SGK1 inhibition attenuated the action potential duration in patient- and genotype-specific re-engineered heart cells with congenital long QT syndrome

Background Long QT syndrome (LQTS) stems from pathogenic variants in KCNQ1 (LQT1), KCNH2 (LQT2), or SCN5A (LQT3) and is characterized by action potential duration (APD) prolongation. Inhibition of serum and glucocorticoid regulated kinase-1 (SGK1) is proposed as a novel therapeutic for LQTS. Objective The study sought to test the efficacy of novel, selective SGK1 inhibitors in induced pluripotent stem cell–derived cardiomyocyte (iPSC-CM) models of LQTS. Methods The mexiletine (MEX)-sensitive SCN5A-P1332L iPSC-CMs were tested initially compared with a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 SCN5A-P1332L variant–corrected isogenic control (IC). The SGK1-I1 therapeutic efficacy, compared with MEX, was tested for APD at 90% repolarization (APD90) shortening in SCN5A-P1332L, SCN5A-R1623Q, KCNH2-G604S, and KCNQ1-V254M iPSC-CMs using FluoVolt. Results The APD90 was prolonged in SCN5A-P1332L iPSC-CMs compared with its IC (646 ± 7 ms vs 482 ± 23 ms; P < .0001). MEX shortened the APD90 to 560 ± 7 ms (52% attenuation, P < .0001). SGK1-I1 shortened the APD90 to 518 ± 5 ms (78% attenuation, P < .0001) but did not shorten the APD90 in the IC. SGK1-I1 shortened the APD90 of the SCN5A-R1623Q iPSC-CMs (753 ± 8 ms to 475 ± 19 ms compared with 558 ± 19 ms with MEX), the KCNH2-G604S iPSC-CMs (666 ± 10 ms to 574 ± 18 ms vs 538 ± 15 ms after MEX), and the KCNQ1-V254M iPSC-CMs (544 ± 10 ms to 475 ± 11ms; P = .0004). Conclusions Therapeutically inhibiting SGK1 effectively shortens the APD in human iPSC-CM models of the 3 major LQTS genotypes. These preclinical data support development of SGK1 inhibitors as novel, first-in-class therapy for patients with congenital LQTS.


Introduction
Long QT syndrome (LQTS) is characterized by delayed repolarization of the myocardium resulting in a prolonged QT interval on the 12-lead electrocardiogram (ECG). Patients with LQTS may present with arrhythmic syncope/ seizures, sudden cardiac arrest, or sudden cardiac death (SCD) often following a precipitating event such as exercise, auditory trigger, or extreme emotion. 1 LQTS occurs in w1 in 2000 people. 2 For patients with LQTS who remain untreated, it is estimated that there is a 50% 10-year mortality in the highestrisk subset. 3 Current therapeutic options include drug therapy (b-blockers), denervation therapy (left cardiac sympathetic denervation surgery), and/or device therapy (implantable cardioverter-defibrillator). These efforts are usually efficacious. 4 However, while many patients with LQTS remain protected while on b-blocker therapy, there is a substantial population of LQTS patients experiencing breakthrough cardiac events including implantable cardioverter-defibrillator shocks and SCD. 4 Furthermore, noncompliance is common with b-blocker therapy due to intolerable side effects. 5 Approximately 80% of LQTS stems from either loss-offunction (LOF) or gain-of-function (GOF) pathogenic variants in 1 of 3 LQTS-susceptibility genes: KCNQ1-encoded I Ks (K v 7.1) potassium channel (LQTS type 1 [LQT1], w35%-40%, LOF), KCNH2-encoded I Kr (K v 11.1) potassium channel (LQTS type 2 [LQT2], w30%-35%, LOF), or SCN5A-encoded I Na (Na v 1.5) sodium channel (LQTS type 3 [LQT3], w5%-10%, GOF). The LOF or GOF of these critical ion channels underlies the pathological prolongation of the ventricular cardiomyocyte's action potential duration (APD). 6 Serum and glucocorticoid regulated kinase-1 (SGK1) is an important regulator of Na v 1.5-mediated I Na in the heart. 7,8 LQT3-causing GOF pathogenic variants in SCN5A typically lead to an increase in late I Na current. Small-molecule inhibitors of SGK1 may be antiarrhythmic in cardiac diseases through attenuation of the abnormally increased late I Na . 8,9 Recently, Bezzerides and colleagues 9 provided a proof of concept for a SGK1 inhibitor (SGK1-I)-based therapeutic for LQT3 by demonstration of an APD-shortening effect on a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model of the LQT3-causing SCN5A-N406K variant following treatment with a novel SGK1-I. 9 Here, we extend this analysis to test the efficacy of 2 new, potent, and selective SGK1-Is (SGK1-I1 and SGK1-I2) in additional patient-specific iPSC-CM models of LQT3 (SCN5A-P1332L and SCN5A-R1623Q) as well as determine for the first time the potential role of an SGK1-I-based treatment strategy for LQT1 and LQT2.

