Atrial Fibrillation and Underlying Structural and Electrophysiological Heterogeneity.

As atrial fibrillation (AF) progresses from initial paroxysmal episodes to the persistent phase, maintaining sinus rhythm for an extended period through pharmacotherapy and catheter ablation becomes difficult. A major cause of the deteriorated treatment outcome is the atrial structural and electrophysiological heterogeneity, which AF itself can exacerbate. This heterogeneity exists or manifests in various dimensions, including anatomically segmental structural features, the distribution of histological fibrosis and the autonomic nervous system, sarcolemmal ion channels, and electrophysiological properties. All these types of heterogeneity are closely related to the development of AF. Recognizing the heterogeneity provides a valuable approach to comprehending the underlying mechanisms in the complex excitatory patterns of AF and the determining factors that govern the seemingly chaotic propagation. Furthermore, substrate modification based on heterogeneity is a potential therapeutic strategy. This review aims to consolidate the current knowledge on structural and electrophysiological atrial heterogeneity and its relation to the pathogenesis of AF, drawing insights from clinical studies, animal and cell experiments, molecular basis, and computer-based approaches, to advance our understanding of the pathophysiology and management of AF.

Genetic risk score of cerebral infarction in atrial fibrillation genome-wide association study.

INTRODUCTION: Stroke is a leading cause of death and the primary cause of adult-acquired disability. Patients with cardiogenic embolic stroke also have higher mortality and recurrence rates than patients with other stroke subtypes. Atrial fibrillation (AF) is a major risk factor for cerebral infarction (CI). The large-scale study identified 32 loci in the MEGASTROKE study. However, few studies have attempted to identify novel stroke risk variants in patients with a history of AF. Our overall aim was to identify novel CI risk variants in AF cases and explore whether their associations with the CI risk were affected by the CHADS2 and CHA2DS2-VASc scores. METHODS: We performed association study with CI using 8181 AF cases in previous genome-wide association study (GWAS) and imputation data without controls. We classified AF cases into those with or without past history of CI, and the genetic associations with the CI risk were examined. RESULTS: GWAS identified eight associated loci. The generated genetic risk score (GRS) for the eight loci was significantly associated with CI in patients with AF (1.46 × 10-8 ). We estimated bivariate logistic regression model which contained GRS and CHADS2 score (GRS: p-Value = 7.41 × 10-9 , CHADS2 score: p-Value <2.0 × 10-16 ) or CHA2DS2-VASc scores (GRS: p-Value = 2.52 × 10-10 , CHA2DS2-VASc score: p-Value <2.0 × 10-16 ). CONCLUSION: We identified eight genetic variants that were potentially associated with the risk of CI of AF cases and the significant GRS, whose associations were independent of the CHADS2 or CHA2DS2-VASc score.

Effect of the recording condition on the quality of a single-lead electrocardiogram.

