Search

Chronic kidney disease (CKD) is a complex condition with a prevalence of 10–15% worldwide. An inverse-graded relationship exists between cardiovascular events and mortality with kidney function which is independent of age, sex, and other risk factors. The proportion of deaths due to heart failure and sudden cardiac death increase with progression of chronic kidney disease with relatively fewer deaths from atheromatous, vasculo-occlusive processes. This phenomenon can largely be explained by the increased prevalence of CKD-associated cardiomyopathy with worsening kidney function. The key features of CKD-associated cardiomyopathy are increased left ventricular mass and left ventricular hypertrophy, diastolic and systolic left ventricular dysfunction, and profound cardiac fibrosis on histology. While these features have predominantly been described in patients with advanced kidney disease on dialysis treatment, patients with only mild to moderate renal impairment already exhibit structural and functional changes consistent with CKD-associated cardiomyopathy. In this review we discuss the key drivers of CKD-associated cardiomyopathy and the key role of hypertension in its pathogenesis. We also evaluate existing, as well as developing therapies in the treatment of CKD-associated cardiomyopathy.

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.

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.

Atrial fibrillation (AF) affects over 1% of the population and is a leading cause of stroke and heart failure in the elderly. A feared side effect of sodium channel blocker therapy, ventricular pro-arrhythmia, appears to be relatively rare in patients with AF. The biophysical reasons for this relative safety of sodium blockers are not known.\ud \ud Our data demonstrates intrinsic differences between atrial and ventricular cardiac voltage-gated sodium currents (INa), leading to reduced maximum upstroke velocity of action potential and slower conduction, in left atria compared to ventricle. Reduced atrial INa is only detected at physiological membrane potentials and is driven by alterations in sodium channel biophysical properties and not by NaV1.5 protein expression. Flecainide displayed greater inhibition of atrial INa, greater reduction of maximum upstroke velocity of action potential, and slowed conduction in atrial cells and tissue.\ud \ud Our work highlights differences in biophysical properties of sodium channels in left atria and ventricles and their response to flecainide. These differences can explain the relative safety of sodium channel blocker therapy in patients with atrial fibrillation.

Aims Emipagliflozin (EMPA) is a potent inhibitor of the renal sodium-glucose co-transporter 2 (SGLT2) and an effective treatment for type-2 diabetes. In patients with diabetes and heart failure, EMPA has cardioprotective effects independent of improved glycaemic control, despite SGLT2 not being expressed in the heart. A number of non-canonical mechanisms have been proposed to explain these cardiac effects, most notably an inhibitory action on cardiac Na+/H+ exchanger 1 (NHE1), causing a reduction in intracellular [Na+] ([Na+]i). However, at resting intracellular pH (pHi), NHE1 activity is very low and its pharmacological inhibition is not expected to meaningfully alter steady-state [Na+]i. We re-evaluate this putative EMPA target by measuring cardiac NHE1 activity. Methods and results The effect of EMPA on NHE1 activity was tested in isolated rat ventricular cardiomyocytes from measurements of pHi recovery following an ammonium pre-pulse manoeuvre, using cSNARF1 fluorescence imaging. Whereas 10 µM cariporide produced near-complete inhibition, there was no evidence for NHE1 inhibition with EMPA treatment (1, 3, 10, or 30 µM). Intracellular acidification by acetate-superfusion evoked NHE1 activity and raised [Na+]i, reported by sodium binding benzofuran isophthalate (SBFI) fluorescence, but EMPA did not ablate this rise. EMPA (10 µM) also had no significant effect on the rate of cytoplasmic [Na+]i rise upon superfusion of Na+-depleted cells with Na+-containing buffers. In Langendorff-perfused mouse, rat and guinea pig hearts, EMPA did not affect [Na+]i at baseline nor pHi recovery following acute acidosis, as measured by 23Na triple quantum filtered NMR and 31P NMR, respectively. Conclusions Our findings indicate that cardiac NHE1 activity is not inhibited by EMPA (or other SGLT2i’s) and EMPA has no effect on [Na+]i over a wide range of concentrations, including the therapeutic dose. Thus, the beneficial effects of SGLT2i’s in failing hearts should not be interpreted in terms of actions on myocardial NHE1 or intracellular [Na+]., Graphical Abstract

No abstract available.

