Patient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.
Four human iPSC cell lines (one Jervell and Lange-Nielsen Syndrome, one Long QT Syndrome-type 1 and two healthy controls) were generated from peripheral blood obtained from donors belonging to the same family. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing OCT3/4, KLF4, SOX2 and cMYC as reprogramming factors) was used to generate all cell lines. The four iPSCs have normal karyotype, express pluripotency markers as determined by RT-PCR and flow cytometry and differentiated spontaneously in vitro into cells of the three germ layers, confirming their pluripotent capacity.
Two gene variants previously associated with LQTS were found in two families clinically diagnosed with LQTS. The prolongation of the QT interval was observed in all family members carrying the mutations. A strategy was developed to identify variants of genes KCNQ1, KCNH2 and SCN5A, making it possible to train technical staff for future application to diagnosis routine.
The influence of genetics on atherosclerosis development remains unclear. Association of single-nucleotide polymorphisms (SNPs) with early-onset coronary artery disease (EOCAD), as well as with carotid intima-media thickness (CIMT), and systemic microvascular reactivity was examined in 52 patients with EOCAD (45 ±3 years old) and 35 age-matched controls. SNPs in ACE, TNF-α (-308G/A, -238 G/A), IFN-γ (+874 A/T), MMP-9 (-1562 C/T), IL-10 (-1082 A/G, -819 C/T, 592 C/A), NOS3 (894 G/T), ApoA1 (rs964184), and TGF-β (codon 10 T/C) genes were genotyped by automatic sequencing. CIMT was determined by ultrasound and capillary density by intravital video-microscopy. Microvascular reactivity was evaluated using laser speckle contrast imaging, cutaneous blood flow was assessed at rest and during post-occlusive reactive hyperemia (PORH), and acetylcholine (Ach) or sodium nitroprusside (SNP) iontophoresis. Genotype distribution was: ACE: 31 DD, 12 II, 43 DI; TNF-α: 56 GG, 20 GA, 3 AA at position -308, and 76 GG, 3 GA at position -238; IFN-γ: 8 subjects with high producer TT genotype; MMP-9: 62 CC, 23 CT; IL-10: 8 high producer haplotype, 35 intermediate, and 43 low; NOS3: 41 GG, 34 GT, 11 TT; ApoA1: 47 CC, 29 CG, 10 GG; TGF-β (cd 25): 75 GG, 10 GC; TGF-β (cd 10): 66 high producer genotype, 19 low producer. IFN-γ, IL-10 haplotype, and TGF-β were significantly related to EOCAD. CIMT was increased (0.93 ±0.02 mm, p<0.0001), while capillary density (87 ±3 capillaries/mm2, p=0.029) was reduced in EOCAD. CC genotype of TGF-β showed higher CIMT (p=0.004) and lower capillary density (p=0.019) than TT. High IL-10 haplotype reduced vasodilator response after PORH (0.32 APU/mmHg, p=0.0316), while high IFN-γ genotype reduced the response induced by both Ach (0.19 APU/mmHg) and SNP (0.13 APU/mmHg) in relation to low producer (0.44, 0.31, and 0.24 APU/mmHg, respectively). TT genotype of TGF-β showed lower vasodilator response after SNP than CC. In conclusion, IFN-γ, IL-10 and TGF-β genotypes were associated with EOCAD, as well as with systemic microvascular response, CIMT, and capillary rarefaction. The results suggest that IFN-γ, IL-10 and TGF-β genotypes could influence EOCAD, affecting microvascular reactivity through endothelium-dependent and -independent pathways.
The purpose of this work was to investigate if iPSC derived cardiomyocytes would reproduce the divergent phenotypes observed in a proband and mother that carried the same likely-pathogenic variant in KCNQ1 gene. A 15 years old proband had syncope and cardiac arrest while swimming being diagnosed with LQTS based on a 12 lead ECG. The mother’s ECG showed no abnormalities. Genomic DNA from blood of daughter and mother was extracted using standard procedures. All coding exons and flanking intronic sequences of KCNQ1 (NM_000218) genes were amplified by polymerase chain reaction. Direct sequencing was performed on a 3500XL Genetic Analyzer. Data was analyzed with Geneious software for identification of mutations. For iPSC generation CD71+ CD36+ erythroblasts were reprogrammed using CytoTune™-iPS 2.0 Sendai Reprogramming Kit on irradiated mouse embryonic fibroblast feeder layers. Differentiation into cardiomyocytes was accomplished using the protocol described by Lian et al. (2012). Electrophysiological recordings were obtained from cardiomyocytes after 30–45 days of differentiation. Cells were transferred to acrylic plates pretreated with Matrigel and kept in culture for 3 days before microelectrode action potential (AP) were registered. The ECG recordings showed a corrected QT interval (QTc) of 516 ms for the daughter and 436 ms for the mother. Sequencing showed that both daughter and mother had a c.1763C>T mutation resulting in p.Ile588Thr substitution. iPSC generated from daughter, mother and an unrelated control displayed normal karyotype and expressed pluripotent genes, besides spontaneously differentiating into cells of the three germ layers. Differentiation into cardiomyocytes was ascertained by immunofluorescence and flow cytometry for troponin T. In all differentiations more than 70% of cells were cardiomyocytes. Action potentials recordings showed that ventricular cardiomyocytes from the daughter exhibited significantly longer action potential durations at 90% repolarization than the mother’s or the unrelated control’s cardiomyocytes. Cardiomyocytes derived from iPS cells reproduced the clinical phenotypes exhibited by daughter and mother.
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