Abstract Body: Introduction/Background: Dilated cardiomyopathy (DCM) is frequently associated with genetic variants affecting sarcomeric proteins. Cardiac troponin T (cTnT), encoded by TNNT2, plays a critical role in sarcomere organization and myocardial contractility. Although several TNNT2 variants have been reported in patients with DCM, functional validation remains limited for many variants, making it difficult to directly link specific variants to disease phenotypes.
Research Questions/Hypothesis: This study hypothesized that the TNNT2-R205W variant is a pathogenic driver responsible for the functional and structural defects observed in DCM cardiomyocytes, and that these phenotypes can be rescued by genomic correction.
Goals/Aims: We aimed to provide a comprehensive variant-to-function validation of the TNNT2-R205W mutation by establishing a causal link between the variant and the DCM phenotype using an isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) model.
Methods/Approach: We generated hiPSCs from a patient carrying the TNNT2-R205W variant and differentiated them into hiPSC-CMs. CRISPR-Cas9 genome editing was used to correct the mutation and generate isogenic control lines. Electrophysiological properties were evaluated using multi-electrode array (MEA) recordings, intracellular Ca²⁺ dynamics were assessed by confocal line-scan imaging with Fluo-4 dye, and sarcomeric ultrastructure was examined using transmission electron microscopy (TEM).
Results/Data: Patient-derived TNNT2-R205W hiPSC-CMs exhibited significant functional abnormalities, including reduced conduction velocity, impaired Ca²⁺ handling, and decreased contractile amplitude. TEM demonstrated clear ultrastructural defects, specifically a markedly increased proportion of Z-band disruption in TNNT2-R205W hiPSC-CMs. Importantly, CRISPR-Cas9–mediated correction of the variant substantially restored electrophysiological function, Ca²⁺ transients, and contractile performance, and markedly reduced the proportion of Z-band disruption, effectively reversing the disease phenotype in vitro.
Conclusions: These findings provide functional evidence supporting the pathogenic role of the TNNT2-R205W variant and demonstrate the utility of patient-specific hiPSC models combined with genome editing for variant-to-function validation in inherited cardiomyopathies.