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): CATCH ME Foundation Leducq BACKGROUND In cardiac myocytes, desmosomal proteins and ion channels form macromolecular complexes important for maintaining cell adhesion and electrical integrity. High serum levels of androgenic anabolic steroids (AAS) promote cardiac muscle growth, but any detrimental impact on atrial gene transcription and/or electrophysiological function is unknown. PURPOSE To investigate the effects of chronic AAS exposure on atria in a mouse model with desmosomal impairment. METHODS Young (8-10 week) male wild-type (WT) and heterozygous plakoglobin-deficient (plako+/-) mice were challenged with the AAS dihydrotestosterone (DHT) or placebo for 6 weeks by osmotic mini pumps. RNA sequencing (n = 3-6 atria/group) revealed effects of genotype and DHT on left atrial (LA) transcription. Membrane-localised cardiac sodium channels (Nav1.5) were visualised using direct STochastic Optical Reconstruction Microscopy (dSTORM, n = 5-11 LA/group, 122 cells in total) and clustering of individual molecules was quantified using persistence-based clustering. Patch clamping of LA cardiac myocytes was used to record whole cell sodium currents (n = 4-5 LA/group, 77 cells in total). LA action potentials and conduction velocity were evaluated using microelectrode and optical mapping techniques (n = 5-9 LA/group). RESULTS DHT increased expression of pro-hypertrophic transcripts, e.g. Igf1, Mtpn, fibrosis-associated transcripts, e.g. Col1a1, Col3a1, Lox and pro-inflammatory transcripts, e.g. Ccl6, C7, in both WT and plako+/- LA. Despite Scn5a transcript levels being maintained, dSTORM identified a 29% reduction (p = 0.042) in the number of Nav1.5 localisations at the membrane of plako+/- DHT LA cardiomyocytes, and 25% fewer localisations (p = 0.005) were found within Nav1.5 clusters, compared to WT DHT. Electrophysiological methods revealed a significant reduction in peak sodium current density, decreased action potential amplitude and conduction slowing in plako+/- LA after exposure to DHT. CONCLUSION This data suggests that a reduction in plakoglobin expression predisposes atrial cardiomyocytes to detrimental electrophysiological effects of high testosterone levels. This is characterised by a perturbed spatial organisation of Nav1.5, decreased sodium current density and conduction slowing. Abstract Figure. Abstract Picture

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): BHF MRC Introduction Cardiac fibroblasts (cFbs) are responsible for deposition of extracellular matrix in the heart, providing support to the contracting myocardium and contributing to a myriad of physiological signalling processes. Prolonged and excessive activation of cFbs, via stimulation by transforming growth factor β (TGF-β), causes conversion of cFbs into myofibroblasts. Myofibroblasts are believed to cause pathological cardiac remodelling and to contribute to heart failure and arrhythmias. Reversion of myofibroblasts into cFbs has been demonstrated in rodent cells; it has yet to be explored in human cells. Purpose To characterise the effects of long-term 2D standard culture on the activation status of human cFbs. To identify the potential for human myofibroblasts to dedifferentiate back to cFbs. Methods Primary human cFbs were cultured in Corning Costar flasks (Young’s modulus E = ∼3GPa) for up to 10 passages. Cells were subsequently plated onto dishes with a Young’s modulus of ∼3GPa, 25kPa and 2kPa in the presence or absence of TGF-β (10ng/ml) and/or TGF-β receptor I inhibitor SD208 (10nM) for up to 4 days. The proliferative capacity of the cells was assessed using the CyQUANT NF® assay. Cells were assessed for mRNA and protein expression of myofibroblast activation markers α-smooth muscle actin (α-SMA) and collagen-1 by qPCR and western blotting. The localised distribution of α-SMA was assessed by confocal microscopy. Results Human cardiac fibroblasts robustly expressed α-SMA. Proliferation was significantly decreased at 2kPa compared to higher Young’s moduli (mean percentage change over 2 days: 2kPa = 115.1, 25kPa = 191.4, 3GPa = 205.9, p 0.05; n = 2-3). TGF-β or SD208 treatment did not affect α-SMA expression when assessed by confocal microscopy. Conclusions Long-term culture of human cFbs in 2D format leads to a robust and persistent activation of myofibroblasts that is unresponsive to TGF-ß activation or inhibition. Ongoing work is focussed on investigating whether human myofibroblast de-differentiation is possible.

