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Abstract Aims Cardiac resynchronization therapy (CRT) is an established treatment for drug‐refractory heart failure (HF) in patients with left bundle branch block (LBBB). Acute haemodynamic improvement after CRT implantation may enable the intensification of HF medication soon thereafter. Immediate pharmacotherapy intensification (IPI) after CRT implantation achieves a synergetic effect, possibly leading to a better prognosis. This study aimed to explore the incidence, characteristics, and impact of IPI on real‐world outcomes among CRT recipients with a history of hospitalization for acute HF. Methods and results This multicentre retrospective study enrolled CRT recipients with LBBB morphology, a QRS width ≥120 ms, a left ventricular ejection fraction ≤35%, and New York Heart Association II–IV HF symptoms. All patients had previous HF hospitalizations within the previous year and received guideline‐directed medical therapy before CRT implantation. Patient baseline characteristics, including HF medication, were collected. IPI was defined as the intensification of beta‐blockers, angiotensin‐converting enzyme inhibitors, angiotensin receptor blockers, and mineralocorticoid receptor antagonists within 30 days of CRT implantation. The primary endpoint was all‐cause death or first hospitalization for HF; the secondary endpoint was all‐cause death. We enrolled 194 patients (75% male; mean age, 65 ± 13 years; 78% with non‐ischaemic cardiomyopathy). One hundred five (54%) patients received IPI. Patients who received IPI exhibited a significantly shorter QRS duration (159 ± 26 vs. 171 ± 32 ms; P = 0.004), higher estimated glomerular filtration rate (55.2 ± 20.0 vs. 47.8 ± 24.7 mL/min/1.73 m2; P = 0.022), and more dilated cardiomyopathy. During a median follow‐up period of 29 months, 70 (36%) patients reached the primary endpoint and 42 (22%) patients died. Patients with IPI showed significantly better outcomes for the primary and secondary endpoints than patients without IPI. The volumetric responder ratio at 6 months after implantation was not significantly different between patients with and without IPI; however, patients who received IPI had reduced mortality even at 6 months after implantation. In the multivariate analysis, IPI was an independent predictor of the primary endpoint (hazard ratio, 0.51; 95% confidence interval, 0.27–0.97; P = 0.043). Conclusions Immediate intensification of HF medication was achieved in 54% of CRT recipients and was significantly higher in patients without excessive QRS prolongation, preserved renal function, and dilated cardiomyopathy than others. In patients with LBBB morphology and QRS ≥ 120 ms, IPI was associated with a significantly better prognosis and fewer HF hospitalizations after CRT implantation than others.

Abstract Hypertrophic cardiomyopathy (HCM) is an inherited disorder characterized by left ventricular hypertrophy and diastolic dysfunction, and increases the risk of arrhythmias and heart failure. Some patients with HCM develop a dilated phase of hypertrophic cardiomyopathy (D-HCM) and have poor prognosis; however, its pathogenesis is unclear and few pathological models exist. This study established disease-specific human induced pluripotent stem cells (iPSCs) from a patient with D-HCM harboring a mutation in MYBPC3 (c.1377delC), a common causative gene of HCM, and investigated the associated pathophysiological mechanisms using disease-specific iPSC-derived cardiomyocytes (iPSC-CMs). We confirmed the expression of pluripotent markers and the ability to differentiate into three germ layers in D-HCM patient-derived iPSCs (D-HCM iPSCs). D-HCM iPSC-CMs exhibited disrupted myocardial sarcomere structures and an increased number of damaged mitochondria. Ca2+ imaging showed increased abnormal Ca2+ signaling and prolonged decay time in D-HCM iPSC-CMs. Cell metabolic analysis revealed increased basal respiration, maximal respiration, and spare-respiratory capacity in D-HCM iPSC-CMs. RNA sequencing also showed an increased expression of mitochondrial electron transport system-related genes. D-HCM iPSC-CMs showed abnormal Ca2+ handling and hypermetabolic state, similar to that previously reported for HCM patient-derived iPSC-CMs. Although further studies are required, this is expected to be a useful pathological model for D-HCM.

Abstract Images obtained from single-photon emission computed tomography for myocardial perfusion imaging (MPI SPECT) contain noises and artifacts, making cardiovascular disease diagnosis difficult. We developed a deep learning-based diagnosis support system using MPI SPECT images. Single-center datasets of MPI SPECT images (n = 5443) were obtained and labeled as healthy or coronary artery disease based on diagnosis reports. Three axes of four-dimensional datasets, resting, and stress conditions of three-dimensional reconstruction data, were reconstructed, and an AI model was trained to classify them. The trained convolutional neural network showed high performance [area under the curve (AUC) of the ROC curve: approximately 0.91; area under the recall precision curve: 0.87]. Additionally, using unsupervised learning and the Grad-CAM method, diseased lesions were successfully visualized. The AI-based automated diagnosis system had the highest performance (88%), followed by cardiologists with AI-guided diagnosis (80%) and cardiologists alone (65%). Furthermore, diagnosis time was shorter for AI-guided diagnosis (12 min) than for cardiologists alone (31 min). Our high-quality deep learning-based diagnosis support system may benefit cardiologists by improving diagnostic accuracy and reducing working hours.

Background: Owing to the minimally invasive nature of transcatheter aortic valve replacement (TAVR), TAVR seems to be preferred in patients with cancer; however, related research on the clinical efficacy and safety of TAVR in patients with cancer and severe aortic stenosis is limited, and conclusions are controversial. This study aimed to evaluate the clinical outcomes of patients with cancer who underwent TAVR. Method and results: We conducted a systematic review and meta-analysis to investigate the clinical outcomes in patients with and without cancer who underwent TAVR. We systematically reviewed and analyzed 15 studies (195,658 patients) published in PubMed and Cochrane Library databases between January 2022 and January 2023. The primary outcomes were short-term (in-hospital or 30-day) and long-term (≥12 months) mortality. The prevalence of current or previous cancer in the patients undergoing TAVR was 19.8 % (38,695 patients). Patients with cancer had a lower risk of short-term mortality (odds ratio [OR] 0.69, 95 % confidence interval [CI] 0.61–0.77, P

