Albin A Bernardin

Sarah Colombani

Antoine Rousselot

Virginie Andry

Yannick Goumon

Côme Pasqualin

Bernard Brugg

Etienne Jacotot

Jean-Luc Pasquié

Alain Lacampagne
email: alain.lacampagne@inserm.fr.

Albano C Meli
email: albano.meli@inserm.fr

Impact of neurons on patient derived-cardiomyocytes using organ-on-a-chip and iPSC biotechnologies

Keywords: organ-on-a-chip, microfluidic system, iPSC, cardiomyocytes, neurons

In the heart, cardiac function is regulated by the autonomic nervous system (ANS) that extends through the myocardium and establish junctions at the sinus node and ventricular levels. Thus, an increase or decrease of neuronal activity acutely affects myocardial function and chronically affects its structure through remodeling processes. The neuro-cardiac junction (NCJ), which is the major structure of this system, is poorly understood and only few cell models allow us to study it. Here we present an innovant neuro-cardiac organ-on-chip model to study this structure to better understand the mechanisms involved in the establishment of NCJ. To create such a system, we used microfluidic devices composed of two separate cells compartment interconnected by asymmetric microchannels. Rat PC12 cells, were differentiated to recapitulate the characteristics of sympathetic neurons, and cultivated with cardiomyocytes derived from human induced pluripotent stem cells (hiPSC). We confirmed the presence of specialized structure between the two cell types that allow neuromodulation and observed that the neuronal stimulation impacts the excitation-contraction coupling properties including the intracellular calcium handling. Finally, we also co-cultivated human neurons (hiPSC-NRs) with human cardiomyocytes (hiPSC-CMs) both obtained from the same hiPSC line. Hence, we have developed a neuro-cardiac compartmentalized in vitro model system that allows to recapitulate structural and functional properties of neuro-cardiac junction and that can be used to better understand interaction between heart and brain in humans, as well as to evaluate the impact of drugs on a reconstructed human neuro-cardiac system.

Introduction

The cardiac function is tightly regulated by the autonomic nervous system (ANS) that extends through the myocardium [START_REF] Crick | Localisation and quantitation of autonomic innervation in the porcine heart ii: Endocardium, myocardium and epicardium[END_REF][START_REF] Rajendran | Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies[END_REF]. The autonomic nervous system is composed of the parasympathetic nervous system (PNS) and sympathetic nervous system (SNS) through the establishment of neuro-cardiac junction (NCJ) at the sinus node and ventricular levels [START_REF] Crick | Localisation and quantitation of autonomic innervation in the porcine heart ii: Endocardium, myocardium and epicardium[END_REF][START_REF] Rajendran | Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies[END_REF][START_REF] Ieda | Sema3a maintains normal heart rhythm through sympathetic innervation patterning[END_REF][START_REF] Kimura | Development, maturation, and transdifferentiation of cardiac sympathetic nerves[END_REF].

NCJs allow the interaction of ANS neurons and cardiomyocytes for rapid regulation of homeostasis and heart integrity. The ANS also exerts a trophic action during the development [START_REF] Dowell | Postnatal development of rat heart during 6-hydroxydopamine or propranolol treatment[END_REF][START_REF] Kreipke | Innervating sympathetic neurons regulate heart size and the timing of cardiomyocyte cell cycle withdrawal[END_REF] and under cardiac pathologies [START_REF] Kimura | Development, maturation, and transdifferentiation of cardiac sympathetic nerves[END_REF]. Thus, an increase or decrease of sympathetic activity directly affects myocardial function and its structure through remodeling processes.

Unlike the neuromuscular junctions, synapses of the neuro-cardiac junction are poorly understood. Rare studies have demonstrated the presence of protein complexes involved in cellular interaction or in exocytosis [START_REF] Shcherbakova | Organization of β-adrenoceptor signaling compartments by sympathetic innervation of cardiac myocytes[END_REF]. The immunostaining has revealed a specialization of the cardiomyocyte membrane, with a large concentration of β1-adrenergic receptor, and a decreased expression of caveolin-3.

The secretion of noradrenalin is involved in the development of the myocardium by the tropism it exerts [START_REF] Zaglia | Cardiac sympathetic neurons provide trophic signal to the heart via beta2-adrenoceptor-dependent regulation of proteolysis[END_REF]. Dowell has demonstrated its involvement during myocardial development. The specific death of sympathetic neurons by the addition of 6hydroxydopamine is directly correlated with a decrease in myocardial size [START_REF] Dowell | Postnatal development of rat heart during 6-hydroxydopamine or propranolol treatment[END_REF]. Kreipke and Birren showed that noradrenalin is involved in the transition between hyperplastic and hypertrophic stages [START_REF] Kreipke | Innervating sympathetic neurons regulate heart size and the timing of cardiomyocyte cell cycle withdrawal[END_REF] and directly impact the size and number of cardiomyocytes (CMs).

Several heart conditions are associated with changes in the intracardiac neuronal network that can lead to cardiac arrhythmias or an increased risk of myocardial infarction. These attacks can be induced by neuronal degeneration associated with diabetes [START_REF] Mustafa | Decreased myocardial perfusion reserve in diabetic autonomic neuropathy[END_REF], sympathetic hyperinnervation [START_REF] Mann | Mechanisms and models in heart failure: The biomechanical model and beyond[END_REF] or infection of the stellate ganglia [START_REF] Hong | Macrophage activation in stellate ganglia contributes to lung injury induced arrhythmogenesis in male rats[END_REF] as the major sympathetic ganglion for cardiac regulation [START_REF] Gardner | The effects of unilateral stellate ganglion blockade on human cardiac function during rest and exerciseeffects of unilateral stellate ganglion blocka[END_REF].

There are very few models to study the NCJ. The cellular models are based on the use of direct co-cultures in the same compartment [START_REF] Shcherbakova | Organization of β-adrenoceptor signaling compartments by sympathetic innervation of cardiac myocytes[END_REF][START_REF] Oh | Functional coupling with cardiac muscle promotes maturation of hpsc-derived sympathetic neurons[END_REF][START_REF] Prando | Dynamics of neuroeffector coupling at cardiac sympathetic synapses: Dynamics of neurocardiac communication[END_REF]. Those models involve the mixing the cells together in non-optimal conditions with the lack of control on specific cell population.

The development of microfluidic systems allows the establishment of hybrid co-culture and organ-on-a-chip (OOC) with the possibility of cultivating different cell types in separate compartments connected by micro-channels [START_REF] Duc | Human neuromuscular junction on micro-structured microfluidic devices implemented with a custom micro electrode array (mea)[END_REF]. Spatial and fluidic compartmentalization of distal axons and cell bodies replicates the in vivo conditions. Somatic and axonal milieux are (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10. [START_REF] Takeuchi | Sympathetic neurons modulate the beat rate of pluripotent cellderived cardiomyocytes in vitro[END_REF].512703 doi: bioRxiv preprint independently controlled. Compartmentalized devices are used in a variety of experimental models ranging from isolating axons to forming neuronal networks, from creating axon cocultures to engineering axonal behavior. Such protocols have emerged in several areas of research such as angiogenic development [START_REF] Chiew | Bioengineered three-dimensional co-culture of cancer cells and endothelial cells: A model system for dual analysis of tumor growth and angiogenesis: Analysis of tumor and vessel growth in a 3d co-culture[END_REF] or extravasation processes in tumors [START_REF] Arrigoni | In vitro co-culture models of breast cancer metastatic progression towards bone[END_REF].

Other groups have developed similar systems to study the NCJ [START_REF] Oh | Functional coupling with cardiac muscle promotes maturation of hpsc-derived sympathetic neurons[END_REF][START_REF] Takeuchi | Device for co-culture of sympathetic neurons and cardiomyocytes using microfabrication[END_REF][START_REF] Takeuchi | Sympathetic neurons modulate the beat rate of pluripotent cellderived cardiomyocytes in vitro[END_REF], demonstrating its feasibility. The human induced pluripotent stem cells (hiPSC) are self-renewal and have the potential to differentiate into any somatic cells. They nowadays offer great opportunities to create patient-derived OOC and model key physiological and pathophysiological features of human organs [START_REF] Acimovic | Human pluripotent stem cell-derived cardiomyocytes as research and therapeutic tools[END_REF].

In the present study we designed an innovant neuro-cardiac OOC based on microfluidic chip and hiPSC to study the impact of rat sympathetic neurons (differentiated for PC12 cells) or human hiPSC-derived autonomic neurons (hiPSC-NRs) on the functional properties of ventricular-like human hiPSC-derived cardiomyocytes (hiPSC-CMs).

Materials and Methods

Microfluidic devices

Microfluidic chips were produced by standard molding methods using epoxy-based negative photoresists (SU-8) and MicroBrain Biotech proprietary microdesigns (Brainies™, Cat#: MBBT5; Marly le Roi, France). Briefly, Polydimethylsiloxane (Sylgard 184, PDMS; Dow Corning) was mixed with curing agent (9:1 ratio) and degassed under vacuum. The resulting preparation was poured onto a chosen SU8 mold and reticulated at 70°C for 2 at least hours.

