Generation of innervated engineered human myocardium (iEHM) from human induced pluripotent stem cells to elucidate the mechanism underlying neuro-cardiac pathologies

by Lennart Valentin Schneider
Doctoral thesis
Date of Examination:2024-01-22
Date of issue:2024-11-12
Advisor:Dr. Maria-Patapia Zafeiriou
Referee:Dr. Maria-Patapia Zafeiriou
Referee:Prof. Dr. André Fischer
Referee:Prof. Dr. Kao Mei Guan
Persistent Address: http://dx.doi.org/10.53846/goediss-10843

 

 

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Abstract

English

Brain and heart consist of networks of excitable cells, the function of which is orchestrated by a complex interplay of ionic currents. Mutations in ion channels can predispose patients to cardiocerebral channelopathies that manifest as epilepsy in the brain and conduction disorders in the heart. These patients have an elevated risk for sudden unexpected death in epilepsy (SUDEP). Although the underlying mechanism is still incompletely understood, increasing evidence suggests a contribution of the ANS to the development of lethal arrhythmias. In this study, we generated a human induced pluripotent stem cell (hiPSC)-derived organoid to model the interaction of ANS and heart in the physiological and diseased states. This model was based on the sympathetic neuronal organoid (SNO) we recently developed, which contained functional sympathetic neurons (SNs) and physiological levels of noradrenaline (NE). We fused the SNO with engineered human myocardium (EHM) to generate innervated engineered human myocardium (iEHM). SNs densely interlaced with the myocardium within weeks and formed characteristic synaptic varicosities in close proximity to cardiomyocytes (CMs). An extensive vascular network co-developed in the iEHM, which featured basement membrane components and pericytes. SN orientational analysis revealed potential guidance through neural growth factor (NGF) expressing pericytes, similar to the developing heart. Optogenetic and pharmacological stimulation of SNs in iEHM proved functionality of the neuro-cardiac junctions (NCJs) in iEHM. Finally, we employed the iEHM to investigate a channelopathy induced by mutations in hERG, an ion channel expressed in brain and heart. To assess the involvement of the ANS in the development of arrhythmias, we used two hERG loss-of-function mutant lines to generate iEHM. Due to the iEHM’s modular construction, we were able to characterize the cardiac and neural modules independently. Therefore, we assessed the long QT syndrome (LQTS) phenotype in the EHM and CMs and found a significant action potential (AP) prolongation for both mutations. LQTS-EHM exhibited a higher frequency of afterdepolarizations upon -adrenergic stimulation, which was normalized by anti-arrhythmic treatment. Next, we assessed the effect of hERG loss-of-function on autonomic neurons using SNOs. The mutant SNOs demonstrated increased overall firing rates and synchrony. To investigate the impact of SN dysregulation on LQTS-compromised and healthy myocardium, we fused mutant SNOs with mutant and wild-type (WT) EHM (hybrids). Mutant iEHM showed higher beat-to-beat variability upon sympathetic stimulation compared to WT. Hybrid iEHM of one of the mutant lines displayed irregular beating patterns upon SN stimulation. Our data suggests a pro-arrhythmic effect of dysregulated SN activity on the myocardium, which was more pronounced in tissue with underlying LQTS. In conclusion, we present a novel human model of ANS and heart interaction, which features a cellular composition and functionality reminiscent of the neuro-cardiac interface in human. We could demonstrate that the iEHM is suitable for disease modeling of neuro-cardiac diseases and represents a promising tool for pharmacological screenings in the future.
Keywords: Sympathetic neurons; Autonomic nervous system; Organoids; Neuro-cardiac interaction; Induced pluripotent stem cells; SUDEP; Cardiac arrhythmia
 
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