Background There are few data on sex differences in the association between schizophrenia and the development of cardiovascular disease (CVD). We sought to clarify the relationship of schizophrenia with the risk of developing CVDs and to explore the potential modification effect of sex differences. Methods and Results We conducted a retrospective analysis using the JMDC Claims Database between 2005 and 2022. The study population included 4 124 508 individuals aged 18 to 75 years without a history of CVD or renal replacement therapy. The primary end point is defined as a composite end point that includes myocardial infarction, angina pectoris, stroke, heart failure, atrial fibrillation, and pulmonary thromboembolism. During a mean follow‐up of 1288±1001 days, we observed 182 158 composite end points. We found a significant relationship of schizophrenia with a greater risk of developing composite CVD events in both men and women, with a stronger association observed in women. The hazard ratio for the composite end point was 1.63 (95% CI, 1.52–1.74) in women and 1.42 (95% CI, 1.33–1.52) in men after multivariable adjustment (P for interaction=0.0049). This sex‐specific difference in the association between schizophrenia and incident CVD was consistent for angina pectoris, heart failure, and atrial fibrillation. Conclusions Our analysis using a large‐scale epidemiologic cohort demonstrated that the association between schizophrenia and subsequent CVD events was more pronounced in women than in men, suggesting the clinical importance of addressing schizophrenia and tailoring the CVD prevention strategy based on sex‐specific factors.

A 24-year-old woman with chronic active Epstein-Barr virus (CAEBV) infection successfully underwent coronary artery bypass grafting for triple coronary arteries with chronic total occlusion and aneurysms. This case underscores the importance of accurate assessment and treatment of coronary artery lesions in patients with CAEBV infection.

Abstract Although glucose metabolism and atrial fibrillation (AF) have complex interrelationships, the impact of catheter ablation of AF on glucose status has not been well evaluated. Continuous glucose monitoring (CGM) with a FreeStyle Libre Pro (Abbott) was performed for 48 h pre-procedure, during the procedure, and for 72 h post-procedure in 58 non-diabetes mellitus (DM) patients with symptomatic AF and 20 patients with supraventricular or ventricular arrhythmias as a control group. All ablation procedures including pulmonary vein isolation were performed successfully. Glucose levels during procedures consistently increased in the AF and control groups (83.1 ± 16.1 to 110.0 ± 20.5 mg/dL and 83.3 ± 14.7 to 98.6 ± 16.3 mg/dL, respectively, P

BackgroundRecent studies suggested a relationship between Takotsubo syndrome (TTS) and malignancy. However, clinical outcomes of TTS associated with cancer have not been assessed completely. This study was aimed to investigate the outcomes of patients with TTS and cancer.MethodsWe performed a systematic review and meta-analysis to evaluate the clinical outcomes of TTS in patients with and without malignancy. We systematically reviewed and analyzed 14 studies (189,210 patients) published in PubMed and Cochrane Library databases until December 2022. The primary outcome was all-cause mortality at the longest follow-up.ResultsThe prevalence of current or previous malignancy in patients with TTS was 8.7% (16,461 patients). Patients with TTS and malignancy demonstrated a higher risk of mortality at the longest follow-up than those with TTS alone (odds ratio [OR], 2.41; 95% confidence interval [CI]; 1.95–2.98; P

Background Prognostic implications of transcatheter aortic valve implantation (TAVI) in low‐gradient (LG) aortic stenosis (AS) remain controversial. The authors hypothesized that differences in cardiac functional recovery may solve this ongoing controversy. The aim was to evaluate clinical outcomes and the response of left ventricular (LV) function following TAVI in patients with LG AS. Methods and Results This multicenter retrospective study included 1742 patients with severe AS undergoing TAVI between January 2015 and March 2019. Patients were subdivided into low‐flow (LF) LG, normal‐flow (NF) LG, LF high‐gradient, and NF high‐gradient AS groups according to the mean gradient of the aortic valve (LG

Summary: Myoblast determination protein 1 (MyoD) dynamics define the activation status of muscle stem cells (MuSCs), aiding in muscle tissue regeneration after injury. However, the lack of experimental platforms to monitor MyoD dynamics in vitro and in vivo has hampered the investigation of fate determination and heterogeneity of MuSCs. Herein, we report a MyoD knock-in (MyoD-KI) reporter mouse expressing tdTomato at the endogenous MyoD locus. Expression of tdTomato in MyoD-KI mice recapitulated the endogenous MyoD expression dynamics in vitro and during the early phase of regeneration in vivo. Additionally, we showed that tdTomato fluorescence intensity defines MuSC activation status without immunostaining. Based on these features, we developed a high-throughput screening system to assess the effects of drugs on the behavior of MuSCs in vitro. Thus, MyoD-KI mice are an invaluable resource for studying the dynamics of MuSCs, including their fate decisions and heterogeneity, and for drug screening in stem cell therapy.

Background: Catheter ablation (CA) benefits atrial fibrillation (AF) patients with heart failure (HF). Brain natriuretic peptide (BNP), a marker of left-ventricular pressure load, may serve as a potential surrogate for predicting quality of life (QOL) in a broader range of patients. Methods: Within the multicenter KiCS-AF registry, 491 AF patients underwent CA without clinical HF (e.g., documented history of HF, left ventricular ejection fraction ≤ 40%, or BNP levels ≥ 100 pg/mL). Participants, aged 61 ± 10 years, were categorized by baseline BNP quartiles. Atrial Fibrillation Effect on QualiTy-of-Life (AFEQT) questionnaire assessments were assessed at baseline and 1 year. Results: A lower baseline BNP correlated with reduced AFEQT scores. Post CA, all groups showed significant AFEQT score improvements. The lower-BNP group displayed notable enhancements (18.2 ± 1.2, 15.0 ± 1.1, 12.6 ± 1.2, 13.6 ± 1.2, p < 0.005), especially in symptom and treatment concern areas. Even those with normal BNP levels (≤18.4 pg/mL) exhibited significant QOL improvements. Comparing paroxysmal AF (PAF) and non-PAF groups, the PAF group, especially with higher BNP levels, showed greater AFEQT score improvements. Conclusions: This study establishes BNP as a predictive marker for QOL enhancement in non-HF patients undergoing CA for AF. BNP levels represent AF stages, with individuals in earlier stages, especially within normal BNP levels, experiencing greater QOL improvements.

