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ABSTRACT: 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.
Journal of Molecular and Cellular Cardiology 01/2009; 46(4):474-81. · 5.17 Impact Factor
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ABSTRACT: The major effect of Na/Ca exchange (NCX) on the systolic Ca transient is secondary to its effect on the Ca content of the sarcoplasmic reticulum (SR). SR Ca content is controlled by a mechanism in which an increase of SR Ca produces an increase in the amplitude of the systolic Ca transient. This, in turn, increases Ca efflux on NCX as well as decreasing entry on the L-type current resulting in a decrease of both cell and SR Ca content. This control mechanism also changes the response to other maneuvers that affect excitation-contraction coupling. For example, potentiating the opening of the SR Ca release channel (ryanodine receptor, RyR) with caffeine produces an immediate increase in the amplitude of the systolic Ca transient. However, this increases efflux of Ca from the cell on NCX and then decreases SR Ca content until a new steady state is reached. Changing the activity of NCX (by decreasing external Na) changes the level of SR Ca reached by this mechanism. If the cell and SR are overloaded with Ca then Ca waves appear during diastole. These waves activate the electrogenic NCX and thereby produce arrhythmogenic-delayed afterdepolarizations. A major challenge is how to remove this arrhythmogenic Ca release without compromising the normal systolic release. We have found that application of tetracaine to decrease RyR opening can abolish diastolic release while simultaneously potentiating the systolic release.
Annals of the New York Academy of Sciences 04/2007; 1099:315-25. · 3.15 Impact Factor
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Cardiovascular Research 02/2007; 73(1):247-8; author reply 249-50. · 6.06 Impact Factor
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Annals of the New York Academy of Sciences 12/2006; 639(1):444 - 452. · 3.15 Impact Factor
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ABSTRACT: The aim of this work was to investigate whether it is possible to remove arrhythmogenic Ca2+ release from the sarcoplasmic reticulum that occurs in calcium overload without compromising normal systolic release. Exposure of rat ventricular myocytes to isoproterenol (1 micromol/L) resulted in an increased amplitude of the systolic Ca2+ transient and the appearance of waves of diastolic Ca2+ release. Application of tetracaine (25 to 50 micromol/L) decreased the frequency or abolished the diastolic Ca2+ release. This was accompanied by an increase in the amplitude of the systolic Ca2+ transient. Cellular Ca2+ flux balance was investigated by integrating Ca2+ entry (on the L-type Ca2+ current) and efflux (on Na-Ca2+ exchange). Isoproterenol increased Ca2+ influx but failed to increase Ca2+ efflux during systole (because of the abbreviation of the duration of the Ca2+ transient). To match this increased influx the bulk of Ca2+ efflux occurred via Na-Ca2+ exchange during a diastolic Ca2+ wave. Subsequent application of tetracaine increased systolic Ca2+ efflux and abolished the diastolic efflux. The increase of systolic efflux in tetracaine resulted from both increased amplitude and duration of the systolic Ca2+ transient. In the presence of isoproterenol, those Ca2+ transients preceded by diastolic release were smaller than those where no diastolic release had occurred. When tetracaine was added, the amplitude of the Ca2+ transient was similar to those in isoproterenol with no diastolic release and larger than those preceded by diastolic release. We conclude that tetracaine increases the amplitude of the systolic Ca2+ transient by removing the inhibitory effect of diastolic Ca2+ release.
Circulation Research 06/2006; 98(10):1299-305. · 9.49 Impact Factor
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ABSTRACT: [Ca2+]i is used as a signal in many tissues. In this review we discuss the mechanisms that regulate [Ca2+]i and, importantly, what determines their stability. Brief mention is made of the effects of feedback gain and delays on stability. The control of cytoplasmic Ca concentration is shown to be generally stable as Ca pumping is essentially an instantaneous function of [Ca2+]i. In contrast, regulation of the Ca content of intracellular stores may be less stable. One example of this is instability in the control of sarcoplasmic reticulum (SR) Ca content in cardiac muscle. An increase of SR Ca content increases the systolic Ca transient amplitude. This in turn decreases Ca influx into the cell and increases efflux, thereby restoring SR Ca to control levels. This feedback system has an inherent delay and is potentially unstable if the gain is increased beyond a certain level. This instability produces Ca transients of alternating amplitude and may contribute to the clinical syndrome of pulsus alternans.