The elastomeric polymer print was detached, and 2 reservoirs were punched for each chamber. The polymer print and a glass cover slip were cleaned with isopropanol, dried, and treated for 3 minutes in an air plasma generator (98% power, 0.6 mBar, Diener Electronic) and bonded together. Brainies™ MBBT5 is a chip with a design containing 4 neuronal diodes. One neuronal diode includes 2 rectangular culture chambers (volume ~1 µL) each connected to 2 reservoirs and separated by a series of 500 µm-long asymmetrical microchannels (3 µm high, tapering from 15 µm to 3 µm).

Rat PC12 culture

Rat adrenal gland (phaeochromocytoma) PC12 cells from European Collection of Authenticated Cell Cultures (ECACC, 88022401) were seeded on collagen-coated 60mm petri dish in complete medium. Complete medium was composed of RPMI 1640 (Gibco, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.19.512703 doi: bioRxiv preprint 21875091) supplemented with 10% horse serum (Gibco, 26050088), 5% Foetal Bovine Serum (Gibco, 26140079) and 12.5µg/mL gentamicin (Sigma, G1397). PC12 differentiation was induced by switching from complete medium to differentiation medium. Differentiation medium was composed of RPMI 1640 supplemented with 1% horse serum, 12.5µg/mL gentamicin and 50ng/mL nerve growth factor (NGF, ABIN804475).

hiPSCs maintenance and differentiation

hiPSC cell lines were obtained from healthy male control. hiPSC were maintained in StemFlex medium (Gibco, A3349401) on Matrigel hESC-qualified matrix (Corning, 354277)coated petri dishes at 37°c in 5% CO2 incubator between passage 17 to 27. When the cells reached confluence, cell monolayer was dissociated using Tryple express enzyme (Gibco, 12604013) to obtain single cell solution. We differentiated patient-specific hiPSC-derived cardiomyocytes as previously published [START_REF] Moreau | Deciphering dsc2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ecg and tailored drug therapy[END_REF][START_REF] Sleiman | Modeling polymorphic ventricular tachycardia at rest using patient-specific induced pluripotent stem cell-derived cardiomyocytes[END_REF]. hiPSC were differentiated in ventricular-like cardiomyocytes using monolayer protocol and sandwich approaches [START_REF] Zhang | Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cellsnovelty and significance: The matrix sandwich method[END_REF]. The hiPSC-CMs were purified using the lactate-based protocol in glucose-and pyruvate-free conditions supplemented with 4mM Na-lactate as done previously [START_REF] Tohyama | Distinct metabolic flow enables largescale purification of mouse and human pluripotent stem cell-derived cardiomyocytes[END_REF].

The hiPSC-derived autonomic neurons were obtained from the same cell-line and differentiated following protocols described by Montgomery group [START_REF] Winbo | Functional coculture of sympathetic neurons and cardiomyocytes derived from human induced pluripotent stem cells[END_REF]. Neuronal induction was launched on a cell monolayer cultured on culture dishes coated on Matrigel hESCqualified matrix. Neuronal induction was conducted for 12 days to obtain a population of neurons. These neurons are seeded in microfluidic chips and matured for 42 days in co-culture with hiPSC-CMs.

Neurotransmitter release quantification

The acetylcholine neurotransmitter, released in cell medium was quantified using fluorometric kit (Acetylcholine, Abcam, ab65345). The four neurotransmitters were quantified in supernatant of 14 days differentiated PC12 cells harvested in conditioned medium after addition of Carbachol (1.5mM) in the medium.

Mass spectrometry was performed as follow. The cell secretion media were collected. Cells were recovered in 50µl of H2O containing 0.1mM ascorbic acid. Cells were sonicated ( (20,000g, 30min, 4°C), supernatants were collected and protein concentrations were accessed using the Protein Assay kit (Bio-Rad). An isotopic dilution approach was used for the absolute quantification. 20µl of the cell extracts or 50µl of the secretion medium were mixed with 10µl of internal standards containing 20pM of D4-Dopamine, C6-Noradrenalin, D6-Adrenalin, L-DOPA in 0.1mM ascorbic acid1. Then, 40µl of borate buffer and 10µl AccQtag Ultra reagent (AccQ-Tag Ultra derivatization kit, Waters, Guyancourt, France) were added to cell extracts and secretion media. The mixture was incubated 10min at 55°C under agitation. 500µl of ice-cold acetonitrile (ACN) were added and samples were centrifuged (20,000g, 30min, 4°C). The resulting supernatants were dried under vacuum and suspended in 20µl of H 2 O containing 0.1% formic acid (v/v).

Analyses were performed on a Dionex Ultimate 3000 HPLC system (Thermo Scientific) coupled with an Endura triple quadrupole mass spectrometer (Thermo Electron). The system was controlled by Xcalibur v. 2.0 software (Thermo Electron). 5µl of samples were loaded into reverse phase Zorbax column (SB-C18, #863600-902; 1mm x 150mm, 3.5µm, Agilent Technologies). Elution of the compounds was performed at a flow rate of 90 µl/min, at 40°C (see supplemental table 1 for conditions). Buffer A corresponded to H2O 98.9%/ACN 1%/formic acid 0.1% (v/v/v) and buffer B was ACN 99.9%/ formic acid 0.1% (v/v). Dopamine, adrenalin and noradrenalin are measured using the multiple reaction monitoring mode (MRM) according to the settings. The targeted compounds are detailed in supplement tables 2 and 3. The selection of the monitored transitions and the optimization of the collision energy (CE) were manually determined. The identification of the compounds was based on precursor ions, daughter ions and retention times obtained for dopamine, adrenalin and noradrenalin and their corresponding internal standards. Amounts of neurotransmitters were quantified according to the isotopic dilution method [START_REF] Liu | Selecting an appropriate isotopic internal standard for gas chromatography/mass spectrometry analysis of drugs of abuse-pentobarbital example[END_REF].

PC12/hiPSC-CMs co-culture

The day before neuronal seeding, chips were UV-sterilized for 20 min, then coated with a solution of poly-D-lysine (10 µg/ml, Sigma, P6407), incubated overnight (37°C, 5% CO 2 ), and rinsed 3 times with Dulbecco's phosphate buffer saline (PBS) (Sigma, D8537). Then, 4 hours before cell seeding, chips were treated with a solution of Laminin (10 µg/mL: Sigma) in PBS. Undifferentiated PC12 cells were seeded in microfluidic devices in differentiation medium supplemented with 50ng/mL of NGF and differentiated for 5 days. During this phase, PC12 started to differentiate with the formation of neurites and started projecting into micro-(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.19.512703 doi: bioRxiv preprint channels and invaded cardiac compartment. At day 5, hiPSC-CMs aged between 11 days and 20 days were seeded on Matrigel hESC-qualified matrix with a density of 20 000 cells/compartment in B27 medium supplemented with 10 µM Rock inhibitor and NGF at 150ng/ml. Complete medium changes with corresponding NGF concentrations were performed once a week to avoid cell detachment.

For neuro-cardiac OOC between hiPSC-NRs and hiPSC-CMs, autonomic neurons are seeded in neuronal compartment on Matrigel hESC-qualified and matured for 7 days before cardiomyocytes seeding without addition of NGF. Once the cardiomyocytes seeded, coculture started for 30 days to allow neurites to invade cardiac compartment and create interaction with CMs.

Immunocytochemistry

Immunocytochemistry (ICC) were performed 14 days after cells seeding on chips. Classical ICC protocols have been adapted to microfluidic chips to avoid liquid flux and cell detachment. Cells in neuronal and cardiac compartments were fixed with 4% PFA for 15 minutes at room temperature then rinsed 3 times with PBS. Cell membranes were permeabilized with 0.1% Triton for neuronal cells and 0.01% Triton for cardiac cells in PBS, 10min at room temperature followed by blockade solution (1% BSA in PBS) for 30min at room temperature. Primary antibodies were diluted in PBS supplemented with 0.1% BSA and incubated at 4°C overnight and rinse three times with PBS. Secondary antibodies were diluted in PBS and 0.1% BSA and incubated 2 hours at room temperature supplemented with 40,6diamidino-2-phenylindole (DAPI, 1:2000). Antibodies are listed in supplemental table 4. The chips were stored hydrated at 4°C before use. Staining was obtained with inverted microscope Axio Observer Z1 coupled with laser scanning confocal microscope LSM800 piloted with Zen 2.5 software, all provided by Zeiss. ICC images were acquired in 3D with z-stack option and reconstructed with ImageJ.

Intracellular calcium dynamics

Intracellular calcium signaling was studied to confirm the modulation of cardiac functional properties after neuronal stimulation. Calcium transient (CaT) were recorded with cytosolic free calcium probe Fluo-4 AM (Molecular Probes™, F14201) as published before [27,[START_REF] Lacampagne | Post-translational remodeling of ryanodine receptor induces calcium leak leading to alzheimer's disease-like pathologies and cognitive deficits[END_REF].