Abstract Amiodarone and its main metabolite, desethylamiodarone (DEA), are highly distributed to serum lipoproteins such as very‐low‐density lipoprotein (VLDL) and low‐density lipoprotein (LDL), which are the carriers of triglyceride and cholesterol. This study aimed to investigate the association of serum concentrations of amiodarone and DEA with the levels of serum lipids in terms of drug distribution to lipoprotein fractions in patients with hyperlipidemia. Total serum concentrations of amiodarone and DEA were examined in 116 patients receiving amiodarone for tachyarrhythmias. The concentration‐to‐dose (C/D) ratio of amiodarone positively correlated with the level of serum triglyceride (rs = 0.541, p

BackgroundAtrial fibrillation (AF) is the most common arrhythmia and often recurs despite catheter ablation. The recurrence of AF is often underdiagnosed by standard 24-hour electrocardiogram (ECG) because of its transient and silent nature. A garment-style ECG with a highly conductive textile electrode made of poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate)(PEDOTPSS) and nanofiber (Garment ECG) has been developed that can provide longer-term continuous monitoring. This study investigated whether 2-week Garment ECG can reveal instances of AF recurrence in patients who are diagnosed as remaining in sinus rhythm by 24-hour Holter ECG.MethodsThe open-label randomized crossover study enrolled 67 patients (63.1±10.6 years old, 53 men) who had undergone initial AF ablation. Three months after ablation, patients were randomly assigned to group 1 (n = 35), 2-week Garment ECG followed by 24-hour Holter ECG, or group 2 (n = 32), 24-hour Holter ECG followed by 2-week Garment ECG. The detection of AF recurrence was compared between the two devices.ResultsThe Garment ECG showed AF recurrence in 12 patients (18%) compared to 4 patients for the Holter ECG (6%, p = 0.008). The ECG acquisition rate was higher for Holter ECG than for Garment ECG (100.0% [interquartile range 100.0-100.0%] versus 82.4% [71.1-91.0%], pConclusionsDespite the lower ECG acquisition rate, the 2-week Garment ECG revealed instances of AF recurrence after ablation in patients who were underdiagnosed by 24-hour Holter ECG.Trial registrationClinical Trial Registration: URL: https://jrct.niph.go.jp/en-latest-detail/jRCTs032180018 Unique Identifier: jRCTs032180018.

BackgroundThere is growing evidence indicating a close relationship between inflammation and atrial fibrillation (AF). Although underlying inflammatory atrial cardiomyopathy may contribute to the development of AF, the arrhythmogenic remodeling caused by atrial inflammation has not been elucidated in detail. Herein, we examined electrical, structural, and autonomic changes in the atria in a mouse model of autoimmune myocarditis.MethodsBALB/c mice were immunized with cardiac myosin peptide (MyHC-α614–629) conjugated with complete Freund’s adjuvant on days 0 and 7. Susceptibility to AF was assessed using right-atrial burst pacing.ResultsThe mice immunized with MyHC-α614–629 showed an inflammatory atrial cardiomyopathy phenotype, with enlarged atria; a high degree of inflammatory cell infiltration primarily consisting of CD4+ T cells, CD8+ T cells, Ly6GlowCD11b+ macrophages, and CD11c+ dendritic cells; and severe interstitial fibrosis with collagen deposition. These mice demonstrated significantly enhanced susceptibility to AF, as indicated by their increased AF induction rate and duration. In addition, the expression of potassium channels (Kcnh2, Kcnd3, and Kcnj2) and calcium handling-associated genes (Cacna1c, Camk2, Ryr2, and Atp2a2) was downregulated. Connexin 40 expression was significantly downregulated, leading to frequent lateralization to the inflamed atrium. Sympathetic and parasympathetic innervation and neurotrophin expression (nerve growth factor and brain-derived neurotrophic factor) were upregulated in the inflamed atria.ConclusionInflammatory atrial cardiomyopathy promotes susceptibility to AF via arrhythmogenic electrical, structural, and autonomic remodeling of the atria.

Abstract Background Nilotinib, a second-generation BCR-ABL tyrosine kinase inhibitor (TKI), is highly effective in the treatment of patients with chronic myeloid leukemia (CML), despite being more vasculotoxic than older TKIs such as imatinib. Herein, we present a case of nilotinib-associated vasospastic angina confirmed by an acetylcholine spasm provocation test. Case presentation A 62-year-old CML patient treated with 300 mg nilotinib twice daily complained of several episodes of rest angina and was hospitalized at our institution. Coronary angiography revealed no severe organic stenosis, and the acetylcholine spasm provocation test confirmed the diagnosis of vasospastic angina. Although treatment with a calcium channel blocker and nicorandil reduced the frequency of chest pain, angina symptoms continued to occur. At 10 months post discharge, the patient complained of increased frequency of angina; therefore, the nilotinib dosage was reduced to 150 mg twice daily. Consequently, the patient reported a significant improvement in chest symptoms. Conclusions This case report highlights the potential vasculotoxic effects of nilotinib. Cardiologists and hematologists should be vigilant for coronary artery spasm as a possible vascular adverse event caused by nilotinib.