Experimental Physiology 02/2005; 90(1):3-12. · 3.21 Impact Factor
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The 4th Mammalian Myocardium MeetingThe 4th Mammalian Myocardium Meeting, Bristol University; 01/2005
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Biophysical Society Meeting AbstractsBiophysical Society Meeting Abstracts; 01/2005
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The Journal of Physiology 09/2004; 501(1):3 - 16. · 4.72 Impact Factor
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The Journal of Physiology 09/2004; 502(3):471 - 479. · 4.72 Impact Factor
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ABSTRACT: Waves of calcium-induced calcium release occur in a variety of cell types and have been implicated in the origin of cardiac arrhythmias. We have investigated the effects of inhibiting the SR Ca(2+)-ATPase (SERCA) with the reversible inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on the properties of these waves. Cardiac myocytes were voltage clamped at a constant potential between -65 and -40 mV and spontaneous waves evoked by increasing external Ca(2+) concentration to 4 mm. Application of 100 microm TBQ decreased the frequency of waves. This was associated with increases of resting [Ca(2+)](i), the time constant of decay of [Ca(2+)](i) and the integral of the accompanying Na(+)-Ca(2+) exchange current. There was also a decrease in propagation velocity of the waves. There was an increase of the calculated Ca(2+) efflux per wave. The SR Ca(2+) content when a wave was about to propagate decreased to 91.7 +/- 3.2%. The period between waves increased in direct proportion to the Ca(2+) efflux per wave meaning that TBQ had no effect on the Ca(2+) efflux per unit time. We conclude that (i) decreased wave frequency is not a direct consequence of decreased Ca(2+) pumping by SERCA between waves but, rather, to more Ca(2+) loss on each wave; (ii) inhibiting SERCA increases the chance of spontaneous Ca(2+) release propagating at a given SR content.
The Journal of Physiology 09/2004; 559(Pt 1):121-8. · 4.72 Impact Factor
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S. C. O'Neill
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ABSTRACT: We have investigated the role of changes of intracellular pH (pHi) in the effects of metabolic blockade (cyanide plus 2-deoxyglucose) on Ca2+ release from the sarcoplasmic reticulum (SR) in rat ventricular myocytes. pHi and cell length were measured simultaneously. Metabolic blockade decreased the frequency of Ca2+ waves, an effect previously shown to be due to inhibition of Ca2+ release from the SR. This was accompanied by an intracellular acidification. Intracellular acidification was produced in the absence of metabolic inhibition by application of sodium butyrate. A maintained intracellular acidosis produced a decrease of wave frequency. A hysteresis between pHi and wave frequency was observed such that during the onset of the acidification the wave frequency decreased more than in the steady state. Comparison of the steady state relationship between pHi and wave frequency showed that the decrease of wave frequency produced by metabolic blockade was greater than could be accounted for simply by the accompanying decrease of pHi. In other experiments the buffering power of the solution was increased. Under these conditions, metabolic blockade produced no change of pHi but the decrease of wave frequency persisted. We conclude that, although intracellular acidification occurs during metabolic blockade, it is not responsible for most of the inhibition of Ca2+ release from the SR.
The Journal of Physiology 07/2004; 550(2):413 - 418. · 4.72 Impact Factor
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ABSTRACT: Calcium release from the sarcoplasmic reticulum (SR) in cardiac muscle occurs through a specialised release channel, the ryanodine receptor, RyR, via the process of Ca-induced Ca release (CICR). The open probability of the RyR is increased by elevation of cytoplasmic Ca concentration ([Ca(2+)](i)). However, in addition to Ca, other modulators affect the RyR open probability. Agents which increase the RyR opening during systole produce a transient increase of systolic [Ca(2+)](i) followed by a return to the initial level due to a compensating decrease of SR Ca content. Increasing RyR opening during diastole decreases SR Ca content and thereby decreases systolic [Ca(2+)](i). We therefore conclude that potentiation of RyR opening will, if anything, decrease systolic [Ca(2+)](i). The effects of specific examples of modulators of the RyR, such as phosphorylation, metabolic changes, heart failure and polyunsaturated fatty acids, are discussed.
Cell Calcium 07/2004; 35(6):583-9. · 3.77 Impact Factor
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ABSTRACT: n-3 polyunsaturated fatty acids (PUFAs) can prevent life-threatening arrhythmias but the mechanisms responsible have not been established. There is strong evidence that part of the antiarrhythmic action of PUFAs is mediated through inhibition of the Ca(2+)-release mechanism of the sarcoplasmic reticulum (SR). It has also been shown that PUFAs activate protein kinase A (PKA) and produce effects in the cardiac cell similar to beta-adrenergic stimulation. We have investigated whether the inhibitory effect of PUFAs on the Ca(2+)-release mechanism is caused by direct inhibition of the SR Ca(2+)-release channel/ryanodine receptor (RyR) or requires activation of PKA. Experiments in intact cells under voltage-clamp show that the n-3 PUFA eicosapentaenoic acid (EPA) is able to reduce the frequency of spontaneous waves of Ca(2+)-release while increasing SR Ca(2+) content even when PKA activity is inhibited with H-89. This suggests that the EPA-induced inhibition of SR Ca(2+)-release is not dependent on activation of PKA. Consistent with this, single-channel studies demonstrate that EPA (10-100 microM), but not saturated fatty acids, reduce the open probability (Po) of the cardiac RyR incorporated into phospholipid bilayers. EPA also inhibited the binding of [3H]ryanodine to isolated heavy SR. Our results indicate that direct inhibition of RyR channel gating by PUFAs play an important role in the overall antiarrhythmic properties of these compounds.
