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Aims Cardiac electrophysiological heterogeneity includes: (i) regional differences in action potential (AP) waveform, (ii) AP waveform differences in cells isolated from a single region, (iii) variability of the contribution of individual ion currents in cells with similar AP durations (APDs). The aim of this study is to assess intra-regional AP waveform differences, to quantify the contribution of specific ion channels to the APD via drug responses and to generate a population of mathematical models to investigate the mechanisms underlying heterogeneity in rabbit ventricular cells. Methods and results APD in ∼50 isolated cells from subregions of the LV free wall of rabbit hearts were measured using a voltage-sensitive dye. When stimulated at 2 Hz, average APD90 value in cells from the basal epicardial region was 254 ± 25 ms (mean ± standard deviation) in 17 hearts with a mean interquartile range (IQR) of 53 ± 17 ms. Endo-epicardial and apical-basal APD90 differences accounted for ∼10% of the IQR value. Highly variable changes in APD occurred after IK(r) or ICa(L) block that included a sub-population of cells (HR) with an exaggerated (hyper) response to IK(r) inhibition. A set of 4471 AP models matching the experimental APD90 distribution was generated from a larger population of models created by random variation of the maximum conductances (Gmax) of 8 key ion channels/exchangers/pumps. This set reproduced the pattern of cell-specific responses to ICa(L) and IK(r) block, including the HR sub-population. The models exhibited a wide range of Gmax values with constrained relationships linking ICa(L) with IK(r), ICl, INCX, and INaK. Conclusion Modelling the measured range of inter-cell APDs required a larger range of key Gmax values indicating that ventricular tissue has considerable inter-cell variation in channel/pump/exchanger activity. AP morphology is retained by relationships linking specific ionic conductances. These interrelationships are necessary for stable repolarization despite large inter-cell variation of individual conductances and this explains the variable sensitivity to ion channel block.

S-palmitoylation is an essential lipid modification catalysed by zDHHC-palmitoyl acyltransferases that regulates the localisation and activity of substrates in every class of protein and tissue investigated to date. In the heart, S-palmitoylation regulates sodiumcalcium exchanger (NCX1) inactivation, phospholemman (PLM) inhibition of the Na+/K+ ATPase, Nav1.5 influence on membrane excitability and membrane localisation of heterotrimeric G-proteins. The cell surface localised enzyme zDHHC5 palmitoylates NCX1 and PLM and is implicated in injury during anoxia/reperfusion. Information is lacking about how palmitoylation remodels in cardiac diseases. We investigated expression of zDHHC5 in animal models of left ventricular hypertrophy (LVH) and heart failure (HF), along with HF tissue from humans. zDHHC5 expression was rapidly elevated during onset of LVH, whilst HF was associated with decreased zDHHC5 expression. Paradoxically, palmitoylation of the zDHHC5 substrate NCX1 was significantly reduced in LVH but increased in human HF. Overexpression of zDHHC5 in rabbit ventricular cardiomyocytes was not sufficient to drive changes in palmitoylation of zDHHC5 substrates or overall cardiomyocyte contractility, suggesting changes in zDHHC5 expression in disease may not be a primary driver of pathology. zDHHC5 itself is regulated by post-translational modifications, including palmitoylation in its Cterminal tail, and we found the palmitoylation of zDHHC5 may be increased in heart failure in the same manner as NCX1, suggesting additional regulatory mechanisms such as acyl-CoA availability may be involved. Importantly, this study provides the first evidence that palmitoylation of cardiac substrates is altered in the setting of HF, and that expression of zDHHC5 is dysregulated in both hypertrophy and HF.

Because cardiotoxicity is one of the leading causes of drug failure and attrition, the design of new protocols and technologies to assess proarrhythmic risks on cardiac cells is in continuous development by different laboratories. Current methodologies use electrical, intracellular Ca2+, or contractility assays to evaluate cardiotoxicity. Increasingly, the human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are the in vitro tissue model used in commercial assays because it is believed to recapitulate many aspects of human cardiac physiology. In this work, we demonstrate that the combination of a contractility and voltage measurements, using video-based imaging and fluorescence microscopy, on hiPSC-CMs allows the investigation of mechanistic links between electrical and mechanical effects in an assay design that can address medium throughput scales necessary for drug screening, offering a view of the mechanisms underlying drug toxicity. To assess the accuracy of this novel technique, 10 commercially available inotropic drugs were tested (5 positive and 5 negative). Included were drugs with simple and specific mechanisms, such as nifedipine, Bay K8644, and blebbistatin, and others with a more complex action such as isoproterenol, pimobendan, digoxin, and amrinone, among others. In addition, the results provide a mechanism for the toxicity of itraconazole in a human model, a drug with reported side effects on the heart. The data demonstrate a strong negative inotropic effect because of the blockade of L-type Ca2+ channels and additional action on the cardiac myofilaments. We can conclude that the combination of contractility and action potential measurements can provide wider mechanistic knowledge of drug cardiotoxicity for preclinical assays.

Animal models are 78% accurate in determining whether drugs will alter contractility of the human heart. To evaluate the suitability of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for predictive safety pharmacology, we quantified changes in contractility, voltage, and/or Ca2+ handling in 2D monolayers or 3D engineered heart tissues (EHTs). Protocols were unified via a drug training set, allowing subsequent blinded multicenter evaluation of drugs with known positive, negative, or neutral inotropic effects. Accuracy ranged from 44% to 85% across the platform-cell configurations, indicating the need to refine test conditions. This was achieved by adopting approaches to reduce signal-to-noise ratio, reduce spontaneous beat rate to ≤ 1 Hz or enable chronic testing, improving accuracy to 85% for monolayers and 93% for EHTs. Contraction amplitude was a good predictor of negative inotropes across all the platform-cell configurations and of positive inotropes in the 3D EHTs. Although contraction- and relaxation-time provided confirmatory readouts forpositive inotropes in 3D EHTs, these parameters typically served as the primary source of predictivity in 2D. The reliance of these “secondary” parameters to inotropy in the 2D systems was not automatically intuitive and may be a quirk of hiPSC-CMs, hence require adaptations in interpreting the data from this model system. Of the platform-cell configurations, responses in EHTs aligned most closely to the free therapeutic plasma concentration. This study adds to the notion that hiPSC-CMs could add value to drug safety evaluation.

