Enhancing Functionality of Atrial and Ventricular Heart-on-chip Via Mechanical Stimulation to Develop Models for Cardiotoxicity Screening

Cardiotoxicity assessment still represents a critical step in drug development process. At present, preclinical investigation mostly relies on in vitro and animal experimentations, known for lack of specificity and poor recapitulation of human heart behavior. Notably, the development of mature, chamber-specific (Atrial/Ventricular) cardiac models using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) remains a significant challenge. Hence, advancing these models holds great potential. Here we present a chamber specific(Atrial/Ventricular) human functional 3D cardiac model developed within a beating Organ-on-Chip (OoC) platform, named uHeart, integrating fit-to-purpose assays for assessing microtissue contractility and electrophysiology and exploited for detecting drug-induced functional alterations. The model was developed by culturing Atrial/Ventricular human induced pluripotent stem cells derived cardiomyocytes(h-iPSC-CMs, AXOL Bioscience) combined with human cardiac fibroblasts (h-CFs, Innoprot) in a 75%–25% ratio, embedded in fibrin hydrogel (100 × 106 cells/mL) and cultured for up to 11 days in static and dynamic conditions. Dynamically-cultured microtissues were subjected to mechanical stimulation through a patented technology, an actuating mechanism integrated in uHeart that provides a physiological uniaxial cyclic strain (i.e., 10% strain, 1 Hz, 50% duty cycle). Following biological characterization, a toxicological evaluation was performed, assessing microtissues' contractility and electrophysiological changes upon 4-Aminopyridine and Dofetilide administration. Real-time q-PCR showed increased gene expression of light-chain-myosin MYL7 and ratio of heavy-chain-myosins MYH7/MYH6 in all dynamically-cultured microtissues. Atrial microtissues also featured increased gene expression of MYL2, sodium (SCN5A), potassium (KCNH2) and calcium (CACNA1C) channels. Immunofluorescence demonstrated chamber-specific phenotypes maintenance (atrial microtissues expressed atrial Sarcolipin, ventricular microtissues exhibited ventricular Light-Chain-Myosin). Video analysis of spontaneously beating microtissues showed greater contraction synchronicity for dynamically-cultured microtissues (Correlation Coefficient: atrial-static = 0.36,atrial-dynamic = 0.81;ventricular-static = 0.44,ventricular-dynamic = 0.93). Electrophysiological characterization showed that ventricular microtissues had a shorter beating period (BP-ventricular = 1.7 s), increased spike amplitude (SA-ventricular = 219 μV) and field potential duration (FPD-ventricular = 0.83 s) than atrial microtissues (BP-atrial = 2.9 s,SA-atrial = 144 μV,FPD-atrial = 0.66 s). Toxicological evaluation revealed that 4-Aminopyridine and Dofetilide prolonged the atrial and ventricular FPD, respectively (FPD variation respect to control: FDP-atrial = 22.4% at C_4AP = 100 μM,FPD-ventricular = 24% at C_Dofetilide = 2 nM). The study suggests that mechanical stimulation benefits atrial and ventricular microtissues' functionality. The preliminary pharmacological tests demonstrated the suitability of atrial and ventricular uHeart to be exploited for cardiotoxicity testing.