Although many wearable single-lead electrocardiogram (ECG) monitoring devices have been developed, information regarding their ECG quality is limited. This study aimed to evaluate the quality of single-lead ECG in healthy subjects under various conditions (body positions and motions) and in patients with arrhythmias, to estimate requirements for automatic analysis, and to identify a way to improve ECG quality by changing the type and placement of electrodes. A single-lead ECG transmitter was placed on the sternum with a pair of electrodes, and ECG was simultaneously recorded with a conventional Holter ECG in 12 healthy subjects under various conditions and 35 patients with arrhythmias. Subjects with arrhythmias were divided into sinus rhythm (SR) and atrial fibrillation (AF) groups. ECG quality was assessed by calculating the sensitivity and positive predictive value (PPV) of the visual detection of QRS complexes (vQRS), automatic detection of QRS complexes (aQRS), and visual detection of P waves (vP). Accuracy was defined as a 100% sensitivity and PPV. We also measured the amplitude of the baseline, P wave, and QRS complex, and calculated the signal-to-noise ratio (SNR). We then focused on aQRS and estimated thresholds to obtain an accurate aQRS in more than 95% of the data. Finally, we sought to improve ECG quality by changing electrode placement using offset-type electrodes in 10 healthy subjects. The single-lead ECG provided 100% accuracy for vQRS, 87% for aQRS, and 74% for vP in healthy subjects under various conditions. Failure for accurate detection occurred in several motions in which the baseline amplitude was increased or in subjects with low QRS or P amplitude, resulting in low SNR. The single-lead ECG provided 97% accuracy for vQRS, 80% for aQRS in patients with arrhythmias, and 95% accuracy for vP in the SR group. The AF group showed higher baseline amplitude than the SR group (0.08 mV vs. 0.02 mV, P < 0.01) but no significant difference in accuracy for aQRS (79% vs. 81%, P = 1.00). The thresholds to obtain an accurate aQRS were a QRS amplitude > 0.42 mV and a baseline amplitude < 0.20 mV. The QRS amplitude was significantly influenced by electrode placement and body position (P < 0.01 for both, two-way analysis of variance), and the maximum reduction by changing body position was estimated as 30% compared to the sitting posture. The QRS amplitude significantly increased when the inter-electrode distance was extended vertically (1.51 mV for vertical extension vs. 0.93 mV for control, P < 0.01). The single-lead ECG provided at least 97% accuracy for vQRS, 80% for aQRS, and 74% for vP. To obtain stable aQRS in any body positions, a QRS amplitude > 0.60 mV and a baseline amplitude < 0.20 mV were required in the sitting posture considering the reduction induced by changing body position. Vertical extension of the inter-electrode distance increased the QRS amplitude.

Functional Role of the L396R Mutation of Tks5 Identified by an Exome-Wide Association Study in Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia; however, the current treatment strategies for AF have limited efficacy. Thus, a better understanding of the mechanisms underlying AF is important for future therapeutic strategy. A previous study (Exome-Wide Association Study (ExWAS)) identified a rare variant, rs202011870 (MAF=0.00036, GenomAD), which is highly associated with AF (OR=3.617, P<0.0001). rs202011870 results in the replacement of Leu at 396 with Arg (L396R) in a molecule, Tks5; however, the mechanism of how rs202011870 links to AF is completely unknown.Methods and Results:The association of rs202011870 with AF was examined in 3,378 participants (641 control and 2,737 AF cases) from 4 independent cohorts by using an Invader assay. Consequences of rs202011870 in migration ability, podosome formation, and expression of inflammation-related molecules in macrophages were examined using RAW264.7 cells with a trans-well assay, immunocytochemistry, and qPCR assay. Validation of the association of rs202011870 with AF was successful. In vitro studies showed that RAW264.7 cells with L396R-Tks5 increased trans-well migration ability, and enhanced podosome formation. RAW264.7 cells with L396R-Tks5 also increased the expression of several inflammatory cytokines and inflammation-related molecules. CONCLUSIONS: L396R mutation in Tks5 associated with AF enhances migration of macrophages and their inflammatory features, resulting in enhanced susceptibility to AF.

Combined Analysis of Human and Experimental Murine Samples Identified Novel Circulating MicroRNAs as Biomarkers for Atrial Fibrillation.

BACKGROUND: Recent experimental studies have demonstrated that several microRNAs (miRNAs) expressed in atrial tissue promote a substrate of atrial fibrillation (AF). However, because it has not been fully elucidated whether these experimental data contribute to identifying circulating miRNAs as biomarkers for AF, we used a combined analysis of human serum and murine atrial samples with the aim of identifying these biomarkers for predicting AF.Methods and Results:Comprehensive analyses were performed to screen 733 miRNAs in serum from 10 AF patients and 5 controls, and 672 miRNAs in atrial tissue from 6 inducible atrial tachycardia model mice and 3 controls. We selected miRNAs for which expression was detected in both analyses, and their expression levels were changed in the human analyses, the murine analyses, or both. This screening identified 11 candidate miRNAs. Next, we quantified the selected miRNAs using a quantitative RT-PCR in 50 AF and 50 non-AF subjects. The individual assessment revealed that 4 miRNAs (miR-99a-5p, miR-192-5p, miR-214-3p, and miR-342-5p) were significantly upregulated in AF patients. A receiver-operating characteristics curve indicated that miR-214-3p and miR-342-5p had the highest accuracy. The combination of the 4 miRNAs modestly improved the predictive accuracy for AF (76% sensitivity, 80% specificity). CONCLUSIONS: Novel circulating miRNAs were upregulated in the serum of AF patients and might be potential biomarkers of AF.

