Heterocellular electrotonic coupling between myocytes and non-myocytes in cardiac remodeling

Introduction

The heart is composed of cardiomyocytes (CM) and non-myocytes (NM). The latter include fibroblasts (FB), endothelial, and immune cells. FB and macrophages (MΦ) are electrically coupled 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 FB and MΦ microarchitecture in scar tissue, border zone and remote myocardium.

Methods

We used Tcf21-MerCreMer and Cx3cr1-CreERT2 mice to target ChR2-eYFP to FB and MΦ, respectively. We studied hearts 28 days after left ventricular (LV) cryo-ablation or sham surgery. Electrical and optical stimulation of isolated Langendorff-perfused hearts were used to evaluate the effects of ChR2-mediated FB or MΦ depolarization on CM electrophysiology, action potential (AP) shape, restitution curve, conduction velocity (CV), comparing the scar, border zone and remote LV of cryo-ablated hearts to sham-operated hearts and cryo-ablated hearts not expressing ChR2. 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, surface area and fractional volume in near-native tissue in healthy, sham and cryo-ablated hearts.

Results and Conclusions

The main findings are: (i) FB depolarization by ChR2 activation in cryo-ablated hearts prolong CM APD in remote, border and scar area. (ii) Illumination of sham-operated and cryo-ablated hearts not expressing ChR2 did not show any effect on APD. (iii) In contrast to FB, ChR2-mediated depolarization of MФ did not affect ventricular APD. Additionally, we studied the morphology and distributions of FB and MΦ in healthy and post-injury cleared hearts, finding that: (iv) FB in healthy hearts show elongated shapes and thin branches, and form interconnected networks that follow the orientation of CM. (v) MΦ show a very similar morphology compared to FB, but are arranged as solitary cells. (vi) In cryo-ablated hearts, both FB and MΦ are enriched in the scar and scar border zone, whereas only FB are enriched in remote LV myocardium. Accordingly, the fractional volumes occupied by FB and MΦ in healthy ventricles were 2.5±0.3%, n = 10 and, 1.6±0.2%, respectively, n = 8 (mean±SEM, n= analyzed regions). In cryo-ablated hearts, FB and MΦ occupy a volume of 12.5±1.1%, n= 12 and 6.9±0.5%, n = 5 in the scar, and 7.8±0.3%, n = 16 and 2.0±0.1%, n = 5 in remote tissue from the same hearts. Our study show the electrophysiological relevance and structural characteristics of FB and MΦ in situ in post-injury hearts. Future work will be focused on FB and MΦ roles in arrhythmogenesis using optogenetic tools.

References
[1] Fernández MC et al. Channelrhodopsins for Cell-Type Specific Illumination of Cardiac Electrophysiology. Methods Mol Biol 2021(2191:287-307) [PMID:32865751]
[2] Simón-Chica et al. Novel insights into the electrophysiology of murine cardiac macrophages: relevance of voltage-gated potassium channels. Cardiovasc Res 2022(118):798–813 [PMID: 33823533]