Search

Atrial fibrillation (AF) is the most common chronic arrhythmia presenting a heavy disease burden. We report a new approach for generating cardiomyocytes (CMs) resembling atrial cells from human-induced pluripotent stem cells (hiPSCs) using a combination of Gremlin 2 and retinoic acid treatment. More than 40% of myocytes showed rod-shaped morphology, expression of CM proteins (including ryanodine receptor 2, α -actinin-2 and F-actin) and striated appearance, all of which were broadly similar to the characteristics of adult atrial myocytes (AMs). Isolated myocytes were electrically quiescent until stimulated to fire action potentials with an AM profile and an amplitude of approximately 100 mV, arising from a resting potential of approximately −70 mV. Single-cell RNA sequence analysis showed a high level of expression of several atrial-specific transcripts including NPPA , MYL7 , HOXA3 , SLN , KCNJ4 , KCNJ5 and KCNA5 . Amplitudes of calcium transients recorded from spontaneously beating cultures were increased by the stimulation of α -adrenoceptors (activated by phenylephrine and blocked by prazosin) or β -adrenoceptors (activated by isoproterenol and blocked by CGP20712A). Our new approach provides human AMs with mature characteristics from hiPSCs which will facilitate drug discovery by enabling the study of human atrial cell signalling pathways and AF. This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’.

Background Although atrial fibrillation ablation is increasingly used for rhythm control therapy, antiarrhythmic drugs (AADs) are commonly used, either alone or in combination with ablation. The effectiveness of AADs is highly variable. Previous work from our group suggests that alterations in atrial resting membrane potential (RMP) induced by low Pitx2 expression could explain the variable effect of flecainide. Objective The purpose of this study was to assess whether alterations in atrial/cardiac RMP modify the effectiveness of multiple clinically used AADs. Methods The sodium channel blocking effects of propafenone (300 nM, 1 μM), flecainide (1 μM), and dronedarone (5 μM, 10 μM) were measured in human stem cell–derived cardiac myocytes, HEK293 expressing human NaV1.5, primary murine atrial cardiac myocytes, and murine hearts with reduced Pitx2c. Results A more positive atrial RMP delayed INa recovery, slowed channel inactivation, and decreased peak action potential (AP) upstroke velocity. All 3 AADs displayed enhanced sodium channel block at more positive atrial RMPs. Dronedarone was the most sensitive to changes in atrial RMP. Dronedarone caused greater reductions in AP amplitude and peak AP upstroke velocity at more positive RMPs. Dronedarone evoked greater prolongation of the atrial effective refractory period and postrepolarization refractoriness in murine Langendorff-perfused Pitx2c+/– hearts, which have a more positive RMP compared to wild type. Conclusion Atrial RMP modifies the effectiveness of several clinically used AADs. Dronedarone is more sensitive to changes in atrial RMP than flecainide or propafenone. Identifying and modifying atrial RMP may offer a novel approach to enhancing the effectiveness of AADs or personalizing AAD selection.

Cardiovascular disease is the leading cause of global mortality and morbidity. Cardiac dysrhythmias contribute significantly to this disease burden. Atrial fibrillation (AF) is the most common chronic dysrhythmia. Human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-AMs) present an exciting new model for AF but currently fail to reach maturity and so are limited in translational potential currently. We report a new approach using a combination of Gremlin 2 and retinoic acid treatment of human iPSCs for generating cardiomyocytes resembling atrial cells. More than 40% of myocytes generated by this approach showed rod-shaped morphology, expression of cardiomyocyte proteins (including RyR2 receptors, a-actinin-2, F-actin) and typically a striated appearance, all of which were broadly similar to the characteristics of adult atrial myocytes. Isolated myocytes were electrically quiescent until stimulated to fire action potentials with an atrial myocyte profile and an amplitude of approximately 100 mV, arising from a resting potential of approximately −70 mV. Single-cell RNA sequence (scRNASeq) analysis showed a high level of expression of several atrial specific transcripts including NPPA, MYL7, HOXA3, SLN, KCNJ4, KCNJ5 and KCNA5. Amplitudes of calcium transients recorded from spontaneously beating cultures were increased by the stimulation of α-adrenoceptors (activated by phenylephrine and blocked by prazosin) or β-adrenoceptors (activated by isoproterenol and blocked by CGP20712A). Thus, our new method provides an efficient approach for differentiating human atrial myocytes with mature characteristics from hiPSCs. This preparation will be very useful for studying signalling pathways in human atrial myocytes, and provides a valuable model for investigating atrial fibrillation and drug discovery.

