INTRODUCTION: The cardiac autonomic nervous system regulates the heart intrinsic activity (e.g., heart rate), and its dysregulation is involved in a number of cardiac arrhythmias. Dysfunction of parasympathetic neurons has been associated with atrial fibrillation, the most common form of cardiac arrhythmias (2.5% of English population [1]). Unfortunately, the interaction between parasympathetic neurons and atrial cardiomyocytes is particularly difficult to study and, to our knowledge, there are currently no human in vitro models.
AIMS: Our project aims to develop a human in vitro model of the interaction between parasympathetic neurons and atrial cardiomyocytes. It will allow us to investigate the cross-talk between these cells, to manipulate them to study atrial fibrillation (e.g. through genetic manipulation) or to carry out drug screening work.
METHODS: We have generated atrial cardiomyocytes and parasympathetic neurons from induced pluripotent stem cells. We followed published protocols for atrial cardiomyocytes [2] and parasympathetic (vagal) neurons [3]. With a combination of immunostaining and functional recordings (e.g. Ca2+ imaging and patch clamp recordings) we characterised the neurons confirming their function and lineage. Using qPCR and immunostaining we compared the expression of atrial and ventricular specific markers between atrial iPSC-derived cardiomyocytes and standard iPSC-derived cardiomyocytes (Burridge protocol [4]). We co-cultured atrial cardiomyocytes and parasympathetic neurons for 5-7 days and assessed the modulation of cardiomyocyte beating rates by the activation of the neurons with nicotine. We also tested whether the effect could be reversed by a neuronal blocker (a selective Nav1.8 antagonist).
RESULTS: Our data confirm the parasympathetic nature of the iPSC-derived neurons, as they express the autonomic lineage marker Phox2b and the cholinergic marker ChAT. They are also responsive to nicotine, as expected. Atrial iPSC-derived cardiomyocytes present a reduction in ventricular specific markers (e.g. MYL2, IRX4 assessed with qPCR) and increased expression of atrial markers with immunostaining (e.g. MYL7).
When we cocultured the two cell types for 7 days, nicotine (20µM) resulted in a significant reduction of cardiomyocyte beating rate in co-cultures compared to mono-cultures (mono-culture: 111.2±2.729; co-culture: 65.42±6.931; p=0.002; Mann-Whitney test; n= 6 two differentiations of the parasympathetic neurons). Interestingly, the activity-dependent Nav1.8 blocker A803467 (100nM) increased the beating rate in co-culture but not in mono-culture (mono-culture=96.28±0.7658; co-culture=130.8±11.09; p=0.0043; Mann-Whitney test; n=6; two differentiations of the parasympathetic neurons) reversing, to some extent, the effect of nicotine.
CONCLUSIONS: Our preliminary data show a potential to reproduce in vitro some key physiological features of parasympathetic neurons-cardiomyocyte interactions. In future studies, we aim to extend our characterisation and employ our system for disease modelling.