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The electrophysiological properties of the hearts of women and men are different. These differences are at least partly mediated by the actions of circulating estrogens and androgens on the cardiomyocytes. Experimentally, much of our understanding in this field is based on studies focusing on ventricular tissue, with considerably less known in the context of atrial electrophysiology. The aim of this investigation was to compare the electrophysiological properties of male and female atria and assess responses to acute sex steroid exposure. Age-matched adult male and female C57BL/6 mice were anesthetized (4 % isoflurane) and left atria isolated. Atria were loaded with Di-4-ANEPPS voltage sensitive dye and optical mapping performed to assess action potential duration (APD; at 10 %, 20 %, 30 %, 50 %, and 70 % repolarization) and conduction velocity in the presence of 1 nM and 100 nM 17β-estradiol or testosterone. Male and female left atria demonstrated similar baseline action potential duration and conduction velocity, with significantly greater APD70 spatial heterogeneity evident in females. 17β-estradiol prolonged action potential duration in both sexes – an effect that was augmented in females. Atrial conduction was slowed in the presence of 100 nM 17β-estradiol in both males and females. Testosterone prolonged action potential duration in males only and did not modulate conduction velocity in either sex. This study provides novel insights into male and female atrial electrophysiology and its regulation by sex steroids. As systemic sex steroid levels change and intra-cardiac estrogen synthesis capacity increases with aging, these actions may have an increasingly important role in determining atrial arrhythmia vulnerability.

KairoSight-3.0 is a recently released Python-based, open-source software for cardiac optical mapping analysis. Addressing challenges in high-resolution electrophysiological data analysis, KairoSight-3.0 facilitates comprehensive studies of cardiac conduction and excitation-contraction coupling. We compared its performance with ElectroMap, focusing on action potential duration and conduction velocity measurements in mouse heart models subjected to ischaemia and flecainide treatment. Our findings reveal that while both software are effective, inherent methodological differences impact measurement outcomes. KairoSight-3.0's robust analysis capabilities make it a valuable tool in cardiac research. Additionally, future directions for KairoSight-3.0 and other mapping analysis tools are explored. Statement of importance: Open-source methods for analysis of cardiac optical mapping are vital tools in electrophysiological research. Our work directly evaluates the latest version of KarioSight, recently published in JMCC plus, with ElectroMap, an established and widely used tool. Our results show both software are effective in analysis of changes in both conduction and repolarisation. Considering the new features of KairoSight-3.0 and python implementation, our study importantly demonstrates the effectiveness of the software, highlights potential discrepancies between it and ElectroMap, and provides a perspective on future directions for KairoSight-3.0 and other software.

Abstract Activation of cardiac fibroblasts and differentiation to myofibroblasts underlies development of pathological cardiac fibrosis, leading to arrhythmias and heart failure. Myofibroblasts are characterised by increased α-smooth muscle actin (α-SMA) fibre expression, secretion of collagens and changes in proliferation. Transforming growth factor-beta (TGF-β) and increased mechanical stress can initiate myofibroblast activation. Reversibility of the myofibroblast phenotype has been observed in murine cells but has not been explored in human cardiac fibroblasts. In this study, chronically activated adult primary human ventricular cardiac fibroblasts and human induced pluripotent stem cell derived cFbs (hiPSC-cFbs) were used to investigate the potential for reversal of the myofibroblast phenotype using either subculture on soft substrates or TGF-β receptor inhibition. Culture on softer plates (25 or 2 kPa Young’s modulus) did not alter proliferation or reduce expression of α-SMA and collagen 1. Similarly, culture of myofibroblasts in the presence of TGF-β inhibitor did not reverse myofibroblasts back to a quiescent phenotype. Chronically activated hiPSC-cFbs also showed attenuated response to TGF-β receptor inhibition and inability to reverse to quiescent fibroblast phenotype. Our data demonstrate substantial loss of TGF-β signalling plasticity as well as a loss of feedback from the surrounding mechanical environment in chronically activated human myofibroblasts.

