Atrial Tissue Research Articles

Characterization of remodeling processes in the atria of atrioventricular block dogs: Utility as an early-stage atrial fibrillation model

To characterize utility of atrioventricular block (AVB) dogs as atrial fibrillation (AF) model, we studied remodeling processes occurring in their atria in acute (<2 weeks) and chronic (>4 weeks) phases. Fifty beagle dogs were used. Holter electrocardiogram demonstrated that paroxysmal AF occurred immediately after the production of AVB, of which duration tended to be prolonged in chronic phase. Electrophysiological analysis showed that inter-atrial conduction time and duration of burst pacing-induced AF increased in the chronic phase compared with those in the acute phase, but that atrial effective refractory period was hardly altered. Echocardiographic study revealed that diameters of left atrium, right pulmonary vein and inferior vena cava increased similarly in the acute and chronic phases. Histological evaluation indicated that hypertrophy and fibrosis in atrial tissue increased in the chronic phase. Electropharmacological characterization showed that i.v. pilsicainide effectively suppressed burst pacing-induced AF with increasing atrial conduction time and refractoriness of AVB dogs in chronic phase, but that i.v. amiodarone did not exert such electrophysiological effects. Taken together, AVB dogs in chronic phase appear to possess such pathophysiology as developed in the atria of early-stage AF patients, and therefore they can be used to evaluate drug candidates against early-stage AF.

Influence of genetic mutations to atria vulnerability to atrial fibrillation: An in-silico 3D human atria study

Background and ObjectivePersonalized 3D computer models of atria have been extensively implemented in the last yearsas a tool to facilitate the understanding of the mechanisms underlying different forms of arrhythmia, such as atrial fibrillation (AF). Meanwhile, genetic mutations acting on potassium channel dynamics were demonstrated to induce fibrillatory episodes in asymptomatic patients. This research study aims at assessing the effects and the atrial susceptibility to AF of three gain-of-function mutations – namely, KCNH2 T895M, KCNH2 T436M, and KCNE3-V17M – associated with AF outbreaks, using highly detailed 3D atrial models with realistic wall thickness and heterogenous histological properties. MethodsThe 3D atrial model was generated by reconstructing segmented anatomical structures from CT scans of an AF patient. Modified versions of the Courtemanche human atrial myocyte model were used to reproduce the electrophysiological activity of the WT and of the three mutant cells. Ectopic foci (EF) were simulated in sixteen locations across the atrial mesh using an S1-S2 protocol with two S2 basic cycle lengths (BCL) and eleven coupling intervals in order to induce arrhythmias. ResultsThe three genetic mutations at 3D level reduced the APD90. The KCNE3-V17M mutation provoked the highest shortening (55 % in RA and LA with respect to WT), followed by KCNH2 T895M (14 % in RA and 18 % LA with respect to WT)and KCNH2 T436M (7 % in RA and 9 % LA with respect to WT). The KCNE3-V17M mutation led to arrhythmia in 67 % of the cases simulated and in 94 % of ectopic foci considered, at S2 BCL equal to 100 ms. The KCNH2 T436M and KCNH2 T895M mutations increased the vulnerability to AF in a similar way, leading to arrhythmic episodes in 7 % of the simulated conditions, at S2 BCL set to 160 ms. Overall, 60 % of the arrhythmic events generated arise in the left atrium. Spiral waves, multiple rotors and disordered electrical pattern were elicited in the presence of the KCNE3-V17M mutation, exhibiting an instantaneous mean frequency of 7.6 Hz with a mean standard deviation of 1.12 Hz. The scroll waves induced in the presence of the KCNH2 T436M and KCNH2 T895M mutations showed steadiness and regularity with an instantaneous mean frequencies in the range of 4.9 – 5.1 Hz and a mean standard deviation within 0.19 – 0.53 Hz. ConclusionsThe pro-arrhythmogenicity of the KCNE3-V17M, KCNH2 T895M and KCNH2 T436M mutations was studied and proved on personalized 3D cardiac models. The three genetic mutations were demonstrated to increase the predisposition of atrial tissue to the formation of AF-susceptible substrate in different ways based on their effects on electrophysiological properties of the atria.

Impaired Intracellular Calcium Buffering Contributes to the Arrhythmogenic Substrate in Atrial Myocytes From Patients With Atrial Fibrillation.

Alterations in the buffering of intracellular Ca2+, for which myofilament proteins play a key role, have been shown to promote cardiac arrhythmia. It is interesting that although studies report atrial myofibrillar degradation in patients with persistent atrial fibrillation (persAF), the intracellular Ca2+ buffering profile in persAF remains obscure. Therefore, we aimed to investigate the intracellular buffering of Ca2+ and its potential arrhythmogenic role in persAF. Transmembrane Ca2+ fluxes (patch-clamp) and intracellular Ca2+ signaling (fluo-3-acetoxymethyl ester) were recorded simultaneously in myocytes from right atrial biopsies of sinus rhythm (Ctrl) and patients with persAF, alongside human atrial subtype induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs). Protein levels were quantified by immunoblotting of human atrial tissue and induced pluripotent stem cell-derived cardiac myocytes. Mouse whole heart and atrial electrophysiology were measured on a Langendorff system. Cytosolic Ca2+ buffering was decreased in atrial myocytes of patients with persAF because of a depleted amount of Ca2+ buffers. In agreement, protein levels of selected Ca2+ binding myofilament proteins, including cTnC (cardiac troponin C), a major cytosolic Ca2+ buffer, were significantly lower in patients with persAF. Small interfering RNA (siRNA)-mediated knockdown of cTnC (si-cTNC) in atrial iPSC-CM phenocopied the reduced cytosolic Ca2+ buffering observed in persAF. Si-cTnC treated atrial iPSC-CM exhibited a higher predisposition to spontaneous Ca2+ release events and developed action potential alternans at low stimulation frequencies. Last, indirect reduction of cytosolic Ca2+ buffering using blebbistatin in an ex vivo mouse whole heart model increased vulnerability to tachypacing-induced atrial arrhythmia, validating the direct mechanistic link between impaired cytosolic Ca2+ buffering and atrial arrhythmogenesis. Our findings suggest that loss of myofilament proteins, particularly reduced cTnC protein levels, causes diminished cytosolic Ca2+ buffering in persAF, thereby potentiating the occurrence of spontaneous Ca2+ release events and atrial fibrillation susceptibility. Strategies targeting intracellular buffering may represent a promising therapeutic lead in persAF management.

