Background
Long QT syndrome type 7 (LQT7), also called Andersen–Tawil syndrome [
1], is a rare potassium channel disease involving multiple systems that shows autosomal dominant inheritance or arises from sporadic individual mutation [
2,
3]. The prevalence of LQT7 is approximately one in a million [
2,
3], and 70% of cases are related to abnormal
KCNJ2 gene function [
4]. The inward rectifier potassium channel protein Kir2.1, which encoded by the
KCNJ2 gene is mainly expressed in ventricular muscle, skeletal muscle and brain tissue [
5], and plays a crucial role in development. The whole gene knockout mice will have dysplasia after birth and cannot survive for a long time [
6]. Therefore, the "typical" clinical triad observed in LQT7 patients consists of QT interval prolongation and ventricular arrhythmia, periodic paralysis and skeletal dysplasia [
7]. More than 97% of LQT7 patients have cardiac-related abnormalities, which further lead to serious cardiogenic adverse events [
8]. Although inward rectifier potassium channel Kir2.1 may affect the electrophysiological function of myocardial cells [
9], the changes in electrophysiological maturation and chromatin accessibility that affect myocardial development and differentiation are unknown. Herein, we established a cardiac cell model derived from human iPSCs to specifically reproduce the ATS disease phenotype and analyze changes of potential transcription factors (TFs) and related target proteins in the transcriptome and open chromatin status at different stages of cardiac development and differentiation. Then, the overall expression of members of potassium ion-related pathways was analyzed, and key target proteins were further screened and verified to help explain the pathogenesis of LQT7.
Materials and methods
Sample ethics statement
The current study (Ethics No. JS-1233) received approval from the Ethics Committee of the Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, and Peking Union Medical College (Beijing, China). The investigation conformed to the principles outlined in the Declaration of Helsinki.
iPSC culture
Peripheral blood was obtained from LQT7 patients and age- and sex-matched control volunteers with no clinical history after informed consent was obtained. We used the mature method [
10,
11] to verify the extraction of hematopoietic stem cells from PBMCs to establish iPSCs. Our team has used this method to construct stem cells to study other diseases [
12].
An isogenic gene-corrected control was generated using CRISPR/Cas9-mediated HDR with ssODN, which provided the wild-type template for gene correction. To correct the heterozygous c.199C > T mutation in exon 2 of the
KCNJ2 gene, a mutation-specific sgRNA (target site: ATGTGGGTGAGAAGGGGCAA) was designed using CRISPR, as previously described [
13]. sgRNA was generated by in vitro transcription using the GeneArt™ Precision gRNA Synthesis Kit (A29377, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. ssODN was designed to contain 123 nt in total, with 83 nt upstream and 40 nt downstream of the Cas9 cut site (5′-gacaggacgaaagccaggcagaagataaccagcatccaccgccagcgaatgtccacacacgtggtgaagatgtctgcgaggtaTcGttgccccttctcacccacattgatgaactgaacattacag-3′). hiPSCs at 80%-85% confluence were harvested with 0.5 mM EDTA. A total of 1.0 × 10
5 cells were coelectroporated with 400 ng of sgRNA, 2 μg of recombinant Cas9 protein (A36496, TrueCut™ Cas9 Protein v2, Thermo Fisher Scientific, Waltham, MA, USA) and 10 pmol of ssODN using a 10-μl Neon® Tip and a Neon transfection system (MPK5000S, Thermo Fisher Scientific, Waltham, MA, USA) with conditions of 1100 V, 20 ms and 2 pulses, as previously described [
14]. Immediately after electroporation, the cells were transferred into one well of a Matrigel-coated 24-well plate and cultured in 500 μl of mTeSR1 medium containing 5 μM Y-27632 (S1049, Selleck, Houston, TX, USA). The medium was changed daily, and the Y-27632 was removed 24 h later. Three days after electroporation, the cells were dispersed at low density into a Matrigel-coated 60-mm dish in mTeSR1 medium with 5 μM Y-27632, which was removed 24 h later. Approximately 14 days later, large colonies were picked, expanded, and analyzed for the desired base correction by Sanger sequencing of an amplicon spanning the target site of
KCNJ2 gene exon 2. A positive clone was selected for further analysis. Genomic DNA was extracted from the iPSCs using a Genomic DNA Extraction Kit (DV811A, Takara, Japan), followed by PCR amplification using a KAPA Taq EXtra HotStart ReadyMix PCR Kit (KK3604, Merck, New Jersey, USA). PCR was performed on a Peltier thermal cycler (PTC-100, Bio-Rad, Hercules, CA, USA) using the following conditions: 94 °C, 3 min; 30 cycles of 94 °C, 25 s; 55 °C, 15 s; 72 °C, 1 min; and 72 °C, 3 min. The PCR product was subsequently sequenced by Sanger sequencing at Shanghai Personal Biotechnology Co., Ltd. (China) to confirm correction of the heterozygous c.199C > T mutation of the
KCNJ2 gene.
