Tbx20 controls the expression of the KCNH2 gene and of hERG channels

Abstract

Long QT syndrome (LQTS) exhibits great phenotype variability among family members carrying the same mutation, which can be partially attributed to genetic factors. We functionally analyzed the KCNH2 (encoding for Kv11.1 or hERG channels) and TBX20 (encoding for the transcription factor Tbx20) variants found by next-generation sequencing in two siblings with LQTS in a Spanish family of African ancestry. Affected relatives harbor a heterozygous mutation in KCNH2 that encodes for p.T152HfsX180 Kv11.1 (hERG). This peptide, by itself, failed to generate any current when transfected into Chinese hamster ovary (CHO) cells but, surprisingly, exerted "chaperone-like" effects over native hERG channels in both CHO cells and mouse atrial-derived HL-1 cells. Therefore, heterozygous transfection of native (WT) and p.T152HfsX180 hERG channels generated a current that was indistinguishable from that generated by WT channels alone. Some affected relatives also harbor the p.R311C mutation in Tbx20. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), Tbx20 enhanced human KCNH2 gene expression and hERG currents (I hERG ) and shortened action-potential duration (APD). However, Tbx20 did not modify the expression or activity of any other channel involved in ventricular repolarization. Conversely, p.R311C Tbx20 did not increase KCNH2 expression in hiPSC-CMs, which led to decreased I hERG and increased APD. Our results suggest that Tbx20 controls the expression of hERG channels responsible for the rapid component of the delayed rectifier current. On the contrary, p.R311C Tbx20 specifically disables the Tbx20 protranscriptional activity over KCNH2. Therefore, TBX20 can be considered a KCNH2-modifying gene.is characterized by abnormal prolongation of the QT interval of the electrocardiogram (ECG) and is due to delayed ventricular repolarization. LQTS increases the occurrence of ventricular tachyarrhythmias, particularly torsade de pointes, leading to recurrent syncope, seizures, ventricular fibrillation, and sudden cardiac death (SCD) (1). At least 15 genes have been reported in autosomal-dominant forms of LQTS (1). However, mutations in KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3) represent the most frequent forms of LQTS (∼90%) (1, 2).KCNH2 encodes Kv11.1, or hERG, channels, which generate the rapid component of the delayed rectifier current (I Kr ) responsible for ventricular repolarization in humans (3). In a Spanish family of African ancestry suffering LQTS, we identified a frameshift and a missense mutation in KCNH2 that were assumed to be the diseasecausing mutations. However, in some family members, we also identified a missense mutation in TBX20 coding for the transcription factor Tbx20, which is necessary in early stages of heart development (4). Importantly, results in flies and mice demonstrated that Tbx20 is also required for maintaining adult heart function (5, 6).Here we have tested the KCNH2 and TBX20 mutations to establish whether they can account for prolongation of repolarization. Our results dem...

Brugada syndrome trafficking–defective Nav1.5 channels can trap cardiac Kir2.1/2.2 channels

“…Cell culture and transfection. CHO cells (ATCC) were transiently transfected with WT and mutant Nav1.5 or Kir2.x channels by using FUGENE X-tremeGENE (Roche Diagnostics) and cultured as described previously (6,14,15,18). In all experiments, Nav1.5 channels, both WT and mutated, were transfected together with the hNavβ1 subunit.…”

“…Patch clamping. Currents were recorded using the whole-cell patch-clamp technique (6,14,15,18). Series resistance was compensated manually using the compensation unit of the Axopatch amplifier (Molecular Devices); ≥80% compensation was achieved.…”

“…I Na , I K1 , and AP recordings in hiPSC-CMs. Enriched and mature DF19-9-11T hiPSC-CMs generated as described elsewhere (18) and commercial hiPSC-CMs (iCell Cardiomyocytes 2 , Cellular Dynamics) were used and infected with the adenoviral constructs coding WT or mutant Nav1.5 channels. Currents (recorded at 21-23°C) and APs were recorded using the whole-cell patch-clamp technique.…”

“…APs were also recorded in hiPSC-CMs, which exhibited spontaneous automatic activity (Supplemental Figure 6) (18). Infection with Ad-Nav1.5 significantly reduced automatic firing frequency (n ≥ 6, P < 0.05), while infection with Ad-p.G1748D significantly increased it (n ≥ 7, P < 0.05) (Supplemental Figure 6).…”

Human heart disease: lessons from human pluripotent stem cell-derived cardiomyocytes

“…Recently, the use of hiPSC-CMs for modeling LQT2 helped revealing a key role for the transcription factor TBX20 in the regulation of KCNH2 expression [ 98 ]. In this study, Caballero and colleagues investigated the electrophysiological effects of the R311C-TBX20 mutation, which is found in individuals affected by LQTS, in hiPSC-CMs.…”