Key points Spatiotemporal contractility analysis of human induced pluripotent stem cell derived cardiomyocyte (hiPSC-CM) monolayers seeded on conventional, rigid surfaces (glass or plastic) revealed the presence of multiphasic contraction patterns across the monolayer with a high variability, despite action potentials recorded in the same areas being identical. These multiphasic patterns are not present in single cells, in detached monolayers or in monolayers seeded on soft substrates such as a hydrogel, where only 'twitch'-like transients are observed. HiPSC-CM monolayers that display a high percentage of regions with multiphasic contraction have significantly increased contractile duration and a decreased lusotropic drug response. There is no indication that the multiphasic contraction patterns are associated with significant activation of the stress-activated NPPA or NPPB signalling pathways. A computational model of cell clusters supports the biological findings that the rigid surface and the differential cell-substrate adhesion underly multiphasic contractile behaviour of hiPSC-CMs. Abstract Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) in monolayers interact mechanically via cell-cell and cell-substrate adhesion. Spatiotemporal features of contraction were analysed in hiPSC-CM monolayers 1) attached to glass or plastic (Young's modulus (E) >1 GPa), 2) detached (substrate-free) and 3) attached to a flexible collagen hydrogel (E = 22 kPa). The effects of isoprenaline on contraction were compared between rigid and flexible substrates. To clarify underlying mechanisms, further gene expression and computational studies were performed. HiPSC-CM monolayers exhibited multi-phasic contractile profiles on rigid surfaces in contrast to hydrogels, substrate-free cultures or single cells where only simple twitch-like time-courses were observed. Isoprenaline did not change the contraction profile on either surface, but its lusitropic and chronotropic effects were greater in hydrogel compared to glass. There was no significant difference between stiff and flexible substrates in regard to expression of the stress activated genes NPPA and NPPB. A computational model of cell clusters demonstrated similar complex contractile interactions on stiff substrates as a consequence of cell-to-cell functional heterogeneity. Rigid biomaterial surfaces give rise to unphysiological, multi-phasic contractions in hiPSC-CM monolayers. Flexible substrates are necessary for normal twitch-like contractility kinetics and interpretation of inotropic interventions. This article is protected by copyright. All rights reserved.

The immature electrophysiology of human-induced pluripotent stem cell-derived cardiomyocytes (hiCMs) complicates their use for therapeutic and pharmacological purposes. An insufficient inward rectifying current (IK1) and the presence of a funny current (if) cause spontaneous electrical activity. This study tests the hypothesis that the co-culturing of hiCMs with a human embryonic kidney (HEK) cell-line expressing the Kir2.1 channel (HEK-IK1) can generate an electrical syncytium with an adult-like cardiac electrophysiology. The mechanical activity of co-cultures using different HEK-IK1:hiCM ratios was compared with co-cultures using wildtype (HEK–WT:hiCM) or hiCM alone on days 3–8 after plating. Only ratios of 1:3 and 1:1 showed a significant reduction in spontaneous rate at days 4 and 6, suggesting that IK1 was influencing the electrophysiology. Detailed analysis at day 4 revealed an increased incidence of quiescent wells or sub-areas. Electrical activity showed a decreased action potential duration (APD) at 20% and 50%, but not at 90%, alongside a reduced amplitude of the aggregate AP signal. A computational model of the 1:1 co-culture replicates the electrophysiological effects of HEK–WT. The addition of the IK1 conductance reduced the spontaneous rate and APD20, 50 and 90, and minor variation in the intercellular conductance caused quiescence. In conclusion, a 1:1 co-culture HEK-IK1:hiCM caused changes in electrophysiology and spontaneous activity consistent with the integration of IK1 into the electrical syncytium. However, the additional electrical effects of the HEK cell at 1:1 increased the possibility of electrical quiescence before sufficient IK1 was integrated into the syncytium.

Human stem cell-derived cardiomyocytes (hSC-CMs) hold great promise as in vitro models to study the electrophysiological effects of novel drug candidates on human ventricular repolarization. Two recent large validation studies have demonstrated the ability of hSC-CMs to detect drug-induced delayed repolarization and ""cellrhythmias"" (interrupted repolarization or irregular spontaneous beating of myocytes) linked to Torsade-de-Pointes proarrhythmic risk. These (and other) studies have also revealed variability of electrophysiological responses attributable to differences in experimental approaches and experimenter, protocols, technology platforms used, and pharmacologic sensitivity of different human-derived models. Thus, when evaluating drug-induced repolarization effects, there is a need to consider 1) the advantages and disadvantages of different approaches, 2) the need for robust functional characterization of hSC-CM preparations to define ""fit for purpose"" applications, and 3) adopting standardized best practices to guide future studies with evolving hSC-CM preparations. Examples provided and suggested best practices are instructional in defining consistent, reproducible, and interpretable ""fit for purpose"" hSC-CM-based applications. Implementation of best practices should enhance the clinical translation of hSC-CM-based cell and tissue preparations in drug safety evaluations and support their growing role in regulatory filings.