Teratoma assay
For this assay, four-week-old immunocompromised SCID mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were used. Cells (2–4 × 106) were subcutaneously injected into the flank area of each mouse. Tumor growth was monitored weekly by palpation. The mice were euthanized when the tumor volume reached ≥ 2 cm3 after 8 weeks. All animal experiments were approved by the Animal Ethics Committee of the Peking Union Medical College Hospital.
iPSC-CM differentiation and treatments
The mutation, CRISPR and control iPSC lines were used in this study. We referred to a well-developed method used in our previous research [
12], and CM differentiation was initiated using a monolayer differentiation method with a STEMdiff CM Differentiation Kit (05010, STEMCELL Technologies Inc, British Columbia, Canada) according to the manufacturer's instructions. After the iPSC-CMs had grown to a density of 80%, ZNF528 siRNA (siZNF528) was transfected into CMs using lipofectamine 3000 (Invitrogen L3000015, USA). After co-transfection for 48 h, the cells were used for further experiments. The primer of si-ZNF528 were showed in Additional file
1: Table S1.
iPSCs- and iPSC-CM-specific protein immunofluorescence staining and confocal microscopy
Pluripotency markers of iPSCs were measured by using a hESC marker panel (1:1000; ab109884, Abcam plc., Cambridge, UK). The structural composition of iPSC-CMs could be identified by the presence of α-actinin and cardiac contractile proteins. So we used antibodies against Brachyury (ab209665, Abcam plc., Cambridge, UK), α-SMA (A5228, Sigma), NKX2.5 and TNNT-2 kit (Thermo Fisher A25973). Stained beating cells and monolayer cells were observed by using laser-scanning confocal microscopy (LSM 710, Carl Zeiss, Jena, Germany), and captured with the Delta Vision OMX SR imaging system.
Single-cell patch clamp experiment for recording action potentials and quantified current signals
The mature myocardial cells were digested into single cells with digestive enzymes 2 days in advance and adhere to the cover glasses. The whole-cell patch clamp experiment was conducted to measure the Cm with a single beat at 32–35 °C through the EPC-10 patch clamp amplifier (HEKA Electronics, Lambrecht, Germany). The whole-cell Kir2.1 potassium current was recorded with the help of an EPC-10 patch clamp amplifier controlled by PULSE software (HEKA Electronics). At the same time, PatchMaster software (HEKA Electronics) was used to capture data.
Sample collection for RNA-seq, ATAC-seq and Proteomic analysis
We selected 6 specific time points for collection of the mutation and CRISPR groups using Accutase (A1110501, Thermo Fisher Scientific, Waltham, MA, USA) to perform RNA-seq and ATAC-seq. ATAC-seq was performed as previously reported [
15]. Two groups of CMs were collected for further proteome analysis. Quantitative proteomics and mass spectrum analysis were performed by TMT labeling as described in the previous study [
16].