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Leducq Foundation Background The point mutation M1875T in the SCN5A gene, which encodes the pore-forming α-subunit of the cardiac voltage-gated Na+ channel Nav1.5, has been associated with familial atrial fibrillation (AF), but its effects on atrial cardiomyocyte electrophysiology is unclear. Aim To investigate the effect of the point mutation M1875T on atrial electrophysiological parameters. Methods In a novel heterozygous knock-in murine model (Scn5a-M1875T+/-), whole-cell patch clamp electrophysiology was used to investigate Na+ currents in left atrial (LA) cardiomyocytes isolated from hearts of young adult mice (10-16 weeks). LA microelectrode and optical mapping recordings were used to study action potential (AP) characteristics. Cardiac size and function were measured by transthoracic echocardiography. Atrial Scn5a gene and Nav1.5 protein expression were assessed by Rt-PCR and Western blot. Results The Na+ current was increased in cardiomyocytes isolated from Scn5a-M1875T+/- LA (wildtype (WT) -22.7 ± 0.9 pA/pF (N = 14, n = 115); Scn5a-M1875T+/- -28.3 ± 1.1 pA/pF (N = 15, n = 117)). Scn5a-M1875T+/- intact isolated superfused LA had an elevated AP amplitude (100 ms pacing cycle length (PCL): WT 86.4 ± 0.9 mV (N = 8, n = 24); Scn5a-M1875T+/- 91.2 ± 0.7 mV (N = 8, n = 25)) and a faster peak upstroke velocity (100 ms PCL: WT 127.98 ± 3.28 mV/ms; Scn5a-M1875T+/- 142.80 ± 3.98 mV/ms). AP duration (APD) was not different apart from a small APD shortening at slow rates. Echocardiography revealed no difference in size and function at the age of investigation. Atrial Scn5a gene and Nav1.5 protein expression were comparable. When challenged with flecainide (1 µM), Scn5a-M1875T+/- LA showed less conduction slowing than WT (100 ms PCL: WT -10.43 ± 1.27 cm/s (N = 12); Scn5a-M1875T+/- -6.10 ± 1.34 cm/s (N = 12)). 5 µM flecainide caused significant increase in WT refractoriness (7/12 atria lost 1:1 capture at PCL ≤ 120 ms) compared to Scn5a-M1875T+/- (1/12). Conclusion(s): SCN5A point mutation M1875T increases the Na+ current in atrial cardiomyocytes and intact atria, leading to a faster AP upstroke and an attenuated response to flecainide. Abstract Figure 1: Current-Voltage relationship

Cardiac fibroblasts are the primary cell type responsible for deposition of extracellular matrix in the heart, providing support to the contracting myocardium and contributing to a myriad of physiological signaling processes. Despite the importance of fibrosis in processes of wound healing, excessive fibroblast proliferation and activation can lead to pathological remodeling, driving heart failure and the onset of arrhythmias. Our understanding of the mechanisms driving the cardiac fibroblast activation and proliferation is expanding, and evidence for their direct and indirect effects on cardiac myocyte function is accumulating. In this review, we focus on the importance of the fibroblast‐to‐myofibroblast transition and the cross talk of cardiac fibroblasts with cardiac myocytes. We also consider the current use of models used to explore these questions.

