1Universitäts-Herzzentrum Freiburg - Bad Krozingen Institut für Experimentelle Kardiovaskuläre Medizin Freiburg im Breisgau, Deutschland
In the heart, fibroblast (FB) and macrophages (MΦ) can be electrically coupled to cardiomyocytes (CM) via connexin 43. In addition, there is evidence that mechanisms independent of Cx-based gap junctions, such as ephatic coupling, may contribute to electrical impulse propagation between FB and CM in the native heart [1,2]. We are interested in exploring the morphology and distribution of FB and MΦ, and examining how those cells alter CM 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-eYFP) to depolarize FB or MΦ [3].
Methods
We used Tcf21-MerCreMer and Cx3cr1-CreERT2 mice to target Cre-depedent ChR2-eYFP to FB and MΦ, respectively, both in healthy and cryo-injured left ventricles (LV). We performed electrical and optical pacing of isolated Langendorff-perfused hearts to evaluate the effects of ChR2-mediated FB or MΦ depolarization on CM electrophysiology (AP shape, restitution, conduction velocity), comparing the scar, border zone and remote LV of cryo-ablated hearts to sham-operated animals hearts. 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.
Results and Conclusions
Experiments showed that depolarization of FB by ChR2 activation in cryoinjured hearts can alter cardiac conduction by modulating action potential duration and restitution in the scar and in remote myocardium, but not in hearts from sham-operated mice. Furthermore, we studied the morphology and distributions of FB and MΦ in healthy and post-injury cleared hearts. (i) In healthy hearts, FB and MΦ show very similar morphology, they have elongated shapes and thin branches but are arranged differently: FB form networks and MΦ are solitary cells. (ii) Volume and surface area of FB and MΦ show no significant difference in LV of healthy tissue (see Fig1A). (iii) In post-injury hearts, we found both FB and MΦ form complex and dense networks in the lesion. (iv) FB but not MΦ also occupy a greater fractional volume in remote tissue, compared to non-lesioned tissue (see Fig 1B). Our study highlights the electrophysiological relevance and structural complexity of FB and MΦ in cardiac scar tissue in situ. Future work will focus on FB and MΦ roles in arrhythmogenesis.
Figure 1. A) Average of volume and surface of FB and MΦ populations in LV from healthy cleared heart. B) Percentage of fractional volume that occupy FB and MΦ in LV of healthy, cryo-ablated heart, remote and scar areas.
References
[1] Wang Y et al. Science 2023/381(6665): 1480-1487 [PMID: 37769108]
[2] Simon-Chica A et al. Am J. Physiol. Heart Circ. 2023/325(3):H475-H491 [PMID:37417876]
[3] Fernandez MC et al. Methods Mol Biol 2021(2191:287-307) [PMID:32865751]