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

Ricardo Caballeroa,b,1, Raquel G. Utrillaa,b,1, Irene Amorósa,b, Marcos Matamorosa,b, Marta Pérez-Hernándeza,b, David Tinaqueroa,b, Silvia Alfayatea,b, Paloma Nieto-Marína,b, Guadalupe Guerrero-Sernac,d, Qing-hua Liuc,d, Roberto Ramos-Mondragónc,d, Daniela Ponce-Balbuenac,d, Todd Herronc,d, Katherine F. Campbellc,d, David Filgueiras-Ramae,2, Rafael Peinadob,e, José L. López-Sendónb,e, José Jalifec,d,f, Eva Delpóna,b,3,4, and Juan Tamargoa,b,3

aDepartment of Pharmacology, School of Medicine, Instituto de Investigación Sanitaria Gregorio Marañón, Universidad Complutense, 28040 Madrid, Spain; bCentro de Investigación Biomédica en Red (CIBER), Spain; cDepartment of Internal Medicine, Center for Arrhythmia Research, University of Michigan, Ann Arbor, MI 48109; dDepartment of Molecular and Integrative Physiology, Center for Arrhythmia Research, University of Michigan, Ann Arbor, MI 48109; eDepartment of Cardiology, Hospital Universitario La Paz, Instituto de Investigación Sanitaria La Paz (IdiPaz), 28046 Madrid, Spain; and fCardiac Arrhythmia Department, Fundación Centro Nacional de Investigaciones Cardiovasculares (CNIC), 28029 Madrid, Spain

Edited by Richard W. Aldrich, The University of Texas at Austin, Austin, TX, and approved November 23, 2016 (received for review July 27, 2016) Long QT syndrome (LQTS) exhibits great phenotype variability among Here we have tested the KCNH2 and TBX20 mutations to family members carrying the same mutation, which can be partially establish whether they can account for prolongation of re- attributed to genetic factors. We functionally analyzed the KCNH2 polarization. Our results demonstrated that more than “one hit” is (encoding for Kv11.1 or hERG channels) and TBX20 (encoding for the necessary to give rise to LQTS in the affected relatives. Moreover, transcription factor Tbx20) variants found by next-generation sequenc- data reveal that the peptide resulting from the KCNH2 frameshift ing in two siblings with LQTS in a Spanish family of African ancestry. mutation exerts chaperone-like effects by increasing the membrane Affected relatives harbor a heterozygous mutation in KCNH2 that en- expression of WT hERG channels. Conversely, the p.R311C Tbx20 codes for p.T152HfsX180 Kv11.1 (hERG). This peptide, by itself, failed to mutation specifically and markedly decreases KCNH2 expression. generate any current when transfected into Chinese hamster ovary Therefore, our genetic and functional studies suggest that Tbx20 (CHO) cells but, surprisingly, exerted “chaperone-like” effects over na- controls the expression of hERG channels in human myocytes and, PHYSIOLOGY tive hERG channels in both CHO cells and mouse atrial-derived HL-1 thus, TBX20 may be considered a KCNH2-modifying gene. cells. Therefore, heterozygous transfection of native (WT) and p.T152HfsX180 hERG channels generated a current that was indistin- Results guishable from that generated by WT channels alone. Some affected The proband (II:4; Fig. 1A) was a 41-y-old male who experienced relatives also harbor the p.R311C mutation in Tbx20. In human induced syncope when he got out of bed. The ECG showed sinus rhythm at pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), Tbx20 en- 68 beats per min (bpm) with normal PR (168 ms) and QRS (88 ms) KCNH2 I hanced human gene expression and hERG currents ( hERG) but with a low-amplitude and wide T wave (Fig. 1B). Bazzett-cor- and shortened action-potential duration (APD). However, Tbx20 did rected QT value (QTc) was 480 ms. Echocardiography, exercise not modify the expression or activity of any other channel involved in ventricular repolarization. Conversely, p.R311C Tbx20 did not in- Significance crease KCNH2 expression in hiPSC-CMs, which led to decreased IhERG and increased APD. Our results suggest that Tbx20 controls the expres- sion of hERG channels responsible for the rapid component of the Tbx20 is a transcription factor whose critical role in cardiogenesis delayed rectifier current. On the contrary, p.R311C Tbx20 specifically is well-established. Here we functionally analyzed the electro- disables the Tbx20 protranscriptional activity over KCNH2. Therefore, physiological effects produced by a mutation (p.R311C) in Tbx20 TBX20 can be considered a KCNH2-modifying gene. found in some affected individuals belonging to a family with long QT syndrome (an inherited cardiac arrhythmia due to Tbx20 | hERG channels | long QT syndrome | cardiomyocytes | delayed ventricular repolarization). We demonstrated that Tbx20 KCNH2 human induced pluripotent stem cells selectively increases the expression of , which encodes for the channel Kv11.1 (hERG) that generates the main ventricular repolarizing current. Conversely, the p.R311C mutation disables ong QT syndrome (LQTS) is characterized by abnormal pro- the Tbx20 protranscriptional activity over KCNH2,leadingtoa Llongation of the QT interval of the electrocardiogram (ECG) decrease in the hERG current and a prolongation of the action and is due to delayed ventricular repolarization. LQTS increases the potentials recorded in human induced pluripotent stem cell-derived occurrence of ventricular tachyarrhythmias, particularly torsade de cardiomyocytes. Therefore, we propose that Tbx20, besides its pointes, leading to recurrent syncope, seizures, ventricular fibrilla- described role, regulates KCNH2 expression. tion, and sudden cardiac death (SCD) (1). At least 15 genes have

been reported in autosomal-dominant forms of LQTS (1). How- Author contributions: R.C., J.J., E.D., and J.T. designed research; R.C., R.G.U., I.A., M.M., ever, mutations in KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A M.P.-H., D.T., S.A., P.N.-M., G.G.-S., Q.-h.L., R.R.-M., D.P.-B., T.H., K.F.C., J.J., E.D., and J.T. (LQT3) represent the most frequent forms of LQTS (∼90%) (1, 2). performed research; D.F.-R., R.P., and J.L.L.-S. conducted clinical evaluation of family KCNH2 encodes Kv11.1, or hERG, channels, which generate the members; R.C., R.G.U., I.A., M.M., M.P.-H., D.T., S.A., P.N.-M., G.G.-S., Q.-h.L., R.R.-M., D.P.-B., J.J., E.D., and J.T. analyzed data; and R.C., J.J., E.D., and J.T. wrote the paper. rapid component of the delayed rectifier current (IKr) responsible for The authors declare no conflict of interest. ventricular repolarization in humans (3). In a Spanish family of Af- This article is a PNAS Direct Submission. rican ancestry suffering LQTS, we identified a frameshift and a 1R.C. and R.G.U. contributed equally to this work. missense mutation in KCNH2 that were assumed to be the disease- 2Present address: Cardiac Arrhythmia Department, Fundación Centro Nacional de Inves- causing mutations. However, in some family members, we also tigaciones Cardiovasculares (CNIC), 28029 Madrid, Spain. TBX20 identified a missense mutation in coding for the transcription 3E.D. and J.T. contributed equally to this work. factor Tbx20, which is necessary in early stages of development 4To whom correspondence should be addressed. Email: [email protected]. (4). Importantly, results in flies and mice demonstrated that Tbx20 is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. also required for maintaining adult heart function (5, 6). 1073/pnas.1612383114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1612383114 PNAS Early Edition | 1of10 Downloaded by guest on September 25, 2021 A

