Cardiac sympathetic neurons are additional cells affected in genetically determined arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (AC) is a familial cardiac disease, mainly caused by mutations in desmosomal genes, which accounts for most cases of stress-related arrhythmic sudden death, in young and athletes. AC hearts display fibro-fatty lesions that generate the arrhythmic substrate and cause contractile dysfunction. A correlation between physical/emotional stresses and arrhythmias supports the involvement of sympathetic neurons (SNs) in the disease, but this has not been confirmed previously. Here, we combined molecular, in vitro and ex vivo analyses to determine the role of AC-linked DSG2 downregulation on SN biology and assess cardiac sympathetic innervation in desmoglein-2 mutant (Dsg2mut/mut) mice. Molecular assays showed that SNs express DSG2, implying that DSG2-mutation carriers would harbour the mutant protein in SNs. Confocal immunofluorescence of heart sections and 3-D reconstruction of SN network in clarified heart blocks revealed significant changes in the physiologialc SN topology, with massive hyperinnervation of the intact subepicardial layers and heterogeneous distribution of neurons in fibrotic areas. Cardiac SNs isolated from Dsg2mut/mut neonatal mice, prior to the establishment of cardiac innervation, show alterations in axonal sprouting, process development and distribution of varicosities. Consistently, virus-assisted DSG2 downregulation replicated, in PC12-derived SNs, the phenotypic alterations displayed by Dsg2mut/mut primary neurons, corroborating that AC-linked Dsg2 variants may affect SNs. Our results reveal that altered sympathetic innervation is an unrecognized feature of AC hearts, which may result from the combination of cell-autonomous and context-dependent factors implicated in myocardial remodelling. Our results favour the concept that AC is a disease of multiple cell types also hitting cardiac SNs. KEY POINTS: Arrhythmogenic cardiomyopathy is a genetically determined cardiac disease, which accounts for most cases of stress-related arrhythmic sudden death. Arrhythmogenic cardiomyopathy linked to mutations in desmoglein-2 (DSG2) is frequent and leads to a left-dominant form of the disease. Arrhythmogenic cardiomyopathy has been approached thus far as a disease of cardiomyocytes, but we here unveil that DSG2 is expressed, in addition to cardiomyocytes, by cardiac and extracardiac sympathetic neurons, although not organized into desmosomes. AC-linked DSG2 downregulation primarily affect sympathetic neurons, resulting in the significant increase in cardiac innervation density, accompanied by alterations in sympathetic neuron distribution. Our data supports the notion that AC develops with the contribution of several 'desmosomal protein-carrying' cell types and systems.

Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes.

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.

Cardiac multiscale bioimaging: from nano- through micro- to mesoscales.

Cardiac multiscale bioimaging is an emerging field that aims to provide a comprehensive understanding of the heart and its functions at various levels, from the molecular to the entire organ. It combines both physiologically and clinically relevant dimensions: from nano- and micrometer resolution imaging based on vibrational spectroscopy and high-resolution microscopy to assess molecular processes in cardiac cells and myocardial tissue, to mesoscale structural investigations to improve the understanding of cardiac (patho)physiology. Tailored super-resolution deep microscopy with advanced proteomic methods and hands-on experience are thus strategically combined to improve the quality of cardiovascular research and support future medical decision-making by gaining additional biomolecular information for translational and diagnostic applications.

Recent advances and current limitations of available technology to optically manipulate and observe cardiac electrophysiology.

