Charles M. Pearman

Methods for isolating atrial cells from large mammals and humans

The identification of disturbances in the cellular structure, electrophysiology and calcium handling of atrial cardiomyocytes is crucial to the understanding of common pathologies such as atrial fibrillation. Human right atrial specimens can be obtained during routine cardiac surgery and may be used for isolation of atrial myocytes. These samples provide the unique opportunity to directly investigate the effects of human disease on atrial myocytes. However, atrial myocytes vary greatly between patients, there is little if any access to truly healthy controls and the challenges associated with assessing the in vivo effects of drugs or devices in man are considerable. These issues highlight the need for animal models. Large mammalian models are particularly suitable for this purpose as their cardiac structure and electrophysiology are comparable with humans. Here, we review techniques for obtaining atrial cardiomyocytes. We start with background information on solution composition. Agents shown to increase viable cell yield will then be explored followed by a discussion of the use of tissue-dissociating enzymes. Protocols are detailed for the perfusion method of cell isolation in large mammals and the chunk digest methods of cell isolation in humans.

Increased Ca buffering underpins remodelling of Ca2+ handling in old sheep atrial myocytes

Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart. Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur. We report alterations to the atrial systolic Ca2+ transient that have implications for the function of the atrial in the elderly. We describe a novel mechanism by which increased Ca buffering can account for changes to systolic Ca2+ in the old atria. The present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops.

An Excel-based implementation of the spectral method of action potential alternans analysis.

Action potential (AP) alternans has been well established as a mechanism of arrhythmogenesis and sudden cardiac death. Proper interpretation of AP alternans requires a robust method of alternans quantification. Traditional methods of alternans analysis neglect higher order periodicities that may have greater pro‐arrhythmic potential than classical 2:1 alternans. The spectral method of alternans analysis, already widely used in the related study of microvolt T‐wave alternans, has also been used to study AP alternans. Software to meet the specific needs of AP alternans analysis is not currently available in the public domain. An AP analysis tool is implemented here, written in Visual Basic for Applications and using Microsoft Excel as a shell. This performs a sophisticated analysis of alternans behavior allowing reliable distinction of alternans from random fluctuations, quantification of alternans magnitude, and identification of which phases of the AP are most affected. In addition, the spectral method has been adapted to allow detection and quantification of higher order regular oscillations. Analysis of action potential morphology is also performed. A simple user interface enables easy import, analysis, and export of collated results.

Minimally Invasive Epicardial Surgical Ablation Alone Versus Hybrid Ablation for Atrial Fibrillation: A Systematic Review and Meta-Analysis

Maintaining sinus rhythm in patients with non-paroxysmal AF is an elusive goal. Some suggest that hybrid ablation, combining minimally invasive epicardial surgical ablation with endocardial catheter ablation, may be more effective than either modality alone. However, randomised trials are lacking. We investigated whether hybrid ablation is more effective than epicardial ablation alone at preventing recurrent AF by performing a systematic review and meta-analysis. The review was prospectively registered with PROSPERO (CRD42016043389). MEDLINE and EMBASE were searched for studies of standalone minimally invasive epicardial ablation of AF and/or hybrid ablation, identifying 41 non-overlapping studies comprising 2737 patients. A random-effects meta-analysis, meta-regression and sensitivity analysis were performed. Single-procedure survival free from atrial arrhythmias without antiarrhythmic drugs was similar between epicardial-alone and hybrid approaches at 12 months (epicardial alone 71.5 %; [95 % CI 66.1-76.9], hybrid 63.2 %; [95 % CI 51.5-75.0]) and 24 months (epicardial alone 68.5 %; [95 % CI 57.7-79.3], hybrid 57.0 %; [95 % CI 33.6-80.4]). Freedom from atrial arrhythmias with AADs and rates of unplanned additional catheter ablations were also similar between groups. Major complications occurred more often with hybrid ablation (epicardial alone 2.9 %; [95 % CI 1.9-3.9], hybrid 7.3 %; [95 % CI 4.2-10.5]). Meta-regression suggested that bipolar radiofrequency energy and thoracoscopic access were associated with greater efficacy, but adjusting for these factors did not unmask any difference between epicardial-alone and hybrid ablation. Hybrid and epicardial ablation alone appear to be equally effective treatments for AF, although hybrid ablation may be associated with higher complication rates. These data derived from observational studies should be verified with randomised data.

