Generation of cardiomyocytes from human-induced pluripotent stem cells resembling atrial cells with ability to respond to adrenoceptor agonists

Atrial fibrillation (AF) is the most common chronic arrhythmia presenting a heavy disease burden. We report a new approach for generating cardiomyocytes (CMs) resembling atrial cells from human-induced pluripotent stem cells (hiPSCs) using a combination of Gremlin 2 and retinoic acid treatment. More than 40% of myocytes showed rod-shaped morphology, expression of CM proteins (including ryanodine receptor 2, α-actinin-2 and F-actin) and striated appearance, all of which were broadly similar to the characteristics of adult atrial myocytes (AMs). Isolated myocytes were electrically quiescent until stimulated to fire action potentials with an AM profile and an amplitude of approximately 100 mV, arising from a resting potential of approximately −70 mV. Single-cell RNA sequence analysis showed a high level of expression of several atrial-specific transcripts including NPPA, MYL7, HOXA3, SLN, KCNJ4, KCNJ5 and KCNA5. Amplitudes of calcium transients recorded from spontaneously beating cultures were increased by the stimulation of α-adrenoceptors (activated by phenylephrine and blocked by prazosin) or β-adrenoceptors (activated by isoproterenol and blocked by CGP20712A). Our new approach provides human AMs with mature characteristics from hiPSCs which will facilitate drug discovery by enabling the study of human atrial cell signalling pathways and AF. This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’

A protocol for dual calcium-voltage optical mapping in murine sinoatrial preparation with optogenetic pacing

Among the animal models for studying the molecular basis of atrial and sinoatrial node (SAN) biology and disease, the mouse is a widely used species due to its feasibility for genetic modifications in genes encoding ion channels or calcium handling and signaling proteins in the heart. It is therefore highly valuable to develop robust methodologies for studying SAN and atrial electrophysiological function in this species. Here, we describe a protocol for performing dual calcium-voltage optical mapping on mouse sinoatrial preparation (SAP), in combination with an optogenetic approach, for studying SAP membrane potential, intracellular Ca2+ transients, and pacemaker activity. The protocol includes the details for preparing the intact SAP, robust tissue dual-dye loading, light-programmed pacing, and high-resolution optical mapping. Our protocol provides an example of use of the combination of optogenetic and optical mapping techniques for investigating SAP membrane potential and intracellular Ca2+ transients and pacemaker activity with high temporal and spatial resolution in specific cardiac tissues. Thus, our protocol provides a useful tool for studying SAP physiology and pathophysiology in mice

