A comprehensive protocol combining in vivo and ex vivo electrophysiological experiments in an arrhythmogenic animal model.

Ventricular arrhythmias contribute significantly to cardiovascular mortality, with coronary artery disease as the predominant underlying cause. Understanding the mechanisms of arrhythmogenesis is essential to identify proarrhythmic factors and develop novel approaches for antiarrhythmic prophylaxis and treatment. Animal models are vital in basic research on cardiac arrhythmias, encompassing molecular, cellular, ex vivo whole heart, and in vivo models. Most studies use either in vivo protocols lacking important information on clinical relevance or exclusively ex vivo protocols, thereby missing the opportunity to explore underlying mechanisms. Consequently, interpretation may be difficult due to dissimilarities in animal models, interventions, and individual properties across animals. Moreover, proarrhythmic effects observed in vivo are often not replicated in corresponding ex vivo preparations during mechanistic studies. We have established a protocol to perform both an in vivo and ex vivo electrophysiological characterization in an arrhythmogenic rat model with heart failure following myocardial infarction. The same animal is followed throughout the experiment. In vivo methods involve intracardiac programmed electrical stimulation and external defibrillation to terminate sustained ventricular arrhythmia. Ex vivo methods conducted on the Langendorff-perfused heart include an electrophysiological study with optical mapping of regional action potentials, conduction velocities, and dispersion of electrophysiological properties. By exploring the retention of the in vivo proarrhythmic phenotype ex vivo, we aim to examine whether the subsequent ex vivo detailed measurements are relevant to in vivo pathological behavior. This protocol can enhance greater understanding of cardiac arrhythmias by providing a standardized, yet adaptable model for evaluating arrhythmogenicity or antiarrhythmic interventions in cardiac diseases.NEW & NOTEWORTHY Rodent models are widely used in arrhythmia research. However, most studies do not standardize clinically relevant in vivo and ex vivo techniques to support their conclusions. Here, we present a comprehensive electrophysiological protocol in an arrhythmogenic rat model, connecting in vivo and ex vivo programmed electrical stimulation with optical mapping. By establishing this protocol, we aim to facilitate the adoption of a standardized model for investigating arrhythmias, enhancing research rigor and comparability in this field.

Artery Tertiary Lymphoid Organs Control Aorta Immunity and Protect against Atherosclerosis via Vascular Smooth Muscle Cell Lymphotoxin ß Receptors.

Tertiary lymphoid organs (TLOs) emerge during nonresolving peripheral inflammation, but their impact on disease progression remains unknown. We have found in aged Apoe(-/-) mice that artery TLOs (ATLOs) controlled highly territorialized aorta T cell responses. ATLOs promoted T cell recruitment, primed CD4(+) T cells, generated CD4(+), CD8(+), T regulatory (Treg) effector and central memory cells, converted naive CD4(+) T cells into induced Treg cells, and presented antigen by an unusual set of dendritic cells and B cells. Meanwhile, vascular smooth muscle cell lymphotoxin ß receptors (VSMC-LTßRs) protected against atherosclerosis by maintaining structure, cellularity, and size of ATLOs though VSMC-LTßRs did not affect secondary lymphoid organs: Atherosclerosis was markedly exacerbated in Apoe(-/-)Ltbr(-/-) and to a similar extent in aged Apoe(-/-)Ltbr(fl/fl)Tagln-cre mice. These data support the conclusion that the immune system employs ATLOs to organize aorta T cell homeostasis during aging and that VSMC-LTßRs participate in atherosclerosis protection via ATLOs.

Unlocking Personalized Biomedicine and Drug Discovery with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Fit for Purpose or Forever Elusive?

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.

Conventional rigid 2D substrates cause complex contractile signals in monolayers of human induced pluripotent stem cell-derived cardiomyocytes.

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 the underlying mechanisms, further gene expression and computational studies were performed. HiPSC-CM monolayers exhibited multiphasic 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 with 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, multiphasic contractions in hiPSC-CM monolayers. Flexible substrates are necessary for normal twitch-like contractility kinetics and interpretation of inotropic interventions. 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.

The Use of Voltage Sensitive Dye di-4-ANEPPS and Video-Based Contractility Measurements to Assess Drug Effects on Excitation-Contraction Coupling in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

ABSTRACT: 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.

Electrophysiology of hiPSC-Cardiomyocytes Co-Cultured with HEK Cells Expressing the Inward Rectifier Channel.

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.

MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo.

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.

Prolongation of atrio-ventricular node conduction in a rabbit model of ischaemic cardiomyopathy: Role of fibrosis and connexin remodelling.

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) 8weeks post-MI. In vivo studies established that the PQ interval increases by approximately 7ms (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 14ms (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 20ms) 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.

