Measurement Of Calcium Transients Research Articles

Mechanical and Biochemical Characteristics of Human Stem Cell-Derived Cardiomyocytes

Cell-based cardiac repair following myocardial infarction has gained considerable interest recently, and the human pluripotent stem cell is an attractive cell source due its efficient differentiation into immature but functional cardiomyocytes. We examined the biophysical characteristics of cardiomyocytes generated from human embryonic stem cells (hESC-CMs) by measuring calcium transients, single cell contractions, and actomyosin interactions via flash photolysis. Furthermore, we compared these characteristics with those obtained from a second promising but still poorly characterized cell type, the human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM). We hypothesized that understanding fundamental biochemical and mechanical characteristics of these cells would provide insight into potential strategies to induce further cell maturation in vitro.Our results suggest that hESC-CMs and hiPSC-CMs exhibit spontaneous contractions and calcium transients with similar kinetics, including time to peak [Ca2+]i (116±34ms vs. 155±40ms) and time to 50% [Ca2+]i decay (352±87ms vs. 296±49ms). Furthermore, quantitative videomicroscopy of resulting single cell contractions suggests that cardiomyocytes from both sources demonstrate similar resting cell size (17.1±1.4um vs. 16.5±4.6um), contraction amplitude (4.2±1.6% vs. 4.4±2.1%), time to peak contraction (0.346±0.135sec vs. 0.339±0.214sec), maximum contraction velocity (6.34±3.50um/sec vs. 7.46±4.81um/sec), and maximum relaxation velocity (3.21±2.49um/sec vs. 3.40±2.49um/sec).We have also successfully isolated and purified 20 ug of myosin per million hESC-CMs. Using flash photolysis to liberate ATP in a solution of actomyosin, we have shown that the myosin binds actin and is dissociated from the complex by ATP with the expected 2nd order rate constant (∼1 uM−1sec−1).In summary, the contractile properties of hESC-CMs and hiPSC-CMs are similar to each other but differ from values published for adult human cardiomyocytes, suggesting that they are functionally immature and may benefit from in vitro maturation efforts.Support appreciated from NIH HL064387/HL080431, NHLBI T32GMO7266-35S1, and the Wellcome Trust.

How do synapses measure milliseconds?

How do synapses measure milliseconds?

A Comparison of the Effects of Commercially Available Hawthorn Preparations on Calcium Transients of Isolated Cardiomyocytes

We studied the potential cardiac effects of two alcohol extracts of commercially available hawthorn using rat cardiomyocytes and measuring calcium transients by real-time fluorescence spectrophotometry. One preparation was a blend of hawthorn flowers, leaves, and berries (extract #1), and the other (extract #2) was from a "berries-only" preparation. Fluorescent images and calcium transients were acquired concurrently. Addition of extract #1 resulted in the initiation of robust calcium transients and eventual calcium overload, while addition of extract #2 caused increased calcium sparking, initiation of calcium transients, and an increased beating rate but no calcium overload. To identify the mechanisms of increased calcium influx, adult rat cardiomyocytes were challenged with 10 microM ouabain, a Na(+),K(+)-ATPase inhibitor, and the calcium channel blocker nifedipine. The findings revealed that equal volumes of the two readily available hawthorn preparations demonstrated markedly different effects on isolated adult rat cardiomyocytes, suggesting important implications for patients who are using these preparations to supplement or even replace their prescribed cardiac medications as to which preparation(s) to use, and potential dire consequences, particularly in cardiac patients. Our study indicates that the mechanism of cardiac activity of hawthorn is via the Na(+),K(+)-ATPase and intracellular calcium concentrations are influenced.

