Epicardioid single-cell genomics uncovers principles of human epicardium biology in heart development and disease.

The epicardium, the mesothelial envelope of the vertebrate heart, is the source of multiple cardiac cell lineages during embryonic development and provides signals that are essential to myocardial growth and repair. Here we generate self-organizing human pluripotent stem cell-derived epicardioids that display retinoic acid-dependent morphological, molecular and functional patterning of the epicardium and myocardium typical of the left ventricular wall. By combining lineage tracing, single-cell transcriptomics and chromatin accessibility profiling, we describe the specification and differentiation process of different cell lineages in epicardioids and draw comparisons to human fetal development at the transcriptional and morphological levels.

High-throughput optical action potential recordings in hiPSC-derived cardiomyocytes with a genetically encoded voltage indicator in the locus.

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) represent an excellent model in cardiovascular research. Changes in their action potential (AP) dynamics convey information that is essential for disease modeling, drug screening and toxicity evaluation. High-throughput optical AP recordings utilizing intramolecular Förster resonance energy transfer (FRET) of the voltage-sensitive fluorescent protein (VSFP) have emerged as a substitute or complement to the resource-intensive patch clamp technique.

Human BIN1 isoforms grow, maintain, and regenerate excitation-contraction couplons in adult rat and human stem cell-derived cardiomyocytes.

Aims: In ventricular myocytes, transverse-tubules (T-tubules) are instrumental for excitation-contraction (EC)coupling and their disarray is a hallmark of cardiac diseases. BIN1 is a key contributor to their biogenesis. Our study set out to investigate the role of human BIN1 splice variants in the maintenance and regeneration of EC-coupling in rat adult ventricular myocytes and human-induced pluripotent stem cell-derived cardiac myocytes (hiPS-CMs).

Apparent calcium spark properties and fast-scanning 2D confocal imaging modalities.

Ca sparks are instrumental to understand physiological and pathological Ca signaling in the heart. High-speed two spatially dimensional (2D) confocal imaging (>120 Hz) enables acquisition of sparks with high-content information, however, owing to a wide variety of different acquisition modalities the question arises: how much they reflect the "true" Ca spark properties. To address this issue, we compared a fast point and a 2D-array scanner equipped with a range of different detectors.

Transcriptional signatures regulated by TRPC1/C4-mediated Background Ca entry after pressure-overload induced cardiac remodelling.

Aims: After summarizing current concepts for the role of TRPC cation channels in cardiac cells and in processes triggered by mechanical stimuli arising e.g. during pressure overload, we analysed the role of TRPC1 and TRPC4 for background Ca entry (BGCE) and for cardiac pressure overload induced transcriptional remodelling.

Large scale, unbiased analysis of elementary calcium signaling events in cardiac myocytes.

The identification of spatiotemporally restricted Ca signals, Ca sparks, was instrumental for our understanding of cardiac Ca homeostasis. High-speed 2D confocal imaging enables acquisition of such Ca sparks with high-content information but their full appreciation is constrained by the lack of unbiased and easy-to-use analysis tools. We developed a software toolset for unbiased and automatic Ca spark analysis for huge data sets of subcellular Ca signals.

DREADD technology reveals major impact of Gq signalling on cardiac electrophysiology.

Aims: Signalling via Gq-coupled receptors is of profound importance in many cardiac diseases such as hypertrophy and arrhythmia. Nevertheless, owing to their widespread expression and the inability to selectively stimulate such receptors in vivo, their relevance for cardiac function is not well understood. We here use DREADD technology to understand the role of Gq-coupled signalling in vivo in cardiac function.

Aberrant Deactivation-Induced Gain of Function in TRPM4 Mutant Is Associated with Human Cardiac Conduction Block.

A gain-of-function mutation in the Ca-activated transient receptor potential melastatin member 4 (TRPM4) is linked to life-threatening cardiac conduction disturbance, but the underlying mechanism is unclear. For deeper insights, we used photolysis of caged Ca, quantitative Ca, and electrophysiological measurements. TRPM4's 2-fold larger membrane current was associated with 50% decreased plasma membrane expression.

Suppression of Arrhythmia by Enhancing Mitochondrial Ca Uptake in Catecholaminergic Ventricular Tachycardia Models.

Cardiovascular disease-related deaths frequently arise from arrhythmias, but treatment options are limited due to perilous side effects of commonly used antiarrhythmic drugs. Cardiac rhythmicity strongly depends on cardiomyocyte Ca handling and prevalent cardiac diseases are causally associated with perturbations in intracellular Ca handling. Therefore, intracellular Ca transporters are lead candidate structures for novel and safer antiarrhythmic therapies.

An adaptation of astronomical image processing enables characterization and functional 3D mapping of individual sites of excitation-contraction coupling in rat cardiac muscle.

In beating cardiomyocytes, synchronized localized Ca transients from thousands of active excitation-contraction coupling sites (ECC couplons) comprising plasma and sarcoplasmic reticulum membrane calcium channels are important determinants of the heart's performance. Nevertheless, our knowledge about the properties of ECC couplons is limited by the lack of appropriate experimental and analysis strategies. We designed CaCLEAN to untangle the fundamental characteristics of ECC couplons by combining the astronomer's CLEAN algorithm with known properties of calcium diffusion.

Subtype-specific promoter-driven action potential imaging for precise disease modelling and drug testing in hiPSC-derived cardiomyocytes.

Aims: Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are increasingly used in disease modelling and drug evaluation. However, they are typically a heterogeneous mix of ventricular-, atrial-, and nodal-like cells based on action potentials (APs) and gene expression. This heterogeneity and the paucity of methods for high-throughput functional phenotyping hinder the full exploitation of their potential.

