High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes

blique plane microscopy (OPM) is a form of light sheet microscopy that uses a single high numerical aperture microscope objective for both fluorescence excitation and collection. In this paper, measurements of the relative collection efficiency of OPM are presented. An OPM system incorporating two sCMOS cameras is then introduced that enables single isolated cardiac myocytes to be studied continuously for 22 seconds in two dimensions at 667 frames per second with 960 × 200 pixels and for 30 seconds with 960 × 200 × 20 voxels at 25 volumes per second. In both cases OPM is able to record in two spectral channels, enabling intracellular calcium to be studied via the probe Fluo-4 AM simultaneously with the sarcolemma and transverse tubule network via the membrane dye Cellmask Orange. The OPM system was then applied to determine the spatial origin of spontaneous calcium waves for the first time and to measure the cell transverse tubule structure at their point of origin. Further results are presented to demonstrate that the OPM system can also be used to study calcium spark parameters depending on their relationship to the transverse tubule structure

Hierarchical statistical techniques are necessary to draw reliable conclusions from analysis of isolated cardiomyocyte studies

Aims It is generally accepted that post-MI heart failure (HF) changes a variety of aspects of sarcoplasmic reticular Ca2+ fluxes but for some aspects there is disagreement over whether there is an increase or decrease. The commonest statistical approach is to treat data collected from each cell as independent, even though they are really clustered with multiple likely similar cells from each heart. In this study, we test whether this statistical assumption of independence can lead the investigator to draw conclusions that would be considered erroneous if the analysis handled clustering with specific statistical techniques (hierarchical tests). Methods and results Ca2+ transients were recorded in cells loaded with Fura-2AM and sparks were recorded in cells loaded with Fluo-4AM. Data were analysed twice, once with the common statistical approach (assumption of independence) and once with hierarchical statistical methodologies designed to allow for any clustering. The statistical tests found that there was significant hierarchical clustering. This caused the common statistical approach to underestimate the standard error and report artificially small P values. For example, this would have led to the erroneous conclusion that time to 50% peak transient amplitude was significantly prolonged in HF. Spark analysis showed clustering, both within each cell and also within each rat, for morphological variables. This means that a three-level hierarchical model is sometimes required for such measures. Standard statistical methodologies, if used instead, erroneously suggest that spark amplitude is significantly greater in HF and spark duration is reduced in HF. Conclusion Ca2+ fluxes in isolated cardiomyocytes show so much clustering that the common statistical approach that assumes independence of each data point will frequently give the false appearance of statistically significant changes. Hierarchical statistical methodologies need a little more effort, but are necessary for reliable conclusions. We present cost-free simple tools for performing these analyses

Flecainide reduces Ca2+ spark and wave frequency via inhibition of the sarcolemmal sodium current

AIMS: Ca(2+) waves are thought to be important in the aetiology of ventricular tachyarrhythmias. There have been conflicting results regarding whether flecainide reduces Ca(2+) waves in isolated cardiomyocytes. We sought to confirm whether flecainide inhibits waves in the intact cardiomyocyte and to elucidate the mechanism. METHODS AND RESULTS: We imaged spontaneous sarcoplasmic reticulum (SR) Ca(2+) release events in healthy adult rat cardiomyocytes. Variation in stimulation frequency was used to produce Ca(2+) sparks or waves. Spark frequency, wave frequency, and wave velocity were reduced by flecainide in the absence of a reduction of SR Ca(2+) content. Inhibition of I(Na) via alternative pharmacological agents (tetrodotoxin, propafenone, or lidocaine) produced similar changes. To assess the contribution of I(Na) to spark and wave production, voltage clamping was used to activate contraction from holding potentials of −80 or −40 mV. This confirmed that reducing Na(+) influx during myocyte stimulation is sufficient to reduce waves and that flecainide only causes Ca(2+) wave reduction when I(Na) is active. It was found that Na(+)/Ca(2+)-exchanger (NCX)-mediated Ca(2+) efflux was significantly enhanced by flecainide and that the effects of flecainide on wave frequency could be reversed by reducing [Na(+)](o), suggesting an important downstream role for NCX function. CONCLUSION: Flecainide reduces spark and wave frequency in the intact rat cardiomyocyte at therapeutically relevant concentrations but the mechanism involves I(Na) reduction rather than direct ryanodine receptor (RyR2) inhibition. Reduced I(Na) results in increased Ca(2+) efflux via NCX across the sarcolemma, reducing Ca(2+) concentration in the vicinity of the RyR2

Non-Linear Optical Microscopy Sheds Light on Cardiovascular Disease

Copyright © 2013 Caorsi et al. Many cardiac diseases have been associated with increased fibrosis and changes in the organization of fibrillar collagen. The degree of fibrosis is routinely analyzed with invasive histological and immunohistochemical methods, giving a limited and qualitative understanding of the tissue's morphological adaptation to disease. Our aim is to quantitatively evaluate the increase in fibrosis by three-dimensional imaging of the collagen network in the myocardium using the non-linear optical microscopy techniques Two-Photon Excitation microscopy (TPE) and Second Harmonic signal Generation (SHG). No sample staining is needed because numerous endogenous fluorophores are excited by a two-photon mechanism and highly non-centrosymmetric structures such as collagen generate strong second harmonic signals. We propose for the first time a 3D quantitative analysis to carefully evaluate the increased fibrosis in tissue from a rat model of heart failure post myocardial infarction. We show how to measure changes in fibrosis from the backward SHG (BSHG) alone, as only backward-propagating SHG is accessible for true in vivo applications. A 5-fold increase in collagen I fibrosis is detected in the remote surviving myocardium measured 20 weeks after infarction. The spatial distribution is also shown to change markedly, providing insight into the morphology of disease progression.Royal Society (Newton International fellowship to VC); Biotechnology and Biological Sciences Research Council [grant number BB/I019448/1] and the Wellcome Trust [grant numbers 091460/Z/10/Z, 092852/Z/10/Z]. ARL was supported by a British Heart Foundation Intermediate Research Fellowship (FS/11/67/28954) and the National Institute for Health Research-funded Cardiovascular Biomedical Research Unit at the Royal Brompton Hospital

