The heart is composed of cardiomyocytes (CM) and non-myocytes (NM), the latter including stromal, endothelial, and immune cells. NM have long been suspected to electrically couple to CM in native myocardium [1]. However, only with the advent of optogenetics – a method to observe and manipulate cell-type specific electrophysiology with light – heterocellular interactions could be directly assessed in intact tissue [2,3]. Using optogenetic experiments electrotonic coupling was functionally confirmed for cardiac fibroblasts (FB) and macrophages (MΦ), and we are thus interested in exploring how FB and MΦ affect the electrical and mechanical activity during myocardial remodeling in response to heart disease and injury. Methods: We used Cre-loxP recombination to selectively express fluorescent reporter proteins and optogenetic actuators in NM populations of murine hearts [4]. We optically cleared hearts using X-CLARITY and imaged them with super-resolution confocal microscopy to visualize fluorescently labelled NM populations. This allowed us to reconstruct 3D models of FB and MΦ in situ, and to assess their morphology, distribution, interconnectivity, and surface area. We  characterized the electrophysiological properties of resident cardiac MΦ, using RNA sequencing, single-cell patch-clamp recordings and pharmacological interventions, showing functional expression of Cx43 and different voltage-gated K+ channels. Based on our structural and functional data, we developed a computational model describing cardiac MΦ electrophysiology, which we then used to quantitatively assess CM-MΦ coupling in silico. In on-going experiments, we utilize the light-gated cation channel channel­rhodopsin-2 and the voltage-sensitive fluorescent protein VSFP2.3 to study heterocellular coupling between CM, FB and MΦ, with a focus on NM effects on cardiac activity in developing scars following cardiac injury (ischemia-reperfusion injury and cryoablation). Results: In healthy cleared ventricles, we found that FB networks consist of elongated, thin strands of interconnected cells, which appear to wrap around CM with finger-like nano-protrusions that may be related to tunneling nanotubes – as seen with electron microscopy in post-cryoinjury murine myocardium [2]. In the atria, on the other hand, FB display sheet-like morphologies. Quantitatively, the volume and surface area of 3D reconstructed FB does not differ statistically between atrial and ventricular walls (average surface area in right atrial and left ventricular myocardium are 2,130±280 μm2 [n=276 nuclei, N=3 hearts] and 1,770±190 µm2 [n=126 nuclei, N=3], respectively). Unlike FB, resident cardiac MΦ appear as solitary cells in intact ventricular myocardium with an average surface area of 1,160±80 µm2 (n=35 cells, N=3). In isolated and cultured cells, we found that passive electrophysiological properties of MΦ such as capacitance and membrane resistance scale with surface area (inverse relation for resistance). In silico models of CM-MΦ coupling illustrate that those parameters are directly linked to the electrical load of MΦ on coupled CM [5]. Conclusions: FB and tissue-resident MΦ exhibit surprisingly similar cell dimensions in intact tissue, although they differ in their distribution and electrophysiology. Upon injury, these cell populations undergo changes (local proliferation, recruitment, activation) leading to larger NM numbers in the scar and scar border zone, increasing the likelihood of electrotonic coupling. The functional effects of this are a matter of ongoing research.