Dapagliflozin: A sodium–glucose cotransporter 2 inhibitor, attenuates angiotensin II-induced atrial fibrillation by regulating atrial electrical and structural remodeling

AimAtrial fibrillation (AF), the most common arrhythmia, is characterized by atrial electrical and structural remodeling. Previous studies have found that sodium–glucose cotransporter 2 inhibitor (SGLT2i) can protect myocardium in a glucose independent mechanism. But the role of SGLT2i in regulating AF remains largely unknown. This study, we aimed to investigate the effect of Dapagliflozin (DAPA) in reducing AF susceptibility via inhibiting electrical and structural remodeling. MethodThe mouse model was established by Angiotensin II (2000 ng/kg/min) infusion for 3 weeks, and an in vitro model was generated by stimulating HL-1 and primary mouse fibroblast with Ang II (1 μM) for 24 h. Programmed electrical stimulation, ECG and whole-cell patch clamp were used to detect DAPA effect on atrial electrical remodeling induced by Ang II. To observe DAPA effect on atrial structural remodeling induced by Ang II, we used echocardiographic, H&E and Masson staining to evaluate atrial dilation. To further explore the protective mechanism of DAPA, we adopt in silico molecular docking approaches to investigate the binding affinity of Ang II and CaMKII at Met-281 site. Western blot was to detect expression level of CaMKII, ox-CaMKII, Nav1.5, Kv4.3, Kv4.2, Kchip2, Kir2.1 and Cx40. ResultsAng II induced AF, atrial dilatation and fibrosis, led to atrial electrical and structural remodeling. However, these effects were markedly abrogated by DAPA treatment, a specific SGLT2i. Our observation of atrial electrical activity in mice revealed that DAPA could rescue the prolonged action potential duration (APD) and the abnormal currents of IK1, Ito and INaL triggered by Ang II infusion. DAPA could reduce the binding affinity of Ang II and CaMKII at Met-281 site, which indicated that DAPA may directly alleviate the activation of CaMKII caused by Ang II. DAPA could reduce the upregulation of ox-CaMKII caused by Ang II infusion in atrial tissues. Moreover, DAPA also ameliorated the aberrant expression levels of electrical activity related proteins (Nav1.5, Kv4.3, Kv4.2, Kchip2, Kir2.1 and Cx40) and fibrosis related signal pathways (TGF-β1, p-smad/smad) caused by Ang II. Furthermore, we confirmed that DAPA, as well as other SGLT2i (EMPA, CANA), could reverse these abnormalities caused by Ang II incubation in HL-1 cells and primary mouse fibroblasts, respectively. ConclusionOverall, our study identifies DAPA, a widely used SGLT2i, contributes to inhibiting Ang II-induced ox-CaMKII upregulation and electrical and structural remodeling to reduce AF susceptibility, suggesting that DAPA may be a potential therapy of treating AF.

Distribution of peak frequency and omnipolar voltage in electrograms across the atrial body and thoracic veins in a normal heart.

The innovative peak frequency mapping facilitates the quantification of electrogram sharpness. However, reference values for normal atrial tissue are currently undefined. In this study, we explored the distribution of peak frequency and omnipolar peak-to-peak voltage (V-max) in a normal heart. Twenty-two patients with structurally normal heart were included. Either the right atrium (RA) and superior vena cava (SVC) or the left atrium (LA) and pulmonary veins (PVs) were mapped during sinus rhythm. In total, 13,654 points in the RA and 4143 points in the SVC from 15 patients and 4662 points in the LA and 2761 points in PVs from 7 patients were analyzed. The correlation between peak frequency and V-max was weak (R = 0.223). The median peak frequency was larger in the SVC than in the RA (441 [358-524] Hz vs. 358 [291-441] Hz, P < 0.0001) and in PVs than in the LA (346 [253-441] Hz vs. 323 [262-397] Hz, P < 0.0001). Conversely, the median V-max was smaller in the SVC than in the RA (1.96 [0.77-3.75] mV vs. 4.11 [2.10-6.83] mV, P < 0.0001) and in PVs than in the LA (1.16 [0.33-3.17] mV vs. 4.42 [2.63-6.84] mV, P < 0.0001). More than 95% of peak frequencies were > 174Hz in the RA and > 185Hz in the LA, and > 95% of V-maxes were > 0.52 and > 1.07mV in the RA and LA, respectively. Given the limited correlation between peak frequency and V-max, and recognizing their potential to provide distinct information, they can be used complementarily. Employing these parameters to extract varied insights can provide comprehensive understandings of tissue characteristics.

In Silico TRials guide optimal stratification of ATrIal FIbrillation patients to Catheter Ablation and pharmacological medicaTION: the i-STRATIFICATION study.

