Reprogramming cardiomyocyte mechanosensing by crosstalk between integrins and hyaluronic acid receptors

Department of Biomedical Engineering, Drexel University, Philadelphia, PA 19102, USA.
Journal of Biomechanics (Impact Factor: 2.75). 12/2011; 45(5):824-31. DOI: 10.1016/j.jbiomech.2011.11.023
Source: PubMed


The elastic modulus of bioengineered materials has a strong influence on the phenotype of many cells including cardiomyocytes. On polyacrylamide (PAA) gels that are laminated with ligands for integrins, cardiac myocytes develop well organized sarcomeres only when cultured on substrates with elastic moduli in the range 10 kPa-30 kPa, near those of the healthy tissue. On stiffer substrates (>60 kPa) approximating the damaged heart, myocytes form stress fiber-like filament bundles but lack organized sarcomeres or an elongated shape. On soft (<1 kPa) PAA gels myocytes exhibit disorganized actin networks and sarcomeres. However, when the polyacrylamide matrix is replaced by hyaluronic acid (HA) as the gel network to which integrin ligands are attached, robust development of functional neonatal rat ventricular myocytes occurs on gels with elastic moduli of 200 Pa, a stiffness far below that of the neonatal heart and on which myocytes would be amorphous and dysfunctional when cultured on polyacrylamide-based gels. The HA matrix by itself is not adhesive for myocytes, and the myocyte phenotype depends on the type of integrin ligand that is incorporated within the HA gel, with fibronectin, gelatin, or fibrinogen being more effective than collagen I. These results show that HA alters the integrin-dependent stiffness response of cells in vitro and suggests that expression of HA within the extracellular matrix (ECM) in vivo might similarly alter the response of cells that bind the ECM through integrins. The integration of HA with integrin-specific ECM signaling proteins provides a rationale for engineering a new class of soft hybrid hydrogels that can be used in therapeutic strategies to reverse the remodeling of the injured myocardium.

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    • "Even though we have previously shown that fibroblasts respond to substrate elasticity on the gels used here in respect to their adhesion and migration behavior [21], we cannot exclude the possibility that differences in cell adhesive coating or underlying polymer structure are responsible for the presence or lack of correlation between marker uptake and substrate elasticity. Indeed, a dependence of ECM type on internalization and transfection efficiency has been reported [51] and the extent of cell responses on substrate elasticity greatly depends on the substrate material [52] and the type of cell-adhesive coating [21], [30], [53]–[55]. Future studies should expand the validity of our results on the stiffness dependencies in endocytosis to cells exposed to varying insoluble biochemical signals. "
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    ABSTRACT: Substrate mechanical properties have emerged as potent determinants of cell functions and fate. We here tested the hypothesis that different forms of endocytosis are regulated by the elasticity of the synthetic hydrogels cells are cultured on. Towards this objective, we quantified cell-associated fluorescence of the established endocytosis markers transferrin (Tf) and cholera toxin subunit B (CTb) using a flow-cytometry based protocol, and imaged marker internalization using microscopy techniques. Our results demonstrated that clathrin-mediated endocytosis of Tf following a 10-minute incubation with a fibroblast cell line was lower on the softer substrates studied (5 kPa) compared to those with elasticities of 40 and 85 kPa. This effect was cancelled after 1-hour incubation revealing that intracellular accumulation of Tf at this time point did not depend on substrate elasticity. Lipid-raft mediated endocytosis of CTb, on the other hand, was not affected by substrate elasticity in the studied range of time and substrate elasticity. The use of pharmacologic contractility inhibitors revealed inhibition of endocytosis for both Tf and CTb after a 10-minute incubation and a dissimilar effect after 1 hour depending on the inhibitor type. Further, the internalization of fluorescent NPs, used as model drug delivery systems, showed a dependence on substrate elasticity, while transfection efficiency was unaffected by it. Finally, an independence on substrate elasticity of Tf and CTb association with HeLa cells indicated that there are cell-type differences in this respect. Overall, our results suggest that clathrin-mediated but not lipid-raft mediated endocytosis is potentially influenced by substrate mechanics at the cellular level, while intracellular trafficking and accumulation show a more complex dependence. Our findings are discussed in the context of previous work on how substrate mechanics affect the fundamental process of endocytosis and highlight important considerations for future studies.
    Full-text · Article · May 2014 · PLoS ONE
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    • "Interestingly , there is a matrix-dependent response of CMs to substrate stiffness. Hyaluronic acid (HA) has the potential to alter CMs' integrin-mediated response to stiffness, because when cultured on extremely soft substrates in the presence of HA, CMs maintain their ability to form organized sarcomeres [43]. Further work is needed to understand the mechanism by which substrate composition mediates the cellular response to stiffness. "
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    ABSTRACT: The extracellular matrix is no longer considered a static support structure for cells, but a dynamic signaling network with the power to influence cell, tissue and whole organ physiology. In the myocardium, cardiac fibroblasts are the primary cell type responsible for the synthesis, deposition and degradation of matrix proteins and they therefore play a critical role in the development and maintenance of functional heart tissue. This review will summarize the extensive research conducted in vivo and in vitro demonstrating the influence of both physical and chemical stimuli on cardiac fibroblasts and how these interactions impact both cardiomyocytes and the extracellular matrix. This work is of considerable significance given that cardiovascular diseases are marked by extensive remodeling of the extracellular matrix, which ultimately impairs the functional capacity of the heart. We seek to summarize the unique role of cardiac fibroblasts in normal cardiac development and the most prevalent cardiac pathologies including congenital heart defects, hypertension, hypertrophy, and the remodeled heart following myocardial infarction. We will conclude by identifying existing holes in the research that, if answered, have the potential to dramatically improve current therapeutic strategies for the repair and regeneration of damaged myocardium via mechanotransductive signaling.
    Full-text · Article · May 2013 · Journal of Biomechanical Engineering
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    • "Rather than that, cells kept a round shaped morphology and did not interconnect to build up a contracting tissue construct. This was not surprising, since alginate and hyaluronic acid display low cell-adhesiveness [59] [60], which might also interfere with cell migration and tissue formation. Nevertheless , Live/Dead stainings demonstrated excellent cell viability within hydrogels, which highlights the value of this system for potential approaches like cell/hydrogel injection or tissue engineering of constructs with low cell density like cartilage and bone. "
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    ABSTRACT: Despite recent major advances including reprogramming and directed cardiac differentiation of human cells, therapeutic application of in vitro engineered myocardial tissue is still not feasible due to the inability to construct functional large vascularized contractile tissue patches based on clinically applicable and fully defined matrix components. Typical matrices with preformed porous 3D structure cannot be applied due to the obvious lack of migratory capacity of cardiomyocytes (CM). We have therefore developed a fully defined in situ hydrogelation system based on alginate (Alg) and hyaluronic acid (HyA), in which their aldehyde and hydrazide-derivatives enable covalent hydrazone cross-linking of polysaccharides in the presence of viable myocytes. By varying degrees of derivatization, concentrations and composition of blends in a modular system, mechanophysical properties of the resulting hydrogels are easily adjustable. The hydrogel allowed for the generation of contractile bioartificial cardiac tissue from CM-enriched neonatal rat heart cells, which resembles native myocardium. A combination of HyA and highly purified human collagen I led to significantly increased active contraction force compared to collagen, only. Therefore, our in situ cross-linking hydrogels represent a valuable toolbox for the fine-tuning of engineered cardiac tissue's mechanical properties and improved functionality, facilitating clinical translation toward therapeutic heart muscle reconstruction.
    Full-text · Article · Nov 2012 · Biomaterials
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