High Atrial Defibrillation Threshold With Internal Cardioversion Indicates Arrhythmogenicity of Superior Vena Cava in Non-Long-Standing Persistent Atrial Fibrillation.

BACKGROUND: The purpose of this study was to clarify the relation between atrial defibrillation threshold (ADFT) for internal cardioversion (IC) and arrhythmogenicity of the superior vena cava (SVC). METHODS AND RESULTS: A total of 159 consecutive patients (139 male, age 59.9±10.3 years) who underwent radiofrequency catheter ablation of atrial fibrillation (AF) were assessed. IC was performed in 50 patients with non-long-standing persistent AF (non-LSAF) with a purpose-built cardioversion catheter in which direct current is delivered between the right atrium and the coronary sinus. SVC arrhythmogenicity was defined as SVC firing initiating AF, SVC associated with maintenance of AF, or frequent ectopy in the SVC. In all 50 non-LSAF patients, AF termination was obtained on IC during the procedure except in 1 patient with SVC AF. In the patients with ADFT >10 J (n=10), SVC arrhythmogenicity was observed more often than in those with ADFT ≤10 J (n=40; 60% vs. 13%; P=0.004). There were no significant differences between the 2 groups in left atrial diameter (40.8±7.6 vs. 40.6±6.3 mm; P=0.92), persistent AF (33% vs. 50%; P=0.46), or other clinical parameters. The patients who underwent SVC isolation, however, had higher ADFT before SVC isolation than those who did not (15.5±8.8 vs. 9.2±4.4 J; P=0.01). CONCLUSIONS: High IC ADFT is associated with SVC arrhythmogenicity in non-LSAF patients.

Association of intramural fat deposition in the interatrial septum with focal atrial tachyarrhythmias originating near the atrioventricular node.

We describe a case with three focal atrial tachycardias (ATs) and focal atrial fibrillation (AF) originating from the interatrial septum (IAS) near the atrioventricular node (AVN). Contrast-enhanced computed tomography demonstrated the association of fat deposition within the anterior IAS near the AVN with successful ablation sites of these ATs and AF. This is the first report that the intramural fat deposition in the IAS could be associated with the formation of AT and AF re-entry circuits originating near the AVN.

Sacubitril/valsartan attenuates atrial conduction disturbance and electrophysiological heterogeneity with ameliorating fibrosis in mice.

Background: Sacubitril/valsartan (SacVal) has been shown to improve the prognosis of heart failure; however, whether SacVal reduces the occurrence of atrial fibrillation (AF) in heart failure has not yet been elucidated. In this study, we aimed to determine whether SacVal is effective in reducing the occurrence of AF in heart failure and identify the underlying mechanism of its electrophysiological effect in mice. Methods: Adult male mice underwent transverse aortic constriction, followed by SacVal, valsartan, or vehicle treatment for two weeks. Electrophysiological study (EPS) and optical mapping were performed to assess the susceptibility to AF and the atrial conduction properties, and fibrosis was investigated using heart tissue and isolated cardiac fibroblasts (CFs). Results: EPS analysis revealed that AF was significantly less inducible in SacVal-treated mice than in vehicle-treated mice. Optical mapping of the atrium showed that SacVal-treated and valsartan-treated mice restored the prolonged action potential duration (APD); however, only SacVal-treated mice showed the restoration of decreased conduction velocity (CV) compared to vehicle-treated mice. In addition, the electrophysiological distribution analysis demonstrated that heterogeneous electrophysiological properties were rate-dependent and increased heterogeneity was closely related to the susceptibility to AF. SacVal attenuated the increased heterogeneity of CV at short pacing cycle length in atria, whereas Val could not. Histological and molecular evaluation showed that SacVal exerted the anti-fibrotic effect on the atria. An in vitro study of CFs treated with natriuretic peptides and LBQ657, the metabolite and active form of sacubitril, revealed that C-type natriuretic peptide (CNP) combined with LBQ657 had an additional anti-fibrotic effect on CFs. Conclusions: Our results demonstrated that SacVal can improve the conduction disturbance and heterogeneity through the attenuation of fibrosis in murine atria and reduce the susceptibility of AF in heart failure with pressure overload, which might be attributed to the enhanced function of CNP.

