New Methods and Insights for 3D Imaging and Isolation of Cardiac Cells

Introduction: The heart is criss-crossed with autonomic nerve fibres that regulate cardiac function on a beat-to-beat basis. Cardiovascular diseases cause damage to nerve fibres, leading to neuronal re-structuring of the heart with severe complications like ventricular tachycardia and fibrillation. The cells that make up and support these nerve fibres, which include neurons and their axons, glial cells and endothelial cells, play critical roles in cardiac function and response to injury. However, little is known about these various cell types in the heart and how they respond to heart disease, due to the lack of adequate methods to study them. Therefore, our study aimed to establish robust protocols to enable the 3D visualisation and isolation of these cardiac nerve fibre associated cell-types.

Methods and Results: To enable immunofluorescent labelling of these cardiac cells in 3D, we tested the efficiency of multiple clearing methods, a variety of antibodies, and several tissue clearing workflows. The resulting protocol is based on aqueous solutions that enabled us to make the first 3D reconstruction of several cardiac nerve fibre associated cell-types in a mouse heart using light sheet fluorescence microscopy, comprising between 100 and 200 thousand nerve-fibre associated cells in total. We also demonstrated that these cell types can be effectively fluorescently labelled in human heart tissues using an extended protocol.

In order to establish a process to isolate fluorescently labelled cardiac cells, we tested several dissociation reagents and protocols, and sifted cells using fluorescence-activated cell sorting. Combined, our methods demonstrated that the number and vitality of these cells in murine hearts decrease after induction of a myocardial infarction.

Conclusion: In our study, we have established key methods to reliably study several cardiac nerve fibre associated cell-types, which revealed their abundance in the heart and changes after heart disease. The protocols and data serve as a foundation for follow-up studies studying these cardiac cells as novel therapeutic targets to protect neuronal function and decrease pathological remodelling of the heart.