Human in vitro neurocardiac coculture ( iv NCC ) assay development for evaluating cardiac contractility modulation

Abstract

Two of the most prominent organ systems, the nervous and the cardiovascular systems, are intricately connected to maintain homeostasis in mammals. Recent years have shown tremendous efforts toward therapeutic modulation of cardiac contractility and electrophysiology by electrical stimulation. Neuronal innervation and cardiac ganglia regulation are often overlooked when developing in vitro models for cardiac devices, but it is likely that peripheral nervous system plays a role in the clinical effects. We developed an in vitro neurocardiac coculture ( iv NCC) model system to study cardiac and neuronal interplay using human induced pluripotent stem cell (hiPSC) technology. We demonstrated significant expression and colocalization of cardiac markers including troponin, α‐actinin, and neuronal marker peripherin in neurocardiac coculture. To assess functional coupling between the cardiomyocytes and neurons, we evaluated nicotine‐induced β‐adrenergic norepinephrine effect and found beat rate was significantly increased in iv NCC as compared to monoculture alone. The developed platform was used as a nonclinical model for the assessment of cardiac medical devices that deliver nonexcitatory electrical pulses to the heart during the absolute refractory period of the cardiac cycle, that is, cardiac contractility modulation (CCM) therapy. Robust coculture response was observed at 14 V/cm (5 V, 64 mA), monophasic, 2 ms pulse duration for pacing and 20 V/cm (7 V, 90 mA) phase amplitude, biphasic, 5.14 ms pulse duration for CCM. We observed that the CCM effect and kinetics were more pronounced in coculture as compared to cardiac monoculture, supporting a hypothesis that some part of CCM mechanism of action can be attributed to peripheral nervous system stimulation. This study provides novel characterization of CCM effects on hiPSC‐derived neurocardiac cocultures. This innervated human heart model can be further extended to investigate arrhythmic mechanisms, neurocardiac safety, and toxicity post‐chronic exposure to materials, drugs, and medical devices. We present data on acute CCM electrical stimulation effects on a functional and optimized coculture using commercially available hiPSC‐derived cardiomyocytes and neurons. Moreover, this study provides an in vitro human heart model to evaluate neuronal innervation and cardiac ganglia regulation of contractility by applying CCM pulse parameters that closely resemble clinical setting. This iv NCC platform provides a potential tool for investigating aspects of cardiac and neurological device safety and performance.

Human in vitro assay for irreversible electroporation cardiac ablation

“…These approach has been validated in multi-site studies ( Blinova et al, 2018 ) including chronic studies ( Narkar et al, 2022a ), and the best practice recommendations for use of these methods in drug safety assessment has been published ( Gintant et al, 2020 ). More recently, hiPSC-CMs have been proposed as a valid alternative to animal models in the evaluation of cardiac electrophysiology medical devices ( Casciola et al, 2020 ; Feaster et al, 2021 ; Casciola et al, 2022 ; Feaster et al, 2022 ; Narkar et al, 2022b ). Here, we establish a novel, species-relevant, standard laboratory protocol to evaluate and optimize PEF cardiac ablation parameters using hiPSC-CMs in a high-throughput monolayer format.…”

Nonclinical evaluation of chronic cardiac contractility modulation on 3D human engineered cardiac tissues

“…Cryopreserved hiPSC-CMs (iCell Cardiomyocytes 2 01434, R1017 Fujifilm Cellular Dynamic, Inc.) were thawed and plated, as previously described according to the manufacturer's instructions. 7,10,11,[17][18][19][20] HiPSC-CMs used in this study were derived from a hiPSC line, which was reprogrammed from fibroblast donor tissue, isolated from an apparently healthy normal Caucasian female, <18 years old. 10,17 ECT were generated with modifications to a previously described protocol.…”

“…For both groups, control group and CCM group, electrical pulses were delivered through the carbon electrodes resulting in field stimulation as previously described. 7,[10][11][12]26 ECTs were field stimulated at ∼3 times the capture threshold using monophasic square wave pulses at 1 Hz, 5 ms pulse duration, and ∼20 V/cm amplitude (Figure 1D). CCM was delivered at 1 Hz as 2 biphasic pulses, 5.14 ms phase duration (20.56 ms total duration), 28 V/cm pulse amplitude (phaseamplitude), and an interphase interval of zero.…”

“…2,26,36,37 We have previously shown in 2D hiPSC-CM models that acute CCM effects are likely induced by a mixed mechanism, including sympathetic stimulation through neuronal ganglion and β-adrenergic signaling pathways. 7,10,11 Pharmacological inotropes that act via β-adrenergic receptors, such as isoproterenol, exert their effects by increasing the amount of intracellular calcium and cAMP-dependent pathways resulting in positive inotropy, lusitropy, and chronotropy. Following chronic CCM, ECTs displayed positive inotropy including a twofold (p = .0151) increase in the contraction-time interval (AUC) (Supporting Information:…”

“…9 Toward that end, we have shown that hiPSC-CM models are sufficient to evaluate various cardiac electrophysiology device signals in vitro, including novel cardiac ablation modalities and acute CCM parameters. 7,[10][11][12][13][14] Human 3D cardiac microphysiological systems, including hiPSC-CM engineered cardiac tissues (ECTs), cardiac microtissues or cardiac microbundles, are established models for the acute evaluation of cardiac electrophysiology medical device signals and cardiac drugs. 12,15 However, such models may not be appropriate for long-term chronic in vitro studies.…”