Cells were incubated with 1µM probe for 15 minutes at 37°C in Tyrode solution (in mM: 135 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.19.512703 doi: bioRxiv preprint NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, 0.1 acid ascorbic, pH 7.4).

Calcium transients were recorded for 7 seconds in line-scan mode using LSM800 confocal microscope (Zeiss) and analyzed using Peakinspector as done before [START_REF] Moreau | Deciphering dsc2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ecg and tailored drug therapy[END_REF][START_REF] Sleiman | Modeling polymorphic ventricular tachycardia at rest using patient-specific induced pluripotent stem cell-derived cardiomyocytes[END_REF][START_REF] Jelinkova | Dmd pluripotent stem cell derived cardiac cells recapitulate in vitro human cardiac pathophysiology[END_REF].

Measurement of contractile function using video-edge capture

To assess the contractile properties of the hiPSC-CMs connected with the neurons, we perform the video-edge capture as done before [START_REF] Moreau | Deciphering dsc2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ecg and tailored drug therapy[END_REF][START_REF] Sleiman | Modeling polymorphic ventricular tachycardia at rest using patient-specific induced pluripotent stem cell-derived cardiomyocytes[END_REF] on microfluidic chip after 14 days of co-culture. Activity of connected cardiomyocytes were recorded for 25 seconds. Transmitted light video at 63frame/s (fps) were obtained with LSM800 confocal microscope (Zeiss) at x63 magnification with ORCA-Flash4 camera (Hamamatsu). Experiments were performed in Tyrode solution (in mM: 135 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, 0.1 acid ascorbic, pH 7.4). The movement of each pixel in the image was evaluated using a patented video analysis script (Autobeats) via MATLAB software [START_REF] Moreau | Deciphering dsc2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ecg and tailored drug therapy[END_REF][START_REF] Sleiman | Modeling polymorphic ventricular tachycardia at rest using patient-specific induced pluripotent stem cell-derived cardiomyocytes[END_REF]. This analysis provides a contraction versus time curve from which contractile properties are extracted.

Statistical analysis

All data are expressed as the mean±SEM. Statistical analysis was performed with GraphPad Prism 8.0.1 (GraphPad Software Inc.). Experimental groups were compared by using nonparametric Kruskal-Wallis tested followed by Dunn's multiple comparisons test were performed. P<0.05 was considered statistically significant.

Results

Molecular characterization of the PC12 neurons connected with hiPSC-CMs

Rat PC12 cells were differentiated for 14 days in classic petri dish. Neurotransmitter production was confirmed by ICC showing the expression of enzymes involved in neurotransmitter production: choline acetyltransferase (ChAT) for acetylcholine, tyrosine hydroxylase for all the catecholamines (TH) and dopamine β-hydroxylase (DBH) for noradrenalin. ICC showed that PC12 expressed both TH and ChAT in neurites and DBH in their soma (Figure 1A), suggesting a production of these neurotransmitters. Despite these staining, we were unable to measure catecholamines with ELISA kits (Abnova, KA3836) compared to acetylcholine, which could be measured with fluorometric kits (Abcam, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.19.512703 doi: bioRxiv preprint ab65345). The neurotransmitter quantification was performed in cell supernatant after addition of 1.5mM Carbachol. Medium was collected 2min after neuronal stimulation. Our results showed a larger secretion of acetylcholine (24.39 10 3 pM) than catecholamines or their precursors (noradrenalin: 3.7 pM, L-DOPA: 3.8 pM, dopamine: 214.6 pM) in PC12 (Figure 1B), suggesting a dominant parasympathetic profile of these neurons.

As published before [START_REF] Moreau | Deciphering dsc2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ecg and tailored drug therapy[END_REF][START_REF] Sleiman | Modeling polymorphic ventricular tachycardia at rest using patient-specific induced pluripotent stem cell-derived cardiomyocytes[END_REF][START_REF] Souidi | Oxygen is an ambivalent factor for the differentiation of human pluripotent stem cells in cardiac 2d monolayer and 3d cardiac spheroids[END_REF] we confirmed that we generated ventricular-like hiPSC-CMs through the expression of the sarcomeric cardiac markers including α-actinin and cardiac troponin I (cTnI) at 30-days of differentiation (Figure 1C). quantified as described in material and method section. (C) hiPSCs differentiate in cardiomyocytes (hiPSC-CMs) after 30 days following the so-called "matrix sandwich method" of differentiation. hiPSC-CMs were fixed and immunolabelled for sarcomeric cardiac markers. Representative immunofluorescence microscopy images show Cardiac troponin (in green) and the α-sarcomeric actinin (in red). Scale bars: 20µm.

Microfluidic devices allow a neuro-cardiac OOC.

Twenty year ago, Taylor et al., used a microfabricated silicon wafer to mold a silicone elastomer (namely polydimethylsiloxane) that featured two chambers connected by parallel microgrooves. Once sealed against a glass coverslip microgroove formed microchannels [START_REF] Taylor | A microfluidic culture platform for cns axonal injury, regeneration and transport[END_REF][START_REF] Taylor | Microfluidic multicompartment device for neuroscience research[END_REF]. The resulting devices were developed in many ways by multiples research groups to compartmentalize and study neurons as well as neuronal networks in various physiological and pathological contexts [START_REF] Habibey | Microfluidics for neuronal cell and circuit engineering[END_REF].

Neuronal diodes are fluidic microsystems, inspired by those of Taylor et al., conceived to impose directionality when connecting two neuronal populations [START_REF] Peyrin | Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers[END_REF]. Neuronal diodes are made up of two separate cellular micro-compartments interconnected by a series of asymmetric microchannels, each of whose openings were of different sizes (Figure 2A). Here we have used a slightly modified and standardized design of the two-chamber neuronal diode (Figure 1; Brainies® MBBT5) and seeded neurons in the compartment where the microchannels openings were widest (15µm) and cardiomyocytes in the second compartment, where the openings were very narrow (3 µm). The expectation of such cocultures is to separate the (somato-dendritic) neuronal compartment from the cardiomyocytes compartment, and to obtain axonal projections through the microchannel to analyze axon-dependent neuron to cardiomyocytes interactions. The characteristics of the device combined with the controlled filling of each reservoirs allow fluidic isolation between chamber and the so-called compartmentalized pharmacology where treatment can be added selectively in one chamber but not the opposite.

PC12/hiPSC-CMs OOC involves neuro-cardiac junctions and synapse-like junctions

Undifferentiated PC12 cells were seeded in neuronal compartment with 50ng/mL of NGF.

Cells started differentiating in neuron-like cells through neurites from the soma. NGF gradient concentration was created between neuronal (50ng/ml) and cardiac compartment (150ng/ml) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.19.512703 doi: bioRxiv preprint to attract neurites. At day 5 post PC12 seeding, we observed neurites projecting into microchannel and started invading the cardiac compartment (Figure 2B). hiPSC-CMs were seeded at this day and OOC are co-cultured for 14 days. This procedure allowed us to obtain reproductible and transposable co-cultures protocols. During the 14 days of co-culture, PC12 neurites continued to develop in cardiac compartment and projected in every direction to make numerous interactions with cardiomyocytes (Figure 2C). At the end of microchannels, PC12 projections were organized in network of large neurites. This network divided in several smaller neurites systems that projected in all directions and interacted with the hiPSC-CMs.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2. Neuronal diodes fluidic microsystems as a tool to establish compartmentalized neurocardiac coculture on chip. (A) Structural characteristics of a two-chamber neuronal diode also known as Brainies®. Left panel: 3D-model of the micro-structured PDMS-based block (grey), including cy-lindric wells (diameter: 4 mm;

height: 5 mm), rectangular cell culture chambers (vol. ~1 µL; height: 55 µm), and series of asymmetrical microchannels (length: 500 µm; height: 3 µm; width: tapering from 15 µm to 3 µm). The compartment where neurons are seeded is represented in blue and the compartment where cardiomyocytes are seeded is represented in green. Small in-sert shows a picture of one Brainies® that contains four neuronal diodes. Right panel: micrograph of a portion of the funnel-shaped microchannels. (B) In this fluorescence microscopy image of a microfluidic chip section, the neural compartment is located to the left and the cardiac compartment to the right. Previous studies have shown that the neurocardiac junction is a specialized structure that exhibits a specific cellular organization of neuronal and cardiac cells [START_REF] Shcherbakova | Organization of β-adrenoceptor signaling compartments by sympathetic innervation of cardiac myocytes[END_REF][START_REF] Prando | Dynamics of neuroeffector coupling at cardiac sympathetic synapses: Dynamics of neurocardiac communication[END_REF]. In vivo, sympathetic neurons project along cardiac tissue and form buds with cardiomyocytes. This morphology is referred as the pearl necklace structure [START_REF] Kruepunga | Development of the sympathetic trunks in human embryos[END_REF]. These axonal sprouting are also called varicosity, which contain the cardiac synapse releasing the neurotransmitter. Due to their secretory activity and interaction with cardiomyocytes, varicosities express a panel of proteins involved in the release of neurotransmitters like synapsin-1 [START_REF] Shcherbakova | Organization of β-adrenoceptor signaling compartments by sympathetic innervation of cardiac myocytes[END_REF] or proteins involved in intercellular interaction like SNARE proteins [START_REF] Hilfiker | Synapsins as regulators of neurotransmitter release[END_REF].

