Sparks of interest in cardiac excitation-contraction coupling.
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Calcium↗
Biomedical subjects
M B Cannell
Publications and source records attributed to M B Cannell.
Class specific inhibition of house dust mite proteinases which cleave cell adhesion, induce cell death and which increase the permeability of lung epithelium.
1. House dust mite (HDM) allergens with cysteine and serine proteinase activity are risk factors for allergic sensitization and asthma. A simple method to fractionate proteinase activity from HDM faecal pellets into cysteine and serine class activity is described. 2. Both proteinase fractions increased the permeability of epithelial cell monolayers. The effects of the serine proteinase fraction were inhibited by 4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride (AEBSF) and soybean trypsin inhibitor (SBTI). The effects of the cysteine proteinase fraction could be inhibited by E-64. No reciprocity of action was found. 3. Treatment of epithelial monolayers with either proteinase fraction caused breakdown of tight junctions (TJs). AEBSF inhibited TJ breakdown caused by the serine proteinase fraction, whereas E-64 inhibited the cysteine proteinase fraction. 4. Agarose gel electrophoresis revealed that the proteinases induced DNA cleavage which was inhibited by the matrix metalloproteinase inhibitor BB-250. Compound E-64 inhibited DNA fragmentation caused by the cysteine proteinase fraction, but was without effect on the serine proteinase fraction. Staining of proteinase-treated cells with annexin V (AV) and propidium iodide (PI) revealed a diversity of cellular responses. Some cells stained only with AV indicating early apoptosis, whilst others were dead and stained with both AV and PI. 5. HDM proteinases exert profound effects on epithelial cells which will promote allergic sensitization; namely disruption of intercellular adhesion, increased paracellular permeability and initiation of cell death. Attenuation of these actions by proteinase inhibitors leads to the conclusion that compounds designed to be selective for the HDM enzymes may represent a novel therapy for asthma.
Animals↗
Cell lines of pulmonary and non-pulmonary origin as tools to study the effects of house dust mite proteinases on the regulation of epithelial permeability.
BACKGROUND: Allergenic and non-allergenic proteinases from house dust mites (HDM) cause loss of adhesion between airway epithelial cells that may result in a loss of functional cohesion between the cells and thus assist in allergen presentation. Improved cellular assay systems are needed to ascertain the mechanisms involved. OBJECTIVES: To survey a series of epithelial cell lines (Calu-3, 16HBE14o-, NCI-H292 and A549 from human airways, and MDCK from dog kidney) and establish their utility for studies of the effects of HDM proteinases from D. pteronyssinus on epithelial permeability. To develop an improved method for measuring changes in epithelial permeability induced by HDM proteinases and other provocants. METHODS: The permeability of epithelial monolayer cultures to mannitol was calculated from measurements of clearance using a technique that permits mathematical estimation and reduction of non-cellular diffusional constraints. Permeability was studied under control conditions and after perturbation of monolayers with HDM proteinases (separated into serine- and cysteine-proteinase classes) or chelation of extracellular Ca2+. Fluorescent antibody staining was used to investigate whether the cells expressed tight junctions (staining of ZO-1), desmosomes (staining of desmoplakin) and zonulae adherentes (staining of E-cadherin). RESULTS: The Calu-3 line was identified as an airway cell line that expressed functional tight junctions, desmosomes and zonulae adherentes. Calu-3 monolayers exhibited a low clearance and permeability to mannitol, similar to that seen in the extensively characterized MDCK cell line. Clearance and permeability were significantly increased by treatment with either HDM proteinase fraction or by calcium chelation. 16HBE14o- cells also had a low permeability to mannitol under control conditions and expressed a similar repertoire of functional proteins from major intercellular junctions. In contrast, NCI-H292 and A549 cell lines were functionally deficient in tight junctions, although they did express desmosomes and zonulae adherentes to a greater extent. Epithelial permeability was found to be a more appropriate and sensitive index of epithelial perturbation than was tracer clearance. CONCLUSION: These results suggest that the Calu-3 and 16HBE14o- cell lines are useful tools in studying the mechanism of HDM proteinases on airway epithelial cell function. HDM proteinases of both cysteine and serine mechanistic classes were found to perturb epithelial adhesion and function.
Adult↗
The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse.
