(W1308) GENE REPLACEMENT USING WILD-TYPE TNNT2 RESCUES THE PHENOTYPES OF IPSC-CARDIOMYOCYTES DERIVED FROM PEDIATRIC DILATED CARDIOMYOPATHY HARBORING THE TNNT2-R151W MUTATION.
graduate student Osaka University Osaka University, Osaka, Japan
Abstract: Dilated cardiomyopathy (DCM) is a common cardiomyopathy characterized by ventricular dilatation and systolic dysfunction. The TNNT2-R151W mutation is known to cause DCM in both human and mouse models, demonstrating associations with Ca2+ desensitization and reduced contractility. However, there are no reports of utilizing patient-derived induced pluripotent stem cells (iPSCs) carrying the TNNT2-R151W mutation, looking at functional improvement after gene replacement. Here, we describe the characteristics of both TNNT2-R151W mutant iPSC-derived cardiomyocytes (R151W-iPSC-CMs) and iPSC-CMs with gene replacement for the mutation in vitro. Two human iPSC lines were generated from blood samples of pediatric DCM patients carrying the TNNT2-R151W mutation, and the TNNT2 overexpression lines derived from these R151W-iPSC lines (TNNT2-OE iPSC) were used to assess Ca2+ handling and sarcomere structures. To investigate contractile dynamics, we fabricated engineered heart tissues (EHTs) using R151W-iPSC-CMs and TNNT2-OE iPSC-CMs. Ca2+ handling analysis revealed that R151W-iPSC-CMs exhibited a significant decrease in maximum fluorescence intensity (amplitude) and a prolongation of time to maximum fluorescence intensity (time to peak), which are characteristic of failing heart tissue. Additionally, Ca2+ reuptake efficacy during relaxation, measured by Ca2+ decay tau, or the time taken for an 80% signal reduction, was also significantly prolonged in R151W-iPSC-CMs. Furthermore, R151W-iPSC-CMs displayed abnormality in sarcomere alignment. Notably, R151W-EHTs displayed diminished contractile force compared to TNNT2-OE-EHTs. These properties were ameliorated in TNNT2-OE iPSC-CMs, indicating that R151W-iPSC-CMs reflect DCM phenotypes in vitro. In conclusion, R151W-iPSC-CMs exhibited Ca2+ desensitization and abnormal sarcomere structure, and impaired contractility. These results suggest that gene therapy with normal TNNT2 may represent a promising therapeutic approach for pediatric DCM patients with TNNT2 mutations.