Patients with persistent atrial fibrillation (AF) experience 50% recurrence despite pulmonary vein isolation (PVI), and no consensus is established for secondary treatments. The aim of our i-STRATIFICATION study is to provide evidence for stratifying patients with AF recurrence after PVI to optimal pharmacological and ablation therapies, through in silico trials. A cohort of 800 virtual patients, with variability in atrial anatomy, electrophysiology, and tissue structure (low-voltage areas, LVAs), was developed and validated against clinical data from ionic currents to electrocardiogram. Virtual patients presenting AF post-PVI underwent 12 secondary treatments. Sustained AF developed in 522 virtual patients after PVI. Second ablation procedures involving left atrial ablation alone showed 55% efficacy, only succeeding in the small right atria (<60 mL). When additional cavo-tricuspid isthmus ablation was considered, Marshall-PLAN sufficed (66% efficacy) for the small left atria (<90 mL). For the bigger left atria, a more aggressive ablation approach was required, such as anterior mitral line (75% efficacy) or posterior wall isolation plus mitral isthmus ablation (77% efficacy). Virtual patients with LVAs greatly benefited from LVA ablation in the left and right atria (100% efficacy). Conversely, in the absence of LVAs, synergistic ablation and pharmacotherapy could terminate AF. In the absence of ablation, the patient's ionic current substrate modulated the response to antiarrhythmic drugs, being the inward currents critical for optimal stratification to amiodarone or vernakalant. In silico trials identify optimal strategies for AF treatment based on virtual patient characteristics, evidencing the power of human modelling and simulation as a clinical assisting tool.

Small non-coding RNA signatures in atrial appendages of patients with atrial fibrillation.

The development of high-throughput technologies has enhanced our understanding of small non-coding RNAs (sncRNAs) and their crucial roles in various diseases, including atrial fibrillation (AF). This study aimed to systematically delineate sncRNA profiles in AF patients. PANDORA-sequencing was used to examine the sncRNA profiles of atrial appendage tissues from AF and non-AF patients. Differentially expressed sncRNAs were identified using the R package DEGseq 2 with a fold change >2 and p < 0.05. The target genes of the differentially expressed sncRNAs were predicted using MiRanda and RNAhybrid. Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. In AF patients, the most abundant sncRNAs were ribosomal RNA-derived small RNAs (rsRNAs), followed by transfer RNA-derived small RNAs (tsRNAs), and microRNAs (miRNAs). Compared with non-AF patients, 656 rsRNAs, 45 miRNAs, 191 tsRNAs and 51 small nucleolar RNAs (snoRNAs) were differentially expressed in AF patients, whereas no significantly differentially expressed piwi-interacting RNAs were identified. Two out of three tsRNAs were confirmed to be upregulated in AF patients by quantitative reverse transcriptase polymerase chain reaction, and higher plasma levels of tsRNA 5006c-LysCTT were associated with a 2.55-fold increased risk of all-cause death in AF patients (hazard ratio: 2.55; 95% confidence interval, 1.56-4.17; p < 0.001). Combined with our previous transcriptome sequencing results, 32 miRNA, 31 snoRNA, 110 nucleus-encoded tsRNA, and 33 mitochondria-encoded tsRNA target genes were dysregulated in AF patients. GO and KEGG analyses revealed enrichment of differentially expressed sncRNA target genes in AF-related pathways, including the 'calcium signaling pathway' and 'adrenergic signaling in cardiomyocytes.' The dysregulated sncRNA profiles in AF patients suggest their potential regulatory roles in AF pathogenesis. Further research is needed to investigate the specific mechanisms of sncRNAs in the development of AF and to explore potential biomarkers for AF treatment and prognosis.

Role of NOD1 in atrial myopathy associated with heart failure

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funding: Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ISCIII [PI17/01344 and PI20/01482]), Ministry of Education (FPU19/01973), SEC, Proyecto Traslacional 2019, FEDER, FSE, CIBER-CV, a network funded by ISCIII Introduction Heart failure (HF) is a complex syndrome that constitutes one of the leading causes of cardiovascular death worldwide. Although multifactorial mechanisms appear to be responsible of atrial and ventricular HF-myopathy, the innate immune system and the derived inflammatory response have gained much attention in the last years. Previous studies from our group have demonstrated that the nucleotide-binding oligomerization domain 1 (NOD1), a receptor of the innate immune system, plays a role in HF left ventricular (LV) dysfunction. However, its role in HF-atrial myopathy remains completely unknown. The main purpose of our study is to determine the role of NOD1 in human and experimental HF-atrial myopathy. Methods and Results The study was performed in two cohorts of individuals who underwent heart valve repair surgery, 51 non-failing individuals (NF) with LV ejection fraction (LVEF)&amp;gt;50% and NT-ProBNP levels&amp;lt;200pg/mL and 41 subjects with medium to severe reduced EF (HF)&amp;lt;50% and NT-ProBNP levels&amp;gt;200pg/mL. An informed consent was signed by all patients. Atrial myocardial tissue was excised from the atrial appendage and prepared for biochemical and histological studies. Echocardiography analysis showed that left and right atrial areas (LA and RA) were significantly increased in the HF group compared to NF, while LA and RA reservoir functions were significantly decreased in HF. Immunostaining demonstrated NOD1 location in atrial cardiomyocytes, being the protein levels of NOD1 and its specific adaptor RIP2 significantly increased in atrial tissue obtained from HF vs. NF. Although the prevalence of atrial fibrillation (AF) was significantly higher in the HF group compared to NF, the upregulation of NOD1 was not conditioned by the presence of AF. Similarly, pigs that underwent transverse aortic constriction (TAC) or proximal left circumflex coronary artery occlusion involving the LA branch (LAI), showed an upregulation of NOD1 pathway in atrial tissue, compared with the sham operated animals. Interestingly, an atrial upregulation of NOD1 axis was also observed in wild-type TAC mice, whereas Nod1-/- knockout mice showed a prevention in both structural and functional atrial remodelling induced by TAC, mainly by a prevention of intracellular calcium mishandling in isolated atrial cardiomyocytes. Finally, wild-type mice injected i.p. for 3 days with 3.3 mg/kg with a selective activator of NOD1, showed increased structural atrial remodelling and intracellular calcium mishandling compared to the vehicle-treated group. Conclusions Our results demonstrate that NOD1 axis is upregulated in human, swine and murine atrial failing myocardium. Genetic deletion of NOD1 prevented HF-atrial structural and functional remodelling, mainly by avoiding intracellular calcium mishandling, whereas its pharmacological activation induced atrial dilation and intracellular Ca2+ mishandling. All these data uncover NOD1 as a new player in atrial HF-remodelling.