ABSTRACT Atrial fibrillation (AF) is the most common cardiac arrhythmia. AF increases the risk of stroke, heart failure, dementia, and hospitalization. Obesity significantly increases AF risk, both directly and indirectly, through related conditions, like hypertension, diabetes, and heart failure. Obesity‐driven structural and electrical remodeling contribute to AF via several reported mechanisms, including adiposity, inflammation, fibrosis, oxidative stress, ion channel alterations, and autonomic dysfunction. In particular, expanding epicardial adipose tissue during obesity has been suggested as a key driver of AF via paracrine signaling and direct infiltration. Weight loss has been shown to reverse these changes and reduce AF risk and recurrence after ablation. However, studies on how obesity affects pharmacologic or interventional AF treatments are limited. In this review, we discuss mechanisms by which obesity mediates AF and treatment outcomes, aiming to provide insight into obesity‐drug interactions and guide personalized treatment for this patient subgroup.

Abstract Optical mapping of animal models is a widely used technique in pre-clinical cardiac research. It has several advantages over other methods, including higher spatial resolution, contactless recording and direct visualisation of action potentials and calcium transients. Optical mapping enables simultaneous study of action potential and calcium transient morphology, conduction dynamics, regional heterogeneity, restitution and arrhythmogenesis. In this dataset, we have optically mapped Langendorff perfused isolated whole hearts (mouse and guinea pig) and superfused isolated atria (mouse). Raw datasets (consisting of over 400 files) can be combined with open-source software for processing and analysis. We have generated a comprehensive post-processed dataset characterising the baseline cardiac electrophysiology in these widely used pre-clinical models. This dataset also provides reference information detailing the effect of heart rate, clinically used anti-arrhythmic drugs, ischaemia-reperfusion and sympathetic nervous stimulation on cardiac electrophysiology. The effects of these interventions can be studied in a global or regional manner, enabling new insights into the prevention and initiation of arrhythmia.

Measurement(s) transmembrane potential • calcium transient Technology Type(s) high-throughput simultaneous optical imaging Sample Characteristic - Organism Mus musculus Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.16910542

Collagen, the most thrombogenic constituent of blood vessel walls, activates platelets through glycoprotein VI (GPVI). In suspension, following platelet activation by collagen, GPVI is cleaved by A Disintegrin And Metalloproteinase (ADAM)10 and ADAM17. In this study, we use single-molecule localization microscopy and a 2-level DBSCAN-based clustering tool to show that GPVI remains clustered along immobilized collagen fibers for at least 3 hours in the absence of significant shedding. Tyrosine phosphorylation of spleen tyrosine kinase (Syk) and Linker of Activated T cells (LAT), and elevation of intracellular Ca2+, are sustained over this period. Syk, but not Src kinase-dependent signaling is required to maintain clustering of the collagen integrin α2β1, whilst neither is required for GPVI. We propose that clustering of GPVI on immobilized collagen protects GPVI from shedding in order to maintain sustained Src and Syk-kinases dependent signaling, activation of integrin α2β1, and continued adhesion.

Biological tissues are naturally three-dimensional (3D) opaque structures, which poses a major challenge for the deep imaging of spatial distribution and localization of specific cell types in organs in biomedical research. Here we present a 3D heart imaging reconstruction approach by combining an improved heart tissue-clearing technique with high-resolution light-sheet fluorescence microscopy (LSFM). We have conducted a three-dimensional and multi-scale volumetric imaging of the ultra-thin planes of murine hearts for up to 2,000 images per heart in x-, y-, and z three directions. High-resolution 3D volume heart models were constructed in real-time by the Zeiss Zen program. By using such an approach, we investigated detailed three-dimensional spatial distributions of two specific cardiomyocyte populations including HCN4 expressing pacemaker cells and Pnmt+ cell-derived cardiomyocytes by using reporter mouse lines Hcn4DreER/tdTomato and PnmtCre/ChR2−tdTomato. HCN4 is distributed throughout right atrial nodal regions (i.e., sinoatrial and atrioventricular nodes) and the superior-inferior vena cava axis, while Pnmt+ cell-derived cardiomyocytes show distinct ventral, left heart, and dorsal side distribution pattern. Our further electrophysiological analysis indicates that Pnmt + cell-derived cardiomyocytes rich left ventricular (LV) base is more susceptible to ventricular arrhythmia under adrenergic stress than left ventricular apex or right ventricle regions. Thus, our 3D heart imaging reconstruction approach provides a new solution for studying the geometrical, topological, and physiological characteristics of specific cell types in organs.

