Membrane polarization at the excitation threshold induced by external electric fields in cardiomyocytes of rats at different developmental stages.

External electric fields (E), used for cardiac pacing and defibrillation/cardioversion, induce a spatially variable change in cardiomyocyte transmembrane potential (ΔVm) that depends on cell geometry and E orientation. This study investigates E-induced ΔVm in cardiomyocytes from rats at different ages, which show marked size/geometry variation. Using a tridimensional numerical electromagnetic model recently proposed (NM3D), it was possible: (a) to evaluate the suitability of the simpler, prolate spheroid analytical model (PSAM) to calculate amplitude and location of ΔVm maximum (ΔVmax) for E = 1 V.cm-1; and (b) to estimate the ΔVmax required for excitation (ΔVT) from experimentally determined threshold E values (ET). Ventricular myocytes were isolated from neonatal, weaning, adult, and aging Wistar rats. NM3D was constructed as the extruded 2D microscopy cell image, while measured minor and major cell dimensions were used for PSAM. Acceptable ΔVm estimates can be obtained with PSAM from paralelepidal cells for small θ. ET, but not ΔVT, was higher for neonate cells. ΔVT was significantly greater in the cell from older animals, which indicate lower responsiveness to E associated with aging, rather than with altered cell geometry/dimensions. ΔVT might be used as a non-invasive indicator of cell excitability as it is little affected by cell geometry/size.

Developmental differences in myocardial transmembrane Na+ transport: implications for excitability and Na+ handling.

Little is currently known about possible developmental changes in myocardial Na+ handling, which may have impact on cell excitability and Ca2+ content. Resting intracellular Na+ concentration ([Na+ ]i ), measured in freshly isolated rat ventricular myocytes with CoroNa green, was not significantly different in neonates (3-5 days old) and adults, but electrical stimulation caused marked [Na+ ]i rise only in neonates. Inhibition of L-type Ca2+ current by CdCl2 abolished not only systolic Ca2+ transients, but also activity-dependent intracellular Na+ accumulation in immature cells. This indicates that the main Na+ influx pathway during activity is the Na+ /Ca2+ exchanger, rather than voltage-dependent Na+ current (INa ), which was not affected by CdCl2 . In immature myocytes, INa density was two-fold greater, inactivation was faster, and the current peak occurred at less negative transmembrane potential (Em ) than in adults. Na+ channel steady-state activation and inactivation curves in neonates showed a rightward shift, which should increase channel availability at diastolic Em , but also require greater depolarization for excitation, which was observed experimentally and reproduced in computer simulations. Ventricular mRNA levels of Nav 1.1, Nav 1.4 and Nav 1.5 pore-forming isoforms were greater in neonate ventricles, while a decrease was seen for the ß1 subunit. Both molecular and biophysical changes in the channel profile may contribute to the differences in INa density and voltage-dependence, and also to the less negative threshold Em , in neonates compared to adults. The apparently lower excitability in immature ventricle may confer protection against the development of spontaneous activity in this tissue. KEY POINTS: Previous studies showed that myocardial preparations from immature rats are less sensitive to electrical field stimulation than adult preparations. Freshly isolated ventricular myocytes from neonatal rats showed lower excitability than adult cells, e.g. less negative threshold membrane potential and greater membrane depolarization required for action potential triggering. In addition to differences in mRNA levels for Na+ channel isoforms and greater Na+ current (INa ) density, Na+ channel voltage-dependence was shifted to the right in immature myocytes, which seems to be sufficient to decrease excitability, according to computer simulations. Only in neonatal myocytes did cyclic activity promote marked cytosolic Na+ accumulation, which was prevented by abolition of systolic Ca2+ transients by blockade of Ca2+ currents. Developmental changes in INa may account for the difference in action potential initiation parameters, but not for cytosolic Na+ accumulation, which seems to be due mainly to Na+ /Ca2+ exchanger-mediated Na+ influx.

Accuracy of electromagnetic models to estimate cardiomyocyte membrane polarization.