Abstract Japan faces an increasing incidence of heart disease, owing to a shift towards a westernized lifestyle and an aging demographic. In cases where conventional interventions are not appropriate, regenerative medicine offers a promising therapeutic option. However, the use of stem cells has limitations, and therefore, “direct cardiac reprogramming” is emerging as an alternative treatment. Myocardial regeneration transdifferentiates cardiac fibroblasts into cardiomyocytes in situ. Three cardiogenic transcription factors: Gata4, Mef2c, and Tbx5 (GMT) can induce direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs), in mice. However, in humans, additional factors, such as Mesp1 and Myocd, are required. Inflammation and immune responses hinder the reprogramming process in mice, and epigenetic modifiers such as TET1 are involved in direct cardiac reprogramming in humans. The three main approaches to improving reprogramming efficiency are (1) improving direct cardiac reprogramming factors, (2) improving cell culture conditions, and (3) regulating epigenetic factors. miR-133 is a potential candidate for the first approach. For the second approach, inhibitors of TGF-β and Wnt signals, Akt1 overexpression, Notch signaling pathway inhibitors, such as DAPT ((S)-tert-butyl 2-((S)-2-(2-(3,5-difluorophenyl) acetamido) propanamido)-2-phenylacetate), fibroblast growth factor (FGF)-2, FGF-10, and vascular endothelial growth factor (VEGF: FFV) can influence reprogramming. Reducing the expression of Bmi1, which regulates the mono-ubiquitination of histone H2A, alters histone modification, and subsequently the reprogramming efficiency, in the third approach. In addition, diclofenac, a non-steroidal anti-inflammatory drug, and high level of Mef2c overexpression could improve direct cardiac reprogramming. Direct cardiac reprogramming needs improvement if it is to be used in humans, and the molecular mechanisms involved remain largely elusive. Further advances in cardiac reprogramming research are needed to bring us closer to cardiac regenerative therapy.

Background In the population with cardiac sarcoidosis (CS), approximately one third lacks extracardiac involvement and is considered to have isolated CS. Recently, the Japanese Circulation Society updated the diagnostic criteria for CS, providing a methodology for diagnosing isolated CS. We aimed to assess the characteristics of isolated CS diagnosed using a multimodal imaging approach according to the updated Japanese Circulation Society guidelines. Methods and Results We retrospectively identified 161 consecutive patients who underwent 18F‐fluorodeoxyglucose positron emission tomography for suspected CS between 2012 and 2019. According to the guidelines, patients were classified as having CS with extracardiac involvement, isolated CS, or no CS. We compared the characteristics of multimodality imaging and the prevalence of major adverse cardiovascular events. The Japanese Circulation Society criteria classified 28 patients (17%) as having CS with 4 (2%) with histological confirmation, 21 (13%) as isolated CS, and 112 (70%) as no CS. Compared with CS, isolated CS showed higher left ventricular volume and reduced left ventricular ejection fraction (P

Dilated cardiomyopathy (DCM) is a refractory heart disease characterized by dilation of the left ventricle and systolic dysfunction. LMNA, the gene encoding lamin A/C (a nuclear envelope protein), is the second leading causative gene associated with familial DCM. LMNA-related DCM is likely to develop severe heart failure, various types of arrhythmias, and poor prognosis. We established a human induced pluripotent stem cell line, derived from a patient with DCM carrying a nonsense mutation in LMNA. This line should be a useful resource for elucidating disease mechanisms and developing fundamental treatments for LMNA-related DCM.

Background: Holter electrocardiogram (ECG) is the gold standard for ambulatory monitoring of atrial fibrillation (AF) but it is insufficient because of its limited recording time. Although several consumer ECG devices provide longer recording time, they generally do not undergo the regulatory process for medical use. Furthermore, current medical-grade devices for longer ECG monitoring are not continuous or too invasive for AF monitoring. A wearable ECG with a medical-grade dry textile electrode is a promising technology to remedy this limitation. This pilot study aimed to simultaneously compare the wearable and Holter ECGs for ambulatory monitoring in a clinical setting. Methods: This prospective observational study enrolled 18 patients who underwent AF ablation. One day after AF ablation, ambulatory ECG was obtained for three hours simultaneously using both the wearable and Holter ECG devices. Automatic ECG interpretations between devices were compared with correlation and agreement analyses. Results: Simultaneous ECG monitoring demonstrated a comparable analysis time and total heart beats between the two devices. Almost complete correlation and agreement were also demonstrated in all clinically relevant testing aspects except in R-wave amplitude (r = 0.743, p < .001). AF was detected in three patients. AF duration was the same in both ECG devices in two patients with continuous AF. In the remaining patient with intermittent AF, AF duration was shortened by 0.6% with the wearable ECG as compared to that with the Holter ECG. Conclusions: Simultaneous ECG comparison revealed a high consistency between the wearable and Holter ECG devices. The results of this study warrant further clinical studies for long-term monitoring of ambulatory ECG after AF ablation.

Background: Immune checkpoint inhibitor (ICI)-related myositis with myocarditis is a rare but potentially fatal immune-related adverse event. However, its clinical features, response to immunosuppressive treatment, and prognosis remain poorly understood. Here, we describe the clinical course of patients with ICI-related myositis overlapping with myocarditis treated at our institution and a systematic review focusing on the response to immunosuppressive therapy.Methods: We identified patients who developed ICI-induced myositis with myocarditis and were treated at our hospital using a retrospective chart review of electronic medical records. For the systematic review, studies reporting ICI-induced myositis with myocarditis were identified using the Cochrane Library and PubMed databases.Results: Of the 625 patients treated with ICIs, four developed myositis with concurrent myocarditis. All the patients received immunosuppressive therapy. We assessed the activity of myocarditis and myositis based on temporal changes in troponin and creatine kinase (CK) levels. In all patients, peak troponin values appeared later than the peak CK values (median, 17 days). The median time from the start of ICI therapy to the peak of troponin and CK levels was 42.5 and 28 days, respectively. In all patients, CK levels decreased rapidly and steadily after the initiation of immunosuppressants. However, troponin levels were unstable and increased. In all patients, CK levels normalized within one month (range, 12–27 days), but troponin levels took several months to normalize (range, 84–161 days). Fourteen cases of ICI-related myositis with myocarditis were included in the systematic review. Of the 14 cases, 12 (86%) had their CK level decreased after the initial steroid treatment, but the troponin level increased and was higher than that before the start of treatment. In addition, the peak troponin values appeared later than the peak CK values (a median of 6.5 days). Eight (89%) of 9 long-term follow-up patients had troponin levels above the normal range even after CK normalization.Conclusion: In most cases of ICI-related myositis with myocarditis, troponin levels increased after the initial steroid treatment despite decreased CK levels, and exceeded pre-steroid levels. In addition, troponin remained elevated for several months after CK normalized.

