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 _<i>HR</i> 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 _<i>HR</i> cells. Impulse conduction along preparations stimulated at 2.5 Hz was assessed with multielectrode arrays. The relative density of 3T3 _<i>HR</i> cells was determined by dividing the area showing eYFP fluorescence by the area covered with cardiomyocytes [coverage factor (CF)]. Compared to cardiomyocytes, 3T3 _<i>HR</i> cells exhibited a depolarized membrane potential (-34 mV) that was shifted to -104 mV during activation of halorhodopsin. Without illumination, 3T3 _<i>HR</i> 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 _<i>HR</i> 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. The finding that RL did not affect θ at CFs ≤ 0.3 is explained by the circumstance that, at the respective moderate levels of cardiomyocyte depolarization, supernormal conduction stabilized propagation. The findings provide experimental estimates for the dependence of θ on membrane capacitance in general and suggest that the myocardium can absorb moderate numbers of electrotonically coupled NECs without showing substantial alterations of θ.