External electric fields (E) induce a spatially heterogeneous variation in the membrane potential (ΔVm) of cardiomyocytes that, if sufficiently large, results in an action potential and contraction. Insights into the phenomenon of ΔVm induction by E have been classically gained with electromagnetic models due to the lack of adequate experimental approaches. However, it is not clear yet how reliable these models are. To assess the accuracy of commonly used models, a reference 3D numerical model for cardiomyocytes (NMReal) was developed, consisting of the cell membrane shell reconstructed from rendered confocal microscopy images of freshly isolated ventricular myocytes. NMReal was used to estimate the E-induced maximum ΔVm values (ΔVmax), which were compared with estimates from seven other electromagnetic models. Accurate ΔVmax estimates (average error < 2%) were obtained with a less complex 3D model (NM3D) based on the extruded 2D image of the cell longitudinal section. Acceptable ΔVmax estimates (average error < 5%) were obtained with the prolate spheroid analytical model (PSAM) when the angle of E incidence and the cell major axis was < 30°. In this case, PSAM, a much simpler model requiring only the measurement of the longitudinal and transversal cell dimensions, can be a suitable alternative for ΔVmax calculation. Graphical abstract (A) Confocal images of the cell were used to reconstruct the realistic geometry of cardiomyocytes (NMReal). (B) NMReal was used to estimate the maximum variation in the transmembrane potential (ΔVmax) induced by an external electric field (E) applied at different angles with respect to the cell major axis. Plus (anode) and minus (cathode) signs indicate electrode position (E direction is from minus to plus). (C) Relative error (vs. NMReal) of ΔVmax estimation with simplified electromagnetic models, presented in descending order of accuracy (left-to-right, top-to-bottom). NM2D: 2D numerical model based on the longitudinal cell image; NM3D: numerical model based on the z extrusion of NM2D; EAM, PSAM, and CAM: ellipsoidal, prolate spheroidal, and cylindrical analytical models, respectively; PNM and CNM: parallelepipedal and cylindrical numerical models, respectively.

High Thyrotropin Is Critical for Cardiac Electrical Remodeling and Arrhythmia Vulnerability in Hypothyroidism.

Background: Hypothyroidism, the most common endocrine disease, induces cardiac electrical remodeling that creates a substrate for ventricular arrhythmias. Recent studies report that high thyrotropin (TSH) levels are related to cardiac electrical abnormalities and increased mortality rates. The aim of the present work was to investigate the direct effects of TSH on the heart and its possible causative role in the increased incidence of arrhythmia in hypothyroidism. Methods: A new rat model of central hypothyroidism (low TSH levels) was created and characterized together with the classical propylthiouracil-induced primary hypothyroidism model (high TSH levels). Electrocardiograms were recorded in vivo, and ionic currents were recorded from isolated ventricular myocytes in vitro by the patch-clamp technique. Protein and mRNA were measured by Western blot and quantitative reverse transcription polymerase chain reaction in rat and human cardiac myocytes. Adult human action potentials were simulated in silico to incorporate the experimentally observed changes. Results: Both primary and central hypothyroidism models increased the L-type Ca2+ current (ICa-L) and decreased the ultra-rapid delayed rectifier K+ current (IKur) densities. However, only primary but not central hypothyroidism showed electrocardiographic repolarization abnormalities and increased ventricular arrhythmia incidence during caffeine/dobutamine challenge. These changes were paralleled by a decrease in the density of the transient outward K+ current (Ito) in cardiomyocytes from animals with primary but not central hypothyroidism. In vitro treatment with TSH for 24 hours enhanced isoproterenol-induced spontaneous activity in control ventricular cells and diminished Ito density in cardiomyocytes from control and central but not primary hypothyroidism animals. In human myocytes, TSH decreased the expression of KCND3 and KCNQ1, Ito, and the delayed rectifier K+ current (IKs) encoding proteins in a protein kinase A-dependent way. Transposing the changes produced by hypothyroidism and TSH to a computer model of human ventricular action potential resulted in enhanced occurrence of early afterdepolarizations and arrhythmia mostly in primary hypothyroidism, especially under ß-adrenergic stimulation. Conclusions: The results suggest that suppression of repolarizing K+ currents by TSH underlies most of the electrical remodeling observed in hypothyroidism. This work demonstrates that the activation of the TSH-receptor/protein kinase A pathway in the heart is responsible for the cardiac electrical remodeling and arrhythmia generation seen in hypothyroidism.