Abstract In deep burns, early wound closure is important for healing, and skin grafting is mainly used for wound closure. However, it is difficult to achieve early wound closure in extensive total body surface area deep burns due to the lack of donor sites. Dermal fibroblasts, responsible for dermis formation, may be lost in deep burns. However, fat layers composed of adipocytes, lying underneath the dermis, are retained even in such cases. Direct reprogramming is a novel method for directly reprograming some cells into other types by introducing specific master regulators; it has exhibited appreciable success in various fields. In this study, we aimed to assess whether the transfection of master regulators (ELF4, FOXC2, FOXO1, IRF1, PRRX1, and ZEB1) could reprogram mouse adipocytes into dermal fibroblast-like cells. Our results indicated the shrinkage of fat droplets in reprogrammed mouse adipocytes and their transformation into spindle-shaped dermal fibroblasts. Reduced expression of PPAR-2, c/EBP, aP2, and leptin, the known markers of adipocytes, in RT-PCR, and enhanced expression of anti-ER-TR7, the known anti-fibroblast marker, in immunocytochemistry, were confirmed in the reprogrammed mouse adipocytes. The dermal fibroblast-like cells, reported here, may open up a new treatment mode for enabling early closure of deep burn wounds.

Summary: Direct cardiac reprogramming, in which fibroblasts are converted into induced cardiomyocytes (iCMs) with cardiogenic transcription factors, may be a promising approach for myocardial regeneration. Here, we present a protocol for cardiac reprogramming using a handmade hydrogel culture system. This system can recapitulate substrate stiffness comparable to that of the native myocardium. This protocol features improved efficiency of cardiac reprogramming by generating threefold more beating iCMs on the Matrigel-based hydrogel culture system compared to that on conventional polystyrene dishes.For complete details on the use and execution of this protocol, please refer to Kurotsu et al. (2020)

Abstract Atypical aortic coarctation is a rare condition associated with Takayasu arteritis, and is characterized by symptoms caused either by hypotension in the lower half of the body or secondary hypertension in the upper half of the body, and heart failure. We report a rare case of axillofemoral bypass to improve congestive heart failure for atypical aortic coarctation complicating Takayasu arteritis. Augmented vascular bed and retrograde renal blood flow after axillofemoral bypass surgery could achieve effective blood pressure control and improve renal function and cardiac function (LVEF: 30% → 55%, BNP: 2943 pg/mL → 128 pg/mL). There were two contributing factors for improvement of heart failure such as the increased vascular bed and the increase in retrograde renal blood flow. We believe that axillofemoral bypass is effective for Takayasu arteritis patients with refractory heart failure. In daily practice, careful attention should be paid to an impact of cardiorenal‐aorta interaction in atypical aortic coarctation complicating Takayasu arteritis.

Background: Ablation of the pulmonary vein (PV) carina is occasionally required for PV isolation (PVI). Marshall bundle and epicardial connections between the right-sided PV (RtPV) carina and right atrium (RA) may be one of the mechanisms that necessitates carina ablation. Objective: We sought to clarify anatomical characteristics predictive of the necessity of carina ablation. Methods: Forty-five consecutive patients undergoing radiofrequency catheter ablation of atrial fibrillation were prospectively included in this study. Left atrial (LA) and PV size and morphology, and interatrial distance in the posterior aspect, were measured on cardiac computed tomography (CT) images. Results: For right-sided PVI, the patients were divided into 2 groups based on the necessity of RtPV carina ablation, Carina-ABL group (n = 21) and Non-Carina-ABL group (n = 24). The distance between the anterior portion of the RtPV carina and RA was shorter in the Carina-ABL group vs in the Non-Carina-ABL group (7.7 ± 1.7 mm/m2 vs 9.5 ± 2.3 mm/m2; P = .005), whereas other anatomical parameters (LA and RA volumes, right inferior PV angle, and ostial diameters of the RtPVs) did not differ between the groups. For left-sided PVI, the ostial diameter and circumference of the left superior PV were smaller in the Carina-ABL group (n = 13) vs the Non-Carina-ABL group (n = 32) (8.6 ± 2.1 mm/m2 vs 7.3 ± 1.5 mm/m2; P = .044, and 34.9 ± 6.0 mm/m2 vs 30.1 ± 5.1 mm/m2; P = .017, respectively). Conclusions: A shorter interatrial distance for right-sided PVI and a smaller PV ostium for left-sided PVI were associated with the necessity of additional carina ablation. The presence and location of the epicardial fibers may be affected by the atrial and PV geometry.

Using 3-dimensional speckle-tracking echocardiography-derived activation imaging system, we visualized interventricular dyssynchrony in a repaired tetralogy of Fallot case with pacing-induced left ventricular dysfunction. The activation imaging system visualized interventricular dyssynchrony and resynchronization after cardiac resynchronization therapy and may be useful to assess electromechanical disturbance in complicated congenital heart diseases. (Level of Difficulty: Intermediate.)

Fibroblasts can be directly reprogrammed to cardiomyocytes, but reprogramming is less efficient for adult compared to embryonic fibroblasts. Here, the authors find that inhibition of inflammation and Cox-2-prostaglandin-cAMP-IL-1β signaling enhances reprogramming efficiency of adult, but not embryonic fibroblasts.

Abstract Background Cardiac tamponade is a serious complication of catheter ablation for atrial fibrillation (AF). However, the outcomes of catheter ablation in patients of cardiac tamponade are unknown. Methods We performed catheter ablation in 2467 sessions of AF or a recurrence of AF between January 2007 and January 2016. Of these, 29 events in 27 patients (1.18%: 22 men; 64.5 ± 10.4 years; 17 with paroxysmal AF) of cardiac tamponade during or after the procedure were recorded. The clinical characteristics and outcomes of these 29 events were studied in detail. Results Of the 19 events where the ablation procedure was completed, seven events developed acute recurrence of AF (36.8%). Of the 10 events with an incomplete procedure, 10 exhibited AF recurrence (100.0%). Direct oral anticoagulants were used in seven events, and clinical outcomes were not significantly different compared to the remaining 21 events that were prescribed warfarin. Pericarditis occurred in 10 events (34.5%) after the procedure, and the incidence rate was lower in patients receiving prophylactic nonsteroidal anti‐inflammatory drugs or steroids (2/15, 13.3% vs 8/14, 57.1%; P = 0.013). Repeated sessions were performed in 12 events (two with a complete initial procedure, 10 with an incomplete initial procedure). Freedom from atrial arrhythmias was observed in 27 events (93.1%, 9 with antiarrhythmic drugs) over midterm follow‐up (3.1 ± 2.6 years). Conclusion Although cardiac tamponade caused by catheter ablation led to a high rate of acute AF recurrence and pericarditis, the midterm recurrence rates of AF are unaffected if the procedure can be completed.

