D. A. Eisner

The steady state TTX-sensitive ("window") sodium current in cardiac Purkinje fibres

Voltage clamp experiments on isolated sheep Purkinje fibres showed an increase of the steady state outward membrane current, over the potential range −65 mV to −15 mV, in the presence of tetrodotoxin (TTX, 3 · 10−5 M). This “window” current is considered to be the steady state component of the fast sodium current (INa), resulting from the crossover of the activation and inactivation curves which govern the opening of the sodium channel.TTX had no significant effect on the reversal potential, activation curve, kinetics or instantaneous I–V relationship of the pacemaker currentIK2.The window found in these experiments extends to potentials well into the range of the action potential plateau. Consequently small changes of the steady stateINa might have large effects on the action potential duration. The effects of TTX and local anaesthetics are discussed in this context.

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.

Sarcoplasmic Reticulum Ca2+ and Heart Failure: Roles of Diastolic Leak and Ca2+ Transport

Heart failure (HF) is a leading cause of death and enormous effort has focused at understanding the molecular and cellular mechanisms of the decreased cardiac contractility. While changes of other components contribute, it is generally agreed that much of the contractile deficit is due to reduced myocyte Ca2+ transients.1,2 Alterations in Ca2+ current ( I Ca) and action potential characteristics are also seen in HF, but a central factor limiting Ca2+ transient amplitude is a decrease of sarcoplasmic reticulum (SR) Ca2+ content.3–6 HF is extremely complex, but it is easy to appreciate how reduced SR Ca2+ content would reduce SR Ca2+ release, myofilament activation, and contractility. Despite agreement that SR Ca2+ content is reduced in HF, controversy exists about why SR content is low. SR Ca2+ content reflects the balance between Ca2+ uptake (via SERCA) and Ca2+ efflux via ryanodine receptor (RyR). Thus, reduced SR content in HF must be due to reduced Ca2+ pumping by SERCA or increased SR Ca2+ leak via RyRs. Both are supported by experimental data (below). Transsarcolemmal Ca2+ fluxes also affect SR Ca2+ load. That is, reduced Ca2+ influx (eg, via I Ca) or enhanced Ca2+ extrusion via Na+-Ca2+ exchange (NCX) can unload the SR. Results are not unanimous, but most groups find little alteration in peak I Ca density in HF, while many find evidence of enhanced NCX expression and function.1,2 Increased NCX function can compete with SERCA during [Ca2+]i decline, extruding more Ca2+ from the cell and depleting the SR. In the new steady state, a larger fraction of activating Ca2+ also enters the cell at each beat in HF (eg, smaller Ca2+ release causes less …Heart failure (HF) is a leading cause of death and enormous effort has focused at understanding the molecular and cellular mechanisms of the decreased cardiac contractility. While changes of other components contribute, it is generally agreed that much of the contractile deficit is due to reduced myocyte Ca2+ transients.1,2 Alterations in Ca2+ current ( I Ca) and action potential characteristics are also seen in HF, but a central factor limiting Ca2+ transient amplitude is a decrease of sarcoplasmic reticulum (SR) Ca2+ content.3–6 HF is extremely complex, but it is easy to appreciate how reduced SR Ca2+ content would reduce SR Ca2+ release, myofilament activation, and contractility. Despite agreement that SR Ca2+ content is reduced in HF, controversy exists about why SR content is low. SR Ca2+ content reflects the balance between Ca2+ uptake (via SERCA) and Ca2+ efflux via ryanodine receptor (RyR). Thus, reduced SR content in HF must be due to reduced Ca2+ pumping by SERCA or increased SR Ca2+ leak via RyRs. Both are supported by experimental data (below). Transsarcolemmal Ca2+ fluxes also affect SR Ca2+ load. That is, reduced Ca2+ influx (eg, via I Ca) or enhanced Ca2+ extrusion via Na+-Ca2+ exchange (NCX) can unload the SR. Results are not unanimous, but most groups find little alteration in peak I Ca density in HF, while many find evidence of enhanced NCX expression and function.1,2 Increased NCX function can compete with SERCA during [Ca2+]i decline, extruding more Ca2+ from the cell and depleting the SR. In the new steady state, a larger fraction of activating Ca2+ also enters the cell at each beat in HF (eg, smaller Ca2+ release causes less …

An estimate of the calcium content of the sarcoplasmic reticulum in rat ventricular myocytes