Role of calcium mobilising messengers in cardiac arrhythmias

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent calcium ion (Ca2+) activating second messenger due to its ability to release Ca2+ from intracellular acidic stores at nanomolar concentrations. NAADP has been known to play an important role in controlling excitation-contraction coupling in the heart. The ADP-ribosyl cyclase (ARC) CD38, is believed to be responsible for the production of NAADP in the heart. The first part of the thesis assesses the role of NAADP pathway in calcium handling of cardiac atrial tissues by genetically (CD38-/- mice) and pharmacologically (bafilomycin A1) impairing NAADP pathway. Optical mapping techniques were used to observe the calcium transients and action potentials of mouse atrial tissues. At baseline conditions (without the activation of β-adrenoceptors), no contribution of the NAADP signalling pathway was observed in cardiac atria with respect to its calcium handling, action potential characteristics and resting heart rate. However, under the effect of the β- adrenoceptor agonist isoprenaline, CD38-/- mouse atria and bafilomycin A1 treated wildtype mouse atria showed a blunted increase in calcium transient amplitudes and heart rate as compared to control wildtype atria. The second part of the thesis focuses on understanding the association of NAADP signalling pathway with the sarcoplasmic reticulum (SR), using a Ca2+ indicator to directly observe the SR Ca2+ content. NAADP was observed to increase the Ca2+ content of the SR in wildtype and CD38-/- mice ventricular cardiomyocytes in a bafilomycin A1 sensitive manner. Moreover, stimulating β-adrenergic response using cyclic adenosine monophosphate (cAMP) showed a reduced effect on SR Ca2+ increase in CD38-/- myocytes as compared to wildtype myocytes and disruption of lysosomes using bafilomycin A1 in wildtype myocytes also reduced the SR Ca2+ increase in response to cAMP. These observations provide further evidence that the NAADP signalling pathway has a prominent role in β-adrenergic response in the heart. Although CD38 has been suggested as an enzyme that can produce both NAADP and cyclic adenosine diphosphate ribose (cADPR), several studies have attributed a non- CD38 ARC to catalyse the production of cADPR in the heart. The third part of the thesis, therefore, attempted to characterise the ARC present in sheep cardiac SR membrane preparations (SRMP). Functional and molecular biology experiments in this thesis strongly suggests that the ARC found in sheep SRMP is CD38. Finally, in the last section of this thesis, a protocol for combining optical mapping with optogenetic pacing is discussed to study intracellular Ca2+ transients in atrial tissues of mice genetically modified to express channelrhodopsin 2 (ChR2) on phenylethanolamine N-methyltransferase (Pnmt)-derived cardiomyocytes (PdCMs). On optogenetic pacing, a shift in primary pacemaker sites could be seen towards the left atrium where the PdCMs were localised with minimal disturbance on the Ca2+ handling properties of the atrial tissue. Although this cell type-specific optogenetic pacing strategy targeting PdCMs as candidates for biopacemaking is still at an early stage of development, it could potentially lay foundation for an exciting prospect of clinical applications in treating heart rhythm disorders in the future.</p

Role of calcium mobilising messengers in cardiac arrhythmias

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent calcium ion (Ca2+) activating second messenger due to its ability to release Ca2+ from intracellular acidic stores at nanomolar concentrations. NAADP has been known to play an important role in controlling excitation-contraction coupling in the heart. The ADP-ribosyl cyclase (ARC) CD38, is believed to be responsible for the production of NAADP in the heart. The first part of the thesis assesses the role of NAADP pathway in calcium handling of cardiac atrial tissues by genetically (CD38-/- mice) and pharmacologically (bafilomycin A1) impairing NAADP pathway. Optical mapping techniques were used to observe the calcium transients and action potentials of mouse atrial tissues. At baseline conditions (without the activation of β-adrenoceptors), no contribution of the NAADP signalling pathway was observed in cardiac atria with respect to its calcium handling, action potential characteristics and resting heart rate. However, under the effect of the β- adrenoceptor agonist isoprenaline, CD38-/- mouse atria and bafilomycin A1 treated wildtype mouse atria showed a blunted increase in calcium transient amplitudes and heart rate as compared to control wildtype atria. The second part of the thesis focuses on understanding the association of NAADP signalling pathway with the sarcoplasmic reticulum (SR), using a Ca2+ indicator to directly observe the SR Ca2+ content. NAADP was observed to increase the Ca2+ content of the SR in wildtype and CD38-/- mice ventricular cardiomyocytes in a bafilomycin A1 sensitive manner. Moreover, stimulating β-adrenergic response using cyclic adenosine monophosphate (cAMP) showed a reduced effect on SR Ca2+ increase in CD38-/- myocytes as compared to wildtype myocytes and disruption of lysosomes using bafilomycin A1 in wildtype myocytes also reduced the SR Ca2+ increase in response to cAMP. These observations provide further evidence that the NAADP signalling pathway has a prominent role in β-adrenergic response in the heart. Although CD38 has been suggested as an enzyme that can produce both NAADP and cyclic adenosine diphosphate ribose (cADPR), several studies have attributed a non- CD38 ARC to catalyse the production of cADPR in the heart. The third part of the thesis, therefore, attempted to characterise the ARC present in sheep cardiac SR membrane preparations (SRMP). Functional and molecular biology experiments in this thesis strongly suggests that the ARC found in sheep SRMP is CD38. Finally, in the last section of this thesis, a protocol for combining optical mapping with optogenetic pacing is discussed to study intracellular Ca2+ transients in atrial tissues of mice genetically modified to express channelrhodopsin 2 (ChR2) on phenylethanolamine N-methyltransferase (Pnmt)-derived cardiomyocytes (PdCMs). On optogenetic pacing, a shift in primary pacemaker sites could be seen towards the left atrium where the PdCMs were localised with minimal disturbance on the Ca2+ handling properties of the atrial tissue. Although this cell type-specific optogenetic pacing strategy targeting PdCMs as candidates for biopacemaking is still at an early stage of development, it could potentially lay foundation for an exciting prospect of clinical applications in treating heart rhythm disorders in the future.</p

