[Show abstract][Hide abstract] ABSTRACT: Background
Gap junctions (GJs) are the principal membrane structures that conduct electrical impulses between cardiac myocytes while interstitial collagen (IC) can physically separate adjacent myocytes and limit cell-cell communication. Emerging evidence suggests that both GJ and interstitial structural remodeling are linked to cardiac arrhythmia development. However, automated quantitative identification of GJ distribution and IC deposition from microscopic histological images has proven to be challenging. Such quantification is required to improve the understanding of functional consequences of GJ and structural remodeling in cardiac electrophysiology studies.
Methods and Results
Separate approaches were employed for GJ and IC identification in images from histologically stained tissue sections obtained from rabbit and human atria. For GJ identification, we recognized N-Cadherin (N-Cad) as part of the gap junction connexin 43 (Cx43) molecular complex. Because N-Cad anchors Cx43 on intercalated discs (ID) to form functional GJ channels on cell membranes, we computationally dilated N-Cad pixels to create N-Cad units that covered all ID-associated Cx43 pixels on Cx43/N-Cad double immunostained confocal images. This approach allowed segmentation between ID-associated and non-ID-associated Cx43. Additionally, use of N-Cad as a unique internal reference with Z-stack layer-by-layer confocal images potentially limits sample processing related artifacts in Cx43 quantification. For IC quantification, color map thresholding of Masson's Trichrome blue stained sections allowed straightforward and automated segmentation of collagen from non-collagen pixels. Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts. The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.
PLoS ONE 08/2014; 9(8):e104357. DOI:10.1371/journal.pone.0104357 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Renewed interest in the four-electrode method for identification of passive electrical properties in cardiac tissue has been sparked by a recognition that measurements made with sensors in close proximity are frequency dependent. Therefore, resolution of four-electrode microimpedance spectra (4EMS) may provide an opportunity for routine identification of passive electrical properties for the interstitial and intracellular compartments using only interstitial access. The present study documents a structural framework in which the tissue resistivity (ρt) and reactivity (xt) that comprise spectra are computed using interstitial and intracellular microimpedance distributions that account for differences in compartment size, anisotropic electrical properties in each compartment and electrode separations. We used this framework to consider 4EMS development with relatively wide (d=1 mm) and fine (d=250 μm) electrode separations and sensors oriented along myocyte axes, across myocyte axes and intermediate between those axes.
[Show abstract][Hide abstract] ABSTRACT: A sinusoidal oscillator is the stimulation source of a micro-impedance spectroscopy. The accuracy of its output frequency and waveform dictates the accuracy of the measured complex tissue impedance. In this study, an area-efficient low-power sinusoidal oscillator is developed for applications requiring low-power high-density bio-impedance measurements. Instead of using traditional LC tank based topology, the proposed sinusoidal oscillator implements a series of hyperbolic tangent functions to approximate the sine function. To achieve the continuous sinusoidal oscillation, a sine waveform shaper, a ramp signal generator, and a fully differential driver are developed for the proposed sinusoidal oscillator. To minimize the power dissipation, a low power supply voltage, 0.5 V, is used in the circuit. Designed in a standard 0.13-μm CMOS process, the proposed sinusoidal oscillator can generate continuous sinusoidal signal with a tuning range of 10 Hz-4 kHz. It dissipates power of <; 3 μW and occupies an area of 0.07 mm<;sup>2<;/sup>.
[Show abstract][Hide abstract] ABSTRACT: This study was designed to test the hypothesis that a complex composite impedance (uCI) spectra develops when stimulation and recording of cardiac muscle with sufficiently fine spatial resolution in a four-electrode configuration is employed. With traditional (mm scale) separations, the ratio between the recorded interstitial central potential difference and total supplied interstitial current is constant at all frequencies. This occurs because the fraction of supplied current that redistributes to the intracellular compartment depends on effective membrane resistance between electrodes, which is low, to a much greater extent than effective membrane capacitance. The spectra should therefore change with finer separations at which effective membrane resistance increases, as supplied current will remaining primarily interstitial at lower frequencies and redistribute between compartments at higher frequencies. To test this hypothesis, we built arrays with sensors separated (d) by 804 μm, 452 μm and 252 μm, positioned those arrays across myocyte axes on rabbit ventricular epicardium, and resolved spectra in terms of resistivity (ρt) and reactivity (χt) over the 10 Hz to 4000 Hz range. With all separations, we measured comparable spectra to predictions from passive membrane simulations that used a three-dimensional structural framework in which intracellular, interstitial and membrane properties were prescribed based on the limited data available from the literature. At the finest separation, we found mean ρt at 100 Hz and 4000 Hz that lowered from 395 ohm-cm to 236 ohm-cm, with maximal mean χt of 160 ohm-cm.