Between the two compartments are the microchannels (not visible in the image, scale bar: 100µm). PC12 cells differentiate directly into their compartments and their axons project into the cardiac compartment through the channels. (C) and (D): Images of neuro-cardiac interactions in cardiac compartments after 14 days of coculture. Tyrosine hydroxylase (in Green) appear apposed along axons. Axons (β3-tubulin; in white) harbor varicosities in the interaction zones (White arrows). (Blue

To confirm the presence of synapses between PC12 neurites and hiPSC-CMs, we performed immunofluorescence experiments and acquired images in transmission light. Our results revealed that PC12 neurites interacted densely with hiPSC-CMs and form varicosity-like structures (Figures 2D and3A). To confirm the presence of synapses, we revealed expression of synapsin-1 and β3-tubulin following 14 days of co-culture using fluorescence microscopy.

Our immuno-staining showed that neurites reproduced pearl necklace organization close to the hiPSC-CMs with the specific expression of synaspin-1 localized in the buds (Figure 3B).

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 3. Structural characterization of the neuro-cardiac junction on a 14-day-old OOC. Images showing regions of local interaction between PC12 axons and hiPSC-CM in transmission light (A) or in immunofluorescence (B). PC12 neurites express the protein synapsin-1 colocalized with structural staining (β3tubulin; green) in regions of interaction with cardiomyocytes and neurites. (C): Co-localization of tyrosine hydroxylase and synapsin-1 in the varicosities outside the neurocardiac junctions. (In this figure: blue: DAPI,

red: synapsin-1, Green: β3-tubulin, Grey: sarcomeric α-actinin, scale bar: 20µm).

Neurons modulate the intracellular calcium handling in hiPSC-CMs

We then hypothesized that PC12 neuronal cells could modulate the ECC of the hiPSC-CMs via NCJ. We focused on the intracellular Ca 2+ handling and contractile properties as two major aspects of the ECC. We designed a protocol to apply drugs independently in the neuronal and cardiac compartments (Figure 4A). Using Fluo4-AM and fluorescent microscopy, we acquired CaT at rest and in presence of Carbachol (muscarinic receptor agonist) in the neuronal compartment or Atropine (muscarinic receptor antagonist) in the cardiac compartment (Figure 4B). Their effects were studied just after addition of drugs in the corresponding compartments. We focused on the relative CaT amplitude as indicator of the (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.19.512703 doi: bioRxiv preprint sarcoplasmic reticulum (SR) Ca 2+ content, velocity of Ca 2+ release through type 2 ryanodine receptor (RyR2), Ca 2+ reuptake duration and CaT frequency (Figure 4C).

Release of neurotransmitter after stimulation of PC12 cells by carbachol in neuronal

compartment affects the Ca 2+ kinetics in cardiomyocytes (Figure 4D-F). The CaT frequency (Rest: 0.48±0.04Hz, Carbachol: 0.38±0.03Hz, Atropine: 0.63±0.09Hz, Atropine + Carbachol: 0.53±0.07Hz, p=ns) was not affected by the different experimental conditions in cardiomyocytes (Figure 4F). Addition of carbachol did not modulates the CaT amplitude (Rest: 2.28±0.13, Carbachol: 2.03±0.14, p=ns, Figure 4C) but affected Ca 2+ release velocity after PC12 stimulation (Rest: 11.41±0.86, Carbachol: 7.76±0.73, p<0.01, Figure 4D) and Ca 2+ reuptake duration (Rest: 0.87±0.04s, Carbachol: 1.04±0.07s, p<0.05, Figure 4E).

Blockade of M2 muscarinic receptor by the application of atropine in the cardiac compartment induced an increase of Ca 2+ release velocity in hiPSC-CMs compared to rest condition (Rest: 11.41±0.86, Atropine: 21.54±3.72, p<0.05), after addition of carbachol (Carbachol: 7.76±0.73, Atropine: 21.54±3.72, p<0.001) and after addition of both molecules combined (Atropine: 21.54±3.71, Atropine + Carbachol: 14.14±2.10, p<0.05) (Figure 4D). Addition of atropine also induced a decrease of Ca 2+ reuptake duration compared to rest (Rest: 0.87±0.04s, Atropine: 0.66±0.05s, p<0.05) or to carbachol-treated PC12 cells (Carbachol:

1.04±0.07s, Atropine: 0.660±0.047 seconds, p<0.001) or both combined (Carbachol:

1.042±0.068seconds, Atropine + Carbachol: 0.71± 0.05s, p<0.001) (Figure 4E). Finally, we also observed that addition of atropine significantly increased Ca 2+ transient amplitude compared to rest (Rest: 2.28±0.13, Atropine: 3.30±0.28, p<0.001) and to carbachol condition (Carbachol: 2.09±0.17, Atropine: 3.30±0.28, p<0.001) (Figure 4C).

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Neurons do not impact the contractile properties in hiPSC-CMs

We next evaluated whether neurons could modulate the contractile properties of the hiPSC-CMs using the video-edge capture, as published before [START_REF] Moreau | Deciphering dsc2 arrhythmogenic cardiomyopathy electrical instability: From ion channels to ecg and tailored drug therapy[END_REF][START_REF] Sleiman | Modeling polymorphic ventricular tachycardia at rest using patient-specific induced pluripotent stem cell-derived cardiomyocytes[END_REF], in microfluidic chips in transmitted light at rest (Figure 5A) and in the presence of Carbachol and/or Atropine. The contractility was first measured at rest for 25s, then after addition of Carbachol or Atropine.

An example of the obtained contraction/relaxation cycle is shown in Figure 5B.

Acetylcholine release from PC12 neurites following the addition of Carbachol has no effects on contraction parameters (Figure 5 C-H). The contraction frequency in beats/min (Rest: 16.28±1.39bpm, Carbachol: 17.85±1.63bpm, p=ns, Figure 5C), contractile amplitude (Rest: 0.57±0.07, Carbachol: 0.54± 0.05, p=ns, Figure 5D), contraction velocity (Rest: 2.94±0.34m/s, Carbachol: 2.94±0.39m/s, p=ns, Figure 5E), relaxation velocity (Rest:

1.87±0.24m/s, Carbachol: 2.23±0.44m/s, p=ns, Figure 5F), contraction homogeneity (Rest:

1.68±0.05, Carbachol: 1.79±0.07, p=ns, Figure 5G) and the resting time (Rest: 5310±663.6ms, Carbachol: 5304±1318ms, p=ns, Figure 5H). Surprisingly, addition of Atropine had no effects on the contractile properties compared to the results obtained with calcium experiments (Figure 4C-F) suggesting that spontaneous acetylcholine release is not sufficient to modulate the contraction frequency (Rest: 16.28±1.39bpm, Atropine: 21.15± 2.34bpm, Atropine + Carbachol: 20.05± 2.57bpm, p=ns, Figure 5C), the amplitude of contraction (Rest: 0.57± 0.07, Atropine: 0.44±0.02, Atropine + Carbachol: 0.48±0.03, p=ns, Figure 5D), the contraction velocity (Rest: 2.94±0.34m/s, Atropine: 2.28±0.22m/s, Atropine + Carbachol: 2.85±0.37 m/s, p=ns, Figure 5E), the relaxation velocity (Rest: 1.87±0.24 m/s, Atropine: 1.79±0.18m/s, Atropine + Carbachol: 2.08±0.25m/s, p=ns, Figure 5F), the contraction homogeneity (Rest: 1.68±0.05, Atropine: 1.73 ± 0.05, Atropine + Carbachol:

1.82± 0.05, p=ns, Figure 5G) and resting time (Rest: 5310±663.6ms, Atropine: 2880 ± 518.9ms, Atropine + Carbachol: 3433± 1082ms, p=ns, Figure 5H).

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Patient-specific neuro-cardiac OOC using hiPSC-NRs and hiPSC-CMs

In order to confirm the feasibility of such neuro-cardiac OOC using the same hiPSC line to generate neurons and cardiomyocytes, we differentiated TH-positive hiPSC-NRs that we cocultivated with hiPSC-CMs in microfluidic devices (Figure 6A). The hiPSC-NRs were seeded in microfluidic compartment at the beginning of neuronal maturation 1 week before cardiomyocytes seeding. They were able to create numerous interactions with the projection of other neurons (Figure 6C) and to project into microchannels to invade cardiac compartment without NGF addition (Figure 6B). These neurons generated numerous interactions with one or several cardiomyocytes after 14 days of co-culture (Figure 6D).

