M. E. Díaz

Integrative Analysis of Calcium Cycling in Cardiac Muscle

Abstract— The control of intracellular calcium is central to regulation of contractile force in cardiac muscle. This review illustrates how analysis of the control of calcium requires an integrated approach in which several systems are considered. Thus, the calcium content of the sarcoplasmic reticulum (SR) is a major determinant of the amount of Ca2+ released from the SR and the amplitude of the Ca2+ transient. The amplitude of the transient, in turn, controls Ca2+ fluxes across the sarcolemma and thence SR content. This control of SR content influences the response to maneuvers that modify, for example, the properties of the SR Ca2+ release channel or ryanodine receptor. Specifically, modulation of the open probability of the ryanodine receptor produces only transient effects on the Ca2+ transient as a result of changes of SR content. These interactions between various Ca2+ fluxes are modified by the Ca2+ buffering properties of the cell. Finally, we predict that, under some conditions, the above interactions can result in instability (such as alternans) rather than ordered control of contractility.

Sarcoplasmic Reticulum Calcium Content Fluctuation Is the Key to Cardiac Alternans

Abstract— The aim of this work was to investigate whether beat-to-beat alternation in the amplitude of the systolic Ca2+ transient (Ca2+ alternans) is due to changes of sarcoplasmic reticulum (SR) Ca2+ content, and if so, whether the alternans arises due to a change in the gain of the feedback controlling SR Ca2+ content. We found that, in rat ventricular myocytes, stimulating with small (20 mV) depolarizing pulses produced alternans of the amplitude of the Ca2+ transient. Confocal measurements showed that the larger transients resulted from propagation of Ca2+ waves. SR Ca2+ content (measured from caffeine-evoked membrane currents) alternated in phase with the alternans of Ca2+ transient amplitude. After a large transient, if SR Ca2+ content was elevated by brief exposure of the cell to a Na+-free solution, then the alternans was interrupted and the next transient was also large. This shows that changes of SR Ca2+ content are sufficient to produce alternans. The dependence of Ca2+ transient amplitude on SR content was steeper under alternating than under control conditions. During alternation, the Ca2+ efflux from the cell was also a steeper function of SR Ca2+ content than under control. We attribute these steeper relationships to the fact that the larger responses in alternans depend on wave propagation and that wave propagation is a steep function of SR Ca2+ content. In conclusion, alternans of systolic Ca2+ appears to depend on alternation of SR Ca2+ content. This, in turn results from the steep dependence on SR Ca2+ content of Ca2+ release and therefore Ca2+ efflux from the cell as a consequence of wave propagation.

Measurement of sarcoplasmic reticulum Ca2+ content and sarcolemmal Ca2+ fluxes in isolated rat ventricular myocytes during spontaneous Ca2+ release.

1 Intracellular calcium concentration ([Ca2+]i) and Na+–Ca2+ exchange currents were measured in calcium‐overloaded voltage‐clamped rat ventricular myocytes loaded with the Ca2+sensitive fluorescent indicator indo‐1. Sarcoplasmic reticulum (SR) Ca2+ content was measured from the integral of the caffeineevoked current. In cells that had spontaneous SR Ca2+ release in 1 mm external Ca2+ concentration ([Ca2+]o), raising [Ca2+]o increased the frequency of release with no effect on SR Ca2+ content. In quiescent cells, increased [Ca2+]o produced spontaneous Ca2+ release associated with increased SR Ca2+ content. Further increase of [Ca2+]o had no effect on SR Ca2+ content. The amount of Ca2+ leaving the cell during each release was constant over a wide range of frequencies and [Ca2+]o values. It appears there is a maximum level of SR Ca2+ content, perhaps because spontaneous Ca2+ release results when the content reaches a threshold. 2 From the relationship between [Ca2+]i and Na+–Ca2+ exchange current during a caffeine response, it is possible to estimate the changes in Na+–Ca2+ exchange current expected from a change of [Ca2+]i. The data show that the calcium oscillations contribute a significant fraction of the total extra Ca2+ efflux induced by increasing [Ca2+]o. Raising [Ca2+]o decreased the rate of calcium removal from the cell as measured from the rate of decay of the caffeine response, suggesting that both inhibition of Ca2+ efflux and increased Ca2+ entry account for the Ca2+ overload at elevated [Ca2+]o. 3 Inhibiting spontaneous SR Ca2+ release increases resting [Ca2+]i. The Ca2+ efflux is identical to that in the presence of release. It is concluded that spontaneous release of calcium, although potentially arrhythmogenic, is an effective way to activate Ca2+ efflux in overloaded conditions and minimizes any increase of diastolic tension.