Accumulating evidence suggests that the breakdown of immune tolerance plays an important role in the development of myocarditis triggered by cardiotropic microbial infections. Genetic deletion of immune checkpoint molecules that are crucial for maintaining self-tolerance causes spontaneous myocarditis in mice, and cancer treatment with immune checkpoint inhibitors can induce myocarditis in humans. These results suggest that the loss of immune tolerance results in myocarditis. The tissue microenvironment influences the local immune dysregulation in autoimmunity. Recently, tenascin-C (TN-C) has been found to play a role as a local regulator of inflammation through various molecular mechanisms. TN-C is a nonstructural extracellular matrix glycoprotein expressed in the heart during early embryonic development, as well as during tissue injury or active tissue remodeling, in a spatiotemporally restricted manner. In a mouse model of autoimmune myocarditis, TN-C was detectable before inflammatory cell infiltration and myocytolysis became histologically evident; it was strongly expressed during active inflammation and disappeared with healing. TN-C activates dendritic cells to generate pathogenic autoreactive T cells and forms an important link between innate and acquired immunity.

The authors wish to make the following corrections to this paper [...]

Inhibition of K+-conductance through the human ether-a-go-go related gene (hERG) channel leads to QT prolongation and is associated with cardiac arrhythmias. We previously reported that physiological concentrations of some estrogens partially suppress the hERG channel currents by interacting with the S6 residue F656 and increase the sensitivity of hERG blockade by E-4031. Although these studies suggested that clinically used synthetic estrogens with similar structures have the marked potential to alter hERG functions, the hERG interactions with synthetic estrogens have not been assessed. We therefore examined whether ethinylestradiol (EE2), a synthetic estrogen used in oral contraceptives, affects hERG function and blockade by drugs. Supratherapeutic concentrations of EE2 did not alter amplitudes or kinetics of the hERG currents elicited by train pulses at 20 mV (0.1 Hz). On the other hand, EE2 at therapeutic concentrations reduced the degree of hERG current suppression by E-4031. The administration of EE2 followed by E-4031 blockade reversed the current suppression, suggesting that the interaction of EE2 and E-4031 alters hERG at the drug-binding site. The effects of EE2 on hERG blockade raised the possibility that other estrogens, including synthetic estrogens, can alter hERG blockade by drugs that cause QT prolongation and ventricular arrhythmias.

Thrombus embolization is a rare consequence of ultrasonographic examination of the vessels of the lower limbs. We present a case of a 77-year-old woman with lung cancer who developed pulmonary embolism originating from the right femoral deep vein thrombus during the compression maneuver of the ultrasonographic examination of the lower limbs. (Level of Difficulty: Beginner.)

Immune checkpoint inhibitors (ICIs) have changed the treatment landscape of advanced cancers. Unfortunately, these agents can induce a wide spectrum of immune-related adverse events (irAEs) through activation of immune responses in non-target organs, including the heart. As the clinical use of ICI therapy increases rapidly, management of irAEs is becoming extremely important. The most commonly presented cardiac irAE is myocarditis. Histopathologically, T-cell (with a predominance of CD8+ cells) and macrophage infiltration in the myocardium is typically observed in ICI-associated myocarditis. Other presentations of cardiac irAEs include congestive heart failure, Takotsubo cardiomyopathy, pericardial disease, arrhythmias, and conduction disease. Although cardiac irAEs are relatively rare, they can be life-threatening. Hence, cardiologists and oncologists should be vigilant for these presentations.

As the number of cancer survivors increases, cardiac management in anthracycline-treated patients has become more important. We planned to conduct a prospective multicenter registry study for comprehensive echocardiographic and biomarker data collection and an evaluation of the current practice in terms of diagnosis and management of anthracycline-induced cardiotoxicity (AIC registry). To examine the feasibility of this registry study, we analyzed the 1-year follow-up data of 97 patients registered during the first year of this registry. The AIC registry was launched in July 2016. Data on echocardiographic parameters (e.g., two-and three-dimensional [(2- and 3-D) left ventricular ejection fraction (LVEF) and global longitudinal strain (GLS)) and biomarkers (e.g., troponin T and brain natriuretic peptide) were collected before anthracycline treatment, every 3 months during the first year after starting anthracycline, and every 6 months during the second year. Eighty-three patients (86%) completed a 1-year follow-up. The measurable rates of 2D LVEF, 3D LVEF, and GLS on each visit were nearly optimal (100%, 86–93%, and 84–94%, respectively). During the 1-year follow-up, 5 patients (6.0%) developed cardiotoxicity (a reduction in LVEF ≥ 10 percentage points from baseline and

Neutrophils are recruited into the heart at an early stage following a myocardial infarction (MI). These secrete several proteases, one of them being neutrophil elastase (NE), which promotes inflammatory responses in several disease models. It has been shown that there is an increase in NE activity in patients with MI; however, the role of NE in MI remains unclear. Therefore, the present study aimed to investigate the role of NE in the pathogenesis of MI in mice. NE expression peaked on day 1 in the infarcted hearts. In addition, NE deficiency improved survival and cardiac function post-MI, limiting fibrosis in the noninfarcted myocardium. Sivelestat, an NE inhibitor, also improved survival and cardiac function post-MI. Flow cytometric analysis showed that the numbers of heart-infiltrating neutrophils and inflammatory macrophages (CD11b+F4/80+CD206low cells) were significantly lower in NE-deficient mice than in wild-type (WT) mice. At the border zone between intact and necrotic areas, the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells was lower in NE-deficient mice than in WT mice. Western blot analyses revealed that the expression levels of insulin receptor substrate 1 and phosphorylation of Akt were significantly upregulated in NE-knockout mouse hearts, indicating that NE deficiency might improve cardiac survival by upregulating insulin/Akt signaling post-MI. Thus, NE may enhance myocardial injury by inducing an excessive inflammatory response and suppressing Akt signaling in cardiomyocytes. Inhibition of NE might serve as a novel therapeutic target in the treatment of MI.