Sources of Ca2+ for contraction of the heart tube of Tenebrio molitor (Coleoptera: Tenebrionidae).

Insect and vertebrate hearts share the ability to generate spontaneously their rhythmic electrical activity, which triggers the fluid-propelling mechanical activity. Although insects have been used as models in studies on the impact of genetic alterations on cardiac function, there is surprisingly little information on the generation of the inotropic activity in their hearts. The main goal of this study was to investigate the sources of Ca2+ for contraction in Tenebrio molitor hearts perfused in situ, in which inotropic activity was assessed by the systolic variation of the cardiac luminal diameter. Increasing the pacing rate from 1.0 to 2.5 Hz depressed contraction amplitude and accelerated relaxation. To avoid inotropic interference of variations in spontaneous rate, which have been shown to occur in insect heart during maneuvers that affect Ca2+ cycling, experiments were performed under electrical pacing at near-physiological rates. Raising the extracellular Ca2+ concentration from 0.5 to 8 mM increased contraction amplitude in a manner sensitive to L-type Ca2+ channel blockade by D600. Inotropic depression was observed after treatment with caffeine or thapsigargin, which impair Ca2+ accumulation by the sarcoplasmic reticulum (SR). D600, but not inhibition of the sarcolemmal Na+/Ca2+ exchanger by KB-R7943, further depressed inotropic activity in thapsigargin-treated hearts. From these results, it is possible to conclude that in T. molitor heart, as in vertebrates: (a) inotropic and lusitropic activities are modulated by the heart rate; and (b) Ca2+ availability for contraction depends on both Ca2+ influx via L-type channels and Ca2+ release from the SR.

Macrophage-dependent IL-1ß production induces cardiac arrhythmias in diabetic mice.

Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1ß in DM mice. IL-1ß causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1ß-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1ß axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1ß as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.

Atrial chronotropic reactivity to catecholamines in neonatal rats: Contribution of ß-adrenoceptor subtypes.

Although increase in heart rate is a crucial determinant for enhancement of cardiac output in the neonate, information on the chronotropic reactivity to catecholamines during postnatal development is scarce. The present study was aimed at investigating the role of ß-adrenoceptor subtypes and catecholamine removal mechanisms in the adrenergic chronotropic response during the early post-natal period. Right atria isolated from immature (0-21 day old) and adult (4-6 month old) rats were used for determination of the responsiveness to agonists and quantitation of the transcripts of proteins involved in ß-adrenergic signaling. The main results were: (a) the maximum response (Rmax) to norepinephrine increased with age, whereas sensitivity decreased; (b) age-dependent differences in sensitivity to norepinephrine were abolished by inhibition of the neuronal norepinephrine transporter; (c) Rmax to isoproterenol was similar in immature and adult atria, and depressed only in the former by ß2-adrenoceptor blockade with ICI118,551; (d) neonatal atria showed greater ß2-adrenoceptor mRNA levels, and more prominent positive chronotropic response to the ß2- and ß3-adrenoceptor agonists zinterol and YM178, respectively (nanomolar range); (e) in atria of immature rats, transcript levels of the extraneuronal monoamine transporter were lower, and its inhibition did not affect sensitivity to isoproterenol; and (f) reactivity to forskolin and 3-isobutyl-1-methylxanthine was not affected by age. The increased ß2- and ß3-adrenoceptor participation in the adrenergic chronotropic response, in addition to weaker catecholamine removal, may compensate for the immature cardiac innervation and the apparently reduced efficiency of ß1-adrenoceptor signaling in the neonate, increasing the responsiveness to endogenous and exogenous ß2-adrenoceptor agonists.

Chasing cardiac physiology and pathology down the CaMKII cascade.