B C p.T152HfsX180 p.Q1068R II:1 II:1

II:4 III:2

D +60 mV E 125 135 404 666 748 872 1159 -60 mV WT -80 mV N-CapPAS Proximal S1-S6 C-linker cNBD Distal N-terminus Transmembrane C-terminus segments

WT (0.5 g) WT (1 g) 113533225 152 p.T152HfsX180 N-CapPAS Changed aa 10 pA/pF 2 s 2 s P<0.05 F P<0.05 p.T152HfsX180 WT (0.5 g) + P>0.05 (1 g) p.T152HfsX180 (0.5 g) 30 P<0.01

20 tail HERG

I 10 density (pA/pF) 0

10 pA/pF HERG WT ( g) 0.5 1 0 0.5 2 s 2 s p.T152HfsX180 ( g) 0010.5

Fig. 1. (A) Pedigree of the studied family. The arrow indicates the proband. Circles and squares represent females and males, respectively. + and – represent subjects with and without the p.T152HfsX180 and p.Q1068R hERG variants, respectively. (B) Twelve-lead electrocardiogram of the proband (paper speed 25 mm/s). (C) DNA sequence chromatograms depicting the heterozygous c.453dupC and the c.3203A>G changes of the KCNH2 gene in different family members. (D) Traces were obtained by applying the protocol (Top) for currents recorded in CHO cells transfected with WT, p.T152HfsX180, and WT/p.T152HfsX180 hERG channels. (E) Schematic representation of the WT and p.T152HfsX180 hERG protein domains. (F) hERG tail current density recorded in CHO cells transfected with WT, p.T152HfsX180, and WT/p.T152HfsX180 hERG channels after pulses to +60 mV (n ≥ 6). Each bar represents mean ± SEM of the data (n ≥ 8cells).

test, and Holter were completely normal. Thereafter, bisoprolol not reveal any structural disease or arrhythmias. However, she treatment was started. No new episodes have been documented to suffers from lupus with a mild decrease of kidney function and is date. Evaluation of the family identified two sisters who died sud- under treatment with prednisone, hydroxychloroquine, myco- denly: one at age 19, in the postpartum period (II:2), and another at phenolate, and spironolactone (25 mg daily). Her most recent + age 17 (II:5). Both had been diagnosed with epilepsy and treated serum K concentration was 5.2 mEq per L. Sister II:6 is also with phenobarbital until death. Interestingly, II:2, who exhibited a asymptomatic; her ECG showed normal PR (140 ms) and QRS QTc of 440 ms, underwent an adrenaline test that was negative. (90 ms) intervals but sinus bradycardia (48 bpm) and low-voltage Sister II:1 also presented syncopal episodes since she was 13, wide QT waves (QTc 460 ms) (Fig. S1). An adrenaline test was when she was diagnosed with epilepsy and treated with phenobar- positive: on ECG the QTc was prolonged to 618 ms, and there bital. After a syncopal episode at rest, the ECG showed a QTc of were T-wave amplitude alternans (Fig. S1) and polymorphic 560 ms, and ECG monitoring documented a polymorphic ventric- ventricular extrasystoles. Therefore, an ICD was implanted and ular tachycardia. At age 43, she experienced an aborted SCD de- bisoprolol treatment was started. The proband’smother(66yold), spite atenolol treatment (50 mg twice daily) and pacemaking at who had previously been asymptomatic with a normal ECG, was 75 bpm with a dual chamber pacemaker (DDD). Thereafter, a also diagnosed with LQTS after an aborted SCD episode during dual-chamber cardioverter defibrillator (ICD) was implanted. She antibiotic therapy (piperacillin/tazobactam, ciprofloxacin, and has been asymptomatic since then. tobramycin) in the context of chemotherapy (idarubicin and Sister II:3 has no cardiac symptoms. Her ECG showed sinus cytarabine) for the treatment of acute leukemia. An ICD was rhythm at 68 bpm with normal PR, QRS, and QTc values. implanted but she died shortly thereafter from the leukemia. The Echocardiography, ergometry, Holter, and adrenaline test did proband’s father is still alive, asymptomatic, and with a normal

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1612383114 Caballero et al. Downloaded by guest on September 25, 2021 ECG. The proband has two nephews (Fig. 1A). III:1 was studied CHO cells HL-1 cells PNAS PLUS

(ECG, echocardiography, ergometry, and Holter) when he was a +60 mV child; the results revealed an electrically and structurally normal A WT (0.5 g) WT (1 g) C 20 -40 mV -60 mV heart. Afterward, an adrenaline test was conducted that was p.T152HfsX180 (1 g) negative when he was 23 y old. Conversely, III:2 has experienced 15 WT (0.5 g) + p.T152HfsX180 (0.5 g) p.T152HfsX180 (1 g) epileptic crises since he was 2 yo. He has been treated with 10 p.T152HfsX180 (-) 5 density (pA/pF) oxcarbazepine since he was 6 y old, and no new episodes have * * * * 2 pA/pF

0 2 pA/pF hERG been documented (he is 14 now). His ECG, Holter, echocardio- I * * * * * * * * 1 s 1 s gram, and stress test are normal. -100 -75 -50 -25 0 25 50 75 Membrane potential (mV)

KCNH2 Variants and Functional Analysis. Next-generation sequenc- B WT (0.5 g) WT (1 g) D p.T152HfsX180 (-), n=13 ing of 82 genes (Table S1) demonstrated that the proband and p.T152HfsX180 (1 g) p.T152HfsX180 (0.5 g), n=10 30 sister II:1 carried a heterozygous frameshift mutation in the WT (0.5 g) +p.T152HfsX180 (0.5 g) 5 p.T152HfsX180 (1 g), n=12 4 KCNH2 C 20 gene (NM_000238.3:c.453dupC) (Fig. 1 ) encoding for * * * 3 * * * * * p.T152HfsX180 hERG. This variant is also present in sister II:6. * * * * * 10 2 * * * * * * * * * Sisters II:1 and II:3 and both nephews carry another variant in the tail density (pA/pF) 1

**** tail density (pA/pF) KCNH2 > 0 * * ** gene (NM_000238.3:c.3203A G) encoding for p.Q1068R Kr 0 hERG I I hERG (Fig. 1C). Because recombination is a very rare event after -100 -75 -50 -25 0 25 50 75 -100 -75 -50 -25 0 25 50 75 fertilization, expression of the p.T152HfsX180 mutation in one Membrane potential (mV) Membrane potential (mV) allele and p.Q1068R in the other allele is more likely to represent F n=12 Grandi-Bers 5 * # the condition of sister II:1 (compound heterozygosity). 4 n=10 Mathematical Model * “ ” 3

p.Q1068R is considered a rare control variant (7) that appears tail

Kr n=13 E I with a 0.03% frequency in the African population (Table S2). WT+ 2 p.T152HfsX180 Functional analysis has demonstrated that the p.Q1068R mutation WT (pA/pF)density 1 0 accelerates both inactivation and recovery from inactivation, whose 00.51 ∼ 0.1 pA/pF p.T152HfsX180 ( g) time constants suffer 10- to 20-mV shifts in their voltage de- 100 ms G pendence (8). However, the p.Q1068R variant has not been con- 30 PHYSIOLOGY sidered pathogenic by itself (7). In contrast, p.T152HfsX180 hERG Epi is a 332-aa peptide (Fig. 1E) that we considered to be highly 0 mV 20 Endo