Hyperaldosteronism induces left atrial systolic and diastolic dysfunction.

Patients with hypertension and hyperaldosteronism show an increased risk of stroke compared with patients with essential hypertension. Aim of the study was to assess the effects of aldosterone on left atrial function in rats as a potential contributor to thromboembolism. Osmotic mini-pumps delivering 1.

Cardiac N-methyl D-aspartate Receptors as a Pharmacological Target.

This study focuses on characterization of the cardiac N-methyl D-aspartate receptors (NMDARs) as a target for endogenous and synthetic agonists and antagonists. Using isolated perfused rat hearts, we have shown that intracoronary administration of the NMDAR agonists and antagonists has a pronounced effect on autonomous heart function. Perfusion of rat hearts with autologous blood supplemented with NMDAR agonists was associated with induction of tachycardia, sinus arrhythmia, and ischemia occurring within physiological plasma concentration range for glutamate and glycine.

Endothelin-1-induced remodelling of murine adult ventricular myocytes.

The precise role of hormones binding to Gαq protein-coupled receptors (H-GαqPCRs) in chronic heart diseases remains poorly understood. To address this, we used a model of cultured adult rat ventricular myocytes stimulated with endothelin-1 (ET-1) or phenylephrine (PE) over a period of 8 days in vitro (DIV). Chronically treated cells showed an increased number of arrhythmogenic Ca(2+) transients when electrically paced at 0.

Genetically Encoded Voltage Indicators in Circulation Research.

Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available.

A background Ca2+ entry pathway mediated by TRPC1/TRPC4 is critical for development of pathological cardiac remodelling.

Aims: Pathological cardiac hypertrophy is a major predictor for the development of cardiac diseases. It is associated with chronic neurohumoral stimulation and with altered cardiac Ca(2+) signalling in cardiomyocytes. TRPC proteins form agonist-induced cation channels, but their functional role for Ca(2+) homeostasis in cardiomyocytes during fast cytosolic Ca(2+) cycling and neurohumoral stimulation leading to hypertrophy is unknown.

Arrhythmia causes lipid accumulation and reduced glucose uptake.

Atrial fibrillation (AF) is characterized by irregular contractions of atrial cardiomyocytes and increased energy demand. The aim of this study was to characterize the influence of arrhythmia on glucose and fatty acid (FA) metabolism in cardiomyocytes, mice and human left atrial myocardium. Compared to regular pacing, irregular (pseudo-random variation at the same number of contractions/min) pacing of neonatal rat cardiomyocytes induced shorter action potential durations and effective refractory periods and increased diastolic [Ca(2+)]c.

Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity.

During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors.

Confocal FLIM of genetically encoded FRET sensors for quantitative Ca2+ imaging.

Fluorescence lifetime imaging (FLIM) is a powerful imaging mode that can be combined with confocal imaging. Changes in the fluorescence decay time of a donor in an intramolecular Förster resonance energy transfer (FRET)-based biosensor provide intrinsic quantitative data. Here, we describe a protocol using both the Ca(2+) sensor TN-XL, which uses troponin C, as the Ca(2+)-sensing unit, and the FLIM technology based on time-correlated single-photon counting.

Two-dimensional imaging of fast intracellular Ca2+ release.

Asynchronous release of calcium (Ca(2+))-for example, the generation of Ca(2+) alternans in cardiac myocytes-is a phenomenon important in the development of cardiac arrhythmogenesis. The development of a failure to release Ca(2+) at individual release sites can be regarded as a major contributor to cardiac pathologies such as hypertrophy. Although confocal linescans provide sufficient temporal resolution to investigate the physiological and pathological cardiac excitation-contraction (EC) coupling, linescans can only image ∼1.

Genetically encoded Ca2+ indicators in cardiac myocytes.

Genetically encoded Ca(2+) indicators constitute a powerful set of tools to investigate functional aspects of Ca(2+) signaling in isolated cardiomyocytes, cardiac tissue, and whole hearts. Here, we provide an overview of the concepts, experiences, state of the art, and ongoing developments in the use of genetically encoded Ca(2+) indicators for cardiac cells and heart tissue. This review is supplemented with in vivo viral gene transfer experiments and comparisons of available genetically encoded Ca(2+) indicators with each other and with the small molecule dye Fura-2.

Morphologically homogeneous red blood cells present a heterogeneous response to hormonal stimulation.

Red blood cells (RBCs) are among the most intensively studied cells in natural history, elucidating numerous principles and ground-breaking knowledge in cell biology. Morphologically, RBCs are largely homogeneous, and most of the functional studies have been performed on large populations of cells, masking putative cellular variations. We studied human and mouse RBCs by live-cell video imaging, which allowed single cells to be followed over time.

Calcium dysregulation in ventricular myocytes from mice expressing constitutively active Rac1.

Increased Rac1 activity and its concomitant elevation of reactive oxygen species (ROS) levels is believed to be involved in the development of cardiac diseases such as hypertrophy and arrhythmia. To study the effects of activated Rac1 on the properties of isolated ventricular myocytes we used a transgenic mouse model (RacET) expressing constitutively active Rac1. Concurrent with dilated cardiomyopathy global Ca(2+) handling as well as single cell contractility was substantially decreased.

Noise-free visualization of microscopic calcium signaling by pixel-wise fitting.

Rationale: Our insights into physiological and pathophysiological cardiac excitation-contraction coupling has greatly benefited from significant advancement in optical technologies such as high-speed confocal microscopy. This has pushed pixel dwell times into the time domain of nanoseconds, resulting in low signal-to-noise ratios, which have limited data analysis and interpretation.

Objective: Line scan imaging has been and still is dominant in high speed confocal recording.