Accelerated inactivation of cardiac L-type calcium channels triggered by anaesthetic-induced preconditioning

Abstract

Background and purpose: Cardioprotection against ischaemia by anaesthetic-induced preconditioning (APC) is well established. However, the mechanism underlying Ca(2+) overload attenuation by APC is unknown. The effects of APC by isoflurane on the cardiac L-type Ca channel were investigated.

Experimental approach: In a model of in vivo APC, Wistar rats were exposed to isoflurane (1.4%), delivered via a vaporizer in an enclosure, prior to thoracotomy. The Dahl S rats were similarly preconditioned to determine strain-dependent effects. Whole-cell patch clamp using cardiac ventricular myocytes was used to determine the L-type Ca(2+) current (I(Ca,L)) characteristics and calmodulin (CaM) levels were determined by Western blot analysis. Cytosolic Ca(2+) levels were monitored using fluo-4-AM. Action potential (AP) simulations examined the effects of APC.

Key results: In Wistar rats, APC significantly accelerated I(Ca,L) inactivation kinetics. This was abolished when external Ca(2+) was replaced with Ba(2+), suggesting that Ca(2+)-dependent inactivation of I(Ca,L) was modulated by APC. Expression levels of CaM, a determinant of I(Ca,L) inactivation, were not affected. Attenuation of cytosolic Ca(2+) accumulation following oxidative stress was observed in the APC group. Simulations showed that the accelerated inactivation of I(Ca,L) resulted in a shortening of the AP duration. The Dahl S rat strain was resistant to APC and changes in I(Ca,L) inactivation were not observed in cardiomyocytes prepared from these rats.

Conclusions and implications: APC triggered persistent changes in the inactivation of cardiac L-type Ca channels. This can potentially lead to a reduction in Ca(2+) influx and attenuation of Ca(2+) overload during ischaemia/reperfusion.

Figure 1

Effects of in vivo APC on cardiomyocyte survival after oxidative stress. Myocytes were isolated from Wistar rat hearts. In the non-APC group, oxidative stress significantly increased the percentage of cell death compared with the time control (TC) group. This effect of oxidative stress was significantly attenuated by in vivo APC. *P < 0.05 vs. TC, ^†P < 0.05 vs. non-APC. n = 7–10 per group.

Figure 2

Effects of in vivo APC on the biophysical profile of I_Ca,L obtained in myocytes from non-APC and APC hearts. (A) Representative whole-cell I_Ca,L recorded from an isolated cardiomyocyte obtained from a non-APC heart. Arrow indicates zero current level. (B) Current-voltage relationship. The current-voltage relationship for the non-APC and APC groups are shown. No significant differences were observed (n = 21–22 per group). (C) Steady-state activation curves. Data were fitted with the Boltzmann function. No significant shifts in the steady-state activation curves were observed between the two groups (n = 21–22 per group). (D) Steady-state inactivation curves. Similarly to (C), data were fitted with the Boltzmann function. No significant shifts in the steady-state inactivation curves were observed between the two groups (n = 22 per group). (E) Recovery from fast inactivation. The inset depicts the standard two-pulse protocol that was utilized as described in Methods (n = 19 per group). (F) Recovery from slow inactivation. The inset depicts the two pulse protocol that was utilized as described in Methods (n = 4 per group). There were no significant differences in the recoveries from fast and slow inactivations between the non-APC and APC groups.

Figure 3

Effects of in vivo APC on voltage- and Ca²⁺-dependent inactivation of the cardiac L-type Ca channel. (A) Sample whole-cell I_Ca,L traces recorded from myocytes in the non-APC and APC groups monitored at 0 mV from a holding potential of −80 mV are shown. The current traces were superimposed. Arrow indicates zero current level. (B) The fast and slow time constants of the inactivation kinetics of I_Ca,L in myocytes from the non-APC and APC groups are plotted against test potentials. Compared with the non-APC group, APC significantly decreased τ_fast in the range from −20 to +10 mV and τ_slow in the range from −20 to +40 mV. (C) The fraction of channels that ‘fast inactivated’ was determined from the double exponential fit to the inactivating I_Ca,L traces. In the APC group, the fraction in the fast inactivated state was significantly increased compared to the non-APC group. (D) Changes in I_Ca,L inactivation was correlated to changes in the total influx of charge by integrating the area of I_Ca,L traces during a 100 ms period. The charge transfer was significantly decreased by APC in the range from −20 to +30 mV. *P < 0.05 vs. non-APC. n = 21–22 per group.

