The disease-specific phenotype in cardiomyocytes derived from induced pluripotent stem cells of two long QT syndrome type 3 patients

Abstract

Long QT syndromes (LQTS) are heritable diseases characterized by prolongation of the QT interval on an electrocardiogram, which often leads to syncope and sudden cardiac death. Here we report the generation of induced pluripotent stems (iPS) cells from two patients with LQTS type 3 carrying a different point mutation in a sodium channel Nav1.5 (p.V240M and p.R535Q) and functional characterization of cardiomyocytes (CM) derived from them. The iPS cells exhibited all characteristic properties of pluripotent stem cells, maintained the disease-specific mutation and readily differentiated to CM. The duration of action potentials at 50% and 90% repolarization was longer in LQTS-3 CM as compared to control CM but this difference did not reach statistical significance due to high variations among cells. Sodium current recordings demonstrated longer time to peak and longer time to 90% of inactivation of the Na(+) channel in the LQTS-3 CM. This hints at a defective Na(+) channel caused by deficiency in open-state inactivation of the Na(+) channel that is characteristic of LQTS-3. These analyses suggest that the effect of channel mutation in the diseased CM is demonstrated in vitro and that the iPS cell-derived CM can serve as a model system for studying the pathophysiology of LQTS-3, toxicity testing and design of novel therapeutics. However, further improvements in the model are still required to reduce cell-to-cell and cell line-to-cell line variability.

Figure 1. Characterization of LQTS-3 iPS cells.

V240M-LQTS-3 iPS cell colony derived from patient NP0016 showing typical human ES cell-like morphology (A) and positive staining for alkaline phosphatase (B). The V240M-LQTS-3 iPS cell colonies expressed pluripotency markers OCT4, SOX2, NANOG, SSEA4, TRA-1-80 and TRA-1-60 as determined by flow cytometry (C) and immmunostaining (D). Expression of pluripotency markers in V240M-LQTS-3 iPS cells by qRT-PCR. Expression values were normalized to GAPDH and are presented as mean +SEM (n=3) relative to corresponding transcript levels in human ES cells (E). Methylation levels of promoter regions of OCT4 and NANOG genes in three different clones of R535Q-LQTS-3 iPS cells at passage 7 and R535Q-LQTS-3 fibroblasts (F). The LQTS-3 iPS colonies derived from dermal fibroblasts of patient NP0012 were verified for the presence of a heterozygous SCN5A point mutation p.R535Q by DNA sequencing (G).

Figure 2. Expression of cardiac markers in R535Q-LQTS-3 iPS cell-derived CM.

(A) Immunocytochemical analysis of cardiac markers α-actinin (red) and troponin I (green). Nuclei were counterstained with DAPI (blue). Scale bars: 20 μm (α-actinin) and 100 μm (troponin I). (B) The analysis of expression levels of cardiospecific transcripts was done by RT-PCR showing that the R535Q-LQTS-3 iPS cell-derived cardiomyocytes express cardiac transcripts at a similar level as their human ES cell-derived counterparts.

Figure 3. Patch clamp recordings of action potentials in R535Q-LQTS-3 iPS cell-derived CM.

(A) Overlay of trace from R535Q-LQTS-3 and healthy cells representing the difference in action potential duration at 50% of repolarization (APD50) in ventricular (left) and atrial (right) like cells. (B) Statistical analysis of the differences in APD90 and APD50 of control and diseased cells.

Figure 4. Sodium current measurements in control and LQTS-3 cells.

Voltage-gated Na⁺ channel currents were recorded in a voltage-clamp mode. Values for current density (A), time of 90% inactivation (B) and time to peak (C) are shown for human ES cell derived CM (n=8), control iPS cell-derived CM (n=11) and for LQTS-3 iPS cell-derived CM from patients NP0012 (p.R535Q, n=13) and NP0016 (p.V240M, n=12). (D,E) Exemplary current traces with an increased persistent sodium current typical of LQTS-3 in diseased R535Q-LQTS-3 CM but not in healthy ES cell-derived CM. Symbols: # p<0.05, * p<0.02, ** p<0.01, ***p<0.005. Differences between all other groups were not statistically significant.