[Show abstract][Hide abstract] ABSTRACT: Contraction of the heart results from an increase of cytoplasmic Ca concentration ([Ca2+]i), the so-called systolic Ca transient. Most of this results from the release of Ca from the sarcoplasmic reticulum (SR) through the Ryanodine Receptor (RyR). In turn, the amplitude of this Ca transient determines the contractility of the heart. In this lecture, I consider the factors which govern the size and stability of this Ca release. The amplitude of the Ca transient is a steep function of SR Ca resulting in a requirement for very precise beat-to-beat regulation of SR Ca content. This is achieved by a simple negative feedback mechanism in which an increase of SR Ca content increases the size the Ca transient resulting in a decrease of Ca influx on the L-type Ca current and an increase of efflux on Na-Ca exchange. Changing the activity of any of the Ca cycling proteins will change the steady state SR Ca content. This feedback mechanism has many consequences including the fact that a change of RyR open probability has a only a temporary effect on the amplitude of the Ca transient due to a compensating change of SR Ca content. The remainder of the article considers the link between intracellular Ca waves and arrhythmias. This is done in the context of catecholaminergic polymorphic ventricular tachycardia (CPVT). This is an inherited arrhythmia syndrome, in many cases due to a RyR mutation, where arrhythmias occur during exercise as a result of β-adrenergic stimulation. Ca waves only occur when the SR Ca content exceeds a threshold level. Our data shows that the threshold is reduced by the RyR mutation and the that adrenergic stimulation increases SR Ca content.This article is protected by copyright. All rights reserved
Preview · Article · Aug 2014 · Experimental physiology
[Show abstract][Hide abstract] ABSTRACT: The incidence of atrial fibrillation (AF) has a steep relationship with age. AF prevalence in persons aged over 85 exceeds 15% in European cohorts. The basis for the exaggerated vulnerability of the atria to fibrillation in the elderly is incompletely understood. Previous work has shown that ageing is associated with a prolongation of action potential duration, a finding that could be expected to protect against re-entrant arrhythmias. Alternating beat to beat variation in action potential duration and amplitude occurs at high heart rates and has been associated with a history of AF in humans. We investigated whether isolated atrial myocytes from young and old sheep would exhibit differences in alternans behaviour.
No preview · Article · Jun 2014 · Heart (British Cardiac Society)
[Show abstract][Hide abstract] ABSTRACT: Cardiomyocytes in heart failure have increased ryanodine receptor (RyR) open probability (po) leading to calcium leak from the sarcoplasmic reticulum. This process results in calcium waves, delayed after-depolarisations, and subsequent ventricular arrhythmias. However, this leak will decrease the calcium content of the sarcoplasmic reticulum which will in turn decrease the occurrence of waves. We therefore hypothesised that with extreme RyR leak (such as in end-stage heart failure) the calcium content of the sarcoplasmic reticulum might be too low to sustain calcium waves and that part correction of severe leak could lead to a resurgence of calcium waves.
[Show abstract][Hide abstract] ABSTRACT: Over the last two decades, understanding of the mechanisms that underlie heart failure has grown enormously. One of the key concepts is that heart failure is associated with profound alterations in myocardial calcium handling and excitation-contraction coupling. Myocardial Ca handling Most of the calcium that activates contraction comes from the sarcoplasmic reticulum (SR). It leaves the SR through a specialized release channel known as the ryanodine receptor (RyR). The probability that a RyR is open and can therefore allow Ca to leave the SR into the cytoplasm is increased by an increase in the concentration of either cytosolic or SR (luminal) Ca concentration. During the normal heartbeat sarcolemmal Ca channels open and some of the entering Ca binds to the RyRs making them open thereby triggering the release of a much greater amount of Ca from the SR into the cytosol. This Ca release causes a rapid rise of cytosolic Ca to levels that activate the myofilaments and initiate contraction. After termination of release of Ca from the SR (because of closure of RyRs), cytosolic Ca levels decline rapidly and relaxation occurs. Ca is rapidly removed from the cytosol by two major Ca removal systems: the sarcoendoplasmic reticulum Ca ATPase (SERCA) and the sarcolemmal sodium /calcium exchanger (NCX). SERCA pumps Ca back into the SR while NCX pumps 1 Ca(2+) ion out in exchange for the influx of 3 Na(+) ions into the cell. This rapid cycle of Ca release and reuptake is known as the systolic Ca transient and it is one of the main factors that control force of contraction in the heart. It is worth emphasizing that the normal Ca transient depends on the RyRs being virtually closed in diastole, opening very briefly to produce the systolic increase of Ca and then closing to allow Ca to fall to resting levels.