SummaryThe aim of this paper was to estimate the Ca content of the sarcoplasmic reticulum (s.r.) and to compare this with the amount of Ca which enters the cell via the calcium current in systole. The s.r. Ca content was measured electrophysiologically in voltage-clamped rat ventricular myocytes. Rapid application of caffeine produced a transient increase of [Ca2+]i which was accompanied by a transient inward Na-Ca exchange current. The integral of this current gives a measure of the Ca2+ pumped out of the cell by Na-Ca exchange. Ni2+ (5 mM) inhibited the current and decreased the rate of fall of [Ca2+]i to 32% of the control suggesting that Na-Ca exchange is responsible for 68% of Ca removal from the cytoplasm following the addition of caffeine. Correcting for the Na-Ca independent Ca removal suggests that the s.r. Ca content is equivalent to about 120 μmol per litre cell. Furthermore we estimate that, during systole, Ca entry into the cell via the sarcolemmal calcium current is equal to about 6 % of the Ca content of the s.r.

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.

Increasing Ryanodine Receptor Open Probability Alone Does Not Produce Arrhythmogenic Calcium Waves Threshold Sarcoplasmic Reticulum Calcium Content Is Required

Diastolic waves of Ca2+ release have been shown to activate delayed afterdepolarizations as well as some cardiac arrhythmias. The aim of this study was to investigate whether increasing ryanodine receptor open probability alone or in the presence of β-adrenergic stimulation produces diastolic Ca release from the sarcoplasmic reticulum (SR). When voltage-clamped rat ventricular myocytes were exposed to caffeine (0.5 to 1.0 mmol), diastolic Ca2+ release was seen to accompany the first few stimuli but was never observed in the steady state. We attribute the initial phase of diastolic Ca2+ release to a decrease in the threshold SR Ca2+ content required to activate Ca2+ waves and its subsequent disappearance to a decrease of SR content below this threshold. Application of isoproterenol (1 &mgr;mol/L) increased the amplitude of the systolic Ca2+ transient and also the SR Ca2+ content but did not usually produce diastolic Ca2+ release. Subsequent addition of caffeine, however, resulted in diastolic Ca2+ release. We estimated the time course of recovery of SR Ca2+ content following recovery from emptying with a high (10 mmol/L) concentration of caffeine. Diastolic Ca2+ release recommenced only when SR content had increased back to its final level. We conclude that increasing ryanodine receptor open probability alone does not produce arrhythmogenic diastolic Ca2+ release because of the accompanying decrease of SR Ca2+ content. β-Adrenergic stimulation increases SR content and thereby allows the increased ryanodine receptor open probability to produce diastolic Ca2+ release. The implications of these results for arrhythmias associated with abnormal ryanodine receptors are discussed.

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.

Increasing ryanodine receptor open probability alone does not produce arrhythmogenic Ca waves: threshold SR Ca content is required

Diastolic waves of Ca2+ release have been shown to activate delayed afterdepolarizations as well as some cardiac arrhythmias. The aim of this study was to investigate whether increasing ryanodine receptor open probability alone or in the presence of β-adrenergic stimulation produces diastolic Ca release from the sarcoplasmic reticulum (SR). When voltage-clamped rat ventricular myocytes were exposed to caffeine (0.5 to 1.0 mmol), diastolic Ca2+ release was seen to accompany the first few stimuli but was never observed in the steady state. We attribute the initial phase of diastolic Ca2+ release to a decrease in the threshold SR Ca2+ content required to activate Ca2+ waves and its subsequent disappearance to a decrease of SR content below this threshold. Application of isoproterenol (1 &mgr;mol/L) increased the amplitude of the systolic Ca2+ transient and also the SR Ca2+ content but did not usually produce diastolic Ca2+ release. Subsequent addition of caffeine, however, resulted in diastolic Ca2+ release. We estimated the time course of recovery of SR Ca2+ content following recovery from emptying with a high (10 mmol/L) concentration of caffeine. Diastolic Ca2+ release recommenced only when SR content had increased back to its final level. We conclude that increasing ryanodine receptor open probability alone does not produce arrhythmogenic diastolic Ca2+ release because of the accompanying decrease of SR Ca2+ content. β-Adrenergic stimulation increases SR content and thereby allows the increased ryanodine receptor open probability to produce diastolic Ca2+ release. The implications of these results for arrhythmias associated with abnormal ryanodine receptors are discussed.

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.