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Discussion

In this study, we built-up a neuro-cardiac OOC to investigate the functional impact of neurons on patient-derived cardiomyocytes. OOC approaches emerge with different protocols in order to study the effect of interaction between cardiac and neurons with the use of direct co-culture [START_REF] Oh | Functional coupling with cardiac muscle promotes maturation of hpsc-derived sympathetic neurons[END_REF][START_REF] Prando | Dynamics of neuroeffector coupling at cardiac sympathetic synapses: Dynamics of neurocardiac communication[END_REF][START_REF] Kowalski | Sympathetic neurons regulate cardiomyocyte maturation in culture[END_REF][START_REF] Hakli | Human neurons form axon-mediated functional connections with human cardiomyocytes in compartmentalized microfluidic chip[END_REF] or microfluidic devices. Compared to direct co-culture, microfluidic chips allow the culture of each cell-lines in their optimal conditions. Fluidic isolation of each chamber also allows to ensure cell-type specific pharmacological treatments. Here, our results indicate that neurons seeded in one compartment are able to further differentiate and spread axons into microchannels to reach the cardiac compartment containing hiPSC-CMs. The width and length of microchannels only allows axon to reach the second compartment, whereas neither dendrites and soma, nor cardiomyocytes can access the opposite compartment.

Our data show that axons form a large network at the end of microchannels. Via a large spread, they subdivide in thin filaments which spread in all the directions to create interaction with hiPSC-CMs. This axonal network is the central element of neurocardiac regulation [START_REF] Freeman | In situ threedimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging[END_REF].

In physiological conditions, it has been shown that neurites spread on long distances from one cardiomyocyte to another and interact with them by forming buddings along axons. These "en passant" structures, also called varicosities, are the functional neurotransmitter release site that enables rapid and axial neurotransmitter release to several cardiomyocytes at the same time [START_REF] Freeman | In situ threedimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging[END_REF]. As published by other groups, these varicosities are a specialized and ordered structure on either sides of the synaptic cleft that induces rapid activation of β-adrenergic signaling pathway [START_REF] Prando | Dynamics of neuroeffector coupling at cardiac sympathetic synapses: Dynamics of neurocardiac communication[END_REF]. In neuromuscular junction, these structures are also observed with high concentration of nicotinic receptors at the post-synaptic side [START_REF] Huh | Clustering of nicotinic acetylcholine receptors: From the neuromuscular junction to interneuronal synapses[END_REF].

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. To confirm development of functional synapse between PC12 cells and hiPSC-CMs, we performed structural and functional characterization of the neuro-cardiac junction. The NCJ and its proteins are not very well known. In our study, we focused on the presence of varicosities in neuronal network. We confirmed that expression of synapsin-1, a key synapse protein, at the neuro-cardiac interaction zone. Synapsin-1 is a protein expressed in neuronal synapse to organize vesicles pools, maturation and neurotransmitter release [START_REF] Hilfiker | Synapsins as regulators of neurotransmitter release[END_REF][START_REF] Fornasiero | Synapsins contribute to the dynamic spatial organization of synaptic vesicles in an activity-dependent manner[END_REF][START_REF] Rosahl | Essential functions of synapsins i and ii in synaptic vesicle regulation[END_REF]. In our neuro-cardiac OOC, we found that some structures similar to varicosities co-localized with synapsin-1. These observations strongly suggest that PC12 axons and cardiomyocytes form a NCJ-like structure and synapses in vitro. Further experiments using electronic microscopy could confirm such specialized structure by showing organized cell membrane in neurons and cardiomyocytes.

Our findings revealed that PC12 cells express both adrenergic (DBH and TH) and cholinergic (ChAT) enzymes but secrete a larger amount of acetylcholine compared to catecholamine resulting rather in a parasympathetic than sympathetic neurotransmitters pattern. This observed phenotype makes sense with our results on the intracellular Ca 2+ handling properties we acquired. Using Carbachol and Atropine, to activate and inhibit the muscarinic receptors, respectively, we monitored the impact on the CaT properties and found only slight consequences. We found that stimulation of PC12 cells by Carbachol modulates calcium release velocity and Ca 2+ reuptake duration but did not affect the CaT of the hiPSC-CMs.

Moreover, inhibition of M2 muscarinic receptors in hiPSC-CMs using Atropine causes increased CaT amplitude, Ca 2+ release velocity via RyR2 and decreased Ca 2+ reuptake duration via SERCA2a. These results indicated that acetylcholine is spontaneously secreted by PC12 neurites under non stimulating condition and binds to muscarinic receptors to modulate Ca 2+ kinetics affecting the SR Ca 2+ cycling in hiPSC-CMs. Surprisingly, addition of carbachol or atropine has no effects on the contractile properties compared to the results obtained with Ca 2+ experiments suggesting that spontaneous acetylcholine release is not sufficient to modulate contractility. This absence of effect is possibly due to excessive spontaneous secretion which would reduce the stock of neurotransmitter. During stimulation of PC12 cells by carbachol, this stock of neurotransmitter may be too low and insufficient to significantly impact the hiPSC-CMs contraction. This spontaneous secretion could also constitutively activate the cardiac receptors which could no longer respond to a new release of neurotransmitters. Another explanation could be that our neurocardiac OOC does not have enough interactions between the two cell types to allow a deep modulation of the contractile (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.19.512703 doi: bioRxiv preprint properties. Our data may also suggest that PC12 cell may reveal limitations to study the effect of autonomic nervous system on cardiac function due to their abnormal neurotransmitter secretion phenotype and the co-expression of cholinergic and catecholaminergic enzyme.

The use of neurons derived from hiPSC is the next step. Even if we here, as well as other group [START_REF] Oh | Functional coupling with cardiac muscle promotes maturation of hpsc-derived sympathetic neurons[END_REF][START_REF] Kowalski | Sympathetic neurons regulate cardiomyocyte maturation in culture[END_REF] demonstrated the feasibility of neuro-cardiac OOC from hiPSC in microfluidic chips. Direct co-culture improves the maturity of peripheral neurons derived from hiPSC by improving their Ca 2+ kinetics and the expression of peripheral marker such as TH and DBH [START_REF] Oh | Functional coupling with cardiac muscle promotes maturation of hpsc-derived sympathetic neurons[END_REF]. The cardiac maturity was also improved by the increased expression of contractile protein α-actinin and proteins involved in Ca 2+ signaling such as RyR2 and phospholamban [START_REF] Kowalski | Sympathetic neurons regulate cardiomyocyte maturation in culture[END_REF]. Another group showed that culture of CMs with cortical neurons both from hiPSC in microfluidic devices are able to create NCJ with limited functional effects [START_REF] Hakli | Human neurons form axon-mediated functional connections with human cardiomyocytes in compartmentalized microfluidic chip[END_REF]. These results show that without pure autonomic neuronal population, we are limited to study NCJ and their implications.

In this context, two groups recently published ANS differentiation protocols to obtain sympathetic and/or cholinergic neurons from hiPSC. In these articles, they demonstrated that autonomic neurons are able to modulate beating rate of cardiomyocytes co-cultured with them. Despite these important progresses, they did not go further and observed these effects on other functional properties like calcium signaling and contractile properties. Moreover, it seems unclear if these neurons secrete noradrenalin or acetylcholine as desired. Further experiments will be needed to obtain specific hiPSC-NRs able to impact the hiPSC-CMs properties in healthy or disease-related conditions.

Conclusions

In this work, we built-up a neuro-cardiac OOC allowing to study the physiological interactions between the ANS and heart during development and pathologies. We revealed the presence of specialized zone between neurons and cardiomyocytes, in particular with synapsin-1, a protein involved in the release of neurotransmitter-containing vesicles and varicosities. Our neuro-cardiac OOC modulates the intracellular Ca 2+ cycling of the patientderived cardiomyocytes but does not change their contractile properties. Overall, this work brings evidence of the functionality of a human neurocardiac OOC to further study the relationship between nervous system and heart in the dish.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 1 .

 1 Figure 1. Molecular characterization of neuro-cardiac OOC composed of PC12 cells and hiPSC-CMs. (A) PC12 cells differentiate into parasympathetic neurons. PC12 were cultured for 14 days in the presence of 50ng/ml NGF, fixed and immunolabelled for sympathetic markers. Representative immunofluorescence microscopy images show choline acetyltransferase (left panel; in red), tyrosine hydroxylase (central panel; in green), dopamine β-hydroxylase (Right panel; in green), and β3-tubulin (Right panel; in red). (B) Quantification of neurotransmitters in supernatant of PC12 cells. PC12 cells were differentiated for 14 days and subjected to addition of 1.5mM Carbachol for 2 min. Then, medium was collected and the indicated neurotransmitters were

  : DAPI, Red: sarcomeric α-actinin, Green: Tyrosine hydroxylase, White: β3-tubulin, scale bar: 20µm).

Figure 4 .

