The reverse mode of the Na+/Ca2+ exchanger contributes to the pacemaker mechanism in rabbit sinus node cells

Figures

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  Figure 1. Characterization of experimental groups and the effect of 1 μM ORM-10962. Representative original current traces of control (black), 1 μM ORM-10962 (green) and 10 mM NiCl2 (pink) obtained under a conventional voltage ramp protocol (see inset). The intracellular Ca2+ was set to ~ 100 nM, K+ and Ca2+ currents were inhibited. In the presence of 2 mM [Na]pip (panel a, left side, repeated measures ANOVA) we did not find neither ORM- nor NiCl2-sensitive currents in the outward direction. In contrast, the outward component is clearly observable when 8 mM NaCl was used in the pipette (panel a, right side, repeated measures ANOVA). Panel (b) represents the ORM-10962 sensitive currents obtained from the experiment demonstrated in panel A. Current–voltage diagram (panel c) of the NiCl2-sensitive current demonstrates the lack of reverse current when 2 mM NaCl was employed in the internal solution (blue graph, independent t-test). Panel (d) illustrates comparison of the ORM-effect between experimental groups, and we found that 1 μM ORM-10962 similarly reduces the forward component of the NCX independent from the Nai level that may exclude pharmacological interactions between the applied Nai…

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  Figure 2. Characterization of the NCX current as a Ni2+-sensitive current under a canonical SN action potential (panel a). The intracellular Ca2+ was buffered with 10 mM EGTA, K+ and Ca2+ currents were inhibited. Left side of the Figure demonstrates 2 mM [Na]pip, right side shows 8 mM [Na]pip. Panel (b) illustrates the control (black) and the NiCl2-treated currents (pink) and panel (c) depicts the subtracted currents.

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  Figure 3. An estimation of Ca2+ transients under a canonical SN action potential command potential in the presence of 2 mM (left) and 8 mM (right) NaCl in the pipette solution. The compositions of the extra and intracellular solutions ensured that K-currents, If, and Na/K pump were inhibited during these measurements. The intracellular Ca2+ was unbuffered. Panel (a) represents a canonical AP command potential. The amplitude of the parallel measured Ca2+ transients (panel b and c) was larger in the presence of 8 mM [Na]pip compared to 2 mM [Na]pip group while the diastolic Ca2+ remained unchanged (panel c *refers to the difference of transient amplitudes between 2 and 8 mM [Na]pip and means p < 0.05). Similarly, the half relaxation time of the transient decay was slower when 8 mM [Na]pip was applied (panel d *refers to the half relaxation time in comparison between 2 and 8 mM [Na]pip groups, and means p < 0.05). Data shown as mean ± SEM, n = 14–14, p value was calculated by independent t-test.

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  Figure 4. Measurement of the SR Ca2+ content by rapid application of 10 mM caffeine. Caffeine was applied after 10 consecutive conditioning pulses to reach a steady-state load of the SR Ca2+ content (panel a). Panel (b) illustrates original caffeine induced inward current, while panel (c) illustrates the Ca2+ transients in the presence of 2 (blue trace) and 8 mM (red trace) pipette Na+. We found larger SR Ca2+ content in presence of 8 mM pipette Na+ (independent t-test, panel b). In contrast, we did not find statistically significant difference between the half-relaxation times of the Ca2+ transients (independent t-test, panel c). Data shown as mean ± SEM, n = 11 (panel b) and n = 17 (panel c), *means p < 0.05.

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  Figure 5. Evaluation of the Ca2+-transients after application of 10 mM caffeine. The step-by-step increasing Ca2+ transients (panel a) reached their steady-state value after the 10th stimulus where the diastolic Ca2+ transient (panel b) was not changed while the transient amplitude (panel c) was higher in the presence of 8 mM pipette NaCl (red graphs). The bar graph in panel (c) indicates faster change of transient amplitude between 1st and 2nd beats when pipette Na+ was adjusted to 8 mM while in the case of diastolic Ca2+ level it was found identical refilling kinetics between 2 (blue column) and 8 (red column) pipette NaCl (bar graph, panel b). Independent t-test, *means p < 0.05.

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  Figure 6. Comparison of spontaneous AP (panel a, upper traces) and CaT characteristics (panel a, lower traces) between 2 mM (blue traces) and 8 mM (red traces) [Na]pip groups, measured by whole-cell patch clamp technique. Bar graphs indicate that the cycle length of spontaneous APs (panel b) was shorter, the diastolic slope (panel c) was steeper, the APD (panel d) was shorter and CaT amplitude (panel e) was larger in the 8 mM [Na]pip group (red columns) compared to 2 mM [Na]pip group (blue columns). Data shown as mean ± SEM, n = 8–8, independent t-test, *means p < 0.05.

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  Figure 7. Evaluation of spontaneous AP and CaT characteristic measured by patch clamp technique in perforated cells. Panel a indicates that in the presence of 1 µM strophantin (green curve) both the AP cycle length (upper part) and the Ca2+-transient amplitude (lower part) was increased compared to control (black curve). Bar graphs illustrate statistically significant change of the cycle length and diastolic slope in the presence of strophantin (panel b). In contrast, when the cells were pre-treated with 1 µM ORM-10962 (panel c, pink curves) both the action potential cycle length (upper part) and the Ca2+ transient amplitude (lower part) remained unchanged. As bar graphs show none of the investigated parameters changed when the NCX was inhibited prior to strophantin employment (panel d). Data shown as mean ± SEM, n = 6, paired t-test, *means p < 0.05.

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  Figure 8. Computational simulation of Na-dependence of SN pacemaking by using the Maltsev–Lakatta minimal model14 in the presence (blue traces) and in the absence (brown traces) of reverse NCX. Brown traces represent NCX function when only the forward (negative current) mode was active. Intracellular Na+-concentration was changed from 2 to 10 mM (vertical columns) and the behaviour of the action potential (panel a first row), NCX-current (panel a second row), global intracellular Ca2+ (panel a third row), SR Ca2+ content (panel a fourth row) and ICaL (panel a last row) were investigated. In the presence of reverse NCX (blue curves), increase of Na+i enhances SN pacemaking with parallel increase of outward NCX and network SR Ca2+ content. In the absence of reverse mode, the SN pacemaking failed at all Na+i levels (panel a) and a considerable loss in the SR Ca2+ content was observed.