Size controlled hydroxyapatite and calcium carbonate particles: synthesis and their application as templates for SERS platform.

An elegant route for hydroxyapatite (HA) particle synthesis via ionic exchange reaction is reported. Calcium carbonate particles (CaCO3) were recrystallized into HA beads in water solution with phosphate ions. The size of initial CaCO3 particles was controlled upon the synthesis by varying the amount of ethylene glycol (EG) in aqueous solution. The average size of HA beads ranged from 0.6±0.1 to 4.3±1.1µm. Silver nanoparticles were deposited on the surface of HA and CaCO3 particles via silver mirror reaction. Surface enhanced Raman scattering of silver functionalized beads was demonstrated by detecting Rhodamine B. CaCO3 and HA particles have a great potential for design of carrier which can provide diagnostic and therapeutic functions.

New techniques in linear and non-linear laser optics in muscle research.

This review proposes a brief summary of two applications of lasers to muscle research. The first application (laser tweezers), is now a well-established technique in the field, adopted by several laboratories in the world and producing a constant stream of original data, fundamental for our improved understanding of muscle contraction at the level of detail that only single molecule measurements can provide. As an example of the power of this technique, here we focus on some recent results, revealing the performance of the working stroke in at least two distinct steps also in skeletal muscle myosin. A second laser-based technique described here is second-harmonic generation; the application of this technique to muscle research is very recent. We describe the main results obtained thus far in this area and the potentially remarkable impact that this technology may have in muscle research.

Global versus local linear beat-to-beat analysis of the relationship between arterial pressure and pulse transit time during dynamic exercise.

Global linear analysis has been traditionally performed to verify the relationship between pulse transit time (PTT) and systolic arterial pressure (SAP) at the level of their spontaneous beat-to-beat variabilities: PTT and SAP have been plotted in the plane (PTT,SAP) and a significant linear correlation has been found. However, this relationship is weak and in specific individuals cannot be found. This result prevents the utilization of the SAP-PTT relationship to derive arterial pressure changes from PTT measures on an individual basis. We propose a local linear approach to study the SAP-PTT relationship. This approach is based on the definition of short SAP-PTT sequences characterized by SAP increase (decrease) and PTT decrease (increase) and on their search in the SAP and PTT beat-to-beat series. This local approach was applied to PTT and SAP series derived from 13 healthy humans during incremental supine dynamic exercise (at 10, 20 and 30% of the nominal individual maximum effort) and compared to the global approach. While global approach failed in some subjects, local analysis allowed the extraction of the gain of the SAP-PTT relationship in all subjects both at rest and during exercise. When both local and global analyses were successful, the local SAP-PTT gain is more negative than the global one as a likely result of noise reduction.

Cell imaging and manipulation by nonlinear optical microscopy.

Advances in the technologies for labeling and imaging biological samples drive a constant progress in our capability of studying structures and their dynamics within cells and tissues. In the last decade, the development of numerous nonlinear optical microscopies has led to a new prospective both in basic research and in the potential development of very powerful noninvasive diagnostic tools. These techniques offer large advantages over conventional linear microscopy with regard to penetration depth, spatial resolution, three-dimensional optical sectioning, and lower photobleaching. Additionally, some of these techniques offer the opportunity for optically probing biological functions directly in living cells, as highlighted, for example, by the application of second harmonic generation to the optical measurement of electrical potential and activity in excitable cells. In parallel with imaging techniques, nonlinear microscopy has been developed into a new area for the selective disruption and manipulation of intracellular structures, providing an extremely useful tool of investigation in cell biology. In this review we present some basic features of nonlinear microscopy with regard both to imaging and manipulation, and show some examples to illustrate the advantages offered by these novel methodologies.

Second-harmonic generation sensitivity to transmembrane potential in normal and tumor cells.

Second-harmonic generation (SHG) is emerging as a powerful tool for the optical measurement of transmembrane potential in live cells with high sensitivity and temporal resolution. Using a patch clamp, we characterize the sensitivity of the SHG signal to transmembrane potential for the RH 237 dye in various normal and tumor cell types. SHG sensitivity shows a significant dependence on the type of cell, ranging from 10 to 17% per 100 mV. Furthermore, in the samples studied, tumor cell lines display a higher sensitivity compared to normal cells. In particular, the SHG sensitivity increases in the cell line Balb/c3T3 by the transformation induced with SV40 infection of the cells. We also demonstrate that fluorescent labeling of the membrane with RH 237 at the concentration used for SHG measurements does not induce any measurable alteration in the electrophysiological properties of the cells investigated. Therefore, SHG is suitable for the investigation of outstanding questions in electrophysiology and neurobiology.