Background Large-scale screening for atrial fibrillation (AF) requires reliable methods to identify at-risk populations. Using an experimental semi-quantitative biomarker assay, B-type natriuretic peptide (BNP) and fibroblast growth factor 23 (FGF23) were recently identified as the most suitable biomarkers for detecting AF in combination with simple morphometric parameters (age, sex, and body mass index [BMI]). In this study, we validated the AF model using standardised, high-throughput, high-sensitivity biomarker assays. Methods and findings For this study, 1,625 consecutive patients with either (1) diagnosed AF or (2) sinus rhythm with CHA2DS2-VASc score of 2 or more were recruited from a large teaching hospital in Birmingham, West Midlands, UK, between September 2014 and February 2018. Seven-day ambulatory ECG monitoring excluded silent AF. Patients with tachyarrhythmias apart from AF and incomplete cases were excluded. AF was diagnosed according to current clinical guidelines and confirmed by ECG. We developed a high-throughput, high-sensitivity assay for FGF23, quantified plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) and FGF23, and compared results to the previously used multibiomarker research assay. Data were fitted to the previously derived model, adjusting for differences in measurement platforms and known confounders (heart failure and chronic kidney disease). In 1,084 patients (46% with AF; median [Q1, Q3] age 70 [60, 78] years, median [Q1, Q3] BMI 28.8 [25.1, 32.8] kg/m2, 59% males), patients with AF had higher concentrations of NT-proBNP (median [Q1, Q3] per 100 pg/ml: with AF 12.00 [4.19, 30.15], without AF 4.25 [1.17, 15.70]; p < 0.001) and FGF23 (median [Q1, Q3] per 100 pg/ml: with AF 1.93 [1.30, 4.16], without AF 1.55 [1.04, 2.62]; p < 0.001). Univariate associations remained after adjusting for heart failure and estimated glomerular filtration rate, known confounders of NT-proBNP and FGF23. The fitted model yielded a C-statistic of 0.688 (95% CI 0.656, 0.719), almost identical to that of the derived model (C-statistic 0.691; 95% CI 0.638, 0.744). The key limitation is that this validation was performed in a cohort that is very similar demographically to the one used in model development, calling for further external validation. Conclusions Age, sex, and BMI combined with elevated NT-proBNP and elevated FGF23, quantified on a high-throughput platform, reliably identify patients with AF. Trial registration Registry IRAS ID 97753 Health Research Authority (HRA), United Kingdom, Winnie Chua and colleagues identify and validate biomarkers for atrial fibrillation, Author summary Why was this study done? Atrial fibrillation (AF) is the most commonly sustained arrhythmia in the world, markedly increasing the risk of stroke, heart failure, and cardiovascular death. AF episodes can be transient and go undetected by ECG for a long time, leading to untreated AF and avoidable complications such as stroke. Blood biomarkers are an easy and affordable way to enable screening for undiagnosed, silent AF. An earlier study using a dual-antibody-based research assay identified a simple model, consisting of age, sex, body mass index, and 2 biomarkers, FGF23 and NT-proBNP, that identified patients with AF. What did the researchers do and find? We measured 2 biomarkers, NT-proBNP and FGF23, using high-throughput, high-sensitivity assays that are ready for clinical use. The FGF23 assay was developed for this study. We incorporated these biomarkers into a model including age, sex, and body mass index, which was derived from previous work where research assays were used to identify biomarkers to detect patients with AF. We demonstrated that the model using the standardised assays performed consistently to identify patients with AF. What do these findings mean? Elevated concentrations of FGF23 and NT-proBNP, in addition to older age, female sex, and high body mass index, identify patients who are likely to have AF. In the future, point-of-care testing can be easily performed using these biomarkers in combination with 3 simple pieces of information (age, sex, and body mass index) to target intensive screening, for example, using ECG monitoring.

Chronic kidney disease is highly prevalent, affecting 10% to 15% of the adult population worldwide and is associated with increased cardiovascular morbidity and mortality. As chronic kidney disease worsens, a unique cardiovascular phenotype develops characterized by heart muscle disease, increased arterial stiffness, atherosclerosis, and hypertension. Cardiovascular risk is multifaceted, but most cardiovascular deaths in patients with advanced chronic kidney disease are caused by heart failure and sudden cardiac death. While the exact drivers of these deaths are unknown, they are believed to be caused by uremic cardiomyopathy: a specific pattern of myocardial hypertrophy, fibrosis, with both diastolic and systolic dysfunction. Although the pathogenesis of uremic cardiomyopathy is likely to be multifactorial, accumulating evidence suggests increased production of fibroblast growth factor‐23 and αKlotho deficiency as potential major drivers of cardiac remodeling in patients with uremic cardiomyopathy. In this article we review the increasing understanding of the physiology and clinical aspects of uremic cardiomyopathy and the rapidly increasing knowledge of the biology of both fibroblast growth factor‐23 and αKlotho. Finally, we discuss how dissection of these pathological processes is aiding the development of therapeutic options, including small molecules and antibodies, directly aimed at improving the cardiovascular outcomes of patients with chronic kidney disease and end‐stage renal disease.