Objectives: We aimed to establish which of sitting height (SH) and total height (Ht) is most appropriately used in the scaling of adult body mass (BM), waist circumference (WC), and forced vital capacity (FVC), considering likely explanations and proposing a suitable index for each. Methods: Data were from the US Third National Health and Nutrition Survey for white and black American women and men aged 20 to 50 years. Statistical analysis involved mainly correlation coefficients, the multiple regression of BM, WC, or FVC on SH and leg length (LL), and fitting of allometric regression equations relating each of BM, WC, and FVC to SH or Ht. Results: BM and WC correlated more strongly with SH than with Ht, and FVC correlated more strongly with Ht. Associations with LL were negative for WC, negative or nonsignificant for BM, and positive for FVC. Using round‐number exponents for Ht and SH, the allometric relationships indicated that appropriate indices are BM/SH3, WC/SH, and FVC/Ht2. Conclusions: Contrary to usual practice, BM and WC are better scaled in relation to SH than to Ht. FVC is slightly better scaled in relation to Ht, as is conventional. Interpretations involve the small influence of LL on BM and the influence both of gluteo‐femoral fatness on measured SH and of childhood health and nutrition on adult LL, WC, and FVC. It is evident that SH should be measured more often for research purposes.

Aims Treatment of arrhythmias evoked by hypothermia/rewarming remains challenging, and the underlying mechanisms are unclear. This in vitro experimental study assessed cardiac electrophysiology in isolated rabbit hearts at temperatures occurring in therapeutic and accidental hypothermia. Methods and results Detailed ECG, surface electrogram, and panoramic optical mapping were performed in isolated rabbit hearts cooled to moderate (31°C) and severe (17°C) hypothermia. Ventricular activation was unchanged at 31°C while action potential duration (APD) was significantly prolonged (176.9 ± 4.2 ms vs. 241.0 ± 2.9 ms, P Conclusions Moderate hypothermia does not significantly change ventricular conduction time but prolongs repolarization and is pro-arrhythmic. Further cooling to severe hypothermia causes parallel changes in ventricular activation and repolarization, changes which are anti-arrhythmic. Therefore, relative changes in QRS and QT intervals (QR/QTc) emerge as an ECG-biomarker of pro-arrhythmic activity. Risk for ventricular fibrillation appears to be linked to the relatively low temperature sensitivity of ventricular transmural conduction, a conclusion supported by the anti-arrhythmic effect of heptanol at 31°C.

In recent decades, drug development costs have increased by approximately a hundredfold, and yet about 1 in 7 licensed drugs are withdrawn from the market, often due to cardiotoxicity. This review considers whether technologies using human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) could complement existing assays to improve discovery and safety while reducing socioeconomic costs and assisting with regulatory guidelines on cardiac safety assessments. We draw on lessons from our own work to suggest a panel of 12 drugs that will be useful in testing the suitability of hiPSC-CM platforms to evaluate contractility. We review issues, including maturity versus complexity, consistency, quality, and cost, while considering a potential need to incorporate auxiliary approaches to compensate for limitations in hiPSC-CM technology. We give examples on how coupling hiPSC-CM technologies with Cas9/CRISPR genome engineering is starting to be used to personalize diagnosis, stratify risk, provide mechanistic insights, and identify new pathogenic variants for cardiovascular disease.

The pulmonary veins have an external sleeve of cardiomyocytes that are a widely recognised source of ectopic electrical activity that can lead to atrial fibrillation. Although the mechanisms behind this activity are currently unknown, changes in intracellular calcium (Ca2+) signalling are purported to play a role. Therefore, the intracellular Ca2+ concentration was monitored in the pulmonary vein using fluo-4 and epifluorescence microscopy. Electrical field stimulation evoked a synchronous rise in Ca2+ in neighbouring cardiomyocytes; asynchronous spontaneous Ca2+ transients between electrical stimuli were also present. Immediately following termination of electrical field stimulation at 3 Hz or greater, the frequency of the spontaneous Ca2+ transients was increased from 0.45 ± 0.06 Hz under basal conditions to between 0.59 ± 0.05 and 0.65 ± 0.06 Hz (P

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and higher throughput platforms have emerged as potential tools to advance cardiac drug safety screening. This study evaluated the use of high bandwidth photometry applied to voltage-sensitive fluorescent dyes (VSDs) to assess drug-induced changes in action potential characteristics of spontaneously active hiPSC-CM. Human iPSC-CM from 2 commercial sources (Cor.4U and iCell Cardiomyocytes) were stained with the VSD di-4-ANEPPS and placed in a specialized photometry system that simultaneously monitors 2 wavebands of emitted fluorescence, allowing ratiometric measurement of membrane voltage. Signals were acquired at 10 kHz and analyzed using custom software. Action potential duration (APD) values were normally distributed in cardiomyocytes (CMC) from both sources though the mean and variance differed significantly (APD90: 229 ± 15 ms vs 427 ± 49 ms [mean ± SD, P < 0.01]; average spontaneous cycle length: 0.99 ± 0.02 s vs 1.47 ± 0.35 s [mean ± SD, P < 0.01], Cor.4U vs iCell CMC, respectively). The 10-90% rise time of the AP (Trise) was ∼6 ms and was normally distributed when expressed as 1/[Formula: see text] in both cell preparations. Both cell types showed a rate dependence analogous to that of adult human cardiac cells. Furthermore, nifedipine, ranolazine, and E4031 had similar effects on cardiomyocyte electrophysiology in both cell types. However, ranolazine and E4031 induced early after depolarization-like events and high intrinsic firing rates at lower concentrations in iCell CMC. These data show that VSDs provide a minimally invasive, quantitative, and accurate method to assess hiPSC-CM electrophysiology and detect subtle drug-induced effects for drug safety screening while highlighting a need to standardize experimental protocols across preparations.