Causal transfer function analysis to describe closed loop interactions between cardiovascular and cardiorespiratory variability signals.

Although the concept of transfer function is intrinsically related to an input-output relationship, the traditional and widely used estimation method merges both feedback and feedforward interactions between the two analyzed signals. This limitation may endanger the reliability of transfer function analysis in biological systems characterized by closed loop interactions. In this study, a method for estimating the transfer function between closed loop interacting signals was proposed and validated in the field of cardiovascular and cardiorespiratory variability. The two analyzed signals x and y were described by a bivariate autoregressive model, and the causal transfer function from x to y was estimated after imposing causality by setting to zero the model coefficients representative of the reverse effects from y to x. The method was tested in simulations reproducing linear open and closed loop interactions, showing a better adherence of the causal transfer function to the theoretical curves with respect to the traditional approach in presence of non-negligible reverse effects. It was then applied in ten healthy young subjects to characterize the transfer functions from respiration to heart period (RR interval) and to systolic arterial pressure (SAP), and from SAP to RR interval. In the first two cases, the causal and non-causal transfer function estimates were comparable, indicating that respiration, acting as exogenous signal, sets an open loop relationship upon SAP and RR interval. On the contrary, causal and traditional transfer functions from SAP to RR were significantly different, suggesting the presence of a considerable influence on the opposite causal direction. Thus, the proposed causal approach seems to be appropriate for the estimation of parameters, like the gain and the phase lag from SAP to RR interval, which have a large clinical and physiological relevance.

Multiphoton multifocal microscopy exploiting a diffractive optical element.

Multiphoton multifocal microscopy (MMM) usually has been achieved through a combination of galvo scanners with microlens arrays, with rotating disks of microlens arrays, and cascaded beam splitters with asynchronous rastering of scanning mirrors. Here we describe the achievement of a neat and compact MMM by use of a high-diffraction-efficiency diffractive-optic element that generates a multiple-spot grid of uniform intensity to achieve higher fidelity in imaging of live cells at adequate speeds.

Experimental approach for testing the uncoupling between cardiovascular variability series.

In cardiovascular variability analysis, the significance of the coupling between two series is commonly assessed by defining a zero level on the magnitude-squared coherence (MSC). Although the use of the conventional value of 0.5 does not consider the dependence of MSC estimates on the analysis parameters, a theoretical threshold Tt is available only for the weighted covariance (WC) estimator. In this study, an experimental threshold for zero coherence Te was derived by a statistical test from the sampling distribution of MSC estimated on completely uncoupled time series. MSC was estimated by the WC method (Parzen window, spectral bandwidth B = 0.015, 0.02, 0.025, 0.03 Hz) and by the parametric autoregressive (AR) method (model order M= 4, 8, 12, 16), on time series with length L = 180, 300, 420, 540 s. Te decreased with increasing B and L and with decreasing M (range: 0.11-0.54 for WC estimator, 0.06-0.46 for AR estimator). Values for the typical parameter settings of WC and AR estimation (B = 0.025 Hz; M = 8; L = 300 s) were, respectively, 0.24 and 0.17. Moreover, Tt was always higher (range: 0.12-0.65) and the results were less dependable than those for Te in defining the zero level of MSC. Thus, with the proposed method, the hypothesis of uncoupling is rejected by accounting for the parameters that affect the confidence of spectral and cross-spectral estimates. The broad applicability of this approach should favour its introduction for assessing the significance of the coupling between cardiovascular variability series.

Continuous monitoring of myocardial acid-base status during intermittent warm blood cardioplegia.