Asprosin as a potential new paracrine regulator in the heart

Abstract Funding Acknowledgements None. Background Asprosin was recently described as a novel hormone that is released by white adipose tissue upon fasting. However, a functional role of asprosin in human cardiovascular tissues has not been yet investigated at the tissue level. Asprosin is the C-terminal propeptide of the extracellular matrix (ECM) glycoprotein fibrillin-1 and is cleaved off from the pro-fibrillin-1 precursor by the endopeptidase furin. Fibrillin-1 is expressed in the heart and aorta, and FBN1 mutations are causative for aortic aneurysm formation and cardiomyopathy. Whether asprosin is also present in cardiac tissues and utilized by cardiac resident cells remains unknown. Purpose In the present study, we investigated the localization and distribution of asprosin protein in human atrial and ventricular heart tissue. Methods Human heart tissue was obtained from patients undergoing aortic valve replacement. In addition, we used ventricular tissue from mice. Cryosections from human (n=8) and mouse (n=8) heart samples were analysed by immunohistochemistry. The anti-asprosin antibodies were raised against human or mouse asprosin respectively and applied in combination with anti-CD31/PECAM, an endothelial cell marker, or anti-sarcomeric alpha-actinin, a Z-line marker. The immunofluorescence of incubated specimens was examined by confocal microscopy. Results Our data suggest that asprosin is localized in myocardium in different cell types: cardiomyocytes (Figure 1 a-d), arterial smooth muscle cells (Figure 1 a), micro- and vascular endothelial cells (Figure 1 d), and with the lowest intensity, in periarterial located epicardial adipocytes (Figure 1 e). In cardiomyocytes, incubation with anti-asprosin resulted in a high signal intensity. Asprosin immunoreactivity was observed: in intercalary discs (Figure 1 b-d), as striated pattern in the sarcoplasm, and as weak signal in the perinuclear area (Figure 1 b, c). In human tissue, the anti-asprosin signal was about four times higher in intercalated discs than in the sarcoplasm. The distribution pattern in ventricular cardiomyocytes of mice were similar. Conclusion Our results show that in human myocardium, asprosin localises mainly intracellular. Asprosin accumulations were found in intercalary discs. In short, asprosin was found at locations that are known to be crucial for regulation of cardiac function. Intracellular presence of asprosin may indicate cellular utilization by uptake from extracellular asprosin pools. Whether these pools are replenished exclusively by resident cells or by other tissues such as WAT is currently unknown. However, further research is needed to prove this hypothesis. Figure. 1. Confocal microscopy of asprosin localization in human myocardium. Cryosections incubated with anti-asprosin (red) or combined with anti-alpha actinin (sarcomeric) or anti-CD31/PECAM (green) antibodies and DAPI (blue, nuclei). SM: vascular smooth muscle cells, BV: blood vessels, Arrowheads: Intercalated disks.

Functional substrate analysis in patients with persistent atrial fibrillation.

The aim of this study was to describe the correlation between atrial electrogram duration map (AEDUM), spatiotemporal electrogram dispersion (STED) and low voltage areas (LVA) in patients with persistent atrial fibrillation (PsAF). The degree of left atrial (LA) tissue remodelling and augmented anisotropic conduction is one of the major issues related to PsAF ablation outcome. This study enrolled consecutive patients with PsAF undergoing pulmonary vein isolation. In all patients, voltage, AEDUM and STED maps were created, and the correlation was reported between these three mapping methods. A total of 40 patients with PsAF were enrolled. The mean age was 62.2 ± 7.4years, and males were 72.5% (n = 29). The overall bipolar voltage of the LA was 3.06 ± 1.87mV. All patients had at least one AEDUM area (overall AEDUM area: 21.8 ± 8.2 cm2); the mean longest electrogram (EGMs) duration was 90 ± 19ms. STED areas with < 120ms was 46.3 ± 20.2 cm2 which covered 45 ± 22% of the LA surface. AEDUM and STED areas were most frequently reported on the roof, the anterior wall and the septum. The extension of the AEDUM areas was significantly smaller than STED areas with CL < 120ms (21.8 ± 8.2 vs 46.3 ± 20.2; p-value < 0.0001). In 24 patients (60%), AEDUM areas was entirely included in the STED areas with CL < 120ms. In the three (7.5%) patients with LVA, no correspondence with STED and AEDUM was noted. AEDUM and STED maps allow to identify areas of conductive dysfunction as a possible atrial substrate even if a normal voltage is detected.

