Engineering cardiac microphysiological systems to model pathological extracellular matrix remodeling

Abstract

Many cardiovascular diseases are associated with pathological remodeling of the extracellular matrix (ECM) in the myocardium. ECM remodeling is a complex, multifactorial process that often contributes to declines in myocardial function and progression toward heart failure. However, the direct effects of the many forms of ECM remodeling on myocardial cell and tissue function remain elusive, in part because conventional model systems used to investigate these relationships lack robust experimental control over the ECM. To address these shortcomings, microphysiological systems are now being developed and implemented to establish direct relationships between distinct features in the ECM and myocardial function with unprecedented control and resolution in vitro. In this review, we will first highlight the most prominent characteristics of ECM remodeling in cardiovascular disease and describe how these features can be mimicked with synthetic and natural biomaterials that offer independent control over multiple ECM-related parameters, such as rigidity and composition. We will then detail innovative microfabrication techniques that enable precise regulation of cellular architecture in two and three dimensions. We will also describe new approaches for quantifying multiple aspects of myocardial function in vitro, such as contractility, action potential propagation, and metabolism. Together, these collective technologies implemented as cardiac microphysiological systems will continue to uncover important relationships between pathological ECM remodeling and myocardial cell and tissue function, leading to new fundamental insights into cardiovascular disease, improved human disease models, and novel therapeutic approaches.

Keywords: biomaterials; contractility; electrophysiology; metabolism; microfabrication.

Fig. 1.

Engineering cardiac microphysiological systems to define the functional impacts of pathological extracellular matrix remodeling. A: distinct features of native healthy and diseased/fibrotic myocardium, such as myocyte shape, tissue alignment, extracellular matrix (ECM) rigidity, and cell demographics, are used as design templates for engineering cardiac microphysiological systems. B: features of native healthy and diseased/fibrotic myocardium are replicated by combining appropriate biomaterials and microfabrication techniques. C: engineered cardiac cells and tissues are interrogated with functional assays to quantify contractility, electrophysiology, and metabolism as a function of their microenvironment. Collectively, these approaches implemented as cardiac microphysiological systems can identify the functional impact of ECM remodeling to streamline mechanistic studies and therapeutic development. Images in C were adapted from Ref. (top left), Ref. with permission from The Royal Society of Chemistry (bottom left), Ref. (top middle), and Ref. (bottom middle) with permission from Elsevier.

Fig. 2.

Dictating two-dimensional tissue architecture with substrate micropatterning. Customizable polydimethylsiloxane (PDMS) stamps (gray) are fabricated with photolithography and soft lithography. A: in one example of microcontact printing, stamps are coated with proteins (red) such as Matrigel and inverted onto glass coverslips (blue), transferring the pattern to the coverslip. These patterned coverslips are inverted onto polyacrylamide hydrogel prepolymer solution to transfer the extracellular matrix protein pattern to the hydrogel as it polymerizes. B: micromolding is performed by inverting PDMS stamps onto a hydrogel prepolymer solution (yellow) such as methacrylated tropoelastin (MeTro). The stamp remains in position during hydrogel polymerization, which is initiated by ultraviolet (UV) light in this example, to mold the pattern from the PDMS stamp into the hydrogel. Images were adapted from Ref. with permission from the National Academy of Sciences (A) and from Ref. with permission from John Wiley & Sons (B).

Fig. 3.

Quantifying contractility in engineered two-dimensional cardiac myocytes and tissues. A: micropillar substrates can be used to quantify contractile forces generated by cardiac myocytes. In this example, Ca²⁺ transients were also measured in engineered tissues on micropillar substrates. hESC-CM, human embryonic stem cell-derived cardiomyocytes. FEM, finite-element modeling. B–D: traction force microscopy and microcontact-printed polyacrylamide hydrogels have been implemented to quantify the combined effects of extracellular matrix elasticity and cell/tissue architecture on peak systolic traction stress generated by single cardiac myocytes (B), cardiac myocyte pairs (C), and multicellular cardiac tissues (D). Scale bars = 10 μm. In B, white denotes α-actinin. In C, red denotes actin, green denotes β-catenin, and blue denotes nuclei. In D, red denotes α-actinin, green denotes actin, and blue denotes nuclei. E: muscular thin film assay entails culturing cardiac myocytes on precut polymer cantilevers (shown here, micromolded gelatin hydrogels), which are released from the substrate at the time of analysis. Stress is calculated based on cantilever deflection. Images were adapted from Ref. with permission from the American Chemical Society (A), Ref. (B), Ref. with permission from the National Academy of Sciences (C), Ref. with permission from the Royal Society of Chemistry (D), and Ref. with permission from Elsevier (E).

Fig. 4.

Metabolic profiling of cardiac myocytes and engineered two-dimensional cardiac tissues. A: cardiac myocytes isolated from adult rats subjected to sham surgery or aortocaval fistula (ACF) and tagged for mitochondrial membrane potential (TMRM) or reactive oxygen species (CM-DCF). B: microcontact-printed polydimethylsiloxane (PDMS) disks of distinct elasticities were fabricated in a multistep process (i–vi), transferred to the wells of modified microplates (vi), and seeded with neonatal rat ventricular myocytes (vii). These plates were inserted into an extracellular flux analyzer to quantify the effects of extracellular matrix elasticity and tissue architecture on oxygen consumption rates (OCRs). C and D: representative OCR profiles for isotropic (C) and aligned (D) tissues demonstrate distinct profiles due to extracellular matrix rigidity and tissue alignment. Images were adapted from Ref. (A) and Ref. (B–D).

Fig. 5.

Engineering and functionally assessing engineered three-dimensional cardiac tissues. A: aligned three-dimensional cardiac tissues are fabricated by filling a custom polydimethylsiloxane (PDMS) mold with a mixture of cells and an extracellular matrix hydrogel prepolymer solution, which gradually solidifies and compacts into a tissue supported by the PDMS pillars. B: the electrical and mechanical functionality of “iWire” engineered three-dimensional cardiac tissue constructs are quantified using patch clamp and optical recording of a flexible probe, respectively. Images were adapted from Refs. (A) and Ref. with permission from Elsevier (B).