TAD boundary deletion causes PITX2-related cardiac electrical and structural defects.

While 3D chromatin organization in topologically associating domains (TADs) and loops mediating regulatory element-promoter interactions is crucial for tissue-specific gene regulation, the extent of their involvement in human Mendelian disease is largely unknown. Here, we identify 7 families presenting a new cardiac entity associated with a heterozygous deletion of 2 CTCF binding sites on 4q25, inducing TAD fusion and chromatin conformation remodeling. The CTCF binding sites are located in a gene desert at 1 Mb from the Paired-like homeodomain transcription factor 2 gene (PITX2). By introducing the ortholog of the human deletion in the mouse genome, we recapitulate the patient phenotype and characterize an opposite dysregulation of PITX2 expression in the sinoatrial node (ectopic activation) and ventricle (reduction), respectively. Chromatin conformation assay performed in human induced pluripotent stem cell-derived cardiomyocytes harboring the minimal deletion identified in family#1 reveals a conformation remodeling and fusion of TADs. We conclude that TAD remodeling mediated by deletion of CTCF binding sites causes a new autosomal dominant Mendelian cardiac disorder.

Clinical characterization of type 1 long QT syndrome caused by C-terminus Kv7.1 variants.

BACKGROUND: Variants in the KCNQ1 gene, encoding the α-subunit of the slow component of delayed rectifier K+ channel Kv7.1, cause long QT syndrome (LQTS) type 1. The location of variants may be one of the factors in determining prognosis. However, detailed genotype-phenotype relationships associated with C-terminus variants remain unelucidated. OBJECTIVE: We investigated the clinical characteristics and variant-specific arrhythmic risks in patients with LQTS carrying Kv7.1 C-terminus variants. METHODS: The study comprises 202 consecutive patients with LQTS (98 probands and 104 family members) who carry a rare heterozygous variant in the Kv7.1 C-terminus. Their clinical characteristics and arrhythmic events were investigated. RESULTS: We identified 36 unique C-terminus variants (25 missense and 11 non-missense). The p.R366W variant was identified in 8 families, and p.T587M was identified in 21 families in large numbers from northwestern Japan. As for the location of the variant, we found that the variants in highly conserved regions and nonhelical domains were associated with longer QTc intervals compared with the variants in other regions. Both p.R366W and p.T587M variants are located in the highly conserved and functionally pivotal regions close to helices A and D, which are associated with calmodulin binding and channel assembly (tetramerization), respectively. The probands carrying p.T587M and p.R366W variants had worse arrhythmia outcomes compared with those with other C-terminus variants. The haplotype analysis of p.T587M families was suggestive of a founder effect. CONCLUSION: The arrhythmic risk of C-terminus variants in Kv7.1 in LQTS is not homogeneous, and locations of variants can be a determining factor for prognosis.

KUS121, a VCP modulator, has an ameliorating effect on acute and chronic heart failure without calcium loading via maintenance of intracellular ATP levels.

AIMS: As heart failure (HF) progresses, ATP levels in myocardial cells decrease, and myocardial contractility also decreases. Inotropic drugs improve myocardial contractility but increase ATP consumption, leading to poor prognosis. Kyoto University Substance 121 (KUS121) is known to selectively inhibit the ATPase activity of valosin-containing protein, maintain cellular ATP levels, and manifest cytoprotective effects in several pathological conditions. The aim of this study is to determine the therapeutic effect of KUS121 on HF models. METHODS AND RESULTS: Cultured cell, mouse, and canine models of HF were used to examine the therapeutic effects of KUS121. The mechanism of action of KUS121 was also examined. Administration of KUS121 to a transverse aortic constriction (TAC)-induced mouse model of HF rapidly improved the left ventricular ejection fraction and improved the creatine phosphate/ATP ratio. In a canine model of high frequency-paced HF, administration of KUS121 also improved left ventricular contractility and decreased left ventricular end-diastolic pressure without increasing the heart rate. Long-term administration of KUS121 to a TAC-induced mouse model of HF suppressed cardiac hypertrophy and fibrosis. In H9C2 cells, KUS121 reduced ER stress. Finally, in experiments using primary cultured cardiomyocytes, KUS121 improved contractility and diastolic capacity without changing peak Ca2+ levels or contraction time. These effects were not accompanied by an increase in cyclic adenosine monophosphate or phosphorylation of phospholamban and ryanodine receptors. CONCLUSIONS: KUS121 ameliorated HF by a mechanism totally different from that of conventional catecholamines. We propose that KUS121 is a promising new option for the treatment of HF.

