Analysis of factors affecting Ca(2+)-dependent inactivation dynamics of L-type Ca(2+) current of cardiac myocytes in pulmonary vein of rabbit

Abstract

L-type Ca(2+) channels (ICaLs) are inactivated by an increase in intracellular [Ca(2+)], known as Ca(2+)-dependent inactivation (CDI). CDI is also induced by Ca(2+) released from the sarcoplasmic reticulum (SR), known as release-dependent inhibition (RDI). As both CDI and RDI occur in the junctional subsarcolemmal nanospace (JSS), we investigated which factors are involved within the JSS using isolated cardiac myocytes from the main pulmonary vein of the rabbit. Using the whole-cell patch clamp technique, RDI was readily observed with the application of a pre-pulse followed by a test pulse, during which the ICaLs exhibited a decrease in peak current amplitude and a slower inactivation. A fast acting Ca(2+) chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), abolished this effect. As the time interval between the pre-pulse and test pulse increased, the ICaLs exhibited greater recovery and the RDI was relieved. Inhibition of the ryanodine receptor (RyR) or the SR Ca(2+)-ATPase (SERCA) greatly attenuated RDI and facilitated ICaL recovery. Removal of extracellular Na(+),which inhibits the Na(+)-Ca(2+) exchange (Incx), greatly enhanced RDI and slowed ICaL recovery, suggesting that Incx critically controls the [Ca(2+)] in the JSS. We incorporated the Ca(2+)-binding kinetics of the ICaL into a previously published computational model. By assuming two Ca(2+)-binding sites in the ICaL, of which one is of low-affinity with fast kinetics and the other is of high-affinity with slower kinetics, the new model was able to successfully reproduce RDI and its regulation by Incx. The model suggests that Incx accelerates Ca(2+) removal from the JSS to downregulate CDI and attenuates SR Ca(2+) refilling. The model may be useful to elucidate complex mechanisms involved in excitation–contraction coupling in myocytes.

Figure 1. Inactivation of ICaLs with 0.1 mm EGTA (A) or 10 mm BAPTA (B) as the Ca²⁺ chelator

The rate of inactivation was much slower with BAPTA than with EGTA.

Figure 2. Characteristics of release-dependent inactivation (RDI)

A, repetitive step pulses separated by 30 s intervals did not induce any change in the ICaLs. B, when a pre-pulse was applied, the inward current activated by Ca²⁺ appeared, after which RDI of the ICaLs was observed with a reduced peak current and slower rate of inactivation.

Figure 3. Effect of altering the change in intracellular [Ca²⁺] on release-dependent inactivation (RDI)

A, suppression of the intracellular [Ca²⁺] change by 10 mm BAPTA eliminated the RDI. B, suppression of Ca²⁺ release from the SR by ryanodine caused a significant decrease in the RDI.

Figure 4. Effects of increasing the time interval between pre-pulse and test pulse on the extent of RDI

The traces from subsequent experiments are overlapped to illustrate the trend. A, the amplitude of the ICaL current initially decreased as a result of the pre-pulse; as the time interval between the pre-pulse and test pulse increased, the amplitude of the ICaL current recovered to that of the control, and the inactivation rate also increased. B, under conditions of Na⁺ deprivation (Na⁺-free solution), the current amplitude was smaller and recovery was slower than in normal physiological (NT) solution. C, the inactivation time constant for the ICaL was always less in the Na⁺-free solution than in NT solution, meaning the rate of inactivation of ICaL was faster in the absence of Na⁺. D, the decrease in current amplitude attributable to RDI was larger and recovered more slowly in Na⁺-free solution than in NT solution.

Figure 5. Effect of fast Ca²⁺ chelation by BAPTA on the time-dependent change in release-dependent inactivation (RDI)

A and B, with the intracellular [Ca²⁺] change suppressed by 10 mm BAPTA, the L-type Ca²⁺ channel (ICaL) was little changed, as seen by a series of step pulses, regardless of the presence (A) or absence (B) of extracellular Na⁺. C, the inactivation time constant of the ICaL was unchanged. D, the amplitude of the ICaL current was slightly decreased when the time interval between the pre-pulse and test pulse was less than 20 ms; this effect may be due to voltage-dependent inactivation (see text).

Figure 6. Time-dependent changes in release-dependent inactivation (RDI) when SR function is suppressed

A, in the presence of 150 μm ryanodine, the peak current amplitude was little changed, similar to the pattern seen in the presence of BAPTA (see Fig. 5C), although the inactivation time constant was smaller and decreased slightly as the time interval between the pre-pulse and test pulse increased. Removal of extracellular Na⁺ decreased the inactivation time further. B, similar findings were observed in the presence of 10 μm thapsigargin.

