Variants of the promoter of MYH6 gene in congenital isolated and sporadic patent ductus arteriosus: case-control study and cellular functional analyses.

Patent ductus arteriosus (PDA) is a common form of congenital heart disease. The MYH6 gene has important effects on cardiovascular growth and development, but the effect of variants in the MYH6 gene promoter on ductus arteriosus is unknown. DNA was extracted from blood samples of 721 subjects (428 patients with isolated and sporadic PDA and 293 healthy controls) and analyzed by sequencing for MYH6 gene promoter region variants. Cellular function experiments with three cell lines (HEK-293, HL-1, and H9C2 cells) and bioinformatics analyses were performed to verify their effects on gene expression. In the MYH6 gene promoter, 11 variants were identified. Four variants were found only in patients with PDA and 2 of them (g.3434G>C and g.4524C>T) were novel. Electrophoretic mobility shift assay showed that the transcription factors bound by the promoter variants were significantly altered in comparison to the wild-type in all three cell lines. Dual luciferase reporter showed that all the 4 variants reduced the transcriptional activity of the MYH6 gene promoter (P < 0.05). Prediction of transcription factors bound by the variants indicated that these variants alter the transcription factor binding sites. These pathological alterations most likely affect the contraction of the smooth muscle of ductus arteriosus, leading to PDA. This study is the first to focus on variants at the promoter region of the MYH6 gene in PDA patients with cellular function tests. Therefore, this study provides new insights to understand the genetic basis and facilitates further studies on the mechanism of PDA formation.

Nuclear AGO2 promotes myocardial remodeling by activating ANKRD1 transcription in failing hearts.

Heart failure (HF) is manifested by transcriptional and posttranscriptional reprogramming of critical genes. Multiple studies have revealed that microRNAs could translocate into subcellular organelles such as the nucleus to modify gene expression. However, the functional property of subcellular Argonaute2 (AGO2), the core member of the microRNA machinery, has remained elusive in HF. AGO2 was found to be localized in both the cytoplasm and nucleus of cardiomyocytes, and robustly increased in the failing hearts of patients and animal models. We demonstrated that nuclear AGO2 rather than cytosolic AGO2 overexpression by recombinant adeno-associated virus (serotype 9) with cardiomyocyte-specific troponin T promoter exacerbated the cardiac dysfunction in transverse aortic constriction (TAC)-operated mice. Mechanistically, nuclear AGO2 activates the transcription of ANKRD1, encoding ankyrin repeat domain-containing protein 1 (ANKRD1), which also has a dual function in the cytoplasm as part of the I-band of the sarcomere and in the nucleus as a transcriptional cofactor. Overexpression of nuclear ANKRD1 recaptured some key features of cardiac remodeling by inducing pathological MYH7 activation, whereas cytosolic ANKRD1 seemed cardioprotective. For clinical practice, we found ivermectin, an antiparasite drug, and ANPep, an ANKRD1 nuclear location signal mimetic peptide, were able to prevent ANKRD1 nuclear import, resulting in the improvement of cardiac performance in TAC-induced HF.

Carrying both the heterozygous Myh6-R453C and Tnnt2-R92W mutations aggravate the hypertrophic cardiomyopathy phenotype in mice.

Hypertrophic cardiomyopathy (HCM) is an inherited disease of the heart muscle that is dominated by variations in eight genes encoding sarcomere proteins. Although there are clinical or basic research reports that carrying double mutations can lead to more severe HCM phenotypes, there are also research reports that after reanalyzing the reported mutations, the severity of clinical symptoms in patients with double mutations did not significantly increase compared to patients with only one mutation. To determine whether double pathogenic mutations can aggravate the phenotype of hypertrophic cardiomyopathy in mice, we constructed mice carrying single pathogenic heterozygous mutation Myh6-R453C or Tnnt2-R92W and mice carrying both pathogenic heterozygous mutations. Our results showed that mice with double heterozygous mutations exhibited significant hypertrophic cardiomyopathy phenotypes at 4 weeks of age, and the degree of hypertrophy was significantly higher than that of single heterozygous mutant mice of the same age. Our study suggests that carrying the two pathogenic heterozygous mutations simultaneously can aggravate the phenotype of HCM in mice, which provides experimental evidence for the genotype-phenotype relationship of double pathogenic mutations and provides reference significance for clinical risk stratification of HCM patients.