Calmodulinopathy in Japanese Children　- Their Cardiac Phenotypes Are Severe and Show Early Onset in Fetal Life and Infancy.

BACKGROUND: Cardiac calmodulinopathy, characterized by a life-threatening arrhythmia and sudden death in the young, is extremely rare and caused by genes encoding calmodulin, namely calmodulin 1 (CALM1), CALM2, and CALM3.Methods and Results: We screened 195 symptomatic children (age 0-12 years) who were suspected of inherited arrhythmias for 48 candidate genes, using a next-generation sequencer. Ten probands were identified as carrying variants in any of CALM1-3 (5%; median age 5 years), who were initially diagnosed with long QT syndrome (LQTS; n=5), catecholaminergic polymorphic ventricular tachycardia (CPVT; n=3), and overlap syndrome (n=2). Two probands harbored a CALM1 variant and 8 probands harbored 6 CALM2 variants. There were 4 clinical phenotypes: (1) documented lethal arrhythmic events (LAEs): 4 carriers of N98S in CALM1 or CALM2; (2) suspected LAEs: CALM2 p.D96G and D132G carriers experienced syncope and transient cardiopulmonary arrest under emotional stimulation; (3) critical cardiac complication: CALM2 p.D96V and p.E141K carriers showed severe cardiac dysfunction with QTc prolongation; and (4) neurological and developmental disorders: 2 carriers of CALM2 p.E46K showed cardiac phenotypes of CPVT. Beta-blocker therapy was effective in all cases except cardiac dysfunction, especially in combination with flecainide (CPVT-like phenotype) and mexiletine (LQTS-like). CONCLUSIONS: Calmodulinopathy patients presented severe cardiac features, and their onset of LAEs was earlier in life, requiring diagnosis and treatment at the earliest age possible.

Non-missense variants of KCNH2 show better outcomes in type 2 long QT syndrome.

AIMS: More than one-third of type 2 long QT syndrome (LQT2) patients carry KCNH2 non-missense variants that can result in haploinsufficiency (HI), leading to mechanistic loss-of-function. However, their clinical phenotypes have not been fully investigated. The remaining two-thirds of patients harbour missense variants, and past studies uncovered that most of these variants cause trafficking deficiency, resulting in different functional changes: either HI or dominant-negative (DN) effects. In this study, we examined the impact of altered molecular mechanisms on clinical outcomes in LQT2 patients. METHODS AND RESULTS: We included 429 LQT2 patients (234 probands) carrying a rare KCNH2 variant from our patient cohort undergoing genetic testing. Non-missense variants showed shorter corrected QT (QTc) and less arrhythmic events (AEs) than missense variants. We found that 40% of missense variants in this study were previously reported as HI or DN. Non-missense and HI-groups had similar phenotypes, while both exhibited shorter QTc and less AEs than the DN-group. Based on previous work, we predicted the functional change of the unreported variants-whether they cause HI or DN via altered functional domains-and stratified them as predicted HI (pHI)- or pDN-group. The pHI-group including non-missense variants exhibited milder phenotypes compared to the pDN-group. Multivariable Cox model showed that the functional change was an independent risk of AEs (P = 0.005). CONCLUSION: Stratification based on molecular biological studies enables us to better predict clinical outcomes in the patients with LQT2.

