The Role of Membrane Capacitance in Cardiac Impulse Conduction: An Optogenetic Study With Non-excitable Cells Coupled to Cardiomyocytes

Abstract

Non-excitable cells (NECs) such as cardiac myofibroblasts that are electrotonically coupled to cardiomyocytes affect conduction velocity (θ) by representing a capacitive load (CL: increased membrane to be charged) and a resistive load (RL: partial depolarization of coupled cardiomyocytes). In this study, we untangled the relative contributions of both loading modalities to NEC-dependent arrhythmogenic conduction slowing. Discrimination between CL and RL was achieved by reversibly removing the RL component by light activation of the halorhodopsin-based hyperpolarizing membrane voltage actuator eNpHR3.0-eYFP (enhanced yellow fluorescent protein) expressed in communication-competent fibroblast-like NIH3T3 cells (3T3 HR cells) that served as a model of coupled NECs. Experiments were conducted with strands of neonatal rat ventricular cardiomyocytes coated at increasing densities with 3T3 HR cells. Impulse conduction along preparations stimulated at 2.5 Hz was assessed with multielectrode arrays. The relative density of 3T3 HR cells was determined by dividing the area showing eYFP fluorescence by the area covered with cardiomyocytes [coverage factor (CF)]. Compared to cardiomyocytes, 3T3 HR cells exhibited a depolarized membrane potential (−34 mV) that was shifted to −104 mV during activation of halorhodopsin. Without illumination, 3T3 HR cells slowed θ along the preparations from ∼330 mm/s (control cardiomyocyte strands) to ∼100 mm/s (CF = ∼0.6). Illumination of the preparation increased the electrogram amplitudes and induced partial recovery of θ at CF > 0.3. Computer simulations demonstrated that the θ deficit observed during illumination was attributable in full to the CL represented by coupled 3T3 HR cells with θ showing a power-law relationship to capacitance with an exponent of −0.78 (simulations) and −0.99 (experiments). The relative contribution of CL and RL to conduction slowing changed as a function of CF with CL dominating at CF ≤ ∼0.3, both mechanisms being equally important at CF = ∼0.5, and RL dominating over CL at CF > 0.5.

Generation, functional analysis and applications of isogenic three-dimensional self-aggregating cardiac microtissues from human pluripotent stem cells

“…For example, we demonstrated that modelling Arrhythmogenic Cardiomyopathy (ACM) caused by a mutation in plakophilin 2, a protein of the desmosome, only required mutated CFs for arrhythmias to appear in paced cardiac MTs, indicating CFs and not just the CMs contribute to the disease 11 . Individual CMs and CFs are known to couple and communicate through homo-and heterocellular gap junctions to control electrical activity 58 and conduction in the heart 59 . This is also evident in the cardiac MT model.…”

Advancing mechanobiology by performing whole-cell patch clamp recording on mechanosensitive cells subjected simultaneously to dynamic stretch events

“…Proof-of-concept recordings with NIH3T3 cells showed robust strain-induced inward currents when cells were clamped to their resting membrane potential ( Chen et al., 1988 ; De Simone et al., 2020 ). At ε ≥ 6%, MA currents increased with increasing strain amplitudes with some signals showing a sigmoidal dependence on strain, whereas others failed to saturate at the maximal strain levels provided by the LSSE system.…”

Myofibroblasts impair myocardial impulse propagation by heterocellular connexin43 gap-junctional coupling through micropores

“…Besides the resting membrane-potential effects by MFs, the relatively large size of MFs compared with that of CMs could also potentially contribute to the conduction slowing in the CM-MF model by acting as a large electrical sink; CMs of smaller capacitance than MF can be charged by coupled MFs having large capacitance (De Simone et al, 2020), resulting in a relative reduction of the depolarizing current for firing of CMs (Miragoli, et al, 2006), and thereby reducing the action potential upstroke and conduction velocity. Notably, we observed that seeding of MFs at 3 × 10 6 cells on the reverse side, nearly identical cell number to that of the upper CMs, failed to evoke excitatory Ca 2+ transients by electrical stimulation in all 7 preparations (obtained from 3 cases of isolation) tested, consistent with the MF-load-dependent conduction block (Xie et al, 2009).…”

“…This observation suggests marked resting membrane depolarization of the CMs. Thus, in addition to the electrotonic effects, the capacitive loading of MFs on CMs (De Simone et al, 2020) would also contribute to the slowing of impulse propagation in our CM-MF model.…”