Protease-activated Receptor-2 Exerts Contrasting Model-specific Effects on Acute Experimental Pancreatitis

Protease-activated receptor-2 (PAR2) is a 7-transmembrane G-protein-coupled tethered ligand receptor that is expressed by pancreatic acinar and ductal cells. It can be physiologically activated by trypsin. Previously reported studies (Namkung, W., Han, W., Luo, X., Muallem, S., Cho, K. H., Kim, K. H., and Lee, M. G. (2004) Gastroenterology 126, 1844-1859; Sharma, A., Tao, X., Gopal, A., Ligon, B., Andrade-Gordon, P., Steer, M. L., and Perides, G. (2005) Am. J. Physiol. 288, G388-G395) have shown that PAR2 activation exerts a protective effect on the experimental model of pancreatitis induced by supramaximal secretagogue (caerulein) stimulation. We now show that PAR2 exerts a worsening effect on a different model of experimental pancreatitis, i.e. one induced by retrograde pancreatic ductal infusion of bile salts. In vitro studies using freshly prepared pancreatic acini show that genetic deletion of PAR2 reduces bile salt-induced pathological calcium transients, acinar cell injury, and activation of c-Jun N-terminal kinase, whereas genetic deletion of PAR2 has the opposite or no effect on these pancreatitis-related events when they are elicited, in vitro, by caerulein stimulation. Studies employing a combination of trypsin inhibition and activation of PAR2 with the activating peptide SLIGRL show that all these differences indeed depend on the activation of PAR2. These studies are the first to report that a single perturbation can have model-specific and opposite effects on pancreatitis, and they underscore the importance of performing mechanistic pancreatitis studies using two dissimilar models of the disease to detect idiosyncratic, model-specific events. We suggest PAR2 activation exerts a worsening effect on the severity of clinical pancreatitis and that interventions interfering with PAR2 activation may be of benefit in the treatment of patients with severe pancreatitis.

Generation and Escape of Local Waves from the Boundary of Uncoupled Cardiac Tissue

We aim to understand the formation of abnormal waves of activity from myocardial regions with diminished cell-to-cell coupling. En route to this goal, we studied the behavior of a heterogeneous myocyte network in which a sharp coupling gradient was placed under conditions of increasing network automaticity. Experiments were conducted in monolayers of neonatal rat cardiomyocytes using heptanol and isoproterenol as means of altering cell-to-cell coupling and automaticity, respectively. Experimental findings were explained and expanded using a modified Beeler-Reuter numerical model. The data suggest that the combination of a heterogeneous substrate, a gradient of coupling, and an increase in oscillatory activity of individual cells creates a rich set of behaviors associated with self-generated spiral waves and ectopic sources. Spiral waves feature a flattened shape and a pin-unpin drift type of tip motion. These intercellular waves are action-potential based and can be visualized with either voltage or calcium transient measurements. A source/load mismatch on the interface between the boundary and well-coupled layers can lock wavefronts emanating from both ectopic sources and rotating waves within the inner layers of the coupling gradient. A numerical approach allowed us to explore how 1), the spatial distribution of cells, 2), the amplitude and dispersion of cell automaticity, and 3), the speed at which the coupling gradient moves in space affect wave behavior, including its escape into well-coupled tissue.

Spatiotemporal dynamics of calcium and calmodulin at the spine

Calcium levels in spines play a significant role in determining the sign and magnitude of synaptic plasticity. Recent experiments using calcium sensitive dyes have allowed measurements of calcium transients in whole spines, however experimental resolution does not allow imaging of the spatial distribution of calcium within the spine [1,2,5]. Calcium can activate Calcineurin or bind to CaM and consequently activate CaMKII which is key mediator of synaptic plasticity. A main source of calcium influx into the spine is from the NMDA receptors. There are four different subtypes of obligatory NR2 subunits of NMDA receptors, NR2A/B/C/D. In the mature cortex the majority of the synaptic NMDA receptors are constituted by NR1/NR2A and in the immature cortex by NR1/NR2B. Experiments have shown that the subunit composition of NMDA receptors has an influence on the sign of synaptic plasticity, but different experiments resulted in different and possibly conflicting results [3,4]. NR2B has slower kinetics and higher affinity for Glutamate than that of NR2A. In addition NR2B receptors have a binding site for CaMKII.

