Recently, we have published (Kamkin et al. 2000) that local axial stretch can activate whole-cell currents (I_SAC) through non-selective cation channels (SACs; for review, see Bett & Sachs, 1999). Here, we measured changes in intracellular sodium concentration ([Na⁺]_i) as they were expected from the simultaneously recorded I_SAC.

Ventricular myocytes were superfused by a solution composed of (mM): 150 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.2 MgCl₂, 10 glucose and 5 Hepes/NaOH (pH 7.4, 37 °C). Patch pipettes were filled with 140 KCl, 5 Na₂ATP, 5.5 MgCl₂, 10 BAPTA and 10 Hepes/KOH (pH 7.2). Local mechanical stimuli (m.s.) were applied via a glass stylus whose distance to the patch electrode was increased by approximately 20 %. Voltage dependence of mechanosensitive currents was evaluated from I-V curves obtained by a series of 20 voltage clamp pulses (1 Hz) that started from -45 mV (holding potential) and went to potentials between -100 and +50 mV (170 ms), current components were separated by comparing I-V curves before and after m.s. Stretch-induced changes in intracellular sodium concentration [Na⁺]_i were analysed by 3-D imaging of ventricular myocytes that had been loaded with 20 mM cell-permeant sodium green or by electronprobe microanalysis of cryosections (Wendt-Gallitelli et al. 1993).

Axial stretch of ventricular myocytes prolonged the action potential, depolarised the resting membrane and caused extra systoles. The most prominent stretch-sensitive current component was a stretch-activated inward current (I_SAC) that followed modest outward rectification, reversed close to 0 mV and was blocked by 8 mM GdCl₃ or 30 mM streptomycin, respectively. Long periods of stretch (< 5 min) induced a nearly voltage-independent outward current (Cs⁺ substitution for extra- and intracellular K⁺ ions); the sensitivity of this current to removal of e.c. Cs⁺ or to strophanthidin let us attribute this current to intracellular Na⁺ accumulation followed by electrogenic Na⁺ extrusion.

Pseudo-ratiometric imaging of sodium green fluorescence indicated that a 2 min axial stretch can double [Na⁺]_i, which is an increment in [Na⁺]_i twice as big as the one induced by a 1 Hz stimulation. Stretch increased [Na⁺]_i with spatial heterogeneities, in ‘hot spots’ [Na⁺]_i could be as high as 50 mM. Co-imaging with ANEPPS localized most of the [Na⁺]_i hot spots close to the surface membrane. Co-imaging with tetramethyl rhodamine indicated that [Na⁺]_i hot spots were outside the mitochondria. After the stretch, the [Na⁺]_i hot spots dissipated with a time constant of approximately 1 min, suggesting that Na⁺ accumulated in a space of restricted diffusion.

Electron probe microanalysis (EPMA, lateral resolution of 20 nm X 20 nm). Myocytes were shock-frozen during axial stretch. EPMA of cryosections (Wendt-Gallitelli et al. 1993) indi-cated that a 2 min stretch increased the total concentration [Na] (sum of free [Na⁺]_i plus bound [Na]_i) by a factor of approximately 2. In the cytosol close (100 nm) to the sarcolemma (100 nm) [Na] increased from 26 to 58 mmol l^-1 and in the central cytosol from 19 to 36 mM. In mitochondria underneath the sarcolemma [Na] increased from 21 to 44 mM and in central mitochondria from 10 to 20 mM. [Na] increased from 25 to 44 mM in the nuclear envelope and from 25 to 37 mM in the nuclear matrix.

Local axial stretch can increase the intracellular sodium content; this is indicated by the increase in the free and total sodium concentration. The increments in [Na] can be attributed to Na⁺ influx through SACs (charge -400 pA X 240 s) on the assumption that only a third of the influxing Na⁺ is extruded by the Na⁺,K⁺-ATPase. The result that [Na] and [Na⁺] were higher underneath the sarcolemma than in the centre of the cell is discussed as a result of restricted diffusion of Na⁺ ions in a ‘fuzzy space’ delimited by membranes of sarcolemma and subsarcolemmal SR or sarcolemma and subsarcolemmal mitochondria, respectively.

This work was supported by the DFG Transregio 02 TP A1 and A3.