Cross-talk between cAMP and Ca2+ signalling in cardiac pacemaker cells involves IP3-evoked Ca2+ release and stimulation of adenylyl cyclase 1

Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP3 signalling pathway has been proposed as an atrial specific target for AF therapy, and atrial IP3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. Here we investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sinoatrial node (SAN) cells to the α-adrenoceptor agonist phenylephrine (PE) using the selective AC1 inhibitor ST034307. The maximum rate change of spontaneously beating mouse right atrial tissue exposed to PE was reduced from 14.46 % to 8.17% (P = 0.005) in the presence of 1 μM ST034307, whereas the increase in tension generated in paced left atrial tissue in the presence of PE was not inhibited by ST034307 (Control = 14.20 %, ST034307 = 16.32 %; P > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transient amplitude (CaT) generated by 10μM PE in the presence and absence of 1μM ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; P = 0.004), but did not result in an inhibition of CaT amplitude increase in response to PE in atrial cells. The results presented here demonstrate the involvement of AC1 in the downstream response of atrial pacemaker activity to α-adrenoreceptor stimulation and IP3R calcium release.

Atrial fibrillation driver identification through regional mutual information networks: a modeling perspective

Purpose Effective identification of electrical drivers within remodeled tissue is a key for improving ablation treatment for atrial fibrillation. We have developed a mutual information, graph-based approach to identify and propose fault tolerance metric of local efficiency as a distinguishing feature of rotational activation and remodeled atrial tissue. Methods Voltage data were extracted from atrial tissue simulations (2D Karma, 3D physiological, and the Multiscale Cardiac Simulation Framework (MSCSF)) using multi-spline open and parallel regional mapping catheter geometries. Graphs were generated based on varied mutual information thresholds between electrode pairs and the local efficiency for each graph was calculated. Results High-resolution mapping catheter geometries can distinguish between rotational and irregular activation patterns using the derivative of local efficiency as a function of increasing mutual information threshold. The derivative is decreased for rotational activation patterns comparing to irregular activations in both a simplified 2D model (0.0017 ± 1 × 10−4 vs. 0.0032 ± 1 × 10−4, p < 0.01) and a more realistic 3D model (0.00092 ± 5 × 10−5 vs. 0.0014 ± 4 × 10−5, p < 0.01). Average local efficiency derivative can also distinguish between degrees of remodeling. Simulations using the MSCSF model, with 10 vs. 90% remodeling, display distinct derivatives in the grid design parallel spline catheter configuration (0.0015 ± 5 × 10−5 vs. 0.0019 ± 6 × 10−5, p < 0.01) and the flower shaped open spline configuration (0.0011 ± 5 × 10−5 vs. 0.0016 ± 4 × 10−5, p < 0.01). Conclusion A decreased derivative of local efficiency characterizes rotational activation and varies with atrial remodeling. This suggests a distinct communication pattern in cardiac rotational activation detectable via high-resolution regional mapping and could enable identification of electrical drivers for targeted ablation.

A mathematical model for electrical activity in pig atrial tissue

Atrial fibrillation (AF) is the most common sustained form of cardiac arrhythmia occurring in humans. Its effective treatment requires a detailed understanding of the underlying mechanisms at the genetic, molecular, cellular, tissue and organ levels. To study the complex mechanisms underlying the development, maintenance and termination of cardiac arrhythmias, we need preclinical research models. These models range from in vitro cell cultures to in vivo small and large animal hearts. However, translational research requires that the results of these animal experiments are understood in the context of human subjects. Currently, this is achieved through simulations with state-of-the-art mathematical models for human and animal heart tissue. In the context of AF, a model that is extensively used by experimentalists, is that of the pig atria. However, until now, an ionically detailed mathematical model for pig atrial tissue has been lacking, and researchers have been forced to rely on mathematical models from other animal species to understand their experimental observations. In this paper, we present the first ionically detailed mathematical model of porcine atrial electrophysiology. To build the model, we first fitted experimental patch-clamp data from literature to describe the individual currents flowing across the cell membrane. Later, we fine-tuned the model by fitting action potential duration restitution (APDR) curves for different repolarisation levels. The experimental data for the APDR studies was produced in N. Voigt’s lab. We extended our model to the tissue level and demonstrated the ability to maintain stable spiral waves. In agreement with previous experimental results, our model shows that early repolarisation is primarily driven by a calcium-mediated chloride current, IClCa, which is completely inactivated at high pacing frequencies. This is a condition found only in porcine atria. The model shows spatiotemporal chaos with reduced repolarisation.