Human Genetics of Tricuspid Atresia and Univentricular Heart.

Tricuspid atresia (TA) is a rare congenital heart condition that presents with a complete absence of the right atrioventricular valve. Because of the rarity of familial and/or isolated cases of TA, little is known about the potential genetic abnormalities contributing to this condition. Potential responsible chromosomal abnormalities were identified in exploratory studies and include deletions in 22q11, 4q31, 8p23, and 3p as well as trisomies 13 and 18. In parallel, potential culprit genes include the ZFPM2, HEY2, NFATC1, NKX2-5, MYH6, and KLF13 genes. The aim of this chapter is to expose the genetic components that are potentially involved in the pathogenesis of TA in humans. The large variability in phenotypes and genotypes among cases of TA suggests a genetic network that involves many components yet to be unraveled.

Human Genetics of Atrial Septal Defect.

Although atrial septal defects (ASD) can be subdivided based on their anatomical location, an essential aspect of human genetics and genetic counseling is distinguishing between isolated and familiar cases without extracardiac features and syndromic cases with the co-occurrence of extracardiac abnormalities, such as developmental delay. Isolated or familial cases tend to show genetic alterations in genes related to important cardiac transcription factors and genes encoding for sarcomeric proteins. By contrast, the spectrum of genes with genetic alterations observed in syndromic cases is diverse. Currently, it points to different pathways and gene networks relevant to the dysregulation of cardiomyogenesis and ASD pathogenesis. Therefore, this chapter reflects the current knowledge and highlights stable associations observed in human genetics studies. It gives an overview of the different types of genetic alterations in these subtypes, including common associations based on genome-wide association studies (GWAS), and it highlights the most frequently observed syndromes associated with ASD pathogenesis.

Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

Myh6 promoter-driven Cre recombinase excises floxed DNA fragments in a subset of male germline cells.

Myh6-Cre transgenic mouse line was known to express Cre recombinase only in the heart. Nevertheless, during breeding Myh6-Cre to Rosa26fstdTom reporter (tdTom) mouse line, we observed that a significant part of their F2 tdTom/+ offspring had tdTom reporter gene universally activated. Our results show that Myh6-Cre transgenic mice have Cre recombinase activity in a subpopulation of the male germline cells, and that Myh6 gene transcripts are enriched in the interstitial Leydig cells and the undifferentiated spermatogonia stem cells. In summary, the current study confirms that the previously known "heart-specific" Myh6 promoter drives Cre expression in the testis.

The G4 resolvase RHAU regulates ventricular trabeculation and compaction through transcriptional and post-transcriptional mechanisms.

The G-quadruplex (G4) resolvase RNA helicase associated with AU-rich element (RHAU) possesses the ability to unwind G4 structures in both DNA and RNA molecules. Previously, we revealed that RHAU plays a critical role in embryonic heart development and postnatal heart function through modulating mRNA translation and stability. However, whether RHAU functions to resolve DNA G4 in the regulation of cardiac physiology is still elusive. Here, we identified a phenotype of noncompaction cardiomyopathy in cardiomyocyte-specific Rhau deletion mice, including such symptoms as spongiform cardiomyopathy, heart dilation, and death at young ages. We also observed reduced cardiomyocyte proliferation and advanced sarcomere maturation in Rhau mutant mice. Further studies demonstrated that RHAU regulates the expression levels of several genes associated with ventricular trabeculation and compaction, including the Nkx2-5 and Hey2 that encode cardiac transcription factors of NKX2-5 and Hey2, and the myosin heavy chain 7 (Myh7) whose protein product is MYH7. While RHAU modulates Nkx2-5 mRNA and Hey2 mRNA at the post-transcriptional level, we uncovered that RHAU facilitates the transcription of Myh7 through unwinding of the G4 structures in its promoter. These findings demonstrated that RHAU regulates ventricular chamber development through both transcriptional and post-transcriptional mechanisms. These results contribute to a knowledge base that will help to understand the pathogenesis of diseases such as noncompaction cardiomyopathy.

