S. C. O'Neill

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.

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.

Omega-3 Fatty Acid Supplementation Does Not Reduce Risk of Atrial Fibrillation After Coronary Artery Bypass Surgery A Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Background—Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been reported to reduce the risk of sudden cardiac death presumed to be due to fatal ventricular arrhythmias, but their effect on atrial arrhythmias is unclear. Methods and Results—Patients (n=108) undergoing coronary artery bypass graft surgery were randomly assigned to receive 2 g/d n-3 PUFA or placebo (olive oil) for at least 5 days before surgery (median, 16 days; range, 12 to 21 days). Phospholipid n-3 PUFA were measured in serum at study entry and at surgery and in right atrial appendage tissue at surgery. Echocardiography was used to assess left ventricular function and left atrial dimensions. Postoperative continuous ECG monitoring (Lifecard CF) for 5 days or until discharge, if earlier, was performed with a daily 12-lead ECG and clinical review if patients remained in the hospital beyond 5 days. Lifecard recordings were analyzed for episodes of atrial fibrillation (AF) ≥30 seconds (primary outcome). Clinical AF, AF burden (% time in AF), hospital stay, and intensive care/high dependency care stay were measured as secondary outcomes. One hundred three patients completed the study (51 in the placebo group and 52 in the n-3 PUFA group). There were no clinically relevant differences in baseline characteristics between groups. n-3 PUFA levels were higher in serum and right atrial tissue in the active treatment group. There was no significant difference between groups in the primary outcome of AF (95% confidence interval [CI], −6% to 30%, P=0.28) in any of the secondary outcomes or in AF-free survival. Conclusions—Omega-3 PUFA do not reduce the risk of AF after coronary artery bypass graft surgery. Clinical Trial Registration—www.ukcrn.org.uk. Identifier: 4437.

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.

Fluorescence measurements of cytoplasmic and mitochondrial sodium concentration in rat ventricular myocytes.

1. The fluorescent Na+ indicator SBFI was incorporated into isolated ventricular myocytes using the acetoxymethyl (AM) ester. 2. The excitation spectrum was found to be shifted about 20 nm in the cell compared to in vitro. In the cell, an increase of [Na+] decreased fluorescence at 380 nm (F380) and had no effect at 340 nm (F340). The ratio (R = F340/F380) was used as a measure of [Na+]i. 3. In vivo calibration of SBFI for [Na+]i was obtained by equilibrating [Na+] across the plasma membrane with a divalent‐free solution in the presence of gramicidin D. 4. Selective removal of the surface membrane with saponin or digitonin released only about 50% of the indicator. Following saponin treatment, cyanide or carbonylcyanide m‐chlorphenylhydrazone (CCCP) increased the apparent [Na+] measured by the remaining (presumably mitochondrial) SBFI. It is suggested that mitochondrial [Na+] is normally less than cytoplasmic. 5. Attempts to examine the effects of metabolic inhibition on [Na+]i were hampered by changes of autofluorescence due to changes of [NADH]. It is shown that this effect can be corrected for using the isosbestic signal (excited at 340 nm). 6. Inhibition of both aerobic metabolism (with CN‐) and glycolysis (glucose removal or iodoacetate) produced a gradual increase of [Na+]i. This began before the resting contracture developed and may (via Na(+)‐Ca2+ exchange) account for some of the rise of diastolic [Ca2+]i seen in previous work. The rise of [Na+]i began at about the same time as the decrease of systolic contraction and therefore at a time when [ATP]i had begun to fall.

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.

The effect of tetracaine on supontaneous Ca2+ release and sarcoplasmic reticulum calcium content in rat ventricular myocytes

1 The effects of tetracaine were studied on voltage‐clamped rat ventricular myocytes, which exhibited Ca2+ overload as identified by supontaneous Ca2+ release from the sarcoplasmic reticulum (SR) as shown by the associated contractions. This Ca2+ release was initially abolished by tetracaine before returning at a lower frequency, but greater amplitude, than the control. On removal of tetracaine, there was a burst of supontaneous Ca2+ release activity. All these effects were dose dependent, from 25 to 200 μm tetracaine. 2 The supontaneous Ca2+ release activated an inward Na+–Ca2+ exchange current as Ca2+ was pumped out of the cell. The integral of this current (i.e. the Ca2+ efflux) was increased in the presence of tetracaine. The calcium efflux per unit time was unaffected by tetracaine. 3 The SR Ca2+ content was increased by tetracaine, as shown by the integral of the caffeine‐evoked Na+–Ca2+ exchange current. The increase of SR Ca2+ content was equal to the extra Ca2+ lost from the cell during the burst on removal of tetracaine, and to estimates of the extra calcium gained over the quiescent period following addition of tetracaine. 4 It is concluded that partial inhibition of calcium‐induced calcium release increases SR Ca2+ content. In the steady state, cell Ca2+ balance is maintained as the lower frequency of supontaneous release (that activates efflux) is compensated for by their greater size.