Novel Calmodulin Variant p.E46K Associated With Severe Catecholaminergic Polymorphic Ventricular Tachycardia Produces Robust Arrhythmogenicity in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BACKGROUND: CaM (calmodulin) is a ubiquitously expressed, multifunctional Ca2+ sensor protein that regulates numerous proteins. Recently, CaM missense variants have been identified in patients with malignant inherited arrhythmias, such as long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the exact mechanism of CaM-related CPVT in human cardiomyocytes remains unclear. In this study, we sought to investigate the arrhythmogenic mechanism of CPVT caused by a novel variant using human induced pluripotent stem cell (iPSC) models and biochemical assays. METHODS: We generated iPSCs from a patient with CPVT bearing CALM2 p.E46K. As comparisons, we used 2 control lines including an isogenic line, and another iPSC line from a patient with long QT syndrome bearing CALM2 p.N98S (also reported in CPVT). Electrophysiological properties were investigated using iPSC-cardiomyocytes. We further examined the RyR2 (ryanodine receptor 2) and Ca2+ affinities of CaM using recombinant proteins. RESULTS: We identified a novel de novo heterozygous variant, CALM2 p.E46K, in 2 unrelated patients with CPVT accompanied by neurodevelopmental disorders. The E46K-cardiomyocytes exhibited more frequent abnormal electrical excitations and Ca2+ waves than the other lines in association with increased Ca2+ leakage from the sarcoplasmic reticulum via RyR2. Furthermore, the [3H]ryanodine binding assay revealed that E46K-CaM facilitated RyR2 function especially by activating at low [Ca2+] levels. The real-time CaM-RyR2 binding analysis demonstrated that E46K-CaM had a 10-fold increased RyR2 binding affinity compared with wild-type CaM which may account for the dominant effect of the mutant CaM. Additionally, the E46K-CaM did not affect CaM-Ca2+ binding or L-type calcium channel function. Finally, antiarrhythmic agents, nadolol and flecainide, suppressed abnormal Ca2+ waves in E46K-cardiomyocytes. CONCLUSIONS: We, for the first time, established a CaM-related CPVT iPSC-CM model which recapitulated severe arrhythmogenic features resulting from E46K-CaM dominantly binding and facilitating RyR2. In addition, the findings in iPSC-based drug testing will contribute to precision medicine.

Disrupted CaV1.2 selectivity causes overlapping long QT and Brugada syndrome phenotypes in the CACNA1C-E1115K iPS cell model.

BACKGROUND: A missense mutation in the α1c subunit of voltage-gated L-type Ca2+ channel-coding CACNA1C-E1115K, located in the Ca2+ selectivity site, causes a variety of arrhythmogenic phenotypes. OBJECTIVE: We aimed to investigate the electrophysiological features and pathophysiological mechanisms of CACNA1C-E1115K in patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). METHODS: We generated iPSCs from a patient carrying heterozygous CACNA1C-E1115K with overlapping phenotypes of long QT syndrome, Brugada syndrome, and mild cardiac dysfunction. Electrophysiological properties were investigated using iPSC-CMs. We used iPSCs from a healthy individual and an isogenic iPSC line corrected using CRISPR-Cas9-mediated gene editing as controls. A mathematical E1115K-CM model was developed using a human ventricular cell model. RESULTS: Patch-clamp analysis revealed that E1115K-iPSC-CMs exhibited reduced peak Ca2+ current density and impaired Ca2+ selectivity with an increased permeability to monovalent cations. Consequently, E1115K-iPSC-CMs showed decreased action potential plateau amplitude, longer action potential duration (APD), and a higher frequency of early afterdepolarization compared with controls. In optical recordings examining the antiarrhythmic drug effect, late Na+ channel current (INaL) inhibitors (mexiletine and GS-458967) shortened APDs specifically in E1115K-iPSC-CMs. The AP-clamp using a voltage command obtained from E1115K-iPSC-CMs with lower action potential plateau amplitude and longer APD confirmed the upregulation of INaL. An in silico study recapitulated the in vitro electrophysiological properties. CONCLUSION: Our iPSC-based analysis in CACNA1C-E1115K with disrupted CaV1.2 selectivity demonstrated that the aberrant currents through the mutant channels carried by monovalent cations resulted in specific action potential changes, which increased endogenous INaL, thereby synergistically contributing to the arrhythmogenic phenotype.

Targeted deep sequencing analyses of long QT syndrome in a Japanese population.