Dilated cardiomyopathy (DCM) is a potentially lethal disorder characterized by progressive impairment of cardiac function. Chronic myocarditis has long been hypothesized to be one of the causes of DCM. However, owing to the lack of suitable animal models of chronic myocarditis, its pathophysiology remains unclear. Here, we report a novel mouse model of chronic myocarditis induced by recombinant bacille Calmette-Guérin (rBCG) expressing a CD4+ T-cell epitope of cardiac myosin heavy chain-α (rBCG-MyHCα). Mice immunized with rBCG-MyHCα developed chronic myocarditis, and echocardiography revealed dilation and impaired contraction of ventricles, similar to those observed in human DCM. In the heart, CD62L−CD4+ T cells were increased and produced significant amounts of IFN-γ and IL-17 in response to cardiac myosin. Adoptive transfer of CD62L−CD4+ T cells induced myocarditis in the recipient mice, which indicated that CD62L−CD4+ T cells were the effector cells in this model. rBCG-MyHCα-infected dendritic cells produced proinflammatory cytokines and induced MyHCα-specific T-cell proliferation and Th1 and Th17 polarization. This novel chronic myocarditis mouse model may allow the identification of the central pathophysiological and immunological processes involved in the progression to DCM.

Programmed death ligand 2 (PD-L2) is the second ligand of programmed death 1 (PD-1) protein. In autoimmune myocarditis, the protective roles of PD-1 and its first ligand programmed death ligand 1 (PD-L1) have been well documented; however, the role of PD-L2 remains unknown. In this study, we report that PD-L2 deficiency exacerbates myocardial inflammation in mice with experimental autoimmune myocarditis (EAM). EAM was established in wild-type (WT) and PD-L2-deficient mice by immunization with murine cardiac myosin peptide. We found that PD-L2-deficient mice had more serious inflammatory infiltration in the heart and a significantly higher myocarditis severity score than WT mice. PD-L2-deficient dendritic cells (DCs) enhanced CD4+ T cell proliferation in the presence of T cell receptor and CD28 signaling. These data suggest that PD-L2 on DCs protects against autoreactive CD4+ T cell expansion and severe inflammation in mice with EAM.

Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors, including Gata4, Mef2c, and Tbx5; however, this process is inefficient under serum-based culture conditions, in which conversion of partially reprogrammed cells into fully reprogrammed functional iCMs has been a major hurdle. Here, we report that a combination of fibroblast growth factor (FGF) 2, FGF10, and vascular endothelial growth factor (VEGF), termed FFV, promoted cardiac reprogramming under defined serum-free conditions, increasing spontaneously beating iCMs by 100-fold compared with those under conventional serum-based conditions. Mechanistically, FFV activated multiple cardiac transcriptional regulators and converted partially reprogrammed cells into functional iCMs through the p38 mitogen-activated protein kinase and phosphoinositol 3-kinase/AKT pathways. Moreover, FFV enabled cardiac reprogramming with only Mef2c and Tbx5 through the induction of cardiac reprogramming factors, including Gata4. Thus, defined culture conditions promoted the quality of cardiac reprogramming, and this finding provides new insight into the mechanism of cardiac reprogramming.

Background and objectives: Pulmonary vein (PV) reconnection is a major reason for recurrence after catheter ablation of paroxysmal atrial fibrillation (PAF). However, the timing of the recurrence varies between patients, and recurrence >1 year after ablation is not uncommon. We sought to elucidate the characteristics of atrial fibrillation (AF) that recurred in different follow-up periods. Materials and Methods: Study subjects comprised 151 consecutive patients undergoing initial catheter ablation of PAF. Left atrial volume index (LAVi) and atrial/brain natriuretic peptide (ANP/BNP) levels were systematically measured annually over 3 years until AF recurred. Results: Study subjects were classified into four groups: non-recurrence group (n = 84), and short-term- (within 1 year) (n = 30), mid-term- (1–3 years) (n = 26), and long-term-recurrence group (>3 years) (n = 11). The short-term-recurrence group was characterized by a higher prevalence of diabetes mellitus (hazard ratio 2.639 (95% confidence interval, 1.174–5.932), p = 0.019 by the Cox method), frequent AF episodes (≥1/week) before ablation (4.038 (1.545–10.557), p = 0.004), and higher BNP level at baseline (per 10 pg/mL) (1.054 (1.029–1.081), p < 0.0001). The mid-term-recurrence group was associated with higher BNP level (1.163 (1.070–1.265), p = 0.0004), larger LAVi (mL/m2) (1.033 (1.007–1.060), p = 0.013), and longer AF cycle length at baseline (per 10 ms) (1.194 (1.058–1.348), p = 0.004). In the long-term-recurrence group, the ANP and BNP levels were low throughout follow-up, as with those in the non-recurrence group, and AF cycle length was shorter (0.694 (0.522–0.924), p = 0.012) than those in the other recurrence groups. Conclusions: Distinct characteristics of AF were found according to the time to first recurrence after PAF ablation. The presence of secondary factors beyond PV reconnections could be considered as mechanisms for the recurrence of PAF in each follow-up period.

Aging and obesity are the most prominent risk factors for onset of atrial fibrillation (AF). Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme that catalyzes nicotinamide adenine dinucleotide (NAD) activity. Nampt and NAD are essential for maintenance of cellular redox homeostasis and modulation of cellular metabolism, and their expression levels decrease with aging and obesity. However, a role for Nampt in AF is unknown. The present study aims to test whether there is a role of Nampt/NAD axis in the pathogenesis of obesity-induced AF. Male C57BL/6J (WT) mice and heterozygous Nampt knockout (NKO) mice were fed with a normal chow diet (ND) or a high-fat diet (HFD). Electrophysiological study showed that AF inducibility was significantly increased in WT+HFD, NKO+ND, and NKO+HFD mice compared with WT+ND mice. AF duration was significantly longer in WT+HFD and NKO+ND mice and further prolonged in NKO+HFD mice compared with WT+ND mice and the calcium handling pathway was altered on molecular level. Also, treatment with nicotinamide riboside, a NAD precursor, partially restored the HFD-induced AF perpetuation. Overall, this work demonstrates that partially deletion of Nampt facilitated HFD-induced AF through increased diastolic calcium leaks. The Nampt/NAD axis may be a potent therapeutic target for AF.