Patients with heart failure often develop cardiac arrhythmias. The mechanisms and interrelations linking heart failure and arrhythmias are not fully understood. Historically, research into arrhythmias has been performed on affected individuals or in vivo (animal) models. The latter however is constrained by interspecies variation, demands to reduce animal experiments and cost. Recent developments in in vitro induced pluripotent stem cell technology and in silico modelling have expanded the number of models available for the evaluation of heart failure and arrhythmia. An agnostic approach, combining the modalities discussed here, has the potential to improve our understanding for appraising the pathology and interactions between heart failure and arrhythmia and can provide robust and validated outcomes in a variety of research settings. This review discusses the state of the art models, methodologies and techniques used in the evaluation of heart failure and arrhythmia and will highlight the benefits of using them in combination. Special consideration is paid to assessing the pivotal role calcium handling has in the development of heart failure and arrhythmia.

BackgroundRe-entrant ventricular tachycardia may be non-inducible or haemodynamically compromising, requiring assessment of the electrophysiological properties of the myocardium during sinus rhythm (i.e., substrate mapping). Areas of heart tissue with slow conduction can act as a critical isthmus for re-entrant electrical excitation and are a potential target for ablation therapy.AimTo develop and validate a novel metric of local conduction delay in the heart, the amplitude-normalized electrogram area (norm_EA).MethodsA computational model of a propagating mouse action potential was used to establish the impact of altering sodium channel conductance, intracellular conductivity, fibrosis density, and electrode size/orientation on bipolar electrogram morphology. Findings were then validated in experimental studies in mouse and guinea pig hearts instrumented for the recording of bipolar electrograms from a multipolar linear mapping catheter. norm_EA was calculated by integrating the absolute area of a bipolar electrogram divided by the electrogram amplitude. Electrogram metrics were correlated with the local conduction delay during sodium channel block, gap junction inhibition, and acute ischemia.ResultsIn computational simulations, reducing sodium channel conductance and intracellular conductivity resulted in a decrease in signal amplitude and increase in norm_EA (reflecting a broadening of electrogram morphology). For larger electrodes (3 mm diameter/7.1 mm2 area), the change in norm_EA was essentially linear with the change in local conduction delay. Experimental studies supported this finding, showing that the magnitude of change in norm_EA induced by flecainide (1–4 μM), carbenoxolone (10–50 μM), and low-flow ischemia (25% of initial flow rate) was linearly correlated with the local conduction delay in each condition (r2 = 0.92). Qualitatively similar effects were observed in guinea pig hearts perfused with flecainide. Increasing fibrosis density in the computational model also resulted in a decrease in signal amplitude and increase in norm_EA. However, this remains to be validated using experimental/clinical data of chronic infarct.Conclusionnorm_EA is a quantitative measure of local conduction delay between the electrode pair that generates a bipolar electrogram, which may have utility in electrophysiological substrate mapping of non-inducible or haemodynamically compromising tachyarrhythmia.

IntroductionThe upstroke of optical action potentials (APs) recorded from intact hearts are generally recognized to be slower than those recorded with microelectrodes. This is thought to reflect spatial signal averaging within the volume of tissue that makes up the optical signal. However, to date, there has been no direct experimental study on the relationship between conduction velocity (CV) and optical AP upstroke morphology in the intact heart. Notably, it is known that sodium channel block and gap junction inhibition, which both slow CV, exert differential effects on the upstroke velocity of microelectrode-recorded APs. Whether such differences are evident in optical APs is not known. The present study sought to determine the relationship between tissue CV and optical AP upstroke velocity in intact mouse hearts.Materials and MethodsIsolated, perfused mouse hearts were stained with the potentiometric dye Rh-237. Fluorescent signals were recorded from across the anterior surface of the left and right ventricles during constant pacing. Maximum rate of change in fluorescence (dF/dtmax) and tissue CV were assessed in control conditions, during an acute period of low-flow ischemia, and following perfusion of flecainide (1–3 μmol/L), a sodium channel blocker, or carbenoxolone (10–50 μmol/L), a gap junction inhibitor.ResultsDuring epicardial pacing, an anisotropic pattern was observed in both activation and dF/dtmax maps, with more rapid optical AP upstroke velocities orientated along the fastest conduction paths (and vice versa). Low-flow ischemia resulted in a time-dependent slowing of ventricular CV, which was accompanied by a concomitant reduction in optical AP upstroke velocity. All values returned to baseline on tissue reperfusion. Both flecainide and carbenoxolone were associated with a concentration-dependent reduction in CV and decrease in optical AP upstroke velocity, despite distinct mechanisms of action. Similar responses to carbenoxolone were observed for low- (156 μm pixel with) and high- (20 μm pixel width) magnification recordings. Comparison of data from all interventions revealed a linear relationship between CV and upstroke dF/dt.ConclusionIn intact mouse hearts, slowing of optical AP upstroke velocity is directly proportional to the change in CV associated with low-flow ischemia, sodium channel block, and gap junction inhibition.

