Abstract
Intercellular electrical coupling is an essential means of communication between cells. It is important to obtain quantitative knowledge of such coupling between cardiomyocytes and non-excitable cells when, for example, pathological electrical coupling between myofibroblasts and cardiomyocytes yields increased arrhythmia risk or during the integration of donor (e.g., cardiac progenitor) cells with native cardiomyocytes in cell-therapy approaches. Currently, there is no direct method for assessing heterocellular coupling within multicellular tissue. Here we demonstrate experimentally and computationally a new contactless assay for electrical coupling, OptoGap, based on selective illumination of inexcitable cells that express optogenetic actuators and optical sensing of the response of coupled excitable cells (e.g., cardiomyocytes) that are light-insensitive. Cell–cell coupling is quantified by the energy required to elicit an action potential via junctional current from the light-stimulated cell(s). The proposed technique is experimentally validated against the standard indirect approach, GapFRAP, using light-sensitive cardiac fibroblasts and non-transformed cardiomyocytes in a two-dimensional setting. Its potential applicability to the complex three-dimensional setting of the native heart is corroborated by computational modelling and proper calibration. Lastly, the sensitivity of OptoGap to intrinsic cell-scale excitability is robustly characterized via computational analysis.
Highlights
Intercellular electrical coupling is an essential means of communication between cells
The gap junctional resistance is proportional to the imposed voltage clamp, divided by the measured compensatory current. (b) Using low-molecular weight fluorescent dyes (1), GapFRAP infers coupling from the recovery of fluoresce in a target cell after it is subjected to photobleaching (2,3); the gap-junctional resistance is directly proportional to the time constant of recovery due to dye diffusion from neighbouring cells (4)
The method is applicable to 2D multicellular settings. (c,d) Optogenetic methods offer new ways for assessing heterocellular coupling in the native tissue setting. (c) In the “optogenetic-sensor” variant, coupling is typically confirmed by measuring membrane potential fluctuations in nCMs ( VnCM), expressing genetically-encoded voltage indicators (GEVIs)/Genetically-encoded calcium indicators (GECI) indicator and connected to CMs undergoing excitation
Summary
No direct method exists for quantification of coupling in multicellular tissue. For the baseline configuration discussed in the prior section (i.e., ChR2-cFB donor cell; Fig. 5a), we gradually decreased the excitability of the coupled host cell (normal human ventricular myocyte) by simulating progressive blockade of the fast sodium channel ( INa) This modification did not alter the shape of the E e,th vs Ggj relationship (i.e., weaker coupling led to higher thresholds for optogenetic stimulation) but markedly improved the assay sensitivity. It is important to note that, in some cases, our simulations predict that changing donor cell excitability can degrade assay sensitivity or abolish the monotonic relationship that is essential to proper interpretability of OptoGap. The most striking examples are for cases with 20 and 30% I Na in donor cells (green and blue lines in Fig. 5c,d, respectively), for which σ values are reduced and the E e,th vs Ggj relationship becomes U-shaped, making it impossible to use optogenetic stimulus strength as a measure of intercellular coupling. Care must be taken to ensure the relationship between E e,th vs. G gj is properly interpreted
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