Atrial fibrillation (AF) is the most prevalent arrhythmia in the general population. There is a close association between chronic kidney disease (CKD) and AF. In recent years, attention has been focused on the relationship between AF and uremic toxins, including indoxyl sulfate (IS). Several animal studies have shown that IS promotes the development and progression of AF. IS has been shown to cause fibrosis and inflammation in the myocardium and exacerbate AF by causing oxidative stress and reducing antioxidative defense. Administration of AST-120, an absorbent of uremic toxins, decreases uremic toxin-induced AF in rodents. We have recently reported that patients with a higher serum IS level exhibit a higher rate of AF recurrence after catheter ablation, with serum IS being a significant predictor of AF recurrence. In this review, we discuss the possible mechanisms behind the AF-promoting effects of uremic toxins and summarize the reported clinical studies of uremic toxin-induced AF.

Natriuretic peptides (NPs) have become important diagnostic and prognostic biomarkers in cardiovascular diseases, particularly in heart failure (HF). Diagnosis and management of coronary artery disease and atrial fibrillation (AF) can also be guided by NP levels. When interpreting NP levels, however, the caveat is that age, sex, body mass index, renal dysfunction, and race affect the clearance of NPs, resulting in different cut-off values in clinical practice. In AF, NP levels have been associated with incident AF in the general population, recurrences after catheter ablation, prediction of clinical prognosis, and the risk of stroke. In this article, we first review and summarize the current evidence and the roles of B-type NP and atrial NP in HF and coronary artery disease and then focus on the increasing utility of NPs in the diagnosis and management of and the research into AF.

Cardiac diseases are among the most common causes of death globally. Cardiac muscle has limited proliferative capacity, and regenerative therapies are highly in demand as a new treatment strategy. Although pluripotent reprogramming has been developed, it has obstacles, such as a potential risk of tumor formation, poor survival of the transplanted cells, and high cost. We previously reported that fibroblasts can be directly reprogrammed to cardiomyocytes by overexpressing a combination of three cardiac-specific transcription factors (Gata4, Mef2c, Tbx5 (together, GMT)). We and other groups have promoted cardiac reprogramming by the addition of certain miRNAs, cytokines, and epigenetic factors, and unraveled new molecular mechanisms of cardiac reprogramming. More recently, we discovered that Sendai virus (SeV) vector expressing GMT could efficiently and rapidly reprogram fibroblasts into integration-free cardiomyocytes in vitro via robust transgene expression. Gene delivery of SeV-GMT also improves cardiac function and reduces fibrosis after myocardial infarction in mice. Through direct cardiac reprogramming, new cardiomyocytes can be generated and scar tissue reduced to restore cardiac function, and, thus, direct cardiac reprogramming may serve as a powerful strategy for cardiac regeneration. Here, we provide an overview of the previous reports and current challenges in this field.

Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.

Background: Holter electrocardiogram (ECG) is the gold standard for ambulatory monitoring of atrial fibrillation (AF) but it is insufficient because of its limited recording time. Although several consumer ECG devices provide longer recording time, they generally do not undergo the regulatory process for medical use. Furthermore, current medical-grade devices for longer ECG monitoring are not continuous or too invasive for AF monitoring. A wearable ECG with a medical-grade dry textile electrode is a promising technology to remedy this limitation. This pilot study aimed to simultaneously compare the wearable and Holter ECGs for ambulatory monitoring in a clinical setting. Methods: This prospective observational study enrolled 18 patients who underwent AF ablation. One day after AF ablation, ambulatory ECG was obtained for three hours simultaneously using both the wearable and Holter ECG devices. Automatic ECG interpretations between devices were compared with correlation and agreement analyses. Results: Simultaneous ECG monitoring demonstrated a comparable analysis time and total heart beats between the two devices. Almost complete correlation and agreement were also demonstrated in all clinically relevant testing aspects except in R-wave amplitude (r = 0.743, p < .001). AF was detected in three patients. AF duration was the same in both ECG devices in two patients with continuous AF. In the remaining patient with intermittent AF, AF duration was shortened by 0.6% with the wearable ECG as compared to that with the Holter ECG. Conclusions: Simultaneous ECG comparison revealed a high consistency between the wearable and Holter ECG devices. The results of this study warrant further clinical studies for long-term monitoring of ambulatory ECG after AF ablation.

Background: Holter electrocardiogram (ECG) is the gold standard for ambulatory monitoring of atrial fibrillation (AF) but it is insufficient because of its limited recording time. A wearable ECG with a medical-grade dry textile electrode is a promising technology to remedy this limitation. This pilot study aimed to simultaneously compare the wearable and Holter ECGs for ambulatory monitoring in a clinical setting. Methods: This prospective observational study enrolled 18 patients who underwent AF ablation. One day after AF ablation, ambulatory ECG was obtained for three hours simultaneously using both the wearable and Holter ECG devices. Automatic ECG interpretations between devices were compared with correlation and agreement analyses. Results: Simultaneous ECG monitoring demonstrated a comparable analysis time and total heart beats between the two devices. Almost complete correlation and agreement were also demonstrated in all clinically relevant testing aspects except in R-wave amplitude (r = 0.743, p < .001). AF was detected in three patients. AF duration was the same in both ECG devices in two patients with continuous AF. In the remaining patient with intermittent AF, AF duration was shortened by 0.6% with the wearable ECG as compared to that with the Holter ECG. Conclusions: Simultaneous ECG comparison revealed a high consistency between the wearable and Holter ECG devices. The results of this study warrant further clinical studies for long-term monitoring of ambulatory ECG after AF ablation.