Heteroduplex oligonucleotide technology boosts oligonucleotide splice switching activity of morpholino oligomers in a Duchenne muscular dystrophy mouse model.

The approval of splice-switching oligonucleotides with phosphorodiamidate morpholino oligomers (PMOs) for treating Duchenne muscular dystrophy (DMD) has advanced the field of oligonucleotide therapy. Despite this progress, PMOs encounter challenges such as poor tissue uptake, particularly in the heart, diaphragm, and central nervous system (CNS), thereby affecting patient's prognosis and quality of life. To address these limitations, we have developed a PMOs-based heteroduplex oligonucleotide (HDO) technology. This innovation involves a lipid-ligand-conjugated complementary strand hybridized with PMOs, significantly enhancing delivery to key tissues in mdx mice, normalizing motor functions, muscle pathology, and serum creatine kinase by restoring internal deleted dystrophin expression. Additionally, PMOs-based HDOs normalized cardiac and CNS abnormalities without adverse effects. Our technology increases serum albumin binding to PMOs and improves blood retention and cellular uptake. Here we show that PMOs-based HDOs address the limitations in oligonucleotide therapy for DMD and offer a promising approach for diseases amenable to exon-skipping therapy.

Embryological Classification of Arrhythmogenic Triggers Initiating Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is a prevalent multifactorial arrhythmia associated with specific single-nucleotide polymorphisms (SNPs). Pulmonary vein (PV) isolation is an established treatment for AF; however, recurrence risk remains caused by AF triggers beyond the PVs. Understanding the embryological origins of these triggers could improve treatment outcomes. OBJECTIVES: This study aimed to investigate the association between embryologically categorized AF triggers, clinical and genetic backgrounds, and postablation prognosis. METHODS: In cohort 1, comprising 3,067 patients with AF undergoing PV isolation, the clinical characteristics and outcomes were analyzed. Among them, 815 patients underwent genetic analysis using AF-associated SNPs (cohort 2). Patients were delineated based on the developmental origin of the AF triggers: common PV, sinus venosus (SV), and primitive atrium (PA). RESULTS: SV-origin extra-PV AF triggers occurred in 20.3% (n = 622) of patients, whereas PA-origin triggers occurred in 11.9% (n = 365) of patients in cohort 1. Multivariate analysis of cohort 2 revealed that female sex, lower body mass index, absence of hypertension, rs2634073 near PITX2, and rs6584555 in NEURL1 were associated with SV-AF, whereas nonparoxysmal AF and rs2634073 near PITX2 were predictors of PA-AF. The PA group had a significantly higher arrhythmia recurrence rate after repeated procedures than the common PV (HR: 1.75; 95% CI: 1.34-2.29; P < 0.001) and SV-AF (HR: 1.31; 95% CI: 1.19-1.45; P < 0.001) groups with more de novo AF triggers. However, the incidence of adverse events did not differ significantly among the 3 groups. CONCLUSIONS: SV-derived AF triggers may have hereditary factors with a favorable postablation prognosis, whereas PA-derived triggers are linked to AF persistence and poor ablation response. Variants near PITX2 may play a pivotal role in extra-PV triggers.

Risk stratification of atrial fibrillation and stroke using single nucleotide polymorphism and circulating biomarkers.

