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

Abstract

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.

Traction force microscopy of engineered cardiac tissues

“…In conclusion, the tissue-level assay presented here completes a consistent suite of TFM-based assays that can enable thorough multi-scale studies of the coupling between metabolism, mechanotransduction, and contractility in primary and stem cell-derived myocytes [ 5 – 8 ]. Two recent studies from Prof. McCain’s group addressed similar topics: tissue-level cardiac TFM [ 46 ] and NRMV metabolism as a function of substrate stiffness [ 47 ]. While these studies are consistent with our findings, a few important differences exist.…”

“…While these studies are consistent with our findings, a few important differences exist. For example, they engineered NRVM in square-shaped tissues on normal and stiff gels using fibronectin and laminin cues [ 46 ] we used larger, diamond-shaped Fibronectin-only cues to improve cellular alignment in mTissues cultured on soft, normal, and stiff gels. Consistent with our study, stiffening of the substrate caused a sharp decline in cell-induced deformation and contractile work in engineered tissues, independently from the chosen ECM ligand.…”

Engineering cardiac microphysiological systems to model pathological extracellular matrix remodeling

“…To induce myocyte adhesion, ECM proteins can be directly mixed into the hydrogel prepolymer solution before cross-linking (59,176) or attached to the surface of the hydrogel using sulfo-SANPAH and ultraviolet light followed by ECM protein coating (10,47,69). Polyacrylamide hydrogels can also be doped with streptavidin-acrylamide to enable surface attachment of biotinylated ECM proteins such as fibronectin (119) or laminin (11). Polyethylene glycol (PEG) hydrogels have also been used as mechanically tunable synthetic substrates for in vitro cardiac models.…”

“…These substrates were seeded with primary neonatal rat ventricular myocytes, which also contain a small population of cardiac fibroblasts. Thus, these "micromyocardium" tissues consist of both cardiac myocytes and fibroblasts with ratios that are relatively tunable, adding another level of microenvironmental control to this platform (11). Tunable PDMS substrates can also be microcontact printed and used to control both tissue alignment and ECM elasticity for engineered cardiac tissues (111), although PDMS generally offers less fine control over elastic modulus in lower ranges compared with hydrogels.…”

“…With this system, a weakened relationship between cytoskeletal organization and force generation and defects in mechanical cell-cell coupling was identified on stiffer hydrogels compared with softer hydrogels (118), suggesting that remodeling of the ECM can directly affect cell-cell junction formation. To probe the combined effects of ECM rigidity, ECM ligand, and fibroblasts on cardiac tissue contractility, aligned MicroMyocardium with distinct myocyte-to-fibroblast ratios was engineered on polyacrylamide hydrogels microcontact printed with fibronectin or laminin (11) (Fig. 3D).…”