Calcium dynamics is central in cardiac physiology, as the key event leading to the excitation-contraction coupling (ECC) and relaxation processes. The primary function of Ca(2+) in the heart is the control of mechanical activity developed by the myofibril contractile apparatus. This key role of Ca(2+) signaling explains the subtle and critical control of important events of ECC and relaxation, such as Ca(2+) influx and SR Ca(2+) release and uptake. The multifunctional Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a signaling molecule that regulates a diverse array of proteins involved not only in ECC and relaxation but also in cell death, transcriptional activation of hypertrophy, inflammation, and arrhythmias. CaMKII activity is triggered by an increase in intracellular Ca(2+) levels. This activity can be sustained, creating molecular memory after the decline in Ca(2+) concentration, by autophosphorylation of the enzyme, as well as by oxidation, glycosylation, and nitrosylation at different sites of the regulatory domain of the kinase. CaMKII activity is enhanced in several cardiac diseases, altering the signaling pathways by which CaMKII regulates the different fundamental proteins involved in functional and transcriptional cardiac processes. Dysregulation of these pathways constitutes a central mechanism of various cardiac disease phenomena, like apoptosis and necrosis during ischemia/reperfusion injury, digitalis exposure, post-acidosis and heart failure arrhythmias, or cardiac hypertrophy. Here we summarize significant aspects of the molecular physiology of CaMKII and provide a conceptual framework for understanding the role of the CaMKII cascade on Ca(2+) regulation and dysregulation in cardiac health and disease.

Electrocardiogram, heart movement and heart rate in the awake gecko (Hemidactylus mabouia).

The electrocardiogram (ECG) is the simplest and most effective non-invasive method to assess the electrical activity of the heart and to obtain information on the heart rate (HR) and rhythm. Because information on the HR of very small reptiles (body mass <10 g) is still scarce in the literature, in the present work we describe a procedure for recording the ECG in non-anesthetized geckos (Hemidactylus mabouia, Moreau de Jonnès, 1818) under different conditions, namely manual restraint (MR), spontaneous tonic immobility (TI), and in the non-restrained condition (NR). In the gecko ECG, the P, QRS and T waves were clearly distinguishable. The HR was 2.83 ± 0.02 Hz under MR, which was significantly greater (p < 0.001) than the HR under the TI (1.65 ± 0.09 Hz) and NR (1.60 ± 0.10 Hz) conditions. Spontaneously beating isolated gecko hearts contracted at 0.84 ± 0.03 Hz. The in vitro beating rate was affected in a concentration-dependent fashion by adrenoceptor stimulation with noradrenaline, as well as by the muscarinic cholinergic agonist carbachol, which produced significant positive and negative chronotropic effects, respectively (p < 0.001). To our knowledge, this is the first report on the ECG morphology and HR values in geckos, particularly under TI. The methodology and instrumentation developed here are useful for non-invasive in vivo physiological and pharmacological studies in small reptiles without the need of physical restraint or anesthesia.

Rapidly switching multidirectional defibrillation: reversal of ventricular fibrillation with lower energy shocks.

OBJECTIVES: Cardiac arrest after open surgery has an incidence of approximately 3%, of which more than 50% of the cases are due to ventricular fibrillation. Electrical defibrillation is the most effective therapy for terminating cardiac arrhythmias associated with unstable hemodynamics. The excitation threshold of myocardial microstructures is lower when external electrical fields are applied in the longitudinal direction with respect to the major axis of cells. However, in the heart, cell bundles are disposed in several directions. Improved myocardial excitation and defibrillation have been achieved by applying shocks in multiple directions via intracardiac leads, but the results are controversial when the electrodes are not located within the cardiac chambers. This study was designed to test whether rapidly switching shock delivery in 3 directions could increase the efficiency of direct defibrillation. METHODS: A multidirectional defibrillator and paddles bearing 3 electrodes each were developed and used in vivo for the reversal of electrically induced ventricular fibrillation in an anesthetized open-chest swine model. Direct defibrillation was performed by unidirectional and multidirectional shocks applied in an alternating fashion. Survival analysis was used to estimate the relationship between the probability of defibrillation and the shock energy. RESULTS: Compared with shock delivery in a single direction in the same animal population, the shock energy required for multidirectional defibrillation was 20% to 30% lower (P < .05) within a wide range of success probabilities. CONCLUSIONS: Rapidly switching multidirectional shock delivery required lower shock energy for ventricular fibrillation termination and may be a safer alternative for restoring cardiac sinus rhythm.