PDE5 inhibition improves symptom-free survival and restores transverse tubule loss and catecholamine responsiveness in heart failure

Heart failure is characterized by poor survival, a loss of catecholamine reserve and cellular structural remodeling in the form of disorganization and loss of the transverse tubule network. Indeed, survival rates for heart failure are worse than many common cancers and have not improved over time. Tadalafil is a clinically relevant drug that blocks phosphodiesterase 5 with high specificity and is used to treat erectile dysfunction. Using a sheep model of advanced heart failure, we show that tadalafil treatment results in a marked improvement in symptom-free survival, reverses transverse tubule loss and restore cardiac myocyte systolic calcium transient amplitude and the hearts chronotropic and contractile response to catecholamines. These effects are independent of changes in myocardial cGMP content and are associated with upregulation of both monomeric and dimerized forms of protein kinase G and of the cGMP hydrolyzing phosphodiesterase 2 and 3. We propose that the molecular switch for the loss of transverse tubules in heart failure and their restoration following tadalafil treatment involves the BAR domain protein Amphiphysin II (BIN1) and the restoration of catecholamine sensitivity is through reductions in G-protein receptor kinase 2, protein phosphatase 1 and protein phosphatase 2A abundance following phosphodiesterase 5 inhibition.

A classic textbook flutter

The following trace was recorded during ablation of typical counterclockwise atrial flutter using a two-catheter setup. Having performed ablation along the cavotricuspid isthmus starting at the ventricular end, the ablation catheter was placed at the inferior vena caval end of the line (Figure 1). What electrograms can be identified from the trace recorded from the ablation catheter andwhat can be inferred from them?

When you have eliminated the impossible

1Department of Cardiology, LiverpoolHeart andChestHospital, Liverpool, UK 2Unit of Cardiac Physiology,Manchester AcademicHealth SciencesCentre, University ofManchester,Manchester, UK Correspondence SimonModi,MBBS,DepartmentofCardiology, LiverpoolHeart andChestHospital, ThomasDrive, Liverpool, L143PE,UK. Email: simon.modi@lhch.nhs.uk Contributions: S.M. collected thedata anddrafted themanuscript,C.M., Z.B., and J.W. critically revised themanuscript.

Calcium in the pathophysiology of atrial fibrillation and heart failure

Atrial fibrillation (AF) is commonly associated with heart failure. A bidirectional relationship exists between the two—AF exacerbates heart failure causing a significant increase in heart failure symptoms, admissions to hospital and cardiovascular death, while pathological remodeling of the atria as a result of heart failure increases the risk of AF. A comprehensive understanding of the pathophysiology of AF is essential if we are to break this vicious circle. In this review, the latest evidence will be presented showing a fundamental role for calcium in both the induction and maintenance of AF. After outlining atrial electrophysiology and calcium handling, the role of calcium-dependent afterdepolarizations and atrial repolarization alternans in triggering AF will be considered. The atrial response to rapid stimulation will be discussed, including the short-term protection from calcium overload in the form of calcium signaling silencing and the eventual progression to diastolic calcium leak causing afterdepolarizations and the development of an electrical substrate that perpetuates AF. The role of calcium in the bidirectional relationship between heart failure and AF will then be covered. The effects of heart failure on atrial calcium handling that promote AF will be reviewed, including effects on both atrial myocytes and the pulmonary veins, before the aspects of AF which exacerbate heart failure are discussed. Finally, the limitations of human and animal studies will be explored allowing contextualization of what are sometimes discordant results.

