Three-dimensional image reconstruction of distribution of Pnmt+ cell-derived cells in murine heart

Abstract

Elucidating the function of specific cell types in a highly complex multicellular system such as the heart often requires detailed anatomic reconstruction. We recently described a distinctive class of phenylethanolamine n-methyltransferase (Pnmt⁺) cell-derived cardiomyocytes (PdCMs), a new cardiomyocyte population with a potential endocrine role. In this dataset, a 3D reconstruction was carried out to visualise the distribution of PdCMs throughout the murine heart. Rigid registration (stiff rotation and translation) was applied to properly align the fused heart slice images based on landmarks using TrakEM2, an open source plug-in in Fiji. The registered slices were then analysed and reconstructed using MATLAB (MATLAB^®. Version 8.3.0.532). The final reconstructed 3D volume was 561×866×48 pixels (corresponding to spatial resolutions of 5.8, 8.9 and 2.5 mm in the x-, y- and z-direction respectively), and visualised in Paraview. The reconstruction allows for detailed analyses of morphology, projections and cellular features of different cell types, enabling further geometrical and topological analyses. Image data can be accessed and viewed through Figshare.

Figure 1. A schematic overview of the workflow of the study.

Left panel: the processes of heart cryosectioning and immmunohistology. Middle: imaging process. Fluorescence images for detecting ChR2/tdTomato positive cells were obtained using Olympus FV1000 Confocal or Zippy Moving-stage Fluorescent microscopes. Right: diagram illustrating the procedures of the 3D reconstruction. Raw images were first manually improved and registered; an intensity-based classification was applied to obtain the positively stained cells; reconstructed 3D volume data was visualised in Paraview following Gaussian smoothing and down-sampling.

Figure 2. Representative images of the coronal section and selected regions from the section of an adult ChR2/tdTomato mouse heart showing fluorescence and morphology of the ChR2/tdTomato positive cells.

Representative images of the coronal section and selected regions from the section of an adult ChR2/tdTomato mouse heart showing fluorescence and morphology of the ChR2/tdTomato positive cells. (a) A representative coronal section from an adult ChR2/tdTomato mouse heart; (b) inserts of zoom-in views showing tdTomato fluorescence in different regions of the heart; the labelling of the inserts indicates the corresponding locations as marked in a. AVN: atrioventricular node; ASEP: atrial septum; LA: left atrium; LV: left ventricle; SAN: sinoatrial node; VSEP: ventricular septum; RA, right atrium, RV: right ventricle.

Figure 3. 3D reconstruction of the distribution of ChR2/tdTomato positive cells in different regions of the heart using customised computer programs and scripts to generate 3D representations of the ChR2/tdTomato staining patterns.

Multiple views are given.

Figure 4. Pnmt staining and tdTomato fluorescence in adrenal medulla tissue.

(i) Immunostaining of Pnmt with anti-Pnmt antibody; (ii) tdTomato fluorescence in a representative section of Pnmt^Cre/ChR2 adrenal medulla; (iii) overlay of i and ii, showing the co-localisation of Pnmt and tdTomato fluorescence; (iv) without Pnmt staining.