Electrotonic loading of anisotropic cardiac monolayers by unexcitable cells depends on connexin type and expression level

Abstract

Understanding how electrotonic loading of cardiomyocytes by unexcitable cells alters cardiac impulse conduction may be highly relevant to fibrotic heart disease. In this study, we optically mapped electrical propagation in confluent, aligned neonatal rat cardiac monolayers electrotonically loaded with cardiac fibroblasts, control human embryonic kidney (HEK-293) cells, or HEK-293 cells genetically engineered to overexpress the gap junction proteins connexin-43 or connexin-45. Gap junction expression and function were assessed by immunostaining, immunoblotting, and fluorescence recovery after photobleaching and were correlated with the optically mapped propagation of action potentials. We found that neonatal rat ventricular fibroblasts negative for the myofibroblast marker smooth muscle alpha-actin expressed connexin-45 rather than connexin-43 or connexin-40, weakly coupled to cardiomyocytes, and, without significant depolarization of cardiac resting potential, slowed cardiac conduction to 75% of control only at high (>60%) coverage densities, similar to loading effects found from HEK-293 cells expressing similar levels of connexin-45. In contrast, HEK-293 cells with connexin-43 expression similar to that of cardiomyocytes significantly decreased cardiac conduction velocity and maximum capture rate to as low as 22% and 25% of control values, respectively, while increasing cardiac action potential duration to 212% of control and cardiac resting potential from -71.6 +/- 4.9 mV in controls to -65.0 +/- 3.8 mV. For all unexcitable cell types and coverage densities, velocity anisotropy ratio remained unchanged. Despite the induced conduction slowing, none of the loading cell types increased the proportion of spontaneously active monolayers. These results signify connexin isoform and expression level as important contributors to potential electrical interactions between unexcitable cells and myocytes in cardiac tissue.

Optical mapping of cryoinjured rat myocardium grafted with mesenchymal stem cells

“…Moreover, it has been shown that MSCs are able to functionally couple with other cells (21,32). Studies from our group (5,6) and others (12,15,23) have shown the importance of connexin expression in engrafted cells. This may explain the electrotonic influence we observed.…”

Abolishing Myofibroblast Arrhythmogeneicity by Pharmacological Ablation of α-Smooth Muscle Actin Containing Stress Fibers

“…24,25 Given the possibility to pharmacologically ablate ␣-SMA-SFs and, as a result, backtransform MFBs to fibroblasts, we investigated to which extent this intervention might affect MFB-dependent slow conduction and ectopic activity. Irrespective of the type of actin-targeting drug used, ablation of ␣-SMA-SFs consistently led to complete suppression of MFB arrhythmogeneicity.…”

“…This conclusion is supported by a recent study in which cultured cardiac fibroblasts that did not express ␣-SMA-SFs showed only little adverse effects on the electrophysiology of cocultured cardiomyocytes. 25 …”

The Effects of Fibroblasts on Wave Dynamics in a Mathematical Model for Human Ventricular Tissue

“…Zlochiver, et al [15] have shown the expression of Cx43 between fibroblasts and myocytes in a monolayer of myocytes and fibroblasts of neonatal rats; Miragoli, et al [16] have reported that Cx43 and Cx45 are expressed among fibroblasts and between fibroblasts and myocytes in cultured fibroblasts coated over rat-ventricular-myocyte strands. Both experimental and computational studies have shown that such coupling between myocytes and fibroblasts enhances electrical-signal propagation in cardiac tissue [7,[15][16][17][18]; this enhancement increases with N f , the number of fibroblasts that are attached to a myocyte. In cell-culture experiments, Miragoli, et al [16] have found that the conduction velocity (CV) decreases with an increase in the density of fibroblasts in cultured strands of neonatal-rat myocytes coated by fibroblasts; studies by McSpadden, et al [17] have found that CV decreases as the fibroblast number increases on the top of a myocyte layer in a monolayer of neonatal-rat cardiac myocytes, which are electrotonically loaded with a layer of cardiac fibroblasts.…”

“…Both experimental and computational studies have shown that such coupling between myocytes and fibroblasts enhances electrical-signal propagation in cardiac tissue [7,[15][16][17][18]; this enhancement increases with N f , the number of fibroblasts that are attached to a myocyte. In cell-culture experiments, Miragoli, et al [16] have found that the conduction velocity (CV) decreases with an increase in the density of fibroblasts in cultured strands of neonatal-rat myocytes coated by fibroblasts; studies by McSpadden, et al [17] have found that CV decreases as the fibroblast number increases on the top of a myocyte layer in a monolayer of neonatal-rat cardiac myocytes, which are electrotonically loaded with a layer of cardiac fibroblasts. Zlochiver, et al [15] have shown that CV decreases as (i) the gapjunctional conductance increases or (ii) the fibroblasts density increases in their experiments with fibroblasts of neonatal rats; they have also obtained similar result in their computational studies in a two-dimensional (2D) sheet of myocyte tissue in the dynamic Luo-Rudy (LRd) model [19,20] by inserting fibroblasts.…”