Abstract-The aim of this work was to investigate whether beat-to-beat alternation in the amplitude of the systolic Ca 2ϩ transient (Ca 2ϩ alternans) is due to changes of sarcoplasmic reticulum (SR) Ca 2ϩ content, and if so, whether the alternans arises due to a change in the gain of the feedback controlling SR Ca 2ϩ content. We found that, in rat ventricular myocytes, stimulating with small (20 mV) depolarizing pulses produced alternans of the amplitude of the Ca 2ϩ transient. Confocal measurements showed that the larger transients resulted from propagation of Ca 2ϩ waves. SR Ca 2ϩ content (measured from caffeine-evoked membrane currents) alternated in phase with the alternans of Ca 2ϩ transient amplitude. After a large transient, if SR Ca 2ϩ content was elevated by brief exposure of the cell to a Na ϩ -free solution, then the alternans was interrupted and the next transient was also large. This shows that changes of SR Ca 2ϩ content are sufficient to produce alternans. The dependence of Ca 2ϩ transient amplitude on SR content was steeper under alternating than under control conditions. During alternation, the Ca 2ϩ efflux from the cell was also a steeper function of SR Ca 2ϩ 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 Ca 2ϩ content.

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 Ca 2ϩ released from the SR and the amplitude of the Ca 2ϩ transient. The amplitude of the transient, in turn, controls Ca 2ϩ 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 Ca 2ϩ release channel or ryanodine receptor. Specifically, modulation of the open probability of the ryanodine receptor produces only transient effects on the Ca 2ϩ transient as a result of changes of SR content. These interactions between various Ca 2ϩ fluxes are modified by the Ca 2ϩ 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.

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. 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. 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.

Abstract-Sarcoplasmic reticulum (SR) Ca 2ϩ release, through the ryanodine receptor (RyR), is essential for the systolic Ca 2ϩ transient and thus the cardiac contractile function. The aim of this study was to examine the effects on the spatial organization of the systolic Ca 2ϩ transient of depressing RyR open probability (P o ) with tetracaine or intracellular acidification. Voltage-clamped, fluo-3-loaded myocytes were studied using confocal microscopy. Depressing RyR P o increased the variability of the Ca 2ϩ 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 Ca 2ϩ transient became biphasic. The initial phase was constant but the second was variable and propagated as a wave through part of the cell. transient as one of the main causes contributing to impaired performance of the heart during cardiac hypertrophy and failure. [5][6][7] A decrease in the number of RyRs activated may underlie some models of heart failure, 8 but other work 5 did not report changes of RyR expression and found that the Ca 2ϩ transients were reduced in amplitude due to decreased ability of the L-type Ca 2ϩ current to trigger SR Ca 2ϩ release. A qualitatively different result was observed in a study of cells from a region bordering an infarct, where dyssynchronous Ca 2ϩ release was observed. 9 In atrial cells, glycolytic inhibition results in alternans, and it was suggested that this arose due to metabolic effects on SR Ca 2ϩ release. 10,11 This possible linkage between alternans and SR Ca 2ϩ release is interesting inasmuch as alternans is prominent in heart failure 12 Given the complexity of the conditions reviewed above, it is important to know what the effects on the spatial organization of the Ca 2ϩ transient are of only changing RyR P o . We have therefore examined the effects of tetracaine and acidosis. We find that, in addition to changes in amplitude, there are marked alterations in both the spatial and temporal properties of the Ca 2ϩ transient. Materials and MethodsAll experiments were performed on single ventricular myocytes isolated from Wistar rats by collagenase/protease digestion as described. 13 Intracellular Ca 2ϩ was measured after loading myocytes with the acetoxymethyl ester of fluo-3 or fluo-4 (5 mol/L) for 5 minutes. Confocal Ca 2ϩ measurements were made using a BioRad 1024 confocal microscope in line-scan mode and were synchronized with membrane current measurements. Cells were stimulated with 100-ms pulses (from Ϫ40 to 0 mV) at 0.5 Hz. Voltage clamp (switch-clamp mode) was imposed using the perforated patch technique with amphotericin-B. Electrodes (1.5 to 3 M⍀ resistance) were Original

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. 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. 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. 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. 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.