BACKGROUND Genomic and experimental studies suggest a role for PITX2 in atrial fibrillation (AF). To assess if this association is relevant for recurrent AF in patients, we tested whether left atrial PITX2 affects recurrent AF after AF ablation. METHODS mRNA concentrations of PITX2 and its cardiac isoform, PITX2c, were quantified in left atrial appendages (LAAs) from patients undergoing thoracoscopic AF ablation, either in whole LAA tissue (n = 83) or in LAA cardiomyocytes (n = 52), and combined with clinical parameters to predict AF recurrence. Literature suggests that BMP10 is a PITX2-repressed, atrial-specific, secreted protein. BMP10 plasma concentrations were combined with 11 cardiovascular biomarkers and clinical parameters to predict recurrent AF after catheter ablation in 359 patients. RESULTS Reduced concentrations of cardiomyocyte PITX2, but not whole LAA tissue PITX2, were associated with AF recurrence after thoracoscopic AF ablation (16% decreased recurrence per 2–(ΔΔCt) increase in PITX2). RNA sequencing, quantitative PCR, and Western blotting confirmed that BMP10 is one of the most PITX2-repressed atrial genes. Left atrial size (HR per mm increase [95% CI], 1.055 [1.028, 1.082]); nonparoxysmal AF (HR 1.672 [1.206, 2.318]), and elevated BMP10 (HR 1.339 [CI 1.159, 1.546] per quartile increase) were predictive of recurrent AF. BMP10 outperformed 11 other cardiovascular biomarkers in predicting recurrent AF. CONCLUSIONS Reduced left atrial cardiomyocyte PITX2 and elevated plasma concentrations of the PITX2-repressed, secreted atrial protein BMP10 identify patients at risk of recurrent AF after ablation. TRIAL REGISTRATION ClinicalTrials.gov NCT01091389, NL50069.018.14, Dutch National Registry of Clinical Research Projects EK494-16. FUNDING British Heart Foundation, European Union (H2020), Leducq Foundation., Building on genomic and experimental findings, low PITX2 in left atrial cardiomyocytes and high BMP10 in plasma predict recurrent atrial fibrillation in patients after ablation.

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.

Cardiac arrhythmias of both atrial and ventricular origin are an important feature of cardiovascular disease. Novel antiarrhythmic therapies are required to overcome current drug limitations related to effectiveness and pro-arrhythmia risk in some contexts. Cardiomyocyte culture models provide a high-throughput platform for screening antiarrhythmic compounds, but comparative information about electrophysiological properties of commonly used types of cardiomyocyte preparations is lacking. Standardization of cultured cardiomyocyte microelectrode array (MEA) experimentation is required for its application as a high-throughput platform for antiarrhythmic drug development. The aim of this study was to directly compare the electrophysiological properties and responses to isoproterenol of three commonly used cardiac cultures. Neonatal rat ventricular myocytes (NRVMs), immortalized atrial HL-1 cells, and custom-generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured on microelectrode arrays for 48–120 h. Extracellular field potentials were recorded, and conduction velocity was mapped in the presence/absence of the β-adrenoceptor agonist isoproterenol (1 µM). Field potential amplitude and conduction velocity were greatest in NRVMs and did not differ in cardiomyocytes isolated from male/female hearts. Both NRVMs and hiPSC-CMs exhibited longer field potential durations with rate dependence and were responsive to isoproterenol. In contrast, HL-1 cells exhibited slower conduction and shorter field potential durations and did not respond to 1 µM isoproterenol. This is the first study to compare the intrinsic electrophysiologic properties of cultured cardiomyocyte preparations commonly used for in vitro electrophysiology assessment. These findings offer important comparative data to inform methodological approaches in the use of MEA and other techniques relating to cardiomyocyte functional screening investigations of particular relevance to arrhythmogenesis.

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.

Ever since British Physician William Withering first described the use of foxglove extract for treatment of patients with congestive heart failure in 1785, cardiotonic steroids have been used clinically to treat heart failure and more recently atrial fibrillation. Due to their ability to bind and inhibit the ubiquitous transport enzyme sodium potassium pump, thus regulating intracellular Na+ concentration in every living cell, they are also an essential tool for research into the sodium potassium pump structure and function. Exogenous CTS have been clearly demonstrated to affect cardiovascular system through modulation of vagal tone, cardiac contraction (via ionic changes) and altered natriuresis. Reports of a number of endogenous CTS, since the 1980s, have intensified research into their physiologic and pathophysiologic roles and opened up novel therapeutic targets. Substantive evidence pointing to the role of endogenous ouabain and marinobufagenin, the two most prominent CTS, in development of cardiovascular disease has accumulated. Nevertheless, their presence, structure, biosynthesis pathways and even mechanism of action remain unclear or controversial. In this review the current state-of-the-art, the controversies and the remaining questions surrounding the role of endogenous cardiotonic steroids in health and disease are discussed.

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.