Long QT syndrome (LQTS) is one of the most common inherited arrhythmias and multiple genes have been reported as causative. Presently, genetic diagnosis for LQTS patients is becoming widespread and contributing to implementation of therapies. However, causative genetic mutations cannot be detected in about 20% of patients. To elucidate additional genetic mutations in LQTS, we performed deep-sequencing of previously reported 15 causative and 85 candidate genes for this disorder in 556 Japanese LQTS patients. We performed in-silico filtering of the sequencing data and found 48 novel variants in 33 genes of 53 cases. These variants were predicted to be damaging to coding proteins or to alter the binding affinity of several transcription factors. Notably, we found that most of the LQTS-related variants in the RYR2 gene were in the large cytoplasmic domain of the N-terminus side. They might be useful for screening of LQTS patients who had no known genetic factors. In addition, when the mechanisms of these variants in the development of LQTS are revealed, it will be useful for early diagnosis, risk stratification, and selection of treatment.

Gradient-based parameter optimization method to determine membrane ionic current composition in human induced pluripotent stem cell-derived cardiomyocytes.

Premature cardiac myocytes derived from human induced pluripotent stem cells (hiPSC-CMs) show heterogeneous action potentials (APs), probably due to different expression patterns of membrane ionic currents. We developed a method for determining expression patterns of functional channels in terms of whole-cell ionic conductance (Gx) using individual spontaneous AP configurations. It has been suggested that apparently identical AP configurations can be obtained using different sets of ionic currents in mathematical models of cardiac membrane excitation. If so, the inverse problem of Gx estimation might not be solved. We computationally tested the feasibility of the gradient-based optimization method. For a realistic examination, conventional 'cell-specific models' were prepared by superimposing the model output of AP on each experimental AP recorded by conventional manual adjustment of Gxs of the baseline model. Gxs of 4-6 major ionic currents of the 'cell-specific models' were randomized within a range of ± 5-15% and used as an initial parameter set for the gradient-based automatic Gxs recovery by decreasing the mean square error (MSE) between the target and model output. Plotting all data points of the MSE-Gx relationship during optimization revealed progressive convergence of the randomized population of Gxs to the original value of the cell-specific model with decreasing MSE. The absence of any other local minimum in the global search space was confirmed by mapping the MSE by randomizing Gxs over a range of 0.1-10 times the control. No additional local minimum MSE was obvious in the whole parameter space, in addition to the global minimum of MSE at the default model parameter.

Increased CaV1.2 late current by a CACNA1C p.R412M variant causes an atypical Timothy syndrome without syndactyly.

Timothy syndrome (TS) is a rare pleiotropic disorder associated with long QT syndrome, syndactyly, dysmorphic features, and neurological symptoms. Several variants in exon 8 or 8a of CACNA1C, a gene encoding the α-subunit of voltage-gated Ca2+ channels (Cav1.2), are known to cause classical TS. We identified a p.R412M (exon 9) variant in an atypical TS case. The aim of this study was to examine the functional effects of CACNA1C p.R412M on CaV1.2 in comparison with those of p.G406R. The index patient was a 2-month-old female infant who suffered from a cardio-pulmonary arrest in association with prolonged QT intervals. She showed dysmorphic facial features and developmental delay, but not syndactyly. Interestingly, she also presented recurrent seizures from 4 months. Genetic tests identified a novel heterozygous CACNA1C variant, p.R412M. Using heterologous expression system with HEK-293 cells, analyses with whole-cell patch-clamp technique revealed that p.R412M caused late Ca2+ currents by significantly delaying CaV1.2 channel inactivation, consistent with the underlying mechanisms of classical TS. A novel CACNA1C variant, p.R412M, was found to be associated with atypical TS through the same mechanism as p.G406R, the variant responsible for classical TS.

Exacerbation of ventricular arrhythmias by continuous positive airway pressure treatment in idiopathic dilated cardiomyopathy.