Right Atrial Morphology in Coronary Heart Disease and Degenerative Aortic Valve Stenosis. Summary of the Doctoral Thesis

One of the main forms of cardiovascular diseases is coronary heart disease (CHD) but degenerative aortic valve (AoV) stenosis is the most frequent native valve disease. Both CHD and degenerative AoV stenosis have common risk factors such as age, high blood cholesterol, diabetes, smoking, high blood pressure, inflammation, and metabolic syndrome. Not only risk factors, but also pathophysiological changes, especially in the early stages of degenerative aortic valve stenosis, are similar to atherosclerosis - endothelial damage, lipid deposition, focal sclerosis, inflammatory cell infiltration, cytokine release and calcification. However, these conditions are not always observed at the same time. This confirms the existence of risk and pathogenesis factors specific to each disease. Although these heart diseases have been known for a long time and are intensively studied, there is still a lack of reliable markers that could help predict disease progression, the need for further surgery and mortality, therefore the pathophysiological processes involved in disease pathogenesis should be re-evaluated. Tissue changes in these diseases are complex and include cell death, cardiac innervation, tissue ischemia, regulators of metabolism and homeostasis, markers of inflammation and anti-inflammation, and other changes that are still not fully understood. Aim of the study: to determine the prevalence of markers of apoptosis, homeostasis regulating factors, innervation, ischemia and inflammation in right atrial tissue in cases of coronary heart disease and degenerative aortic valve stenosis. The tissue material used in the study – fragments of the right atrial appendage collected during elective open heart surgeries. A total of 36 patients with acquired heart diseases were included in the study – 24 patients with coronary heart disease and 12 patients with degenerative aortic valve stenosis. Samples of right atrial tissue from 5 patients with congenital heart disease operated at an early age were used as the study control group. Tissues were stained with hematoxylin and eosin for routine light microscopy, treated with the biotin-streptovidine method for immunohistochemical detection of tissue markers and by the TUNEL method for the detection of apoptotic cells. The following markers were identified in right atrial tissue by immunohistochemistry: atrial natriuretic peptide (ANUP), PGP 9.5- containing innervation, vascular endothelial growth factor (VEGF), chromogranin A (ChgA), endothelin 1 (ET-1), interleukin 1α (Il-1α ), interleukin 10 (II-10), β defensins 2, 3 and 4 (βD2, βD3 and βD4, respectively). Right atrial tissue in both CHD and degenerative AoV stenosis is characterized by non-specific degenerative morphological changes – pronounced vacuolization as well as changes in the shape and size of cardiomyocytes and their nuclei. In addition, these patients have a high proportion of apoptotic cardiomyocytes. Although there were no significant lesions in the coronary arteries in patients with AoV stenosis, connective tissue ingrowth and vascular sclerosis were observed in some patients in both groups. In the case of CHD and degenerative AoV stenosis, activation of the right atrial endocardial endothelial cells occurs, characterized by a change of shape from flat to cubic and rich release of ChgA, ET-1, Il-1α, Il-10, βD2 and βD3. Patients with CHD and AoV stenosis in the right atrial tissue had statistically significant higher numbers of ANUP-positive cardiomyocytes, all types of IL-10 positive cells and βD2 and βD3-positive endocardial endothelial cells, but fewer ChgA-positive cells than controls or patients with congenital heart disease. Thus, in both cases of acquired heart disease, an anti-inflammatory response prevails in the right atrial tissue, but increased activity of the neuroendocrine system is more common in patients with congenital heart disease at an early age. Although some tendencies were observed, for example, in the CHD group, there were slightly more VEGF, ET-1, Il-1α positive endocardial endothelial cells, Il-10 positive cardiomyocytes, connective tissue and endothelial cells, but in AoV stenosis group, there were slightly more ChgA-positive endocardial endothelial cells, however, these differences did not reach statistical significance. The most striking finding in our study was the rich expression of antimicrobial peptides, such as human β defensins 2 and 3, in the right atrial tissues in patients with CHD, degenerative AoV stenosis and congenital heart disease.

Integration of Scandinavian genetic data with UK biobank data implicates the RBM20 gene with atrial fibrillation pathogenesis