Conduction abnormalities are frequently associated with cardiac disease, though the mechanisms underlying the commonly associated increases in PQ interval are not known. This study uses a chronic left ventricular (LV) apex myocardial infarction (MI) model in the rabbit to create significant left ventricular dysfunction (LVD) 8 weeks post-MI. In vivo studies established that the PQ interval increases by approximately 7 ms (10%) with no significant change in average heart rate. Optical mapping of isolated Langendorff perfused rabbit hearts recapitulated this result: time to earliest activation of the LV was increased by 14 ms (16%) in the LVD group. Intra-atrial and LV transmural conduction times were not altered in the LVD group. Isolated AVN preparations from the LVD group demonstrated a significantly longer conduction time (by approximately 20 ms) between atrial and His electrograms than sham controls across a range of pacing cycle lengths. This difference was accompanied by increased effective refractory period and Wenckebach cycle length, suggesting significantly altered AVN electrophysiology post-MI. The AVN origin of abnormality was further highlighted by optical mapping of the isolated AVN. Immunohistochemistry of AVN preparations revealed increased fibrosis and gap junction protein (connexin43 and 40) remodelling in the AVN of LVD animals compared to sham. A significant increase in myocyte–non-myocyte connexin co-localization was also observed after LVD. These changes may increase the electrotonic load experienced by AVN muscle cells and contribute to slowed conduction velocity within the AVN., Highlights • Chronic myocardial infarction (MI) causes changes in atrio-ventricular node (AVN) function. • Isolated hearts post-MI show delays in ventricular activation due to slowed conduction via the AVN. • Isolated AVN preparations demonstrated AVN electrical remodelling post-MI. • Electrical remodelling is associated with fibrosis and altered expression of connexins in the AVN. • AVN dysfunction post-MI is caused by localized functional and structural remodelling.

Quantification of contraction is essential to the study of cardiac diseases, injury, and responses to drugs. While there are many techniques to assess contractility, most rely on costly, dedicated hardware and advanced informatics, and can only be used in specific experimental models. We have developed an automated open-source software tool (MUSCLEMOTION) for use with standard imaging equipment, to assess contractility in vitro and in vivo and quantify responses to drugs and diseases. We describe high-speed and disturbance-free acquisition of images from either electrically paced or non-paced human pluripotent stem cell-derived cardiomyocytes, isolated adult cardiomyocytes, zebrafish hearts, and human echocardiograms. Recordings are then used as input for automated batch analysis by the MUSCLEMOTION software tool configured with specific settings and parameters tailored to the recording technique. Details on accuracy, interpretation, and troubleshooting are discussed. Acquisition duration depends on the experimental setup and aim, but quantification of drug or disease responses in an in vitro muscle model can typically be completed within a few hours. © 2018 by John Wiley & Sons, Inc.

Contraction of muscle reflects its physiological state. Methods to quantify contraction are often complex, expensive and tailored to specific models or recording conditions, or require specialist knowledge for data extraction. Here we describe an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes and pharmacological responses. MUSCLEMOTION allowed rapid and easy measurement of contractility in (i) single cardiomyocytes from primary adult heart and human pluripotent stem cells, (ii) multicellular 2D-cardiomyocyte cultures, 3D engineered heart tissues and cardiac organoids/microtissues in vitro and (iii) intact hearts of zebrafish and humans in vivo. Good correlation was found with conventional measures of contraction in each system. Thus, using a single method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell- and animal models and human echocardiograms.

We describe a novel two-photon (2P) laser scanning microscopy (2PLSM) protocol that provides ratiometric transmural measurements of membrane voltage (Vm) via Di-4-ANEPPS in intact mouse, rat and rabbit hearts with subcellular resolution. The same cells were then imaged with Fura-2/AM for intracellular Ca2+ recordings. Action potentials (APs) were accurately characterized by 2PLSM vs. microelectrodes, albeit fast events ( 1ms) could be accurately described by 2PLSM. In conclusion, Vm - and Ca2+-sensitive dyes can be 2P excited within the cardiac muscle wall to provide AP and Ca2+ signals to ∼400 µm. (© 2013 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim)

Background— Electric excitability in the ventricular wall is influenced by cellular electrophysiology and passive electric properties of the myocardium. Action potential (AP) rise time, an indicator of myocardial excitability, is influenced by conduction pattern and distance from the epicardial surface. This study examined AP rise times and conduction velocity as the depolarizing wavefront approaches the epicardial surface. Methods and Results— Two-photon excitation of di-4-aminonaphthenyl-pyridinum-propylsulfonate was used to measure electric activity at discrete epicardial layers of isolated Langendorff-perfused rabbit hearts to a depth of 500 μm. Endo-to-epicardial wavefronts were studied during right atrial or ventricular endocardial pacing. Similar measurements were made with epi-to-endocardial, transverse, and longitudinal pacing protocols. Results were compared with data from a bidomain model of 3-dimensional (3D) electric propagation within ventricular myocardium. During right atrial and endocardial pacing, AP rise time (10%–90% of upstroke) decreased by ≈50% between 500 and 50 μm from the epicardial surface, whereas conduction velocity increased and AP duration was only slightly shorter (≈4%). These differences were not observed with other conduction patterns. The depth-dependent changes in rise time were larger at higher pacing rates. Modeling data qualitatively reproduced the behavior seen experimentally and demonstrated a parallel reduction in peak I Na and electrotonic load as the wavefront approaches the epicardial surface. Conclusions— Decreased electrotonic load at the epicardial surface results in more rapid AP upstrokes and higher conduction velocities compared with the bulk myocardium. Combined effects of tissue depth and pacing rate on AP rise time reduce conduction safety and myocardial excitability within the ventricular wall.