Cardiovascular Research 12/2003; 60(2):337-46. · 6.06 Impact Factor
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ABSTRACT: We have investigated the role of changes of intracellular pH (pHi) in the effects of metabolic blockade (cyanide plus 2-deoxyglucose) on Ca2+ release from the sarcoplasmic reticulum (SR) in rat ventricular myocytes. pHi and cell length were measured simultaneously. Metabolic blockade decreased the frequency of Ca2+ waves, an effect previously shown to be due to inhibition of Ca2+ release from the SR. This was accompanied by an intracellular acidification. Intracellular acidification was produced in the absence of metabolic inhibition by application of sodium butyrate. A maintained intracellular acidosis produced a decrease of wave frequency. A hysteresis between pHi and wave frequency was observed such that during the onset of the acidification the wave frequency decreased more than in the steady state. Comparison of the steady state relationship between pHi and wave frequency showed that the decrease of wave frequency produced by metabolic blockade was greater than could be accounted for simply by the accompanying decrease of pHi. In other experiments the buffering power of the solution was increased. Under these conditions, metabolic blockade produced no change of pHi but the decrease of wave frequency persisted. We conclude that, although intracellular acidification occurs during metabolic blockade, it is not responsible for most of the inhibition of Ca2+ release from the SR.
The Journal of Physiology 08/2003; 550(Pt 2):413-8. · 4.72 Impact Factor
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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 (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 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 P(o) alone can produce disarray of the Ca2+ release process and initiate alternans.
Circulation Research 11/2002; 91(7):585-93. · 9.49 Impact Factor
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ABSTRACT: This review discusses the control of the amplitude of the cardiac systolic Ca transient. The Ca transient arises largely from release from the sarcoplasmic reticulum (SR). Release is triggered by calcium-induced calcium release (CICR) whereby the entry of a small amount of Ca on the L-type Ca current, "the trigger", results in the release of much more Ca from the SR. There are three potential control points: (1) the Ca content of the SR; (2) the properties of the SR Ca release channel or ryanodine receptor (RyR); (3) the amplitude of the L-type Ca current. The data reviewed show that the Ca content of the SR has pronounced effects on systolic [Ca2+]i and, reciprocally, the amount of Ca released from the SR affects sarcolemmal Ca fluxes thereby "autoregulating" SR content. Modulation of the ryanodine receptor has no steady-state effect due to compensating changes of SR Ca content. An increase of the L-type Ca current results in an abrupt increase of systolic [Ca2+]i with little change of SR content. This is because of a coordinated increase of both the trigger and loading function of the Ca current. These results emphasise the importance of considering all aspects of Ca handling in the context of SR Ca release and thus the regulation of the systolic Ca transient and contraction in cardiac muscle.
Frontiers in Bioscience 05/2002; 7:d843-52. · 3.52 Impact Factor
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ABSTRACT: Measurements were made of trans-sarcolemmal Ca(2+) fluxes and intracellular [Ca(2+)](i) in rat ventricular myocytes loaded with Indo-1 to determine how the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) suppresses spontaneous waves of Ca(2+) release. We report that in 10 microM EPA, the Ca(2+) efflux generated by individual waves increased by 11.3 +/- 4.9 % over control levels. However, wave-generated efflux per unit time fell overall by 19 +/- 5.3 %. On removal of EPA, wave frequency increased transiently such that Ca(2+) efflux was greater than normal and the cell lost 28.0 +/- 10.6 micromol l(-1) Ca(2+). This probably represents the loss of extra Ca(2+) accumulated by the sarcoplasmic reticulum (SR), while Ca(2+) release was inhibited. These results are evidence of inhibition of the SR Ca(2+)-release mechanism and reduced availability of Ca(2+) to the SR. From the relationship between average intracellular Ca(2+) and the frequency of spontaneous waves, we have calculated the relative contributions of these different mechanisms to the lower frequency of waves. In EPA, the frequency of spontaneous waves fell by 37.5 +/- 8.1 %, the majority of this (29.2 +/- 8.8 %) is due to inhibition of the Ca(2+)-release mechanism. In EPA, the rate of fall of Ca(2+) in the caffeine response (an indicator of surface membrane Ca(2+) efflux pathway activity) was not altered. We conclude, therefore, that the lower resting level of Ca(2+) observed in EPA is due to a lower influx of Ca(2+) across the surface membrane rather than increased activation of efflux pathways. How these effects might contribute to the anti-arrhythmic actions of EPA is discussed.