 4 Figure 4. Monitoring of the intracellular calcium cycling properties on 14-day-old neuro-cardiac OOC using PC12 and hiPSC-CMs. (A) Schematic representation of neurocardiac OOCs with cellular compartments showing where the different molecules are added. The M2 muscarinic receptor antagonist atropine is added to the cardiac compartment and the muscarinic receptor agonist Carbachol is added to the neuronal compartment. (B) CaT are recorded using Fluo4-AM. Representation of CaT in hiPSC-CMs recorded in line-scan configuration with the corresponding plot below from which calcium kinetics are obtained. (C) Parameters of normalized amplitude of CaT, (D) calcium release velocity, (E) duration of Ca 2+ reuptake and (F) frequency of CaT are presented in resting condition, after addition of carbachol and or atropine on the cardiomyocytes located in the cardiac compartment after 14 days of co-culture. Data are presented as mean±SEM, Rest n=145,

Figure 5 .

 5 Figure 5. Contractile properties of hiPSC-CMs recorded in 14-day-old neuro-cardiac OOC by video-edge capture. (A) Picture of PC12 neurites connected (marked with black arrows) to hiPSC-CMs in transmitted light.Spontaneous contraction is recorded on cardiac compartment for 25 seconds. (B) Representation of contraction/relaxation cycle obtained from movies. (C-H) Measurement of contractile parameters on neurocardiac OOC at rest, in the presence of Carbachol, Atropine, or in the presence of both. (C) Beating frequency, (D) amplitude of contraction, (E) contraction and (F) relaxation velocity, (G) contraction homogeneity and (H)average resting time are obtained from series of images as shown in Figure 5A. Data are presented as mean ± SEM, Rest n=49, Carbachol n=23, Atropine n=23, Carbachol + Atropine n=19, Dunn's multiple comparisons test were performed.

(

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.19.512703 doi: bioRxiv preprint

Figure 6 .

 6 Figure 6. Neuro-cardiac OOC using neurons and cardiomyocytes derived from the same patient-derived hiPSC line. (A) Schematic presentation of the production of patient-specific neuro-cardiac OOC using similar control hiPSC line to generate autonomic neurons and cardiomyocytes. (B) Global view of neuro-cardiac interaction after 14 days in microfluidic chips of autonomic hiPSC-NRs seeded in neuronal compartment (C) projecting to the cardiac compartment. (D) Autonomic neurons expressing TH create projection that cross the microchannels and interact with cardiomyocyte (Blue: DAPI, Red: sarcomeric α-actinin, White: β3-tubulin, Green: TH, scale bar: 20µm).

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2022.10.19.512703	this version posted October 21, 2022.	The copyright holder for this preprint
					4 x
	10sec, 100W; Model 505 Sonic Dismembrator; Fisher Scientific). After centrifugation

(

  (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2022.10.19.512703	this version posted October 21, 2022.	The copyright holder for this preprint

  The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.19.512703 doi: bioRxiv preprint

Acknowledgments:

We thanks Dr. Hélène Delanoë-Ayari for her precious advices on the video-edge capture analysis, and Gullen Lacin (MicroBrain BT) for chips microfabrication.

Funding: This work was supported by grants of the "Fondation Coeur et Recherche", AssociationNationale pour la Recherche Technologique (ANRT ; CIFRE N°2018/003 to MicroBrain Biotech)the "Institut National pour la Santé et la Recherche Médicale" (INSERM), MUSE CIBSEEA and ANR MUSAGE. Albin Bernardin received a doctoral fellowship from "Le Fonds Marion Elisabeth Brancher".


Ousmane Ly
email: ousmane.ly@kaust.edu.sa

Aurelien Manchon
email: aurelien.manchon@univ-amu.fr

Spin-orbit coupling induced ultrahigh-harmonic generation from magnetic dynamics

The generation of nonlinear high-frequency response in solids from powerful optical pumps has gained momentum over the past decade. High-harmonic generation in solids can be obtained from strong-field laser excitation, usually restricted to optical frequencies and limited both in amplitude and in harmonic order. Here, we demonstrate that high-harmonic emission can be achieved by exploiting conventional spin pumping, without the need for optical excitation. Considering a noncentrosymmetric (ferro-or antiferro-)magnet, we show that when the spin-orbit coupling strength is close to the s-d exchange energy, a strongly nonlinear regime resulting from resonant spin flip scattering occurs leading to the generation of a large number of harmonics at realistic precession frequencies, thereby enhancing both spin and charge dynamics by orders of magnitude. This mechanism could be used to trigger high-frequency emission deep in the THz regime.

Introduction -

The recent boost in data transfer rates puts a daring strain on information technology. Sustaining such a growth rate requires the development of sources, detectors and systems working in the so-called TeraHertz (THz) gap covering the frequency window from 0.1 to 10 THz (1 THz = 10 12 Hz). This gap represents a challenge for conventional electronic devices due to carrier transit delays (∼1-10ps), as well as for photonic devices due to thermal fluctuations (300K∼6THz). Nonetheless, designing efficient, room-temperature THz sources would constitute a key enabler to applications spanning from high-resolution imaging to extreme wide band wireless communication. The grand challenge posed by the THz gap is that it is located on the high-end of electronic processes and on the low-end of optical excitations. It is therefore difficult to emit THz electromagnetic field using purely electronic mechanisms because the scattering time of the electronic carriers is typically in the THz range. A successful strategy exploited in quantum cascade lasers is to use semiconductor superlattices with small gaps in order to generate the required frequency [START_REF] Benjamin | Terahertz quantum-cascade lasers[END_REF]. Since the efficiency is low, one needs to multiply the number of gaps (hence, the quantum cascade) and work at low temperature to quench thermal fluctuations. Another strategy is to exploit optical rectification of a femtosecond laser pulse in semiconductors like ZnTe [START_REF] Tonouchi | Cutting-edge terahertz technology[END_REF]. An alternative approach uses an optically-generated ultrafast spin current, i.e., a charge-neutral current carrying spin angular momentum [START_REF] Seifert | Efficient metallic spintronic emitters of ultrabroadband terahertz radiation[END_REF]. In this geometry, a femtosecond laser pulse impinges on the surface of a thin ferromagnetic film and excites a superdiffusive spin current [START_REF] Battiato | Superdiffusive Spin Transport as a Mechanism of Ultrafast Demagnetization[END_REF]. This spin current then penetrates into an adjacent layer possessing large spin-orbit coupling and is subsequently converted into a charge current via either spin Hall effect [START_REF] Sinova | Spin Hall effect[END_REF] or Rashba-Edelstein effect [START_REF] V M Edelstein | Spin Polarization of conduction electrons induced by electric current in two-dimensional asymmetric electron systems[END_REF], depending on the considered heterostructure. This apparatus enables the conversion of a femtosecond laser pulse into a THz electromagnetic field [START_REF] Seifert | Efficient metallic spintronic emitters of ultrabroadband terahertz radiation[END_REF][START_REF] Kampfrath | Terahertz spin current pulses controlled by magnetic heterostructures[END_REF][START_REF] Huisman | Femtosecond control of electric currents in metallic ferromagnetic heterostructures[END_REF]. Although promising this solution relies on optical pumping and therefore FIG. 1. (Color online) Spin-flip scattering driven highharmonic generation: In the presence of a precessing magnetization order (gray line) the spin-orbit interaction leads to spin-flip scattering. Subsequently, a phase corresponding to the fundamental frequency is accumulated in the electronic wave-function. Under adiabatic magnetization dynamics, the spin flip time (τ sf ) becomes very small compared to the period of the precessing magnetic order, and higher harmonics appear as a consequence of subsequent spin flip scattering events during one fundamental cycle.

lacks scalability.

In this letter, we demonstrate that extremely efficient and ultrafast THz emission can be obtained by exploiting the internal dynamics of the spin-to-charge conversion to generate high-harmonics in a device solely powered by conventional magnetic resonance. The driving force is given by adiabatic spin pumping [START_REF] Brataas | Spin battery operated by ferromagnetic resonance[END_REF][START_REF] Saitoh | Conversion of spin current into charge current at room temperature: Inverse spin-Hall effect[END_REF], a widely used technique to generate pure spin currents out of ferromagnetic resonance, while the high-harmonic generation itself relies on the spin scattering events induced by spinorbit coupling in the presence of a precessing magnetic order.

Spin pumping [START_REF] Brataas | Spin battery operated by ferromagnetic resonance[END_REF] is an adiabatic process by which a precessing magnetic order m(t) (ferromagnetic or antiferromagnetic alike) injects a spin current into an adjacent metallic layer. This spin current possesses two components, a rectified one whose spin polarization is aligned along the precession axis J s ∼ m , and an os-cillating contribution whose spin polarization is aligned perpendicular to the precession axis J s ∼ m × ∂ t m. In a conventional spin pumping experiment, once injected in the normal metal, this spin current is converted into a charge current via spin-orbit coupling [START_REF] Saitoh | Conversion of spin current into charge current at room temperature: Inverse spin-Hall effect[END_REF][START_REF] Wei | Spin Hall voltages from a.c. and d.c. spin currents[END_REF][START_REF] Rojas-Sánchez | Spin-to-charge conversion using Rashba coupling at the interface between non-magnetic materials[END_REF], which leads to the dc and ac components depicted on Fig. 2(a). This spin-to-charge conversion does not affect the spin dynamics itself as long as the spin-orbit coupling is negligible compared to the s-d exchange, and thereby only generates a harmonic charge current.