OBJECTIVE: Intermittent warm blood cardioplegia (IWBC) is a well-established technique for myocardial protection during cardiac operations. According to standardized protocols, IWBC administration is currently performed every 15-20 min regardless of any individual variable and in the absence of any instrumental monitoring. We devised a new system for continuous measurement of the acid-base status of coronary sinus blood for on-line evaluation of myocardial oxygenation during IWBC. METHODS: In 19 patients undergoing cardiac surgery for coronary artery bypass graft and/or valve surgery and receiving IWBC (34-37 degrees C) by antegrade induction (3 min) and retrograde or antegrade maintenance (2 min) every 15 min, continuous monitoring of myocardial oxygenation and acid/base status was performed by means of a multiparameter PO(2), PCO(2), pH, and temperature sensor (Paratrend7 (R), Philips Medical System) inserted into the coronary sinus. RESULTS: Mean cross-clamping time was 76+/-26 min; ischemic time was 13+/-0.2 min. pH decline was not linear, showing an initial fast decline, a point of flexus, and a progressive slow decline. After every ischemic period, the pH adaptation curve showed a complex pattern reaching step-by-step lower minimum levels (7.28+/-0.14 during the first ischemic period, to 7.16+/-0.19 during the third ischemic period - P=0.003). PO(2) decreased rapidly at 90% in 5.0+/-1.2 min after every reperfusion. During ischemia, PCO(2) increased steadily at 1.6+/-0.1 mmHg per minute, with progressively incomplete removal after successive reperfusion, and progressive increase of maximal level (42+/-12 mmHg during the first ischemic period, to 53+/-23 mmHg during the third ischemic period - P=0.05). CONCLUSIONS: Myocardial oxygen, carbon dioxide, and pH show marked changes after repeated IWBC. Myocardial ischemia is not completely reversed by standardized reperfusions, as reflected by steady deterioration of PCO(2) and pH after each reperfusion. Progressive increase of reperfusion durations or direct monitoring of myocardial oxygenation could be advisable in cases of prolonged cross-clamping time.

Different patterns of atrial activation in idiopathic atrial fibrillation: simultaneous multisite atrial mapping in patients with paroxysmal and chronic atrial fibrillation.

OBJECTIVES: We aimed to evaluate: 1) the behavior of electrical activity simultaneously in different atrial regions during atrial fibrillation (AF); 2) the difference of atrial activation between paroxysmal and chronic AF; 3) the atrial refractoriness dispersion; and 4) the correlation between the effective refractory periods (ERPs) and the FF intervals. BACKGROUND: Little data exist on the electrophysiologic characteristics of the different atrial regions in patients with AF. A more detailed knowledge of the electrical activity during AF may provide further insights to improve treatment of AF. METHODS: Right and left atria were extensively mapped in 30 patients with idiopathic AF (18 paroxysmal and 12 chronic). In different atrial locations, we analyzed 1) the FF interval duration; and 2) the grade of organization and, in case of organized electrical activity, the direction of atrial activation. Furthermore, in patients with paroxysmal AF, we determined the atrial ERP, evaluated the ERP dispersion and assessed the presence of a correlation between the ERPs and the FF intervals. RESULTS: In patients with chronic AF, we observed a shortening of the FF intervals and a greater prevalence of disorganized activity in all the atrial sites examined. In patients with paroxysmal AF, a significant dispersion of refractoriness was observed. The right lateral wall showed longer FF intervals and more organized atrial activity and, unexpectedly, the shortest mean ERPs. In contrast, the septal area showed shorter FF intervals, greater disorganization and the longest mean ERPs. CONCLUSIONS: Electrical activity during AF showed a significant spatial inhomogeneity, which was more evident in patients with paroxysmal AF. The mean FF intervals did not correlate with the mean ERPs.

Principal component analysis and cluster analysis for measuring the local organisation of human atrial fibrillation.

The distribution of atrial electrogram types has been proposed to characterise human atrial fibrillation. The aim of this study was to provide computer procedures for evaluating the local organisation of intracardiac recordings during AF as an alternative to off-line manual classification. Principal component analysis (PCA) reduced the data set to a few representative activations, and cluster analysis (CA) measured the average dissimilarity between consecutive activations of an intracardiac signal. The data set consisted of 106 bipolar signals recorded on 11 patients during electrophysiological studies for catheter ablation. Performances of PCA and CA in distinguishing between organised (type I) and disorganised (type II/III, Wells criteria) were assessed, in comparison with manual reading, by evaluating the predictive parameters of the classification analysis. Both methods gave high accuracy (92% for PCA and 89% for CA), confirming the feasibility of on-line characterisation of AF. Sensitivity was lower than specificity (81% against 98% for PCA, and 77% against 97% for CA), with seven out of eight misclassifications of PCA in common with CA. Differences between manual and computer analysis may be related to the higher resolution of PCA and CA in the measurement of the organisation of atrial activations. These procedures are suitable for providing automatic (by CA) or semi-automatic (by PCA) measures of the extent of local organisation of AF in the pre-ablation treatment phase.

Reproducibility of spontaneous initiations of atrial fibrillation.

