Abstract Body: Background: Micro-heart tissues (μHTs), generated by seeding iPSC-cardiomyocytes (iPSC-CMs) around elastomeric posts, allow for medium-throughput pharmacology analysis. Mimicking afterload by increasing post bending stiffness (resistance to systolic force) enhances functional maturation; we hypothesized this includes improved Excitation-Contraction Coupling (ECC).

Methods: We differentiated iPSC-CMs from a GCaMP6f (Ca2+ indicator) knock-in WTC line and formed µHTs (Fig 1). To assess if afterload-induced maturation can improve ECC, we developed Python-based software tracking fluorescence intensity and post deflection (via dense Farneback optical flow). To track texture on changing intensity, we Gaussian-blurred frames and contrast gated low-signal regions. To validate ECC outputs, we treated µHTs with mavacamten. Tunable elastic modulus poly(dimethyl siloxane) yielded soft (bending stiffness: 0.42 N/m) or stiff (1.96 N/m) posts.

Results: Automated FS measurements were validated manually. Mavacamten treatment of µHTs showed a paired decrease in FS (P=.0018) and no change in ΔF/F0 (background-corrected peak Ca2+ amplitude) (P=.9165) (Fig 1). Consistent with our hypothesis, stiff post µHTs showed an increase in slope at 50% Ca rise for both Ca2+-force and Ca2+-FS loops compared to soft µHTs (P=.0009, .0008) (Fig 2). However, stiff µHTs also exhibited a longer delay between peak Ca2+ and peak force (P=.0021) (Fig 2), suggesting afterload adaptations involve improved ECC and longer force generation time, signifying a more mature phenotype.

Conclusion: Afterload drives physiological maturation of iPSC-CMs in engineered µHTs, reflected in improved ECC.