Suguru Nishiuchi

Allele-specific ablation rescues electrophysiological abnormalities in a human iPS cell model of long-QT syndrome with a CALM2 mutation

&NA; Calmodulin is a ubiquitous Ca2+ sensor molecule encoded by three distinct calmodulin genes, CALM1‐3. Recently, mutations in CALM1‐3 have been reported to be associated with severe early‐onset long‐QT syndrome (LQTS). However, the underlying mechanism through which heterozygous calmodulin mutations lead to severe LQTS remains unknown, particularly in human cardiomyocytes. We aimed to establish an LQTS disease model associated with a CALM2 mutation (LQT15) using human induced pluripotent stem cells (hiPSCs) and to assess mutant allele‐specific ablation by genome editing for the treatment of LQT15. We generated LQT15‐hiPSCs from a 12‐year‐old boy with LQTS carrying a CALM2‐N98S mutation and differentiated these hiPSCs into cardiomyocytes (LQT15‐hiPSC‐CMs). Action potentials (APs) and L‐type Ca2+ channel (LTCC) currents in hiPSC‐CMs were analyzed by the patch‐clamp technique and compared with those of healthy controls. Furthermore, we performed mutant allele‐specific knockout using a CRISPR‐Cas9 system and analyzed electrophysiological properties. Electrophysiological analyses revealed that LQT15‐hiPSC‐CMs exhibited significantly lower beating rates, prolonged AP durations, and impaired inactivation of LTCC currents compared with control cells, consistent with clinical phenotypes. Notably, ablation of the mutant allele rescued the electrophysiological abnormalities of LQT15‐hiPSC‐CMs, indicating that the mutant allele caused dominant‐negative suppression of LTCC inactivation, resulting in prolonged AP duration. We successfully recapitulated the disease phenotypes of LQT15 and revealed that inactivation of LTCC currents was impaired in CALM2‐N98S hiPSC model. Additionally, allele‐specific ablation using the latest genome‐editing technology provided important insights into a promising therapeutic approach for inherited cardiac diseases.

Patient-specific Human Induced Pluripotent Stem Cell Model Assessed with Electrical Pacing Validates S107 as a Potential Therapeutic Agent for Catecholaminergic Polymorphic Ventricular Tachycardia

Introduction Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. However, it is not invariably successful to recapitulate the disease phenotype because of the immaturity of hiPSC-derived cardiomyocytes (hiPSC-CMs). The purpose of this study was to establish and analyze iPSC-based model of catecholaminergic polymorphic ventricular tachycardia (CPVT), which is characterized by adrenergically mediated lethal arrhythmias, more precisely using electrical pacing that could promote the development of new pharmacotherapies. Method and Results We generated hiPSCs from a 37-year-old CPVT patient and differentiated them into cardiomyocytes. Under spontaneous beating conditions, no significant difference was found in the timing irregularity of spontaneous Ca2+ transients between control- and CPVT-hiPSC-CMs. Using Ca2+ imaging at 1 Hz electrical field stimulation, isoproterenol induced an abnormal diastolic Ca2+ increase more frequently in CPVT- than in control-hiPSC-CMs (control 12% vs. CPVT 43%, p<0.05). Action potential recordings of spontaneous beating hiPSC-CMs revealed no significant difference in the frequency of delayed afterdepolarizations (DADs) between control and CPVT cells. After isoproterenol application with pacing at 1 Hz, 87.5% of CPVT-hiPSC-CMs developed DADs, compared to 30% of control-hiPSC-CMs (p<0.05). Pre-incubation with 10 μM S107, which stabilizes the closed state of the ryanodine receptor 2, significantly decreased the percentage of CPVT-hiPSC-CMs presenting DADs to 25% (p<0.05). Conclusions We recapitulated the electrophysiological features of CPVT-derived hiPSC-CMs using electrical pacing. The development of DADs in the presence of isoproterenol was significantly suppressed by S107. Our model provides a promising platform to study disease mechanisms and screen drugs.

