Inactivation of ICa-L is the major determinant of use-dependent facilitation in rat cardiomyocytes

Abstract

Two models have been proposed to explain facilitation of the L-type calcium current (ICa-L). A positive feedback model proposes that calcium released during a conditioning pulse (I1) facilitates the subsequent pulse (I2) via calmodulin/calmodulin kinase II (CaMKII) mechanisms. The negative feedback model proposes that the calcium release of each pulse feeds back on itself via calcium-dependent inactivation. The relative physiological roles were evaluated in rat ventricular myocytes. Paired pulses (450 ms interpulse interval) elicited facilitation (I2 of 872 +/- 145 versus I1 of 777 +/- 132 pA, P < 0.01). Inactivation time (T0.37) was prolonged for I2 versus I1 (22 +/- 2 and 16 +/- 2 ms, P > 0.01). Evidence for the negative feedback mechanism includes: (a) ryanodine (0.3 mM ) eliminated facilitation, surprisingly by increasing the amplitude of I1 more than that of I2 (1039 +/- 216 and 977 +/- 186 pA) and eliminated the difference in T0.37 between I2 and I1 (33.1 +/- 4.5 versus 32.5 +/- 4.6 ms); (b) an outward I2, which does not trigger sarcoplasmic reticulum (SR) Ca2+ release, eliminated facilitation even when it was conditioned by an inward I1; (c) facilitation decayed as the I1-I2 interval lengthened (time constant (tau) = 16.9 +/- 1.4 s); (d) thapsigargin (0.1 microM ) slowed this decay (tau = 43.8 +/- 11.7 s) whereas isoproterenol accelerated it (tau = 5.6 +/- 1.4 s, P < 0.01) and T0.37 paralleled this decay; and (e) the magnitude of ICa-L was negatively correlated with the sodium-calcium exchange current (INa/Ca) elicited by the SR-Ca2+ release. In conclusion, Ca2+-dependent inactivation of ICa-L is the major mechanism underlying facilitation.

Figure 2. Ryanodine has no direct effects on I_Ca-L

In the setting of pretreatment with thapsigargin, representative current traces (A), mean magnitudes of I_Ca-L (B) and mean T_0.37 (C) before (black bars) and after ryanodine (open bars) in SR- Ca²⁺-depleted cells. The current traces of I_Ca-L before and after ryanodine completely overlapped (A). The voltage clamp protocol is the same as that shown in Fig. 1.

Figure 1. The effects of high concentration of ryanodine on I_Ca-L

Ryanodine decreased I_Ca-L and eliminated use-dependent facilitation. A, representative paired I_Ca-L current traces in control (C) and 0.3 mm ryanodine (R). The current traces between two I_Ca-L were cut off. The arrows indicate the peak level of I₁ and I₂ in control. The broken line indicates the zero current level. B, the time course of amplitude change of I₁ (•) and I₂ ([ww3]) within the experiment in paired I_Ca-L protocol. The arrows indicate the point in time at which the examples in A were obtained Inset of B, voltage clamp protocol. C and D, the mean amplitude (C) and T_0.37 (D) of I_Ca-L in control (black bars) and ryanodine (open bars). *P < 0.01; #P < 0.05 compared to I₁.

Figure 3. Outward currents through L-type calcium channel do not elicit facilitation

Representative current traces of inward (A) and outward (B) Ca²⁺ channel currents conditioned by a preceding inward I₁ (labelled and equivalent to I₂) and not conditioned by a preceding inward I_Ca-L (labelled and equivalent to I₁, solid trace). The current traces recorded by same protocol in 0.2 mm Cd²⁺ are also superimposed in B. Insets show the voltage clamp protocols. The statistical values of the current magnitudes and T_0.37 are shown in C and D.

Figure 4. Alteration of the SERCA 2A pump activity changes the facilitation delay

A, superimposed current traces of I_Ca-L after different resting intervals in control (CONT), during treatment with 10 nm isoproterenol (ISO) and during treatment with 0.1 μm thapsigargin (THAP, a different cell). I₁ indicates the ICa I_Ca-L after a 3 min rest. I₂ indicates the I_Ca-L 0.5 s after I₁ (first row of raw data) or 20 s after I₁ (second row of data). B, the recovery time courses of the facilitation ratio (FR) in control (•), 10 nm isoproterenol ([ww3]) and in 0.1 μm thapsigargin ([ww5]). The smooth curves in B were theoretical curves of data fitted by a single exponential function. C, relationship of inactivation time (T_0.37) of the I_Ca-L with its the pre-intervals. NB: a log time scale was used.

Figure 5. The negative linear relationship between I_Na/Ca and I_Ca-L

A, representative current traces of I_Ca-L and following I_Na/Ca of I₁ (•), I₂ (□) in control and that after 0.3 mm ryanodine ([ww3]). I₁–I₂ interval was 350 ms. B, the I_Na/Ca was integrated and the ratios of integrated I_Na/Ca-2/I_Na/Ca-1 were plotted against ratios of I₂/I₁at varied I₁–I₂ interval in nine cells. The linear regression showed y = 1.26 − 0.23x, r = 0.56, P < 0.05.