[Show abstract][Hide abstract] ABSTRACT: This study was designed to test the feasibility of using sinusoidal approximation in combination with a new instrumentation approach to resolve complex impedance (uCI) spectra from heart preparations. To assess that feasibility, we applied stimuli in the 10 Hz to 4000 Hz range and recorded potential differences (uPDs) in a four-electrode configuration that allowed identification of probe constants (Kp) during calibration that were in turn used to measure total tissue resistivity (t) from rabbit ventricular epicardium. Simultaneous acquisition of a signal proportional to the supplied current (Vstim) with uPD allowed identification of the voltage-to-current ratio needed for t measurement, as well as the phase shift from Vstim to uPD needed for uCI spectra resolution. Performance with components integrated to reduce noise in cardiac electrophysiologic experiments, in particular, and provide accurate electrometer-based measurements, in general, was first characterized in tests using passive loads. Load tests showed accurate uCI recovery with mean uPD signal-to-noise ratios (SNRs) between 101 and 103 measured with supplied currents as low as 10 nA. Comparable performance characteristics were identified during calibration of 9 arrays built with 250 μm Ag/AgCl electrodes, with uCIs that matched analytic predictions and no apparent effect of frequency (F=0.12,P=0.99). The potential ability of parasitic capacitance in the presence of the electrode-electrolyte interface associated with the small sensors to influence the uCI spectra was therefore limited by the instrumentation. Resolution of uCI spectra in rabbit ventricle allowed measurement of t=134±53 - cm. The rapid identification available with this strategy provides an opportunity for new interpretations of the uCI spectra to improve quantification of disease-, region-, tissue- and speciesdependent intercellular uncoupling in hearts.
[Show abstract][Hide abstract] ABSTRACT: Purkinje-to-ventricular (P-to-V) propagation and electrotonic modulation of repolarization at discrete Purkinje-ventricular junctions (PVJs) depend on differences in the ionic currents and tissue structure of the P network and the V myocardium. We used computer simulations to assess these membrane and tissue level contributions to P-V interactions. At the membrane level, we used the DiFrancesco-Noble membrane equations to model P ionic kinetics and the Luo-Rudy dynamic membrane equations to model V ionic kinetics. At the tissue level, we modeled the P network as a layer of branching cables, and we modeled a single myocardial sheet with an anisotropic layer of excitable cells. P-to-V propagation was enhanced at the tissue level when multiple wavefronts in the branching P network collided at the PVJ. At the membrane level, P-to-V propagation was enhanced by a reduced transient outward current (Ito) in the P layer. Repolarization at the PVJ was also modulated by both membrane and tissue level contributions. Under nominal conditions, action potential duration (APD) shortened in the P layer and prolonged in the V layer. However, when the V mass was reduced, both P and V cell APDs shortened during coupling with nominal Ito. Subsequent Ito inhibition restored coupling-induced prolongation of the V action potential in the reduced V mass. These results suggest that under physiologic conditions, both membrane and tissue level contributions to P-V interactions are important, while membrane level contributions become even more important under pathologies that reduce the difference in P and V tissue size, particularly in the setting of healed myocardial infarction.
Journal of Biological Systems 11/2011; 07(04). DOI:10.1142/S0218339099000280 · 0.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intramural measurements of V(m) and Ca(i)(2+) are important in the studies of cardiac arrhythmias and defibrillation. We developed a new design of an "optrode" (bundle of optical fibers) for use in intramural cardiac mapping. The optrodes are made from seven optical fibers with the fiber ends polished at 45° angle and coated with mirror surfaces. The optrodes are enclosed in smooth epoxy resin cast, which protects mirror surfaces from damage and ensures constant optrode diameter along its length. The optrodes are strong enough to be easily inserted into heart muscle, can be reused multiple times, and they may reduce artifacts in the measurements of the effects of defibrillation shocks on V(m).