Figure 4

Effects of in vivo APC on the voltage-dependent inactivation of the cardiac L-type Ca channel. (A) Representative traces of I_Ca,L and I_Ba recorded at 0 mV from a holding potential of −80 mV using Ca²⁺ and Ba²⁺ as charge carriers, respectively, are shown. The currents were scaled and superimposed. Arrow indicates zero current level. Ba²⁺ as the charge carrier removed Ca²⁺-dependent inactivation of the L-type Ca channel. (B) The fast and slow time constants of the inactivation kinetics of I_Ba in the non-APC and APC groups are plotted against test potentials. No significant differences were observed in τ_fast and τ_slow between the non-APC and APC groups (n = 10 per group).

Figure 5

Cytosolic calmodulin (CaM) levels. Effects of in vivo APC on cytosolic CaM levels were determined by Western blot. (A) Representative results from one heart each from the non-APC and APC groups are shown. β-actin was used as the loading control. (B) Summary of the CaM/β-actin ratio in the non-APC and APC groups are shown. No significant differences were observed between two groups (n = 6 hearts per group).

Figure 6

Cytosolic Ca²⁺ accumulation under stress conditions. Isolated cardiomyocytes were loaded with the cytosolic Ca²⁺ indicator, fluo-4-AM, and exposed to oxidative stress, followed by perfusion with glucose-free Tyrode solution. Fluo-4 fluorescence was monitored over time. Myocytes from both the non-APC and APC groups showed increases in cytosolic Ca²⁺ following stress as indicated by the increases in fluo-4 fluorescence. However, the increase in fluo-4 fluorescence was significantly reduced in the APC-treated myocytes. *P < 0.05 vs. non-APC. n = 10–12 per group.

Figure 7

Simulation of the action potential. (A) Using a modified FMG model as described in Methods, inactivation kinetics were accelerated by 23% in the simulated APC I_Ca,L compared with the Control I_Ca,L. (B) Simulated changes in the AP (control vs. APC) are shown as the corresponding consequence of the accelerated I_Ca,L inactivation. The APD₅₀ and APD₉₀ were 83.0 and 98.1 ms, respectively, in the control-simulated AP, and 77.8 and 92.9 ms in the APC-simulated AP.

Figure 8

Effect of Ca²⁺ sensitivity on I_Ca,L inactivation. The effect of altering the sensitivity of L-type Ca channels to intracellular Ca²⁺ on I_Ca,L inactivation was simulated using the modified FMG model. Ca²⁺ sensitivity was enhanced by 28% (dashed lines) relative to the control. The exponential fits are depicted by the dotted lines. This enhancement of Ca²⁺ sensitivity induced only 5% change in the acceleration of the I_Ca,L inactivation.

Figure 9

Effects of in vivo APC on the Dahl S strain of rats. (A) Cell survival study. Oxidative stress significantly increased the percentage of cell death in the non-APC group compared with the time control (TC) group. In the APC group, cell survival following oxidative stress was not significantly different from the APC group, indicating resistance to cardioprotection in the Dahl S rats. *P < 0.05 vs. TC. n = 5–9 per group. (B) Representative whole-cell I_Ca,L traces from Dahl S myocytes in the non-APC and APC groups. Currents were recorded at a test potential of 0 mV from a holding potential of −80 mV. The traces shown were superimposed. Arrow indicates zero current level. (C) I_Ca,L inactivation kinetics. The fast and slow time constants of current inactivation in myocytes from the non-APC and APC groups showed no significant differences in the Dahl S rats (n = 9 per group).