Mouse sperm-egg fusion was examined using two-photon and confocal microscopy. A delay of several minutes occurred between the first observable event of fusion (which was the diffusion of Ca2+-sensitive dyes from egg into sperm) and any change in egg cytoplasmic Ca2+. When indo-1 dextran was used to obtain ratiometric two-photon images, there was no detectable local increase in egg cytoplasmic Ca2+ near the site of sperm fusion. However, the sperm underwent a Ca2+ transient which appeared to be coincident with the egg cytoplasm Ca2+ transient, which suggested that there was a high permeability pathway for Ca2+ between egg and sperm. To exclude this pathway from providing trigger Ca2+ for the egg transient, we reduced bathing [Ca2+] to approx. 18 microM and 13nM (with EGTA). In these conditions the first egg Ca2+ transient was not prevented, which makes an obligatory role for extracellular Ca2+ in the initiation of the egg Ca2+ transient unlikely. Both FITC-albumin (70 kDa) and 10 kDa dextran-linked Ca2+ indicators were able to diffuse into the sperm from the egg. In addition, phycoerythrin (240 kDa) rapidly diffused into the sperm shortly after fusion (but before any changes in Ca2+ occurred). This suggests that the 'pore(s)' that form during sperm-egg fusion must be at least 8 nm in diameter. These data are compatible with the idea that a diffusible sperm protein could trigger the observed changes in intracellular Ca2+ in the egg, but do not exclude the possibility that other second messengers are generated during sperm-egg fusion.
Animals↗
Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure.
Cardiac hypertrophy and heart failure caused by high blood pressure were studied in single myocytes taken from hypertensive rats (Dahl SS/Jr) and SH-HF rats in heart failure. Confocal microscopy and patch-clamp methods were used to examine excitation-contraction (EC) coupling, and the relation between the plasma membrane calcium current (ICa) and evoked calcium release from the sarcoplasmic reticulum (SR), which was visualized as "calcium sparks." The ability of ICa to trigger calcium release from the SR in both hypertrophied and failing hearts was reduced. Because ICa density and SR calcium-release channels were normal, the defect appears to reside in a change in the relation between SR calcium-release channels and sarcolemmal calcium channels. beta-Adrenergic stimulation largely overcame the defect in hypertrophic but not failing heart cells. Thus, the same defect in EC coupling that develops during hypertrophy may contribute to heart failure when compensatory mechanisms fail.
Adrenergic beta-Agonists↗
Changes in subsarcolemmal sodium concentration measured by Na-Ca exchanger activity during Na-pump inhibition and beta-adrenergic stimulation in guinea-pig ventricular myocytes.
Measurement of Na-Ca exchange activity was used to examine subsarcolemmal sodium levels ([Na+]s) in single, voltage-clamped guinea-pig cardiac myocytes while Na-K pump activity was modulated pharmacologically. Changes in Nas were evaluated from phase-plane analysis of the changes in intracellular calcium, measured using the fluorescent indicators Fura-red and Fluo-3. Activation of beta-adrenoceptors with 1 microM isoprenaline resulted in activation of the cAMP-dependent chloride current, but had no effect on the calcium transient mediated via the Na-Ca exchanger, regardless of whether the Na-K pump was active or inhibited (with strophanthidin). The ability of Na-Ca exchange activity to report [Na+]s was demonstrated by the effect of changing the extracellular rubidium concentration from 1 to 5.4 mM to modulate Na-K pump activity. We suggest that beta-adrenergic stimulation does not directly affect either the Na-K pump or the Na-Ca exchanger and that the Na-Ca exchanger can be used as a sensitive indicator of changes in [Na+]s and Na-K pump activity.
Adrenergic beta-Agonists↗
Numerical simulation of local calcium movements during L-type calcium channel gating in the cardiac diad.
Computer simulation was used to investigate the calcium levels after sarcolemmal calcium influx through L-type calcium channels (DHPRs) into the narrow diadic space of cardiac muscle. The effect of various cytosolic and membranebound buffers, diad geometry, DHPR properties (open time and current), and surface charge were examined. The simulations showed that phospholipid binding sites on the sarcolemmal membrane are the major buffer affecting free calcium ([Ca2+]) levels in the diad. The inclusion of surface charge effects calculated from Gouy-Chapman theory resulted in a marked decrease in [Ca2+] levels at all times and a faster decay of [Ca2+] after termination of DHPR influx. For a DHPR current of 200 fA, [Ca2+] at the center of the diad reached peak levels of approximately 73 microM. In larger diads (> or = 400 nm diameter), [Ca2+] decayed more slowly than in smaller diads (100-200 nm diameter), although peak [Ca2+] levels reached during typical DHPR open times were similar. For a wide range of DHPR single-channel current magnitudes (Ica = 25-200 fA), [Ca2+] levels in the diad were approximately proportional to ICa. The decrease in calculated [Ca2+] levels due to the effects of surface charge can be interpreted as resulting from an effective "volume expansion" of the diad space. Furthermore, the layer of increased [Ca2+] close to the sarcolemmal membrane can act as a fast buffer.
