Establishment of the KCNQ1-knockout and KCNQ1-mutant hESC-CM models
We used the CRISPR/Cas9 system to establish a KCNQ1-deficient H9 hESC cell model. We designed a highly specific sgRNA targeting KCNQ1 and electroporated hESC H9 cells with a plasmid containing sgRNA and Cas9 element. Subsequently, the transfected cells were screened using puromycin, and the genotype of the surviving clones was identified using Sanger sequencing. Sequencing results showed that a homozygous clone with a 2-bp mutation in KCNQ1 was obtained (KCNQ1R190Q/+) (Fig. S1A). The pluripotency of KCNQ1R190Q/+ was identified using the pluripotency markers, and the karyotype and tumorigenic characteristics of stem cells did not change (Fig.S1B, C, D, and 1E)
. KCNQ1R190Q/+ and other hESCs (KCNQ1L114P/+ and KCNQ1−/−) established in our previous work were induced to differentiate into CMs using small molecules with clear chemical compositions (Fig. S2A)21. Immunofluorescence staining of CMs for 30 days showed normal expression of troponin T (TNNT2) and α-actin (Fig. S2B). Flow cytometry showed that the TNNT2 positivity rate of H9 hESC wild-type (WT) and KCNQ1−/− CMs was close to 86% (Fig. S2C and S2D). Western blotting confirmed the absence of the KCNQ1 protein in KCNQ1−/− CMs (Fig. 1A and 1B). Furthermore, the expression of KCNQ1 in KCNQ1L114P/+ CMs significantly decreased compared with that in WT CMs and KCNQ1R190Q/+ CMs, suggesting that the KCNQ1 transport of KCNQ1L114P/+ CMs was abnormal.
KCNQ1−/−, KCNQ1L114P/+, and KCNQ1R190Q/+ CM models can reflect the LQT phenotype
Differences between KCNQ1−/−, KCNQ1L114P/+, and KCNQ1R190Q/+ CMs were observed at the multicellular level using the high-throughput Maestro Edge microelectrode array (MEA) system22,23. The field potential duration (FPD) was calculated as the time between the depolarization and repolarization, noted by the beat time and the repolarization peak or T-wave, respectively. The FPD can reflect the duration of myocardial QT interval. FPD statistics showed no difference between KCNQ1R190Q/+ and WT CMs. Moreover, the FPD of KCNQ1L114P/+ CMs was slightly prolonged, whereas that of KCNQ1−/− CMs was significantly prolonged (Fig. 1C ,D). The prolongation of the FPD of KCNQ1L114P/+ CMs may be due to the lack of repolarization IKs caused by abnormal KCNQ1 transport, which is consistent with the decreased expression of KCNQ1 in KCNQ1L114P/+ CMs shown in Western blotting results. Irregular rhythm and EADs are precursors of ventricular arrhythmias in LQT; therefore, we also used the MEA system to analyze the rhythm of the three models. The results showed that compared with WT CMs, KCNQ1−/− CMs showed obvious arrhythmia, and the proportion of EADs significantly increased. KCNQ1L114P/+ and KCNQ1R190Q/+ CMs also showed obvious arrhythmia (Fig. 1E ,F).
Response to IKs-specific blocker
Moreover, we tested the effects of the IKs-specific blocker chromanol 293B (293B) on the FPD of the three models24. We used the ratio of the FPD after dosing treatment to baseline FPD (FDP'/FDP) to represent the degree of FPD change. A value greater than 1 indicates that the FPD is prolonged, and a value less than 1 indicates that FPD is shortened. Interestingly, the FPD of WT, KCNQ1L114P/+, and KCNQ1R190Q/+ CMs showed prolongation after treatment with 100-mM 293B. In contrast, the FPD of KCNQ1−/− CMs did not significantly prolong as that of KCNQ1L114P/+ and KCNQ1R190Q/+ CMs after treatment with 293B (Fig. 2A and 2B). Meanwhile, the FDP'/FDP of WT, KCNQ1L114P/+, and KCNQ1R190Q/+ CMs was significantly higher than that of KCNQ1−/− CMs; however, the prolongation of the FPD of knockout CMs is the least obvious (Fig. 2C). These results indicated that the KCNQ1 mutation and knockout models were successfully established.
