Glycopeptide-based supramolecular hydrogel supports the alignment and contractility of cardiomyocytes

Introduction/Objectives: Cardiovascular diseases are the leading cause of death worldwide, with myocardial infarction and cardiac ischemia as the most common contributors to heart failure. The full recovery of the heart function after these injuries is still challenging. Tissue engineering/regenerative medicine offers potential alternatives for in-situ regeneration of myocardial tissue.1 Herein, we report the supramolecular hydrogels derived from a glycopeptide building block (FmocFFGlcN6S) that copycat the biofunctionality of the glycoproteins present in the heart’s extracellular matrix (ECM). 2 We explore micropatterning of hydrogels to guide cardiomyocytes alignment and to develop a glycopeptide-based biofunctional hydrogel patch that promotes the regeneration of the myocardium. Methods: The glycopeptide FmocFFGlcN6S was synthesized using the carbodiimide coupling chemistry to conjugate GlcN6S to FmocFF. FmocFFGlcN6S were dissolved in water and heated (90°C). After cooling to room temperature, viscous solutions were poured into inserts, and sol-gel transition occurred by contact with the medium. The hydrogels’ surface was micropatterned using PDMS molds. SEM and AFM were used to assess morphology and micropatterning. Conductivity measurements were performed to study the ability to propagate electrical signals. Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) were cultured on the micropatterned hydrogels, and viability and morphology were assessed. The contractility and the propagation of Ca2+ fluxes (Fluo-3 AM) were determined. Expression of cardiac markers was evaluated by immunostaining and Western Blot. Results: The synthesized glycopeptide formed a stable hydrogel with aligned surface grooves (width: 15lm, depth: 5lm, spacing: 12lm). GlcN6S moiety enhanced hydrogels conductivity (400 - 600mS/m), similar to native tissue (30 - 600mS/m). 3 Nanoindentation experiments revealed a rigidity of ～41 kPa. The alignment of hiPSC-CMs was confirmed by cell and nuclei morphology analyses. Live-cell fluorescence imaging showed Ca 2+ flux propagation throughout the hiPSC-CMs monolayer and synchronized beating. Immunostaining analyses confirmed the expression of the Troponin-T and Connexin-43 associated with contractility. WB showed a 1.5-fold increase in the expression of Connexin-43 in the patterned hydrogels. Conclusions: The glycopeptide amphiphile (FmocFFGlcN6S) forms a hydrated nanofiber mesh mimicking the organization of the glycoproteins present in the heart’s ECM. The hydrogels have mechanical and conductive properties comparable to native heart tissue. Micropatterned grooves on these hydrogels induced an alignment of hiPSC-CMs, while preserving their phenotype. Finally, the developed hydrogels are able to promote the contractility of hiPSC-CMs. Altogether these data support the potential of micropatterned glycopeptide-based supramolecular hydrogels for advancing cardiac tissue regeneration.