New method for determination of tubular membrane capacity in intact cardiomyocytes
| Authors | |
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| Year of publication | 2017 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | Introduction: Transverse tubular system (TTS) plays a key role in the fast transmission of action potential from the cell surface to the depth of cardiomyocytes, thus, near to calcium stores and contractile machinery. The function of TTS is highly dependent on the presence of transport proteins whose distribution considerably differs in TTS and surface cell membrane. Knowledge of TTS capacity is required for the proper analysis of TTS function. Determination of TTS capacity and its contribution to the total cell capacity may be done by measurement of the membrane capacity before and after the so called detubulation, i.e. detachment of TTS caused by osmotic shock. However, this method is irreversible and, moreover, may result in wrong data due to incomplete TTS detachment. Purpose: The aim of this work was to suggest an alternative method for determination of TTS capacity that preserves the measured cardiomyocyte intact and, thus, enables repetitive measurements in the same cell. Method: It is impossible to distinguish TTS capacity and surface membrane capacity during measurement of the cell membrane capacity in normal conditions because both membrane systems are connected closely one to another. The newly developed method is based on the alternative electrical cell circuit with two membrane systems separated by electrical resistance of TTS. Transient application of isotonic sucrose solution (ISS) on the measured cell causes a significant increase of TTS resistance and, therefore, electrical separation of the two systems. Analysis of current responses to the imposed rectangular voltage pulses (by a new quantitative model) then enables to reveal capacity of the surface membrane and TTS separately. In this work, the membrane capacities were determined on enzymatically isolated rat ventricular myocytes. Results: After ISS application, a transitory phenomenon was observed. It was registered by changes of the membrane current at the imposed constant voltage. The analysed parameters gradually changed, first only the surface membrane parameters and, with a delay, also TTS parameters. It enabled to visualize the process of solution diffusion into TTS by a newly suggested quantitative model. Capacities of TTS and surface membranes (CTTS = 68.0±4.8 pF a CS = 99.8±3.9 pF, respectively; n = 16) were determined in the steady-state from approximation of the responses to rectangular pulses by the sum of two exponential waveforms. Ratio of both surfaces was determined as SS/STTS = 0.40±0.02, assuming the same specific capacity. Conclusion: The newly suggested method leaves the measured cell intact and allows repetitive measurements. Determined value of the tubular capacity fraction (Ct/Cm = 0.43) falls into the range of published data obtained by various methods (0.21-0.64). Recent estimates are based on the model of cell membrane with concentrated parameters. Further improvement of this method may be achieved after implementation of TTS description as a system with distributed parameters. |
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