pathogenic and responsible for the LQTS in this family. Thus, we shortening (%) 10 WT + WT 90 conducted a functional analysis by transfecting CHO cells with p.T152HfsX180 50 mV50 0 cDNA encoding either WT (n = 7) or p.T152HfsX180 hERG (n = APD 0 1 2 3 6) channels (1 μg). hERG channels generated a slowly activating 100 ms Frequency (Hz) current whose amplitude progressively increased with pulses up to Fig. 2. (A and B) Maximum current density (current density–voltage rela- 0 mV and then progressively decreased at potentials >0mVowing tionships) (A) and tail currents (activation curves) (B) generated by WT and to the fast C-type inactivation (9), resulting in the bell-shaped cur- p.T152HfsX180 hERG channels alone or when they are cotransfected in CHO rent density–voltage curve typical of hERG channels (Fig. 2A). Fig. cells, as a function of the membrane potential. In B, solid lines represent the fit < μ ≥ 1D shows that, as expected, p.T152HfsX180 hERG channels did of a Boltzmann equation. *P 0.05 vs. hERG WT (1 g) (n 6). (C) IKr traces not generate any current. To simulate the heterozygous condition of recorded in IKr-predominant HL-1 cells transfected or not with p.T152HfsX180 all of the mutation carriers, cells (n = 17) were transfected with WT hERG. (D) IKr tail current densities recorded in HL-1 cells transfected or not with + μ p.T152HfsX180 hERG. (E) Simulated IKr traces (Top)andAPs(Bottom) obtained at plus p.T152HfsX180 hERG channels (0.5 0.5 g). Surprisingly, 0.1HzbyusingtheGrandi–Bers mathematical model of human ventricular en- maximum current amplitudes generated by depolarizing pulses (Fig. docardial cells by introducing the modifications produced by p.T152HfsX180 hERG 2A) and tail currents recorded upon repolarization to −60 mV on the I .(F) I tail current densities recorded in HL-1 cells transfected or not with F B Kr Kr (Figs. 1 and 2 ) were not statistically different from those gen- p.T152HfsX180 hERG after pulses to +60 mV. (G) Percentage of APD90 shortening erated by WT hERG channels (1 μg) (P > 0.05). We surmised that in APs simulated at different frequencies in epicardial and endocardial cells. Points/ the p.T152HfsX180 hERG peptide could exert a “chaperone-like” bars represent mean ± SEM of the data. In D and F, n,numberofcells;*P < 0.05 vs. # effect by increasing membrane expression of WT hERG channels. nontransfected cells; P < 0.05 vs. p.T152HfsX180 0.5 μg transfected cells. In fact, Fig. 1F demonstrates that addition of the peptide (0.5 μg) to hERG WT (0.5 μg) generated significantly greater currents than those generated by hERG channels alone (P < 0.05). Furthermore, for endocardial and epicardial cells at different frequencies ranging between 0.1 and 3 Hz. The voltage- and time-dependent character- p.T152HfsX180 hERG did not modify the voltage dependence of + hERG activation (Fig. 2B) but slowed deactivation (Table S3). To istics of currents generated by WT p.T152HfsX180 hERG channels confirm this chaperone-like effect, we transfected mouse atrial- were incorporated into the model to simulate mutationeffects.Fig. E derived HL-1 cells with the p.T152HfsX180 hERG. Some HL-1 2 shows superimposed human endocardial APs driven at 0.1 Hz generated by WT and WT+p.T152HfsX180 hERG channels. As can cells (36%) exhibited IKr as the main repolarizing current (IKr-pre- be observed, the duration of the heterologous mutant case AP dominant cells), whereas in other cells (36%) IKr overlapped with the slow component of the delayed rectifier current (IKs)(in- (APD; action-potential duration) was slightly briefer. Further- termediate cells). Thus, IKr was measured in IKr-predominant and more, APD measured at 90% of repolarization (APD90) of sim- -intermediate cells as the dofetilide-sensitive current, because it was ulated WT+p.T152HfsX180 endocardial and epicardial cells was completely inhibited by this selective Kr blocker (1 μmol/L) (10). onlyslightlyabbreviated(∼3%) at either driving frequency (Fig. 2G). Fig. 2C demonstrates that p.T152HfsX180 hERG significantly in- Overall, these results suggested that the heterozygous I creases both the maximum and tail amplitudes of IKr (P < 0.05). p.T152HfsX180 hERG mutation produced subtle effects over the Kr, Furthermore, the tail current increase and the slowing of tail cur- even when confirmation on a more physiological setting is needed. rent deactivation depended on the amount of cDNA transfected (Fig. 2 D and F and Table S3). TBX20 Mutation and Functional Analysis. Next-generation sequencing We used a previously validated in silico model of the human of the proband also identified the heterozygous mutation ventricular action potential (AP) (11) to test for the effects of the NM_001077653.2:c.931C>TattheTBX20 gene (Table S2), which heterozygous p.T152HfsX180 hERG mutation. The model was run was confirmed by Sanger analysis (Fig. 3B). The mutation leads to