What role does modulation of the ryanodine receptor play in cardiac inotropy and arrhythmogenesis

In this article we review the role of the Ryanodine Receptor (RyR) in cardiac inotropy and arrhythmogenesis. Most of the calcium that activates cardiac contraction comes from the sarcoplasmic reticulum (SR) from where it is released through the RyR. The amplitude of the systolic Ca transient depends steeply on the SR Ca content and it is therefore important that SR content be regulated. This regulation occurs via changes of SR Ca content affecting systolic Ca and thence sarcolemmal Ca fluxes. In the steady state, the cardiac myocyte must be in Ca flux balance on each beat and this has implications for understanding even simple inotropic manoeuvres. The main part of the review considers the effects of modulating the RyR on systolic Ca. Potentiation of RyR opening produces an increase of the amplitude of the Ca transient but this effect disappears within a few beats because the increased sarcolemmal efflux of Ca decreases SR Ca content. We conclude that it is therefore unlikely that potentiation of the RyR by phosphorylation plays a dominant role in the actions of positive inotropic agents such as beta-adrenergic stimulation. Some cardiac arrhythmias result from release of Ca from the SR in the form of waves. This is best known to occur when the SR is overloaded with calcium. Mutations in the RyR also produce cardiac arrhythmias attributed to Ca waves due to leaky RyRs and a similar leak has been suggested to contribute to arrhythmias in heart failure. We show that, due to compensatory changes of SR Ca content, simply making the RyR leaky does not produce Ca waves in the steady state and that SR Ca content is critical in determining whether Ca waves occur.

The effect of tetracaine on stimulated contractions, sarcoplasmic reticulum Ca2+ content and membrane current in isolated rat ventricular myocytes

1 The effects of tetracaine were examined on rat ventricular myocytes. In both field‐stimulated and voltage‐clamped cells tetracaine (100–200 μM) produced an initial decrease of contraction before a recovery towards the control level. Removal of tetracaine produced a transient overshoot of contraction to levels greater than the control. 2 The transient decrease of contraction produced by tetracaine was accompanied by a small transient increase in the integral of the L‐type Ca2+ current and a larger transient decrease of the Na+‐Ca2+ exchange current on repolarization. These are attributed to decreased systolic release of Ca2+. On removal of tetracaine there was an increase of the Na+‐Ca2+ exchange current. Before the addition of tetracaine, calculated Ca2+ influx and efflux across the sarcolemma were approximately equal. On adding tetracaine, efflux was transiently less than influx and, on removal of tetracaine, efflux was greater than influx. 3 These changes in Ca2+ fluxes result in an increase of cell Ca2+ during exposure to tetracaine. The calculated magnitude of this increase was equal to that measured directly by applying caffeine (20 mM) to release sarcoplasmic reticulum (SR) Ca2+ and integrating the resulting Na+‐Ca2+ exchange current. 4 It is concluded that the effects of tetracaine can be accounted for by depression of calcium‐induced Ca2+ release (CICR). The response is transient because the inhibition is compensated for by an increase of SR Ca2+ content such that there is no steady‐state effect on the magnitude of the systolic Ca2+ transient. The consequences of this result for the effects of other modulators of CICR are discussed.

Inhibition of sarcoplasmic reticulum function by polyunsaturated fatty acids in intact, isolated myocytes from rat ventricular muscle

1 We have studied the effects of two polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on spontaneous and electrically stimulated contractions in single, isolated ventricular myocytes from rat hearts. 2 The frequency of spontaneous waves of calcium release and contraction (induced by elevation of the bathing calcium concentration) is reduced in the presence of EPA. At the same time the resting level of intracellular calcium falls, the resting cell length increases and the amplitude of shortening decreases. All these effects are reversed on removal of EPA. 3 Imaging of the waves of calcium release shows that the amplitude and the rate of propagation of the wave is increased in EPA. Consistent with the increased amplitude, integration of the caffeine‐induced Na+‐Ca2+ exchange current (a measure of the sarcoplasmic reticulum (SR) calcium content) is increased by both EPA and DHA. 4 EPA has a maintained negative inotropic effect on voltage clamped myocytes. This seems to be entirely due to inhibition of the L‐type calcium current. Smaller depolarising pulses in control conditions that elicit the same calcium current as in EPA also activate the same level of contraction. This is in spite of the increased SR calcium content in EPA. 5 It is concluded that PUFAs have two effects on the SR; they reduce the availability of calcium for uptake and they inhibit the release mechanism. Both of these effects should lower the frequency of spontaneous waves of calcium release. As spontaneous release of calcium can initiate arrhythmias, some of the anti‐arrhythmic action of PUFAs must be exerted at the level of the SR.