3D structure of fibroblasts and macrophages in the healthy and cryo-ablated heart

IntroductionCardiac non-myocytes (NM) play important roles in heart development, homeostasis, and lesion repair. To assess the relevance of different NM populations for cardiac (patho)physiology, a quantitative assessment of their abundance and structure in the different heart chambers is an essential prerequisite. We here present an experimental approach to determine the distribution, dimensions, and 3D morphology of fibroblasts (FB) and macrophages (M{Phi}) in healthy and pathologically remodelled hearts. Methods and resultsWe used Cre-loxP recombination to selectively target channelrhopsin-2 (ChR2)-eYFP to either FB or M{Phi} in healthy and cryo-ablated mouse hearts. Hearts were optically cleared using X-CLARITY and membrane-bound eYFP fluorescence was recorded by confocal microscopy. The resulting image stacks were segmented to generate 3D reconstructions of labelled cell populations in near native tissue. In doing so, we show that FB and M{Phi} have similar surface areas, volumes and morphologies, but that FB occupy larger fractional volumes than M{Phi} in all chambers of healthy murine hearts. Furthermore, M{Phi} appear primarily as single cells, whereas FB form extended networks of interconnected cells. In left-ventricular tissue following cryo-ablation, we observed large disordered networks of FB in the scar area with an increased volume occupied by FB both in the scar and remotely. In cryo-ablated ventricles, M{Phi} form comparatively small, but dense networks in the scar without changing their abundance in remote myocardium. ConclusionsOur study assesses the 3D distribution and structure of fluorescently labelled FB and M{Phi} in healthy and lesioned murine hearts. Based on 3D reconstructions of FB and M{Phi} networks, we quantified the surface areas and volumes of individual non-myocytes in the different chambers of the heart and in ventricular scar tissue, thus providing important quantitative data serving as basis for computational modelling of non-myocyte contributions to cardiac structure and physiology.