Prenatal diagnosis of recurrent hypoplastic left heart syndrome associated with MYH6 variants: a case report.

BACKGROUND: Hypoplastic left heart syndrome (HLHS) is a rare but genetically complex and clinically and anatomically severe form of congenital heart disease (CHD). CASE PRESENTATION: Here, we report on the use of rapid prenatal whole-exome sequencing for the prenatal diagnosis of a severe case of neonatal recurrent HLHS caused by heterozygous compound variants in the MYH6 gene inherited from the (healthy) parents. MYH6 is known to be highly polymorphic; a large number of rare and common variants have variable effects on protein levels. We postulated that two hypomorphic variants led to severe CHD when associated in trans; this was consistent with the autosomal recessive pattern of inheritance. In the literature, dominant transmission of MYH6-related CHD is more frequent and is probably linked to synergistic heterozygosity or the specific combination of a single, pathogenic variant with common MYH6 variants. CONCLUSIONS: The present report illustrates the major contribution of whole-exome sequencing (WES) in the characterization of an unusually recurrent fetal disorder and considered the role of WES in the prenatal diagnosis of disorders that do not usually have a genetic etiology.

Phenomapping for classification of doxorubicin-induced cardiomyopathy in rats.

Cardiomyopathy resistant to treatment is the most serious adverse effect of doxorubicin (dox). The mechanisms of dox-induced cardiomyopathy (DCM) have been extensively studied in dilated forms of DCM. However, efficient treatment did not emerge. The aim of the present work was to revisit the experimental model of DCM in rats, to define phenotype/s and associate them to the changes in cardiac transcriptome. Male Wistar rats equipped with radiotelemetry device, were randomized in DOX group (5 mg/0,5 mL/kg, IV dox; n = 18) and CONT group (0,5 mL/kg IV saline; n = 6). Echocardiography, autonomic spectral markers and baroreceptor reflex evaluation was performed prior to, and after treatment. Blood samples were collected at the end of experimentation. Cardiac, renal and hepatic tissues were analysed post-mortem by histology. Changes in expression of key cardiac genes affected by dox were assessed by RT-qPCR. Phenotypes were identified by clustering non-redundant features using four different algorithms averaged by evidence accumulation cluster technique. The results emphasize the existence of two major phenotypes of DCM with comparably high mortality rates: phenotype 1 characterized by, left ventricular (LV) dilatation, thinning of LV posterior wall, reduced LV ejection fraction (LVEF) and fractional shortening (LVFS), decreased HR variability (HRV), decreased baroreceptor effectiveness index (BEI) and increased NT-proBNP; and phenotype 2 with LV hypertrophy - increased LV mass, preserved LVEF, LVFS, no changes in HRV and BEI and moderate NT-proBNP increase. Both phenotypes exhibited a genetic shift to a new-born program.

Marked lateral ST-segment elevation with inferior ST-segment depression in an asymptomatic 12 year-old girl: A normal variant?

Significant ST-segment changes raise concern for myocardial ischemia, cardiomyopathy or myocardial inflammation and therefore, warrant an extensive and often invasive cardiovascular evaluation. We report a 12 year-old asymptomatic African-American girl with marked ST-segment elevation in leads I and aVL and ST-segment depression in inferior leads II, III and aVF. Extensive cardiovascular evaluation did not reveal any abnormality suggesting that these findings, which have previously not been reported, are likely benign, at least in this young girl.

Analysis of Cre-mediated genetic deletion of Gdf11 in cardiomyocytes of young mice.