Continuous positive airway pressure (CPAP) therapy can be beneficial in patients with obstructive sleep apnea (OSA) and cardiovascular diseases, reducing arrhythmia frequency and improving cardiac function. We describe a case of moderate OSA with idiopathic dilated cardiomyopathy, in which the frequency of premature ventricular contraction (PVC) and non-sustained ventricular tachycardia (NSVT) increased immediately after initiating CPAP therapy. Although PVC and NSVT are benign cardiac arrhythmias, they are associated with an increased risk of sustained lethal ventricular tachyarrhythmias. Therefore, when initiating CPAP therapy, the possibility of increased arrhythmia should be considered.

Oral Adrenergic Agents Produced Ventricular Fibrillation and QT Prolongation in an Elderly Patient Carrying an RYR2 Variant.

Mutant cardiac ryanodine receptor channels (RyR2) are "leaky," and spontaneous Ca2+ release through these channels causes delayed afterdepolarizations that can deteriorate into ventricular fibrillation. Some patients carrying RYR2 mutations in type 1 catecholaminergic polymorphic ventricular tachycardia exhibit QT prolongation and are initially diagnosed with long QT syndrome. However, none have been reported to cause drug-induced ventricular fibrillation in patients with RYR2 variants. We describe the first case of an elderly woman with drug-induced QT prolongation and ventricular fibrillation who carried a novel RYR2 variant but no other mutations related to long QT syndrome. Oral adrenergic agents might induce QT prolongation and subsequent ventricular fibrillation in patients carrying an RYR2 variant. Screening for RYR2 could be valuable in patients with suspected drug-induced long QT syndrome.

Association Between Deleterious SCN5A Variants and Ventricular Septal Defect in Young Patients With Brugada Syndrome.

OBJECTIVES: This study aimed to investigate the clinical characteristics of young patients with Brugada syndrome (BrS) with ventricular septal defect (VSD) and explore their genetic backgrounds. BACKGROUND: VSD is the most frequently occurring congenital heart disease among children. In contrast, BrS is a rare hereditary disease that is responsible for ventricular fibrillation and sudden cardiac death. Owing to their low incidence, the genetic background and clinical characteristics of patients with BrS with VSD have not been elucidated yet. METHODS: This study enrolled 36 individuals who were diagnosed with BrS when they were <20 years of age and performed genetic screening for SCN5A. The functional alteration in mutant Na+ channels was confirmed by patch clamp technique. RESULTS: Among the 36 patients with BrS, 5 had been diagnosed with VSD. This study found 14 heterozygous SCN5A variants in 15 unrelated patients. The 5 patients with VSD carried SCN5A variants, including R367S, R535∗, R893C, W1345C, and G1743R. The 3 missense variants (R893C, W1345C, and G1743R) have been proved to reduce peak Na+ current to <10%. A functional analysis of SCN5A R367S was performed and the variant was found to be nonfunctional. CONCLUSIONS: This study identified 5 loss-of-function SCN5A variants in 5 young patients with BrS with VSD. The study hypothesizes that altered blood flow in the right ventricular outflow tract leads to fibrosis and electrophysiological changes, predisposing the patients to earlier clinical presentation of BrS. In patients with VSD and ST-segment elevation in the right precordial leads, BrS should be considered and appropriate screening should be pursued accordingly.

Impact of cascade screening for catecholaminergic polymorphic ventricular tachycardia type 1.

OBJECTIVE: Human cardiac ryanodine receptor 2 (RYR2) shows autosomal-dominant inheritance in catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1); however, de novo variants have been observed in sporadic cases. Here, we investigated CPVT1-related RYR2 variant inheritance and its clinical significance between familial and de novo cases. METHODS: We enrolled 82 independent CPVT1 probands (median age: 10.0 (7.0-13.0) years; 45 male) carrying the RYR2 variants and whose biological origin could be confirmed by parental genetic analysis: assured familial inheritance (familial group: n=24) and de novo variants (de novo group: n=58). We examined the clinical characteristics of the probands and their family members carrying the RYR2 variants. RESULTS: In the de novo group, the RYR2 variants were more likely located in the C-terminus domain and less likely in the N-terminus domain than those in the familial group. The cumulative incidence of the first cardiac events (syncope and cardiac arrest (CA) or CA only) of the probands at the age of 5 and 10 years was higher in the de novo group than in the familial group. Nearly half of the probands in both groups experienced CA events before diagnosis. Only 37.5% of their genotype-positive parents had symptoms; however, at least 66.7% of the genotype-positive siblings were symptomatic. CONCLUSIONS: CPVT1 probands harbouring de novo RYR2 variants showed an earlier onset of symptoms than those with assured familial inheritance. Cascade screening may enable early diagnosis, risk stratification and prophylactic therapeutic intervention to prevent sudden cardiac death of probands and potential genotype-positive family members.

