Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB030

Poster Communications

The Transverse Tubular System is Preserved by Activation of the Glucocorticoid Receptor in Cultured Rat Cardiomyocytes

T. Seidel1, D. Fiegle1, T. Baur1, A. Ritzer1, S. Nay1, R. H. Oakley2, J. A. Cidlowski2, T. Volk1

1. Institute of Cellular and Molecular Physiology, University of Erlangen-Nürnberg (FAU), Erlangen, Germany. 2. Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, North Carolina, United States.

The transverse tubular system (t-system) of ventricular cardiomyocytes is essential for excitation-contraction coupling. It is remodeled in cardiac disease, contributing to impaired cardiac contractility. However, mechanisms leading to t-system remodeling are incompletely understood. Because previous studies suggest upregulation of excitation-contraction coupling by glucocorticoid receptor (GR) activation, we investigated effects of GR activation on t-system structure. Ventricular cardiomyocytes were isolated from adult female Wistar rats and kept in culture for 3 days in either M199 + vehicle or M199 + corticosteroid agonist/antagonist. Freshly after isolation and after 3 days in culture, membranes in living cells were stained and imaged by confocal microscopy. T-system distance, volume fraction and orientation were analyzed. Additionally, cells were fixed and stained for JPH2, RyR2 and L-type Ca2+channels (LTCC). Cell capacitance and LTCC currents were measured by whole-cell voltage clamping. JPH2 and BIN1 mRNA expression was quantified by RT-PCR. In tissue from cardiac-specific GR knockout mice, we measured RyR2-LTCC distances by confocal and STED microscopy. T-system density was reduced after 3 days in culture, as indicated by increased intracellular t-system distance (0.53±0.02 µm vs. 0.66±0.02 µm, n≥67, p<0.01) and reduced volume fraction (8.7±0.2% vs. 7.7±0.2%, p<0.05). The fraction of longitudinally oriented components was increased (14.5±0.9% vs. 21.7±0.7%, p<0.01). Both dexamethasone (DEX, 1 µM) and corticosterone (1 µM) treatment prevented the changes in t-system density. Blocking the GR by mifepristone (10 µM) abrogated the effects of DEX, whereas spironolactone (10 µM), an inhibitor of the mineralocorticoid receptor, did not. Cell capacitance, which is proportional to cell membrane area including the t-system, was higher in myocytes treated with DEX than in vehicle after 3 days (83.3±4.1 pF vs. 115.5±4.5 pF, n≥25, p<0.001), confirming results obtained from microscopic imaging. Furthermore, LTCC current density was substantially larger in DEX-treated cells (7.8±0.6 pA/pF vs. 14.3±1.0 pA/pF, n≥25, p<0.001). Fluorescent staining revealed increased relative amounts of JPH2 near the surface sarcolemma after 3 days in culture (11.7±2.6% vs. 19.7±2.3%, p<0.01), which was not prevented by DEX. However, DEX treatment decreased RyR2-LTCC distance (0.58±0.02 vs 0.71±0.02, p<0.01), and GR knockout mice showed greater RyR2-LTCC distance than control mice. Surprisingly, JPH2 and BIN1 mRNA levels were downregulated by DEX in cultured rat myocytes (p<0.01). Dexamethasone preserves t-system density in primary culture of adult rat left-ventricular cardiomyocytes, most likely via activation of the GR. GR knockout mice exhibited reduced RyR2-LTCC coupling. We suggest that glucocorticoid signaling is involved in t-system regulation.

Where applicable, experiments conform with Society ethical requirements