Stress and high heart rate provoke ventricular tachycardia in mice expressing triadin

Reduced function of the cardiac ryanodine receptor or calsequestrin causes catecholaminergic ventricular tachycardia (VT). These proteins regulate sarcoplasmic Ca 2+ release in close conjunction with two accessory proteins, triadin and junctin. Based on data from cardiomyocytes, we hypothesized that enhanced triadin expression could cause VT. We assessed arrhythmias and electrophysiological changes in vivo and in the beating heart in mice expressing junctin, triadin, or both proteins (TRD×JCN), and measured calcium transients in isolated ventricular cardiomyocytes. TRD×JCN mice were studied to compensate the down-regulation of junctin expression in triadin-expressing mice. Exercise or stress provoked repetitive VT in freely roaming TRD×JCN mice whenever heart rate increased above approximately 600 bpm ( p < 0.05 vs. the three other genotypes). TRD×JCN mice expressed total triadin 2.9-fold ( p < 0.05) and total junctin not different to wildtype ( p = ns). Left ventricular systolic function was not different between lineages. β-adrenoreceptor stimulation (orciprenaline 1.7 μM) provoked early-coupled ventricular ectopy and repetitive VT in isolated, Langendorff-perfused TRD×JCN hearts ( p < 0.05). Under conditions associated with VT (high pacing rate, catecholamine stimulation), action potential duration was shorter in TRD×JCN with VT than in the other genotypes and shorter than in TRD×JCN hearts without VT ( p < 0.05). Ca 2+ transient duration was prolonged in Indo1-loaded TRD×JCN cardiomyocytes under VT-provoking conditions. Action potential prolongation by mexiletine (2 μM or 4 μM) or clarithromycine (150 μM) suppressed VT. Expression of triadin provokes stress- and tachycardia-related ventricular arrhythmias in mice. An imbalance between prolonged intracellular calcium release and shortening of the ventricular action potential may contribute to genesis of arrhythmias in this model.

In vivo calcium imaging from genetically specified target cells in mouse cerebellum

Genetically encoded fluorescent calcium indicator proteins provide the potential to monitor activity from genetically specified target cells without a need for single cell resolution. Here we report the use of transgenic mice expressing the fluorescent calcium indicator protein GCaMP2 in cerebellar granule cells to image parallel fiber activity transcranially in vivo. We demonstrated reliable measurements of calcium transients from beams of parallel fibers in response to electrical stimulation in the molecular layer through the intact skull. These parallel fiber calcium transients differed from intrinsic postsynaptic autofluorescence signals in their faster kinetics and resistance to blockers of synaptic transmission. Finally, we used 2P laser-scanning microscopy to demonstrate reliable measurements of calcium transients from beams of parallel fibers at high spatial resolution in living mice. We expect that genetically targeted fluorescent calcium indicator proteins along with optical imaging techniques will be instrumental for the construction of macroscopic and microscopic maps of the function of specific brain circuits.

Pore forming polyalkylpyridinium salts from marine sponges versus synthetic lipofection systems: distinct tools for intracellular delivery of cDNA and siRNA

BackgroundHaplosclerid marine sponges produce pore forming polyalkylpyridinium salts (poly-APS), which can be used to deliver macromolecules into cells. The aim of this study was to investigate the delivery of DNA, siRNA and lucifer yellow into cells mediated by poly-APS and its potential mechanisms as compared with other lipofection systems (lipofectamine and N4,N9-dioleoylspermine (LipoGen)). DNA condensation was evaluated and HEK 293 and HtTA HeLa cells were used to investigate pore formation and intracellular delivery of cDNA, siRNA and lucifer yellow.ResultsPoly-APS and LipoGen were both found to be highly efficient DNA condensing agents. Fura-2 calcium imaging was used to measure calcium transients indicative of cell membrane pore forming activity. Calcium transients were evoked by poly-APS but not LipoGen and lipofectamine. The increases in intracellular calcium produced by poly-APS showed temperature sensitivity with greater responses being observed at 12°C compared to 21°C. Similarly, delivery of lucifer yellow into cells with poly-APS was enhanced at lower temperatures. Transfection with cDNA encoding for the expression enhanced green fluorescent protein was also evaluated at 12°C with poly-APS, lipofectamine and LipoGen. Intracellular delivery of siRNA was achieved with knockdown in beta-actin expression when lipofectamine and LipoGen were used as transfection reagents. However, intracellular delivery of siRNA was not achieved with poly-APS.ConclusionPoly-APS mediated pore formation is critical to its activity as a transfection reagent, but lipofection systems utilise distinct mechanisms to enable delivery of DNA and siRNA into cells.

