CRISPR interference-mediated electrical rescue of calmodulinopathy restores electromechanical function and is accompanied by early structural remodeling in human iPSC-derived engineered cardiac tissues

Calmodulinopathies, caused by mutations such as D130G-CALM2, produce life-threatening arrhythmias by disrupting Ca²⁺-dependent inactivation of cardiac L-type Ca²⁺ channels, resulting in prolonged and unstable repolarization. Whether CALM2 suppression can rescue these phenotypes after formation of structured human cardiac tissues remains uncertain. To determine whether CRISPR interference (CRISPRi)-mediated CALM2 suppression rescues electrophysiological and mechanical dysfunction in patient-specific hiPSC-derived cardiac models and to characterize associated transcriptional and ultrastructural changes. Two-dimensional monolayers, three-dimensional cardiospheres, and aligned cardiac microtissues were generated from D130G-CALM2 hiPSC-derived cardiomyocytes and treated with lentiviral CRISPRi targeting CALM2. Phenotypic rescue was evaluated by optical mapping, global GCaMP6f imaging, force measurement, qRT-PCR, and transmission electron microscopy. CRISPRi selectively reduced CALM2 expression, shortened prolonged action potentials, suppressed electrical, global Ca²⁺-amplitude, and mechanical alternans, lowered the minimum capture cycle length, and enhanced force generation. CRISPRi-treated tissues also showed transcriptional and ultrastructural changes consistent with improved organization and early structural remodeling, including a Z-line-aligned membrane invagination suggestive of an early T-tubule-like feature. In engineered in vitro cardiac models, CRISPRi-mediated CALM2 suppression provided proof-of-concept rescue of electromechanical dysfunction associated with calmodulinopathic long QT syndrome. The associated ultrastructural and transcriptional changes are consistent with early structural remodeling and require validation using additional maturation assays and clinically applicable delivery systems.