BACKGROUND: The collagen receptor glycoprotein VI (GPVI) is an attractive antiplatelet target due to its critical role in thrombosis but minor involvement in hemostasis.OBJECTIVE: To investigate GPVI receptor involvement in platelet activation by collagen-I and atherosclerotic plaque using novel blocking and non-blocking anti-GPVI nanobodies (Nbs).METHODS: Nb effects on GPVI-mediated signaling and function were assessed by western blot and whole blood thrombus formation under flow. GPVI clustering was visualized in thrombi using fluorescently labeled Nb28.RESULTS: Under arterial shear, inhibitory Nb2 blocks thrombus formation and platelet activation on collagen and plaque, but only reduces adhesion on plaque. In contrast, adhesion on collagen, but not plaque, is decreased by blocking integrin α2β1. Adhesion on plaque is maintained despite inhibition of integrins αvβ3, α5β1, α6β1, and αIIbβ3. Only combined αIIbβ3 and α2β1 blockade inhibits adhesion and thrombus formation to the same extent as Nb2 alone. Nb2 prevents GPVI signaling, with loss of Syk, Lat, and PLCɣ2 phosphorylation, especially to plaque stimulation. Non-blocking fluorescently labeled Nb28 reveals distinct GPVI distribution patterns on collagen and plaque, with GPVI clustering clearly apparent on collagen fibers and less frequent on plaque. Clustering on collagen fibers is lost in the presence of Nb2.CONCLUSIONS: This work emphasizes the critical difference in GPVI-mediated platelet activation by plaque and collagen; it highlights the importance of GPVI clustering for downstream signaling and thrombus formation. Labeled Nb28 is a novel tool for providing mechanistic insight into this process and the data suggest Nb2 warrants further investigation as a potential anti-thrombotic agent.

Androgenic anabolic steroids (AAS) are commonly abused by young men. Male sex associates with earlier manifestation of common and rare cardiac conditions including atrial fibrillation and arrhythmogenic right ventricular cardiomyopathy (ARVC). Clinical data suggest an atrial involvement in ARVC. The disease is caused by desmosomal gene defects such as reduced plakoglobin expression. Analysis of clinical records from 146 ARVC patients identified male preponderance and increased prevalence of atrial arrhythmias in patients with definite ARVC. Definite patients displayed ECG changes suggesting atrial remodelling. To study mechanisms of atrial remodelling due to desmosomal vulnerability and AAS, young adult male mice, heterozygously deficient for plakoglobin (Plako+/-) and wildtype (WT) littermates, were chronically exposed to 5α-dihydrotestosterone (DHT) or placebo. DHT increased atrial expression of pro-hypertrophic, fibrotic and inflammatory transcripts. DHT caused atrial conduction slowing, decreased peak sodium current density, reduced action potential amplitude and lowered the peak depolarisation rate in Plako+/- but not WT atria. Super-resolution microscopy revealed a reduction in Nav1.5 clustering in Plako+/- atrial cardiomyocytes following DHT exposure. These data reveal that AAS combined with plakoglobin deficiency cause pathological atrial electrical remodelling in young male hearts. AAS abuse may increase the risk of atrial myopathy in males with desmosomal gene variants.

Aims Genetically altered mice are powerful models to investigate mechanisms of atrial arrhythmias, but normal ranges for murine atrial electrophysiology have not been robustly characterized. Methods and results We analyzed results from 221 electrophysiological (EP) studies in isolated, Langendorff-perfused hearts of wildtype mice (114 female, 107 male) from 2.5 to 17.7 months (mean 7 months) with different genetic backgrounds (C57BL/6, FVB/N, MF1, 129/Sv, Swiss agouti). Left atrial monophasic action potential duration (LA-APD), interatrial activation time (IA-AT), and atrial effective refractory period (ERP) were summarized at different pacing cycle lengths (PCLs). Factors influencing atrial electrophysiology including genetic background, sex, and age were determined. LA-APD70 was 18 ± 0.5 ms, atrial ERP was 27 ± 0.8 ms, and IA-AT was 17 ± 0.5 ms at 100 ms PCL. LA-APD was longer with longer PCL (+17% from 80 to 120 ms PCL for APD70), while IA-AT decreased (−7% from 80 to 120 ms PCL). Female sex was associated with longer ERP (+14% vs. males). Genetic background influenced atrial electrophysiology: LA-APD70 (−20% vs. average) and atrial ERP (−25% vs. average) were shorter in Swiss agouti background compared to others. LA-APD70 (+25% vs. average) and IA-AT (+44% vs. average) were longer in 129/Sv mice. Atrial ERP was longer in FVB/N (+34% vs. average) and in younger experimental groups below 6 months of age. Conclusion This work defines normal ranges for murine atrial EP parameters. Genetic background has a profound effect on these parameters, at least of the magnitude as those of sex and age. These results can inform the experimental design and interpretation of murine atrial electrophysiology.

