In the heart, Na+ is a key modulator of the action potential, Ca2+ homeostasis, energetics, and contractility. Because Na+ currents and cotransport fluxes depend on the Na+ concentration in the submembrane region, it is necessary to accurately estimate the submembrane Na+ concentration ([Na+]sm). Current methods using Na+-sensitive fluorescent indicators or Na+ -sensitive electrodes cannot measure [Na+]sm. However, electrophysiology methods are ideal for measuring [Na+]sm. In this article, we develop patch-clamp protocols and experimental conditions to determine the upper bound of [Na+]sm at the peak of action potential and its lower bound at the resting state. During the cardiac cycle, the value of [Na+]sm is constrained within these bounds. We conducted experiments in rabbit ventricular myocytes at body temperature and found that 1) at a low pacing frequency of 0.5 Hz, the upper and lower bounds converge at 9 mM, constraining the [Na+]sm value to ∼9 mM; 2) at 2 Hz pacing frequency, [Na+]sm is bounded between 9 mM at resting state and 11.5 mM; and 3) the cells can maintain [Na+]sm to the above values, despite changes in the pipette Na+ concentration, showing autoregulation of Na+ in beating cardiomyocytes.
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