BACKGROUND: Atrial fibrillation (AF) is the most common sustained arrhythmia, and it causes a high rate of complications such as stroke. It is known that AF begins as paroxysmal form and gradually progresses to persistent form, and sometimes it is difficult to identify paroxysmal AF (PAF) before having stroke. The aim of this study is to evaluate the risk of PAF and stroke using genetic analysis and circulating biomarkers. MATERIALS AND METHODS: A total of 600 adult subjects were enrolled (300 from PAF and control groups). Peripheral blood was drawn to identify the genetic variation and biomarkers. Ten single nucleotide polymorphisms (SNPs) were analyzed, and circulating cell-free DNA (cfDNA) was measured from plasma. Four microRNAs (miR-99a-5p, miR-192-5p, miR-214-3p, and miR-342-5p) were quantified in serum using quantitative RT-PCR. RESULTS: Genotyping identified 4 single nucleotide polymorphisms (SNPs) that were significantly associated with AF (rs6817105, rs3807989, rs10824026, and rs2106261), and the genetic risk score using 4 SNPs showed the area under the curve (AUC) of 0.631. Circulating miRNAs and cfDNA did not show significant differences between PAF and control groups. The concentration of cfDNA was significantly higher in patients with a history of stroke, and the AUC was 0.950 to estimate the association with stroke. CONCLUSION: The risk of AF could be assessed by genetic risk score. Furthermore, the risk of stroke might be evaluated by plasma cfDNA level.

Endoplasmic Reticulum Selective Autophagy Alleviates Anthracycline-Induced Cardiotoxicity.

Background: The administration of anthracycline drugs induces progressive and dose-related cardiac damage through several cytotoxic mechanisms, including endoplasmic reticulum (ER) stress. The unfolded protein response plays a crucial role for mitigating misfolded protein accumulation induced by excessive ER stress. Objectives: We aimed to clarify whether endoplasmic reticulum-selective autophagy machinery (ER-phagy) serves as an alternative system to protect cardiomyocytes from ER stress caused by anthracycline drugs. Methods: Primary cultured cardiomyocytes, H9c2 cell lines, and cardiomyocyte-specific transgenic mice, all expressing ss-RFP-GFP-KDEL proteins, were used as ER-phagy reporter models. We generated loss-of-function models using RNA interference or gene-trap mutagenesis techniques. We assessed phenotypes and molecular signaling pathways using immunoblotting, quantitative polymerase chain reaction, cell viability assays, immunocytochemical and histopathological analyses, and cardiac ultrasonography. Results: The administration of doxorubicin (Dox) activated ER-phagy in ss-RFP-GFP-KDEL-transduced cardiomyocytes. In addition, Dox-induced cardiomyopathy models of ER-phagy reporter mice showed marked activation of ER-phagy in the myocardium compared to those of saline-treated mice. Quantitative polymerase chain reaction analyses revealed that Dox enhanced the expression of cell-cycle progression gene 1 (CCPG1), one of the ER-phagy receptors, in H9c2 cells. Ablation of CCPG1 in H9c2 cells resulted in the reduced ER-phagy activity, accumulation of proapoptotic proteins, and deterioration of cell survival against Dox administration. CCPG1-hypomorphic mice developed more severe deterioration in systolic function in response to Dox compared to wild-type mice. Conclusions: Our findings highlight a compensatory role of CCPG1-driven ER-phagy in reducing Dox toxicity. With further study, ER-phagy may be a potential therapeutic target to mitigate Dox-induced cardiomyopathy.

Role of Inflammation in the Pathogenesis of Atrial Fibrillation.

Atrial fibrillation (AF) is one of the most common arrhythmias encountered in clinical practice. AF is a major risk factor for stroke, which is associated with high mortality and great disability and causes a significant burden on society. With the development of catheter ablation, AF has become a treatable disease, but its therapeutic outcome has been limited so far. In persistent and long-standing AF, the expanded AF substrate is difficult to treat only by ablation, and a better understanding of the mechanism of AF substrate formation will lead to the development of a new therapeutic strategy for AF. Inflammation is known to play an important role in the substrate formation of AF. Inflammation causes and accelerates the electrical and structural remodeling of the atria via pro-inflammatory cytokines and other inflammatory molecules, and enhances the AF substrate, leading to the maintenance of AF and further inflammation, which forms a vicious spiral, so-called "AF begets AF". Breaking this vicious cycle is expected to be a key therapeutic intervention in AF. In this review, we will discuss the relationship between AF and inflammation, the inflammatory molecules included in the AF-related inflammatory process, and finally the potential of those molecules as a therapeutic target.