[Show abstract][Hide abstract] ABSTRACT: The microcirculation is the site of gas and nutrient exchange. Control of central or local signals acting on the myocytes, pericytes and endothelial cells within it, is essential for health. Due to technical problems of accessibility, the mechanisms controlling Ca(2+) signalling and contractility of myocytes and pericytes in different sections of microvascular networks in situ have not been investigated. We aimed to investigate Ca(2+) signalling and functional responses, in a microcirculatory network in situ. Using live confocal imaging of ureteric microvascular networks, we have studied the architecture, morphology, Ca(2+) signalling and contractility of myocytes and pericytes. Ca(2+) signals vary between distributing arcade and downstream transverse and precapillary arterioles, are modified by agonists, with sympathetic agonists being ineffective beyond transverse arterioles. In myocytes and pericytes, Ca(2+) signals arise from Ca(2+) release from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate-induced Ca(2+) release and not via ryanodine receptors or Ca(2+) entry into the cell. The responses in pericytes are less oscillatory, slower and longer-lasting than those in myocytes. Myocytes and pericytes are electrically coupled, transmitting Ca(2+) signals between arteriolar and venular networks dependent on gap junctions and Ca(2+) entry via L-type Ca(2+) channels. Endothelial Ca(2+) signalling inhibits intracellular Ca(2+) oscillations in myocytes and pericytes via L-arginine/nitric oxide pathway and intercellular propagating Ca(2+) signals via EDHF. Increases of Ca(2+) in pericytes and myocytes constrict all vessels except capillaries. These data reveal the structural and signalling specializations allowing blood flow to be regulated by myocytes and pericytes.
[Show abstract][Hide abstract] ABSTRACT: The atria contribute 25 % to ventricular stroke volume and are the site of the commonest cardiac arrhythmia, atrial fibrillation (AF). The initiation of contraction in the atria is similar to that in ventricle involving a systolic rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). There are, however, substantial inter-species differences in the way systolic Ca(2+) is regulated in atrial cells. These differences are a consequence of a well-developed and functionally relevant transverse (t) - tubule network in the atria of large mammals including man and its virtual absence in smaller laboratory species such as the rat. Where t-tubules are absent the systolic Ca(2+) transient results from a 'fire-diffuse-fire' sequential recruitment of Ca(2+) release sites from the cell edge to the centre and hence marked spatiotemporal heterogeneity of systolic Ca(2+). Conversely the well-developed t-tubule network in large mammals ensures a near synchronous rise of [Ca(2+)](i). In addition to synchronizing the systolic rise of [Ca(2+)](i) the presence of t-tubules in the atria of large mammals, by virtue of localization of the L-type Ca(2+) channels and Na(+)-Ca(2+) exchanger antiporters on the t-tubules, may serve to respectively accelerate changes in the amplitude of the systolic Ca(2+) transient during inotropic manoeuvres and lower diastolic [Ca(2+)](i). On the other hand, the presence of t-tubules and thence wider cellular distribution of the Na(+)-Ca(2+) exchanger may predispose the atria of large mammals to Ca(2+) dependent delayed after-depolarisations (DADs); this may be a determining factor in why the atria of large mammals spontaneously develop and maintain AF.
No preview · Article · Feb 2013 · Cardiovascular Research
[Show abstract][Hide abstract] ABSTRACT: Phosphodiesterase type 5A inhibition with sildenafil improves cardiac function in heart failure. In addition, sildenafil in animal models of myocardial infarction has direct cardioprotective and antiarrhythmic effects. Sildenafil reduces L-type calcium current (Ica-L) and attenuates adrenergically driven inotropism, but effects on calcium handling are largely undetermined.Isolated adult rat ventricular myocytes were voltage clamped and calcium fluorescence measured with the indicator fura-2. Cells were paced at 0·5 Hz with depolarisations from −60 mV to +10 mV. Sarcoplasmic reticulum (SR) content was determined by application of caffeine (10–20 mmol/L) and integration of inward sodium-calcium exchanger current. Rate constants for calcium extrusion from the cell (Kcaff) and calcium uptake into the SR (KSERCA) were determined by fitting first order exponentials to decay phases of the respective calcium transients. Following the initial control protocol, a therapeutically relevant dose of sidenafil (1 μM) was applied. Differences were determined with student's paired t tests.Sildenafil reduced SR content by 26·5% (n=9, p<0·01). To a lesser extent, sildenafil also reduced calcium transient amplitude (by −13·6%, n=9, p<0·05); this was not accompanied by a reduction in KSERCA (–2.3% with sildenafil, p=0·97, n=5). Peak and integrated Ica-L were also reduced with sildenafil (–9·1% and −6·0%, respectively, n=9, p<0·05). The effect on Ica-L was also seen in adult dog ventricular myocytes (reducing peak and integrated Ica-L by 15·9% and 26·4%, respectively, p<0·05 and p<0·01, n=6, 23°C). These effects cannot be attributed to run-down effects.Sildenafil substantially reduced SR content with no reduction in KSERCA, and thus may be mediated through ryanodine receptor modulation. Such reduction in SR load may reduce proarrhythmic SR calcium release, indicating a novel mechanism through which sildenafil exerts an antiarrhythmic effect. Acute reductions in calcium transient amplitude and Ica-L with sildenafil indicate acute negative inotropic effects and may contribute to our understanding of its cardioprotective effects in the setting of hyperadrenergic drive in heart failure.FundingBritish Heart Foundation.