Methods
To prevent the reidentification of patients included in this study, individual patient data will not be made available to other researchers. The authors declare that all supporting data are available within the article and its Supplementary Appendix.

Generation of patient-specific IPSCs
Following written informed consent for this Mayo Clinic Institutional Review Board-approved study (09-006465), iPSCs were generated from peripheral blood mononuclear cells from 4 unrelated patients diagnosed with LQTS; each with a different LQTS-causative pathogenic variant in KCNQ1 (c.760G.A, p.V254M), KCNH2 (c.1810G.A, p.G604S) or SCN5A (c.3965C.T, p.P1332L and c.4868G.A, p.R1623Q). The peripheral blood mononuclear cells were reprogrammed by Sendai virus transduction using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, Waltham, MA; A16517) as described previously. 10 Colonies were picked within 21 days postinfection and clonally expanded for further analysis. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene-edited/variant-corrected, isogenic control (IC) iPSC lines were engineered by Applied StemCell (Milpitas, CA). All iPSC clones were confirmed to express Tra-1-60, SSEA-3, OCT4, and Nanog pluripotent markers and demonstrated to have a normal karyotype. The presence of the heterozygous pathogenic variant in patient-derived iPSC lines and the genetic correction of the specific variant to wild type in the IC lines were confirmed by Sanger sequencing.
-Therapeutically inhibiting SGK1 with our novel SGK1 inhibitor effectively shortens the action potential duration (the cellular surrogate for the QT interval) in human iPSC-CM models of all 3 major long QT syndrome genotypes.
-As a result of this preclinical data, clinical trials with a novel SGK1 inhibitor in patients with congenital long QT syndrome are anticipated to begin enrolling patients later this year. If this new drug demonstrates appropriate safety and efficacy, it could become the first Food and Drug Administration-approved medication indicated to reduce arrhythmic risk in patients with congenital long QT syndrome.
concentration) from days 0 to 2. On day 2, medium was changed to RPMI-B27-minus insulin containing IWP2 (Tocris Bioscience; 3533, 5mM as working concentration) and incubated until day 4. On day 4, the medium was changed back to normal RPMI GlutaMAX-B27-minus insulin and cells were maintained in this media until beating cardiomyocytes appeared, typically around day 6 or day 8. After beating was seen, iPSC-CMs were maintained in RPMI GlutaMAX medium with B27 serum-free supplement (Life Technologies; 17504-044).

Dissociation of iPSC-CMs
At day 21, iPSC-CMs were washed with phosphate-buffered saline (PBS) (without Ca/Mg) and then subjected to STEMdiff cardiomyocyte dissociation medium (STEMCELL Technologies; 05026). After 5 minutes at 37 C, the iPSC-CMs were pelleted at 300 g for 5 minutes and resuspended in plating medium (Dulbecco's modified Eagle medium, no phenol red, 20% charcoal stripped fetal bovine serum) for subsequent assays. From the following day and onward, the iPSC-CMs were cultured in CM maintenance medium (Dulbecco's modified Eagle medium, no phenol red, 2% charcoal stripped fetal bovine serum) and media were changed every 2 to 3 days.

Live cell imaging for APD measurement
iPSC-CMs were cultured on 35-mm glass-bottom dishes (MatTek, Ashland, MA; P35G-1.5-10-C) that were precoated with Matrigel matrix at 37 C, 5% CO 2 . For imaging, cells were incubated at 37 C, 5% CO 2 for 20 minutes in Tyrode solution containing a volage-sensitive fluorescent dye, Fluo-Volt, that responds to changes in membrane potential (Thermo Fisher Scientific; F10488). The cells were then washed 3 times in fresh Tyrode solution. During imaging, the dishes were kept in a heated 37 C stage-top environment chamber supplied with 5% CO 2 . Imaging of voltageindicated cellular APD was taken under a !40 water objective using a Nikon Eclipse Ti light microscope (Nikon, Tokyo, Japan). Time-lapse videos of multiple, individual beating iPSC-CMs, paced at 1 Hz were recorded at a speed of 20 ms per frame for 20 seconds at 10% LED power. Single regions of interest were selected for every beating iPSC-CM captured in the recordings. The raw data were exported to Excel software (Microsoft, Redmond, WA) and then analyzed with an "in-lab" developed Excel-based program. 11

Statistical analysis
All data points are shown as the mean value, and bars represent the SEM. Student's t test was performed to determine statistical significance between 2 groups, and a 1-way analysis of variance and Tukey-Kramer post hoc test were performed for comparisons among 3 or more groups. A P , .05 was considered significant.