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 Ca

Background Studying cardiac electrophysiology in isolated perfused beating murine hearts is a well-established method. The ranges of normal values for left atrial (LA) action potential durations (LA-APD), activation times (LA-AT) and effective refractory periods (atrial ERP) in murine wildtype (WT) are not well known. Purpose This study aimed to establish reference values for LA-APD, LA-AT and atrial ERP and to identify the influence of genetic background, sex and age on these electrophysiological parameters in WT mice. Method We combined results from isolated beating heart Langendorff experiments carried out in WT mice between 2005 and 2019 using an octopolar catheter inserted into the right atrium and a monophasic action potential electrode recording from the LA epicardium. Electrophysiological parameters (LA-APD at 50%, 70%, 90% repolarization (APD50, APD70, APD90), LA-AT and atrial ERP) at different pacing cycle lengths (PCL) were summarized. We analysed effects of PCL, genetic background, age, gender, heart weight to body weight ratio (HW/BW), LA weight to body weight ratio (LAW/BW) as well as coronary flow and temperature as experimental conditions. Results Electrophysiological parameters from 222 isolated hearts (114 female, mean age 6.6±0.25 months, range 2.47–17.7 months) of different backgrounds (77 C57BL/6, 23 FVB/N, 33 MF1, 69 129/Sv and 20 Swiss agouti) were combined. Coronary flow rate, flow temperature and start of isolation to cannulation time were constant experimental conditions over the timespan of experiments. LA-APD was longer while LA-AT decreased with longer PCL throughout all genetic backgrounds (Figure 1A). Genetic background showed strong effects on all electrophysiological parameters. LA-APD70 and atrial ERP were significantly shorter in Swiss agouti background compared to others. LA-APD70 was also significantly prolonged in 129/Sv background compared to MF1 (Figure 1B). LA activation was delayed in 129/Sv compared to other backgrounds (Figure 1C). Atrial ERP was longer in FVB/N compared to other backgrounds. Atrial ERP was also significantly prolonged (+ 3.4 ms, + 13.5%) in female mice compared to males (Figure 1D). Age effects were compared in groups. Atrial ERP was significantly longer in mice younger than 3 months compared to older mice (Figure 1E). Conclusion This dataset summarises left atrial electrophysiological parameters in the beating mouse heart and can serve as a reference for design and interpretation of electrophysiological experiments in murine models of commonly used genetic backgrounds. We demonstrate that PCL, genetic background, age and gender affect atrial electrophysiological parameters. Awareness of these will support successful experimental design. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): This work was partially supported by the European Commission (grant agreements no. 633196 [CATCH ME]) to LF and PK, Deutsche Forschungsgemeinschaft DFG FA413, British Heart Foundation (FS/13/43/30324 to LF and PK; AA/18/2/34218 to LF and PK).The Institute of Cardiovascular Sciences has received the British Heart Foundation (BHF) Accelerator Award (AA/18/2/34218). JO has received financial support for abroad studies within his scholarship of the Studienstiftung des deutschen Volkes (German Academic Scholarship Foundation).

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 immobilised collagen fibres 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 signalling is required to maintain clustering of the collagen integrin α2β1, whilst neither is required for GPVI. We propose that clustering of GPVI on immobilised collagen protects GPVI from shedding in order to maintain sustained Src and Syk-kinases dependent signalling, activation of integrin α2β1 and continued adhesion.

Transmural and regional gradients in membrane potential and Ca2+ transient in the murine heart are largely unexplored. Here, we developed and validated a robust approach which combines transverse ultra‐thin cardiac slices and high resolution optical mapping to enable systematic analysis of transmural and regional gradients in transmembrane potential (Vm) and intracellular Ca2+ transient (CaT) across the entire murine ventricles. The voltage dye RH237 or Ca2+ dye Rhod‐2 AM were loaded through the coronary circulation using a Langendorff perfusion system. Short‐axis slices (300 μm thick) were prepared from the entire ventricles (from the apex to the base) by using a high‐precision vibratome. Action potentials (APs) and CaTs were recorded with optical mapping during steady‐state baseline and rapid pacing. Significant transmural gradients in Vm and CaT were observed in the left ventricle, with longer AP duration (APD50 and APD75) and CaT duration (CaTD50 and CaTD75) in the endocardium compared with that in the epicardium. No significant regional gradients were observed along the apico‐basal axis of the left ventricle. Interventricular gradients were detected with significantly shorter APD50, APD75 and CaTD50 in the right ventricle compared with left ventricle and ventricular septum. During rapid pacing, AP and CaT alternans were observed in most ventricular regions, with significantly greater incidence in the endocardium in comparison with epicardium. In conclusion, these observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in Vm and CaT in murine ventricular tissue.

