Kinematic-calcium loops unravel impaired excitation–contraction coupling in MELAS-affected cardioids

: Impaired Ca2+ handling, and in particular leakage from the sarcoplasmic reticulum, is a critical mechanism in metabolic diseases affecting the heart. Phase-plane loop analysis provides an integrated assessment of excitation-contraction coupling (ECC) by capturing the dynamic relation between Ca2 + transients and mechanical contraction, exceeding standard time analysis limitations. Here, we investigated how mitochondrial encephalopathy, lactic acidosis and stroke-like episodes metabolic disorder (MELAS) impairs the ECC using cardiac spheroids from diseased human induced pluripotent stem cells (m3243A>G mutation) and matched control (mtDNA mutation <10%). High-speed dual-mode imaging at 200 fps enabled simultaneous acquisition of Ca2 + dynamics and spheroid kinematics. To uncover disease-specific mechanisms, supra-threshold electric field stimulation was applied to simulate increased energy demand. After signal extraction, we built our interpretation of phase-plane loops, comprising kinematic-calcium (Ki-Ca) loops, to quantify ECC efficiency. Time-domain analysis demonstrated that MELAS cardiac spheroids showed significant reduction in beat duration at kinematics (1.068 ± 0.066 s vs. 0.775 ± 0.094 s), as well as decreased Ca2+ transient duration (0.984 ± 0.049 s vs. 0.664 ± 0.042 s). Critically, Ki-Ca loop analysis provided a more complete picture where MELAS samples displayed visibly different loops and a significant reduction of their area compared to controls (0.129 ± 0.056 vs. 0.082 ± 0.163). These findings demonstrate that Ki-Ca loops provide a sensitive and integrative metric for detecting ECC dysfunction in human in vitro cardiac models. This approach offers mechanistic insight into how mitochondrial metabolic disorders, such as MELAS, compromise the coupling between Ca2 + cycling and contractility. KEY POINTS: Phase-plane Ki-Ca loops effectively contribute to understanding the excitation-contraction coupling (ECC) efficiency. Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes metabolic disorder (MELAS) impairs ECC in cardioid models. Cardiac challenge pacing protocol highlights beating anomaly in MELAS spheroids, uncovering ECC failure.