Introduction                                                                                                                                         

The heart is composed of cardiomyocytes (CM) and non-myocytes (NM). The latter include fibroblasts, endothelial, and immune cells. Cardiac fibroblasts (FB) and macrophages (MΦ) have recently been shown to electrically couple to CM in the native heart [1,2]. We are interested in how FB and MΦ electrotonically couple to myocytes and may alter electrical activity during myocardial remodeling in response to disease and injury.

Methods

We used Cre-Lox tamoxifen inducible recombination to selectively express the light-gated cation channel Channelrhodopsin-2 (ChR2) or fluorescent reporter proteins in NM population in the mouse heart. Periostin and Tcf21 were used as drivers for FB and Cx3cr1 was used as driver for cardiac resident MΦ [3].  Adult Tcf21-, Periostin- and Cx3cr1-ChR2 mice were subjected to left ventricular cryoinjury to generate a non-transmural scar. We optically cleared hearts using X-CLARITY and imaged them with deconvolution-based super-resolution confocal microscopy. This allowed us to reconstruct 3D models of FB and MΦ and assess their morphology; distribution, interconnectivity, and surface area in near-native tissue. Additionally, immunohistochemical staining of cryosections of scar and remote areas from mouse hearts were used to quantify the ratio of NM to CM in injured heart.

Results and Conclusions

In healthy cleared hearts, we found that FB have elongated shapes and thin branches which form interconnected networks that appear to wrap around CM with finger-like nano-protrusions similar to tunneling nanotubes seen with electron microscopy in post-infarct murine myocardium [1]. Surprisingly, resident cardiac MΦ show a similar morphology, although they are located as solitary cells. Volume and surface area of FB and MΦ show no significant differences (FB volume from networks 1,094 µm³, surface area 1,140 µm², n = 10 networks; and MΦ volume 1,358 µm³ and surface area 1,395 µm² n = 52 cells).  In on-going experiments, we utilize ChR2 in combination with our Cre driver lines to study the functional relevance of FB and MΦ for cardiac electrophysiology in mouse, with a focus on heterocellular electrotonic coupling in left-ventricular scar following cryoinjury hearts.