Dynamic Loading of Human Engineered Heart Tissue Enhances Contractile Function and Drives Desmosome-linked Disease Phenotype

Abstract

The role mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes in the heart. However, most EHT systems are unable to model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained fractional shortening of >10%.To do this, 3D EHTs are integrated with an elastic polydimethylsiloxane (PDMS) strip that provides mechanical pre-and afterload to the tissue in addition to enabling contractile force measurements based on strip bending. Our results demonstrate in wild-type EHTs that dynamic loading is beneficial based on the magnitude of the forces, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we use hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy (ACM) due to mutations in desmoplakin. We demonstrate that manifestation of this desmosome-linked disease state requires the dyn-EHT conditioning and that it cannot be induced using 2D or standard 3D EHT approaches. Thus, dynamic loading strategy is necessary to provoke a disease phenotype (diastolic lengthening, reduction of desmosome counts, and reduced contractility), which are akin to primary endpoints of clinical disease, such as chamber thinning and reduced cardiac output.Studying the effects of mechanical loading on adaptive and maladaptive changes in heart structure and function is challenging in human patients, driving the need to develop new approaches. Animals have been used to model a wide range of human cardiovascular disease states, but there are inherent differences in physiology, gene expression and pharmacokinetics that limit the ability to replicate complex pathology (6). In vitro 2-dimensional (2D) culture of cardiomyocytes on microengineered surfaces have been used to study structure-function relationships and to create region-specific ventricular myocardium (7,8) Muscular thin films (MTFs), consisting of cells cultured on a flexible film, have enabled these 2D platforms to measure contractility (9,10), and combined with patient-specific human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes can model cardiomyopathies such as Barth's syndrome (11). However, 2D cardiomyocytes are adhered to a surface (12) and thus do not have the same mechanical cell-cell coupling as found in the native heart (13). To address this, researchers have developed more physiologically-relevant 3D engineered heart tissues (EHTs) in the form papillary muscle-like linear bundles (14-18) myocardium-like sheets (19,20), and ventricle-like chambers (21,22). Incorporating human induced pluripotent stem cell (hi...

Engineered Human Cardiac Microtissues: The State-of-the-(He)art

“…However, it is unclear if modeling preload and afterload enables disease manifestation, or if it is the improved maturation in the system that brings about the defects observed. 40 There are other approaches that are suitable for modeling afterload. A key parameter that can be manipulated with respect to mechanical loading is substrate stiffness.…”

“…While most heart‐on‐a‐chip setups do not replicate both preload (pressure from ventricular filling) and afterload (pressure the ventricle must work against to pump blood) on cardiac microtissues, a recent study has used a bent silicone rod to mimic aspects of both preload and afterload, while also enabling force measurements by bending of the same rod (Figure 1D). 40 This system was shown to improve the maturation of hiPSC‐CMs, demonstrated by gene expression and improved conduction velocity. The application of load also resulted increased fidelity of sarcomeric alignment compared with having constrained rods (no load).…”

“…Staining revealed decreased desmoplakin levels, and transmission electron microscopy revealed a lower number of desmosomes, with the ones present being less protein dense. However, it is unclear if modeling preload and afterload enables disease manifestation, or if it is the improved maturation in the system that brings about the defects observed 40 …”

FRESH™ 3D Bioprinted Cardiac Tissue, a Bioengineered Platform for in vitro Toxicology and Pharmacology

“…Cardiac spheroids and organoids provide an ability to place hiPSC-CMs in a 3D structure, and this clearly provides some advantages, however the structure and function still recapitulates the embryonic state at best 20 . Engineered heart tissues (EHTs) have been developed to better recreate the 3D structure and function of the heart at the tissue and organ scale, leading to more physiologically data-rich assays [21][22][23][24][25][26][27][28][29][30] . Current 3D fabrication approaches for hiPSC-CMs through molding of cell-laden hydrogels, seeding on fiber-based scaffolds and 3D bioprinting have been effective in creating contractile cardiac tissues in a dish.…”

“…FRESH™ 3D bioprinting works by extruding cell-laden bioinks within a gelatin microparticle support bath that provides mechanical support to the bioink while it gels and then can be non-destructively removed by melting at 37°C 31 . We have previously demonstrated that FRESH™ 3D bioprinting can be used to engineer cardiac tissues in a range of complex 3D structures such as ventricle-like constructs and heart tubes with advanced functionality 29,30 . To expand upon this, we created a process that enables EHTs to be FRESH™ 3D printed around custom-designed tissue fixtures within 24-well plates and then performed structural, functional, and pharmacologic assays to demonstrate platform capabilities.…”