Administration of active growth differentiation factor 11 (GDF11) to aged mice can reduce cardiac hypertrophy, and low serum levels of GDF11 measured together with the related protein, myostatin (also known as GDF8), predict future morbidity and mortality in coronary heart patients. Using mice with a loxP-flanked ("floxed") allele of Gdf11 and Myh6-driven expression of Cre recombinase to delete Gdf11 in cardiomyocytes, we tested the hypothesis that cardiac-specific Gdf11 deficiency might lead to cardiac hypertrophy in young adulthood. We observed that targeted deletion of Gdf11 in cardiomyocytes does not cause cardiac hypertrophy but rather leads to left ventricular dilation when compared with control mice carrying only the Myh6-cre or Gdf11-floxed alleles, suggesting a possible etiology for dilated cardiomyopathy. However, the mechanism underlying this finding remains unclear because of multiple confounding effects associated with the selected model. First, whole heart Gdf11 expression did not decrease in Myh6-cre; Gdf11-floxed mice, possibly because of upregulation of Gdf11 in noncardiomyocytes in the heart. Second, we observed Cre-associated toxicity, with lower body weights and increased global fibrosis, in Cre-only control male mice compared with flox-only controls, making it challenging to infer which changes in Myh6-cre;Gdf11-floxed mice were the result of Cre toxicity versus deletion of Gdf11. Third, we observed differential expression of cre mRNA in Cre-only controls compared with the cardiomyocyte-specific knockout mice, also making comparison between these two groups difficult. Thus, targeted Gdf11 deletion in cardiomyocytes may lead to left ventricular dilation without hypertrophy, but alternative animal models are necessary to understand the mechanism for these findings. NEW & NOTEWORTHY We observed that targeted deletion of growth differentiation factor 11 in cardiomyocytes does not cause cardiac hypertrophy but rather leads to left ventricular dilation compared with control mice carrying only the Myh6-cre or growth differentiation factor 11-floxed alleles. However, the mechanism underlying this finding remains unclear because of multiple confounding effects associated with the selected mouse model.

Ventricular remodeling of single-chambered myh6-/- adult zebrafish hearts occurs via a hyperplastic response and is accompanied by elastin deposition in the atrium.

Zebrafish (Danio rerio) is widely used as an animal model to understand the pathophysiology of cardiovascular diseases. Here, we present the adult cardiac phenotype of weak atrium, myh6-/-, which carry mutations in the zebrafish atrial myosin heavy chain. Homozygous mutants survive to adulthood and are fertile despite their initial weak atrial beat. In adult mutants, the atrium remains hypoplastic and shows elastin deposition while mutant ventricles exhibit increased size. In mammals, hypertrophy is the most common mechanism resulting in cardiomegaly. Using immunohistochemistry and confocal microscopy to measure cardiomyocyte cell size, density and proliferation, we show that the enlargement of the myh6-/- ventricle is predominantly due to hyperplasia. However, we identified similar transcriptional profiles to the mammalian hypertrophy response via RT-PCR of the hyperplastic ventricles. Furthermore, we show activation of the ER-stress pathway by western blot analysis. In conclusion, we can assume, based on our model, that molecular signaling pathways associated with hypertrophy in mammals, in combination with ER-stress activation, result in hyperplasia in zebrafish. In addition, to our knowledge, this is the first time to report elastin deposition in the atrium.

Whole-exome sequencing identifies R1279X of MYH6 gene to be associated with congenital heart disease.

BACKGROUND: Myosin VI, encoded by MYH6, is expressed dominantly in human cardiac atria and plays consequential roles in cardiac muscle contraction and comprising the cardiac muscle thick filament. It has been reported that the mutations in the MYH6 gene associated with sinus venosus atrial septal defect (ASD type III), hypertrophic (HCM) and dilated (DCM) cardiomyopathies. METHODS: Two patients in an Iranian family have been identified who affected to Congenital Heart Disease (CHD). The male patient, besides CHD, shows that the thyroglossal sinus, refractive errors of the eye and mitral stenosis. The first symptoms emerged at the birth and diagnosis based on clinical features was made at about 5 years. The family had a history of ASD. For recognizing mutated gene (s), whole exome sequencing (WES) was performed for the male patient and variants were analyzed by autosomal dominant inheritance mode. RESULTS: Eventually, by several filtering processes, a mutation in MYH6 gene (NM_002471.3), c.3835C > T; R1279X, was identified as the most likely disease-susceptibility variant and then confirmed by Sanger sequencing in the family. The mutation frequency was checked out in the local databases. This mutation results in the elimination of the 660 amino acids in the C-terminal of Myosin VI protein, including the vital parts of the coiled-coil structure of the tail domain. CONCLUSIONS: Our study represents the first case of Sinus venosus defect caused directly by MYH6 stop codon mutation. Our data indicate that by increase haploinsufficiency of myosin VI, c.3835C > T mutation with reduced penetrance could be associated with CHD.