AimEvaluation of stress and its causes in UK gastroenterologists,DesignQuestionnaire emailed to all 1932 medical members of the British Society of Gastroenterology.ResultsOf 567 respondents (29%), 107 (20%) graded their stress level as 4 or 5 out of 5. Stress levels correlated inversely with self-reported happiness levels (r=−0.60; pThe main causes of stress were excessive clinical work (ranked highest by 47% and most commonly patient-related administration), working conditions beyond control (ranked highest by 15% and most commonly inadequate information technology systems, workspace and secretarial staff) and conflict (ranked highest by 9%). Of eight potential factors, happiness with work showed the closest associations with overall happiness (positive) and overall stress (negative) levels. Talking to someone at work about stress was ranked difficult or impossible by 35%. The highest ranked suggested solutions were relief from some duties and mentoring.ConclusionsStress is common and has objective correlates in UK gastroenterologists. The main cause is excessive workload.

Thin living tissue slices have recently emerged as a new tissue model for cardiac electrophysiological research. Slices can be produced from human cardiac tissue, in addition to small and large mammalian hearts, representing a powerful in vitro model system for preclinical and translational heart research. In the present protocol, we describe a detailed mouse heart transverse slicing and optical imaging methodology. The use of this technology for high-throughput optical imaging allows study of electrophysiology of murine hearts in an organotypic pseudo two-dimensional model. The slices are cut at right angles to the long axis of the heart, permitting robust interrogation of transmembrane potential (Vm) and calcium transients (CaT) throughout the entire heart with exceptional regional precision. This approach enables the use of a series of slices prepared from the ventricles to measure Vm and CaT with high temporal and spatial resolution, allowing (i) comparison of successive slices which form a stack representing the original geometry of the heart; (ii) profiling of transmural and regional gradients in Vm and CaT in the ventricle; (iii) characterization of transmural and regional profiles of action potential and CaT alternans under stress (e.g., high frequency pacing or β-adrenergic stimulation) or pathological conditions (e.g., hypertrophy). Thus, the protocol described here provides a powerful platform for innovative research on electrical and calcium handling heterogeneity within the heart. It can be also combined with optogenetic technology to carry out optical stimulation; aiding studies of cellular Vm and CaT in a cell type specific manner.

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

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.

The ability to record and analyse electrical behaviour across the heart using optical and electrode mapping has revolutionised cardiac research. However, wider uptake of these technologies is constrained by the lack of multi-functional and robustly characterised analysis and mapping software. We present ElectroMap, an adaptable, high-throughput, open-source software for processing, analysis and mapping of complex electrophysiology datasets from diverse experimental models and acquisition modalities. Key innovation is development of standalone module for quantification of conduction velocity, employing multiple methodologies, currently not widely available to researchers. ElectroMap has also been designed to support multiple methodologies for accurate calculation of activation, repolarisation, arrhythmia detection, calcium handling and beat-to-beat heterogeneity. ElectroMap implements automated signal segmentation, ensemble averaging and integrates optogenetic approaches. Here we employ ElectroMap for analysis, mapping and detection of pro-arrhythmic phenomena in silico, in cellulo, animal model and in vivo patient datasets. We anticipate that ElectroMap will accelerate innovative cardiac research and enhance the uptake, application and interpretation of mapping technologies leading to novel approaches for arrhythmia prevention.