Optical and electrical recordings from isolated coronary-perfused ventricular wedge preparations.

The electrophysiological heterogeneity that exists across the ventricular wall in the mammalian heart has long been recognized, but remains an area that is incompletely understood. Experimental studies of the mechanisms of arrhythmogenesis in the whole heart often examine the epicardial surface in isolation and thereby disregard transmural electrophysiology. Significant heterogeneity exists in the electrophysiological properties of cardiomyocytes isolated from different layers of the ventricular wall, and given that regional heterogeneities of membrane repolarization properties can influence the electrophysiological substrate for re-entry, the diversity of cell types and characteristics spanning the ventricular wall is important in the study of arrhythmogenesis. For these reasons, coronary-perfused left ventricular wedge preparations have been developed to permit the study of transmural electrophysiology in the intact ventricle. Since the first report by Yan and Antzelevitch in 1996, electrical recordings from the transmural surface of canine wedge preparations have provided a wealth of data regarding the cellular basis for the electrocardiogram, the role of transmural heterogeneity in arrhythmogenesis, and differences in the response of the different ventricular layers to drugs and neurohormones. Use of the wedge preparation has since been expanded to other species and more recently it has also been widely used in optical mapping studies. The isolated perfused wedge preparation has become an important tool in cardiac electrophysiology. In this review, we detail the methodology involved in recording both electrical and optical signals from the coronary-perfused wedge preparation and review the advances in cardiac electrophysiology achieved through study of the wedge.

Initiation of ventricular arrhythmia in the acquired long QT syndrome.

AIMS: Long QT syndrome (LQTS) carries a risk of life-threatening polymorphic ventricular tachycardia (Torsades de Pointes, TdP) and is a major cause of premature sudden cardiac death. TdP is induced by R-on-T premature ventricular complexes (PVCs), thought to be generated by cellular early-afterdepolarisations (EADs). However, EADs in tissue require cellular synchronisation, and their role in TdP induction remains unclear. We aimed to determine the mechanism of TdP induction in rabbit hearts with acquired LQTS (aLQTS). METHODS AND RESULTS: Optical mapping of action potentials (APs) and intracellular Ca2+ was performed in Langendorff-perfused rabbit hearts (n = 17). TdP induced by R-on-T PVCs was observed during aLQTS (50% K+/Mg++ & E4031) conditions in all hearts (P < 0.0001 vs. control). Islands of AP prolongation bounded by steep voltage gradients (VGs) were consistently observed before arrhythmia and peak VGs were more closely related to the PVC upstroke than EADs, both temporally (7 ± 5 ms vs. 44 ± 27 ms, P < 0.0001) and spatially (1.0 ± 0.7 vs. 3.6 ± 0.9 mm, P < 0.0001). PVCs were initiated at estimated voltages of ∼ -40 mV and had upstroke dF/dtmax and Vm-Ca2+ dynamics compatible with ICaL activation. Computational simulations demonstrated that PVCs could arise directly from VGs, through electrotonic triggering of ICaL. In experiments and the model, sub-maximal L-type Ca2+ channel (LTCC) block (200 nM nifedipine and 90% gCaL, respectively) abolished both PVCs and TdP in the continued presence of aLQTS. CONCLUSION: These data demonstrate that ICaL activation at sites displaying steep VGs generates the PVCs which induce TdP, providing a mechanism and rationale for LTCC blockers as a novel therapeutic approach in LQTS.

Inhibition of sarcoplasmic reticulum Ca(2+)-ATPase decreases atrioventricular node-paced heart rate in rabbits.

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) (P < 0.01, n = 10). Isoprenaline (100 nm) increased AVN rate to 187.8 ± 12.0 beats min(-1) after 1 min of application (P < 0.01, 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 [P < 0.05, 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 (P < 0.01). 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.

Electrophysiological heterogeneity in large populations of rabbit ventricular cardiomyocytes.

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.

Dynamic but discordant alterations in zDHHC5 expression and palmitoylation of its substrates in cardiac pathologies.

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 sodium-calcium 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. Little is known 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 increased rapidly 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, while palmitoylation of the zDHHC5 substrate PLM was unchanged in all settings. Overexpression of zDHHC5 in rabbit ventricular cardiomyocytes did not alter palmitoylation of its 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 C-terminal tail. We found that in HF palmitoylation of zDHHC5 changed in the same manner as palmitoylation of NCX1, suggesting additional regulatory mechanisms may be involved. This study provides novel 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.

Alternans of action potential duration and amplitude in rabbits with left ventricular dysfunction following myocardial infarction.