Results
The SGK1 activity of the novel SGK1-Is tested was measured in a biochemical assay that assesses the binding and displacement of a wild-type SGK1 active site-directed fluorescent probe and NanoBRET target engagement cellbased assay. The whole-cell SGK1 half maximal inhibitory concentration (IC 50 ) of SGK1-I1 and SGK1-I2 was ,100 nM. Additionally, their selectivity was assessed in a biochemical assay that assessed the kinase reactivity in a panel of 50 kinases and showed very high selectivity. In addition, there were no effects on cardiac ion channels at ,100 times the free tested exposures.

Generation of patient-specific iPSCs and CRISPR/ Cas9-engineered IC iPSCs
Experiments were performed on a mexiletine (MEX)-sensitive SCN5A-P1332L iPSC-CM model that was derived from a 4.5-year-old female patient who presented an ECG rate-corrected QT (QTc) of 583 ms. A CRISPR/Cas9 SCN5A-P1332L variant-corrected IC iPSC-CM was used as a control. The pluripotency of undifferentiated SCN5A-P1332L iPSCs were demonstrated by immunofluorescence staining of pluripotent markers (NANOG, SSEA4, OCT4, and TRA-1-60) (Supplemental Figure 1A). The patientspecific iPSC line had a normal female karyotype (Supplemental Figure 1B). The presence of the heterozygous SCN5A-P1332L variant in patient-derived iPSC lines and the genetic correction of SCN5A-P1332L variant to wild type in the IC iPSC line (Supplemental Figure 1C) were confirmed by Sanger sequencing. Subsequent efficacy studies were performed on a second LQT3-associated patient-derived iPSC-CM model (SCN5A-R1623Q) as well as LQT1 (KCNQ1-V254M)and LQT2 (KCNH2-G604S)-associated iPSC-CM models. The quality control analysis of the SCN5A-R1623Q iPSCs, including proper pluripotent marker immunofluorescence staining, normal karyotype, and Sanger sequence confirmation of the variant are shown in Supplemental Figure 2. The quality control analysis for KCNQ1-V254M and KCNH2-G604S iPSCs were reported previously. 12,13 Patient demographic information is shown in Supplemental Table 1.
Interestingly, the SGK activity in the SCN5A-P1332L iPSC-CMs was upregulated by about 2-fold compared with the IC iPSC-CMs, determined by immunoblotting with an antibody against p-GSK3b (phospho [Ser9]-glycogen synthase kinase beta), a well-established SGK1 substrate ( Figure 1B).