Gene therapy to terminate tachyarrhythmias.

INTRODUCTION: To date, the treatment option for tachyarrhythmia is classified into drug therapy, catheter ablation, and implantable device therapy. However, the efficacy of the antiarrhythmic drugs is limited. Although the indication of catheter ablation is expanding, several fatal tachyarrhythmias are still refractory to ablation. Implantable cardioverter-defibrillator increases survival, but it is not a curable treatment. Therefore, a novel therapy for tachyarrhythmias refractory to present treatments is desired. Gene therapy is being developed as a promising candidate for this purpose, and basic research and translational research have been accumulated in recent years. AREAS COVERED: This paper reviews the current state of gene therapy for arrhythmias, including susceptible arrhythmias, the route of administration to the heart, and the type of vector to use. We also discuss the latest progress in the technology of gene delivery and genome editing. EXPERT OPINION: Gene therapy is one of the most promising technologies for arrhythmia treatment. However, additional technological innovation to achieve safe, localized, homogeneous, and long-lasting gene transfer is required for its clinical application.

Local Injection of Hydroxyapatite Electret Ameliorated Infarct Size After Myocardial Infarction.

Background: Previous studies showed that hydroxyapatite electret (HAE) accelerates the regeneration of vascular endothelial cells and angiogenesis. This study investigated the effects of HAE in myocardial infarction (MI) model mice. Methods and Results: MI was induced in mice by ligating the left anterior descending artery. Immediately after ligation, HAE, non-polarized hydroxyapatite (HAN), or water (control) was injected into the infarct border myocardium. Functional and histological analyses were performed 2 weeks later. Echocardiography revealed that HAE injection preserved left ventricular systolic function and the wall thickness of the scar, whereas HAN-injected mice had impaired cardiac function and thinning of the wall, similar to control mice. Histological assessment showed that HAE injection significantly attenuated the length of the scar lesion. There was significant accumulation of CD31-positive cells and increased expression of vascular endothelial growth factor (Vegf), intercellular adhesion molecule-1 (Icam1), vascular cell adhesion molecule-1 (Vcam1), hypoxia-inducible factor-1α (Hif1a), and C-X-C motif chemokine ligand 12 (Cxcl12) genes in the infarct border zone of HAE-injected mice. These effects were not induced by HAN injection. Anti-VEGFR2 antibody canceled the beneficial effect of HAE. In vitro experiments in a human cardiovascular endothelial cell line showed that HAE dose-dependently increased VEGFA expression. Conclusions: Local injection of HAE attenuated infarct size and improved cardiac function after MI, probably due to angiogenesis. The electric charge of HAE may stimulate angiogenesis via HIF1α-CXCL12/VEGF signaling.

I536T variant of RBM20 affects splicing of cardiac structural proteins that are causative for developing dilated cardiomyopathy.

RBM20 is one of the genes predisposing to dilated cardiomyopathy (DCM). Variants in the RS domain have been reported in many DCM patients, but the pathogenicity of variants within the RNA-recognition motif remains unknown. Two human patients with the I536T-RBM20 variant without an apparent DCM phenotype were identified in sudden death cohorts. A splicing reporter assay was performed, and an I538T knock-in mouse model (Rbm20I538T) was generated to determine the significance of this variant. The reporter assay demonstrated that the human I536T variant affected the TTN splicing pattern compared to wild-type. In the mouse experiments, Rbm20I538T mice showed different splicing patterns in Ttn, Ldb3, Camk2d, and Ryr2. The expressions of Casq1, Mybpc2, and Myot were upregulated in Rbm20I538T mice, but Rbm20I538T mice showed neither DCM nor cardiac dysfunction on histopathological examination and ultrasound echocardiography. The I536T-RBM20 (I538T-Rbm20) variant changes gene splicing and affects gene expression, but the splicing and expression changes in Ttn and Ca handling genes such as Casq1, Camk2d, and Ryr2 do not cause DCM morphology in the mouse model. KEY MESSAGES: • Two human patients with the I536T-RBM20 variant without a DCM phenotype were identified. • A splicing reporter assay demonstrated that the variant affected the TTN splicing. • Rbm20I538T mice showed neither DCM nor cardiac dysfunction. • Rbm20I538T mice showed different splicing patterns and the gene expressions.

