Introduction
The heart is composed of cardiomyocytes (CM) and non-myocytes (NM). The latter include interstitial, 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 affect the electrical activity during myocardial remodeling in response to injury. In order to study NM-CM interactions, we developed an optogenetic approach based on cell-type specific expression of the light-gated cation channel Channelrhodopsin-2 (ChR2). This allows us to not only study cell-specific contributions to overall cardiac electrophysiology, but also enables 3D reconstruction of NM in scar tissue, border zone and remote myocardium.

Methods
We used Cre-Lox tamoxifen inducible recombination to target ChR2-eYFP to NM in the mouse heart. More specifically, inducible Tcf21-Cre and Cx3cr1-Cre lines were used for ChR2 targeting to FB and cardiac resident MΦ, respectively [3]. Adult Tcf21-Cre and Cx3cr1-Cre ChR2 mice were subjected to left ventricular cryoinjury to generate a non-transmural scar. We performed electrical and optical pacing of isolated Langendorff-perfused hearts to evaluate the effect of NM depolarization on CM electrophysiology when ChR2 is activated in the scar and/or border zone. To visualize the fluorescently labelled cells in intact tissue, we optically cleared hearts using X-CLARITY and imaged them with confocal microscopy. This allowed us to reconstruct 3D models of FB and MΦ, and to assess their morphology, distribution, interconnectivity, and surface area in near-native tissue. Additionally, immunohistochemical staining of cryosections from healthy and injured mouse hearts were used to quantify the ratio of NM to CM.

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]. Resident cardiac MΦ show a somewhat similar morphology, although they are located as solitary cells, not usually contacting other MΦ. 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 containing at least 126 cells; and MΦ volume 1,358 µm³ and surface area 1,395 µm², n = 52 cells). We observed an increased number of ChR2-EYFP expressing FB and MΦ in the scar and border zone following ventricular cryoinjury, and found that FB change their morphology and distribution in the scar.  Additionally, preliminary experiments indicate that depolarization of FB and MΦ by ChR2 activation can alter electrical conduction by modulating action potential duration and restitution in the scar and scar border.

References

[1] Quinn TA et al. Proc Natl Acad Sci 2016/113(51): 14852–7 [PMID: 27930302]
[2] Hulsmans M et al. Cell 2017/169: 510-522 [PMID: 28431249]        
[3] Fernández M. C. et al. Methods Mol Biol 2021/2191:287-307. [PMID: 32865751]