Leaky RyR2 channels as pro-arrhythmic trigger in the PKP2-deficient atrial myocardium

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): American Heart Association National Institutes of Health Introduction Atrial arrhythmias, like atrial fibrillation, occur in 20–40% of patients with Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). Atrial fibrillation in ARVC patients is often associated with an increased risk of sustained ventricular arrhythmias and inappropriate ICD shocks. However, the pathophysiology behind the development of atrial arrhythmias in ARVC is far from clear. A genome-wide association study, based on UK-biobank data, revealed a link between atrial fibrillation and variants in the desmosomal gene plakophilin-2 (PKP2). Previous studies show that PKP2 is necessary to maintain the transcription of genes that control intracellular calcium (Ca2+) cycling and that loss of PKP2 expression in adult mouse ventricular tissue leads to disruption of intracellular Ca2+ homeostasis. In this study, we test the hypothesis that loss of PKP2 expression leads to pro-arrhythmic changes in atrial myocytes. Methods Experiments were carried out using a cardiac-specific, tamoxifen (TAM)-activated PKP2 knockout murine model (PKP2-cKO). Cre-negative, flox-positive, TAM-injected littermates were used as controls. We used RNA sequencing, quantitative imaging modalities, as well as biochemical and electrophysiological methods to study the functional and structural properties of atrial PKP2cKO tissue. Studies were carried out 21 days post-tamoxifen injection, a time point at which an arrhythmogenic cardiomyopathy of right ventricular predominance is manifest. Results In contrast to the previously-reported observations in PKP2cKO ventricular myocytes, atrial PKP2cKO myocytes presented no significant differences in Ca2+ transient dynamics, SR load and action potential duration. However, PKP2cKO atrial myocytes showed an increased frequency and amplitude of Ca2+ sparks, in combination with increased diastolic Ca2+ levels as well as an increase in cellular ROS levels. RNAseq data showed an under-representation of mRNA for Integrinα7, a stabilizer of Ryr2 that reduces its phosphorylation. Separate studies showed that PKP2cKO atrial myocytes present impaired relaxation and enhanced sarcomere shortening, a finding consistent with a downregulation in the expression of Myosin Binding Protein C. Isoproterenol (ISO) exposure further increased the frequency of Ca2+ sparks and the levels of diastolic Ca2+, and these effects were reversed by acute (30 minute) in vivo treatment with Ryanodex (medical grade Dantrolene), a known RyR blocker. Conclusion Loss of PKP2 expression affects cellular Ca2+ handling and electrophysiology differently in the atrial versus ventricular myocardium. We speculate that an increased probability of opening of Ryr2 channels, caused by ROS-induced RyR2 oxidation and/or Integrinα7-induced RyR2 phosphorylation, serve as pro-arrhythmic trigger in the PKP2-deficient atrial myocardium, that these effects can be amplified by a catecholaminergic input and that RyR blockers can dampen these pro-arrhythmic effects.

2-Aminoethoxydiphenyl borate prolongs atrial and shortens ventricular action potential duration through modulation of ion channels in the sarcolemma

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): PPP allowance, Health holland ZonmW Background 2-Aminoethoxydiphenyl Borate (2-APB) has been shown to prevent atrial fibrillation in animal models, supposedly through its inhibitory action on the intracellular IP3 receptor and subsequent calcium regulation. However, 2-APB may modulate other ion channels or transporters and the cellular mechanisms associated with the prevention of atrial fibrillation are not well established. We aimed at unravelling the cellular effects of 2-APB on atrial and ventricular action potential and currents. Methods Rabbit (New Zealand White) left atrial and ventricular cardiomyocytes were isolated from Langendorff-perfused hearts by enzymatic dissociation. Adult human atrial cardiomyocytes were obtained by enzymatic dissociation from left atrial appendages (LAA) routinely removed from AF patients undergoing minimally invasive surgical pulmonary vein isolation. Action potentials (APs) and K+ currents were recorded at 36.5 °C with the amphotericin-B-perforated or whole cell patch clamp technique. RNA was isolated from left atrial and ventricular tissue and sequenced on an Illumina HiSeq4000 platform. Results In rabbit atrial cardiomyocytes, extracellular application of 5 uM 2-APB had no effect on action potential duration (APD), but extracellular application of 50 uM 2-APB prolonged the APD by 40% and inhibited an outward K+-current. Inversely, extracellular application of 5 uM 2-APB shortened the APD of rabbit left ventricular cardiomyocytes by 20% while extracellular application 50 uM 2-APB did not prolong the APD. Additionally, 2-APB did not modulate K+ currents in ventricular rabbit cardiomyocytes. Intracellular application (in the patch pipet) of 50 uM 2-APB did not have any effects on atrial APD, suggesting that IP3 receptor inhibition is not involved. However, subsequent application of 50 uM 2-APB in the bath prolonged the APD by 40% indicating that 2-APB prolongs atrial APD by inhibiting one or more K+ channel types in the sarcolemmal membrane from the extracellular site. RNA sequencing analysis revealed potential targets channel including TRP and Kv channels differentially expressed in atria versus ventricle. In human atrial cardiomyocytes from AF patients, application of 50 uM 2-APB prolonged APD by 25%. Conclusions 2-APB prolongs rabbit and human atrial APD and shortens rabbit ventricular APD in a concentration dependent manner, most likely due to modulation of sarcolemmal K+ channels, differentially expressed in the two cell types. Pharmacological studies with 2-APB may reveal novel cellular mechanisms identifying potential new targets for the treatment of atrial fibrillation.