[Show abstract][Hide abstract] ABSTRACT: This article reviews the consequences of the need for the cardiac cell to be in calcium flux balance in the steady state. We first discuss how this steady state condition affects the control of resting [Ca(2+)](i). The next section considers how sarcoplasmic retirculum (SR) Ca content is controlled by a feedback mechanism whereby changes of SR Ca affect the amplitude of the Ca transient and this, in turn, controls sarcolemmal Ca fluxes. Subsequent sections review the effects of altering the activity of individual Ca handling proteins. Increasing the activity of the SR Ca-ATPase (SERCA) increases both the amplitude and rate constant of decay of the systolic Ca transient. The Ca flux balance condition requires that this must be achieved with no change of Ca efflux placing constraints on the magnitude of change of amplitude and decay rate. We analyze the quantitative dependence of Ca transient amplitude and SR content on SERCA activity. Increasing the open probability of the RyR during systole is predicted to have no steady state effect on the amplitude of the systolic Ca transient. We discuss the effects of changing the amplitude of the L-type Ca current in the context of both triggering Ca release from the SR and loading the cell with calcium. These manoeuvres are considered in the context of the effects of β-adrenergic stimulation. Finally, we review calcium flux balance in the presence of Ca waves.
No preview · Article · Dec 2012 · Journal of Molecular and Cellular Cardiology
[Show abstract][Hide abstract] ABSTRACT: Rationale:
Spontaneous Ca(2+) release (SCR) from the sarcoplasmic reticulum can cause delayed afterdepolarizations and triggered activity, contributing to arrhythmogenesis during β-adrenergic stimulation. Excessive beat-to-beat variability of repolarization duration (BVR) is a proarrhythmic marker. Previous research has shown that BVR is increased during intense β-adrenergic stimulation, leading to SCR.
We aimed to determine ionic mechanisms controlling BVR under these conditions.
Methods and results:
Membrane potentials and cell shortening or Ca(2+) transients were recorded from isolated canine left ventricular myocytes in the presence of isoproterenol. Action-potential (AP) durations after delayed afterdepolarizations were significantly prolonged. Addition of slowly activating delayed rectifier K(+) current (I(Ks)) blockade led to further AP prolongation after SCR, and this strongly correlated with exaggerated BVR. Suppressing SCR via inhibition of ryanodine receptors, Ca(2+)/calmodulin-dependent protein kinase II inhibition, or by using Mg(2+) or flecainide eliminated delayed afterdepolarizations and decreased BVR independent of effects on AP duration. Computational analyses and voltage-clamp experiments measuring L-type Ca(2+) current (I(CaL)) with and without previous SCR indicated that I(CaL) was increased during Ca(2+)-induced Ca(2+) release after SCR, and this contributes to AP prolongation. Prolongation of QT, T(peak)-T(end) intervals, and left ventricular monophasic AP duration of beats after aftercontractions occurred before torsades de pointes in an in vivo dog model of drug-induced long-QT1 syndrome.
SCR contributes to increased BVR by interspersed prolongation of AP duration, which is exacerbated during I(Ks) blockade. Attenuation of Ca(2+)-induced Ca(2+) release by SCR underlies AP prolongation via increased I(CaL.) These data provide novel insights into arrhythmogenic mechanisms during β-adrenergic stimulation besides triggered activity and illustrate the importance of I(Ks) function in preventing excessive BVR.