Temporal trends and lesion sets for persistent atrial fibrillation ablation: a meta-analysis with trial sequential analysis and meta-regression

BACKGROUND: Ablation for persistent atrial fibrillation (PsAF) has been performed for over 20 years, although success rates have remained modest. Several adjunctive lesion sets have been studied but none have become standard of practice. We sought to describe how the efficacy of ablation for PsAF has evolved in this time period with a focus on the effect of adjunctive ablation strategies. METHODS: Databases were searched for prospective studies of PsAF ablation. We performed meta-regression and trial sequential analysis. RESULTS: A total of 99 studies (15 424 patients) were included. Ablation for PsAF achieved the primary outcome (freedom of atrial fibrillation/atrial tachycardia rate at 12 months follow-up) in 48.2% (5% CI, 44.0-52.3). Meta-regression showed freedom from atrial arrhythmia at 12 months has improved over time, while procedure time and fluoroscopy time have significantly reduced. Through the use of cumulative meta-analyses and trial sequential analysis, we show that some ablation strategies may initially seem promising, but after several randomized controlled trials may be found to be ineffective. Trial sequential analysis showed that complex fractionated atrial electrogram ablation is ineffective and further study of this treatment would be futile, while posterior wall isolation currently does not have sufficient evidence for routine use in PsAF ablation. CONCLUSIONS: Overall success rates from PsAF ablation and procedure/fluoroscopy times have improved over time. However, no adjunctive lesion set, in addition to pulmonary vein isolation, has been conclusively demonstrated to be beneficial. Through the use of trial sequential analysis, we highlight the importance of adequately powered randomized controlled trials, to avoid reaching premature conclusions, before widespread adoption of novel therapies

Direct evidence for microdomain-specific localization and remodeling of functional L-type calcium channels in rat and human atrial myocytes

Background—Distinct subpopulations of L-type calcium channels (LTCCs) with different functional properties exist in cardiomyocytes. Disruption of cellular structure may affect LTCC in a microdomain-specific manner and contribute to the pathophysiology of cardiac diseases, especially in cells lacking organized transverse tubules (T-tubules) such as atrial myocytes (AMs). Methods and Results—Isolated rat and human AMs were characterized by scanning ion conductance, confocal, and electron microscopy. Half of AMs possessed T-tubules and structured topography, proportional to cell width. A bigger proportion of myocytes in the left atrium had organized T-tubules and topography than in the right atrium. Super-resolution scanning patch clamp showed that LTCCs distribute equally in T-tubules and crest areas of the sarcolemma, whereas, in ventricular myocytes, LTCCs primarily cluster in T-tubules. Rat, but not human, T-tubule LTCCs had open probability similar to crest LTCCs, but exhibited ≈40% greater current. Optical mapping of Ca2+ transients revealed that rat AMs presented ≈3-fold as many spontaneous Ca2+ release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca2+ transients were eliminated by either a caveolae-targeted LTCC antagonist or disrupting caveolae with methyl-β-cyclodextrin, with an associated ≈30% whole-cell ICa,L reduction. Heart failure (16 weeks post–myocardial infarction) in rats resulted in a T-tubule degradation (by ≈40%) and significant elevation of spontaneous Ca2+ release events. Although heart failure did not affect LTCC occurrence, it led to ≈25% decrease in T-tubule LTCC amplitude. Conclusions—We provide the first direct evidence for the existence of 2 distinct subpopulations of functional LTCCs in rat and human AMs, with their biophysical properties modulated in heart failure in a microdomain-specific manner

Targeting the ectopy-triggering ganglionated plexuses without pulmonary vein isolation prevents atrial fibrillation

Background Ganglionated plexuses (GPs) are implicated in atrial fibrillation (AF). Endocardial high-frequency stimulation (HFS) delivered within the local atrial refractory period can trigger ectopy and AF from specific GP sites (ET-GP). The aim of this study was to understand the role of ET-GP ablation in the treatment of AF. Methods Patients with paroxysmal AF indicated for ablation were recruited. HFS mapping was performed globally around the left atrium to identify ET-GP. ET-GP was defined as atrial ectopy or atrial arrhythmia triggered by HFS. All ET-GP were ablated, and PVs were left electrically connected. Outcomes were compared with a control group receiving pulmonary vein isolation (PVI). Patients were followed-up for 12 months with multiple 48-h Holter ECGs. Primary endpoint was ≥30 s AF/atrial tachycardia in ECGs. Results In total, 67 patients were recruited and randomized to ET-GP ablation (n = 39) or PVI (n = 28). In the ET-GP ablation group, 103 ± 28 HFS sites were tested per patient, identifying 21 ± 10 (20%) GPs. ET-GP ablation used 23.3 ± 4.1 kWs total radiofrequency (RF) energy per patient, compared with 55.7 ± 22.7 kWs in PVI (p = <.0001). Duration of procedure was 3.7 ± 1.0 and 3.3 ± 0.7 h in ET-GP ablation group and PVI, respectively (p = .07). Follow-up at 12 months showed that 61% and 49% were free from ≥30 s of AF/AT with PVI and ET-GP ablation respectively (log-rank p = .27). Conclusions It is feasible to perform detailed global functional mapping with HFS and ablate ET-GP to prevent AF. This provides direct evidence that ET-GPs are part of the AF mechanism. The lower RF requirement implies that ET-GP targets the AF pathway more specifically