Loss of pitx2c causes early alterations in atrial calcium handling in zebrafish

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science, Innovation and Universities, Spain (PID2019-111456RB-100); Consejería de Educación, Cultura y Deportes, Junta de Comunidades de Castilla-La Mancha (SBPLY/19/180501/000223, co-funded by EU FEDER-ERDF) and grants for research groups from University of Castilla-La Mancha (2019-GRIN-27019, 2020-GRIN-29186 and 2022-GRIN-34301, co-funded by EU FEDER-ERDF) Introduction Atrial fibrillation (AF) is the most common cardiac arrhythmia in humans. Genome-wide association studies revealed that AF is related to sequence variants in chromosomic regions in close proximity to the transcription factor PITX2, involved in cardiac development and asymmetry, and in morphogenesis of pacemaker regions such as the sinoatrial region and the atrioventricular canal, which receive significant input from the autonomic nervous system. AF patients showed reduced PITX2 mRNA levels. PITX2-/- atrial tissue showed lower expression of CACNA1C accompanied by lower L-type Ca2+ current density. A recent study revealed AF-like phenotypes, including fibrosis, structural and metabolic alterations, arrhythmia, and cardiac conduction defects in adult pitx2c -/- zebrafish. Nevertheless, Ca2+ handling alterations remain unexplored in this zebrafish model. Purpose In this study we investigated cardiac Ca2+ handling in pitx2c deficient zebrafish. Methods We used the transgenic zebrafish line Tg(myl7:Twitch-4) expressing a fluorescent Ca2+ indicator in the heart. We outcrossed pitx2c+/- fish with Tg(myl7:Twitch-4) pitx2c+/- and studied Ca2+ dynamics and cardiac function by videomicroscopy in the beating hearts of 5 days post fertilization (dpf) larvae. Larvae were treated with 100 µM carbachol to assess differential response to parasympathetic stimulation in pitx2c deficient larvae. Results We observed sinoatrial pauses in 40 % of pitx2c+/- larvae accompanied by an increase in heart rate variability. In addition, pitx2c+/- and pitx2c-/- larvae at 5 dpf showed differential response to carbachol, a cholinergic agonist, compared to their wild-type siblings. Moreover, carbachol significantly decreased heart rate in pitx2c+/- and pitx2c-/- 5dpf larvae compared to siblings. Furthermore, we also observed a reduction of the amplitude of Ca2+ transients due to a decrease in the systolic Ca2+ levels in the atrium of pitx2c+/- larvae. The rise time of the atrial Ca2+ transient was prolonged, with a decrease in the rise slope. These data suggest a lower Ca2+ entry in the atrium. Interestingly, pitx2c+/- and wild-type larvae showed similar ventricular Ca2+ handling and contractility. Conclusion These results suggest that loss of pitx2c induce early Ca2+ alterations in the atrium as well as sinoatrial dysfunction in zebrafish during embryogenesis. Furthermore, pitx2c deficiency in zebrafish larvae possibly alters autonomic innervation leading to differentiated cardiac response to carbachol.

The Relationship Between Cardiac CT-based Left Atrial Structure and Epicardial Adipose Tissue and Postablation Atrial Fibrillation Recurrence Within 2 Years.

The aim of this study was to explore the association of cardiac CT-based left atrium (LA) structural and functional parameters and left atrial epicardial adipose tissue (LA-EAT) parameters with postablation atrial fibrillation (AF) recurrence within 2 years. Contrast-enhanced cardiac CT images of 286 consecutive AF patients (median age: 65 y; 97 females) who underwent initial ablation between June 2018 and June 2020 were retrospectively analyzed. Structural and functional parameters of LA, including maximum and minimum volume and ejection fraction of LA and left atrial appendage (LAA), and LA-EAT volume, were measured. The body surface area indexed maximum and minimum volume of LA (LAVImax, LAVImin) and LAA (LAAVImax, LAAVImin), and LA-EAT volume index (LA-EATVI) were calculated. Independent predictors of AF recurrence were determined using Cox regression analysis. The clinical predictors were added to the imaging predictors to build a combined model (clinical+imaging). The predictive performance of the clinical, imaging, and combined models was assessed using the area under the receiver operating characteristics curve (AUC). A total of 108 (37.8%) patients recurred AF within 2 years after ablation at a median follow-up of 24 months (IQR=11, 32). LA and LAA size and LA-EAT volume were significantly increased in patients with AF recurrence (P<0.05). After the multivariable regression analysis, LA-EATVI, LAAVImax, female sex, AF duration, and stroke history were independent predictors for AF recurrence. The combined model exhibited superior predictive performance compare to the clinical model (AUC=0.712 vs. 0.641, P=0.023) and the imaging model (AUC=0.712 vs. 0.663, P=0.018). Cardiac CT-based LA-EATVI and LAAVImax are independent predictors for postablation AF recurrence within 2 years and may provide a complementary value for AF recurrence risk assessment.

Evaluation of left atrial function and tissue after pulmonary vein isolation - initial results of The EVALUATE-PVI STUDY

Abstract Background Pulmonary vein (PV) isolation (PVI) by catheter ablation is a well-established treatment for atrial fibrillation (AF). Currently, the direct and long-term impact of ablation injury on left atrial (LA) function and tissue is poorly understood. Purpose The aim of this study is to evaluate the acute and long-term impact of radiofrequency (RF) PVI ablation on LA function and corresponding tissue changes as assessed with cardiac magnetic resonance imaging (CMR). Methods Seventeen patients (76.5% male, 60.8±9.7 years) with AF who underwent primo RF PVI as part of the EVALUATE-PVI STUDY underwent three sequential CMR scans prior to PVI, and &amp;lt;72 hours and 3 months after PVI ablation. T2-weighted turbo spin echo imaging was applied to assess post-ablation edema. High-resolution three-dimensional (3D) late gadolinium-enhancement (LGE) images were acquired to study acute LA wall injury and chronic fibrosis formation. LA volume and function were derived from two-chamber and four-chamber SSFP cine images. Maximum and minimal LA volume was indexed to body surface area to compute LAVimax and LAVimin. Longitudinal LA strain was calculated using feature tracking, and subdivided into reservoir, conduit, and contractile strain. Results Acutely post-PVI, T2w images showed increased signal intensity and thickening throughout the entire LA wall. LGE images showed diffuse increased signal intensity, without distinct focal high signal intensity areas (Fig. 1). Functional assessment at this stage showed significantly reduced LA emptying fraction (EF), LA reservoir, and contractile strain compared to pre-PVI (54.8±11.1% vs. 48.5±7.9%, p=0.01; 18.6±5.6% vs. 14.6±2.0%, p&amp;lt;0.01; 8.5±3.5% vs. 5.2±1.8%, p&amp;lt;0.001, respectively), while LA conduit strain, LAVimin, and LAVimax did not differ significantly (Fig. 2). The LGE images acquired at 3 months, displayed clear, focal, high signal intensity areas surrounding the PVs. At this stage post-PVI, a significant increase in LA strain was observed (LA reservoir strain 14.6±2.0% vs. 16.9±3.5%, p&amp;lt;0.01; and contractile strain 5.2±1.8% vs. 6.7±2.9%, p&amp;lt;0.01; respectively). LAVimax was significantly reduced at 3 months compared to acutely post-PVI (Fig. 2). LAVimin and LA conduit strain did not differ between the pre- and post-PVI stages. Conclusion PVI ablation resulted in temporary reduced LA function acutely post-PVI, likely due to edema and thickening of the LA wall. At three months, LGE images revealed clearly visible increased signal intensity in the ablated segments. At this stage, LA function improved, though full recovery did not occur as evidenced by the persistent slightly reduced contractile strain, possibly due to the ablation-induced loss of LA myocardium and formation of fibrosis. The decrease in LAVimax indicates reverse remodeling as a result of the ablation.