Modulation of CICR has no maintained effect on systolic Ca2+: simultaneous measurements of sarcoplasmic reticulum and sarcolemmal Ca2+ fluxes in rat ventricular myocytes

1 The effects of modulating Ca2+‐induced Ca2+ release (CICR) in single cardiac myocytes were investigated using low concentrations of caffeine (< 500 μm) in reduced external Ca2+ (0.5 mm). Caffeine produced a transient potentiation of systolic [Ca2+]i (to 800 % of control) which decayed back to control levels. 2 Caffeine decreased the steady‐state sarcoplasmic reticulum (SR) Ca2+ content. As the concentration of caffeine was increased, both the potentiation of the systolic Ca2+ transient and the decrease in SR Ca2+ content were increased. At higher concentrations, the potentiating effect decayed more rapidly but the rate of recovery on removal of caffeine was unaffected. 3 A simple model in which caffeine produces a fixed increase in the fraction of SR Ca2+ which is released could account qualitatively but not quantitatively for the above results. 4 The changes in total [Ca2+] during systole were obtained using measurements of the intracellular Ca2+ buffering power. Caffeine initially increased the fractional release of SR Ca2+. This was followed by a decrease to a level greater than that under control conditions. The fraction of systolic Ca2+ which was pumped out of the cell increased abruptly upon caffeine application but then recovered back to control levels. The increase in fractional loss is due to the fact that, as the cytoplasmic buffers become saturated, a given increase in systolic total[Ca2+] produces a larger increase in free [Ca2+] and thence of Ca2+ efflux. 5 These results confirm that modulation of the ryanodine receptor has no maintained effect on systolic Ca2+ and show the interdependence of SR Ca2+ content, cytoplasmic Ca2+ buffering and sarcolemmal Ca2+ fluxes. Such analysis is important for understanding the cellular basis of inotropic interventions in cardiac muscle.

Depressed Ryanodine Receptor Activity Increases Variability and Duration of the Systolic Ca2+ Transient in Rat Ventricular Myocytes

Abstract— Sarcoplasmic reticulum (SR) Ca2+ release, through the ryanodine receptor (RyR), is essential for the systolic Ca2+ transient and thus the cardiac contractile function. The aim of this study was to examine the effects on the spatial organization of the systolic Ca2+ transient of depressing RyR open probability (Po) with tetracaine or intracellular acidification. Voltage-clamped, fluo-3–loaded myocytes were studied using confocal microscopy. Depressing RyR Po increased the variability of the Ca2+ transient amplitude between different regions of the cell. This variability often produced alternans with a region producing large and small transients alternately. In addition, the raising phase of the Ca2+ transient became biphasic. The initial phase was constant but the second was variable and propagated as a wave through part of the cell. That both phases involved SR Ca2+ release was shown by their reduction by caffeine. Regional [Ca2+]i alternans was accompanied by a much smaller degree of alternans at the whole cell level. We suggest that, in tetracaine or acidosis, the initial phase of the Ca2+ transient results from Ca2+ release via RyRs directly activated by adjacent L-type Ca2+ channels. At some sites, this will activate neighboring RyRs and a Ca2+ wave will propagate via activation of other RyRs. This work is the first demonstration that decreased RyR Po alone can produce disarray of the Ca2+ release process and initiate alternans.

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes

Abstract This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca2+]i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca2+]i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114±18 µmol l–1 and Kd of 0.59±0.17 µM (n=8). During the systolic rise of [Ca2+]i, the measured changes of [Ca2+]i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca.

Coordinated Control of Cell Ca2+ Loading and Triggered Release From the Sarcoplasmic Reticulum Underlies the Rapid Inotropic Response to Increased L-Type Ca2+ Current

Abstract — The aim of this study was to investigate how sarcoplasmic reticulum (SR) Ca2+ content and systolic Ca2+ are controlled when Ca2+ entry into the cell is varied. Experiments were performed on voltage-clamped rat and ferret ventricular myocytes loaded with fluo-3 to measure intracellular Ca2+ concentration ([Ca2+]i). Increasing external Ca2+ concentration ([Ca2+]o) from 1 to 2 mmol/L increased the amplitude of the systolic Ca2+ transient with no effect on SR Ca2+ content. This constancy of SR content is shown to result because the larger Ca2+ transient activates a larger Ca2+ efflux from the cell that balances the increased influx. Decreasing [Ca2+]o to 0.2 mmol/L decreased systolic Ca2+ but produced a small increase of SR Ca2+ content. This increase of SR Ca2+ content is due to a decreased release of Ca2+ from the SR resulting in decreased loss of Ca2+ from the cell. An increase of [Ca2+]o has two effects: (1) increasing the fraction of SR Ca2+ content, which is released on depolarization and (2) increasing Ca2+ entry into the cell. The results of this study show that the combination of these effects results in rapid changes in the amplitude of the systolic Ca2+ transient. In support of this, the changes of amplitude of the transient occur more quickly following changes of [Ca2+]o than following refilling of the SR after depletion with caffeine. We conclude that the coordinated control of increased Ca2+ entry and greater fractional release of Ca2+ is an important factor in regulating excitation-contraction coupling.