In the presence of spin-orbit coupling though, successive spin flip events occur within one magnetic precession cycle, leading to the appearance of higher frequencies in the charge current signal (see Fig. 1). Subsequently, a phase corresponding to the dynamic frequency is accumulated as a consequence of angular momentum conservation. When the spin-orbit coupling energy becomes comparable to the s-d exchange parameter, the full harmonic spectrum is excited with extremely strong amplitudes. This is in close analogy with the high-harmonic generation mechanism from gaseous media under Laser excitation [START_REF] Corkum | Plasma perspective on strong field multiphoton ionization[END_REF][START_REF] Corkum | Attosecond science[END_REF][START_REF] Midorikawa | Ultrafast dynamic imaging[END_REF], where the electron wave-packets undergo harmonic emission as they re-collide to their parent atoms after a fraction of the Laser oscillation cycle. When the magnetization dynamics operates at small frequencies the emission of a huge number of harmonics is predicted.

Interestingly, our numerical simulations suggest the existence of a resonance condition associated to maximally excited high-harmonics. This corresponds to the regime where the s-d exchange energy is very close to the spin-orbit splitting. While the effect is not restricted to a particular type of spin-orbit interaction, the Rashbalike spin-orbit coupling constitutes a central paradigm to demonstrate the proposed effect. In fact, Rashba spinorbit interaction has been found in a broad range of magnetic interfaces, from transition metal interfaces [START_REF] Saitoh | Conversion of spin current into charge current at room temperature: Inverse spin-Hall effect[END_REF], to the surface of topological insulators [START_REF] Shiomi | Spin-Electricity Conversion Induced by Spin Injection into Topological Insulators[END_REF] or in oxide heterostructures [START_REF] Caviglia | Tunable Rashba Spin-Orbit Interaction at Oxide Interfaces[END_REF], and has the major advantage of being electrically tunable [START_REF] Diogo | Mapping spin-charge conversion to the band structure in a topological oxide two-dimensional electron gas[END_REF], offering a powerful means to control the high-harmonic generation.

Numerical simulations -To demonstrate the highharmonic generation, we consider the setup depicted on Fig. 2(b). The numerical calculations are performed using the time dependent quantum transport package T-KWANT [START_REF] Weston | Linear-scaling source-sink algorithm for simulating time-resolved quantum transport and superconductivity[END_REF][START_REF] Gaury | Numerical simulations of time-resolved quantum electronics[END_REF][START_REF] Kloss | Tkwant: a software package for time-dependent quantum transport[END_REF] where the stationary scattering properties are obtained from the dc transport package KWANT [START_REF] Groth | Kwant: A software package for quantum transport[END_REF]. We consider a two dimensional tight binding model including both exchange and Rashba spin-orbit coupling. The underlying Hamiltonian is given by

H(t) = H 0 (t) + H R , (1)
where, with h.c standing for hermitian conjugate, m(r, t) being the time dependent magnetization which also depends on position in the case of the antiferromagnetic dynamics. J is the s-d exchange energy between the conduction electrons and the local magnetic order. The operator σ represents the vector of Pauli matrices and γ the tight binding hopping energy. The operators ĉ † r and ĉr are respectively the creation and annihilation operators at position r given by the coordinates x and y. The Rashba Hamiltonian is given by

H 0 (t) = J r ĉ † r ( σ•m(r, t))ĉ r -γ r,r ĉ † r ĉr + h.c , (2)
H R = iγ α R 2 r ĉ † x,y (σ y ĉx+1,y -σx ĉx,y+1 ) + h.c, (3)
with α R the Rashba parameter. The system is connected to two transversal normal leads that allows for probing the pumped currents, see Fig. 2

(b).

To compute the non-equilibrium charge current, the stationary scattering modes of the tight binding system at different energies are obtained using KWANT. Subsequently, they are evolved forward in time according to the time dependent Schrödinger equation,

i ∂ t Ψ ηmε (t) = H(t)Ψ ηmε (t), (4)
where η stands for the lead from which the state originates, m is the mode index and ε is the transport energy. Furthermore, the time dependent current at temperature T is obtained as

I(t) = ηm dε 2π f (ε, T)Ψ † ηmε (t)Ψ ηmε (t), (5)
with f (ε, T) being the Fermi distribution function at energy ε and temperature T.

All the numerical calculations are performed at a chemical potential of 100 meV. Therefore, the calculated currents are summed from the bottom of the bands to the considered energy. The s-d exchange coupling and the temperature of the system are set to J = 500 meV and T = 0 K respectively, except otherwise stated. To obtain the charge currents in the frequency domain, a discrete fast Fourier transform of the time dependent signal is performed. In the data presented throughout the text I ω represents the absolute value of the normalized Fourier transform of I(t) given by Eq. ( 5). Further, we emphasize that the present effect is independent on the nature of the magnetic resonance and is only governed by the ratio of the spin-orbit coupling strength to the dynamic frequency. In fact, the high-harmonic generation is obtained for both ferromagnetic and antiferromagnetic resonances (see Supplemental information [START_REF]Supplemental Materials[END_REF]). Nonetheless, in the numerical method we use, the simulation time is set by the electron's energy and is of the order of the hopping parameter γ (typically of the order of 0.1 eV). In order to keep the computational cost reasonable, we have to consider a magnetic system whose resonance frequency is only two orders of magnitude smaller than the internal dynamics of the conduction spin. To comply with the numerical constraints, we therefore consider antiferromagnetic resonance (typically ∼meV) rather than ferromagnetic resonance (typically ∼ µeV). We stress out that this does not affect the generality of our results.

In antiferromagnetic resonance the two sublattice magnetization vectors, m 1 and m 2 , undergo different precession modes of opposite chirality [START_REF] Cheng | Spin pumping and spin-transfer torques in antiferromagnets[END_REF]. Without loss of generality, we consider the right-handed polarization of the order parameter, in which case both sublattice vectors rotate anticlockwise [START_REF] Keffer | Theory of Antiferromagnetic Resonance[END_REF]. As a result, the antiferromagnet order parameter n = (m 1 -m 2 )/2 precesses with the same chirality and a non-zero, albeit small, in-plane magnetization m = (m 1 + m 2 )/2 develops. As the sublattice magnetizations precess in time, the spin current pumped out of the antiferromagnet reads [START_REF] Cheng | Spin pumping and spin-transfer torques in antiferromagnets[END_REF],

J s = {g ↑↓ } (m × ∂ t m + n × ∂ t n) -{g ↑↓ }∂ t m, (6)
where g ↑↓ is the interfacial spin mixing conductance. The symbols {...} and {...} stand for real and imaginary parts, respectively. In contrast to the ferromagnetic case, the spin current pumped out of an antiferromagnetic metal decomposes into a couple of a.c. spin currents: a ferromagnetic like contribution J s m along the precession cone axis, given by the first and last terms of Eq. ( 6), and a staggered contribution J s n rotating in the (x, y) plane, given by the second term of Eq. ( 6). In the region subjected to the spin-orbit interaction, the two a.c. spin current contributions are converted into a.c. charge currents in both y and z directions according to the inverse spin Hall effect [START_REF] Wei | Spin Hall voltages from a.c. and d.c. spin currents[END_REF].

High-harmonic generation from adiabatic pumping -The time dependence of the collected current is displayed Here, an exchange coupling of J = 500meV is taken and the precession angle is set at θ = 10 • . Considering a lattice spacing of 5 Å the value of αR at αRkF/J = 1 corresponds to 3.57 eV Å, which is very close to its value in Bi/Ag alloys [START_REF] Ast | Giant Spin Splitting through Surface Alloying[END_REF] or in other bismuth based topological insulators [START_REF] Zhang | Topological insulators in bi2se3, bi2te3 and sb2te3 with a single dirac cone on the surface[END_REF].

on Fig. 3 for different Rashba strengths. We immediately identify three main regimes. For α R k F /J 1, the current response is dominated by oscillations of frequency ω [Fig. 3(a)]. When the Rashba parameter becomes comparable to the s-d exchange, α R k F /J ∼ 1, the magnitude of the a.c. current substantially increases whereas involving oscillations with higher frequencies. Upon further increasing the Rashba parameter, α R k F /J > 1, the amplitude of the signal decreases substantially with very weak components of the higher harmonics. Further numerical simulations suggest the reappearance of the highly excited harmonics regime when α R k F /J 1. However, the underlying parameter space corresponds to unrealistic spin-orbit coupling strengths.