INTRODUCTION: It has been suggested that some atrial regions may play a role in the maintenance of atrial fibrillation (AF), whereas little is known about the presence of critical areas for the initiation of AF. It is conceivable that the identification of such critical areas may lead to more localized and selective strategies of ablative therapy. METHODS AND RESULTS: A patient suffering from paroxysmal AF was extensively mapped in both the atria with a multielectrode basket catheter in right atrium and two decapolar catheters placed in the coronary sinus and along the left septum. During the mapping, seven identical patterns of initiation of AF were recorded. AF was initiated by an atrial premature beat (APB) arising from the superior right septum, followed by a reentrant beat originating from the same area that slowly propagated through the atria and resulted in late activation of the right lateral wall. During sinus rhythm, the majority of the electrograms were single potentials, whereas during the APB, and particularly during the first atrial reentrant beat, a high percentage of fragmented complexes was present, mainly located in the right superior septum. These fragmented complexes were present in the same sites in each initiating episode and remarkably, they showed an almost identical morphology. CONCLUSION: This case suggests that in some patients the initiation of AF could be caused by reentrant circuits localized in specific atrial regions and the reentrant circuits could be identical in the different episodes of AF. This highlights the importance of increasing our understanding of the mechanisms of the initiation of AF so that we can identify any critical area involved in the genesis of AF where selective RF lesions may be effective in curing this arrhythmia.

Performance assessment of standard algorithms for dynamic R-T interval measurement: comparison between R-Tapex and R-T(end) approach.

Three automatic approaches to ventricular repolarisation duration measurement (R-Tapex, R-T(end threshold) and R-T(end fitting) methods) are compared on computer-generated and real ECG signals, in relation to their reliability in the presence of the most common electrocardiographic artefacts (i.e. additive broadband noise and additive and multiplicative periodical disturbances). Simulations permit the evaluation of the amount of R-T beat-to-beat variability induced by the artefacts. The R-T(end threshold) method performs better than the R-T(end fitting) one, and, hence, the latter should be used with caution when R-T(end) variability is addressed. Whereas the R-Tapex method is more robust with regard to broadband noise than the R-T(end threshold) one, the reverse situation is observed in the presence of periodical amplitude modulations. A high level of broadband noise dose not prevent the detection of the central frequency of underlying R-T periodical changes. Comparison between the power spectra of the beat-to-beat R-T variability series obtained from three orthogonal ECG leads (X,Y,Z) is used to assess the amount of real and artefactual variability in 13 normal subjects at rest. The R-Tapex series displays rhythms at high frequency (HF) with a percentage power on the Z lead (57.1 +/- 4.9) greater than that on the X and Y leads (41.9 +/- 4.6 and 46.1 +/- 4.9, respectively), probably because of respiratory-related artefacts affecting the Z lead more remarkably. More uniform HF power distributions over X,Y,Z leads are observed in the R-T(end threshold) series (31.8 +/- 3.8, 39.2 +/- 4.1 and 35.1 +/- 4.2, respectively), thus suggesting minor sensitivity of the R-T(end threshold) measure to respiratory-related artefacts.

Atrial mapping and radiofrequency catheter ablation in patients with idiopathic atrial fibrillation. Electrophysiological findings and ablation results.

BACKGROUND: Knowledge of the electrophysiological substrates and the cure of atrial fibrillation (AF) is still unsatisfactory. The goal of this study was to evaluate the electrophysiological features of idiopathic AF and their relationship to the results of radiofrequency (RF) catheter ablation of AF and the safety and effectiveness of this procedure. METHODS AND RESULTS: Sixteen patients with idiopathic AF underwent atrial mapping during AF and then RF ablation in the right atrium. The atrial activation was simultaneously recorded in four regions in the right atrium: high lateral wall (HL), low lateral wall (LL), high septum (HS), and low septum (LS) and in the left atrium through the coronary sinus (CS). In these regions, we evaluated the atrial fibrillation intervals (FF) and the morphological features of AF recordings by Wells' classification. No complications occurred during RF ablation. Of the 16 patients, 9 (56%) without AF recurrences during the follow-up (11 +/- 4 months) were considered successfully ablated. These patients showed a significantly shorter mean FF interval in the HS and the LS (122 +/- 32 and 126 +/- 28 ms, respectively), than in the HL and LL (159 +/- 24 and 156 +/- 28 ms, respectively). Moreover, the septum had more irregular electrical activity with greater beat-to-beat changes in FF and a higher prevalence of type III AF than the lateral region. The CS had similar behavior to the septum. Conversely, patients with unsuccessful ablation had an irregular atrial activity in the lateral wall, septum, and CS with no significant differences between the different sites. CONCLUSIONS: Right atrial endocardial catheter ablation of AF is a safe procedure and may be effective in some patients with idiopathic AF. The atrial mapping during AF showed a more disorganized right atrial activation in the septum than in the lateral wall in patients with successful ablation.

