Fraction of the T-Tubular Membrane as an Important Parameter in Cardiac Cellular Electrophysiology: A New Way of Estimation

Abstract

The transverse-axial tubular system (t-tubules) plays an essential role in excitation-contraction coupling in cardiomyocytes. Its remodelling is associated with various cardiac diseases. Numerous attempts were made to analyse characteristics essential for proper understanding of the t-tubules and their impact on cardiac cell function in health and disease. The currently available methodical approaches related to the fraction of the t-tubular membrane area produce diverse data. The widely used detubulation techniques cause irreversible cell impairment, thus, distinct cell samples have to be used for estimation of t-tubular parameters in untreated and detubulated cells. Our proposed alternative method is reversible and allows repetitive estimation of the fraction of t-tubular membrane (ft) in cardiomyocytes using short-term perfusion of the measured cell with a low-conductive isotonic sucrose solution. It results in a substantial increase in the electrical resistance of t-tubular lumen, thus, electrically separating the surface and t-tubular membranes. Using the whole-cell patch-clamp measurement and the new approach in enzymatically isolated rat atrial and ventricular myocytes, a set of data was measured and evaluated. The analysis of the electrical equivalent circuit resulted in the establishment of criteria for excluding measurements in which perfusion with a low conductivity solution did not affect the entire cell surface. As expected, the final average ft in ventricular myocytes (0.337 ± 0.017) was significantly higher than that in atrial myocytes (0.144 ± 0.015). The parameter ft could be estimated repetitively in a particular cell (0.345 ± 0.021 and 0.347 ± 0.023 in ventricular myocytes during the first and second sucrose perfusion, respectively). The new method is fast, simple, and leaves the measured cell intact. It can be applied in the course of experiments for which it is useful to estimate both the surface and t-tubular capacitance/area in a particular cell.

A new approach to the determination of tubular membrane capacitance: passive membrane electrical properties under reduced electrical conductivity of the extracellular solution

“…One possibility was the substitution of parallel combination R mt || R t for R ms || R t , which was justified because R mt > > R t and R ms > > R t (as directly confirmed by calculating the resistances R ms , R mt , and R t in the present study). This procedure was recently verified in experiments on rat ventricular and atrial cardiomyocytes [ 17 ]. The above substitution led to the calculation formula where the coefficient k c (0.97 for ventricular and to 0.91 for atrial cardiomyocytes) was introduced as a correction for the mean error caused by the exchange of membrane resistances R mt for R ms in the approximate calculation of C t as justified in [ 17 ].…”

“…introduced in the experimental study [17]. This approach is more general and allows the calculation of the membrane resistances R ms and R mt and the tubular resistance R t in addition to capacitances.…”

“…In addition to the expressions derived so far for R a , C s , and R ms (Eqs. ( 16), (17), and (24)), the remaining elements can be calculated as follows: the most important parameter C t follows from Eq. ( 2) the resistances R t and R mt result from Eqs.…”

“…Enzymatic isolation of cardiomyocytes from the right ventricles of adult male Wistar rats and standard experimental procedure using voltage-clamp method have been described previously [1,17]. Sucrose solution (0.32 M) was prepared by adding sucrose (purity ≥ 99.5%) and CaCl 2 (5 μM) to deionized water (specific conductivity 1.4 µS/cm).…”

“…The derived relationships are verified using a computer model and tested in preliminary experiments. A simplified version of this method limited to the determination of both capacitances was tested in experiments on rat ventricular and atrial cardiomyocytes [ 17 ]. The mean value of the tubular membrane fraction ft = Ct/Cm fitted well within the range of values previously obtained by detubulation approaches and was significantly lower in atrial myocytes than in ventricular myocytes.…”