Purpose Atrial fibrillation (AF) is the most common sustained arrhythmia. It carries a large healthcare burden and is associated with serious complications. The arrhythmia has a substantial genetic component and is associated with several structural genes, including the gene TTN. A recent large genome-wide association study on AF found an association to RBM20. The RBM20 gene is a splicing factor targeting TTN, RYR2 and CAMK2D among other cardiac genes. Using Next-Generation Sequencing and data derived from the UK Biobank, we aimed to reveal the role of RBM20 in AF. Methods and results We examined the burden of rare (Minor allele frequency (MAF)&lt;0.01%) RBM20 loss-of-function (LOF) variants in whole-exome sequencing data from the UK Biobank (n=175,280). AF was defined by ICD9/10, while individuals without AF were used as controls. Association tests aggregating rare variants in RBM20 using the Efficient Variant-Set Mixed Model Association Test (SMMAT) were performed to assess the effect of LOF RBM20 variants, adjusted for age, sex and principal components. We identified 33 LOF variants in RBM20, which were significantly enriched in AF (P=0.0087). To examine the effect of rare missense RBM20 variants in the splicing of TTN, we screened an in-house cohort of 531 Scandinavian early-onset AF patients using targeted sequencing. We filtered for rare (MAF&lt;0.1%) and deleterious (defined as combined annotation dependent depletion score &gt;20) variants and identified nine missense variants and three novel LOF variants in RBM20. To evaluate the effect of these RBM20 variants, we constructed a series of human RBM20 single nucleotide base exchange mutants. The splicing activity of the variants was measured with RT-qPCR on HEK293 cells transfected with a TTN241–3 splicing reporter. Four of these variants resulted in a significantly altered splicing activity in TTN, with the largest effect observed for LOF variants. In order to examine the biological effect of RBM20 variants on structural changes in atrial tissue, we used a Norwegian Brown rat animal model with loss of RBM20. In this model, Transmission Electron Microscopy revealed altered sarcomere and mitochondrial structure in its atrial cardiomyocytes. Furthermore, nanopore RNA sequencing of atrial tissue from the aforementioned animal model indicated altered expression in several key cardiac genes, including TTN and PITX2. Conclusion Rare RBM20 LOF variants are significantly enriched in AF cases, seen in a large population of 175,000 individuals. We demonstrated that the effect of LOF RBM20 on alternative TTN splicing can be detected on an individual level in patients with AF. Studies using an animal model indicates that LOF in RBM20 may affect atrial function through altered expression of several genes in the atria, and may cause structural changes in the atrial cardiomyocytes. This suggests that RBM20 may be involved in AF pathogenesis mediated through an atrial cardiomyopathy. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Novo Nordisk Foundation Pre-Graduate Scholarships (NNF18OC0053094)The Hallas Møller Emerging Investigator grant (Novo Nordisk Foundation (NNF17OC0031204))

Usefulness of a Radiofrequency Transseptal Needle in the Second Puncture of Fontan Extracardiac Conduit

We performed a second puncture of the extracardiac conduit in an 11-year-old Fontan patient to assess the patency of the stent previously deployed in the left pulmonary vein. For the first puncture, a mechanical Brockenbrough needle was selected to puncture the Gore-Tex conduit, an electrical insulator. For the second puncture, the location of that previous hole was detected as an indentation covered with atrial tissue, which is an electrical conductor. The second puncture was performed safely using a radiofrequency transseptal needle.

Homeostasis Regulating Factors, Innervation, Ischemia and Inflammatory Markers in the Right Atrial Tissue from Patients with Degenerative Aortic Valve Stenosis and Coronary Heart Disease

Both coronary heart disease (CHD) and degenerative aortic valve (AoV) stenosis have common risk factors, such as age, high blood cholesterol, diabetes, smoking, high blood pressure, inflammation, and metabolic syndrome. However, these diseases are not always observed together, confirming the existence of risk and pathogenesis factors specific to each disease. The aim of this study was to identify presence and distribution of common and different homeostasis regulating factors, innervation, ischemia and inflammatory markers in the right atrial tissue from patients with degenerative AoV stenosis and CHD. During elective cardiac surgery, right atrial tissue fragments were taken from 20 patients with CHD and from 9 patients with degenerative AoV stenosis. All tissue fragments were stained for immunohistochemical detection of protein-gene peptide 9.5 (PGP 9.5), atrial natriuretic peptide (ANUP), vascular endothelial growth factor (VEGF), chromogranin A, endothelin, interleukin 1 and 10 (Il-1 and Il-10) and β defensins 2, and 3 (βD2 and βD3). For the quantification of structures, a semi-quantitative counting method was used. Mostly numerous Il-10 positive cardiomyocytes and epi-/endocardial endothelial cells were detected in all specimens taken from patients with CHD, and statistically more than in specimens taken from patients with degenerative AoV disease (p = 0.007 and p = 0.016). Also, the number of βD3 positive cardiomyocytes was higher in the coronary heart disease group (p = 0.026). All other tested markers such as PGP 9.5, ANUP, VEGF, endothelin, chromogranin A, Il-1 and βD2 showed similar expression in both groups. Increased production of ANUP in right atrial tissue characterises both CHD and degenerative AoV stenosis. Production of ChgA in right atrial endocardial endothelial cells might represent regulation of sympathetic activity as a compensatory homeostatic response. Increased PGP 9.5-containing innervation is characteristic in patients with degenerative AoV disease and secondary mitral insufficiency. A stable increase of VEGF and variations of endothelin without statistically significant difference suggest influence of ischemia on the local vascular blood supply. Decreased production of Il-1α together with moderate to rich production of Il-10, βD2, and βD3 indicates the dominance of the local immune system over inflammation.