Heterotypic Cardiac Microtissues Exhibit Improved Function and Structural Reorganization in Hyaluronan Supplemented 3D Extracellular Matrix Microenvironment.

Introduction: The cardiac microenvironment is tightly regulated by spatiotemporally controlled biomechanical factors, which include fluid shear forces, cellular responses, and surrounding extracellular matrix (ECM) components. These finely tuned components play a significant role in cardiac development and any abnormalities contribute to disease progression. Collagen I and Hyaluronan are most abundant ECM components in early stage cardiac development and facilitate cell-ECM biomechanical cross talk within cardiac microenvironment. We aim to create a physiologically relevant in vitro model of early cardiac developmental stages by incorporating temporally relevant ECM components (hyaluronan and collagen I) and heterotypic cellular interactions (cardiac muscle and stromal cells) to understand the biomechanical impacts on cardiac function and structural organization.

Materials and

Methods: Human induced pluripotent stem cells (iPSCs) (WTC11 line with encoded Ca+2 GCaMP reporter, Bruce Conklin, Gladstone Institute, and AICS 37 TNNI, Allen Institute) were differentiated into cardiomyocytes (iPS-CM) by modulating the Wnt pathway using small molecules (CHIR, IWP2). Progenitor iPSC-CMs on day 7 were dissociated and combined with fetal cardiac fibroblasts (CF) at a 3:1 ratio and the cell suspension was seeded into 96 well ultralow attachment plates, to form spherical cardiac microtissues (MT) (150-250μm diameter, 5000 cells/aggregate). MTs were transferred to a rotary shaker (65rpm) overnight, where they resumed beating. The cardiac microtissues were suspended in 3D ECM hydrogel containing 1.5mg/ml collagen I (Col. I) and various concentrations of hyaluronic acid (HA) (0.5, 2, 10, and 20 mg/ml). The 0.3ml hydrogel-MT suspension was added to a 24well plate, which was then incubated at 37°C for 60-90 minutes to allow ECM polymerization and beating resumption in MTs. Endothelial cells were added to the surface of the ECM hydrogel. Cultures were maintained for 7 days in the incubator. Structural and functional characterization was performed using fluorescence microscopy and MUSCLE MOTION for assessing changes in structural reorganization and calcium handling in cardiac microtissue.

Results, Conclusions, and Discussions: 3D culture conditions were found to be conducive to robust cardiac beating activity and fibroblast-mediated cardiomyocyte crosstalk. CFs showed migration patterns towards neighboring microtissues, facilitating cellular crosstalk and contractile pairing, resulting in beating synchronization (Fig.1). ECM hydrogel encouraged cardiomyocyte structural alignment in the 3D matrix, and the HA content significantly influenced the cardiac alignment pattern and structure. The contractile function of cardiac alignments was similar to the parent MT that they originated from. HA concentration in the ECM hydrogels also impacted the functional robustness of cardiac MTs assessed through contractile strength and calcium handling dynamics. 10mg/ml HA conditions significantly improved calcium handling by increasing contraction amplitude, peak, and relaxation times whereas a significant decrease was observed in contraction duration indicating stronger and faster individual beats with rapid beating patterns (Fig 2).ECM components affect cell structure and function in the cardiac microenvironment and the mechanosensitive cells sense, respond, and act on the external stimuli by altering functional and structural characteristics.
Our model highlights the role of HA in altering cell behavior and function and embodies key factors in the cardiac microenvironment imperative to understand initial cardiac development. It recapitulates the physiological cell-cell, cell-ECM contact and offers an excellent platform for testing cardiovascular development, disease progression, and new therapeutics.

Acknowledgements (Optional): This work was supported by Fulbright PhD grant, NSF CMMI 1919438 and NIH-R15HL150745.