Caballero et al. PNAS Early Edition | 3of10 Downloaded by guest on September 25, 2021 the substitution of Arg311 by Cys (p.R311C Tbx20). Arg311, (39% and 33%, respectively) were not modified by Tbx20. which is highly conserved among different species (Fig. 3C), is Tbx20 WT significantly increased (P < 0.05) the maximum located in the transactivation region of Tbx20 (Fig. 3D). Two af- outward current recorded upon depolarization (Figs. 3E and fected (II:6 and III:2) and another unaffected (II:3) of the pro- 4A) without modifying the activation kinetics (Table S3). Tbx20 band’s relatives also carry the p.R311C Tbx20 mutation (Fig. 3A). also significantly increased the IKr tail density (Figs. 3E and 4B) The p.R311C Tbx20 variation was annotated with a 0.01% fre- (P < 0.05), whereas it did not modify tail current deactivation quency in Africans. Other nonsynonymous variants identified in (Table S3). Surprisingly, p.R311C Tbx20 was unable to increase I E A the proband are listed in Table S2. maximum outward Kr (Figs. 3 and 4 ) and the tail current Tbx20 binds to the consensus sequence “AGGTGTG” within the density elicited upon repolarization (Figs. 3E and 4B). Consis- DNA of target genes (6). We hypothesized that Tbx20 might reg- tently, the mutated transcription factor did not modify either the ulate the expression of cardiac ion channels involved in the control activation or the deactivation kinetics of the current (Table S3) of human cardiac repolarization as it does in fly and mouse adult (P > 0.05). Importantly, p.R311C Tbx20 did not change the per- I (5, 6). Sequence analysis of mouse and human promoters of centage of Kr-predominant and -intermediate HL-1 cells (39% genes encoding ion-channel subunits revealed that a Tbx20 binding and 28%, respectively). Fig. 4 B and C demonstrate that trans- site appears in both KCNH2 genes (Table S4). Thus, we aimed at fection with Tbx20, either WT or mutated, did not significantly identifying the effects of WT and p.R311C Tbx20 on the expression modify the voltage dependence of Kr channel activation (Table of hERG in HL-1 cells by recording I .TransfectionwithWT S3). Western blot analysis in HL-1 cells showed that WT Tbx20 Kr n = P < (60.8 ± 7.2 pF, n = 72) or p.R311C Tbx20 (65.6 ± 9.4 pF, n = 65) significantly increased ( 5, 0.05), whereas p.R311C Tbx20 D plasmids did not modify HL-1 cell capacitance (55.2 ± 9.0 pF, significantly decreased, the expression of hERG channels (Fig. 4 F n = P < n = 68) (P > 0.05). and )( 5, 0.05). It has been proposed that MiRP1 encoded by KCNE2 is also present in the channels generating the Fig. 3E shows dofetilide-sensitive currents recorded in IKr-pre- IKr in the human heart (12). Fig. 4 E and G demonstrate that dominant HL-1 cells. IKr was recorded again in IKr-predominant and -intermediate HL-1 cells whose distribution percentages Tbx20, either WT or mutated, did not modify MiRP1 expression in HL-1 cells. To further demonstrate the transcriptional effect of Tbx20 over the mouse KCNH2 gene, the IKr density was also assessed in HL-1 A cells, in which endogenous Tbx20 was silenced using lentiviral constructs containing short hairpin RNA (shRNA) for Tbx20 to- gether with GFP. Control cells were infected with a lentivirus containing a scrambled shRNA and GFP. At 48 h postinfection, Tbx20 expression decreased by 58% (Fig. S2). The results demon- strated that the IKr density significantly decreased in Tbx20-silenced cells (Fig. S2)(n≥ 5, P < 0.05). To test whether Tbx20 regulates the expression of human KCNH2, we measured the luciferase activity in HL-1 cells KCNH2 B II:4 C expressing the minimum human promoter. The luciferase assay demonstrated that Tbx20 WT, but not p.R311C, significantly increased (P < 0.05) the transcription of the human KCNH2 gene (Fig. 4H)(n = 8 dishes per group). Similarly, SP1, a transcription factor whose binding site is also present in the minimal promoter of the human KCNH2 gene, significantly increased the transcrip- tion of this gene (Fig. 4H)(n = 8, P < 0.05). We also tested the effects of the combined action of both forms of Tbx20 (WT and D mutated). Fig. 4H shows that in the joint presence of WT and Tbx20 p.R311C Tbx20 the protranscriptional effect was reduced (n = 3, (NP_001071121) P < 0.05 vs. Tbx20 alone), probably because of a competition between the WT and mutated forms for the Tbx20 consensus binding site in the KCNH2 minimal promoter. Interestingly, p.R311C Tbx20 alone did not decrease basal luciferase activity, +60 mV probably because the sensitivity of our system is limited. The -60 mV mouse KCNE2 gene promoter does not exhibit the Tbx20 binding E -80 mV site, whereas the human gene does (Table S3). Unfortunately, it Tbx20 (-) Tbx20 WT Tbx20 p.R311C was not possible to construct the minimal human promoter of the KCNE2 gene for a luciferase assay. To test for the effects of the Tbx20 mutation on human ven- tricular AP characteristics, again the mathematical model was used. Fig. 4I shows superimposed human endocardial APs driven 1pA/pF

1s at 0.1 Hz in control conditions and when cells were transfected with TBX20 either WT or mutated. Tbx20 WT shortened the Fig. 3. (A) Pedigree of the studied family. The arrow indicates the proband. APD measured at 50% of repolarization (APD50) and the Circles and squares represent females and males, respectively. + and – rep- APD90 as a consequence of an IKr increase (Fig. 4I, Top). resent subjects with and without the p.R311C Tbx20 mutation, respectively. Conversely, p.R311C Tbx20 prolonged the APD (by 25% (B) DNA sequence chromatograms of the proband depicting the heterozy- 50 > compared with Tbx20 WT) and APD90 (by 23%), due to the gous change (c.931C T) of the TBX20 gene. (C) Sequence alignment of the I I Top region surrounding R311 in Tbx20 in several species. The box highlights the decrease of the Kr conductance (Fig. 4 , ). conservation of this residue. (D) Schematic representation of the Tbx20 se- quence, indicating the T box, and the transactivation and transrepressor Effects of Tbx20 in Human Induced Pluripotent Stem Cell-Derived

regions. (E) IKr traces recorded in IKr-predominant HL-1 cells transfected or Cardiomyocytes. Next, we determined the effects of the Tbx20 not with either WT or p.R311C Tbx20 by applying the pulse protocol (Top). p.R311C mutation on IKr and AP characteristics in a more

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1612383114 Caballero et al. Downloaded by guest on September 25, 2021 PNAS PLUS ABTbx20 (-) (n=10) C Tbx20 (-) (n=11) Tbx20 (-) Tbx20 W T (n=13) Tbx20 WT (n=12) Tbx20 W T Tbx20 p.R311C (n=14) Tbx20 p.R311C (n=12) Tbx20 p.R311C 3 * * * 1.0 * * 4 * * * Kr * * 3 * 0.8 2 * 0.6 * 2 1 # # # # 0.4 # 1 # #

# tail current # 0.2 0 0 Normalized I

Dofetilide-sensitive 0.0 current density (pA/pF) Dofetilide-sensitive tail -50 -25 0 25 50 75 densitycurrent (pA/pF) -60 -20 20 60 -60 -20 20 60 Membrane potential (mV) Membrane potential (mV) Membrane potential (mV)

DE

N=5 N=3 F 2.0 G 1.2 N=3 N=3 * PHYSIOLOGY 1.5 0.8 N=5 N=5 1.0 # 0.4

* expression expression 0.5 Relative hERG Relative MiRP1 0.0 0.0 Tbx20 Tbx20 Tbx20 Tbx20 Tbx20 Tbx20 (-) WT p.R311C (-) WT p.R311C

Human KCNH2 promoter Tbx20 W T Tbx20 (-) N=8 Tbx20 p.R311C H 1.5 N=8 I ** N=3 ** 0.1 pA/pF N=8 * # 100 ms 1.0 N=8

0mV activity 0.5 Tbx20 p.R311C Tbx20 Tbx20 (-) WT Normalized luciferase 0.0 50 mV Tbx20 WT (-) (+) (-) (+) (-) Tbx20 p.R311C (-) (-) (+) (+) (-) 100 ms SP1 (-) (-) (-) (-) (+)

Fig. 4. (A and B) Maximum IKr density–voltage relationships (A) and activation curves (B) for currents recorded in IKr-predominant and -intermediate HL-1 cells transfected or not with either WT or p.R311C Tbx20. (C) Normalized activation curves for currents recorded in the three experimental groups. In B and C,solid lines represent the fit of a Boltzmann equation. (D and E) Western blot (WB) images and their corresponding stain-free gels showing hERG (arrows in D)and miRP1 (E) expression in HL-1 cells transfected or not with either WT or p.R311C Tbx20. In D, the sample of the last right lane was run in the same gel but was separated (continuous line) when incubating with the primary antibody together with the antigenic peptide. (F and G) Mean densitometric analysis of hERG (F) and MiRP1 (G) levels normalized to total protein. (H) Normalized luciferase activity in HL-1 cells expressing the pLightSwitch_Prom vector carrying the human

KCNH2 promoter cotransfected or not with SP1 and either WT or p.R311C Tbx20. (I)SimulatedIKr traces (Top)andAPs(Bottom) obtained at 0.1 Hz by using the Grandi–Bers mathematical model of human ventricular endocardial cells by introducing the modifications produced by Tbx20 WT and p.R311C on the IKr.Points/ bars represent mean ± SEM of the data. n, number of cells; N, number of dishes. *P < 0.05 vs. Tbx20 (-); #P < 0.05 vs. Tbx20 WT; **P < 0.01 vs. Tbx20 (-).