T-wave alternans may predict the occurrence of ventricular arrhythmias in patients with left ventricular dysfunction and experimental work has linked discordant repolarization alternans to the induction of re-entry. The aim of this study was to examine the occurrence of transmural repolarization alternans and to investigate the link between alternans and ventricular arrhythmia in rabbits with left ventricular dysfunction following myocardial infarction. Optical mapping was used to record action potentials from the transmural surface of left ventricular wedge preparations from normal and post-infarction hearts during a progressive reduction in pacing cycle length at 30 and 37°C. Data were analyzed using custom software, including spectral analysis. There were no significant differences in baseline transmural electrophysiology between the groups. Post-infarction hearts had a lower threshold for both repolarization alternans (286 vs. 333 bpm, p<0.05) and ventricular arrhythmias (79 vs. 19%, p<0.01) during rapid pacing, which was not accounted for by increased transmural discordant alternans. In VF-prone hearts, alternans in optical action potential amplitude was observed and increased until 2:1 block occurred. The degree of optical action potential amplitude alternans (12.0 ± 7.0 vs. 1.8 ± 0.3, p<0.05), but not APD(90) alternans (1.4 ± 0.6 vs. 1.1 ± 0.1, p>0.05) was associated with VF inducibility during rapid pacing. Post-infarction hearts are more vulnerable to transmural alternans and ventricular arrhythmias at rapid rates. Alternans in optical action potential amplitude was associated with conduction block and VF. The data suggest that changes in optical action potential amplitude may underlie a mechanism for alternans-associated ventricular arrhythmia in left ventricular dysfunction.

Effect of activation sequence on transmural patterns of repolarization and action potential duration in rabbit ventricular myocardium.

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.

Blinded, Multicenter Evaluation of Drug-induced Changes in Contractility Using Human-induced Pluripotent Stem Cell-derived Cardiomyocytes.

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.

The link between repolarisation alternans and ventricular arrhythmia: does the cellular phenomenon extend to the clinical problem?

T-wave alternans is considered a potentially useful clinical marker for the risk of ventricular arrhythmia in patients with heart disease. Cellular repolarisation alternans is thought to underlie T-wave alternans, and moreover, to cause re-entrant ventricular arrhythmia. This review examines the experimental and clinical evidence linking repolarisation alternans and T-wave alternans with the occurrence of ventricular arrhythmia. Repolarisation alternans, manifest as alternating changes in action potential duration, is observed in isolated ventricular cardiomyocytes and in multicellular preparations. Its underlying causes are discussed particularly with respect to the role of intracellular Ca(2+). The repolarisation alternans observed at the single cell level is compared to the alternating behaviour observed in isolated multicellular preparations including the perfused ventricular wedge and Langendorff perfused heart. The evidence concerning spatial differences in repolarisation alternans is considered, particularly the situation where adjacent regions of myocardium exhibit repolarisation alternans of different phases. This extreme behaviour, known as discordant alternans, is thought to produce marked gradients of repolarisation that can precipitate unidirectional block and re-entrant ventricular arrhythmias. Finally, the difficulties in extrapolating between experimental models of alternans and arrhythmias and the clinical manifestation are discussed. The areas where experimental evidence is weak are highlighted, and areas for future research are outlined.

Quantification of Muscle Contraction In Vitro and In Vivo Using MUSCLEMOTION Software: From Stem Cell-Derived Cardiomyocytes to Zebrafish and Human Hearts.

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.

Heterogeneity of ventricular fibrillation dominant frequency during global ischemia in isolated rabbit hearts.

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.

Mapping of epicardial activation in a rabbit model of chronic myocardial infarction:.

INTRODUCTION: This study examines the consequences of a large transmural apical infarct on the epicardial electrical activity in isolated rabbit hearts. METHODS AND RESULTS: Hearts were isolated 8 weeks after coronary artery ligation. Membrane voltage from the epicardial surface of the left ventricle (LV) including the infarct was monitored using the voltage sensitive dye RH237. Optical action potentials were detected from the epicardial surface of the infarct; the signal amplitude was approximately 20% of those in the noninfarcted zone (NZ). Epicardial activation mapping of the LV free wall showed that during right atrial (RA) pacing, the activation sequence was not significantly different between infarcted and sham-operated groups. However, direct stimulation of the epicardium in the NZ revealed an area of slow conduction velocity (CV approximately 5 cm/s(-1), approximately 10% of normal values) at the margin of the infarct zone (IZ). Within the IZ, CV was approximately 50% of normal. A prominent endocardial rim of myocardium in the infarct was not the source of epicardial optical signals because chemical ablation of the endocardium did not affect the epicardial activation pattern. CONCLUSION: Therefore, remnant groups of myocytes in the mid-wall and epicardium of the infarct scar support normal electrical activation during RA pacing. Areas of delayed conduction emerge only on epicardial stimulation.