Binding Sites↗
Numerical analysis of ryanodine receptor activation by L-type channel activity in the cardiac muscle diad.
Computer simulations were used to examine the response of ryanodine receptors (RyRs) to the sarcolemmal calcium influx via L-type calcium channels (DHPRs). The effects of ryanodine receptor organization, diad geometry, DHPR single-channel current, and DHPR gating were examined. In agreement with experimental findings, the simulations showed that RyRs can respond rapidly (approximately 0.4 ms) to calcium influx via DHPRs. The responsiveness of the RyR depends on the geometrical arrangement between the RyRs and the DHPR in the diad, with wider diads being generally less responsive. When the DHPR single-channel current is small (approximately 25 fA), the organization of RyRs into small clusters results in an improved responsiveness. With experimentally observed DHPR mean open and closed times (0.17 ms and 4 ms, respectively) it is the first opening of the DHPR that is most likely to activate the RyR. A measure of the efficiency (Q) by which DHPR gating evokes sarcoplasmic reticulum release is defined. Q is at maximum for tau approximately 0.3 ms, and we interpret this finding in terms of the "tuning" of DHPR gating to RyR response. If certain cardiac myopathies are associated with a mismatch in the "tuning," then modification of DHPR gating with drugs to "retune" calcium-induced calcium release should be possible.
Calcium↗
The role of L-type Ca2+ current and Na+ current-stimulated Na/Ca exchange in triggering SR calcium release in guinea-pig cardiac ventricular myocytes.
OBJECTIVE: This study examines the relative ability of sodium current (INa)-stimulated reverse mode Na/Ca exchange and the L-type calcium current (ICa) to trigger calcium-induced calcium release (CICR) in guinea-pig ventricular myocytes. METHODS: Cytosolic Ca2+ transients were recorded from enzymatically dissociated guinea-pig ventricular myocytes using Indo-1. Macroscopic membrane currents were simultaneously recorded using the whole-cell patch-clamp technique. RESULTS: At room temperature (22-25 degrees C) Ca2+ transients were associated with the activation of INa, ICa or INa plus ICa in combination. However, after ICa was blocked by verapamil (10 microM), no Ca2+ transient could be evoked by the activation of INa alone at either -40 or +5 mV. Similar results were obtained with 5 and 8 mM intracellular sodium, and when the temperature of the bathing solution was raised to 35 degrees C and cAMP (10 microM) added to the pipette solution. CONCLUSIONS: From consideration of the relative magnitudes of the Ca2+ influx via ICa and Na/Ca exchange and thermodynamic considerations, we suggest that ICa is the major source of 'trigger' calcium for CICR (and cardiac contraction) under normal conditions. Although the Na/Ca exchanger was incapable of triggering CICR under the conditions of these experiments, we suggest that it may become more important when cytosolic Ca2+ is elevated, a condition which will also lead to decrease the amplitude of ICa.
Animals↗
New insights into cardiac excitation-contraction coupling in normal and hypertension/failure animal models.
Application of the confocal microscope to enzymatically isolated cardiac myocytes has revealed that excitation-contraction coupling is a ¿local control phenomenon'. The whole cell calcium transient is made up of the temporal and spatial summation of a large number of microscopic calcium release events called ¿calcium sparks'. The opening of a single calcium channel in the surface membrane can activate a calcium spark and there is a non-linear relationship between the amplitude of the single calcium channel flux and the probability of activating a calcium spark (Ps). Mathematical modelling shows that the relationship between surface membrane calcium channel gating and the activation of calcium release channels in internal stores is very sensitive to the geometric relationship between these channels. Under normal conditions, the gating behaviour of the surface membrane calcium channels may be near optimal (or well ¿tuned') for activating calcium sparks which will minimise the requirement for calcium influx into the cell. In the spontaneous hypertensive rat (SHR) model of hypertension, the relationship between the calcium channel activity and calcium release from internal stores is altered in a way that results in a reduced contraction strength. The relationship between the calcium channel current and Ps is restored by beta-adrenergic stimulation in the hypertrophy model but not in hearts which are failing. These results suggest that a novel approach to treating certain types of heart failure could be to modify the gating behaviour of the sarcolemmal calcium channel to ¿retune' ability of the sarcolemmal calcium channels to activate calcium release from internal stores, and thereby improve contractility without increasing calcium influx into the cell.