For a more quantitative discussion, we report in Fig. 4 the Fourier transform of the charge current signals for different values of α R . For α R k F /J 1 [Fig. 4(a)], the signal exhibits only one frequency, although we do observe the appearance of the two lowest harmonics. This is consistent with the adiabatic spin pumping theory [START_REF] Brataas | Spin battery operated by ferromagnetic resonance[END_REF][START_REF] Saitoh | Conversion of spin current into charge current at room temperature: Inverse spin-Hall effect[END_REF]. Upon increasing the Rashba strength [Fig. 4(b) and 4(c)], higher harmonics progressively emerge with with amplitudes slightly below the fundamental frequencies. Notice that the amplitudes of the higher harmonics are only one order of magnitude smaller than the fundamental harmonic at frequency ω. For the sake of comparison, Ref. 28 recently reported the optical generation of higher harmonics in graphene driven by internal electron thermalization. The authors observed harmonics up to the seventh order with amplitudes four orders of magnitude smaller than that of the fundamental mode. In contrast, our simulations demonstrate that the first few harmonics remain of the order as the fundamental frequency in this regime. Upon reaching the resonance, even higher number of harmonics are generated. We emphasize that at resonance, our simulations yield signals with amplitudes increasing in time. This peculiar behavior may result from convergence issues related to the resonance condition. Nonetheless, similar features have been recently reported in correlated electron systems [START_REF] Imai | High Harmonic Generation in a Correlated Electron System[END_REF]. Therefore, we believe that this particular parameter space deserves further investigations. Above resonance though, when α R k F /J > 1 [Fig. 4(d)], the current response still display a large number of harmonics.

In order to offer a comprehensive picture of the highharmonic generation, the current output is reported on Fig. 5 as a function of the spin-orbit strength α R k F /J and the harmonic order, the amplitude of the harmonic being given by the color scale. The resonance regime corresponding to the ultrahigh-harmonic generation is centered around α R k F /J = 1 and extends from α R k F /J ≈ 0.8 to α R k F /J ≈ 1.2, indicating that the effect is robust against material's parameters variations and thereby providing a wide region of tunability. As a matter of fact, we observed that the signal bandwidth is proportional to the driving frequency (∝ 1/ω), as shown in Fig. 6(a). Further, it is worth emphasizing that the precession cone opening plays a crucial role in enhancing the amplitudes of the highest harmonics. Although Fig. 6(a) predicts an emission up to the 500th harmonic for the dynamic frequency considered in Fig. 5, only about a hundred of them exhibit strong enough amplitudes for the precession angle considered (here, θ = 10 • ). At larger precession angles all the higher harmonics can be excited with amplitudes comparable to the first harmonic intensity (see [START_REF]Supplemental Materials[END_REF]).

On the contrary, upon reducing the pumping fre- quency, one can substantially enhance the number of harmonics. This is illustrated in [START_REF]Supplemental Materials[END_REF], where the Fourier spectrum of the charge current computed for a driving frequency of 0.4 THz is shown. We notice that a similar decay of the number of the generated harmonics with respect to ω has been reported in different solid state systems under intense laser excitation [START_REF] Ghimire | High-harmonic generation from solids[END_REF][START_REF] Luu | Extreme ultraviolet highharmonic spectroscopy of solids[END_REF][START_REF] Wu | High-harmonic generation from bloch electrons in solids[END_REF].

Discussion -A crucial question we now wish to comment on is the applicability of our single-orbital model to realistic systems. As mentioned in the introduction, adiabatic spin-to-charge conversion has been demonstrated in a wide variety of heterostructures accommodating both Rashba-like spin-orbit coupling and s-d exchange. Transition metal ferromagnets interfaced with topological insulators [START_REF] Shiomi | Spin-Electricity Conversion Induced by Spin Injection into Topological Insulators[END_REF] and oxide heterostructures [START_REF] Diogo | Mapping spin-charge conversion to the band structure in a topological oxide two-dimensional electron gas[END_REF] are among the most promising structures. In these systems, Rashbalike spin-orbit coupling arises from a complex interfacial orbital hybridization scheme that is essentially overlooked in our model. Nonetheless, intense theoretical investigation on spin-orbit physics [START_REF] Manchon | Current-induced spin-orbit torques in ferromagnetic and antiferromagnetic systems[END_REF] (spin-orbit torque and spin-to-charge conversion) has established that in spite of its simplicity, modeling the interfacial spin-orbit coupling by an effective Rashba interaction is sufficient to properly describe the physics at stake and obtain reasonable orders of magnitude. Furthermore, the robustness of the effect at room temperature is of great relevance in terms of the applicability of the effect to real devices.

The present effect opens appealing perspectives for high frequency emission deep into the THz gap. It is worth emphasizing that the effect requires two essential ingredients: strong spin-orbit coupling and oscillating magnetic order or magnetic field with in-plane components. Note that recent simulations based on the present work have reproduced high harmonic generation on 1dimensional magnetic chain, although using a different configuration setup [START_REF] Varela Manjarres | High-harmonic generation in spin and charge current pumping at ferromagnetic or antiferromagnetic resonance in the presence of spin-orbit coupling[END_REF] at low s-d exchange coupling. Now we discuss the possible experimental realization of our proposal. We stress out that conventional spin-tocharge conversion heterostructures are suitable platforms to harness the effect. In fact, the very nature of Rashba spin-orbit coupling makes this perspective quite appealing because it is directly related to the interfacial potential drop and therefore highly sensitive to a gate voltage for instance. Whereas this electrical tuning of the Rashba strength has been demonstrated in several systems, oxide two-dimensional electron gases [START_REF] Diogo | Mapping spin-charge conversion to the band structure in a topological oxide two-dimensional electron gas[END_REF][START_REF] Lesne | Highly efficient and tunable spin-to-charge conversion through Rashba coupling at oxide interfaces[END_REF][START_REF] Noël | Non-volatile electric control of spin âĂŞ charge conversion in a SrTiO3 Rashba system[END_REF] stand out of the most versatile system. The recent demonstration of electrical switching of Rashba coupling [START_REF] Noël | Non-volatile electric control of spin âĂŞ charge conversion in a SrTiO3 Rashba system[END_REF] makes this perspective even more compelling. The Rashba strength can be as large as a few 100 meV, which seems reasonable as the effective s-d exchange experienced on the surface can be tuned by inserting a tunnel barrier for instance. Furthermore, a strong enhancement of spin-orbit coupling strength in graphene to 80 meV has been recently reported [START_REF] Muhammad Afzal | Observation of giant spinâĂŞorbit interaction in graphene and heavy metal heterostructures[END_REF], where the spin-orbit coupling strength can be controlled in a field effect transistor setup. This provides a variety of systems in which the effect can be observed.

FIG. 2 .

 2 FIG. 2. (Color online) Illustration of the spin pumping setup: (a) Schematics of the d.c. and a.c. spin pumping: a magnetic order precessing around its rest direction z induces a spin current composed of a d.c. component, J || s,dc (black), and an a.c. (green) component, J ⊥ s,ac , whose spin polarization is aligned on m × ∂tm. We neglect the contribution from the imaginary part of the spin mixing conductance (∼ ∂tm). Via spin-orbit coupling, these spin currents are converted into a d.c. and an a.c. charge current flowing along y and z, respectively. (b) Corresponding simulation setup: the pumped charge current is collected along the direction about which the magnetic order precesses.

FIG. 3 .

 3 FIG. 3. (Color online) Time domain current signal as a function of Rashba strength: Time dependent currents for different spin-orbit coupling strengths are displayed.Here, an exchange coupling of J = 500meV is taken and the precession angle is set at θ = 10 • . Considering a lattice spacing of 5 Å the value of αR at αRkF/J = 1 corresponds to 3.57 eV Å, which is very close to its value in Bi/Ag alloys[START_REF] Ast | Giant Spin Splitting through Surface Alloying[END_REF] or in other bismuth based topological insulators[START_REF] Zhang | Topological insulators in bi2se3, bi2te3 and sb2te3 with a single dirac cone on the surface[END_REF].

FIG. 4 .

 4 FIG. 4. (Color online) Frequency domain charge current as a function of Rashba strength: Fourier amplitudes of the charge current are shown at different spin-orbit coupling strengths in both perturbative and non perturbative regimes. The antiferromagnetic dynamics parameters are the same as in Fig. 3.

1 I

 1 FIG. 5. (Color online) Charge current amplitude of the harmonic spectrum as a function of spin-orbit coupling strength: The current amplitude (in logarithmic scale) is shown as a function of the Harmonic order as well as the Rashba strength. The frequency of the dynamics as well as the precession angle are the same as in Fig. 3.

The authors acknowledge useful discussions with X. Waintal and B. Nikolic, as well as computing resources on the supercomputer SHAHEEN granted by the KAUST Supercomputing Lab. O. L thanks A. Abbout for useful discussions. This work was supported by the King Abdullah University of Science and Technology (KAUST) through the Office of Sponsored Research (OSR) [Grant Number OSR-2020-CRG8-4048].