Functional analysis of JPH2-knockout cardiomyocytes identifies ECCD as a novel indicator in a human cardiac modelJPH2

Junctophilin-2 (JPH2) is a vital protein in cardiomyocytes, anchoring T-tubule and sarcoplasmic reticulum membranes to facilitate excitation-contraction coupling, a process essential for cardiac contractile function. Dysfunction of JPH2 is associated with cardiac disorders such as heart failure; however, prior studies using mouse models or primary human cardiomyocytes are limited by interspecies differences or poor cell viability, respectively. This study aimed to investigate JPH2's role in human cardiac function and disease using a novel stem cell-derived model, while introducing a new indicator to evaluate related cardiac impairments.

We generated a JPH2-knockout model using human embryonic stem cell-derived cardiomyocytes (hESC-CMs) with CRISPR/Cas9. Cellular morphology, contractile function, calcium dynamics, and electrophysiological properties were assessed via transmission electron microscopy, the CardioExcyte96 system, calcium imaging with Fluo-4 AM, and multi-electrode array recordings, respectively. Wild-type JPH2 was overexpressed through lentiviral transfection to evaluate rescue effects, and two JPH2 variants-one benign (G505S) and one pathogenic (E85K)-were introduced to study mutation-specific effects.

JPH2 knockout disrupted excitation-contraction coupling in hESC-CMs by impairing junctional membrane complex structure, leading to heart failure-like phenotypes with reduced contractility, altered calcium dynamics, and electrophysiological irregularities. Overexpression of wild-type JPH2 restored these functions, affirming its critical role in cardiac physiology. We identified excitation-contraction coupling delay (ECCD) as a novel indicator that precisely quantified coupling impairment severity, with its applicability validated across distinct JPH2 variants (G505S and E85K).

This study demonstrates JPH2's essential role in sustaining excitation-contraction coupling by stabilizing the junctional membrane complex, with its deficiency driving heart failure-like cardiac dysfunction. ECCD is established as a sensitive, comprehensive indicator for assessing JPH2-related impairment severity. These findings advance our understanding of JPH2 in cardiac pathology and position ECCD as a valuable tool for research and potential clinical evaluation, with JPH2 and calcium regulation emerging as promising therapeutic targets.