A series of robust genetic indicators for definitive identification of cardiomyocytes.

Definitively identifying the cell type of newly generated cells in the heart and defining their origins are central questions in cardiac regenerative medicine. Currently, it is challenging to ascertain the myocardial identity and to track myocardial progeny during heart development and disease due to lack of proper genetic tools. This may lead to many misinterpretations of the findings in cardiac regenerative biology. In this study, we developed a set of novel mouse models by inserting double reporter genes nlacZ/H2B-GFP, mGFP/H2B-mCherry into the start codon of Tnnt2 and Myh6. nlacZ (nuclear lacZ) and mGFP (membrane GFP) are flanked by two LoxP sites in these animals. We found that the reporter genes faithfully recapitulated Tnnt2 and Myh6 cardiac expression from embryonic stage and adulthood. The reporter mice provide unprecedented robustness and fidelity for visualizing and tracing cardiomyocytes with nuclear or cell membrane localization signals. These animal models offer superior genetic tools to meet a critical need in studies of heart development, cardiac stem cell biology and cardiac regenerative medicine.

Exome analysis of a family with Wolff-Parkinson-White syndrome identifies a novel disease locus.

Wolff-Parkinson-White (WPW) syndrome is a common cause of supraventricular tachycardia that carries a risk of sudden cardiac death. To date, mutations in only one gene, PRKAG2, which encodes the 5'-AMP-activated protein kinase subunit γ-2, have been identified as causative for WPW. DNA samples from five members of a family with WPW were analyzed by exome sequencing. We applied recently designed prioritization strategies (VAAST/pedigree VAAST) coupled with an ontology-based algorithm (Phevor) that reduced the number of potentially damaging variants to 10: a variant in KCNE2 previously associated with Long QT syndrome was also identified. Of these 11 variants, only MYH6 p.E1885K segregated with the WPW phenotype in all affected individuals and was absent in 10 unaffected family members. This variant was predicted to be damaging by in silico methods and is not present in the 1,000 genome and NHLBI exome sequencing project databases. Screening of a replication cohort of 47 unrelated WPW patients did not identify other likely causative variants in PRKAG2 or MYH6. MYH6 variants have been identified in patients with atrial septal defects, cardiomyopathies, and sick sinus syndrome. Our data highlight the pleiotropic nature of phenotypes associated with defects in this gene.

MYH6 suppresses tumor progression by downregulating KIT expression in human prostate cancer.

Prostate cancer (PRAD) is one of the leading malignancies in men all around the world. Here, we identified Myosin Heavy Chain 6 (MYH6) as a potential tumor suppressor gene in the development of prostate cancer. We found lower expression of MYH6 in prostate cancer tissues, and its lower gene expression was also associated with worse clinical outcomes. In vitro and in vivo assays indicated that overexpressed MYH6 could suppress the proliferation and migration progression of prostate cancer cells. RNA-seq was employed to investigate the mechanism, and KIT Proto-Oncogen (KIT) was determined as the downstream gene of MYH6, which was further confirmed using rescue assays. In all, we provide the evidence that MYH6 could serve as a tumor suppressor in prostate cancer. Our results highlight the potential role of MYH6 in the development of prostate cancer.

Heart proteomic profiling discovers MYH6 and COX5B as biomarkers for sudden unexplained death.