Animals↗
The molecular biology of the Na(+)-Ca2+ exchanger and its functional roles in heart, smooth muscle cells, neurons, glia, lymphocytes, and nonexcitable cells.
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Animals↗
The roles of the sodium and calcium current in triggering calcium release from the sarcoplasmic reticulum.
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Animals↗
Construction of a two-photon microscope and optimisation of illumination pulse duration.
The construction of a two-photon/confocal microscope system is described in detail. For two-photon illumination, a Ti:sapphire modelocked laser generating 62-fs pulses at 715 nm was used. The effect of the optical train on illumination pulse width was examined and the observed increase in pulse duration was almost completely removed by the addition/adjustment of a prism compressor system. The imaging capabilities of the two-photon microscope are demonstrated and it is shown that the imaging performance of the two-photon microscope is similar to that of a conventional confocal microscope. With two-photon illumination, the resolution (full width at half-maximum intensity) was 0.42 microM (x-y) and 0.81 microM axially, while with single-photon illumination (at 488 nm in the same instrument with a confocal pinhole detector) the resolution was 0.3 microM (x-y) and 0.75 microM axially. The results are discussed with regard to the general problem of femtosecond pulse distortion in an optical system and a simple procedure for optimal pulse restoration is described.
Calibration↗
Calcium sparks and [Ca2+]i waves in cardiac myocytes.
Local elevations in intracellular calcium ("Ca2+ sparks") in heart muscle are elementary sarcoplasmic reticulum (SR) Ca(2+)-release events. Ca2+ sparks occur at a low rate in quiescent cells but can also be evoked by electrical stimulation of the cell to produce the cell-wide Ca2+ transient. In this study we investigate how Ca2+ sparks are related to propagating waves of elevated cytosolic Ca2+ induced by "Ca2+ overload." Single ventricular myocytes from rat were loaded with the Ca(2+)-sensitive indicator fluo 3 and imaged with a confocal microscope. After extracellular Ca2+ concentration was increased from 1 to 10 mM to produce Ca2+ overload, the frequency of spontaneous Ca2+ sparks, which occur at the t tubule/SR junction, increased approximately 4-fold, whereas the spark amplitude and spatial size increased 4.1-and 1.7-fold, respectively. In addition, a spectrum of larger subcellular events, including propagating Ca2+ waves, was observed. Ca2+ sparks were seen to occur at the majority (65%) of the sites of wave initiation. For slowly propagating Ca2+ waves, discrete Ca(2+)-release events, similar to Ca2+ sparks, were detected in the wave front. These Ca2+ sparks appeared to recruit other sparks along the wave front so that the wave progressed in a saltatory manner. We conclude that Ca2+ sparks are elementary events that can explain both the initiation and propagation of Ca2+ waves. In addition, we show that Ca2+ waves and electrically evoked Ca2+ transients have the same time course and interact with each other in a manner that is consistent with both phenomena having the same underlying mechanism(s). These results suggest that SR Ca2+ release during Ca2+ waves, like that during normal excitation-contraction coupling, results from the spatial and temporal summation of Ca2+ sparks.
Animals↗
Relation between the sarcolemmal Ca2+ current and Ca2+ sparks and local control theories for cardiac excitation-contraction coupling.
Ca2+ sparks, the elementary events underlying excitation-contraction (E-C) coupling, occur when sarcoplasmic reticulum (SR) Ca2+ release channels open. They are activated locally by Ca2+ influx through sarcolemmal (SL) Ca2+ channels. By measuring the probability of spark occurrence under conditions in which their probability of occurrence is low, we address two important questions raised by our recent work: (1) When a Ca2+ spark is triggered, how many SL Ca2+ channels (at a minimum) contribute to its activation? (2) What is the relation between the subcellular local [Ca2+], produced by the opening of SL Ca2+ channels and the consequent SR Ca2+ release? By comparing the voltage dependence of Ca2+ sparks in rat ventricular myocytes with the Ca2+ current, we show that the opening of a single SL Ca2+ channel can trigger a Ca2+ spark. Furthermore, we deduce that the probability of SR Ca2+ release depends of the square of the local [Ca2+]i produced by SL Ca2+ channel openings. These results are discussed with respect to the properties of Ca2+-induced Ca2+-release (CICR) and the local control theory of excitation-contraction coupling.
