Abstract Body (Do not enter title and authors here): Introduction: During postnatal development, the heart undergoes continuous adaptation to mechanical loads—primarily preload (stretch experienced during chamber filling) and afterload (resistance encountered during blood ejection). Engineered cardiac tissues (ECTs) offer a promising system to investigate cardiac development and diseases in vitro. Here, we present the “crossbow” bioreactor, designed to independently modulate preload and afterload in ECTs. This system enables the application of time-varying mechanical loads, facilitating the study of their individual and combined effects on cardiomyocyte phenotype and function.

Methods: Cylindrically shaped ECTs (“cardiobundles”) are fabricated with neonatal rat ventricular myocytes suspended in a fibrin-matrigel hydrogel. The cardiobundles are mounted onto the crossbow bioreactor, attached on one side to a curved polydimethylsiloxane cantilever arm and on the other side to a ratcheted center beam using a stainless-steel ring. Silk sutures that tether the cantilever arm to the beam are traversed down the beam to deflect the cantilever arm and increase afterload. The position of the stainless-steel ring is also changed to stretch the cardiobundles, increasing preload. In addition to a group with constant mechanical loading, preload, afterload, and combined conditions were assessed using force testing, optical mapping, immunostaining, and bulk RNA-sequencing.

Results: Cardiobundles experiencing increased afterload exhibited the highest contractile forces. Control and loaded tissues exhibited uniform action potential propagation and similar conduction velocities and action potential durations. Increased preload led to increased total muscle mass and greater cardiomyocyte cell cycling. Gene set enrichment analysis revealed significant upregulation of genes related to cell-cycling and downregulation of genes related to oxidative metabolism, action potentials, and cell-cell junctions in mechanically loaded groups compared to controls.

Conclusions: Overall, this work introduces a novel bioreactor system to elucidate the effects of time-varying mechanical loading on ECTs. While increased preload stimulated cardiomyocyte proliferation, increased afterload led to enhanced contractile force generation. The combined effects of preload and afterload yielded distinct cellular and functional adaptations, providing valuable insights for future use of this platform in studies of ECT maturation and drug response.