Andrew W. Trafford

Enhanced Sarcoplasmic Reticulum Ca2+ Leak and Increased Na+-Ca2+ Exchanger Function Underlie Delayed Afterdepolarizations in Patients With Chronic Atrial Fibrillation

Background— Delayed afterdepolarizations (DADs) carried by Na+-Ca2+-exchange current (INCX) in response to sarcoplasmic reticulum (SR) Ca2+ leak can promote atrial fibrillation (AF). The mechanisms leading to delayed afterdepolarizations in AF patients have not been defined. Methods and Results— Protein levels (Western blot), membrane currents and action potentials (patch clamp), and [Ca2+]i (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca2+]i and SR Ca2+ content (integrated INCX during caffeine-induced Ca2+ transient) were unchanged, whereas diastolic SR Ca2+ leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was ≈50% higher in cAF versus control. Single-channel recordings from atrial RyR2 reconstituted into lipid bilayers revealed enhanced open probability in cAF samples, providing a molecular basis for increased SR Ca2+ leak. Calmodulin expression (60%), Ca2+/calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation at Thr287 (87%), and RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 236%) and Ser2814 (CaMKII site, 77%) were increased in cAF. The selective CaMKII blocker KN-93 decreased SR Ca2+ leak, the frequency of spontaneous Ca2+ release events, and RyR2 open probability in cAF, whereas protein kinase A inhibition with H-89 was ineffective. Knock-in mice with constitutively phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca2+ sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca2+]i and INCX density revealed INCX upregulation in cAF. Spontaneous Ca2+ release events accompanied by inward INCX currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca2+]i (diastolic [Ca2+]i–voltage coupling gain) was higher in cAF compared with control. Conclusions— Enhanced SR Ca2+ leak through CaMKII-hyperphosphorylated RyR2, in combination with larger INCX for a given SR Ca2+ release and increased diastolic [Ca2+]i-voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.

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.

How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines

Given that cardiovascular safety liabilities remain a major cause of drug attrition during preclinical and clinical development, adverse drug reactions, and post‐approval withdrawal of medicines, the Medical Research Council Centre for Drug Safety Science hosted a workshop to discuss current challenges in determining, understanding and addressing ‘Cardiovascular Toxicity of Medicines’. This article summarizes the key discussions from the workshop that aimed to address three major questions: (i) what are the key cardiovascular safety liabilities in drug discovery, drug development and clinical practice? (ii) how good are preclinical and clinical strategies for detecting cardiovascular liabilities? and (iii) do we have a mechanistic understanding of these liabilities? It was concluded that in order to understand, address and ultimately reduce cardiovascular safety liabilities of new therapeutic agents there is an urgent need to:

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.

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.

Characterization of an Extensive Transverse Tubular Network in Sheep Atrial Myocytes and its Depletion in Heart Failure

Background—In ventricular myocytes, the majority of structures that couple excitation to the systolic rise of Ca2+ are located at the transverse tubular (t-tubule) membrane. In the failing ventricle, disorganization of t-tubules disrupts excitation contraction coupling. The t-tubule membrane is virtually absent in the atria of small mammals resulting in spatiotemporally distinct profiles of intracellular Ca2+ release on stimulation in atrial and ventricular cells. The aims of this study were to determine (i) whether atrial myocytes from a large mammal (sheep) possess t-tubules, (ii) whether these are functionally important, and (iii) whether they are disrupted in heart failure. Methods and Results—Sheep left atrial myocytes were stained with di-4-ANEPPS. Nearly all control cells had an extensive t-tubule network resulting in each voxel in the cell being nearer to a membrane (sarcolemma or t-tubule) than would otherwise be the case. T-tubules decrease the distance of 50% of voxels from a membrane from 3.35±0.15 to 0.88±0.04 &mgr;m. During depolarization, intracellular Ca2+ rises simultaneously at the cell periphery and center. In heart failure induced by rapid ventricular pacing, there was an almost complete loss of atrial t-tubules. The distance of 50% of voxels from a membrane increased to 2.04±0.08 &mgr;m, and there was a loss of early Ca2+ release from the cell center. Conclusion—Sheep atrial myocytes possess a substantial t-tubule network that synchronizes the systolic Ca2+ transient. In heart failure, this network is markedly disrupted. This may play an important role in changes of atrial function in heart failure.

Differences in intracellular calcium homeostasis between atrial and ventricular myocytes

The role that Ca(2+) plays in ventricular excitation contraction coupling is well defined and much is known about the marked differences in the spatiotemporal properties of the systolic Ca(2+) transient between atrial and ventricular myocytes. However, to date there has been no systematic appraisal of the Ca(2+) homeostatic mechanisms employed by atrial cells and how these compare to the ventricle. In the present study we sought to determine the fractional contributions made to the systolic Ca(2+) transient and the decay of [Ca(2+)](i) by the sarcoplasmic reticulum and sarcolemmal mechanisms. Experiments were performed on single myocytes isolated from the atria and ventricles of the rat. Intracellular Ca(2+) concentration, membrane currents, SR Ca(2+) content and cellular Ca(2+) buffering capacity were measured at 23 degrees C. Atrial cells had smaller systolic Ca(2+) transients (251+/-39 vs. 376+/-41 nmol x L(-1)) that decayed more rapidly (7.4+/-0.6 vs. 5.45+/-0.3 s(-1)). This was due primarily to an increased rate of SR mediated Ca(2+) uptake (k(SR), 6.88+/-0.6 vs. 4.57+/-0.3 s(-1)). SR Ca(2+) content was 289% greater and Ca(2+) buffering capacity was increased approximately 3-fold in atrial cells (B(max) 371.9+/-32.4 vs. 121.8+/-8 micromol x L(-1), all differences P<0.05). The fractional release of Ca(2+) from the SR was greater in atrial cells, although the gain of excitation contraction coupling was the same in both cell types. In summary our data demonstrate fundamental differences in Ca(2+) homeostasis between atrial and ventricular cells and we speculate that the increased SR Ca(2+) content may be significant in determining the increased prevalence of arrhythmias in the atria.

Extracellular matrix profiles in the progression to heart failure. European Young Physiologists Symposium Keynote Lecture-Bratislava 2007.

The myocardial extracellular matrix (ECM), which preserves the geometry and integrity of the myocardium, is a dynamic structure whose component proteins are maintained by a finely controlled homeostatic balance between deposition and degradation. One of the key targets in cardiology is the elucidation of the molecular mechanisms which mediate pathological remodelling of this matrix causing the transition from compensatory hypertrophy to congestive decompensated heart failure. In response to injury or increased workload, cardiac remodelling including myocyte hypertrophy, develops as the heart attempts to compensate for increased wall stresses. Persistence of these stresses over extended time periods leads to disruption of ECM homeostasis resulting in irreversible maladaptive cardiac remodelling, ventricular dilatation and finally heart failure. ECM remodelling is regulated by the matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). Clinical studies and experimental models of cardiac disease states have reported alterations in the balance between the MMPs and TIMPs in the failing heart and crucially at intermediate time points in the progression to failure. This article reviews the recent clinical, genetic and experimental approaches employed to compare ECM, MMP and TIMP profiles in healthy, compensated and failing hearts and identifies common themes in the perturbation of ECM homeostasis in the transition to heart failure.