Systemic DNA/RNA heteroduplex oligonucleotide administration for regulating the gene expression of dorsal root ganglion and sciatic nerve.

Neuropathic pain, a heterogeneous condition, affects 7%-10% of the general population. To date, efficacious and safe therapeutic approaches remain limited. Antisense oligonucleotide (ASO) therapy has opened the door to treat spinal muscular atrophy, with many ongoing clinical studies determining its therapeutic utility. ASO therapy for neuropathic pain and peripheral nerve disease requires efficient gene delivery and knockdown in both the dorsal root ganglion (DRG) and sciatic nerve, key tissues for pain signaling. We previously developed a new DNA/RNA heteroduplex oligonucleotide (HDO) technology that achieves highly efficient gene knockdown in the liver. Here, we demonstrated that intravenous injection of HDO, comprising an ASO and its complementary RNA conjugated to α-tocopherol, silences endogenous gene expression more than 2-fold in the DRG, and sciatic nerve with higher potency, efficacy, and broader distribution than ASO alone. Of note, we observed drastic target suppression in all sizes of neuronal DRG populations by in situ hybridization. Our findings establish HDO delivery as an investigative and potentially therapeutic platform for neuropathic pain and peripheral nerve disease.

Sparsely methylated mitochondrial cell free DNA released from cardiomyocytes contributes to systemic inflammatory response accompanied by atrial fibrillation.

Systemic inflammation is assumed to be the consequence and the cause of atrial fibrillation (AF); however, the underlying mechanism remains unclear. We aimed to evaluate the level of cell-free DNA (cfDNA) in patients with AF and AF mimicking models, and to illuminate its impact on inflammation. Peripheral blood was obtained from 54 patients with AF and 104 non-AF controls, and cfDNA was extracted. We extracted total cfDNA from conditioned medium after rapid pacing to HL-1 cells. Nuclear and mitochondrial DNA were separately extracted and fragmented to simulate nuclear-cfDNA (n-cfDNA) and mitochondrial-cfDNA (mt-cfDNA). The AF group showed higher cfDNA concentration than the non-AF group (12.6 [9.0-17.1] vs. 8.1 [5.3-10.8] [ng/mL], p < 0.001). The copy numbers of n-cfDNA and mt-cfDNA were higher in AF groups than in non-AF groups; the difference of mt-cfDNA was particularly apparent (p = 0.011 and p < 0.001, respectively). Administration of total cfDNA and mt-cfDNA to macrophages significantly promoted IL-1ß and IL-6 expression through TLR9, whereas n-cfDNA did not. Induction of cytokine expression by methylated mt-cfDNA was lower than that by unmethylated mt-cfDNA. Collectively, AF was associated with an increased cfDNA level, especially mt-cfDNA. Sparsely methylated mt-cfDNA released from cardiomyocytes may be involved in sterile systemic inflammation accompanied by AF.

DNA/RNA heteroduplex oligonucleotide technology for regulating lymphocytes in vivo.

Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Although oligonucleotide therapy has been proven to be successful in treating several conditions, efficient in vivo delivery of oligonucleotide to lymphocyte populations remains a challenge. Here, we demonstrate that intravenous injection of a heteroduplex oligonucleotide (HDO), comprised of an antisense oligonucleotide (ASO) and its complementary RNA conjugated to α-tocopherol, silences lymphocyte endogenous gene expression with higher potency, efficacy, and longer retention time than ASOs. Importantly, reduction of Itga4 by HDO ameliorates symptoms in both adoptive transfer and active experimental autoimmune encephalomyelitis models. Our findings reveal the advantages of HDO with enhanced gene knockdown effect and different delivery mechanisms compared with ASO. Thus, regulation of lymphocyte functions by HDO is a potential therapeutic option for immune-mediated diseases.