged myocardial stiffness, Figure 1D). Dynamical mechanical analysis using nanoindentation showed DECIPHER can mimic viscoelastic properties of native tissues, which has been a challenge in classical reconstituted ECM biomaterials. RNA sequencing of CFs cultured on DECIPHER substrates uncovered age-specific global gene expression patterns, with young cells exhibiting ‘cellular aging’ phenotypes, driven by both aged ECM ligands and stiff substrates, while aged cells can be pushed toward ‘rejuvenation’ phenotypes, driven predominately by young ECM ligands. Pathway analysis uncovered CF activation, ECM remodeling, mechanotransduction, and senescence as significantly regulated. Importantly, DECIPHER showed that the pro-fibrotic stiffness cue (~40 kPa) that has been previously reported to drive CF activation and senescence can be overridden by young ECM ligands, as characterized by reduced expression of alpha-smooth muscle actin (a-SMA) and p53 (Figure 1E). Furthermore, DECIPHER enables the elucidation of ligand vs. mechanical regulation mechanisms in cardiac aging due to its ability to decouple these two cues (Figure 1F). These results provide promising targets for mechano- and ligand-targeted rejuvenation therapies. Ultimately, this versatile hybrid biomaterial system can be applied to other tissues and diseases where ECM and mechanics are altered, providing essential insights into mechanobiology research and new therapeutic candidates.

Acknowledgements and/or References (Optional):: Sun, A.R., Ramli, M.F.H., Shen, X., Ramakanth, K.K., Chen, D., Hu, Y., Vidyasekar, P., Foo, R.S., Long, Y., Zhu, J., Ackers-Johnson, M., Young, J.L. Hybrid hydrogel-extracellular matrix scaffolds identify biochemical and mechanical signatures in cardiac aging. Nature Materials (2025, in press)