Determination of depolarisation- and agonist-evoked calcium fluxes on skeletal muscle cells in primary culture

Changes in intracellular calcium concentration ([Ca 2+] i) evoked by prolonged depolarisation (120 mM KCl) or by the application of 15 mM caffeine were measured on skeletal muscle cells in primary culture. The extrusion rate (PV max) of calcium from the myoplasm was determined, which in turn enabled the calculation of the calcium flux (Fl) underlying the measured calcium transients. PV max was found to increase during differentiation, from 107±10 μM/s at the early myotube stage to 596±36 μM/s in secondary myotubes. This was paralleled by a decrease in resting [Ca 2+] i from 99±4 to 51±2 nM. The depolarisation-evoked Fl rose to peak and then ceased despite the continuous presence of KCl. In contrast, the caffeine-induced Fl showed a peak and a clear steady-level with a peak-to-steady ratio of 5.6±1.2. Removal of external calcium suppressed the depolarisation–induced flux by 88±5% indicating that both an influx and a release from the SR underlie the K +-evoked calcium transients. Subsequent applications of caffeine resulted in essentially identical fluxes indicating an efficient refilling of the internal stores. Moreover, if a depolarisation-induced calcium transient preceded the second caffeine-evoked release, the latter was significantly larger than the first suggesting that much of the calcium that entered was stored in the SR rather than extruded.

Nonlinear magic: multiphoton microscopy in the biosciences.

Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.

Action Potential Propagation in Dendrites of Rat Mitral CellsIn Vivo

Odors evoke beta-gamma frequency field potential oscillations in the olfactory systems of awake and anesthetized vertebrates. In the rat olfactory bulb, these oscillations reflect the synchronous discharges of mitral cells that result from both their intrinsic membrane properties and their dendrodendritic interactions with local inhibitory interneurons. Activation of dendrodendritic synapses is purportedly involved in odor memory and odor contrast enhancement. Here we investigate in vivo to what extent action potentials propagate to remote dendrodendritic sites in the entire dendritic tree and if this propagation is changed during discharges at 40 Hz. By combining intracellular recording and two-photon microscopy imaging of intracellular calcium ([Ca2+]i), we show that in remote branches of the apical tuft and basal dendrites, transient Ca2+ changes are triggered by single sodium action potentials. Neither the amplitude of these Ca2+ transients nor that of action potentials obtained from intradendritic recordings showed a significant attenuation as a function of the distance from the soma. Calcium channel density seemed homogeneous; however, propagating action potentials occasionally failed to trigger a Ca2+ transient at a site closer to the soma whereas it did farther. This suggests that measurements of calcium transients underestimate the occurrence of sodium action potentials. During 40 Hz bursts of action potentials, [Ca2+]i increases with the number of action potentials in all dendritic compartments. These results suggest that the presence of release sites in dendrites is accompanied by an "axonal-like behavior" of the entire dendritic tree of mitral cells, including their most distal dendritic branches.

Load dependence of ventricular performance explained by model of calcium-myofilament interactions.

Although a simple concept of load-independent behavior of the intact heart evolved from early studies of isolated, intact blood-perfused hearts, more recent studies showed that, as in isolated muscle, the mode of contraction (isovolumic vs. ejection) impacts on end-systolic elastance. The purpose of the present study was to test whether a four-state model of myofilament interactions with length-dependent rate constants could explain the complex contractile behavior of the intact, ejecting heart. Studies were performed in isolated, blood-perfused canine hearts with intracellular calcium transients measured by macroinjected aequorin. Measured calcium transients were used as the driving function for the model, and length-dependent rate constants yielding the highest concordance between measured and model-predicted midwall stress at different isovolumic volumes were determined. These length-dependent rate constants successfully predicted contractile behavior on ejecting contractions. This, along with additional model analysis, suggests that length-dependent changes in calcium binding affinity may not be an important factor contributing to load-dependent contractile performance in the intact heart under physiological conditions.

Mapping action potentials and calcium transients simultaneously from the intact heart.