Source-sink mismatch and conduction block: simulation study

Abstract Background Source-sink mismatch is one of the key concepts in arrhythmia mechanism such as relationship between pulmonary veins and the left atrium in atrial fibrillation. We sought to find the condition for conduction block in various geometries. Methods To simulate cardiac atrial tissue, the finite element method was used to discretize the governing equation in a 2D mesh. We evaluated under the following conditions: 6 sizes of source width (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mm), 64 types of angle (θ) at the junction (30-330 degrees), 3 conditions of conduction velocity (CV) (x0.5, x0.75, and x1 of normal CV of atrial myocardium). Results Conduction block was not observed with source width &amp;gt; 0.5 mm in any settings. In reduced CV condition, conduction block was not observed in θ &amp;lt; 300 degrees when source width &amp;gt; 0.3 mm. The higher CV, conduction block was observed in the narrower θ. In cases of CV of x1.0 and x0.75, conduction block was observed in θ &amp;lt; 180 degrees when source width &amp;lt; 0.3 mm. The smaller source width, conduction block was observed in the narrower θ. The wider θ, the easier conduction block occurred. Conclusion Conditions for conduction block due to source-sink mismatch are as follows: smaller source width, higher CV, and wider θ. Source with width &amp;gt; 0.5 mm may not be associated with source-sink mismatch in the atrial tissue. CV slowing by antiarrhythmic drugs can be effective in the aspect of source-sink mismatch.

Changes in local endocardial electrograms immediately after PFA show dose-dependent variations

Abstract Background/Introduction Pulsed field ablation (PFA) is a novel nonthermal cardiac ablation technology based on irreversible electroporation. The behaviour of local electrograms after PFA has been poorly characterized and it remains unknown if it is possible to differentiate between different electroporation levels based on local electrogram analysis. Purpose The objective of this study was to characterize the alterations in bipolar and unipolar endocardial electrograms after performing PFA at different doses. Methods Five anesthetized pigs were submitted to PFA treatments. Endovascular access to the right atrium was gained through the femoral vein to create a line of ablation between the superior and inferior vena cava using a focal monopolar catheter. The catheter was switched between a conventional cardiac navigation system (CARTO system) and a custom PFA generator. Two different PFA doses were used: a high dose (HD-1600V) at top and bottom of the line and a low dose (LD-1000V) in the central part of the ablation line. Bipolar and unipolar electrograms were recorded with the ablation catheter before and immediately after each ablation. Additionally, high-density electroanatomical maps were acquired with a multielectrode catheter before and immediately after the procedures. Four weeks later, the animals were remapped again and subsequently euthanized. Atrial tissue was removed and processed to identify the presence of fibrosis in the previously ablated lesions. Results We performed 106 ablations (84 high dose vs. 22 low dose). Four weeks later, histological staining of chronic lesions revealed that the HD lesions were larger and more fibrotic than the LD (Figure 1). Immediately after each ablation R/S-wave amplitudes and their corresponding |dV/dt|max decreased for the bipolar and unipolar electrograms. The percentages of bipolar electrogram reduction HD vs. LD were: R wave 73% vs. 39%, S wave 68% vs. 37% and |dV/dt|max 81% vs. 38%; 1 way-ANOVA p&amp;lt;0.05 for all, Figure 2). The R-wave amplitude in M1-M2 was significantly lower in HD compared to LD (HD vs. LD: 0.3 ± 0.2 mV vs. 0.7 ± 0.6 mV; 1 way-ANOVA p&amp;lt;0.01). S-wave and |dV/dt|max of unipolar electrograms presented a similar behaviour (M2 |dV/dt|max HD vs. LD: -116 ± 118 mV/s vs. -260 ± 267 mV; 1 way-ANOVA p&amp;lt;0.01). Conclusion Local electrograms display PFA dose-dependent variations. This information could result useful to create new metrics to assess in real time the performance of PFA systems.Figure 1.EAM maps and histology studyFigure 2.EGM signals and statistics

Using the impedance map to identify non-conductive structures during cavo-tricuspidal isthmus ablation

