Electrophysiological Determination of Submembrane Na⁺ Concentration in Cardiac Myocytes

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.