The Driving Force of the Na/Ca-Exchanger during Metabolic Inhibition

Abstract

Objective: Metabolic inhibition causes a decline in mechanical performance and, if prolonged, myocardial contracture and cell death. The decline in mechanical performance is mainly due to altered intracellular calcium handling, which is under control of the Na(+)/Ca(2+)-exchanger (NCX) The driving force of the NCX (ΔG(ncx)) determines the activity of NCX. The aim of this study was to describe the relation between ΔG(ncx) and calcium homeostasis during metabolic inhibition.

Methods: In left ventricular rabbit myocytes, during metabolic inhibition (2 mmol/L sodium cyanide), sodium ([Na(+)](i)), calcium ([Ca(2+);](i)), and action potentials were determined with SBFI, indo-1, and the patch clamp technique. Changes of ΔG(ncx) were calculated.

Results: During metabolic inhibition: The first 8 min [Na(+)](i) remained constant, systolic calcium decreased from 532 ± 28 to 82 ± 13 nM, diastolic calcium decreased from 121 ± 12 to 36 ± 10 nM and the sarcoplasmic reticulum (SR) calcium content was depleted for 85 ± 3%. After 8 min [Na(+);](i) and diastolic calcium started to increase to 30 ± 1.3 mmol/L and 500 ± 31 nM after 30 min respectively. The action potential duration shortened biphasically. In the first 5 min it shortened from 225 ± 12 to 153 ± 11 ms and remained almost constant until it shortened again after 10 min. After 14 min action potential and calcium transients disappeared due to unexcitability of the myocytes. This resulted in an increased of the time average of ΔG(ncx) from 6.2 ± 0.2 to 7.7 ± 0.3 kJ/mol during the first 3 min, where after it decreased and became negative after about 15 min.

Conclusion: Metabolic inhibition caused an early increase of ΔG(ncx) caused by shortening of the action potential. The increase of ΔG(ncx) contributed to decrease of diastolic calcium, calcium transient amplitude, SR calcium content, and contractility. The increase of diastolic calcium started after ΔG(ncx) became lower than under aerobic conditions.

Keywords: Na/Ca-exchanger; SR; action potential; calcium; driving force; metabolic inhibition; myocytes; sodium.

Figure 1

Cytosolic [Na⁺]_i in 2 Hz stimulated myocytes (n = 20) during metabolic inhibition. All data expressed as mean ± SEM. *p < 0.05 versus Ctrl (t = 0; one way ANOVA).

Figure 2

(A) Representative examples of [Ca²⁺]_i transients under aerobic conditions (t = 0) and after 2, 5, and 8 min of metabolic inhibition. (B) Systolic (open circles) and diastolic [Ca²⁺]_i (closed circles) in 2 Hz stimulated myocytes (n = 20) during metabolic inhibition. All data expressed as mean ± SEM. *p < 0.05 versus Ctrl (t = 0; one way ANOVA).

Figure 3

(A) Representative examples of action potentials under aerobic conditions (t = 0) and after 2, 5, and 13 min of metabolic inhibition. (B) Action potential duration in 2 Hz stimulated myocytes (n = 20) measured at 90% repolarization (APD₉₀) during metabolic inhibition. All data expressed as mean ± SEM. *p < 0.05 versus Ctrl (t = 0; one way ANOVA).

Figure 4

Representative examples of the dynamic change of ΔG_ncx in 2 Hz stimulated myocytes during one stimulation cycle, under aerobic condition (A) and after 2 (B) and 10 (C) min of metabolic inhibition. The dotted area corresponds to reversed mode operation of NCX. The dotted lines represent time averaged ΔG_ncx over one stimulation cycle.

Figure 5

The time averaged ΔG_exch in 2 Hz stimulated myocytes (n = 20) during metabolic inhibition. All data expressed as mean ± SEM. *p < 0.05 versus Ctrl (t = 0; one way ANOVA).

Figure 6

Top panel: representative examples of the increase of [Ca^2⩲]_i upon rapid cooling (RC) in 2 Hz stimulated myocytes just before metabolic inhibition (t = 0) and 2, 6, 10, and 12. 5 min after metabolic inhibition. Bottom panel: SR calcium content, systolic SR depletion, and SR membrane calcium gradient during metabolic inhibition in 2 Hz stimulated myocytes (n = 10 for each time point). (A) SR calcium content is expressed as the change of cytosolic total calcium (μmol/L) in response to RC. (B) The degree to which SR became depleted during systole (ratio of the calcium transient amplitude expressed as total calcium and SR calcium content. (C) Ratio of SR calcium content and diastolic calcium. All data expressed as mean ± SEM. *p < 0.05 versus Ctrl (t = 0; one way ANOVA).