Engineering micromyocardium to delineate cellular and extracellular regulation of myocardial tissue contractility.

Cardiovascular diseases are a leading cause of death, in part due to limitations of existing models of the myocardium. Myocardium consists of aligned, contractile cardiac myocytes interspersed with fibroblasts that synthesize extracellular matrix (ECM). The cellular demographics and biochemical and mechanical properties of the ECM remodel in many different cardiac diseases. However, the impact of diverse cellular and extracellular remodeling on the contractile output of the myocardium are poorly understood. To address this, we micropatterned 13 kPa and 90 kPa polyacrylamide gels with aligned squares of fibronectin (FN) or laminin (LN). We seeded gels with two concentrations of primary neonatal rat ventricular myocytes, which naturally contain fibroblasts. Cells assembled into aligned "µMyocardia" with fibroblast : myocyte ratios dependent on initial seeding concentration. Using traction force microscopy (TFM), we found that the peak systolic longitudinal cross-sectional force was similar across conditions, but the peak systolic work was significantly lower on 90 kPa gels. This indicates that ECM elasticity dominates over ECM ligand and cell demographics in regulating contractile output. Because our platform provides independent control over cell-cell and cell-matrix interactions, it has many applications for cardiac disease modeling.

Prolonged Culture of Aligned Skeletal Myotubes on Micromolded Gelatin Hydrogels.

In vitro models of skeletal muscle are critically needed to elucidate disease mechanisms, identify therapeutic targets, and test drugs pre-clinically. However, culturing skeletal muscle has been challenging due to myotube delamination from synthetic culture substrates approximately one week after initiating differentiation from myoblasts. In this study, we successfully maintained aligned skeletal myotubes differentiated from C2C12 mouse skeletal myoblasts for three weeks by utilizing micromolded (µmolded) gelatin hydrogels as culture substrates, which we thoroughly characterized using atomic force microscopy (AFM). Compared to polydimethylsiloxane (PDMS) microcontact printed (µprinted) with fibronectin (FN), cell adhesion on gelatin hydrogel constructs was significantly higher one week and three weeks after initiating differentiation. Delamination from FN-µprinted PDMS precluded robust detection of myotubes. Compared to a softer blend of PDMS µprinted with FN, myogenic index, myotube width, and myotube length on µmolded gelatin hydrogels was similar one week after initiating differentiation. However, three weeks after initiating differentiation, these parameters were significantly higher on µmolded gelatin hydrogels compared to FN-µprinted soft PDMS constructs. Similar results were observed on isotropic versions of each substrate, suggesting that these findings are independent of substrate patterning. Our platform enables novel studies into skeletal muscle development and disease and chronic drug testing in vitro.