Rationale: There are several methods to measure cardiomyocyte and muscle contraction, but these require customized hardware, expensive apparatus, and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming, and only specialist researchers can quantify data. Objective: Here, we describe and validate an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes, and pharmacological responses. Methods and Results: MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in (1) 1-dimensional in vitro models, such as isolated adult and human pluripotent stem cell-derived cardiomyocytes; (2) 2-dimensional in vitro models, such as beating cardiomyocyte monolayers or small clusters of human pluripotent stem cell-derived cardiomyocytes; (3) 3-dimensional multicellular in vitro or in vivo contractile tissues, such as cardiac “organoids,” engineered heart tissues, and zebrafish and human hearts. MUSCLEMOTION was effective under different recording conditions (bright-field microscopy with simultaneous patch-clamp recording, phase contrast microscopy, and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement, such as optical flow, post deflection, edge-detection systems, or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. Conclusions: Using a single open-source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell, animal, and human models.

Background: Electrical activity in isolated cardiomyocytes has been widely studied using patch clamping, which offers limited throughput. Using voltage- and calcium-sensitive dyes, the electrical activity, calcium dynamics and contractility profile of cells can be measured simultaneously in approximately 100 cells per heart. This feature provides further insight into the mechanisms of excitation-contraction coupling in the failing heart. Moreover, this high-throughput methodology allows the study of large cell populations in individual hearts, offering a quantitative measure of cellular and regional heterogeneity, otherwise obscured by animal variation and undersampling. Methods: Adult rabbit cardiomyocytes were isolated from the left ventricle of infarcted (MI), control (Sham), and stock rabbits, loaded with the fluorescent, voltage-sensitive dye FluoVolt® (1:6000), and plated on to 35mm glass-bottomed plates (Krebs solution at 37°C; 1.8mM CaCl2). Left ventricle cells were serially paced at 1Hz, 2Hz and 3Hz using field stimulation (2x20mm graphite electrodes; 40V, 2ms pulse width). Separately isolated cells originating from either epicardium or endocardium were paced at 2Hz. A 10s action potential train was recorded from each cell. Results: The methodology corroborates previously published work showing an increase in mean APD90 in MI at slow rates. Mean APD90 in MI (Nanimals=6, ncells=450-800) was 374±11ms, 287±4ms, and 208±4ms at 1Hz, 2Hz and 3Hz, respectively. Mean APD90 in Sham was 336±16ms, 252±6ms, 206±1ms, at 1Hz, 2Hz, and 3Hz, respectively (Mean±S.E.M). Regional analyses (Nanimals=9, ncells=554) found AP90 was shortened in the epicardium vs. the endocardium in stock (21±6ms difference) and sham animals (37±10ms), while this difference was entirely lost in MI (P=0.323). Moreover, epicardial cell populations showed consistently higher variability than endocardial cells, across all experimental groups. This technique consequently provides accurate quantification of biological variation in individual hearts and heart regions, suggesting a strong potential for drug screening assays.

The extent to which exercise-induced changes to postprandial metabolism are dependant on the associated energy deficit is not known. To determine the effects of exercise, with and without energy replacement, on postprandial metabolism. Each subject underwent three 2-day trials in random order. On day 1 of each trial subjects rested (control), walked at 50% maximal oxygen uptake to induce a net energy expenditure of 27 kJ kg−1 body mass (energy-deficit) or completed the same walk with the net energy expended replaced (energy-replacement). On day 2 subjects completed an 8.5-h metabolic assessment. For 3 days prior to day 2, subjects consumed an isocaloric diet, avoided planned exercise (apart from exercise interventions) and alcohol. A total of 13 overweight/obese men (age: 40±8 years, body mass index: 31.1±3.0 kg m−2). Postprandial triglyceride, insulin, glucose, non-esterified fatty acid and 3-hydroxybutyrate concentrations and substrate utilization rates were determined. Energy-deficit lowered postprandial triglyceride concentrations by 14 and 10% compared with control and energy-replacement (P

INTRODUCTION Ventricular fibrillation (VF) studies show that ECG-dominant frequency (DF) decreases as ischemia develops. This study investigates the contribution of the principle ischemic metabolic components to this decline. METHODS AND RESULTS Rabbit hearts were Langendorff-perfused at 40 mL/min with Tyrode's solution and loaded with RH237. Epicardial optical action potentials were recorded with a photodiode array (256 sites, 15 x 15 mm). After 60 seconds of VF (induced by burst pacing), global ischemia was produced by low flow (6 mL/min), or the solution changed to impose hypoxia (95% N2/5% CO2), low pH(o) (6.7, 80% O2/20% CO2), or raised [K+](o) (8 mM). DF of the optical signals was determined at each site. Conduction velocity (CV), action potential duration (APD90), effective refractory period (ERP), activation threshold, dV/dt(max), and membrane potential were measured in separate experiments during ventricular pacing. During VF, ischemia decreased DF in the left ventricle (LV) (to [58 +/- 6]%, P < 0.001), but not the right (RV) ([93 +/- 5]%). Raised [K+](o) reproduced this DF pattern (LV: [67 +/- 12]%, P < 0.001; RV: [95 +/- 9]%). LV DF remained elevated in hypoxia or low pH(o). During ventricular pacing, ischemia decreased CV in LV but not RV. Raised [K+](o) did not change CV in either ventricle. Ischemia and raised [K+](o) shortened APD90 without altering ERP. LV activation threshold increased in both ischemia and raised [K+](o) and was associated with diastolic depolarization and decreased dV/dt(max). CONCLUSIONS These results suggest that during VF, decreased ECG DF in global ischemia is largely due to elevated [K+](o) affecting the activation thresholds in the LV rather than RV.