physiologically relevant setting. Unfortunately, genetically mod- reduced the tail current density by ∼60% (P < 0.05 vs. Tbx20 WT ified mice were not an option, because adult mouse heart does and noninfected cells; n ≥ 7) (Fig. 5B). Tbx20, either WT or not generate IKr (12). Therefore, we drew upon human induced p.R311C, did not modify the voltage dependence of Kr channel pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in- activation (Table S3). APs were recorded in hiPSC-CMs that fected or not with lentiviral constructs encoding Tbx20 WT or exhibited automatic activity (13). In cells infected with Tbx20 mutated. IKr was measured as the dofetilide-sensitive current WT (n = 10), maximum diastolic potential and AP amplitude (1 μmol/L). Fig. 5A shows IKr tail currents recorded at −60 mV averaged −68.4 ± 1.9 and 98.7 ± 14.2 mV, respectively. At 1 Hz, after depolarizing pulses from −60 mV to −40 and +60 mV in a APD50 and APD90 were 108.1 ± 17.4 and 163.2 ± 23.2 ms, re- noninfected hiPSC-CM. Tbx20 WT produced a 1.6-fold increase spectively, and Tbx20 WT shortened whereas Tbx20 p.R311C in IKr tail current density (P < 0.05), whereas Tbx20 p.R311C significantly prolonged the APD90 (Fig. 5C)(n = 10, P < 0.01).

Caballero et al. PNAS Early Edition | 5of10 Downloaded by guest on September 25, 2021 Furthermore, as shown in Fig. 5D, the prolongation of the 33% in the presence of WT and p.R311C Tbx20, respectively). APD90 was greater at slow- (158% at 0.1 Hz) than at fast-driving Consistently, Tbx20 did not modify either the voltage dependence frequencies (102% at 2 Hz). of Ks channel activation or the activation and deactivation kinetics To reproduce the genetic condition of the proband, the math- (Table S3). Western blot analysis (Fig. S3) confirmed that WT and ematical model was run considering both the heterozygous p.R311C Tbx20 were not able to modify the expression of Kv7.1 p.T152HfsX180 hERG mutation together with the p.R311C channels (Fig. 6C)(n ≥ 3, P > 0.05). Sequence analysis of the mouse Tbx20 mutation. Fig. 5E demonstrated that p.R311C Tbx20 would KCNQ1 gene promoter demonstrated that there are two Tbx20 lengthen the APD in the presence of the p.T152HfsX180 hERG consensus binding sites far away from the transcription start site mutation. Furthermore, the prolongation was greater in endo- (−2474 and −1992) (Table S4). Importantly, human KCNQ1 lacks cardial than in epicardial cells and also at slow- than at fast-driving the Tbx20 binding site (Table S4). Consistently, neither WT nor frequencies (Fig. 5F). p.R311C Tbx20 modified the expression of human KCNQ1 mea- sured with a luciferase assay (Fig. 6E). Conversely, SP1, whose con- Effects of p.R311C on the Expression of Other Cardiac K Channels. In the sensus binding site is present in the minimal promoter of the human human ventricular myocardium Kv7.1 (encoded by KCNQ1) gene (16), actually increased KCNQ1 transcription significantly (Fig. homotetramers associate with minK proteins (encoded by KCNE1) 6E). Human and mouse KCNE1 gene promoters exhibit consensus to form the channels that generate IKs (14). Some HL-1 cells (27%) Tbx20 binding sites (Table S4) and, indeed, both WT and p.R311C exhibit IKs as the main repolarizing current (IKs-predominant cells). Tbx20 significantly increased minK expression (Fig. 6D)inHL-1 Thus, IKs was measured in IKs-predominant and -intermediate cells cells and transcription as measured by luciferase assay (Fig. 6F). as dofetilide-resistant current, which was completely inhibited by a Neither human, rat, nor mouse KCNJ2 gene promoters (which + selective Ks channel blocker (HMR-1556; 1 μmol/L) (10). Fig. 6A encode for the inward rectifier K channel Kir2.1) exhibit the shows IKs traces recorded in IKs-predominant cells transfected with Tbx20 consensus binding site (Table S4). Fig. 6G and Fig. S3 WT or mutated Tbx20. Neither WT nor mutant Tbx20 modified the demonstrate that Kir2.1 protein expression was not modified by IKs density (n ≥ 6, P > 0.05) (Fig. 6B). Moreover, neither modified the presence of any of the forms of Tbx20 (n = 3 dishes per group, the percentage of IKs-predominant or -intermediate cells (28% and P > 0.05). However, even when Kir2.1 channels were expressed,

HUMAN iPSC-DERIVED VENTRICULAR MYOCYTES GRANDI-BERS MATHEMATICAL MODEL AC500 n=10 E * # 400 hERG p.T152HfsX180 n=10 hERG p.T152HfsX180+ 300 n=10 p.R311C Tbx20

+60 mV duration (ms) 200 * -60 mV 90 100 -40 mV 0.1 pA/pF

APD 0 Tbx20 (-) WT p.R311C 100 ms Tbx20(-) hERG p.T152HfsX180 Tbx20 (+) hERG p.T152HfsX180+ Tbx20 p.R311C Dofe-sensitive 0mV Tbx20 (-) tail currents 0mV

50 pA Tbx20 p.R311C 40 mV Tbx20 WT 50 mV 250 ms 100 ms Tbx20 (-), n=7 50 ms BDTbx20 WT, n=9 F hERG p.T152HfsX180+Tbx20 p.R311C Tbx20 p.R311C, n=9 vs Tbx20 WT hERG p.T152HfsX180 1.5 * * 800 Tbx20 p.R311C 20 * * * * * 600 Epi 1.0 * Endo 400 * * 10

0.5 duration (ms)

# # # # prolongation (%) # 90 200

* 90 density (pA/pF) * * 0.0 *

APD 0 0

-60 -20 20 60 0 1 2 APD 0 1 2 3 Dofe-sensitive tail current Membrane potential (mV) Frequency (Hz) Frequency (Hz)

Fig. 5. (A) Dofetilide (1 μmol/L)-sensitive (IKr) tail currents obtained by digital subtraction in a noninfected hiPSC-derived cardiomyocytes. (B) IKr density in a hiPSC-derived cardiomyocyte infected or not with either WT or p.R311C Tbx20. Solid lines represent the fit of a Boltzmann equation. (C) Superimposed APs

recorded at 1 Hz in three hiPSC-derived cardiomyocytes infected or not with either WT or p.R311C Tbx20. (Top) The APD90 of each experimental group. (D) APD90 at different stimulation frequencies in cells infected with Tbx20 WT or p.R311C. (E) Simulated IKr traces (Top) and APs (Bottom) obtained at 0.1 Hz by using the Grandi–Bers mathematical model of human ventricular endocardial cells by introducing the modifications produced by heterozygous p.T152HfsX180

hERG alone or in combination with p.R311C Tbx20 on the IKr.(F) Percentage of APD90 prolongation in APs simulated at different frequencies in epicardial and endocardial cells. In B–D, points/bars represent mean ± SEM of ≥7 experiments in each group. *P < 0.05 vs. Tbx20(-); #P < 0.05 vs. Tbx20 WT.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1612383114 Caballero et al. Downloaded by guest on September 25, 2021 +60 mV HL-1 Cells PNAS PLUS -30 mV A -80 mV B Tbx20 (-), n=9 3 Tbx20 WT, n=6 Tbx20 Tbx20 p.R311C, n=6 G H 1.2 N=3 Membrane potential (mV) 2 2.0 I WT N=3 f