Phosphodiesterase-5 inhibition with sildenafil suppresses calcium waves by reducing sarcoplasmic reticulum content

Rationale: Occurrence of diastolic Ca2+ waves in cardiac myocytes leads to arrhythmias by inducing delayed after-depolarisations. Waves are initiated when sarcoplasmic reticulum (SR) content reaches a critical threshold level. The phosphodiesterase-5 inhibitor sildenafil (Sil) is antiarrhythmic in mammalian myocardial ischaemia models, while Sil reduces Ca2+ transient amplitude and sarcoplasmic reticulum (SR) Ca2+ content in rat myocytes. Objective: To determine effects of Sil on propensity to Ca2?+ waves in the large mammal. Methods: Sheep ventricular myocytes were voltage clamped and intracellular Ca2+ measured using Fura-2. Cells were paced at 0.5 Hz with depolarisations from −40 mV to+10 mV. When at steady state, waves were induced with 10–15 mM Ca2+. Upon regular waving, Sil (1µM) was applied. To determine threshold SR content, caffeine (10 mM) was added immediately following a wave, and both wave and caffeine-induced I NCX integrated. Differences between groups were determined using students paired t tests. Results: Increasing external Ca2+ to 10–15 mM increased SR content and induced diastolic waves. Sildenafil abolished waves in 9/15 cells. In cells where Sil terminated waves, SR content was reduced below threshold. In addition, Sil treatment was associated with a reduced rate constant of SERCA (kSERCA−66.0±9.9% of control, p<0.005), an initial (first 4 s) increase in sarcolemmal efflux via the I NCX tail current (+142± 36.4%, p<0.01), and reduced sarcolemmal influx via I Ca-L (–30.5±5.6%, p<0.005). In cells continuing to wave in Sil, SR threshold for waves was unchanged (126.9 µmolL-1 ctrl vs 147.2 µmolL-1 Sil, p=0.6). In unstimulated cells spontaneously waving in 10–15 mM Ca²+, sildenafil reduced wave frequency (6.3 waves per 20 s vs 2.7, p<0.005). The protective effect of sildenafil on both wave models was abolished when cells were pre-incubated with the PKG inhibitor, KT5823. Sildenafil suppression of waves was also observed in cells from animals in end-stage heart failure, while Sil suppressed ventricular ectopy and episodes of torsades de pointes in vivo in a sheep model of LQT2. Conclusions: Sildenafil suppresses waves induced by elevated external Ca2+ via a PKG-dependent mechanism, and mediated by a reduction in SR content, which itself is caused by reduced SERCA function ± reduced I Ca-L. These findings highlight novel antiarrhythmic properties of PDE5 inhibition and translate to suppression of triggered arrhythmias in vivo. Funding: British Heart Foundation.

218 Action Potential Alternans in the Ageing Ovine Atria

Background The incidence of atrial fibrillation (AF) has a steep relationship with age. AF prevalence in persons aged over 85 exceeds 15% in European cohorts. The basis for the exaggerated vulnerability of the atria to fibrillation in the elderly is incompletely understood. Previous work has shown that ageing is associated with a prolongation of action potential duration, a finding that could be expected to protect against re-entrant arrhythmias. Alternating beat to beat variation in action potential duration and amplitude occurs at high heart rates and has been associated with a history of AF in humans. We investigated whether isolated atrial myocytes from young and old sheep would exhibit differences in alternans behaviour. Methods Myocytes from the left and right atrial appendages were isolated from young (<18 months, n = 51 cells, 20 animals) and old (>8 years, n = 51 cells, 16 animals) welsh mountain sheep. Action potentials were recorded using perforated patch clamp in current clamp mode. Myocytes were incrementally stimulated from 0.25 to 8 Hz for 80 cycles. Action potential morphology was analysed and quantification of alternans behaviour was achieved using a discrete fourier transform of action potential trains and subsequent spectral analysis. Action potential duration was assessed at 90% repolarisation (APD90). Results Atrial myocytes from old sheep had a longer APD90 than those from young sheep (488.1 ± 29.3 ms vs. 374.3 ± 31.0 ms in LA at 0.5Hz, p < 0.05). Myocytes from the left and right atria displayed alternans of both phase 0–1 (amplitude) and phase 2–3 (repolarisation). Myocytes from older sheep exhibited alternans behaviour at lower stimulation frequencies than those from young sheep and the oscillations were of a greater magnitude (p < 0.05). Age related changes in alternans were more prominent in the right than the left atrium. Interpretation The longer action potentials seen in atrial myocytes from aged sheep are associated with a decrease in the alternans threshold and an increase in alternans magnitude. These findings help to explain the pro-arrhythmic phenotype of senescence.