An automated method for mapping human tissue permittivities by MRI in hyperthermia treatment planning.

This paper presents an automatic method to obtain tissue complex permittivity values to be used as input data in the computer modelling for hyperthermia treatment planning. Magnetic resonance (MR) images were acquired and the tissue water content was calculated from the signal intensity of the image pixels. The tissue water content was converted into complex permittivity values by monotonic functions based on mixture theory. To obtain a water content map by MR imaging a gradient-echo pulse sequence was used and an experimental procedure was set up to correct for relaxation and radiofrequency field inhomogeneity effects on signal intensity. Two approaches were followed to assign the permittivity values to fat-rich tissues: (i) fat-rich tissue localization by a segmentation procedure followed by assignment of tabulated permittivity values; (ii) water content evaluation by chemical shift imaging followed by permittivity calculation. Tests were performed on phantoms of known water content to establish the reliability of the proposed method. MRI data were acquired and processed pixel-by-pixel according to the outlined procedure. The signal intensity in the phantom images correlated well with water content. Experiments were performed on volunteers' healthy tissue. In particular two anatomical structures were chosen to calculate permittivity maps: the head and the thigh. The water content and electric permittivity values were obtained from the MRI data and compared to others in the literature. A good agreement was found for muscle, cerebrospinal fluid (CSF) and white and grey matter. The advantages of the reported method are discussed in the light of possible application in hyperthermia treatment planning.

Adaptation of the QT interval to heart rate changes in isolated perfused guinea pig heart: influence of amiodarone and D-sotalol.

The inadequacy of the QT interval to shorten following heart rate increase is a feature of the inherited long QT syndrome and may have a role in the genesis of the typical arrhythmias associated with this syndrome (torsade des pointes). The aim of our study was to evaluate whether drugs that prolong the QT interval, such as amiodarone and D-sotalol, may also impair the ability of the QT interval to adapt to sudden heart rate changes. Experiments were carried out on isolated perfused guinea pig hearts (Langendorff preparation). Driving frequency was changed, in steps, every two minutes (Hz: 2.5-3-2.5-3.75-2.5-5-2.5), while epicardial ECG was continuously recorded on magnetic tape. QT interval was automatically measured by means of a beat-by-beat analysis program. D-sotalol was added to the perfusion medium at a concentration of 4 micrograms ml-1, while amiodarone was administered, before in vitro evaluation, for seven days (50 mg kg-1 per day, intraperitoneally). In control experiments two phases of QT adaptation were identified: an abrupt QT shortening at the first beat after frequency change (QT1), followed by a gradual, exponential QT shortening that reached a new steady state in about 1 min (half life: 13 sec). The electrical restitution curve (the relation between QT1 and the corresponding diastolic interval) had a rate constant of 57 +/- 8 ms. Neither drug changed the slow component of QT adaptation. However, both drugs increased the ability of QT to shorten upon premature stimulation: D-sotalol by increasing the rate constant of the restitution curve and amiodarone by decreasing the y-intercept. Our results indicate that D-sotalol and amiodarone do not impair QT shortening during tachycardia but, on the contrary, they may favour QT adaptation, thus reducing the likelihood of the potentially lethal 'R on T phenomenon'. This may be an additional mechanism by which these drugs can exert their antifibrillatory action.

Grading in superficial papillary bladder carcinoma, with an emphasis on nuclear orientation.

OBJECTIVE: To develop a computerized analysis method that could reflect not only cellular anaplasia but also the orientation (PV) of neoplastic elements and could allow the separation of low (grade G 1 and G 2a) and high (G 2b) grades of superficial papillary bladder tumors (SPBTs). STUDY DESIGN: Cellular anaplasia is based on classic morphometric parameters, such as mean and standard deviation of area, perimeter, roundness and aspect ratio (length/width ratio) of 50 papillary bladder tumors (16 G 1, 10 G 2a and 24 G 2b according to Pauwels). Orientation of the major axis of each nucleus with respect to the true basal membrane was calculated using the SD of valve distribution. RESULTS: No statistically significant differences were found between cases showing contiguous grades (G 1 versus G 2a or G 2a versus G 2b). PV, SD of the area and SD of the perimeter were the parameters that could significantly distinguish between G 1 and G 2b and between G 1 plus G 2a and G 2b (P < .05). When comparing G 1 with G 2a plus G 2b, only PV and SD of the area were statistically significant. CONCLUSION: Quantification of the irregularities in the orientation of nuclei with respect to the basal membrane may improve morphometric classification of low grade SPBTs.