WGCNA and ssGSEA
The package of “WGCNA” within R was used for constructing differentially expressed genes coexpression network and modules [
17]. We further evaluated the overall regulation of potassium channel-related pathways at the gene and protein levels via single-sample gene set enrichment analysis (ssGSEA) with the “GSVA” R package [
18].
Expression analysis of target genes and proteins by qPCR and Western blot
The qRT‒PCR and western blot were used to determine the mRNA expression levels of the target proteins. The primers were either obtained from previous studies [
19‐
21] or purchased as a commercial reagent (ORIGENE HP215938) (Additional file
1: Table S1). We chose to isolate total RNA from CMs that exhibited a rate of differentiation greater than 85%, and gene expression was normalized based on the levels of β-actin. Cells with substandard differentiation rates will be discarded. As described in our previous study [
12], the collection, detection, and visualized analysis of sample proteins are carried out. Proteins were detected with antibodies specific to β-actin (1:5000, ab8227, Abcam, Cambridge, UK), KCNJ2 (1:1000, ab85492, Abcam, Cambridge, UK), CTTN (1:2000, ab68438, Abcam, Cambridge, UK), ATP1B1 (1:2000, ab193669, Abcam, Cambridge, UK), and ZNF528 (1:1000, PA5-41182, Thermo, Massachusetts, US).
Resource availability
Further information and requests for resources and reagents should be directed to and will be fulfilled by the first author, Peipei Chen.
Statistical analysis
Numerical data are shown as the mean ± SD. We used Excel software (Windows 2019, Microsoft, Redmond, US) to compare experimental groups through Student’s t test (two tailed). Differences for which p < 0.05 were considered significant.
Discussion
LQT7 is a rare cardiomyopathy with abnormal electrophysiology, and 70% of cases are related to the Kir2.1 inward rectifier potassium channel protein, which is encoded by the KCNJ2 gene, in ventricular muscle, skeletal muscle and brain tissue [
25]. Previous studies in animal and drug-induced cell models have shown the importance of the
KCNJ2 gene, but they exhibit species differences or limitations. For example, whole-gene KCNJ2-knockout mice have a short survival time and there was no Kir2.1 current in their muscle cells [
6,
26]. Although heterozygous Kir2.1−/ + mice exhibited a prolonged action potential duration and a significant increase in QRS and QT intervals, the heart rates of the control group and the disease group were consistent [
27,
28], making the QT intervals of dependence on heart rates impossible to be observed. The canine CMs induced by cesium chloride or calcium chloride can successfully mimic the electrophysiological characteristics of LQT7 (a slightly prolonged QT interval and U wave) [
9], but these primary cells cannot be passaged, exhibit poor repeatability. Furthermore, toad oocytes were used to imitate the electrophysiological characteristics of potassium channels [
29], but they cannot autonomic contraction. However, the hiPSC-derived CM model used in this study avoids the above research problems and it can be further used to explore the developmental differentiation of LQT7 by combined with the different stages of CMs maturation.
In this study, we first compared the specific phenotypes and electrophysiological characteristics of hiPSC-CMs from LQT7 patients with mutation, matched healthy controls and cells after CRISPR-mediated mutation repaired. The successful reprogramming and directional induction of them provide a reliable platform for the exploration (Figs.
1 and
2). Compared with that in the control and CRISPR group, the action potential duration was significantly prolonged in the mutation group, and the inward current of the inward rectifier potassium channel was reduced. After the repair of the mutation site, the electrophysiological characteristics of the CRISPR group tended to be similar to those of the control group, and there was no significant difference in action potential duration or the results of inward rectifier potassium channel patch-clamp experiments between the two groups (Fig.
3). Quantitative detection of iPSC-CMs from LQT7 patients by patch-clamp experiments showed that the Kir2.1 current had a significant negative effect (Fig.