Background Co2nduction velocity is known to be an important determinant of the electrical stability of the heart. Slowing of conduction velocity is implicated in the genesis of re-entrant cardiac arrhythmia and there is substantial interest in the pathological causes of slowed conduction. Despite this, current understanding of the basic physiological mechanisms regulating conduction velocity is poor. It is unclear whether the sympathetic nervous system directly regulates the conduction of electrical impulses within the ventricular myocardium. Interpretation of existing studies, largely performed in vivo, are complicated by non-cardiac effects of catecholamine infusion. To address this, we have examined the effects of bilateral sympathetic nerve stimulation (SNS) on conduction velocity in then ventricles of isolated guinea pig hearts. Objective To determine the effects of SNS on conduction velocity in the guinea pig heart. Methods High resolution spatiotemporal optical mapping was conducted in electrically paced hearts before and during bilateral SNS. Hearts were mechanically uncoupled using blebbistatin (15micromol/L) and stained with commonly used potentiometric dyes (Rh-237 or Di-8-ANEPPS). A region of interest (ROI) spanning from the apex to the mid-wall of the left ventricle was analysed. Pacing stimuli were applied at the epicardial apex and an activation map was generated. This activation map was then used to calculate conduction velocity using the multi-vector method. Results Figure 1 presents data on the regulation of ventricular conduction velocity by SNS in the isolated innervated guinea pig heart. A typical analysis is shown in figure 1A, which shows a change in conduction velocity from 78 to 89 cm/s with SNS and a reduction in the total activation time within the selected ROI from 8.9 to 8.3 ms. This effect was reproducibly seen in hearts stained with Rh-237, as shown in Figure 1B, where conduction velocity is consistently increased by SNS in a rate-independent manner (mean increase of 7.9±0.4%). Comparable data for hearts stained with Di-8-ANEPPS are presented in figure 1C, which shows no observable effect of SNS. Summary data in figure 1D illustrate that Di-8-ANEPPS not only abolishes the response to SNS, but also significantly slows conduction velocity in comparison to Rh-237 stained hearts. Further analysis demonstrated similar changes in heart rate, action potential shortening and heterogeneity of action potential duration with SNS in Rh-237 and Di-8-ANEPPS stained hearts. Thus, the effects of Di-8-ANEPPS on the regulation of conduction velocity by SNS are not due to general inhibition of the actions of the sympathetic nervous system. Rather, Di-8-ANEPPS appears to interfere with the normal physiological regulation of ventricular conduction velocity. Conclusions SNS directly regulates conduction velocity in the ventricular myocardium of the guinea pig heart, but Di-8-ANEPPS inhibits this response.

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.

Background Optical mapping is a powerful research tool that is revolutionising study of cardiac electrophysiology. However, processing and analysis of optical mapping data is computationally challenging to design and implement, a difficulty further enhanced by novel camera technology providing higher temporal and spatial resolution. We have previously developed algorithms capable of objective processing and analysis of electrophysiological parameters acquired using a second-generation complementary metal-oxide semiconductor camera.1 Here we report development of improved algorithms packaged in a user-friendly graphical interface, capable of high-throughput processing and analysis of voltage and calcium optical mapping data from a wide spectrum of cameras. Functionality and processing speed is further improved through automated recognition of pacing frequency and analysis of activation and repolarisation of electrophysiological parameters at high spatiotemporal resolution (200 × 2400 pixels; sampling rate 1 kHz). Processing options allow averaging of multiple beats as well as individual beat segmentation within the whole experimental trace, thus allowing for study of dynamic changes in key parameters such as action potential duration (APD), activation times, conduction velocity (CV) and phase mapping. Methods and results We have compared how our software performs in terms of analysis and processing time with our previously published methods.1Results remain consistent between both methods (APD50=10.41±0.62 vs 10.36±0.63 ms; CV=24.3±1.68 vs 23.3±1.69 cm/s, new software vs published methods; n=5, murine atria). Substantial improvements in processing speed (up to 4 times) are achieved, compared to our previously published methods. This improvement, coupled with automatic recognition and segmentation of whole experiments by cycle length, enables analysis of frequency dependent beat-to-beat changes in APD, CV and activation times. We demonstrate dynamic beat-to-beat frequency-dependent changes in APD50 in isolated superfused murine atria over a long experimental timeframe. As expected, increased frequency reduces APD50 (3 Hz=12.56±0.08; 8.33Hz=11.78±0.29; 10 Hz=11.47±0.3; 12.5Hz=10.88±0.29 ms; n=3), however, extensive beat-to-beat analysis allows investigation of electophysiological changes over whole experimental protocols, potentially providing crucial insight into novel arrhythmic mechanisms. Conclusions We have developed high-throughput software for analysis and processing of optical mapping imaging data compatible with a wide range of optical mapping cameras/systems. Our software offers enhanced processing speeds of key electrophysiological parameters across the heart and allows beat-to-beat analysis of large computationally challenging datasets. Reference 1. Yue et al. Prog Biophys Mol Biol. 2014 Aug;115(2-3):340–8.