Procedures that reduce contraction are used to facilitate optical measurements of membrane potential, but it is unclear to what extent they affect the excitability of the heart. This study has examined the electrophysiological consequences of a range of extracellular [Ca2+] (0.7–2.5 mmol l−1), 2,3-butane-dione monoxime (BDM; 1–20 mmol l−1) and cytochalasin-D (Cyto-D; 1–5 μmol l−1). Methods. Monophasic action potentials (MAPs) were recorded from the basal epicardial surface of the left ventricle of isolated rabbit hearts. Conduction delay (CD) and time to 90% repolarisation of the monophasic action potential (MAPD90) were measured. The effects of BDM and Cyto-D on restitution were studied at a [Ca2+] of 1.9 mmol l−1. Restitution curves for MAPD90 were generated using a standard S1–S2 protocol. Results. All manoeuvres decreased left ventricular developed pressure (LVDP): 0.7 mmol l−1 Ca2+ to 74.0 ± 6.1%, 20 mmol l−1 BDM to 4.5 ± 1.0%, and 5 μmol l−1 Cyto-D to 12.8 ± 3.5% of control value. CD decreased from a control value (33.3 ± 1.0 ms, n= 16) to 93.0 ± 2.2% in 0.7 mmol l−1 Ca2+, but increased to 133.7 ± 10.5% in 20 mmol l−1 BDM and 127.4 ± 10.6% in 5 μmol l−1 Cyto-D. At 350 ms pacing cycle length, MAPD90 (control = 119.6 ± 1.7 ms n= 16) was prolonged by reduced extracellular [Ca2+]. BDM had no effects on MAPD90 at control pacing rates. Cyto-D caused a significant prolongation (to 115.0 ± 3.0% of control, n= 6) at the highest concentration studied (5 μmol l−1). Both BDM (20 mmol l−1) and Cyto-D (3 μmol l−1) flattened the restitution curves but neither agent altered maximum MAPD90. Conclusions. Extracellular [Ca2+] of 1.9 mmol l−1 in conjunction with a moderate dose of Cyto-D (3 μmol l−1) reduced contractility with minimal effects on action potential duration and conduction at a fixed pacing cycle length. However, both BDM and Cyto-D had pronounced effects on electrical restitution.

Acidosis affects the mechanical and electrical activity of mammalian hearts but comparatively little is known about its effects on the function of the atrio-ventricular node (AVN). In this study, the electrical activity of the epicardial surface of the left ventricle of isolated Langendorff-perfused rabbit hearts was examined using optical methods. Perfusion with hypercapnic Tyrode's solution (20% CO2, pH 6.7) increased the time of earliest activation (Tact) from 100.5 ± 7.9 to 166.1 ± 7.2 ms (n = 8) at a pacing cycle length (PCL) of 300 ms (37°C). Tact increased at shorter PCL, and the hypercapnic solution prolonged Tact further: at 150 ms PCL, Tact was prolonged from 131.0 ± 5.2 to 174.9 ± 16.3 ms. 2:1 AVN block was common at shorter cycle lengths. Atrial and ventricular conduction times were not significantly affected by the hypercapnic solution suggesting that the increased delay originated in the AVN. Isolated right atrial preparations were superfused with Tyrode's solutions at pH 7.4 (control), 6.8 and 6.3. Low pH prolonged the atrial-Hisian (AH) interval, the AVN effective and functional refractory periods and Wenckebach cycle length significantly. Complete AVN block occurred in 6 out of 9 preparations. Optical imaging of conduction at the AV junction revealed increased conduction delay in the region of the AVN, with less marked effects in atrial and ventricular tissue. Thus acidosis can dramatically prolong the AVN delay, and in combination with short cycle lengths, this can cause partial or complete AVN block and is therefore implicated in the development of brady-arrhythmias in conditions of local or systemic acidosis.

It is generally accepted that the normal T-wave is the manifestation of repolarisation gradients in the ventricles of the heart in an endocardial- epicardial direction. However, several studies contradict these findings, attributing T-wave polarity to apico-basal and/or interventricular repolarisation gradients. Consequently, the mechanisms responsible for the T-wave under both normal and pathological conditions remain unclear. In this study, isolated Langendorff-perfused rabbit hearts (37oC) were suspended in a customized chamber containing a frame that allowed the heart to be rotated to ±120° from a central position. Epicardial voltage was monitored by staining the heart with voltage sensitive dye (di-4-ANEPPS). Illumination at 480nm was provided by an array of LEDs (OptoLED, Cairn Research Ltd.). Emitted fluorescence was collected, filtered with 590nm long-pass filter and focused on a CCD chip (Redshirt Imaging, Decatur, GA) which acquired images (80x80 pixels) every 1ms. Recordings were made from 3 sides to obtain a panoramic map of cardiac electrical activity. Simultaneously, pseudo-ECG recordings (equivalent to Lead I) were recorded while pacing via electrodes placed on the right atrium. Epicardial ventricular repolarisation patterns were correlated to corresponding ECG recordings. In contrast to some previous studies, less than 5% of the epicardium (right and left ventricles) was repolarised by the peak of the T-wave. In addition, the presence of an apico-basal repolarisation gradient on the left ventricle (LV) developed in the latter half of the T-wave. An inverse relationship between APD and activation time was observed across the LV suggesting that activation pattern is a determinant of epicardial APD. In conclusion, the lack of a prominent epicardial repolarisation gradient during the first half of the T-wave suggests that regions other than the epicardial surface are responsible for the initial deflection.