The Journal of Physiology 02/2002; 538(Pt 1):179-84. · 4.72 Impact Factor
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ABSTRACT: 1. Fluo-3 fluorescence measurements were made in isolated beta-escin permeablised rat cardiac myocytes using confocal microscopy. Perfusion of a mock intracellular solution containing 0.22-0.23 microM Ca(2+) and 5 mM ATP elicited regular waves of Ca(2+) (approximately every 5 s) due to spontaneous release of Ca(2+) from the sarcoplasmic reticulum (SR). 2. An approximately linear relationship was noted between Ca(2+) wave velocity (v) and amplitude (sigma). Under the control conditions the ratio of velocity to amplitude (v/sigma) varied little and was 99.8 +/- 2.5 m s(-1) microM(-1) (n = 78). 3. Reduction of [ATP] in the bathing solution to 0.5 and 0.2 mM ATP progressively decreased Ca(2+) wave frequency and propagation velocity while increasing the amplitude. The changes in Ca(2+) wave characteristics in 0.5 mM ATP were similar to those observed during perfusion with 50 microM tetracaine. In 0.2 mM ATP the decline of [Ca(2+)] during a Ca(2+) wave was slowed suggesting a lowered rate of Ca(2+) re-uptake by the SR Ca(2+) pump. 4. Reduction of [ATP] to 0.1 mM abolished Ca(2+) waves after 15-20 s. Returning the [ATP] to 5 mM caused a burst of high frequency and large amplitude waves. Mean velocity of the first wave on returning to 5 mM ATP was larger than normal but the v/sigma value was 32 +/- 6 % of control (n = 6). In the similar burst on removal of 100 microM tetracaine v/sigma was higher than control (166 +/- 9 %, n = 6). 5. Rapid application of caffeine (10 mM) was used to assess the SR Ca(2+) content. This showed that SR Ca(2+) increased as [ATP] was reduced or [tetracaine] was increased. The highest SR Ca(2+) content was observed after perfusion with 0.1 mM ATP, which was 245 +/- 15 % of control values. 6. Returning [ATP] from 0.1 mM to 5 mM caused a burst of high frequency, large amplitude Ca(2+) waves. But recovery after incubation with 300 microM tetracaine resulted in SR Ca(2+) release with no coherent wave pattern. The reason for this discrepancy is discussed.
The Journal of Physiology 08/2001; 534(Pt 1):37-47. · 4.72 Impact Factor
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ABSTRACT: Changes in the behavior of the sarcoplasmic reticulum (SR) in rat ventricular myocytes were investigated under conditions of metabolic inhibition using laser-scanning confocal microscopy to measure intracellular Ca(2+) and the perforated patch-clamp technique to measure SR Ca(2+) content. Metabolic inhibition had several effects on SR function, including reduced frequency of spontaneous releases of Ca(2+) (sparks and waves of Ca(2+)-induced Ca(2+) release), increased SR Ca(2+) content (79.4+/-5.7 to 115.2+/-6.6 micromol/L cell volume [mean+/-SEM; P:<0.001]), and, after a wave of Ca(2+) release, slower reuptake of Ca(2+) into the SR (rate constant of fall of Ca(2+) reduced from 8.5+/-1.1 s(-)(1) in control to 5.2+/-0.4 s(-)(1) in metabolic inhibition [P:<0.01]). Inhibition of L-type Ca(2+) channels with Cd(2+) (100 micromol/L) did not reproduce the effects of metabolic inhibition on spontaneous Ca(2+) sparks. These results are evidence of inhibition of both Ca(2+) release and reuptake mechanisms. Reduced frequency of release could be attributable to either of these effects, but the increased SR Ca(2+) content at the time of reduced frequency of spontaneous release of Ca(2+) shows that the dominant effect of metabolic inhibition is to inhibit release of Ca(2+) from the SR, allowing the accumulation of greater than normal amounts of Ca(2+). In the context of ischemia, this extra accumulation of Ca(2+) would present a risk of potentially arrhythmogenic, spontaneous release of Ca(2+) on reperfusion of the tissue.
Circulation Research 02/2001; 88(2):181-7. · 9.49 Impact Factor