Optimal observation time after completion of circumferential pulmonary vein isolation for atrial fibrillation to prevent chronic pulmonary vein reconnections

PURPOSE To identify predictors of chronic pulmonary vein (PV) reconnection (CPVR) after successful circumferential PV isolation (CPVI) for atrial fibrillation (AF). MATERIALS AND METHODS A total of 718 PVs from 181 consecutive AF patients (141 males, median age 61 years, 92 paroxysmal AF) who underwent a second ablation procedure for recurrent AF were retrospectively analyzed. RESULTS During the second procedure, a CPVR was observed in 477 PVs (66.4%) among 169 patients. In a multiple logistic regression analysis, the observation time after the final completion of the PVI (OT-final) was a significant negative predictor (odds ratio 0.980; P<0.001). A receiver operating characteristic analysis demonstrated that the greatest area under the curve was for the OT-final (0.670). At an optimal cutoff of 35 min, the sensitivity and specificity for predicting a CPVR were 66.9% and 60.6%, respectively. By Kaplan Meier analysis, CPVR was more frequent in PVs with an OT-final of <35 min than ≥35 min (log-rank test, P=0.018). In a vessel-by-vessel analysis, the OT-final at all PV sites was a significant negative predictor, while male gender in the right PVs and left-inferior PV, number of RF applications for the ipsilateral CPVI in the right PVs and left-superior PV, and major PV diameter in the left-superior PV were significant positive predictors of a CPVR (all P<0.05). CONCLUSIONS An optimal observation time (≥35 min in this study) to determine whether PVI is successfully completed during the initial CPVI for AF may be needed to prevent a CPVR and subsequent AF recurrence thereafter.

Predictors of asymptomatic cerebral infarction associated with radiofrequency catheter ablation for atrial fibrillation using an irrigated-tip catheter.

AIMS Catheter ablation is a potentially curative treatment for atrial fibrillation (AF). However, complications such as ischaemic stroke are more frequent and more severe compared with ablation procedures for other arrhythmias. Irrigated-tip catheters have been reported to reduce the risk of stroke. The present study aimed to evaluate predictors of asymptomatic cerebral infarction (CI) after AF ablation using an irrigated-tip catheter. METHODS AND RESULTS A total of 70 consecutive AF patients who underwent catheter ablation were subjected to brain magnetic resonance imaging (MRI) 1 day after the procedure. In 10 (14.3%) of 70 patients, MRI revealed acute CI, but neither symptoms nor abnormal neurological findings were present in these patients. In univariate analysis, a history of persistent AF, left atrial dimension, presence of spontaneous echo contrast (SEC), procedure duration prior to heparin injection, and electrical cardioversion during the procedure differed significantly between the two groups, those with and without CI (P = 0.02, 0.05, 0.01, 0.01, and 0.05, respectively). Multivariate logistic regression analysis identified SEC [odds ratio (OR), 9.39; 1.60-55.2; P = 0.01] and procedure duration prior to heparin injection (OR, 1.19; 1.04-1.36; P = 0.01) as predictive of acute asymptomatic CI after AF ablation. CONCLUSION The presence of SEC and procedure duration prior to heparin injection are determinants of asymptomatic CI during AF ablation despite the use of an irrigated-tip catheter.

Cardiac sodium channel mutation associated with epinephrine-induced QT prolongation and sinus node dysfunction

BACKGROUND Long-QT syndrome (LQTS) is an inherited arrhythmia characterized by prolonged ventricular repolarization and malignant tachyarrhythmias. LQT1, LQT2, and LQT3 are caused by mutations in KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3), which account for approximately 90% of genotyped LQTS patients. Most cardiac events in LQT1 patients occur during exercise, whereas patients with LQT3 tend to have arrhythmic events during rest or asleep. OBJECTIVE The study aimed to identify a genetic mutation in a Japanese man who presented with sinus node dysfunction and prolonged QT interval on exercise and epinephrine stress tests, as well as to clarify the electrophysiological properties of mutant channels. METHODS LQTS-related genes were screened in this patient. Electrophysiological functional assays were conducted with a heterologous expression system. RESULTS We identified a heterozygous missense SCN5A mutation, V2016M, which changes the last amino acid of the cardiac sodium channel. Electrophysiological analyses revealed that the mutant channels exhibited a loss-of-function feature, decreased peak sodium current densities (wild type 175.2 ± 17.6 pA/pF; V2016M 97.2 ± 16.0 pA/pF; P < .01). In addition, the mutant channels showed gain-of-function features: increased late sodium currents by protein kinase A activation (wild type 0.07 ± 0.01%; V2016M 0.17 ± 0.03%; P < .05) and impaired inactivation of sodium channels by protein kinase A or C activation. CONCLUSION We identified an SCN5A mutation in a patient with sinus node dysfunction and epinephrine-induced QT prolongation, which was an atypical phenotype for LQT3. The electrophysiological properties of the mutant channels might be associated with the overlapping clinical features of the patient.