This study examines the changes in ventricular conduction (CV) and action potential (AP) shape in isolated rat hearts during prolonged exposure to raised intracellular cAMP. Adult male Wistar rats (300-500g) were euthanized and hearts removed and Langendorff perfused with modified Tyrode's solution at 37oC. CV was recorded using custom electrodes placed on the epicardial surface of the left ventricle, which was paced continuously at 8HZ. The delay between the AP wavefront reaching two sequential sets of recording electrodes was used to calculate the CV. AP recordings were made using a voltage sensitive dye, di-4-ANEPPs, via a fiber optic light guide system (3mm diameter). Under control conditions the average CV was 54.9 ± 3.3 cm/s. Treatment with a combination of forskolin and IBMX (fsk+IBMX) increased CV by 7.1±1.7% (p

Time for primary review 34 days. In their classic studies published in 1964, Han, Moe and co-workers [1,2] established an association between nonuniform recovery of excitability and lowered fibrillation threshold. They concluded that “those agencies known to favour the development of ventricular fibrillation were found to increase the temporal dispersion of recovery of excitability, whether the average refractory period was reduced … or increased…. The results emphasise the importance of nonuniformity of excitability and conduction velocity during the relative refractory period in the induction of turbulent impulse propagation.” The purpose of this review is to describe the basis of dispersion in recovery of excitability in the ventricle and its association with arrhythmogenesis. Ventricular tachyarrhythmias are readily generated not only in acute ischaemia/infarction but also in hearts that have undergone remodelling following myocardial infarction [3–5]. Life-threatening arrhythmias are commonly seen in patients with previous myocardial infarction in the absence of new ischaemic events, as evidenced by the ability to initiate sustained ventricular tachycardia by programmed stimulation [6]. The risk of ventricular arrhythmias and sudden death in heart failure is inversely proportional to the left ventricular ejection fraction [7]. However, the Veterans Heart Failure Trial and other studies suggest that the proportion of deaths that are sudden is higher in patients with less severe LV dysfunction [8]. Such individuals are less likely to die from pump failure, hence they are at greater relative (but not absolute) risk of sudden death in comparison with patients with advanced heart failure. The classical prerequisites for the development of reentry are the presence of a potential circuit around an anatomical obstacle, unidirectional block, and sufficiently slow conduction to enable recovery of excitability in time for reexcitation by the depolarizing wavefront [9]. A feature of this type of reentry is the presence … * Corresponding author. Tel.: +44-141-211-4722; fax: +44-141-552-4683 stuart.cobbe{at}clinmed.gla.ac.uk

Patients with congestive heart failure or left ventricular dysfunction (LVD) have a high incidence of ventricular tachyarrhythmias and sudden cardiac death. In addition to structural, metabolic and neuroendocrine changes, mechanoelectrical feedback may play a role in arrhythmogenesis in heart failure. Three groups of rabbits (n = 10 for each) were studied: chronic coronary ligation with ejection fraction (EF)or = 0.45 or0.45, and sham-operated controls. Ventricular fibrillation (VF) thresholds were measured at LV pressures of 0 and 40 mm Hg during modified Langendorff perfusion. Intervals between local activations during VF (VFI) were used as an index of refractoriness. Global dispersion was expressed as coefficient of variation of VFI; local dispersion by maximum difference in VFI between adjacent sites. Median VF threshold was lower at 0 mm Hg in the lower EF group compared to controls (30 vs. 67.5 mA, P0.05). VF threshold in control hearts was lower at 40 mm Hg than at 0 mm Hg (P0.01), but there was no further reduction in threshold in LVD hearts at 40 mm Hg. Global dispersion of VFI did not differ significantly between groups. Local dispersion of VFI in the lower EF group was greater than in controls at 0 mm Hg in the infarct border zone (P0.05). At 40 mm Hg, local dispersion of VFI in zones bordering and remote from the infarct were greater in both LVD groups than in controls (P0.05). Local inhomogeneity of refractoriness is more marked in the infarct border zone, but latent abnormalities are evident in normal myocardium of rabbits with left ventricular dysfunction and are revealed by left ventricular distension.

Objective: To determine the effect of acute left ventricular dilatation on refractoriness in normal hearts. Methods: During sustained ventricular fibrillation (VF) in isolated perfused hearts, recording of local activation time yields VF intervals which provide an index of local refractoriness. Simultaneous measurement from multiple sites enables study of spatial aspects of changes in refractoriness. We studied the effects of stretch on the magnitude and dispersion of changes in VF interval in 10 isolated, Langendorff-perfused rabbit hearts using a flexible epicardial array containing 240 unipolar electrodes. The left ventricular pressure was increased from 0 to 40 mmHg by inflation of an intraventricular balloon during sustained VF. Results: The current threshold for VF induction fell from 64±11 mA to 43±11 mA (mean±SE, P

We describe a novel two-photon (2P) laser scanning microscopy (2PLSM) protocol that provides ratiometric transmural measurements of membrane voltage (Vm ) via Di-4-ANEPPS in intact mouse, rat and rabbit hearts with subcellular resolution. The same cells were then imaged with Fura-2/AM for intracellular Ca(2+) recordings. Action potentials (APs) were accurately characterized by 2PLSM vs. microelectrodes, albeit fast events (1 ms) were sub-optimally acquired by 2PLSM due to limited sampling frequencies (2.6 kHz). The slower Ca(2+) transient (CaT) time course (1ms) could be accurately described by 2PLSM. In conclusion, Vm - and Ca(2+) -sensitive dyes can be 2P excited within the cardiac muscle wall to provide AP and Ca(2+) signals to ∼400 µm.

