Assessment of the fraction of t-tubular membrane in cardiomyocytes: a new and reversible approach
|Year of publication
|MU Faculty or unit
|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 to the stores of Ca2+. A remodelling of t-tubules is associated with various cardiac pathologies including ischemia or heart failure. To understand 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 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 which is fully reversible, allowing repetitive estimation of t-tubular characteristics. A short-term perfusion (tens of seconds) of the measured cell with a low-conductive solution (isotonic sucrose solution, 0.3 M) substantially increases electrical resistance of the t-tubular lumen and electrically separates the surface and t-tubular cell membrane. Fitting of the recorded membrane current activated by a sub-threshold voltage step (20-ms step from -80 to -75 or -70 mV) reveals two current components and following analysis of parameters of this fit allows to identify the t-tubular and surface cell membrane capacitances and, thus, also their ratio, i.e. fraction of the t-tubular membrane (ft). Using the new technique during our whole-cell patch-clamp measurements in enzymatically isolated rat ventricular and atrial myocytes, we were able to estimate the average ft in these cells (0.337 ± 0.017 and 0.144 ± 0.015, respectively). The parameter ft could be estimated repetitively (0.345 ± 0.021 and 0.347 ± 0.023 in ventricular myocytes during the first and second sucrose perfusion, respectively). The new method enabled to detect 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 to properly and repetitively estimate an essential parameter of the t-tubular system ft. Hence, it may be used in studies analysing properties of the cardiac t-tubular system including its transient changes, induced for example by a transient hypoosmolality.