Abstract Background The ablation of the cavo-tricuspidal isthmus (CTI) is often affected by the anatomical variability of structures made of non-conductive tissue (NCS): the Eustachian valve, tricuspid valve leaflet and Tebesio's valve, the presence of which may be associated with failure of the isthmic block despite a large number of erogations and post-ablation recurrences. Since these structures have different conductive properties from atrial tissue, it is plausible that they are characterised by their own tissue impedance, and can therefore be identified through an impedance map. Objectives The objectives of the study were: 1) to characterise CTI by impedance map, in order to identify NCS; 2) to perform ablation of CTI aimed at isolating only conductive tissue by combining impedance map with the Maximum Voltage Gradient (MVG) technique, in order to evaluate the efficacy of this strategy in acute and during follow-up. Methods From 01/2021 to 09/2023 impedance map and CTI substrate map was performed, without the use of fluoroscopy, in all patients undergoing ablation of typical atrial flutter. A 15-ohm impedance reduction from the mean atrial impedance was considered the cut-off for defining the presence of NCS. Substrate maps were constructed according to the MVG technique (0.5-2.5 mV thresholds), in particular looking for high-voltage channels in which the impedance map detected the presence of NCS. Ablation was performed only in the high-voltage channels (W45, AI500). Results Our study included 48 patients with a mean age of 68±13 years, with 92% of them being men. The mean volume of the right atrium was 153 mL, and the mean number of points per map was 1205. With the use of impedence map, we were able to identify the presence of NCS in 25 patients (52%). The distribution of non conductive structures and the mean voltage by quadrants can be seen in Figures B-C. Importantly, we found that the mean number of radiofrequency applications required to achieve isthmic block was significantly lower in patients with identified NCS compared to those without (6.3 vs. 10; p=0.007). In 60% of the patients with NCS, radiofrequency was administered in high-voltage channels that were detected in the quadrant where the NCS was present (odds ratio 3.42, p=0.04). Furthermore, we successfully achieved acute isthmic blockade in all patients, and there were no instances of recurrence during a mean follow-up period of 186 days. Conclusions Our findings demonstrate that the combination of Impedence Map and the Maximum Voltage Gradient technique is an effective approach for achieving isthmic block with a reduced number of radiofrequency applications. The identification of NCS allows for a more precise search for high-voltage channels, enabling ablations to be performed in optimal contact with the tissue. This meticulous approach is crucial for the success of the procedure.

PET-MRI Imaging to assess severity and distribution of atrial fibrosis in atrial fibrillation

Abstract Background Atrial fibrosis is a major determinant of atrial fibrillation (AF) susceptibility and ablation targeting fibrotic areas to suppress AF revealed by magnetic resonance imaging (MRI) was considered a promising approach to treat persistent AF; however, its outcome has been disappointing. We sought to develop a new imaging modality to detect the severity and distribution of atrial fibrosis. Methods This study enrolled 19 healthy volunteers and 85 patients with various cardiovascular (CV) diseases of whom 22 had persistent AF. Each patient underwent 18F-labeled aluminum fluoride targeting fibroblast activation protein inhibitor (18F-AlF-FAPI)-positron emission tomography - magnetic resonance imaging (PET-MRI). Left atrial appendage tissue biopsied at 18F-FAPI-uptake positive and negative sites was stained with Masson trichome to detect fibrosis (N=10). We compared cardiac atrial 18F-AlF-FAPI-PET-MRI imaging among healthy individuals, patients without AF and patients with persistent AF as well as patients developing and not developing postoperative AF (POAF). Results Degree of fibrosis based on Masson staining was consistent with 18F-FAPI-uptake (P&amp;lt;0.05). Whereas 18F-FAPI uptake was essentially undetectable in healthy volunteers, in patients with CV diseases, 18F-FAPI uptake was present in all atrial chambers. Patients with persistent AF had the greatest and most widespread uptake (P&amp;lt;0.001), including left atrium (P=0.001), right atrium (P&amp;lt;0.001), left atrial appendage (P=0.007), right atrial appendage (P&amp;lt;0.001), and left atrial posterior wall (P=0.004). 18F-FAPI-uptake did not differ between those with versus without POAF. Conclusions 18F-AlF-FAPI-PET-MRI imaging detected atrial fibrosis in patients with various CV diseases; patients with persistent AF exhibit severe and widespread fibrosis, while POAF is not associated with increased 18F-FAPI uptake.

Increased circulating BMP10 levels are associated with late postoperative atrial fibrillation after cardiac surgery and left atrial endomysial fibrosis

Abstract Background Serum bone morphogenetic protein 10 (BMP10) levels are a marker for history of atrial fibrillation (AF) and for major adverse cardiovascular events in AF-patients, including stroke, recurrence after catheter ablation, and mortality. The predictive value of BMP10 in patients undergoing cardiac surgery and association with morphological properties of atrial tissue are unknown. Objective To study the correlation between BMP10 levels and pre-operative clinical traits, occurrence of early and late postoperative atrial fibrillation (POAF), and atrial fibrosis in patients undergoing cardiac surgery. Methods Patients with and without preoperative AF history undergoing first cardiac surgery were included (RACE V, n=147). Pre-operative blood biomarkers were analyzed, left (n=114) and right (n=125) atrial appendage biopsies were histologically assessed following WGA-staining, and post-operative rhythm was monitored continuously with implantable loop recorders (n=134, 2.5yrs). Results Multiple logistic regression indicated that BMP10 accurately predicts a history of persistent AF (OR=1.24, 95%CI: 1.10-1.40, p=4.32e-4), similar to NT-pro-BNP. BMP10 levels were associated with increased late-POAF90 occurrence after adjusting for age, sex, AF history, and early-POAF occurrence (HR=1.07 [per 0.1ng/ml increase], 95%CI: 1.00-1.14, p=0.04). Left atrial endomysial fibrosis (St.β=0.22, p=0.04), but not overall fibrosis (St. β= , p=), was associated with circulating BMP10, after adjusting for age, sex, AF history, reduced LVF, and valvular surgery indication. Conclusion Increased BMP10 levels were associated with persistent AF-history, increased late-POAF incidence, and LAA endomysial fibrosis in a diverse population of patients undergoing cardiac surgery.