Living cardiac tissue slices: An organotypic pseudo two-dimensional model for cardiac biophysics research

AbstractLiving cardiac tissue slices, a pseudo two-dimensional (2D) preparation, have received less attention than isolated single cells, cell cultures, or Langendorff-perfused hearts in cardiac biophysics research. This is, in part, due to difficulties associated with sectioning cardiac tissue to obtain live slices. With moderate complexity, native cell-types, and well-preserved cell–cell electrical and mechanical interconnections, cardiac tissue slices have several advantages for studying cardiac electrophysiology. The trans-membrane potential (Vm) has, thus far, mainly been explored using multi-electrode arrays. Here, we combine tissue slices with optical mapping to monitor Vm and intracellular Ca2+ concentration ([Ca2+]i). This combination opens up the possibility of studying the effects of experimental interventions upon action potential (AP) and calcium transient (CaT) dynamics in 2D, and with relatively high spatio-temporal resolution.As an intervention, we conducted proof-of-principle application of stretch. Mechanical stimulation of cardiac preparations is well-established for membrane patches, single cells and whole heart preparations. For cardiac tissue slices, it is possible to apply stretch perpendicular or parallel to the dominant orientation of cells, while keeping the preparation in a constant focal plane for fluorescent imaging of in-slice functional dynamics. Slice-to-slice comparison furthermore allows one to assess transmural differences in ventricular tissue responses to mechanical challenges. We developed and tested application of axial stretch to cardiac tissue slices, using a manually-controlled stretching device, and recorded Vm and [Ca2+]i by optical mapping before, during, and after application of stretch.Living cardiac tissue slices, exposed to axial stretch, show an initial shortening in both AP and CaT duration upon stretch application, followed in most cases by a gradual prolongation of AP and CaT duration during stretch maintained for up to 50 min. After release of sustained stretch, AP duration (APD) and CaT duration reverted to shorter values.Living cardiac tissue slices are a promising experimental model for the study of cardiac mechano-electric interactions. The methodology described here can be refined to achieve more accurate control over stretch amplitude and timing (e.g. using a computer-controlled motorised stage, or by synchronising electrical and mechanical events) and through monitoring of regional tissue deformation (e.g. by adding motion tracking)

A dataset of dual calcium and voltage optical mapping in healthy and hypertrophied murine hearts

Pathological hypertrophy underlies sudden cardiac death due to its high incidence of occurrence of ventricular arrhythmias. The alteration of transmural electrophysiological properties in hypertrophic cardiac murine tissue has never been explored previously. In this dataset, we have for the first time conducted high-throughput simultaneous optical imaging of transmembrane potential and calcium transients (CaT) throughout the entire hypertrophic murine hearts at high temporal and spatial resolution. Using ElectroMap, we have conducted multiple parameters analysis including action potential duration/calcium transient duration, conduction velocity, alternans and diastolic interval. Voltage-calcium latency was measured as time difference between action potential and CaT peak. The dataset therefore provides the first high spatial resolution transmural electrophysiological profiling of the murine heart, allowing interrogation of mechanisms driving ventricular arrhythmias associated with pathological hypertrophy. The dataset allows for further reuse and detailed analyses of geometrical, topological and functional analyses and reconstruction of 2-dimensional and 3-dimentional models