Effects of Time-Varying Mechanical Preload and Afterload in Engineered Cardiac Tissues: Studies Using a Novel Bioreactor

Abstract

Ischemic heart disease remains the leading cause of death worldwide, underscoring the need for additional studies, including in vitro model systems that accurately replicate cardiac pathophysiology and function. Advances in three-dimensional (3D) cardiac tissue engineering have enabled the generation of in vitro tissue equivalents that approximate native myocardium. However, these engineered tissues still fail to replicate the structural, mechanical, and electrophysiological properties of the adult heart. Mechanical loading plays a critical role in heart development and function, with cardiac preload (tissue stretch during chamber filling) and afterload (resistance against which the heart works to eject blood) potentially playing distinct roles in postnatal cardiomyocyte maturation. To investigate the effects of various types of mechanical loading on postnatal cardiomyocytes, we developed a novel “crossbow” bioreactor system capable of independently and dynamically modulating preload and afterload under auxotonic conditions in 3D engineered cardiac tissues. The system employs curved polydimethylsiloxane (PDMS) cantilever arms that increase resistance to cardiac contractions as they are deflected and a ratcheted center beam to allow for control of preload via change in cardiac tissue length. Computational modeling and experimental validation confirmed that cantilever geometry can be tuned to deliver precise ranges of afterload to cardiac tissues while simultaneously and independently modulating their preload. We then cultured cardiobundles, 3D engineered cardiac tissues made from neonatal rat cardiomyocytes embedded in fibrin-based hydrogel, on the crossbow system and progressively increased preload and afterload, both independently or in combination. Cardiobundles maintained or improved electromechanical functionality across all groups, suggesting that the loading protocols applied induced a physiological rather than pathological response. Progressive increase in afterload enhanced contractile force generation, while progressively increased preload promoted cardiomyocyte elongation and DNA synthesis. Structural analysis revealed highly organized and aligned sarcomeres, membrane-localized junctional proteins (N-cadherin and Connexin 43), and frequent evidence for t-tubulogenesis. RNA-sequencing revealed broad transcriptomic changes across conditions, with the greatest divergence from static control observed in cardiobundles experiencing increases in both preload and afterload, underscoring the profound impact of developmentally mimetic mechanical loading on cellular signaling pathways. In summary, this dissertation describes the development of a crossbow bioreactor, a tunable in vitro platform for studying roles of mechanical preload and afterload in engineered cardiac tissue function and maturation. Experiments with the crossbow bioreactor revealed that progressive afterload enhanced cardiomyocyte contractile force, while increased preload promoted cardiomyocyte elongation and proliferation. The crossbow system holds potential for refining our understanding of mechanosensing in cardiac developmental and pathogenic remodeling, making it a valuable tool for in vitro disease modeling, pharmaceutical testing, and regenerative medicine applications.