Comparison of subsarcolemmal and bulk calcium concentration during spontaneous calcium release in rat ventricular myocytes

1. The aim of these experiments was to compare the time course of changes in intracellular Ca2+ concentration ([Ca2+]i) measured in the bulk cytoplasm with those estimated to occur near the sarcolemma. Sarcolemmal Na(+)‐Ca2+ exchange current and [Ca2+]i were measured in single, voltage‐clamped ventricular myocytes. 2. Spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) resulted in a transient inward current. This current developed and decayed more quickly than the accompanying changes in [Ca2+]i (measured with indo‐1) resulting in a hysteresis between [Ca2+]i and current. A similar hysteresis was also observed if [Ca2+]i was elevated with caffeine and was removed if the current was low pass filtered with a time constant of 132 ms. 3. Digital video imaging (using fluo‐3 or calcium green‐1 to measure [Ca2+]i) allowed measurement of [Ca2+]i at all points in the cell during the wave of spontaneous Ca2+ release. The hysteresis between [Ca2+]i and current remained, even after allowing for the spatial and temporal properties of this wave. 4. The hysteresis can be accounted for if there is a barrier to diffusion of Ca2+ ions separating the bulk cytoplasm from the space under the sarcolemma (into which Ca2+ is released from the sarcoplasmic reticulum). The calculated subsarcolemmal [Ca2+] rises and falls more quickly (and reaches a higher peak) than does the bulk [Ca2+]. The delay introduced by this barrier is equivalent to a time constant of 133 ms. 5. The subsarcolemmal space described in this paper may be equivalent to the ‘fuzzy space’ previously suggested to be important in controlling SR Ca2+ release.

Enhanced Ca2+ Current and Decreased Ca2+ Efflux Restore Sarcoplasmic Reticulum Ca2+ Content After Depletion

[Ca2+]i was measured using the fluorescent indicator indo 1 in voltage-clamped ferret and rat ventricular myocytes. The Ca2+ content of the sarcoplasmic reticulum (SR) was estimated from the integral of the Na(+)-Ca2+ exchange current activated by caffeine. Refilling of the SR after caffeine removal was enhanced by stimulation. As the systolic Ca2+ transient recovered, the integral of the L-type Ca2+ current decreased and that of the Na(+)-Ca2+ exchange tail current increased. For the early pulses, the gain of Ca2+ via the Ca2+ current is greater than the loss via the exchanger, and during steady state stimulation, the fluxes are equal. The difference in the integrals gives a measure of the net gain of cell Ca2+ with each pulse. When these are summed, the calculated gain of cell Ca2+ agrees well with the increase of SR Ca2+ produced by stimulation, as measured from the caffeine-evoked currents. There was a nonlinear relationship between SR Ca2+ content and the magnitude of the systolic Ca2+ transient such that at high SR Ca2+ content a given increase of content had a greater effect on the Ca2+ transient than did an increase at low SR content. In conclusion, the effects of systolic Ca2+ on the Ca2+ current and Na(+)-Ca2+ exchange current provide a means to regulate SR Ca2+ content and thence the systolic Ca2+ transient.

Stimulation of Ca-induced Ca release only transiently increases the systolic Ca transient: measurements of Ca fluxes and sarcoplasmic reticulum Ca

OBJECTIVE To investigate the effects of stimulating calcium induced Ca release with low concentrations (100-200 microM) of caffeine and, in particular, to study the cellular mechanisms responsible for the transient responses found previously. METHODS Experiments were performed on isolated rat ventricular myocytes. Intracellular calcium concentration ([Ca2+]i) was measured with Indo-1, the cells were voltage-clamped with the perforated patch technique and sarcoplasmic reticulum (s.r.) Ca content was estimated from the integral of the caffeine-evoked current. RESULTS The systolic Ca transient produced by the first depolarization in the presence of caffeine was larger than the control. Over the next few pulses the magnitude of the Ca transient returned to control levels despite the maintained presence of caffeine. The s.r. Ca content was decreased by 9% after one pulse in caffeine and by 21% after several pulses in caffeine. The first pulse in the low concentration of caffeine was followed by an enhanced inward (Na-Ca exchange) current tail indicating increased efflux of calcium from the cell. The extra loss of calcium calculated from the tail current agreed quantitatively with that from the change of s.r. Ca content. CONCLUSIONS These results show that stimulating calcium induced calcium release produces only a transient increase of the systolic Ca transient. This is due to the larger Ca transient decreasing the s.r. Ca content. It is concluded that any agent whose sole mode of action is stimulation of calcium-induced calcium release will not produce a maintained inotropic effect. The consequences of this for the effects of other modulators of calcium induced calcium release are discussed.