School-based routine screenings of electrocardiograms for the diagnosis of long QT syndrome.

AIMS: School-based routine screenings of electrocardiograms (ECGs) have been performed upon admission to primary school (PS), junior high school (JHS), and high school (HS) in Japan. Though ECGs with prolonged QT intervals are occasionally found, the role of regular ECG screening tests in identifying long QT syndrome (LQTS) remains to be determined. We investigated the usefulness of the ECG screenings by comparing the results of genetic tests between students who showed QT-prolongation in the screenings and patients with LQTS. METHODS AND RESULTS: We genetically screened 341 students (106 PS, 173 JHS, and 62 HS). Of these, 230 subjects showed QT-prolongation during regular screenings (S-S group), and the other 111 patients were clinically consulted with suspected LQTS by paediatricians (C-C group). Genotype-phenotype relationships were compared between the two groups. The positive rates in the genetic tests were comparable among the two groups; however, symptomatic subjects were significantly fewer in the S-S group than the C-C group (3% vs. 70%). Compared to the genotype-negative subjects, the positive subjects showed significantly longer QTc (P < 0.0001) and more frequently presented LQTS risk scores with ≥3.5 points (P < 0.0001). Lethal arrhythmic events (LAE) occurred only in the C-C group; 18 subjects experienced LAE and 83% of them were found to carry variant(s) in the LQTS-related genes. CONCLUSION: The school-based ECG screenings are effective in identifying young patients with LQTS who require genetic analysis. If individuals are screened at a younger age, we can identify patients at risk earlier and provide preventative treatments.

Loss-of-function mutations in cardiac ryanodine receptor channel cause various types of arrhythmias including long QT syndrome.

AIMS: Gain-of-function mutations in RYR2, encoding the cardiac ryanodine receptor channel (RyR2), cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Whereas, genotype-phenotype correlations of loss-of-function mutations remains unknown, due to a small number of analysed mutations. In this study, we aimed to investigate their genotype-phenotype correlations in patients with loss-of-function RYR2 mutations. METHODS AND RESULTS: We performed targeted gene sequencing for 710 probands younger than 16-year-old with inherited primary arrhythmia syndromes (IPAS). RYR2 mutations were identified in 63 probands, and 3 probands displayed clinical features different from CPVT. A proband with p.E4146D developed ventricular fibrillation (VF) and QT prolongation whereas that with p.S4168P showed QT prolongation and bradycardia. Another proband with p.S4938F showed short-coupled variant of torsade de pointes (scTdP). To evaluate the functional alterations in these three mutant RyR2s and p.K4594Q previously reported in a long QT syndrome (LQTS), we measured Ca2+ signals in HEK293 cells and HL-1 cardiomyocytes as well as Ca2+-dependent [3H]ryanodine binding. All mutant RyR2s demonstrated a reduced Ca2+ release, an increased endoplasmic reticulum Ca2+, and a reduced [3H]ryanodine binding, indicating loss-of-functions. In HL-1 cells, the exogenous expression of S4168P and K4594Q reduced amplitude of Ca2+ transients without inducing Ca2+ waves, whereas that of E4146D and S4938F evoked frequent localized Ca2+ waves. CONCLUSION: Loss-of-function RYR2 mutations may be implicated in various types of arrhythmias including LQTS, VF, and scTdP, depending on alteration of the channel activity. Search of RYR2 mutations in IPAS patients clinically different from CPVT will be a useful strategy to effectively discover loss-of-function RYR2 mutations.