Toll-like receptor 4 activation promotes cardiac arrhythmias by decreasing the transient outward potassium current (Ito) through an IRF3-dependent and MyD88-independent pathway.

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca(2+) transients, as well as the L-type Ca(2+) current (ICaL) and the transient outward K(+) current (Ito), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 µg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, Ito amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The Ito decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca(2+) transients and ICaL were not affected by LPS; however, Na(+)/Ca(2+) exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased Ito, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca(2+) efflux via NCX, could explain the presence of arrhythmias in the LPS group.

Muscarinic stimulation and pinacidil produce similar facilitation of tachyarrhythmia induction in rat isolated atria.

Atrial tachyarrhythmias, the most common type of cardiac arrhythmias, are associated with greater stroke risk. Muscarinic cholinergic agonists have been shown to facilitate atrial tachyarrhythmia maintenance in the absence of cardiac disease. This has been attributed to action potential shortening, which enhances myocardial electrical anisotropy, and thus creates a substrate for reentrant excitation. In this study, we describe a similar effect of the ATP-sensitive K(+) channel (KATP) opener pinacidil on tachyarrhythmia induction in isolated rat atria. Pinacidil, which activates a weakly inwardly-rectifying current in isolated atrial myocytes, enhanced arrhythmia induction in the right and left atria. This effect was abolished by the KATP blocker glibenclamide, but not by atropine, which rules out a possible indirect effect due to stimulation of acetylcholine release. However, pinacidil attenuated carbachol-induced tachyarrhythmia facilitation, which may indicate that the action of these agonists converges to a common cellular mechanism. Both agonists caused marked action potential shortening in isolated atrial myocytes. Moreover, during arrhythmia in the presence of pinacidil and carbachol, the atrial vectorelectrographic patterns were similar and consistent with reentrant propagation of the electrical activity. From these results, we conclude that the KATP channel opening is pro-arrhythmic in atrial tissue, which may pose as an additional risk in the scenario of myocardial hypoxia. Moreover, the similarity of the electrophysiological effects of pinacidil and carbachol is suggestive that the sole increase in background K(+) conductance is sufficient for atrial tachyarrhythmia facilitation.

Greater cardiac cell excitation efficiency with rapidly switching multidirectional electrical stimulation.

Electric field (E) stimulation is widely used in experiments with myocardial preparations and in the clinical setting (e.g., defibrillation). As a rule, stimuli are applied in a single direction, which limits excitatory cell recruitment because myocytes are disposed in different directions and their sensitivity to E depends on the stimulus orientation with respect to the cell major axis. Here, we propose a stimulatory approach, namely rapidly switching multidirectional stimulation (RSMS), in which stimuli are delivered in three directions within the electric refractory period. In populations of randomly oriented isolated rat cardiomyocytes, RSMS doubled the percentage of cells excited by near-threshold E (P < 0.001), which was more than the increase in recruitment in a single direction achieved by doubling E intensity. This effect was similar for monophasic and biphasic pulses, but for the latter, a given percent recruitment was obtained with 20-30% lower E intensity ( P < 0.01), so that RSMS with biphasic pulses allowed at least 60% reduction of E intensity for recruitment of >70% of the cells. RSMS can be applied to improve stimulation efficiency in experiments with isolated cardiac myocytes, and may be a promising alternative for decreasing shock intensity requirements for cardioversion and defibrillation.

Estimation of the fractional sarcoplasmic reticulum Ca2+ release in intact cardiomyocytes using integrated Ca2+ fluxes.