Intracellular calcium handling plays an important role in cardiac electrophysiology. Using two fluorescent indicators, we developed an optical mapping system that is capable of measuring calcium transients and action potentials at 256 recording sites simultaneously from the intact guinea pig heart. On the basis of in vitro measurements of dye excitation and emission spectra, excitation and emission filters at 515 +/- 5 and >695 nm, respectively, were used to measure action potentials with di-4-ANEPPS, and excitation and emission filters at 365 +/- 25 and 485 +/- 5 nm, respectively, were used to measure calcium transients with indo 1. The percent error due to spectral overlap was small when action potentials were measured (1.7 +/- 1.0%, n = 3) and negligible when calcium transients were measured (0%, n = 3). Recordings of calcium transients, action potentials, and isochrone maps of depolarization time and the time of calcium transient onset indicated negligible error due to fluorescence emission overlap. These data demonstrate that the error due to spectral overlap of indo 1 and di-4-ANEPPS is sufficiently small, such that optical mapping techniques can be used to measure calcium transients and action potentials simultaneously in the intact heart.

Regulation of cardiac calcium current by NO and cGMP-modulating agents.

Several effects of nitric oxide (NO) on the control of L-type calcium current (ICa) and of calcium handling in cardiomyocytes have been described. Cardiomyocytes have been shown to express in different conditions all types of nitric oxide synthases (NOS), but the role of NO in the regulation of calcium current remains controversial. Previously, we have shown in guinea pig ventricular cells a stimulatory effect of NOS inhibitors on ICa. Here we investigate the intracellular mechanisms involved in the putative inhibitory role of NO on basal ICa in ventricular cells. The stimulatory effect of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) (1 mM) was present also in calcium transient measurements, but only after a preincubation with L-arginine (L-arg, 0.1 mM). The nitric oxide scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 0.5 mM) increased peak ICa in a similar manner to NOS inhibitors in whole-cell voltage-clamp experiments. Also ODQ (1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one, 0.1 mM), a specific inhibitor of a target of NO, the soluble guanylate cyclase, was able to stimulate ICa. The block of type II phosphodiesterase (cGMP-activated) by EHNA (erythro-9-[2-hydroxy-3-nonylladenine, 30 microM) exerted a similar effect on ICa as PTIO and ODQ. Carbachol (CCh, 1 microM) was able to revert the stimulatory effect on ICa observed with PTIO, ODQ, and EHNA. We propose that the increase of basal ICa in guinea pig cardiomyocytes previously observed with L-NMMA depends on the removal of a tonic NO inhibition. This increase of ICa is mimicked by blocking at different steps the cGMP-cascade activated by NO, suggesting a NO-guanylate cyclase mechanism in the basal control of ventricular calcium current.

Localisation and functional significance of ryanodine receptors during beta-adrenoceptor stimulation in the guinea-pig sino-atrial node.

Recent evidence shows that calcium released from the sarcoplasmic reticulum (SR) plays an important role in the regulation of heart rate. The aim of this study was to investigate the subcellular distribution of ryanodine receptors in the guinea-pig sino-atrial (SA) node and to determine their functional role in the regulation of pacemaker frequency in response to beta-adrenoceptor stimulation. Monoclonal antibodies raised against the cardiac ryanodine receptor were used with confocal microscopy to investigate ryanodine receptor distribution in single guinea-pig SA node cells. The functional role of ryanodine receptors was investigated in both multicellular SA node/atrial preparations and in single SA node cells. Ryanodine receptor labelling was observed in all SA node cells studied and showed both subsarcolemmal and intracellular staining. In the latter, labelling appeared as transverse bands with a regular periodicity of approximately 2 microm. This interval resembled that of the expected sarcomere spacing but did not, however, depend on the presence of transverse tubules. The bands of ryanodine receptors appeared to be located in the region of the Z lines, based on co-distribution studies with antibodies to alpha-actinin, myomesin and binding sites for phalloidin. Functional studies on single SA node cells showed that application of ryanodine (2 micromol/l) reduced the rate of firing of spontaneous action potentials (measured using the perforated patch clamp technique) and this was associated with changes in action potential characteristics. Ryanodine also significantly decreased the positive chronotropic actions of isoprenaline in both multicellular and single cell preparations. In single cells exposed to 100 nmol/l isoprenaline, ryanodine caused a decrease in the rate of firing and this was associated with a decrease in the amplitude of the measured calcium transients. These findings are the first to show immunocytochemical evidence for the presence and organisation of ryanodine receptor calcium release channels in mammalian SA node cells. This study also provides evidence of a role for ryanodine sensitive sites in the beta-adrenergic modulation of heart rate in this species.