Discussion
The ubiquitously expressed SGK1 is a PI3K-dependent, serine-threonine kinase that regulates a variety of cellular molecules including ion channels, transporters, enzymes, and signaling molecules. 15,16 While SGK1 is expressed in essentially all tissues, it is strictly transcriptionally and post-transcriptionally regulated in response to many agonists and under several pathological conditions including glucocorticoids, mineralocorticoids, serum, inflammatory cytokines, and increases in cytosolic calcium ion concentration. 15 Notably, SGK1 is activated predominantly under pathological conditions, including hypertension, hypertrophic response, heart failure, and other oxidative and mechanical stressors related to electrical remodeling. 8,15,17 Notably, we demonstrated that SGK1 messenger RNA levels and SGK1 enzymatic activity were increased 2-fold in a human iPSC-CM model of LQT3 compared with its IC model as evident by increased expression of p-GSK3b, a well-known SGK1 substrate. The mechanism responsible for increased SGK1 activity in the iPSC-CMs due to the presence of a pathogenic variant in SCN5A is currently unknown. Whether other pathogenic mutations in LQTS-susceptible genes leading to an arrhythmic substrate and possible primary electrical remodeling at the cellular level in response to functional insult can transcriptionally and posttranslationally regulate SGK1 expression and activity are unknown.
Chronic SGK1 activation in cardiomyocytes is associated with altered sodium flux with pathological late sodium current and hyperpolarizing shift of the steady-state inactivation of the SCN5A-encoded sodium channel, leading to increase in window current I Na , resulting in APD prolongation and increased propensity for afterdepolarization arrhythmic events. 7,8 Moreover, mice with constitutively active SGK1 have a prolonged QTc, spontaneous ventricular tachycardia, and SCD. 8 However, inhibition of SGK1 either by germ-line ablation or dominant-negative inhibition does not lead to a notable pathogenic phenotype under basal conditions and appears to be protective against pathological stress, suggesting that small-molecule inhibitors of SGK1 could be antiarrhythmic in cardiac disease through correction of abnormal I Na and a potential therapeutic for LQTS. 8,9 Recently, using a computer-aided drug discovery platform, Bezzerides and colleagues 9 identified a novel class of SGK1-Is that reduced targeted SGK1 activity in primary cultured cardiomyocytes and reversed the SGK1-induced pathological changes in I Na observed in HEK293 cells coexpressing SCN5A and a constitutively active form of SGK1. The lead compound selectively reduced late I Na without a significant decrease in peak I Na in mammalian cardiomyocytes. In a human iPSC-CM model derived from a patient with the LQT3-causing SCN5A-N406K pathogenic variant, their first-generation SGK1-I shortened significantly the pathologically prolonged APD and corrected the abnormal phenotype. 9 Importantly, the SGK1-I did not shorten the APD of iPSC-CMs derived from a healthy individual. 9 Here, we provide additional data supporting small molecule-based SGK1-Is as a treatment strategy for LQT3 using novel, potent, and selective SGK1-Is. We first tested the efficacy of the novel SGK1-I1 on a MEX-sensitive SCN5A-P1332L iPSC-CM derived from a patient with LQT3 and its CRISPR/Cas9 SCN5A-P1332L variantcorrected IC iPSC-CM line. Interestingly, while MEX attenuated the pathological APD by 52% in this MEX-sensitive variant line, our novel SGK1-I normalized the pathological APD prolongation almost fully (.75%) in the SCN5A-P1332L iPSC-CM model. Akin to the study by Bezzerides and colleagues, 9 SGK1-I1 did not further shorten the APD in the IC iPSC-CM. Importantly, we further validated the efficacy of the SGK1-I by demonstrating its APD-shortening effect in a second LQT3-causing pathogenic variant, SCN5A-R1623Q. Again, the SGK1-I APD-shortening effect was significantly greater than what was observed with MEX.
Our novel SGK1-Is also shortened the pathologically prolonged APD in patient-derived iPSC-CM models of the 2 most common forms of LQTS, KCNQ1-mediated LQT1 and KCNH2-mediated LQT2. Here, while the commercially available SGK1-I EMD638683 failed to shorten the APD, our novel SGK1-Is shortened significantly the APD in both KCNQ1-V254M and KCNH2-G604S iPSC-CM models. Interestingly, Bezzerides and colleagues 9 showed that SGK1 inhibition by SGK1 Morpholino knockdown or injection with SGK1-dominant negative messenger RNA can rescue the 2:1 atrioventricular block phenotype manifestation of prolonged APD in the zebrafish breakdance mutant (bkd -/-) that is due to a mutation in the zebrafish homologue of the KCNH2 gene and recapitulates the human LQT2 phenotype. 9 Additionally, preincubation of homozygous breakdance mutant zebrafish with their lead SGK1-I compound rescued the 2:1 atrioventricular block in a dosedependent manner. 9 While the mechanism is not fully understood, these data suggest that SGK1 inhibition may be potentially therapeutic for the 3 most common LQTS subtypes that collectively account for .80% of LQTS.
Importantly, while we have provided validation of the effectiveness of SGK1 inhibition by small-molecule compounds to shorten the pathological APD in LQT3 iPSC-CM models and have shown for the first time the efficacy of SGK1-I to shorten the APD in patient-specific iPSC-CM models of LQT1 and LQT2, further studies in additional human iPSC-CM models of LQT1, LQT2, and LQT3 with unique pathogenic variants with differing underlying cellular mechanisms of disease are warranted. There may be genotype-and variant-specific effects on APD shortening by SGK1-Is. Preclinical in vivo and ex vivo studies possibly including transgenic animal models will be informative to further advance SGK1-Is as a novel therapeutic strategy for LQTS, though patient studies would be the most definitive.

Conclusion
Therapeutically inhibiting SGK1 effectively shortened the cardiomyocyte APD in human heart cell models of the 3 major LQTS genotypes. These preclinical data support further development of SGK1-Is as a novel, first-in-class therapy for patients with congenital LQTS.
Funding Sources: This work was supported by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program and Thryv Therapeutics, Inc.
Disclosures: Michael J. Ackerman is a consultant for Abbott, Boston Scientific, Bristol Myers Squibb, Daiichi Sankyo, Invitae, Thryv Therapeutics, and Medtronic. Michael J. Ackerman and the Mayo Clinic are involved in an equity/royalty relationship with AliveCor, Anumana, ARMGO Pharma, Pfizer, and UpToDate. These relationships are all modest, and none of these entities have contributed to this study in any manner. Saumya Das is a scientific founder and has received equity for Thryv Therapeutics and Switch Therapeutics and has a consulting relationship with Thryv Therapeutics and Renovacor. Philip T. Sager is a scientific founder and has received equity from Thryv Therapeutics where he is an employee. The other authors have no conflicts of interest to disclose.
Authorship: All authors attest they meet the current ICMJE criteria for authorship.
Patient Consent: Patients provided written informed consent for participation in this study.