Responses to MgCl2
Mg2+ is the main coenzyme for potassium ion transfer inside and outside a cell. Mg2+ supplementation can increase potassium ion transport, increase the intracellular potassium concentration, and increase cell membrane and electrocardiogram stability. Therefore, we observed changes in the FPD of four CMs after MgCl2 treatment. MgCl2 treatment can shorten the FPD of all three models, and the FPD shortening of KCNQ1−/− CMs is the most significant (Fig. 3A,B,C). This suggests that magnesium supplementation is essential for LQT treatment with different mechanisms. However, EAD cannot be eliminated (Fig. 3D,E).
Responses to Isoproterenol
Since the occurrence of LQT1 is often related to sympathetic nerve excitement (e.g., exercise or emotional agitation), sympathetic nerve excitement was simulated using isoproterenol (ISO) treatment25. ISO is a β-agonist that binds β-AR, and activates cAMP–PKA-dependent downstream signals, which can promote the phosphorylation of several target proteins, including L-type calcium channel and lysine receptor. PKA phosphorylates these proteins and increases the calcium concentration in the sarcoplasm. Therefore, the cross-bridge is activated and further enhances the contraction of CMs26. After treatment with 100-μM ISO, the FPD of WT, KCNQ1L114P/+, and KCNQ1−/− CMs was significantly shortened compared with the baseline (Fig. 4A, B, C). This phenotypic change was caused by the agonistic effect of ISO. However, the FPD of KCNQ1R190Q/+ CMs did not appear to be significantly shortened, suggesting that KCNQ1R190Q/+ CMs are not sensitive to ISO. This is consistent with the results of previous studies, which confirmed that mutations in the C-loop, such as KCNQ1R190Q/+ CMs, may indeed inactivate Kv7.1’s response to PKA stimulation12. Furthermore, WT CMs did not show arrhythmia after ISO treatment, whereas KCNQ1L114P/+ CMs and KCNQ1R190Q/+ CMs both showed arrhythmia aggravation (Fig. 4D, E). Both point-mutation cells showed the arrhythmia phenotype under β-adrenergic stimulation, which was consistent with the phenotype in which LQT1 was more easily induced under sympathetic excitation, indicating that the point process model can well reflect the response of the myocardium to sympathetic excitation.
Responses to Propranolol
β-blockers can directly decrease β-adrenergic signaling and have antiarrhythmic effects26. Propranolol, a β-blocker, is one of the most common drugs for the clinical treatment of LQT. Therefore, we used propranolol to treat the CM models and examine their responses. The results showed that the three CM models showed prolongation of the FPD (Fig. 5A, B, C). Moreover, the slowing of heart rhythm and the occurrence of arrhythmia in KCNQ1−/−, KCNQ1L114P/+, and KCNQ1R190Q/+ CMs were significantly decreased compared with the baseline (Fig. 5D, E). Propranolol can better improve the arrhythmia phenotype of the three KCNQ1-mutant myocardial models, including KCNQ1−/− CMs, indicating that propranolol has therapeutic effects on LQT1 with multiple mechanisms.
Responses to Amiodarone
Subsequently, we tested the response of the three models to other common LQT medications in the clinic. Amiodarone is a type III multi-ion channel blocker, which can selectively prolong the repolarization time of the myocardium and is suitable for various ventricular arrhythmias27. After treatment with 100-μM amiodarone, WT, KCNQ1L114P/+, KCNQ1R190Q/+, and KCNQ1−/− CMs showed significant FPD prolongation; however, KCNQ1−/− CMs had the smallest FPD extension (Fig. 6A, B, C). Amiodarone treatment has alleviated the arrhythmia phenotype of KCNQ1L114P/+ CMs but caused the pulsation of KCNQ1−/− CMs to become weak (Fig. 6D,E). These results suggest that amiodarone has a good therapeutic effect on LQT1 with different mutations; however, it is not suitable for treating patients with KCNQ1 large fragment deletion.