Optogenetics, utilising light-reactive proteins to manipulate tissue activity, are a relatively novel approach in the field of cardiac electrophysiology. We here provide an overview of light-activated transmembrane channels (optogenetic actuators) currently applied in strategies to modulate cardiac activity, as well as newly developed variants yet to be implemented in the heart. In addition, we touch upon genetically encoded indicators (optogenetic sensors) and fluorescent dyes to monitor tissue activity, including cardiac transmembrane potential and ion homeostasis. The combination of the two allows for all-optical approaches to monitor and manipulate the heart without any physical contact. However, spectral congestion poses a major obstacle, arising due to the overlap of excitation/activation and emission spectra of various optogenetic proteins and/or fluorescent dyes, resulting in optical crosstalk. Therefore, optogenetic proteins and fluorescent dyes should be carefully selected to avoid optical crosstalk and consequent disruptions in readouts and/or cellular activity. We here present a novel approach to simultaneously monitor transmembrane potential and cytosolic calcium, while also performing optogenetic manipulation. For this, we used the novel voltage-sensitive dye ElectroFluor 730p and the cytosolic calcium indicator X-Rhod-1 in mouse hearts expressing channelrhodopsin-2 (ChR2). By exploiting the isosbestic point of ElectroFluor 730p and avoiding the ChR2 activation spectrum, we here introduce a novel optical imaging and manipulation approach with minimal crosstalk. Future developments in both optogenetic proteins and fluorescent dyes will allow for additional and more optimised strategies, promising a bright future for all-optical approaches in the field of cardiac electrophysiology.

Near-infrared voltage-sensitive dyes based on chromene donor.

Voltage-sensitive dyes (VSDs) are used to image electrical activity in cells and tissues with submillisecond time resolution. Most of these fast sensors are constructed from push-pull chromophores whose fluorescence spectra are modulated by the electric field across the cell membrane. It was found that the substitution of naphthalene with chromene produces a 60 to 80 nm red-shift in absorption and emission spectra while maintaining fluorescence quantum efficiency and voltage sensitivity. One dye was applied to ex vivo murine heart with excitation at 730 nm, by far the longest wavelength reported in voltage imaging. This VSD resolves cardiac action potentials in single trials with 12% ΔF/F per action potential. The well-separated excitation spectra between these long-wavelength VSDs and channelrhodopsin (ChR2) enabled monitoring of action potential propagation in ChR2 hearts without any perturbation of electrical dynamics. Importantly, by employing spatially localized optogenetic manipulation, action potential dynamics can be assessed in an all-optical fashion with no artifact related to optical cross-talk between the reporter and actuator. These new environmentally sensitive chromene-based chromophores are also likely to have applications outside voltage imaging.

Optogenetic manipulation of cardiac repolarization gradients using sub-threshold illumination.

Introduction: Mechanisms underlying cardiac arrhythmias are typically driven by abnormalities in cardiac conduction and/or heterogeneities in repolarization time (RT) across the heart. While conduction slowing can be caused by either electrophysiological defects or physical blockade in cardiac tissue, RT heterogeneities are mainly related to action potential (AP) prolongation or abbreviation in specific areas of the heart. Importantly, the size of the area with altered RT and the difference between the short RT and long RT (RT gradient) have been identified as critical determinators of arrhythmogenicity. However, current experimental methods for manipulating RT gradient rely on the use of ion channel inhibitors, which lack spatial and temporal specificity and are commonly only partially reversible. Therefore, the conditions facilitating sustained arrhythmia upon the presence of RT heterogeneities and/or defects in cardiac conduction remain to be elucidated. Methods: We here employ an approach based on optogenetic stimulation in a low-intensity fashion (sub-threshold illumination), to selectively manipulate cardiac electrical activity in defined areas of the heart. Results: As previously described, subthreshold illumination is a robust tool able to prolong action potentials (AP), decrease upstroke velocity as well as slow cardiac conduction, in a fully reversible manner. By applying a patterned sub-threshold illumination in intact mouse hearts constitutively expressing the light-gated ion channel channelrhodopsin-2 (ChR2), we optically manipulate RT gradients and cardiac conduction across the heart in a spatially selective manner. Moreover, in a proof-of-concept assessment we found that in the presence of patterned sub-threshold illumination, mouse hearts were more susceptible to arrhythmias. Hence, this optogenetic-based approach may be able to mimic conduction slowing and RT heterogeneities present in pathophysiological conditions.

The Role of Crosslinker Molecular Structure on Mechanical and Light-Actuation Properties in Liquid Crystalline Networks.