LMNA cardiomyopathy detected in Japanese arrhythmogenic right ventricular cardiomyopathy cohort

BACKGROUND Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disease. While desmosomal gene mutations are considered major causes of ARVC, LMNA mutations have been reported to be possible causes of ARVC. In this study, we performed extensive genetic screening for LMNA mutations in our Japanese ARVC cohort to assess the prevalence and characteristics of LMNA mutation-positive ARVC cases. METHODS Our study cohort consisted of 57 ARVC probands. Genetic analyses were performed by using direct sequencing and targeted sequencing of LMNA and four desmosomal genes. We compared clinical features of probands with desmosomal gene mutations to those of probands with LMNA mutations. RESULTS Among 57 clinically diagnosed ARVC probands, we identified desmosomal gene mutations in 26 probands (45.6%) and two LMNA mutations in two probands. The first LMNA mutation p.M1K was detected in a 62-year-old male proband, while the second mutation p.W514X was found in a 70-year-old male proband. Compared to the 26 probands with desmosomal gene mutations, in the two probands with LMNA mutations, the mean age at diagnosis was significantly higher, and their heart rate at the diagnosis was significantly slower. While both probands with LMNA mutations underwent pacemaker implantation, only one proband with desmosomal mutations received this treatment (2/2 vs. 1/26). CONCLUSION Genetic screening for LMNA gene is important for ARVC patients, particularly in patients with bradycardia.

Development of a Patient-Derived Induced Pluripotent Stem Cell Model for the Investigation of SCN5A-D1275N-Related Cardiac Sodium Channelopathy

BACKGROUND TheSCN5Agene encodes the α subunit of the cardiac voltage-gated sodium channel, NaV1.5. The missense mutation, D1275N, has been associated with a range of unusual phenotypes associated with reduced NaV1.5 function, including cardiac conduction disease and dilated cardiomyopathy. Curiously, the reported biophysical properties ofSCN5A-D1275N channels vary with experimental system.Methods and Results:First, using a human embryonic kidney (HEK) 293 cell-based heterologous expression system, theSCN5A-D1275N channels showed similar maximum sodium conductance but a significantly depolarizing shift of activation gate (+10 mV) compared to wild type. Second, we generated human-induced pluripotent stem cells (hiPSCs) from a 24-year-old female who carried heterozygousSCN5A-D1275N and analyzed the differentiated cardiomyocytes (CMs). AlthoughSCN5Atranscript levels were equivalent between D1275N and control hiPSC-CMs, both the total amount of NaV1.5 and the membrane fractions were reduced approximately half in the D1275N cells, which were rescued by the proteasome inhibitor MG132 treatment. Electrophysiological assays revealed that maximum sodium conductance was reduced to approximately half of that in control hiPSC-CMs in the D1275N cells, and maximum upstroke velocity of action potential was lower in D1275N, which was consistent with the reduced protein level of NaV1.5. CONCLUSIONS This study successfully demonstrated diminished sodium currents resulting from lower NaV1.5 protein levels, which is dependent on proteasomal degradation, using a hiPSC-based model forSCN5A-D1275N-related sodium channelopathy.