3). Not only were the important conserved genes and transcriptional regulators of myocardial development screened from the same modules (Figs.
4 and
5) but also the pathogenicity-related genes and TFs related to changes in chromatin accessibility were screened from the heterogeneous modules (Fig.
6) by WGCNA. Subsequently, ssGSEA was used to analyze overall potassium-related pathway regulation in mature CMs of the mutation group, and key genes and their encoded proteins were screened by with multiomics, with the results experimentally verified (Fig.
7). The purpose of the above steps was to clarify the molecular changes that occur in the early development and differentiation of CMs in LQT7 and identify key pathogenic molecules.
The results of GO pathway analysis of the six homogeneous modules obtained by WGCNA were consistent with the corresponding stages of CM development and differentiation (Fig.
5 and Table
2). Firstly, the pathways were enriched in the turquoise module were mainly related to mitosis, the cell cycle and stem cell self-renewal. Secondly, the pathways involved at the mesodermal cell stage (green module) were enriched in the formation and development of the mesoderm. Then, the pathways of the cardiac mesodermal precursor cell stage that were enriched in the yellow module were related to the formation and development of embryonic organs in the late mesoderm, and in early pulsating CMs that were enriched in genes in the red module were related to heart formation. In addition, the electrophysiologically mature CM phase (brown module) was mainly related to the processes of mitochondrial ATP synthesis and metabolism, which is consistent with the notion that CMs need to consume a great deal of energy at this stage to grow and develop while maintaining contraction. Finally, the blue module corresponded to the late CM phase of gradual maturation, and their enriched pathways were all related to cardiac function (cardiac contraction, heart rate regulation, action potential regulation, cardiac conduction, and muscle tissue development-related pathways). In addition, our results showed that the TFs matched to the above different phases modules were representative of myocardial growth and development (Fig.
5), which further proved the reliability of our CMs model. For example,
MIXL1 is a key player in Wnt/TGF-β signaling in the early mesodermal differentiation of hESCs [
30,
31]. BHLHE22 is a novel regulator that can direct neural circuit assembly in part through precise regulation [
32,
33]. Previous studies have confirmed that
LHX1 originates from the endoderm and plays an important role in development [
34,
35], and
NKX2 genes are primarily expressed in the mesendoderm-derived organs, such as the heart and gut[
36]. The
DLX gene provides insight into vertebrate genomics and craniofacial evolution [
37,
38], so it was speculated that the craniofacial dysplasia observed in this ATS patients might be related to abnormalities in
DLX 5 and 6. GRHL1 is a conserved transcriptional regulator and key to vertebrate desmosome formation and development [
39,
40]. Embryonic TBX3 form the mature cardiac conduction system [
41], and it also as a key sinoatrial node TF for controlling the development and function of pacemaker[
42,
43]. GATA2 negatively regulates cardiac differentiation at the mesoderm specification stage [
44]. The changes of the above TFs could affect cardiac dysplasia.
HOXB4 regulates endothelial cell turnover and development [
45]. TBX5 is a key regulator of heart development [
46], and TBX5-dependent pathways are associated with heart development, CM function, and congenital heart disease [
47]. Preclinical evidence has reported the therapeutic value of TBX5 protein in regulating adult arrhythmias [
48].
ZEB1 is functionally important for early cardiac differentiation [
49] and to be a regulator of endothelial-mesenchymal transition during development [
50].
GLIS3 can direct the differentiation of hESCs into posterior neural progenitor cells [
51]. These results suggested that cells of different stages could through plasticity regulation and ultimately differentiate into the same cell type (CMs in this study).
The ATAC-seq data showed that the accessibility of the corresponding matching TFs to chromatin decreased at the different stages (Fig.
