3D Computational Modeling of Defective Early Endosome Distribution in Human iPSC-Based Cardiomyopathy Models.

Intracellular cargo delivery via distinct transport routes relies on vesicle carriers. A key trafficking route distributes cargo taken up by clathrin-mediated endocytosis (CME) via early endosomes. The highly dynamic nature of the endosome network presents a challenge for its quantitative analysis, and theoretical modelling approaches can assist in elucidating the organization of the endosome trafficking system. Here, we introduce a new computational modelling approach for assessment of endosome distributions. We employed a model of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with inherited mutations causing dilated cardiomyopathy (DCM). In this model, vesicle distribution is defective due to impaired CME-dependent signaling, resulting in plasma membrane-localized early endosomes. We recapitulated this in iPSC-CMs carrying two different mutations, TPM1-L185F and TnT-R141W (MUT), using 3D confocal imaging as well as super-resolution STED microscopy. We computed scaled distance distributions of EEA1-positive vesicles based on a spherical approximation of the cell. Employing this approach, 3D spherical modelling identified a bi-modal segregation of early endosome populations in MUT iPSC-CMs, compared to WT controls. Moreover, spherical modelling confirmed reversion of the bi-modal vesicle localization in RhoA II-treated MUT iPSC-CMs. This reflects restored, homogeneous distribution of early endosomes within MUT iPSC-CMs following rescue of CME-dependent signaling via RhoA II-dependent RhoA activation. Overall, our approach enables assessment of early endosome distribution in cell-based disease models. This new method may provide further insight into the dynamics of endosome networks in different physiological scenarios.