Why the study?
Calmodulinopathies caused by mutations such as D130G-CALM2 produce life-threatening arrhythmias by disrupting Ca²⁺-dependent inactivation of cardiac L-type Ca²⁺ channels. Whether CALM2 suppression can rescue these electrophysiological and mechanical phenotypes after the formation of structured human cardiac tissues remained uncertain.
Does lentiviral CRISPRi-mediated CALM2 suppression rescue electrophysiological and mechanical dysfunction in human iPSC-derived 2D and 3D cardiac tissue models of D130G-CALM2 calmodulinopathy?
Does lentiviral CRISPRi-mediated CALM2 suppression rescue electrophysiological and mechanical dysfunction in human iPSC-derived 2D and 3D cardiac tissue models of D130G-CALM2 calmodulinopathy?
CRISPRi-mediated suppression of CALM2 provides proof-of-concept rescue of electrophysiological and mechanical dysfunction in 2D and 3D human iPSC-derived cardiac tissue models of calmodulinopathic long QT syndrome, accompanied by early structural and transcriptional remodeling.
May support CRISPRi strategies in calmodulinopathy models; leaves open clinical translation pending in vivo validation.
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.
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Huang et al. (2026) studied Calmodulinopathy-associated long QT syndrome (D130G-CALM2 mutation). Lentiviral CRISPR interference (CRISPRi) targeting CALM2 (dCas9-KRAB and CALM2-targeting gRNA) vs. Scrambled non-targeting gRNA (D130G-CALM2 disease control) was evaluated on Minimum capture cycle length in cardiac microtissues (ms) (p=0.014).
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