Arrhythpy: an automated tool to quantify and classify arrhythmias in Ca ²⁺ transients of iPSC-cardiomyocytes

Arrhythmias constitute an intricate and clinically significant phenomenon of great importance in various research areas. Calcium (Ca²⁺) homeostasis plays a pivotal role in forming rhythmic contractions in the heart, and its dysregulation has emerged as a critical component in the development of arrhythmias. However, the quantification of arrhythmias has been limited to indirect measurements via Ca²⁺ sparks, electrophysiological parameters, or manual classification, which can lead to human bias. We aimed to develop an analysis platform that automatically analyzes arrhythmias in human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Here, we present Arrhythpy, a robust and automated open-source program that quantifies and classifies confocal microscopy-based Fluo-4 Ca²⁺ transients to generate a measure of arrhythmia. In contrast to other automated and semiautomated analysis tools, which measure established parameters such as time-to-peak, Arrhythpy directly analyzes the degree of arrhythmia in a Ca²⁺ transient. We demonstrate its utility in monitoring Ca²⁺ transient-based arrhythmias in atrial and ventricular iPSC-CMs from healthy individuals and patients with cardiac disease, including dilated cardiomyopathy (DCM) and Takotsubo syndrome (TTS). Arrhythpy analysis of iPSC-CMs of patients with TTS recapitulated TTS phenotypes, including atrial arrhythmia that could be normalized with β-blocker treatment. The program's adaptable framework enables the analysis of arrhythmic patterns in various cell types using periodic dye-based line scan measurement techniques, applicable to both single cells and layered cultures.<b>NEW & NOTEWORTHY</b> Arrhythmias in calcium transients are easy to detect by human perception. However, quantifying these arrhythmias in a computer-readable manner remains challenging. To address this, we developed Arrhythpy, an automated tool that measures arrhythmias in iPSC-derived cardiomyocytes by analyzing Ca²⁺ transients. Unlike other tools, Arrhythpy directly evaluates arrhythmia levels. It effectively monitors arrhythmias in healthy and diseased iPSC-CMs, including dilated cardiomyopathy and Takotsubo syndrome. Arrhythpy's flexible framework suits varied cell types and measurement techniques.