The sarcoplasmic reticulum (SR) is the main source of contraction-activating Ca2+ in the adult mammalian myocardium. The fraction of the SR Ca2+ content released at a twitch (fractional SR Ca2+ release, FR) is an important parameter for assessing the efficiency of excitation-contraction coupling under physiological and pathophysiological conditions, as well as for identification of modulators of this process. We here describe and propose an approach for FR quantitation based on the estimation of integrated Ca2+ fluxes mediated by different transporters that remove the ion from the cytosol. These fluxes may be calculated solely from the measurement of cytosolic free Ca2+ concentration ([Ca2+]i) during Ca2+ transients evoked under selective inhibition of the transporters, and from the cell Ca2+ buffering parameters available in the literature. The FR values obtained with this approach in intact rat ventricular myocytes (0.63 ± 0.04; n = 12) were comparable to those estimated in the same cell type with an already established method, based on electrophysiological measurements with the patch-clamp technique, in addition to [Ca2+]i measurement (0.69 ± 0.05; n = 6; p > 0.40). We conclude that the proposed method might be a suitable and technically simpler alternative to the electrophysiological method for FR estimation.

Transthoracic cardiac ultrasonic stimulation induces a negative chronotropic effect.

The objective of this study is to investigate cardiac bioeffects resulting from ultrasonic stimulation using a specific set of acoustical parameters. Ten Sprague-Dawley rats were anesthetized and exposed to 1-MHz ultrasound pulses of 3-MPa peak rarefactional pressure and approximately 1% duty factor. The pulse repetition frequency started slightly above the heart rate and was decreased by 1 Hz every 10 s, for a total exposure duration of 30 s. The control group was composed of five rats. Two-way analysis of variance for repeated measures and Bonferroni post hoc tests were used to compare heart rate and ejection fraction, which was used as an index of myocardial contractility. It was demonstrated for the first time that transthoracic ultrasound has the potential to decrease the heart rate by ~20%. The negative chronotropic effect lasted for at least 15 min after ultrasound exposure and there was no apparent gross damage to the cardiac tissue.

Blood calcium levels in immature rats: influence of extracellular calcium concentration on myocardial calcium handling.

Calcium ions play an important role in several cell functions, from fertilization to cell death. The cytosolic Ca(2+) concentration is much lower than the extracellular concentration ([Ca(2+)](o)). The latter may markedly affect Ca(2+) fluxes across the cell membrane and thus the cellular Ca(2+) load. Thus, when working with preparations in vitro, it is important to keep [Ca(2+)](o) close to the in vivo value. In this study, we determined the calcemia in immature rats, for which values are currently unavailable, and investigated how supraphysiological [Ca(2+)](o) affects myocardial Ca(2+) handling. Blood ionized [Ca(2+)] was similar in neonatal (2-5 days old) and adults Wistar rats (1.28 ± 0.03 and 1.31 ± 0.03 mmol/l; n=6 and 5, respectively, P>0.37), and lower than the [Ca(2+)](o) range often used in experiments with neonatal myocardial preparations. Cytosolic Ca(2+) transients, measured with indo-1 in neonatal ventricular myocytes, were enhanced by an increase in [Ca(2+)](o) from 1.2 to 2 mM, which also increased the Ca(2+) content in the sarcoplasmic reticulum (SR), and changed the pattern of competition between the main transporters that remove Ca(2+) from the cytosol (SR Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger). These observations stress the importance of using physiological [Ca(2+)](o) values for reliability of results. It is expected that the present calcemia data, reported for the first time in immature rats, may contribute to the refinement of in vitro experiments with neonatal rat preparations.

Medical equipment classification: method and decision-making support based on paraconsistent annotated logic.

As technology evolves, the role of medical equipment in the healthcare system, as well as technology management, becomes more important. Although the existence of large databases containing management information is currently common, extracting useful information from them is still difficult. A useful tool for identification of frequently failing equipment, which increases maintenance cost and downtime, would be the classification according to the corrective maintenance data. Nevertheless, establishment of classes may create inconsistencies, since an item may be close to two classes by the same extent. Paraconsistent logic might help solve this problem, as it allows the existence of inconsistent (contradictory) information without trivialization. In this paper, a methodology for medical equipment classification based on the ABC analysis of corrective maintenance data is presented, and complemented with a paraconsistent annotated logic analysis, which may enable the decision maker to take into consideration alerts created by the identification of inconsistencies and indeterminacies in the classification.