Aims: Atrial fibrillation (AF) is increased in patients with heart failure resulting from myocardial infarction (MI). We aimed to determine the effects of chronic ventricular MI in rabbits on the susceptibility to AF, and underlying atrial electrophysiological and Ca2+-handling mechanisms. Methods and results: In Langendorff-perfused rabbit hearts, under beta-adrenergic-stimulation with isoproterenol (1 µM; ISO), 8 weeks MI decreased AF threshold, indicating increased AF-susceptibility. This was associated with increased atrial action potential duration-alternans at 90% repolarisation, by 147%, and no significant change in mean APD or atrial global conduction velocity (n=6-13 non-MI hearts, 5-12 MI). In atrial isolated myocytes, also under beta-stimulation, L-type Ca2+ current (ICaL) density and intracellular Ca2+-transient amplitude were decreased by MI, by 35% and 41%, respectively, and the frequency of spontaneous depolarisations (SDs) was substantially increased. MI increased atrial myocyte size and capacity, and markedly decreased transverse-tubule density. In non-MI hearts perfused with ISO, the ICaL-blocker nifedipine, at a concentration (0.02 µM) causing an equivalent ICaL-reduction (35%) to that from the MI, did not affect AF-susceptibility, and decreased APD. Conclusion: chronic MI in rabbits remodels atrial structure, electrophysiology and intracellular Ca2+-handling. Increased susceptibility to AF by MI, under beta-adrenergic-stimulation, may result from associated production of atrial APD-alternans and SDs, since steady-state APD and global conduction velocity were unchanged under these conditions, and may be unrelated to the associated reduction in whole-cell ICaL. Future studies may clarify potential contributions of local conduction changes, and cellular and sub-cellular mechanisms of alternans, to the increased AF-susceptibility.

Recent data indicate that Ca(2+) cycling in isolated atrioventricular node (AVN) cells contributes to setting spontaneous rate. The aim of the present study was to extend this observation to the intact AVN in situ, by evaluating the effects of inhibiting sarcoplasmic reticulum Ca(2+) uptake with cyclopiazonic acid (CPA) on intact AVN spontaneous activity and its response to isoprenaline. A model of the AVN-paced heart was produced to investigate intact AVN automaticity, by surgical ablation of the sino-atrial node (SAN) in the rabbit Langendorff-perfused heart. Electrograms were recorded from a site close to the AVN (triangle of Koch), an atrial site above the AVN, the left atrium and right ventricle, enabling AVN pacing of the preparation to be confirmed. Before SAN ablation, the heart rate was 166.8 ± 5.4 beats min(-1). Ablation of the SAN was clearly indicated by a sudden and significant decrease of heart rate to 108.6 ± 9.6 beats min(-1) (P0.01, n = 10). Isoprenaline (100 nm) increased AVN rate to 187.8 ± 12.0 beats min(-1) after 1 min of application (P0.01, n = 10). Cyclopiazonic acid (10 and 30 μm) decreased AVN rate to 81.6 ± 4.8 (n = 9) and 77.4 ± 6.0 beats min(-1) (n = 7), respectively [P0.05, 10 or 30 μm CPA versus control (n = 10)] and also reduced the AVN rate increase in response to isoprenaline from 78.8 ± 10.0 to 46.8 ± 6.8 and 26.7 ± 5.3%, respectively (P0.01). These inhibitory effects of CPA on the intact AVN rate and its response to isoprenaline indicate that Ca(2+) cycling is important to the intact AVN spontaneous activity and its acceleration during sympathetic stimulation.

Introduction: Clinically in ventricular fibrillation (VF), ECG amplitude and frequency decrease as ischemia progresses and predict defibrillation success. In-vitro ECG amplitude declines without ischemia, independent of VF frequencies. This study examines the contribution of cellular electrical activity and global organization to ECG amplitude changes during VF. Methods and results: Rabbit hearts were Langendorff-perfused (40mL/min, Tyrode’s solution) and loaded with RH237. During VF, ECG and epicardial optical action potentials were recorded (photodiode array; 256sites, 15×15mm). After 60s of VF, perfusion was either maintained, global ischemia produced by low-flow (6mL/min), or solution [K+]o raised to 8mM. Peak-to-peak amplitude was determined for all signals. During VF, in control, ECG amplitude decreased to a steady-state (~57% baseline), whereas in low-flow steady-state was not reached with the amplitude continuing to fall to 33% of baseline by 600s. Optically, LV amplitude declined more than RV, reaching significance in control (LV vs. RV; 33+/-5% vs. 638%, p

Although transmural heterogeneity of action potential duration (APD) is established in single cells isolated from different tissue layers, the extent to which it produces transmural gradients of repolarization in electrotonically coupled ventricular myocardium remains controversial. The purpose of this study was to examine the relative contribution of intrinsic cellular gradients of APD and electrotonic influences to transmural repolarization in rabbit ventricular myocardium. Transmural optical mapping was performed in left ventricular wedge preparations from eight rabbits. Transmural patterns of activation, repolarization, and APD were recorded during endocardial and epicardial stimulation. Experimental results were compared with modeled data during variations in electrotonic coupling. A transmural gradient of APD was evident during endocardial stimulation, which reflected differences previously seen in isolated cells, with the longest APD at the endocardium and the shortest at the epicardium (endo: 165 ± 5 vs. epi: 147 ± 4 ms; P < 0.05). During epicardial stimulation, this gradient reversed (epi: 162 ± 4 vs. endo: 148 ± 6 ms; P < 0.05). In both activation sequences, transmural repolarization followed activation and APD shortened along the activation path such that significant transmural gradients of repolarization did not occur. This correlation between transmural activation time and APD was recapitulated in simulations and varied with changes in intercellular coupling, confirming that it is mediated by electrotonic current flow between cells. These data suggest that electrotonic influences are important in determining the transmural repolarization sequence in rabbit ventricular myocardium and that they are sufficient to overcome intrinsic differences in the electrophysiological properties of the cells across the ventricular wall.