(G-261) Engineered Cocultures of Induced Pluripotent Cell-Derived Atrial Cardiomyocytes and Atrial Fibroblasts for Modeling Atrial Fibrillation

Introduction:: Although atrial fibrillation (AF) is the most prevalent arrythmia affecting >33M individuals, current drug therapies are not highly effective as they do not target underlying genetic mechanisms. Given species-specific differences in cardiac function, in vitro human atrial models are critical for disease modeling and novel drug discovery. Human atrial cardiomyocytes (aCMs) are very limited due to shortages of human donor tissue and rapidly decline in function/viability. In contrast, induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are a sustainable and patient-specific cell source useful for elucidating genotype-phenotype relationships. However, current protocols yield functionally immature iPSC-aCMs which restricts their use for above applications. Therefore, we sought to test the novel hypothesis that combining micropatterned iPSC-aCMs in precise dimensions to induce sarcomere alignment with co-cultivation with cardiac fibroblasts (CFs) could significantly improve iPSC-aCM maturity within a multiwell plate format to facilitate high-throughput drug screening and disease modeling.


Materials and Methods:: Linear micropatterns of fibronectin (FN) were constructed on tissue culture polystyrene (TCPS) plates using a custom soft-lithography process followed by sequential seeding of iPSC-aCMs and CFs onto the patterned substrates to form patterned co-cultures (PC). For random monoculture (RM) controls, iPSC-aCMs were seeded onto FN-coated TCPS. After 3 weeks of culture, sarcomere structures in iPSC-aCMs were visualized via immunofluorescent (IF) staining for cardiac troponin T (cTnT) and α-actinin, and their lengths were quantified in ImageJ. Electrophysiological signature was analyzed using optical voltage mapping (OVM), performed by treating iPSC-aCMs with a voltage sensitive dye and subsequent fast-acquisition image capture. iPSC-aCM randomly distributed cocultures with atrial or ventricular cardiac fibroblasts (ACFs or VCFs) were cultured for 3 weeks and RNA was isolated via TRIzol^TM. RNA sequencing was performed; sequences were aligned using BioJupies and further analyzed in R and Cytoscape with the ClueGo package. Gene knockdowns were performed on PCs with 20 pmol of siRNA/well and confirmed with qPCR as compared to scrambled siRNA and untreated controls.


Results, Conclusions, and Discussions:: iPSC-aCMs selectively attached to the FN micropatterns (Fig. 1A, left), followed by CF seeding to create PCs (Fig 1A, right). Immunofluorescence revealed sparse and randomly oriented sarcomeres in RMs but aligned and organized sarcomeres in PCs, indicating improved structural maturity (Fig. 1B). Sarcomere length in PC was 1.2-fold higher than RM (Fig. 1C). Use of OVM demonstrated an increase in action potential duration at 90% repolarization (APD₉₀) to a more physiological range in PCs (~550 ms) vs. RMs (~325 ms) (Fig. 1D). Additionally, while verapamil, a drug that blocks both L-type calcium and rapid delayed rectifier potassium ion channels, caused a significant decrease in APD₉₀ in PCs as expected from clinical results, RMs did not respond to the drug, thus indicating a higher sensitivity in PCs (Fig. 1E). iPSC-aCMs cocultured with ACFs or VCFs showed increased expression of CX40 and ephrin signaling pathways in ACF coculture (Fig. 1F). PCs with gene knockdowns for EFNB1 or GJA5 showed significantly decreased contractility than scrambled siRNA controls, thus revealing a novel mechanism underlying iPSC-aCM maturation (Fig. 1G).


We show for the first time that micropatterned co-culture with ACFs significantly improves the structural and functional maturity of human iPSC-aCMs in a multi-well format for high-throughput drug screening and modeling genetic variants associated with AF. Future efforts are focused on incorporating donor-matched iPSC-derived endothelial cells and fibroblasts as well as electrical, mechanical, and fluidic stimuli in the platform.




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