Book of Abstracts - New Frontiers 2022

Abstracts of poster presentations

ASSESSMENT OF THE FRACTION OF T-TUBULAR MEMBRANE IN CARDIOMYOCYTES: A NEW AND REVERSIBLE APPROACH

O. Švecová, M. Bébarová, M. Šimurdová, J. Šimurda

Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

The adequate excitation-contraction coupling in cardiomyocytes requires well developed t-tubular system (t-tubules) that allows fast transmission of excitation to the depth of the cell, near the stores of Ca 2+ . A remodeling of t-tubules is associated with various cardiac pathologies including ischemia or heart failure. To understand the impact of t-tubules on the function of both healthy and diseased cardiomyocytes, several methodological approaches have been developed and applied. Unfortunately, these approaches produce diverse data and the widely used detubulation technique causes an irreversible impairment of the tested cells which disables their repeated use and also paired comparison of the data obtained with and without t tubules. We propose an alternative method that is fully reversible, allowing repetitive estimation of t-tubular characteristics on the same cell. Short-term perfusion (tens of seconds) of the cell measured with a low conductive solution (isotonic sucrose solution, 0.3 M) substantially increases the electrical resistance of the t-tubular lumen and electrically separates the surface and t-tubular cell membrane. Analysis of the recorded membrane current activated by a sub-threshold voltage step (20 ms step from -80 to -75 or -70 mV) reveals two exponential current components. The numerical values of the parameters obtained from the current approximation by a sum of two exponential functions make it possible to calculate the t-tubular and surface membrane capacitances (Ct and Cs) and thus the fraction ft of the t-tubular membrane capacitance (ft=Ct / (Ct +Cs)) which can also be regarded as an indicator of the fraction of tubular membrane area. Using the new technique during our whole-cell patch-clamp measurements in enzymatically isolated rat ventricular and atrial cardiomyocytes, we were able to esti mate ft in these cells 0.337 ± 0.017 in ventricular and 0.144 ± 0.015 in atrial cells). Repetitive measurements in ventricular myocytes during the first and second sucrose perfusion resulted in ft of 0.345 ± 0.021 and 0.347 ± 0.023, respectively. The new m ethod enabled to detection of a significant partial detubulation of ventricular cardiomyocytes using 15-min exposure to 150 µM imipramine and its following wash -out (a decrease of the total cell membrane capacitance by 30.5% and ft by 49.6% in the treated cells). We conclude that our newly proposed method is fast, simple, and fully reversible, enabling us to properly and repetitively estimate an essential parameter of the t-tubular system ft. Hence, it may be used in studies analyzing properties of the cardiac t-tubular system including its transient changes, induced for example by a transient hypoosmolality.

Keywords: t-tubulus, isotonic sucrose solution, surface membrane capacitance, t-tubular membrane capacitance

Funding: Supported by the grant project NU22-02-00348 provided by the Ministry of Health of the Czech Republic and by the Specific University Research Grant of the Masaryk University MUNI/A/1133/2021 provided by the Ministry of Education, Youth and Sports of the Czech Republic.

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