Phase behavior modulation of liquid crystalline molecules can be addressed by structural modification at molecular level. Starting from a rigid rod-like core reduction of the symmetry or increase of the steric hindrance by different substituents generally reduces the clearing temperature. Similar approaches can be explored to modulate the properties of liquid crystalline networks (LCNs)-shape-changing materials employed as actuators in many fields. Depending on the application, the polymer properties have to be adjusted in terms of force developed under stimuli, kinetics of actuation, elasticity, and resistance to specific loads. In this work, the crosslinker modification at molecular level is explored towards the optimization of LCN properties as light-responsive artificial muscles. The synthesis and characterization of photopolymerizable crosslinkers, bearing different lateral groups on the aromatic core is reported. Such molecules are able to strongly modulate the material mechanical properties, such as kinetics and maximum tension under light actuation, opening up to interesting materials for biomedical applications.

Slower Calcium Handling Balances Faster Cross-Bridge Cycling in Human MYBPC3 HCM.

BACKGROUND: The pathogenesis of MYBPC3-associated hypertrophic cardiomyopathy (HCM) is still unresolved. In our HCM patient cohort, a large and well-characterized population carrying the MYBPC3:c772G>A variant (p.Glu258Lys, E258K) provides the unique opportunity to study the basic mechanisms of MYBPC3-HCM with a comprehensive translational approach. METHODS: We collected clinical and genetic data from 93 HCM patients carrying the MYBPC3:c772G>A variant. Functional perturbations were investigated using different biophysical techniques in left ventricular samples from 4 patients who underwent myectomy for refractory outflow obstruction, compared with samples from non-failing non-hypertrophic surgical patients and healthy donors. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and engineered heart tissues (EHTs) were also investigated. RESULTS: Haplotype analysis revealed MYBPC3:c772G>A as a founder mutation in Tuscany. In ventricular myocardium, the mutation leads to reduced cMyBP-C (cardiac myosin binding protein-C) expression, supporting haploinsufficiency as the main primary disease mechanism. Mechanical studies in single myofibrils and permeabilized muscle strips highlighted faster cross-bridge cycling, and higher energy cost of tension generation. A novel approach based on tissue clearing and advanced optical microscopy supported the idea that the sarcomere energetics dysfunction is intrinsically related with the reduction in cMyBP-C. Studies in single cardiomyocytes (native and hiPSC-derived), intact trabeculae and hiPSC-EHTs revealed prolonged action potentials, slower Ca2+ transients and preserved twitch duration, suggesting that the slower excitation-contraction coupling counterbalanced the faster sarcomere kinetics. This conclusion was strengthened by in silico simulations. CONCLUSIONS: HCM-related MYBPC3:c772G>A mutation invariably impairs sarcomere energetics and cross-bridge cycling. Compensatory electrophysiological changes (eg, reduced potassium channel expression) appear to preserve twitch contraction parameters, but may expose patients to greater arrhythmic propensity and disease progression. Therapeutic approaches correcting the primary sarcomeric defects may prevent secondary cardiomyocyte remodeling.

KHz-rate volumetric voltage imaging of the whole Zebrafish heart.

Fast volumetric imaging is essential for understanding the function of excitable tissues such as those found in the brain and heart. Measuring cardiac voltage transients in tissue volumes is challenging, especially at the high spatial and temporal resolutions needed to give insight to cardiac function. We introduce a new imaging modality based on simultaneous illumination of multiple planes in the tissue and parallel detection with multiple cameras, avoiding compromises inherent in any scanning approach. The system enables imaging of voltage transients in situ, allowing us, for the first time to our knowledge, to map voltage activity in the whole heart volume at KHz rates. The high spatiotemporal resolution of our method enabled the observation of novel dynamics of electrical propagation through the zebrafish atrioventricular canal.

Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy.

Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one-photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs).

A junctional cAMP compartment regulates rapid Ca2+ signaling in atrial myocytes.

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca2+-induced Ca2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following ß-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas ß-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca2+-induced Ca2+ release, whereas ß-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.

Optical clearing in cardiac imaging: A comparative study.