Figure 7. Influence of each factor on time-dependent changes in the peak current of L-type Ca²⁺ channels (ICaLs)

A, the current amplitudes were normalised to the control amplitude. The X-axis denoted the time interval between the pre-pulse and the test pulse. Extrapolating to zero time between pre-pulse and test pulse, in BAPTA accessible space, the relative contributions of ICaLs, SR, and inhibition of Incx to the decrease of peak current were approximately 4%, 42% and 54%, respectively. B, relative contributions of the ICaLs (black), SR (blue), and inhibition of Incx (red) to the decrease of ICaL current as a function of the time interval between the pre-pulse and test pulse.

Figure 8. Influence of each factor on the inactivation time constant

A, under physiological (NT) conditions, the changes in the inactivation time constant as a function of the time interval between the pre-pulse and test pulse in the presence of BAPTA (•), ryanodine (○), thapsigargin (▾) and none (▵). B, under Na⁺-free conditions, the changes in the inactivation time constant in the presence of BAPTA (•), ryanodine (○), thapsigargin (▾) and none (▵). C, the control inactivation time constants obtained by the single pulse applied at every 30 s. Black bars denotes NT conditions and grey bars show Na⁺-free conditions. Those were the paired experiments and ** denoted P value < 0.01.

Figure 9. Model structure of cardiac myocytes in the pulmonary vein, showing components involved with the change of Ca²⁺ concentration in the junctional subsarcolemmal space (JSS)

A, the diagram shows half a sarcomere (adapted from Leem et al. 2006). B, sources of Ca²⁺ ingress and egress in the JSS.

Figure 10. Results from a model simulation of release-dependent inactivation (RDI)

The model simulation satisfactorily reproduced the experimental measurements of RDI in the presence of 0.1 mm EGTA/NT (A), 10 mm BAPTA/NT (B), and 0.1 mm EGTA/Ryanodine (C) (see text). Top, depiction of the voltage pulse train given to the L-type Ca²⁺ channels (ICaLs). Bottom, simulated ICaL currents resulting from the voltage pulses.

Figure 11. Model simulation of the recovery from release-dependent inactivation (RDI)

The model simulation satisfactorily reproduced the experimental measurements of RDI recovery in the presence of 0.1 mm EGTA under physiological conditions (A), and 0.1 mm EGTA in Na⁺-free medium (B). Top, depiction of the voltage pulse train given to the L-type Ca²⁺ channels (ICaLs), with varying times between the pre-pulse and test pulse. Bottom, simulated ICaL currents resulting from the voltage pulses. The traces from separate simulations are overlapped to show the trend. Red lines are from the experimental data.

Figure 12. Model simulations of the Ca²⁺ concentration in the junctional subsarcolemmal (A) region ([Ca²⁺]_JSR) or (B) space ([Ca²⁺]_JSS), and (C and D) the corresponding L-type Ca²⁺ channel (ICaL) current due to Ca²⁺ binding at two different types of Ca²⁺-binding sites in a series of double-pulse experiments (see text)

The traces from several simulations at different time intervals between the pre-pulse and test pulse are overlapped to show the trends. A, before the pre-pulse, the Ca²⁺ content of the SR was greater in the Na⁺-free medium (red) than under physiological (NT) conditions (black) because Ca²⁺ refilling was facilitated in the absence of Na⁺. B, the Ca²⁺ released from the SR during the pre-pulse and subsequent pulses generated a much larger increase in [Ca²⁺]_JSS near the ICaL when Na⁺-free medium (red) was used as compared with NT conditions (black). C, the higher [Ca²⁺]_JSS in the absence of Na⁺ (red) as compared with NT conditions (black) decreased the open probability of LAFK sites with bound Ca²⁺, leading to lesser ICaL activity. D, the HASK Ca²⁺-binding site contributed less to the overall ICaL activity because the slow kinetics but determined the availability of ICaLs. Black line: NT, Red line: Na⁺-free.

Figure 13. Model simulation of release-dependent inactivation (RDI) under specific conditions

A, with the SR function removed, RDI did not occur under Na⁺-free conditions, indicating that Na⁺–Ca²⁺ exchange (Incx) had virtually no role in the absence of an active SR. B, with an active SR, the RDI in the absence of Na⁺ (inactive Incx) is greater than that under physiological conditions (active Incx). Top, depiction of the voltage pulse train given to the L-type Ca²⁺ channels (ICaLs), with varying times between the pre-pulse and test pulse. Middle (B), simulated [Ca²⁺] in the junctional subsarcolemmal space. Bottom, simulated ICaL currents resulting from the voltage pulses. The traces from separate simulations are overlapped to show the trends. Red lines are the experimental results from control ICaL current amplitude measurements during RDI.