[Effects of filtering techniques on time-domain analysis of signal-averaged ECG after acute myocardial infarction: a multicenter study, GISS-3 arrhythmia sub-project]. / Effetti delle techniche di filtraggio sull'analisi nel dominio del tempo del signal-averaged ecg dopo infarto miocardico acuto: studio multicentrico, GISSI-3 sottoprogetto aritmie. GISSI-3 Sottoprogetto Aritmie (Elenco dei Centri in ultima pagina).

AIM OF THE STUDY: To evaluate the influence of different filtering techniques on the measurement of ventricular late potentials (VLP) the Sottoprogetto Aritmie of GISSI-3 collected signal-averaged ECG (SAECG) from 647 patients. METHODS: Data were recorded after myocardial infarction (10 +/- 4 days) in 20 Italian Coronary Units. Three main filtering algorithms were used in the different commercial devices: Bidirectional Filter (ART, Aerotel, Fidelity Medical) (BF: 340 Patients), Spectral Filter (Marquette) (SF: 258 Patients) and Del Mar Filter (Del Mar Avionics) (DF: 49 Patients). QRS duration (QRSD), low amplitude signal duration (LAS40) and root mean-square-voltage (RMS40), were measured with various filters set at 40-250 Hz high and low pass frequencies. RESULTS: After correction for clinical variables the measurements of VLP in the three different groups were different. QRSD value obtained by BF (100.6 +/- 13 ms) was shorter than that obtained by SF (109.1 +/- 12 ms). No differences were found in LAS40 and RMS40 values between SF and BF, while DF gave longer LAS40 and lower RMS40 than SF and BF. Residual noise was lower in BF (0.3 +/- 0.1 muV). than in SF and DF (0.5 +/- 0.1 muV). Applying standard criteria DF gave a higher prevalence of VLP (48.9%) than BF (23.8%) and SF (19%) groups. CONCLUSIONS: This study demonstrates that the use of different filters produces discordant result on VLP measurements. For correct application of SAECG analysis in risk stratification after myocardial infarction, normal and abnormal values must be specifically established for the different filter techniques.

Distinct patterns of calcium transients during early and delayed afterdepolarizations induced by isoproterenol in ventricular myocytes.

BACKGROUND: The relation between early afterdepolarizations (EADs) and changes in intracellular Ca2+ concentration ([Ca2+]i) is still unclear. In the present study, we compared spatiotemporal changes in [Ca2+]i related to EADs and delayed afterdepolarizations (DADs) induced by isoproterenol. METHODS AND RESULTS: Isolated patch-clamped guinea pig ventricular myocytes, loaded with fluo-3 acetoxymethyl ester (fluo-3 AM), were paced at 0.1 to 2 Hz. Isoproterenol (100 nmol/L) caused alterations in both phase 2 and phase 4 of the action potential (AP), consistent with EADs and DADs, respectively. During EADs (n = 16), similar to driven APs, increases in [Ca2+]i occurred simultaneously throughout the cell, whereas during DADs (n = 25), they originated in discrete cell sites and propagated as a wave. This difference was confirmed by analysis of eight EADs and DADs coupled to the same beat. Ca2+ transient linked to EADs reached a peak relative fluorescence level (expressed as percentage of the maximal level reached during the last stimulated beat) that was always higher than that reached during the DADs (77 +/- 3% versus 64 +/- 2%, P < .001). Spatial heterogeneity of Ca2+ transients was assessed by the maximal time interval between peaks monitored in different cell regions; this time lag was always greater during DADs than during EADs (290 versus 40 milliseconds, P = .006). CONCLUSIONS: The present study had two main findings. First, even very modest notches occurring during the plateau of the AP may be accompanied by a marked secondary increase in [Ca2+]i. Second, these Ca2+ transients occurring during EADs are synchronous throughout the cell and differ significantly from those observed under identical conditions during DADs.