N=3 density (pA/pF) -150 -110 -70 -30 2 pA/pF 1 -0.5 2 s 0.8 -10 -3.0 Tbx20 0

Dofetilide-resistant 0.4 p.R311C expression

current density (pA/pF) density current -50 -25 0 25 50 75 -5.5 Relative Kir2.1

2 pA/pF Membrane potential (mV) 0.0 -8.0 2 s Tb2x0 Tbx20 Tbx20 Tbx20 (-), n=8 (-) WT p.R311C Tbx20 W T, n=6 Tbx20 p.R311C, n=6 C 1.5 D 3 N=3 N=3 N=3 N=3 * N=3 * 1.0 2 IK1 in Rat Ventricular Myocytes N=3 0.5 expression 1 expression Relative Kv7.1 Relative Relative minK Relative

0.0 0 +40 mV Tbx20 Tbx20 Tbx20 TBX20 TBX20 TBX20 I -40 mV J (-) WT p.R311C (-) WT p.R311C -100 mV 50 ms Membrane potential (mV) I -120 -80 -40 0 40 K1 density (pA/pF)

-5

E Human KCNQ1 promoter F Human KCNE1 promoter 10 pA/pF -10 2.0 N=5 2.0 N=5 N=5 ** Tbx20 WT -15 1.5 1.5 * * N=5 N=5 N=5 N=5 -20 1.0 1.0 Tbx20 (-), n=12 PHYSIOLOGY activity activity Tbx20 WT, n=11 0.5 0.5 Tbx20 p.R311C, n=7 Normalized luciferase 0.0 Normalized luciferase 0.0 Tbx20 WT p.R311C SP1 Tbx20 Tbx20 Tbx20 (-) (+) Tbx20 (+) (-) WT p.R311C Tbx20 p.R311C

Fig. 6. (A) Traces of dofetilide-resistant current (IKs) recorded in IKs-predominant HL-1 cells transfected with WT or p.R311C Tbx20 by applying the pulse protocol (Top). (B) Current density–voltage relationships for IKs recorded in HL-1 cells transfected or not with WT or p.R311C Tbx20. (C and D) Mean den- sitometric analysis of Kv7.1 (C) and minK (D) levels normalized to total protein. (E and F) Normalized luciferase activity in HL-1 cells expressing the pLightSwitch_Prom vector carrying the human KCNQ1 (E)orKCNE1 (F) promoters cotransfected or not with WT or p.R311C Tbx20. (G) Mean densitometric

analysis of Kir2.1 levels normalized to total protein. (H) Current density–voltage relationships for If recorded in HL-1 cells transfected or not with WT or p.R311C Tbx20. (I) IK1 traces recorded in two rat myocytes infected with WT and p.R311C Tbx20. (J) Mean current density–voltage curves for IK1 recorded in rat ventricular myocytes infected or not with lentiviral constructs encoding WT and p.R311C Tbx20. Each point/bar represents mean ± SEM of n cells or N dishes of cells in each group. *P < 0.05 vs. Tbx20 (-); **P < 0.01 vs. Tbx20 (-).

the inward rectifier K current (IK1) could not be recorded in to −20 mV (INaL)(n ≥ 6, P > 0.05). The human SCN2B pro- HL-1 cells. Instead, the (If) predominated moter, which codifies for an Nav1.5 ancillary subunit, exhibits at potentials between −150 and 0 mV but its current density– the Tbx20 binding site (Table S4). Luciferase assays demon- voltage relation was unaltered by either WT or p.R311C Tbx20 strated that Tbx20 WT and p.R311C were unable to modify (Fig. 6H)(n ≥ 6, P > 0.05). This result agrees with the absence human SCN5A gene expression, whereas Tbx20 WT, but not of a Tbx20 consensus binding site in the promoters of the Tbx20 p.R311C, significantly increased the expression of hu- HCN1 HCN3 HCN4 genes ( , ,and ) encoding the channels un- man SCN2B (Fig. 7 F and G). I derlying f in mice and humans (Table S4). Therefore, to assess The mouse, but not the human, CACNA1C gene promoter I theroleofTbx20inKir2.1functional regulation, the K1 was exhibits the Tbx20 binding site (Table S4). Therefore, Tbx20 + recorded in enzymatically dissociated rat ventricular myocytes effects on the L-type Ca2 current (I ) were tested in both that were transfected using a lentiviral construct (17). Fig. 6 I CaL HL-1 cells and hiPSC-CMs. In HL-1 cells, ICaL was measured using and J show that the IK1 density was not modified at any of the 2+ Ba asachargecarrier(IBa) (15, 18). Fig. S4 shows that Tbx20, voltages tested in the presence of either WT or p.R311C Tbx20 both WT and mutated, significantly increased the I (n ≥ 9, P < (n ≥ 7, P > 0.05). Ba 0.05). Furthermore, p.R311C Tbx20 increased the IBa density P > Effects of p.R311C on the Expression of Cardiac Na and Ca Channels. similar to Tbx20 WT ( 0.05 vs. Tbx20 WT). However, neither + SCN5A and CACNA1C genes codify for the α-subunit of the Na WT nor mutated Tbx20 affected the voltage dependence of acti- + (Nav1.5) and L-type Ca2 (Cav1.2) channels, respectively. Mouse vation or inactivation of the channel (Fig. S4 and Table S3). and human SCN5A gene promoters lack a Tbx20 binding site (Table Western blot analysis in HL-1 cells (Fig. S3) demonstrated that + S4). Fig. 7 A and B confirm that the Na current (INa) density was both WT and p.R311C Tbx20 significantly and similarly increase also not modified by WT or p.R311C Tbx20 (n ≥ 8, P > 0.05). Cav1.2 expression (Fig. S4). A I Furthermore, transfection of WT or p.R311C Tbx20 did not modify Fig. 8 shows CaL traces recorded in hiPSC-CMs infected or not the voltage dependence of the INa activation or inactivation or the with the lentiviral constructs encoding for Tbx20, either WT or mu- current kinetics (Fig. 7C and Table S3). Fig. 7 D and E show that tated. Neither WT nor p.R311C Tbx20 modified the ICaL density Tbx20 WT or p.R311C did not modify the amplitude of the sus- (Fig. 8A) at any of the voltages tested (Fig. 8B)(n ≥ 6, P > 0.05). The + tained influx of Na measured at the end of 500-ms depolarizations luciferase assay done using the minimal human CACNA1C promoter

Caballero et al. PNAS Early Edition | 7of10 Downloaded by guest on September 25, 2021 +30 mV Discussion

-120 mV -90 mV Here we functionally describe the consequences of three variants identified in a Spanish family of African ancestry with LQTS. A Tbx20 WT Tbx20 p.R311C Tbx20 (-) The TBX20 mutation selectively decreased the expression of hERG channels, prolonging the AP in hiPSC-CMs. Conversely, the KCNH2 frameshift mutation did not modify IKr density. Our results strongly suggest that, in the adult heart, Tbx20 controls the expression of hERG channels, and thus TBX20 may be

25 pA/pF 5 ms considered an LQTS-modifying gene. The p.T152HfsX180 hERG mutation was found in the proband and in all of the affected relatives that were genotyped but in none B Membrane potential (mV) C 1.0 Tbx20 (-), n=10 of the nonaffected family members. Therefore, the mutation was I