Full-text · Article · Nov 2012 · Circulation Research
[Show abstract][Hide abstract] ABSTRACT: Mammalian ventricular myocytes are characterized by the presence of an extensive transverse (t) tubule network which is responsible for the synchronous rise of intracellular Ca(2+) concentration ([Ca(2+)](i)) during systole. Disruption to the ventricular t-tubule network occurs in various cardiac pathologies and leads to heterogeneous changes of [Ca(2+)](i) which is thought to contribute to the reduced contractility and increased susceptibility to arrhythmias of the diseased ventricle. Here we review evidence that, despite the long-held dogma of atrial cells having no or very few t-tubules, there is indeed an extensive and functionally significant t-tubule network present in atrial myocytes of large mammals including man. Moreover, the atrial t-tubule network is highly plastic in nature and undergoes far more extensive remodeling in heart disease than is the case in the ventricle with profound consequences for the resulting systolic Ca(2+) transient. In addition to considering the functional role of the t-tubule network in the healthy and diseased atria we also provide an overview of recent data concerning the putative factors controlling the formation of t-tubules and conclude by posing some important questions that currently remain to be addressed and whether or not targeting t-tubules offers potential novel therapeutic possibilities for heart disease.
No preview · Article · Nov 2012 · Journal of Molecular and Cellular Cardiology
[Show abstract][Hide abstract] ABSTRACT: The aim of was to investigate the propagation of calcium waves between cells and determine whether this synchronizes alternating Ca release between cells. Experiments were carried out on electrically coupled cell pairs; spontaneous Ca waves were produced by elevating external Ca. There was a significant difference in the ability of these waves to propagate between cells depending on the orientation of the pairs. Although almost all pairs connected by side to side contacts showed propagating Ca release, this was very uncommon in end to end cell pairs. Confocal studies showed that there was a gap at the intercalated disc consisting of cell membranes and a region of cytoplasm devoid of sarcoplasmic reticulum. This gap was 2.3 μm in length and is suggested to interfere with Ca wave propagation. The gap measured was much smaller between side to side contacts: 1.5 μm and so much less likely to interfere with propagation. Subsequent experiments investigated the synchronization between cells of calcium alternans produced by small depolarizing pulses. Although this alternation results from beat to beat alternation of intracellular Ca wave propagation, there was no evidence that propagation of Ca waves between cells contributed to synchronization of this alternans.
Preview · Article · Oct 2012 · The Journal of Physiology
[Show abstract][Hide abstract] ABSTRACT: This study investigated the mechanisms underlying the propagation of cytoplasmic calcium waves and the genesis of systolic Ca(2+) alternans in cardiac myocytes lacking transverse tubules (t-tubules). These correspond to atrial cells of either small mammals or large mammals that have lost their t-tubules due to disease-induced structural remodeling (e.g., atrial fibrillation). A mathematical model was developed for a cluster of ryanodine receptors distributed on the cross section of a cell that was divided into 13 elements with a spatial resolution of 2 μm. Due to the absence of t-tubules, L-type Ca(2+) channels were only located in the peripheral elements close to the cell-membrane surface and produced Ca(2+) signals that propagated toward central elements by triggering successive Ca(2+)-induced Ca(2+) release (CICR) via Ca(2+) diffusion between adjacent elements. Under control conditions, the Ca(2+) signals did not fully propagate to the central region of the cell. However, with modulation of several factors responsible for Ca(2+) handling, such as the L-type Ca(2+) channels (Ca(2+) influx), SERCA pumps (sarcoplasmic reticulum (SR) Ca(2+) uptake), and ryanodine receptors (SR Ca(2+) release), Ca(2+) wave propagation to the center of the cell could occur. These simulation results are consistent with previous experimental data from atrial cells of small mammals. The model further reveals that spatially functional heterogeneity in Ca(2+) diffusion within the cell produced a steep relationship between the SR Ca(2+) content and the cytoplasmic Ca(2+) concentration. This played an important role in the genesis of Ca(2+) alternans that were more obvious in central than in peripheral elements. Possible association between the occurrence of Ca(2+) alternans and the model parameters of Ca(2+) handling was comprehensively explored in a wide range of one- and two-parameter spaces. In addition, the model revealed a spontaneous second Ca(2+) release in response to a single voltage stimulus pulse with SR Ca(2+) overloading and augmented Ca(2+) influx. This study provides what to our knowledge are new insights into the genesis of Ca(2+) alternans and spontaneous second Ca(2+) release in cardiac myocytes that lack t-tubules.
