Direct measurements of SR free Ca reveal the mechanism underlying the transient effects of RyR potentiation under physiological conditions

Abstract

Aims: Most of the calcium that activates contraction is released from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). It is controversial whether activators of the RyR produce a maintained increase in the amplitude of the systolic Ca transient. We therefore aimed to examine the effects of activation of the RyR in large animals under conditions designed to be as physiological as possible while simultaneously measuring SR and cytoplasmic Ca.

Methods and results: Experiments were performed on ventricular myocytes from canine and ovine hearts. Cytoplasmic Ca was measured with fluo-3 and SR Ca with mag-fura-2. Application of caffeine resulted in a brief increase in the amplitude of the systolic Ca transient accompanied by an increase of action potential duration. These effects disappeared with a rate constant of ∼3 s(-1). Similar effects were seen in cells taken from sheep in which heart failure had been induced by rapid pacing. The decrease of Ca transient amplitude was accompanied by a decrease of SR Ca content. During this phase, the maximum (end-diastolic) SR Ca content fell while the minimum systolic increased.

Conclusions: This study shows that, under conditions designed to be as physiological as possible, potentiation of RyR opening has no maintained effect on the systolic Ca transient. This result makes it unlikely that potentiation of the RyR has a maintained role in positive inotropy.

Keywords: Calcium; Ryanodine receptor; Sarcoplasmic reticulum.

Figure 1

The effects of caffeine on systolic [Ca²⁺]_i in sheep ventricular myocytes. (A) Time course of changes in cytoplasmic Ca measured with fura-2. Cells were stimulated with current pulses. Caffeine (500 µM) was applied for the period shown. (B) Specimen records of Ca (top) and membrane potential (bottom) taken at the points indicated on A. (C) Average data for Ca transient amplitude (top) and APD (bottom) for (from left to right); control, first beat in caffeine, steady state in caffeine, first beat on wash, steady-state wash. n = 12 cells from nine animals. Asterisk represents a statically significant difference.

Figure 2

The effects of different caffeine concentrations on systolic [Ca²⁺]_i in control and heart failure sheep ventricular myocytes. (A) Time course of changes in cytoplasmic Ca measured with fura-2 in control sheep. Cells were stimulated with current pulses. Caffeine (125 µM) was applied for the period shown. (B) Mean data showing the concentration-dependence of the increase in the first Ca transient on the application of caffeine n = 7 cells from three animals. (C) Time course of changes in cytoplasmic Ca measured with fluo-3 in sheep with heart failure. Cells were stimulated with current pulses. Caffeine (500 µM) was applied for the period shown. (D) Average data for Ca transient amplitudes in heart failure for (from left to right); control, first beat in caffeine, steady state in caffeine. n = 5 cells from three animals. Asterisk represents a statically significant difference.

Figure 3

The effects of caffeine on systolic [Ca²⁺]_i in canine ventricular myocytes. (A) Time course of changes of cytoplasmic Ca measured with fura-2. Cells were stimulated with current pulses. Caffeine (500 µM) was applied for the period shown. (B) Specimen records of Ca (top) and membrane potential (bottom) taken at the points indicated on A. (C) Average data for Ca transient (top) and APD (bottom) for (from left to right); control, first beat in caffeine, steady state in caffeine, first beat on wash, steady-state wash. n = 12 cells from four animals. Asterisk represents a statically significant difference.

Figure 4

Time course of the onset and recovery of the effects of caffeine in voltage-clamped canine ventricular myocytes. (A) Time course of changes in cytoplasmic Ca measured with fluo-3. The cell was stimulated at 0.5 Hz with a 100 ms duration depolarizing pulse from −40 to 10 mV. Caffeine (500 µM) was applied for the period shown. (B) Specimen records of Ca obtained at the times shown in A. (C) Average data (n = 10 cells from three animals) for the amplitude of the Ca transient. (D and E) Rate constant of recovery of the amplitude of the Ca transient under, respectively, voltage and current clamp. In both panels, the left-hand (red) bar is during caffeine and the right-hand (blue) on removal. Asterisk represents a statically significant difference.

Figure 5

The effects of caffeine on Ca fluxes in voltage-clamped canine ventricular myocytes. (A) Membrane currents in response to a 100 ms duration depolarizing pulse from −40 to 10 mV. The right-hand panel shows expanded records of NCX current on repolarization. Traces show (i) control, (ii) first beat in caffeine, (iii) steady-state in caffeine, (iv) first beat in wash, and (v) steady-state wash. (B) Measurement of total SR Ca content. Traces show (from top to bottom) Ca, membrane current, integrated current. Caffeine (10 mM) was applied as shown by the horizontal solid bars. The three records were obtained: (from left to right) control, steady state in caffeine, and steady-state wash. (C) Average sarcolemmal flux data. The left-hand five bars show influx and the right-hand efflux for six cells from three animals. (D) Average measurement of total SR Ca content for five cells from three animals. Asterisk represents a statically significant difference.

Figure 6

Simultaneous measurement of SR and cytoplasmic Ca. (A) Time course traces showing cytoplasmic Ca (top), measured with fluo-3; and SR Ca (bottom) measured with mag-fura-2. (B) Time course of the entire experiment. The rectangle shows the period covered by A. 10 mM Ca, 1 mM tetracaine, and 10 mM caffeine were applied as shown. (C) Specimen data showing (from top to bottom) cytoplasmic Ca, SR Ca, membrane potential.

Figure 7

Changes of diastolic and systolic SR Ca content. (A) Original data showing SR Ca content measured with mag-fura-2. Caffeine (500 µM) was applied as shown. (B) Dependence of the amplitude of the SR systolic depletion on the diastolic SR Ca content. The data points are taken from the regions shown on A. To permit comparison between different cells, all fluorescence ratios have been normalized to the diastolic ratio in control. (C) Dependence of systolic SR Ca on diastolic SR Ca. The points in B and C are the mean of 12 cells from six animals. (D) Model of the dependence of SR Ca release on diastolic content. The lines show data for r = 1 and r = 0.2 (see text for details). (E) Model of the dependence of systolic SR Ca content on diastolic content. The solid line is for r = 1, and the dashed for r = 0.2. The dotted line is the line of identity.