Sudden unexplained death (SUD) is not uncommon in forensic pathology. Yet, diagnosis of SUD remains challenging due to lack of specific biomarkers. This study aimed to screen differentially expressed proteins (DEPs) and validate their usefulness as diagnostic biomarkers for SUD cases. We designed a three-phase investigation, where in the discovery phase, formalin-fixed paraffin-embedded (FFPE) heart specimens were screened through label-free proteomic analysis of cases dying from SUD, mechanical injury and carbon monoxide (CO) intoxication. A total of 26 proteins were identified to be DEPs for the SUD cases after rigorous criterion. Bioinformatics and Adaboost-recursive feature elimination (RFE) analysis further revealed that three of the 26 proteins (MYH6, COX5B and TNNT2) were potential discriminative biomarkers. In the training phase, MYH6 and COX5B were verified to be true DEPs in cardiac tissues from 29 independent SUD cases as compared with a serial of control cases (n = 42). Receiver operating characteristic (ROC) analysis illustrated that combination of MYH6 and COX5B achieved optimal diagnostic sensitivity (89.7 %) and specificity (84.4 %), with area under the curve (AUC) being 0.91. A diagnostic software based on the logistic regression formula derived from the training phase was then constructed. In the validation phase, the diagnostic software was applied to eight authentic SUD cases, seven (87.5 %) of which were accurately recognized. Our study provides a valid strategy towards practical diagnosis of SUD by integrating cardiac MYH6 and COX5B as dual diagnostic biomarkers.

Contractile abnormalities and altered drug response in engineered heart tissue from Mybpc3-targeted knock-in mice.

Myosin-binding protein C (Mybpc3)-targeted knock-in mice (KI) recapitulate typical aspects of human hypertrophic cardiomyopathy. We evaluated whether these functional alterations can be reproduced in engineered heart tissue (EHT) and yield novel mechanistic information on the function of cMyBP-C. EHTs were generated from cardiac cells of neonatal KI, heterozygous (HET) or wild-type controls (WT) and developed without apparent morphological differences. KI had 70% and HET 20% lower total cMyBP-C levels than WT, accompanied by elevated fetal gene expression. Under standard culture conditions and spontaneous beating, KI EHTs showed more frequent burst beating than WT and occasional tetanic contractions (14/96). Under electrical stimulation (6Hz, 37°C) KI EHTs exhibited shorter contraction and relaxation times and a twofold higher sensitivity to external [Ca(2+)]. Accordingly, the sensitivity to verapamil was 4-fold lower and the response to isoprenaline or the Ca(2+) sensitizer EMD 57033 2- to 4-fold smaller. The loss of EMD effect was verified in 6-week-old KI mice in vivo. HET EHTs were apparently normal under basal conditions, but showed similarly altered contractile responses to [Ca(2+)], verapamil, isoprenaline and EMD. In contrast, drug-induced changes in intracellular Ca(2+) transients (Fura-2) were essentially normal. In conclusion, the present findings in auxotonically contracting EHTs support the idea that cMyBP-C's normal role is to suppress force generation at low intracellular Ca(2+) and stabilize the power-stroke step of the cross bridge cycle. Pharmacological testing in EHT unmasked a disease phenotype in HET. The altered drug response may be clinically relevant.

Changes of ubiquitylated proteins in atrial fibrillation associated with heart valve disease: proteomics in human left atrial appendage tissue.

Background: Correlations between posttranslational modifications and atrial fibrillation (AF) have been demonstrated in recent studies. However, it is still unclear whether and how ubiquitylated proteins relate to AF in the left atrial appendage of patients with AF and valvular heart disease. Methods: Through LC-MS/MS analyses, we performed a study on tissues from eighteen subjects (9 with sinus rhythm and 9 with AF) who underwent cardiac valvular surgery. Specifically, we explored the ubiquitination profiles of left atrial appendage samples. Results: In summary, after the quantification ratios for the upregulated and downregulated ubiquitination cutoff values were set at >1.5 and <1:1.5, respectively, a total of 271 sites in 162 proteins exhibiting upregulated ubiquitination and 467 sites in 156 proteins exhibiting downregulated ubiquitination were identified. The ubiquitylated proteins in the AF samples were enriched in proteins associated with ribosomes, hypertrophic cardiomyopathy (HCM), glycolysis, and endocytosis. Conclusions: Our findings can be used to clarify differences in the ubiquitination levels of ribosome-related and HCM-related proteins, especially titin (TTN) and myosin heavy chain 6 (MYH6), in patients with AF, and therefore, regulating ubiquitination may be a feasible strategy for AF.