Actions of Ketamine and Its Isomers on Contractility and Calcium Transients in Human Myocardium

KUNST, GUDRUN; MARTIN, EIKE; GRAF, BERNHARD M.; HAGL, SIEGFRIED; VAHL, CHRISTINE F. Author Information

C-C and C-X-C chemokines trigger firm adhesion of monocytes to vascular endothelium under flow conditions.

In summary, our findings indicate that specific chemokines that are elaborated by endothelial cells after cytokine or endotoxin activation can play an essential role in monocyte recruitment beyond their chemoattractant activities. We show that this action is to translate initial monocyte tethering into firm adhesion via rapid leukocyte integrin activation. The in vitro model presented here provides a sensitive system for investigating the modulating ability of chemokines and reveals an important biological effect that is not predicted by results in simpler in vitro assays, such as measurement of calcium transients or chemotaxis. The surprising finding that the C-X-C chemokine IL-8 can trigger monocyte firm adhesion to vascular endothelium suggests a potential role for this chemokine in monocyte recruitment and underscores the biological complexity of the chemokine family.

Adrenergic Stimulation Regulates Na+/Ca2+Exchanger Expression in Rat Cardiac Myocytes

K. L. Golden, Q. I. Fan, B. Chen, J. Ren, J. O»Connor and J. D. Marsh. Adrenergic Stimulation Regulates Na+/Ca2+Exchanger Expression in Rat Cardiac Myocytes. Journal of Molecular and Cellular Cardiology (2000) 32, 611–620. The Na/Ca exchanger protein encoded by the NCX1 gene provides the predominant mechanism for calcium efflux during cardiac relaxation. Because β -adrenergic stimulation increases expression of Ca2+channels (Ca2+influx) in cardiac myocytes, we tested the hypothesis that isoproterenol would concomitantly augment expression of NCX1. Four hour treatment of neonatal myocytes with isoproterenol significantly increased NCX1 gene and protein expression, and increased the rate of transcript initiation. Alpha-adrenergic stimulation significantly decreases NCX1 mRNA levels. Calcium transient measurements revealed that for cells that had been pretreated with isoproterenol there was a faster relaxation rate of the Ca2+transient in the presence of thapsigargin, indicating an enhanced rate of intracellular Ca2+removal. We conclude that effectors that increase calcium channel expression in neonatal myocytes also augments NCX1 gene and protein expression over a similar time course, and that this is due to enhanced NCX1 transcription. The regulation of expression of NCX1 by adrenergic pathways may play an important role in regulation of excitation–contraction coupling in cardiac myocytes.

Measurements of calcium transients in ventricular cells during discontinuous action potential conduction.

The L-type calcium current (I(Ca)) is important in sustaining propagation during discontinuous conduction. In addition, I(Ca) is altered during discontinuous conduction, which may result in changes in the intracellular calcium transient. To study this, we have combined the ability to monitor intracellular calcium concentration ([Ca(2+)](i)) in an isolated cardiac cell using confocal scanning laser fluorescence microscopy with our "coupling clamp" technique, which allows action potential propagation from the real cell to a real-time simulation of a model cell. Coupling a real cell to a model cell with a value of coupling conductance (G(C) = 8 nS) just above the critical value for action potential propagation results in both an increased amplitude and an increased rate of rise of the calcium transient. Similar but smaller changes in the calcium transient are caused by increasing G(C) to 20 nS. The increase of [Ca(2+)](i) by discontinuous conduction is less than the increase of I(Ca), which may indicate that much of [Ca(2+)](i) is the result of calcium released from the sarcoplasmic reticulum rather than the integration of I(Ca).