Abstract 4144727: <i>In vitro</i> modelling and functional improvement of TNNT2-R141W mutant pediatric dilated cardiomyopathy <i>.</i>

Introduction: Dilated cardiomyopathy (DCM) is a common cardiomyopathy characterized by ventricular dilatation and systolic dysfunction. The TNNT2-R141W mutation is known to cause DCM in both humans and mice and is associated with Ca 2+ desensitization and reduced contractility. However, there are no reports of utilizing patient-derived induced pluripotent stem cells (iPSCs) carrying the TNNT2-R141W mutation, looking at functional improvement after gene replacement. Here, we describe the characteristics of TNNT2-R141W mutant iPSC-derived cardiomyocytes (iPSC-CMs) in vitro . Methods: Two human iPSC lines were derived from pediatric DCM patient samples, with the TNNT2-R141W mutation (R141W-iPSC), and a R141W-iPSC derived TNNT2 overexpression line (TNNT2-OE iPSC) for comparison. The Ca 2+ handling and sarcomere structures of the differentiated hiPSC-CMs were then assessed. Results: In TNNT2-OE iPSC-CMs, the expression level of TNNT2 was significantly higher than R141W-iPSC-CMs. Evaluation of Ca 2+ handling revealed that R141W-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 (Figure 1a, b, and c). Ca 2+ reuptake efficacy during relaxation, measured by Ca 2+ decay tau, or the time taken for an 80% signal reduction, was also significantly prolonged in R141W-iPSC-CMs (Figure 1d). In addition, R141W-iPSC-CMs displayed abnormality in sarcomere alignment (Figure 2). These properties were corrected in TNNT2-OE iPSC-CMs, indicating that R141W-iPSC-CMs reflect DCM phenotypes in vitro . Conclusions: R141W-iPSC-CMs exhibited Ca 2+ desensitization and abnormal sarcomere structure, further suggesting that gene therapy with normal TNNT2 may be a future therapeutic approach for DCM with TNNT2 mutations.