In-depth Physiological Validation of a Novel 3D Human In Vitro Model of Cardiac Fibrosis

Background: Cardiac fibrosis (CF) is associated with the development of multiple forms of cardiovascular diseases. It impairs proper myocardial function and is a substrate for electrophysiological anomalies potentially resulting in cardiac arrhythmias. Although several preclinical models exist, 3D human in vitro models that allow for longitudinal functional characterization of individual cardiac tissues to study disease progression and to develop novel treatment strategies for CF are scarce.

Methods: For our in vitro CF model, we mixed 1 million highly purified cardiomyocytes (CMs) derived from human iPS cells with 10% human skin fibroblasts (FBs) and collagen type 1 to generate miniaturized 3D bioartificial cardiac tissue (BCT). After an initial in-depth physiological characterization in a custom-made bioreactor system, BCTs were exposed to transforming growth factor-β (TGF-β) for 1 week to induce CF and were then re-assessed.

Results: In comparison to non-TGF-β-treated BCTs, the experimental group developed several CF-specific properties. Maximum contraction forces were significantly reduced by 9% (4.33 ± 0.1 mN versus 3.97 ± 0.16 mN, P < 0.05) accompanied by a more than 40% increase in maximum passive forces (1.74 ± 0.09 ΔmN versus 2.5 ± 0.21 ΔmN, P < 0.05). Furthermore, contraction kinetics were significantly affected by TGF-β treatment indicated by a 7% faster relaxation time of the BCTs (212.3 ± 2.38 milliseconds versus 196.8 ± 5.73 milliseconds, P < 0.05), and spontaneous contraction frequencies increased significantly by almost 40% compared with pre-treatment values (0.26 ± 0.02 Hz versus 0.36 ± 0.04 Hz, P < 0.05). A significant compaction of BCTs’ cross-sectional areas by 17% was observed after treatment (0.77 ± 0.03 mm² versus 0.64 ± 0.03 mm², P < 0.05), and histological analyses revealed a substantially increased abundance in vimentin+/ɑ-smooth muscle actin+ FBs, implying a fibroblast-to-myofibroblast transition. Notably, no differences in CM distribution or abundance were detected.

Conclusion: Our results show that a 1-week exposure to TGF-β is sufficient to robustly induce cardiac fibrosis as indicated by a substantial increase in BCT stiffness accompanied by a significant loss in tissue contractility, an increase in spontaneous contraction frequency, and myocardial remodeling. Extended treatment times might further affect BCTs’ electrophysiology and result in induction of myocardial arrhythmias. In summary, the presented human in vitro model might serve as a valuable tool to study CF development and to test novel treatment strategies to restore the physiological state.