Interplay between SERCA and sarcolemmal Ca2+ efflux pathways controls spontaneous release of Ca2+ from the sarcoplasmic reticulum in rat ventricular myocytes

Abstract

Waves of calcium-induced calcium release occur in a variety of cell types and have been implicated in the origin of cardiac arrhythmias. We have investigated the effects of inhibiting the SR Ca(2+)-ATPase (SERCA) with the reversible inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on the properties of these waves. Cardiac myocytes were voltage clamped at a constant potential between -65 and -40 mV and spontaneous waves evoked by increasing external Ca(2+) concentration to 4 mm. Application of 100 microm TBQ decreased the frequency of waves. This was associated with increases of resting [Ca(2+)](i), the time constant of decay of [Ca(2+)](i) and the integral of the accompanying Na(+)-Ca(2+) exchange current. There was also a decrease in propagation velocity of the waves. There was an increase of the calculated Ca(2+) efflux per wave. The SR Ca(2+) content when a wave was about to propagate decreased to 91.7 +/- 3.2%. The period between waves increased in direct proportion to the Ca(2+) efflux per wave meaning that TBQ had no effect on the Ca(2+) efflux per unit time. We conclude that (i) decreased wave frequency is not a direct consequence of decreased Ca(2+) pumping by SERCA between waves but, rather, to more Ca(2+) loss on each wave; (ii) inhibiting SERCA increases the chance of spontaneous Ca(2+) release propagating at a given SR content.

Figure 1. Inhibition of SERCA reduces the frequency of spontaneous release and prolongs its duration

A, the time course of the effects of addition and removal of TBQ on Fluo-4 fluorescence (upper trace) and membrane current (lower trace; holding potential −55 mV). B, sample records of individual transient inward currents in control (a) and in TBQ (b) from panel A.

Figure 2. TBQ slows the fall of Ca²⁺ and prolongs the duration in a spontaneous wave of Ca²⁺ release

A, the upper panel shows individual linescan images of Ca²⁺ waves in control and TBQ. In the lower panel the Ca²⁺ profile of each wave is shown from the point marked by the arrow. B, percentage change of Ca²⁺ efflux generated by individual waves, the time constant of recovery of the Ca²⁺ transient during a wave and SR Ca²⁺ content. The dashed line marks 100%. In B are shown mean and s.e.m. from 7 cells for wave efflux, 6 cells for time constant and 7 cells for SR content.

Figure 3. TBQ allows spontaneous waves of Ca release at lower SR content

The top trace shows membrane current accompanying a wave and then on application of caffeine (10 mm). The lower trace shows the integral of current expressed in terms of micromoles of Ca²⁺ per litre cell volume. The first deflection represents the loss of Ca²⁺ associated with the wave, the second the remaining SR Ca²⁺ content. The left hand panel was obtained in control, the right in 100 μm TBQ. The holding potential was −40 mV throughout.

Figure 4. Ca²⁺ efflux generated by waves increases as recovery of transient inward current slows in TBQ

A, transient inward currents associated with waves of Ca²⁺ release become less frequent and slower to recover in TBQ (100 μm; holding potential −45 mV). The plot below shows efflux as a function of the time constant of recovery for the five currents in the upper panel. B, efflux as a function of time constant of current recovery for 4 cells. Each has been normalized with respect to control.

Figure 5. Wave period increases as Ca²⁺ efflux generated by waves increases

A, the trace to the left shows the effect of TBQ on transient inward current frequency, on the right the steady state effect of tetracaine (holding potential −55 mV). B, plot of wave period as a function of Ca²⁺ efflux generated by waves in 4 cells. Each symbol type represents an individual cell, the colours code for: 250 μm caffeine (open), control (red), 10 or 50 μm TBQ (green), 200 μm tetracaine (yellow) and 100 μm TBQ (blue).

Figure 6. TBQ reduces transient inward current frequency by increasing wave generated efflux

The upper trace shows the effect of 100 μm TBQ on spontaneous transient inward currents (holding potential −60 mV). The lower trace shows calculations of the changes of SR Ca²⁺ content obtained as follows. The loss of Ca²⁺ from the cell (and therefore the SR) on each wave was calculated from the integral of the associated current. The Ca²⁺ influx was calculated to balance the efflux per unit time (in this experiment 0.76 μmol l⁻¹ s⁻¹).