Animals↗
Ca2+ influx during the cardiac action potential in guinea pig ventricular myocytes.
The relative contributions of L-type Ca2+ current (ICa) and Na+/Ca2+ exchange to Ca2+ influx during the cardiac action potential (AP) are unknown. In this study, we have used an AP recorded under physiological conditions as the command voltage applied to voltage-clamped ventricular myocytes. ICa (measured as nifedipine-sensitive membrane current) had a complex multiphasic time course during the AP. Peak ICa was typically 4 pA/pF, after which it rapidly declined (to about 60% of peak) during the rising phase of the cell-wide Ca2+ transient before increasing to a second, more sustained component. The initial decline in ICa was sensitive to the amount of Ca2+ released by the sarcoplasmic reticulum (SR), and conditions that reduce the amplitude of the Ca2+ transient (such as rest or brief application of caffeine) increased net Ca2+ influx via ICa. Dissection of the Na+/Ca2+ exchange current at the start of the AP suggested that Ca2+ influx via Na+/Ca2+ exchange is less than 30% of that due to ICa. From these data, we suggest that ICa is the primary source of Ca2+ that triggers SR Ca2+ release, even at the highly depolarized membrane potentials associated with the AP. However, Ca2+ influx via Na+/Ca2+ exchange is not negligible and may activate some Ca2+ release from the SR, especially when ICa is reduced. We propose that SR Ca2+ release inhibits ICa within the same beat, thereby providing a negative feedback mechanism that may serve to limit Ca2+ influx as well as to regulate the amount of Ca2+ stored within the SR.
Action Potentials↗
The control of calcium release in heart muscle.
The control of calcium release from intracellular stores (the sarcoplasmic reticulum) in cardiac muscle was examined with the use of a confocal microscope and voltage clamp techniques. Depolarization evoked graded calcium release by altering the extent of spatial and temporal summation of elementary calcium release events called "calcium sparks." These evoked sparks were triggered by local L-type calcium channel currents in a stochastic manner, were similar at different potentials, and resembled spontaneous calcium sparks. Once triggered, the calcium release from the sarcoplasmic reticulum during a calcium spark was independent of the duration of the triggering calcium influx. These results were used to develop a unifying model for cardiac excitation-contraction coupling that explains the large (but paradoxically stable) amplification of the trigger calcium influx by a combination of digital and analog behavior.
Animals↗
Partial inhibition of Ca2+ current by methoxyverapamil (D600) reveals spatial nonuniformities in [Ca2+]i during excitation-contraction coupling in cardiac myocytes.
The laser scanning confocal microscope was used in conjunction with the Ca2+ indicator fluo 3 to examine the spatiotemporal properties of free Ca2+ ([Ca2+]i) transients in isolated rat cardiac myocytes. We show that localized increases in [Ca2+]i (Ca2+ sparks) can be triggered by membrane depolarization in cardiac myocytes when the sarcolemmal Ca2+ current amplitude is reduced by methoxyverapamil (D600). These depolarization-evoked Ca2+ sparks are similar in amplitude and spatiotemporal properties to spontaneous Ca2+ sparks previously observed at rest. These observations support the idea that Ca2+ sparks are the result of the activation of functional elementary units of sarcoplasmic reticulum (SR) Ca2+ release. The synchronous activation of a large number of Ca2+ sparks can explain the increased amplitude and slower time course of the electrically evoked [Ca2+]i transient as well as the presence of spatial nonuniformities in [Ca2+]i during its rise. The data shown here suggest a model for excitation-contraction coupling in which the amplitude of the [Ca2+]i transient is regulated by variations in the probability of recruitment of elementary SR Ca2+ release units as well as the amount of Ca2+ released by each unit. Since the activation of each release unit will depend on the local amplitude of the Ca2+ current, this model can explain the regulation of the amplitude of the [Ca2+]i transient by the Ca2+ current. In addition, these data indicate that caution should be applied to the interpretation of signals obtained with nonlinear Ca2+ indicators during the rising phase of the [Ca2+]i transient, when the nonuniformities in [Ca2+]i are largest.
Animals↗