6D and E); these TFs included MIXL1 (mesoderm stage), GBX1 (cardiac mesodermal precursor cell stage), DLX5 (immature myocardium stage), HOXB4 (electrophysiological mature myocardium stage) and ZEB1 (late myocardium stage). MIXL1 is expressed in the early differentiation of human PSCs toward cardiac mesodermal derivatives [
52]. GBX1 is involved in the development of neurons in the dorsal and ventral spinal cord during embryogenesis [
53], and DLX5 is involved in brain development [
38]. High HOXB4 expression can enhance the differentiation of embryonic stem cells [
54], and HOXB4 is an effective differentiation-promoting transcriptional regulator [
55]. ZEB1 is an important regulator of early cardiac differentiation, especially in the mesoderm stage of the heart [
49]. These changes reflect the features of cardiac differentiation. The chromatin accessibilities of the TFs HOXB4 (day 15) and ZEB1 (day 30) were increased in homologous modules, indicating that the function and regulatory mechanism of CMs at the stage changed from electrophysiological maturity to late functional maturity (Fig.
6D and E). Only pathogenicity-related TF ZNF528 was screened from the heterogeneous tan module. It belongs to the family of zinc finger adjacent-binding domains and is a potential pathogenic gene for neurodevelopmental disorders [
56]. No research has proven the role of the TF ZNF528 in regulating cardiovascular disease, but we verified its pathogenicity from heterogeneity module in this study. ZNF528 began to exhibit chromatin accessibility on day 4 in the CRISPR group, and this accessibility continued to increase over time, while access was reduced at the same time in the mutation group. These results suggest that low ZNF528 expression may be related to the clinical symptoms of LQT7.
In order to further screen out key disease target proteins affected by ZNF528, we jointly analyzed the overall regulatory status of potassium ion-related pathways at the gene and protein levels. The results showed that three inhibited potassium channel-related pathways containing KCNJ2 were related to the whole process by which a potassium ion enters the inward rectifier potassium channel (p < 0.05, Fig.
7E and F). There were the potassium ion enters the cell to exert its effect, which is sensed by the voltage-gated potassium channel complex (GO: 0008076), the potassium ion enters the cell through the membrane (GO: 1990573), and finally, the homeostasis of the potassium ion in the cell is maintained (GO: 030007). This further indicated that expression of these proteins of LQT7 patients was downregulated during this process, which led to the clinical manifestation of QT interval prolongation. The other four downregulated pathways were related to the regulation of potassium transmembrane channels and sodium potassium exchange ATPase, suggesting that the protein or energy demand for regulating potassium influx may be indirectly affected by mutant gene
KCNJ2, thus further aggravating the clinical symptoms. Subsequently, three key sustainably down-regulated target proteins (KCNJ2, CTTN and ATP1B1) were screened in the disease group. KCNJ2 is a known pathogenic mutant protein in LQT7 that was identified in this study, indicating the reliability of our methodology. Therefore, the newly identified CTTN and ATP1B1 proteins may also be related to LQT7 symptoms. CTTN is the actin-binding protein cortactin, and maintains the normal operation of the Kv1.5 channel (delayed rectifier K + current is rapidly activated by atrial muscle) in Purkinje fibroblasts [
57]. Decreased expression of CTTN leads to arrhythmias in mice [
58], so it may inhibit the potassium ion channel Kv1.5 in LQT7 patients for prolonging the APD and QT intervals to aggravate the clinical symptoms. ATP1B1 is a sodium potassium pump β type 1 protein by obtaining energy from ATP under normal conditions, and its low expression leads to cardiac systolic dysfunction in mice [
59]. The decreased expression of the ATP1B1 protein in the mutation group may have prevented K + from being transferred into the cells through the sodium potassium pump, causing the total amount of potassium to decrease, which may aggravate the QT interval extension and abnormal cardiac contraction in LQT7 patients. Finally, the low expression changes in gene and protein levels of these three key targets and one TF were validated in newly collected advanced CMs from the disease group (Fig.
7H–J).
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.