The optical clearing of the cardiac tissue has always been a challenging goal to obtain successful three-dimensional reconstructions of entire hearts. Typically, the developed protocols are targeted at the clearing of the brain; cardiac tissue requires proper arrangements to the original protocols, which are usually tough and time-consuming to figure out. Here, we present the application of three different clearing methodologies on mouse hearts: uDISCO, CLARITY, and SHIELD. For each approach, we describe the required optimizations that we have developed to improve the outcome; in particular, we focus on comparing the features of the tissue after the application of each methodology, especially in terms of tissue preservation, transparency, and staining. We found that the uDISCO protocol induces strong fiber delamination of the cardiac tissue, thus reducing the reliability of structural analyses. The CLARITY protocol confers a high level of transparency to the heart and allows deep penetration of the fluorescent dyes; however, it requires long times for the clearing and the tissue loses its robustness. The SHIELD methodology, indeed, is very promising for tissue maintenance since it preserves its consistency and provides ideal transparency, but further approaches are needed to obtain homogeneous staining of the whole heart. Since the CLARITY procedure, despite the disadvantages in terms of tissue preservation and timings, is actually the most suitable approach to image labeled samples in depth, we optimized and performed the methodology also on human cardiac tissue from control hearts and hearts with hypertrophic cardiomyopathy.

Distributed and Localized Dynamics Emerge in the Mouse Neocortex during Reach-to-Grasp Behavior.

A long-standing question in systems neuroscience is to what extent task-relevant features of neocortical processing are localized or distributed. Coordinated activity across the neocortex has been recently shown to drive complex behavior in the mouse, while activity in selected areas is canonically associated with specific functions (e.g., movements in the case of the motor cortex). Reach-to-grasp (RtG) movements are known to be dependent on motor circuits of the neocortex; however, the global activity of the neocortex during these movements has been largely unexplored in the mouse. Here, we characterized, using wide-field calcium imaging, these neocortex-wide dynamics in mice of either sex engaging in an RtG task. We demonstrate that, beyond motor regions, several areas, such as the visual and the retrosplenial cortices, also increase their activity levels during successful RtGs, and homologous regions across the ipsilateral hemisphere are also involved. Functional connectivity among neocortical areas increases transiently around movement onset and decreases during movement. Despite this global phenomenon, neural activity levels correlate with kinematics measures of successful RtGs in sensorimotor areas only. Our findings establish that distributed and localized neocortical dynamics co-orchestrate efficient control of complex movements.SIGNIFICANCE STATEMENT Mammals rely on reaching and grasping movements for fine-scale interactions with the physical world. In the mouse, the motor cortex is critical for the execution of such behavior, yet little is known about the activity patterns across neocortical areas. Using the mesoscale-level networks as a model of cortical processing, we investigated the hypothesis that areas beyond the motor regions could participate in RtG planning and execution, and indeed a large network of areas is involved while performing RtGs. Movement kinematics correlates mostly with neural activity in sensorimotor areas. By demonstrating that distributed and localized neocortical dynamics for the execution of fine movements coexist in the mouse neocortex during RtG, we offer an unprecedented view on the neocortical correlates of mammalian motor control.

Quantification of Myocyte Disarray in Human Cardiac Tissue.

Proper three-dimensional (3D)-cardiomyocyte orientation is important for an effective tension production in cardiac muscle. Cardiac diseases can cause severe remodeling processes in the heart, such as cellular misalignment, that can affect both the electrical and mechanical functions of the organ. To date, a proven methodology to map and quantify myocytes disarray in massive samples is missing. In this study, we present an experimental pipeline to reconstruct and analyze the 3D cardiomyocyte architecture in massive samples. We employed tissue clearing, staining, and advanced microscopy techniques to detect sarcomeres in relatively large human myocardial strips with micrometric resolution. Z-bands periodicity was exploited in a frequency analysis approach to extract the 3D myofilament orientation, providing an orientation map used to characterize the tissue organization at different spatial scales. As a proof-of-principle, we applied the proposed method to healthy and pathologically remodeled human cardiac tissue strips. Preliminary results suggest the reliability of the method: strips from a healthy donor are characterized by a well-organized tissue, where the local disarray is log-normally distributed and slightly depends on the spatial scale of analysis; on the contrary, pathological strips show pronounced tissue disorganization, characterized by local disarray significantly dependent on the spatial scale of analysis. A virtual sample generator is developed to link this multi-scale disarray analysis with the underlying cellular architecture. This approach allowed us to quantitatively assess tissue organization in terms of 3D myocyte angular dispersion and may pave the way for developing novel predictive models based on structural data at cellular resolution.