[Show abstract][Hide abstract] ABSTRACT: Non-Technical Summary Cardiac contraction is caused by an increase of calcium (Ca2+) concentration in the cells of the heart, the so-called ‘systolic Ca2+ transient’. The majority of this Ca2+ is provided by the sarcoplasmic reticulum (SR), which acts as a Ca2+ store within the cell itself. Before the heart can contract again the Ca2+ store needs to be replenished and so Ca2+ is pumped back into the SR by the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA). We show that a given fractional decrease of SERCA activity produces a much smaller decrease of SR Ca2+ content. This means that changes of SERCA activity can produce large changes of systolic Ca2+ without the need for energetically expensive alterations of SR Ca2+ content.
Abstract Changes of the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) affect the amplitude of the systolic Ca2+ transient and thence cardiac contractility. This is thought to be due to alterations of SR Ca2+ content. Recent work on mice in which the expression of SERCA is decreased found that a large reduction of SERCA expression resulted in a proportionately much smaller decrease of SR Ca2+ content. The aim of the current work was to investigate the quantitative nature of the dependence of both the amplitude of the systolic Ca2+ transient and SR Ca2+ content on SERCA activity during acute partial inhibition of SERCA. Experiments were performed on rat ventricular myocytes. Brief application of thapsigargin (1 μm) resulted in a decrease of SERCA activity as measured from the rate of decay of the systolic Ca2+ transient. This was accompanied by a decrease in the amplitude of the systolic Ca2+ transient which was linearly related to that of SERCA activity. However, the fractional decrease in the SR Ca2+ content was much less than that of SERCA activity. On average SR Ca2+ content was proportional to SERCA activity raised to the 0.38 ± 0.07 power. This shallow dependence of SR content on SERCA activity arises because Ca2+ release is a steep function of SR Ca2+ content. In contrast SR Ca2+ content was increased 4.59 ± 0.40 (n= 8)-fold by decreasing ryanodine receptor opening with tetracaine (1 mm). Therefore a modest decrease of SR Ca2+ content results in a proportionately larger fall of Ca2+ release from the SR which can balance a larger initiating decrease of SERCA. In conclusion, the shallow dependence of SR Ca2+ content on SERCA activity is expected for a system in which small changes of SR Ca2+ content produce larger effects on the amplitude of the systolic Ca2+ transient.
Full-text · Article · Aug 2011 · The Journal of Physiology
[Show abstract][Hide abstract] ABSTRACT: Non-technical summary Heart failure is where the heart is unable to pump sufficient blood in order to meet the requirements of the body. Symptoms of heart failure often first present during exercise. During exercise the blood levels of a hormone, noradrenaline, increase and activate receptors on the muscle cells of the heart known as β-receptors causing the heart to contract more forcefully. We show that in heart failure the response to β-receptor stimulation is reduced and this appears to be due to a failure of the β-receptor to signal correctly to downstream targets inside the cell. However, by-passing the β-receptor and directly activating one of the downstream targets, an enzyme known as adenylyl cyclase, inside the cell restores the function of the muscle cells in failing hearts. These observations provide a number of potential targets for therapies to improve the function of the heart in patients with heart failure.
Abstract Reduced inotropic responsiveness is characteristic of heart failure (HF). This study determined the cellular Ca2+ homeostatic and molecular mechanisms causing the blunted β-adrenergic (β-AR) response in HF. We induced HF by tachypacing in sheep; intracellular Ca2+ concentration was measured in voltage-clamped ventricular myocytes. In HF, Ca2+ transient amplitude and peak L-type Ca2+ current (ICa-L) were reduced (to 70 ± 11% and 50 ± 3.7% of control, respectively, P < 0.05) whereas sarcoplasmic reticulum (SR) Ca2+ content was unchanged. β-AR stimulation with isoprenaline (ISO) increased Ca2+ transient amplitude, ICa-L and SR Ca2+ content in both cell types; however, the response of HF cells was markedly diminished (P < 0.05). Western blotting revealed an increase in protein phosphatase levels (PP1, 158 ± 17% and PP2A, 188 ± 34% of control, P < 0.05) and reduced phosphorylation of phospholamban in HF (Ser16, 30 ± 10% and Thr17, 41 ± 15% of control, P < 0.05). The β-AR receptor kinase GRK-2 was also increased in HF (173 ± 38% of control, P < 0.05). In HF, activation of adenylyl cyclase with forskolin rescued the Ca2+ transient, SR Ca2+ content and SR Ca2+ uptake rate to the same levels as control cells in ISO. In conclusion, the reduced responsiveness of the myocardium to β-AR agonists in HF probably arises as a consequence of impaired phosphorylation of key intracellular proteins responsible for regulating the SR Ca2+ content and therefore failure of the systolic Ca2+ transient to increase appropriately during β-AR stimulation.
Full-text · Article · Mar 2011 · The Journal of Physiology