+20 Na -60 Na Tbx20 WT, n=6 0.8

density (pA/pF) considered pathogenic. Phenotypic manifestations in the family -100 -20 Tbx20 p.R311C 0.6 ’ -25 n=6 match most of the features of LQT2, and three of the proband s 0.4 sisters experienced seizures since they were children. Epilepsy has -50 Tbx20 (-), n=20 0.2 been reported to be more common with LQT2 (39%) than with Normalized I Tbx20 WT, n=14 -75 0.0 other subtypes (10%), possibly because KCNH2 is also expressed in -140 -100 -60 -20 Tbx20 p.R311C, n=8 -100 the brain (21). Interestingly, nephew III:2, who carries the TBX20 Membrane potential (mV) but not the KCNH2 frameshift mutation, has also experienced D E seizures. Additionally, one of the sisters died postpartum, which is a Tbx20 Tbx20 Tbx20 0 (-) WT p.R311C specific trigger of symptoms in LQT2 (22). Functional analysis of -5 Tbx20 (-) 0.0 the p.T152HfsX180 mutation demonstrated that this peptide of 332 -10 Tbx20 WT -0.5 aa, of which only 152 correspond to the hERG sequence, exerts (pA/pF) Tbx20 p.R311C Na chaperone-like effects on WT hERG channels in CHO cells and on I -15 -1.0 IKr recorded in HL-1 cells. Indeed, transfection of p.T152HfsX180 -20 (pA/pF) density -1.5 n=6 “ ” I 0 100 200 300 400 500 n=7 n=9 inHL-1cellsproducesa concentration-dependent increase in Kr. Na,L I -2.0 “ ” Time (ms) As a consequence, current density generated by heterozygous transfection of WT and p.T152HfsX180 hERG channels was not F Human SCN5A promoter G Human SCN2B promoter different from that generated by “homozygous” transfection of WT 1.5 1.5 N=5 hERG channels. This is a somewhat surprising result considering ** N=5 N=5 that, as expected, homozygous transfection of p.T152HfsX180 did N=5 N=5 N=5 1.0 1.0 not generate current at all. We recently demonstrated that the Nav1.5 N-terminal domain, by itself (the 132-aa peptide) (Nter), activity 0.5 activity 0.5 exerts a chaperone-like effect that increases INa and IK1 by en- Normalized luciferase Normalized 0.0 luciferase Normalized 0.0 hancing the expression of Nav1.5 and Kir2.1-Kir2.2 channels in Tbx20 Tbx20 Tbx20 Tbx20 Tbx20 Tbx20 CHO cells and in rat cardiomyocytes (17). We hypothesize that the (-) WT p.R311C (-) WT p.R311C p.T152HfsX180 peptide is able to increase membrane expression of

Fig. 7. (A) INa traces recorded in HL-1 cells transfected or not with WT or p.R311C Tbx20 by applying the pulse protocol (Top). (B and C) Current – density voltage relationships (B) and steady-state inactivation (C)forINa +60 mV recorded in the three experimental groups. (D and E) Superimposed INa -30 mV -50 mV traces (D) recorded in HL-1 cells transfected or not with WT or p.R311C Tbx20 -80 mV by applying 500-ms pulses from −120 to −20 mV and bar graph (E) showing A the mean INaL recorded at 500 ms. (F and G) Normalized luciferase activity in Tbx20 (-) Tbx20 WT Tbx20 p.R311C HL-1 cells expressing the pLightSwitch_Prom vector carrying the human SCN5A (F) or SCN2B (G) promoters cotransfected or not with WT or p.R311C Tbx20. Each point/bar represents the mean ± SEM of n cells or N dishes of cells in each group. **P < 0.01 vs. Tbx20 (-).

5 pA/pF 50 ms confirmed that Tbx20 WT and p.R311C were unable to modify the BC expression of the human CACNA1C gene (Fig. 8C), a result that Membrane potential (mV) Human CACNA1C promoter I I -60 -40 -20 20 40 60 CaL explains the lack of Tbx20 effects over the human CaL density. 1.2 N=5 N=5

density(pA/pF) N=5

Functional Analysis of the c.-66A>G Variation of KCNN3. The pro- -4 0.8 band, sister II:6, and nephew III:1 also present a variation at the activity 5′ UTR of the KCNN3 gene (NM_001204087.1:c.-66A>G) that -8 0.4 α 2+

encodes the -subunit of the small-conductance Ca -activated -12 luciferase Normalized + 0.0 K channel type 3 (SK3) (19). Luciferase experiments demon- Tbx20 (-), n=6 Tbx20 Tbx20 Tbx20 strated that expression of mutated KCNN3 cannot be activated Tbx20 WT, n=9 (-) WT p.R311C by SP1 or Tbx20 (Fig. S5), whose binding sites are present in the Tbx20 p.R311C, n=6

