Abstract 14701: Nonsense Mediated mRNA Decay Mediates the Loss Of IKr in LQT2 Patient-Derived iPS-Cardiomyocytes With W1001X-KCNH2
Type 2 long QT syndrome (LQT2) is characterized by cardiac arrhythmia caused by mutations in the KCNH2-encoded rapidly activating delayed rectifier K+ channel (Kv11.1) that mediates the IKr current and is critical for normal cardiac repolarization. More than 500 LQT2-associated KCNH2 mutations have been reported of which ~30% are nonsense or frame shift mutations that introduce premature termination codons (PTCs). PTC bearing mRNA would make truncated proteins if not degraded via n onsense m ediated mRNA d ecay (NMD). In the present study, we tested the NMD hypothesis in LQT2 patient-derived iPS cardiomyocytes (iPS-CMs). Fibroblasts were obtained from skin biopsy of a 64 yo woman with a KCNH2 nonsense mutation, W1001X. The fibroblasts were reprogrammed using episomal vectors encoding OCT4, SOX2, NANOG, LIN28, c-Myc, KLF4 and T-antigen to generate vector and transgene free iPS cells. Monolayers of W1001X and healthy control (HC) iPS cells were differentiated to iPS-CMs using extracellular matrix and growth factors. CMs contracted spontaneously and expressed cardiac myofilament proteins. The iPS-CMs were studied using whole cell patch clamp. IKr was measured as tail current at -50 mV following a prepulse to 20 mV and was blocked by E4031 (0.5 μM). We observed a 44% loss in IKr peak tail current in W1001X compared to HC iPS-CMs (0.41±0.05 pA/pF and 0.73± 0.06 pA/pF, respectively, n=10, p<0.05). Peak INa for W1001X and HC iPS-CMs was not different (-0.18±0.02 and -0.15±0.04 nA/pF, respectively, n=7, p>0.05). Western blot analyses and confocal imaging indicated a reduction of Kv11.1 protein in W1001X iPS-CMs suggesting haploinsufficiency. Allele specific quantification studies showed a significant decrease in the level of W1001X mutant mRNA compared to that of the wild-type allele. Incubation of W1001X iPS-CMs with the protein translation inhibitor cycloheximide (100 μg/ml) reversed down regulation of the PTC bearing mRNA. Our data suggest that loss of IKr in W1001X iPS-CMs results from allele-specific down regulation of PTC bearing mRNA through NMD. These results provide the first proof of NMD in human cardiomyocytes and enable in vitro assessment of patient-specific therapeutic interventions modulating NMD.
- © 2013 by American Heart Association, Inc.