Among the animal models for studying the molecular basis of atrial and sinoatrial node (SAN) biology and disease, the mouse is a widely used species due to its feasibility for genetic modifications in genes encoding ion channels or calcium handling and signaling proteins in the heart. It is therefore highly valuable to develop robust methodologies for studying SAN and atrial electrophysiological function in this species. Here, we describe a protocol for performing dual calcium-voltage optical mapping on mouse sinoatrial preparation (SAP), in combination with an optogenetic approach, for studying SAP membrane potential, intracellular Ca2+ transients, and pacemaker activity. The protocol includes the details for preparing the intact SAP, robust tissue dual-dye loading, light-programmed pacing, and high-resolution optical mapping. Our protocol provides an example of use of the combination of optogenetic and optical mapping techniques for investigating SAP membrane potential and intracellular Ca2+ transients and pacemaker activity with high temporal and spatial resolution in specific cardiac tissues. Thus, our protocol provides a useful tool for studying SAP physiology and pathophysiology in mice.

Optogenetic control of the heart is an emergent technology that offers unparalleled spatio-temporal control of cardiac dynamics via light-sensitive ion pumps and channels (opsins). This fast-evolving technique holds broad scope in both clinical and basic research setting. Combination of optogenetics with optical mapping of voltage or calcium fluorescent probes facilitates ‘all-optical’ electrophysiology, allowing precise optogenetic actuation of cardiac tissue with high spatio-temporal resolution imaging of action potential and calcium transient morphology and conduction patterns. In this review, we provide a synopsis of optogenetics and discuss in detail its use and compatibility with optical interrogation of cardiac electrophysiology. We briefly discuss the benefits of all-optical cardiac control and electrophysiological interrogation compared to traditional techniques, and describe mechanisms, unique features and limitations of optically induced cardiac control. In particular, we focus on state-of-the-art setup design, challenges in light delivery and filtering, and compatibility of opsins with fluorescent reporters used in optical mapping. The interaction of cardiac tissue with light, and physical and computational approaches to overcome the ‘spectral congestion’ that arises from the combination of optogenetics and optical mapping are discussed. Finally, we summarize recent preclinical work applications of combined cardiac optogenetics and optical mapping approach.

Rationale: Arrhythmogenic cardiac alternans are thought to be an important determinant for the initiation of ventricular fibrillation. There is limited information on the effects of sympathetic nerve stimulation (SNS) on alternans in the intact heart and the conclusions of existing studies, focused on investigating electrical alternans, are conflicted. Meanwhile, several lines of evidence implicate instabilities in Ca handling, not electrical restitution, as the primary mechanism underpinning alternans. Despite this, there have been no studies on Ca alternans and SNS in the intact heart. The present study sought to address this, by application of voltage and Ca optical mapping for the simultaneous study of APD and Ca alternans in the intact guinea pig heart during direct SNS.Objective: To determine the effects of SNS on APD and Ca alternans in the intact guinea pig heart and to examine the mechanism(s) by which the effects of SNS are mediated.Methods and Results: Studies utilized simultaneous voltage and Ca optical mapping in isolated guinea pig hearts with intact innervation. Alternans were induced using a rapid dynamic pacing protocol. SNS was associated with rate-independent shortening of action potential duration (APD) and the suppression of APD and Ca alternans, as indicated by a shift in the alternans threshold to faster pacing rates. Qualitatively similar results were observed with exogenous noradrenaline perfusion. In contrast with previous reports, both SNS and noradrenaline acted to flatten the slope of the electrical restitution curve. Pharmacological block of the slow delayed rectifying potassium current (IKs), sufficient to abolish IKs-mediated APD-adaptation, partially reversed the effects of SNS on pacing-induced alternans. Treatment with cyclopiazonic acid, an inhibitor of the sarco(endo)plasmic reticulum ATPase, had opposite effects to that of SNS, acting to increase susceptibility to alternans, and suggesting that accelerated Ca reuptake into the sarcoplasmic reticulum is a major mechanism by which SNS suppresses alternans in the guinea pig heart.Conclusions: SNS suppresses calcium and action potential alternans in the intact guinea pig heart by an action mediated through accelerated Ca handling and via increased IKs.