Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA037

Poster Communications

Chronic oxidative stress-mediated remodeling of cardiac gap junctions in an in vitro model for arrhythmogenic cardiomyopathies

C. Wahl1, M. Hecker1, N. Ullrich1

1. Institute of Physiology and Pathophysiology, Division of Cardiovascular Physiology, Heidelberg University, Heidelberg, Germany.

Age-related arrhythmogenic cardiomyopathies (ACs), such as atrial fibrillation, are a group of heart diseases characterized by rapid and irregular beating of the heart. Ageing and systemic diseases including diabetes and hypertension are major risk factors for this disease complex. At the molecular level, ACs are associated with elevated levels of reactive oxygen species (ROS) and an altered protein expression pattern at the intercalated disk, predominantly gap junctions built from connexin-43 (Cx43). So far, a causal link between chronic oxidative stress and altered Cx43 expression has not yet been identified. Here, we focus on the characterization of the subcellular signaling pathways that link rapid pacing and enhanced ROS production with gap junction remodeling. We tested the hypothesis that prolonged oxidative stress influences the expression of Cx43 by modifying the activity of a specific microRNA, miR-1, that modulates translation of Cx43 mRNA. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were used as model to investigate the effect of tachypacing, an established experimental technique mimicking the conditions of ACs, on Cx43 expression. Confluent iPSC-CMs were tachypaced at 4 Hz for 24 and 48 hours, and compared with cells paced at 1 Hz and non-paced cells. ROS levels were monitored using CM-H2DCFDA fluorescence and confocal imaging. Levels of miR-1 were quantified and Cx43 expression was assessed by immunocytochemistry and Western blot analyses at different pacing frequencies and after manipulation with a specific miR-1 inhibitor. Our data revealed three effects of tachypacing: First, tachypacing iPSC-CMs for 24 h at 4 Hz significantly increased ROS compared to 1 Hz pacing (mean±SEM: 4.7±0.9 fold increase, n=5, p<0.05, Student's t-test). In addition, tachypacing significantly increased the activity of ROS-producing enzymes (slope: 0.17±0.03 at 4 Hz vs. 0.04±0.01 ΔF/s at 1 Hz, n=3, p<0.05). Second, tachypacing for 48 h at 4 Hz increased miR-1 expression by 20% (n=4). Third, tachypacing for 24 and 48 h at 4 Hz significantly reduced total protein expression of Cx43 (24 h: 0.4±0.2, 48 h: 0.3±0.1 fold decrease, n=3, p<0.05). Next, we investigated the effect of miR-1 on iPSC-CMs. Expression of the specific anti-miR-1 probe significantly increased sarcolemmal Cx43 expression compared to control scrambled miR (2.4±0.2 fold increase, n=4, p<0.05). The effect of anti-miR-1 on Cx43 expression resulted in a significant increase in total Cx43 (1.3±0.1 fold increase, n=3, p<0.05). Our data suggest that chronically increased ROS levels by tachypacing lead to elevated miR-1 expression which reduces Cx43 expression in iPSC-CMs. This is the first experimental evidence that chronic oxidative stress may be causally linked to the pathophysiological remodeling of gap junctions underlying arrhythmogenic diseases.

Where applicable, experiments conform with Society ethical requirements