Abstract 15643: Loss of IKr in LQT2 Patient iPS-derived Cardiomyocytes : Nonsense Mediated Decay as a Potential Mechanism?
Type 2 long QT syndrome (LQT2) is caused by mutations in KCNH2-encoded Kv11.1 K+ channels that underlie the rapidly activating delayed rectifier K+ current (IKr). Most LQT2 mutations are missense and generate trafficking-deficient channel protein to cause loss of IKr by reducing channel density on the cell surface. However, nonsense and frame-shift mutations introduce a premature termination codon (PTC) resulting in degradation of KCNH2 mRNA through nonsense mediated decay (NMD), thus the mutant allele generates little or no protein. In the present work, we investigated the NMD hypothesis by studying induced pluripotent stem cell (iPS) derived cardiac myocytes (CMs) harboring the W1001X nonsense mutation on one allele. Dermal fibroblasts were obtained from skin biopsy of a 64 yo woman expressing KCNH2-W1001X and reprogrammed to iPS cells using episomal vectors encoding OCT4, SOX2, NANOG, LIN28, c-Myc, and KLF4. iPS monolayers were differentiated into CMs by culture in extracellular matrix and growth factors. The iPS-CMs express cardiac myofilament proteins and contract spontaneously. Confocal imaging demonstrated reduced expression of Kv11.1 protein on the cell surface of W1001X iPS-CMs compared to healthy control iPS-CMs. Voltage clamp was performed on iPS-CMs to measure ion channel currents. IKr was measured as tail current at -50 mV following a prepulse to 20 mV, and was blocked by E4031 (0.5 μM). The W1001X iPS-CMs displayed a 44% reduction in peak tail IKr compared to healthy control iPS-CMs; IKr densities were 0.41±0.05 and 0.73± 0.06 pA/pF, respectively (n=10, p<0.05). The Kv11.1 activation half-maximal voltage and slope factor were similar for W1001X and healthy control iPS-CMs. For comparison other ionic currents were measured. Peak INa for the W1001X and healthy control iPS-CMs was not different; INa densities were -0.18±0.02 and -0.15±0.04 nA/pF, respectively (p>0.05, n=7). We also tested PTC124, a drug shown to readthrough mRNA PTCs, and did not detect an increase in IKr in the W1001X iPS-CMs. We conclude that the W1001X mutation, which is predicted to introduce a PTC in KCNH2 mRNA, causes approximately a 50% loss of IKr (haploinsufficiency) in LQT2 patient derived iPS-CMs consistent with nonsense mediated decay of mutant KCNH2 transcripts.
- © 2011 by American Heart Association, Inc.