Novel Optics-Based Approaches for Cardiac Electrophysiology: A Review.

Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 2018, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research.

Mesoscopic Optical Imaging of Whole Mouse Heart.

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

Fast Optical Investigation of Cardiac Electrophysiology by Parallel Detection in Multiwell Plates.

Current techniques for fast characterization of cardiac electrophysiology employ optical technologies to control and monitor action potential features of single cells or cellular monolayers placed in multiwell plates. High-speed investigation capacities are commonly achieved by serially analyzing well after well employing fully automated fluorescence microscopes. Here, we describe an alternative cost-effective optical approach (MULTIPLE) that exploits high-power LED arrays to globally illuminate a culture plate and an sCMOS sensor for parallel detection of the fluorescence coming from multiple wells. MULTIPLE combines optical detection of action potentials using a red-shifted voltage-sensitive fluorescent dye (di-4-ANBDQPQ) with optical stimulation, employing optogenetic actuators, to ensure excitation of cardiomyocytes at constant rates. MULTIPLE was first characterized in terms of interwell uniformity of the illumination intensity and optical detection performance. Then, it was applied for probing action potential features in HL-1 cells (i.e., mouse atrial myocyte-like cells) stably expressing the blue light-activatable cation channel CheRiff. Under proper stimulation conditions, we were able to accurately measure action potential dynamics across a 24-well plate with variability across the whole plate of the order of 10%. The capability of MULTIPLE to detect action potential changes across a 24-well plate was demonstrated employing the selective K v 11.1 channel blocker (E-4031), in a dose titration experiment. Finally, action potential recordings were performed in spontaneous beating human induced pluripotent stem cell derived cardiomyocytes following pharmacological manipulation of their beating frequency. We believe that the simplicity of the presented optical scheme represents a valid complement to sophisticated and expensive state-of-the-art optical systems for high-throughput cardiac electrophysiological investigations.

The HCN channel as a pharmacological target: Why, where, and how to block it.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, expressed in a variety of cell types and in all tissues, control excitation and rhythm. Since their discovery in neurons and cardiac pacemaker cells, they attracted the attention of medicinal chemistry and pharmacology as novel targets to shape (patho)physiological mechanisms. To date, ivabradine represents the first-in-class drug as specific bradycardic agent in cardiac diseases; however, new applications are emerging in parallel with the demonstration of the involvement of different HCN isoforms in central and peripheral nervous system. Hence, the possibility to target specific isoforms represents an attractive development in this field; indeed, HCN1, HCN2 or HCN4 specific blockers have shown promising features in vitro and in vivo, with remarkable pharmacological differences likely depending on the diverse functional role and tissue distribution. Here, we show a recently developed compound with high potency as HCN2-HCN4 blocker; because of its unique profile, this compound may deserve further investigation.

ESC working group on cardiac cellular electrophysiology position paper: relevance, opportunities, and limitations of experimental models for cardiac electrophysiology research.

Cardiac arrhythmias are a major cause of death and disability. A large number of experimental cell and animal models have been developed to study arrhythmogenic diseases. These models have provided important insights into the underlying arrhythmia mechanisms and translational options for their therapeutic management. This position paper from the ESC Working Group on Cardiac Cellular Electrophysiology provides an overview of (i) currently available in vitro, ex vivo, and in vivo electrophysiological research methodologies, (ii) the most commonly used experimental (cellular and animal) models for cardiac arrhythmias including relevant species differences, (iii) the use of human cardiac tissue, induced pluripotent stem cell (hiPSC)-derived and in silico models to study cardiac arrhythmias, and (iv) the availability, relevance, limitations, and opportunities of these cellular and animal models to recapitulate specific acquired and inherited arrhythmogenic diseases, including atrial fibrillation, heart failure, cardiomyopathy, myocarditis, sinus node, and conduction disorders and channelopathies. By promoting a better understanding of these models and their limitations, this position paper aims to improve the quality of basic research in cardiac electrophysiology, with the ultimate goal to facilitate the clinical translation and application of basic electrophysiological research findings on arrhythmia mechanisms and therapies.