Gene-Based Risk Stratification for Cardiac Disorders in LMNA Mutation CarriersCLINICAL PERSPECTIVE

Background— Mutations in LMNA (lamin A/C), which encodes lamin A and C, typically cause age-dependent cardiac phenotypes, including dilated cardiomyopathy, cardiac conduction disturbance, atrial fibrillation, and malignant ventricular arrhythmias. Although the type of LMNA mutations have been reported to be associated with susceptibility to malignant ventricular arrhythmias, the gene-based risk stratification for cardiac complications remains unexplored. Methods and Results— The multicenter cohort included 77 LMNA mutation carriers from 45 families; cardiac disorders were retrospectively analyzed. The mean age of patients when they underwent genetic testing was 45±17, and they were followed for a median 49 months. Of the 77 carriers, 71 (92%) were phenotypically affected and showed cardiac conduction disturbance (81%), low left ventricular ejection fraction (<50%; 45%), atrial arrhythmias (58%), and malignant ventricular arrhythmias (26%). During the follow-up period, 9 (12%) died, either from end-stage heart failure (n=7) or suddenly (n=2). Genetic analysis showed truncation mutations in 58 patients from 31 families and missense mutations in 19 patients from 14 families. The onset of cardiac disorders indicated that subjects with truncation mutations had an earlier occurrence of cardiac conduction disturbance and low left ventricular ejection fraction, than those with missense mutations. In addition, the truncation mutation was found to be a risk factor for the early onset of cardiac conduction disturbance and the occurrence of atrial arrhythmias and low left ventricular ejection fraction, as estimated using multivariable analyses. Conclusions— The truncation mutations were associated with manifestation of cardiac phenotypes in LMNA-related cardiomyopathy, suggesting that genetic analysis might be useful for diagnosis and risk stratification.

Subacute aortic regurgitation due to traumatic tear in the aortic wall

A 37-year-old man presented with heart failure caused by severe aortic regurgitation (AR). He had a history of being involved in a traffic accident 3 months earlier. Imaging tests at admission detected no abnormalities in the aortic valve or aortic wall; however, the left coronary cusp prolapsed slightly on transthoracic echocardiography. He underwent aortic valve replacement because of uncontrolled heart failure and severe AR. Intraoperatively, the intima of the aortic wall just above the commissure of the left and right coronary cusps was torn to the short axial direction. Local aortic tear was the final diagnosis for the subacute AR. <Learning objective: Acute or subacute aortic regurgitation (AR) is comparatively rare, and it is sometimes difficult to clinically recognize. The tear in the aortic wall just above the commissure caused by a traffic accident led to the gradual progression of AR, and the diagnosis of the cause of AR was difficult despite using transesophageal echocardiography and contrast-enhanced computed tomography. We should recognize that the detection of subacute AR caused by a local aortic tear can be challenging.>.

A potential pitfall of the modified 12 lead electrocardiogram (Mason–Likar modification) in catheter ablation of idiopathic ventricular arrhythmias originating from the outflow tract

AIMS The Mason-Likar modified electrocardiogram (ML-ECG) can be interchanged with standard 12 lead ECG electrode positions (standard ECG) without affecting the diagnostic interpretation during sinus rhythm, but the morphological differences during ventricular arrhythmias have not been sufficiently evaluated. This study aimed to elucidate the morphological changes in the ML-ECG precordial leads. METHODS AND RESULTS In 53 consecutive patients with premature ventricular contractions predicted to originate from the outflow tract (OT-PVCs), the arrhythmias were analysed by those two ECG methods. The OT-PVC origin sites, which were predicted by currently published criteria with the respective ECG methods prior to catheter ablation, were compared with the successful ablation sites. Compared with the standard-ECG, S-waves in the ML-ECG became shallower in leads V1-4 (P < 0.05 in lead V1; P < 0.001 in leads V2-4), and pseudo-R-waves in lead V1 appeared in seven patients. The precordial leads transition zone shifted counter-clockwise in 18 patients in the ML-ECG. In leads I and aVL, the negative deflection amplitudes of the ML-ECG were greater than those of the standard ECG (P < 0.001), and polarity reversals in lead I appeared in 18 patients. The R-wave amplitudes in all ML-ECG inferior leads were greater than those in the standard-ECG leads (all for P < 0.001). Those changes had an effect on the diagnostic indexes for the localization, and the specificity of the criteria for the ML-ECG was poorer than that for the standard-ECG. CONCLUSION Great differences were found between those two ECG methods. Predicting OT-PVC origins by diagnostic criteria with the ML-ECG might result in a misdiagnosis and inefficient ablation.