human gene promoter (Table S4). Therefore, the variation Fig. 8. (A) ICaL traces recorded in hiPSC-CMs infected or not with WT or completely abolished its transcription, thus leading to a KCNN3 p.R311C Tbx20 by applying the pulse protocol (Top). (B) Current density- haploinsufficiency. However, because treatment of human mul- voltage relationships for ICaL recorded in the three experimental groups. (C) Normalized luciferase activity in HL-1 cells expressing the pLightSwitch_Prom ticellular ventricular preparations with apamin (a selective SK vector carrying the human CACNA1C promoter cotransfected or not with blocker) does not modify the APD (20), the importance of these WT or p.R311C Tbx20. Each point/bar represents the mean ± SEM of n cells or channels in repolarization seems to be negligible. N dishes of cells in each group.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1612383114 Caballero et al. Downloaded by guest on September 25, 2021 hERG channels. The molecular determinants, and the proteins modifiers (29), including the presence of two or more variants, PNAS PLUS involved in this effect, merit further analysis. The question now is either in the same gene (compound heterozygosity) or in dif- whether this KCNH2 frameshift mutation is, by itself, responsible ferent genes (digenic heterozygosity), and the presence of non- for the LQT phenotype of the family. synonymous single-nucleotide polymorphism (28, 29). All such The proband and relatives II:3, II:6, and III:2 harbor a mu- conditions converge in this Spanish family. The proband and tation in the TBX20 gene. Mutations in Tbx20 have been pre- sister II:6 present digenic heterozygosity (the KCNH2 frameshift viously described and lead to defects in cardiac septation, and the TBX20 mutations), whereas sister II:1 presents a com- valvulogenesis, and chamber growth (23). Indeed, Tbx20 is nec- pound heterozygosity (the frameshift and the variant in KCNH2). essary for proper organogenesis, because it carries strong tran- Interestingly, nephew III:2, who has seizures, carries the benign scriptional activation and repression domains and physically p.Q1068R hERG variant and the TBX20 mutation. interacts with other transcription factors involved in cardiac We are aware of the potential limitations of this study, in- development (4). It is noteworthy that the R311 residue lies in cluding that a better experimental approach would have been to the transcriptional activator domain but none of the mutation analyze in hiPSC-CMs the effects produced by WT and mutated carriers presented any structural cardiac defect. Tbx20 over all of the cardiac currents responsible for the AP Functional analysis demonstrated that Tbx20 does not directly morphology. It would have been even better to analyze the ef- control the expression of the channels that underlie INa, If, IK1, I I fects produced by Tbx20 over cardiomyocytes differentiated and Ks. Regarding the Ks, the results demonstrated that both from hiPSCs derived from each family member. The latter would WT and mutated Tbx20 increased minK expression. Therefore, have allowed directly testing the impact of the variants (and their in HL-1 cells, an I augmentation would have been expected, Ks connection) in a constant genetic background, weighting their because minK increases Kv7.1 conductance (24, 25). However, respective involvement in the phenotypic expression of the simultaneously, minK acts as an endocytic chaperone favoring LQTS. Despite such a limitation, the results strongly suggest that the internalization of the Kv7.1–minK complexes expressed in TBX20 is a potential LQTS modifier gene because WT Tbx20 the membrane (26), an effect that would decrease IKs density. Therefore, the balance between these two opposite actions could increases hERG channel expression. The data show also that some mutations, such as p.R311C, can disable Tbx20 protran- explain why the minK augmentation was not accompanied by a KCNH2 change in I density. Our results confirm previous data dem- scriptional activity over the gene. Therefore, the puta- Ks tive effects of Tbx20 variants on penetrance, expressivity, and onstrating that, in mice, Tbx20 increases ICaL (6). However, Tbx20 did not increase the expression of human CACNA1C, outcome among LQTS patients merit further analysis. PHYSIOLOGY because the canonical Tbx20 binding site is not present in the Methods gene promoter. Accordingly, Tbx20, either WT or mutated, did Clinical Evaluation. Patients were evaluated by the Arrhythmia Unit of the not modify the ICaL in hiPSC-CMs. Our results demonstrated that Tbx20 significantly increases the expression of hERG and Hospital Universitario La Paz. The study was approved by the Investigation I Committee of the hospital and conforms to the principles outlined in the thus Kr in HL-1 cells and hiPSC-CMs. Conversely, in adult flies, Declaration of Helsinki. Each participant gave written informed consent. neuromancer (the invertebrate ortholog of Tbx20) negatively regulates the expression of the invertebrate homolog of the ERG + DNA Sequencing. Genomic DNA was sequenced by means of a HaloPlex channel (eag-like K channel) (5). Moreover, functional analysis custom panel including coding regions and untranslated boundaries of the 82 developed in HL-1 cells and hiPSC-CMs strongly suggested that genes listed in Table S1. Sequencing using the Ion Torrent Personal Genome the p.R311C mutation specifically disables the protranscriptional Machine was carried out at NIMGenetics. Variants identified in KCNH2, activity of Tbx20 on the KCNH2 gene. Therefore, we propose TBX20, and KCNN3 were confirmed by the Sanger method. that in the human adult myocardium, this Tbx20 mutation leads to a prolongation of ventricular repolarization. Cell Culture and Transfection. HL-1 and Chinese hamster ovary cells were Results in flies and mice demonstrated that Tbx20 is a key transiently transfected by using Lipofectamine 2000 and FuGENE X-tremeGENE, determinant of adult cardiac function (5, 6). Indeed, heart-spe- respectively, and cultured as described (15, 17, 18, 30). cific knockdown of the gene that encodes neuromancer in flies (nmr-2) interferes with cardiac performance and disrupts con- Rat Ventricular Myocyte Isolation. Animal studies were approved by the tractile myofibrillar patterning (5). In adult mice, heterozygous Committee on the Use and Care of Animals at Complutense University and conformed to the guidelines from Directive 2010/63/EU of the European loss of TBX20 leads to dilated cardiomyopathy (27), and the Parliament on the protection of animals used for scientific purposes. Single conditional homozygous loss of Tbx20 results in severe cardio- ventricular myocytes isolated from male Sprague–Dawley rats by enzymatic myopathy with associated arrhythmias and death (6). It has been dissociation (17) were infected with lentiviral constructs encoding for human proposed that, in the adult heart, Tbx20 is the pivotal element of Tbx20 WT or p.R311C. a transcriptional cohort (also constituted by Mef2A, Tead1, Esrr, and Creb1) that fine-tunes expression of continuously required Patch Clamping. Currents were recorded using the whole-cell patch-clamp proteins in response to the current myocyte state, availability of technique (15, 17, 18, 30). Series resistance was compensated manually using resources, and contractile requirements (6). Therefore, even the compensation unit of the Axopatch amplifier; ≥80% compensation was when Tbx20 mutant carriers apparently exhibit a mild phenotype achieved. No significant voltage errors (<5 mV) due to series resistance were under basal conditions, we cannot rule out that their myocar- expected with the micropipettes used. dium adapts poorly to more demanding situations (e.g., sympa- I I thetic tone increase or even hormone- or drug-induced decrease Kr, CaL, and AP Recordings in hiPSC-CMs. Enriched and mature DF19-9-11T of the repolarization reserve), because probably the p.R311C hiPSC-CMs were generated as described elsewhere (13) and infected with the mutation affects Tbx20 ability to coordinate adaptive responses lentiviral constructs coding WT or p.R311C Tbx20. Currents were recorded at 21 to 23 °C, and APs were recorded at 35 °C using the whole-cell patch- of the transcriptional cohort. Therefore, the simultaneous pres- clamp technique. ence of KCNH2 and TBX20 mutations probably contributes to the LQTS phenotype in this family. Western Blot Analysis. Cav1.2, Kir2.1, Kv7.1, minK, hERG, MiRP1, and Tbx20 As in other families (28), expressivity of the LQTS phenotype proteins were detected in HL-1 cells transfected or not with Tbx20 WT or in this family ranged from the mild phenotype of the proband to p.R311C by Western blot following previously described procedures (17, 18). the high symptomatic phenotype of sister II:1. Besides de- mographic variables such as gender and age, variable expressivity Luciferase Gene Expression Reporter Assay. Luciferase reporter assays were may be attributed to the concurrence of additional genetic conducted in HL-1 cells transfected with pLightSwitch_Prom luciferase expression

Caballero et al. PNAS Early Edition | 9of10 Downloaded by guest on September 25, 2021 reporter vectors carrying the minimal promoters of human SCN5A, SCN2B, were analyzed with multilevel mixed-effects models. A value of P < 0.05 was CACNA1C, KCNQ1, KCNE1, KCNH2,orKCNN3 (15, 18). considered significant. Additional details are presented in SI Methods.

Tbx20 Silencing. For analysis of Tbx20 silencing, HL-1 cells were infected with ACKNOWLEDGMENTS. We thank Paloma Vaquero, Sandra Sacristán, Lorena lentivirus-encoding shRNA Tbx20 or scrambled shRNA (17). Ondo, and Ainara Albadalejo for their invaluable technical assistance. This work was supported by grants from Comunidad Autónoma de Madrid (S2010/BMD-2374: ITACA); Ministerio de Economía y Competitividad Statistical Analysis. Results are expressed as mean ± SEM. Unpaired t test or – (SAF2014-58769-P); Instituto de Salud Carlos III (PI16/00398, CB16/11/00303, one-way ANOVA followed by Newman Keuls test was used where appro- and CB16/11/00504); ERA-Net for Research on Rare Diseases (AC14/00029); < priate. In small-size samples (n 15), statistical significance was confirmed by Mutua Madrileña and BBVA Foundations; National Heart, Lung, and Blood using nonparametric tests. Comparisons between categorical variables were Institute of the US National Institutes of Health (R01-HL122352 to J.J.); and done using Z test. To take into account repeated sample assessments, data Leducq Foundation (to J.J.).

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