[Show abstract][Hide abstract] ABSTRACT: We developed a new method for ratiometric optical mapping of transmembrane potential (V(m)) in cardiac preparations stained with di-4-ANEPPS. V(m)-dependent shifts of excitation and emission spectra establish two excitation bands (<481 and >481 nm) that produce fluorescence changes of opposite polarity within a single emission band (575-620 nm). The ratio of these positive and negative fluorescence signals (excitation ratiometry) increases V(m) sensitivity and removes artifacts common to both signals. We pulsed blue (450 ± 10 nm) and cyan (505 ± 15 nm) light emitting diodes (LEDs) at 375 Hz in alternating phase synchronized to a camera (750 frames-per-second). Fluorescence was bandpass filtered (585 ± 20 nm). This produced signals with upright (blue) and inverted (cyan) action potentials (APs) interleaved in sequential frames. In four whole swine hearts with motion chemically arrested, fractional fluorescence for blue, cyan, and ratio signals was 1.2 ± 0.3%, 1.2 ± 0.3%, and 2.4 ± 0.6%, respectively. Signal-to-noise ratios were 4.3 ± 1.4, 4.0 ± 1.2, and 5.8 ± 1.9, respectively. After washing out the electromechanical uncoupling agent, we characterized motion artifact by cross-correlating blue, cyan, and ratio signals with a signal with normal AP morphology. Ratiometry improved cross-correlation coefficients from 0.50 ± 0.48 to 0.81 ± 0.25, but did not cancel all motion artifacts. These findings demonstrate the feasibility of pulsed LED excitation ratiometry in myocardium.
[Show abstract][Hide abstract] ABSTRACT: Alterations to cell-to-cell electrical conductance and to the structural arrangement of the collagen network in cardiac tissue are recognized contributors to arrhythmia development, yet no present method allows direct in vivo measurements of these conductances at their true microscopic scale. The present report documents such a plan, which involves interstitial multisite stimulation at a subcellular to cellular size scale, and verifies the performance of the method through biophysical modeling. Although elements of the plan have been analyzed previously, their performance as a whole is considered here in a comprehensive way. Our analyses take advantage of a three-dimensional structural framework in which interstitial, intracellular, and membrane components are coupled to one another on the fine size scale, and electrodes are separated from one another as in arrays we fabricate routinely. With this arrangement, determination of passive tissue resistances can be made from measurements taken on top of the currents flowing in active tissue. In particular, our results show that measurements taken at multiple frequencies and electrode separations provide powerful predictions of the underlying tissue resistances in all geometric dimensions. Because of the small electrode size, separation of interstitial from intracellular compartment contributions is readily achieved.
[Show abstract][Hide abstract] ABSTRACT: Cardiac electric field stimulation is critical for the mechanism of defibrillation. The presence of certain inactive epicardial conductors in the field during defibrillation can decrease the defibrillation threshold. We hypothesized this decrease is due to stimulatory effects of current across the interface between the inactive conductor and the heart during field stimulation. To examine this current and its possible stimulatory effects, we imaged transmittance of indium-tin-oxide (ITO) conductors, tested for indium with X-ray diffraction, created a computer model containing realistic ITO interfacial properties, and optically mapped excitation of rabbit heart during electric field stimulation in the presence of an ITO conductor. Reduction of indium decreased transmittance at the edge facing the anodal shock electrode when trans-interfacial voltage exceeded standard reduction potential. The interfacial current-voltage relationship was nonlinear, producing larger conductances at higher currents. This nonlinearity concentrated the interfacial current near edges in images and in a computer model. The edge current was stimulatory, producing early postshock excitation of rabbit ventricles. Thus, darkening of ITO indicates interfacial current by indium reduction. Interfacial nonlinearity concentrates current near the edge where it can excite the heart. Stimulatory current at edges may account for the reported decrease in defibrillation threshold by inactive conductors.
[Show abstract][Hide abstract] ABSTRACT: Bayesian interpretation of observations began in the early 1700s, and scientific electrophysiology began in the late 1700s. For two centuries these two fields developed mostly separately. In part that was because quantitative Bayesian interpretation, in principle a powerful method of relating measurements to their underlying sources, often required too many steps to be feasible with hand calculation in real applications. As computer power became widespread in the later 1900s, Bayesian models and interpretation moved rapidly but unevenly from the domain of mathematical statistics into applications. Use of Bayesian models now is growing rapidly in electrophysiology. Bayesian models are well suited to the electrophysiological environment, allowing a direct and natural way to express what is known (and unknown) and to evaluate which one of many alternatives is most likely the source of the observations, and the closely related receiver operating characteristic (ROC) curve is a powerful tool in making decisions. Yet, in general, many people would ask what such models are for, in electrophysiology, and what particular advantages such models provide. So to examine this question in particular, this review identifies a number of electrophysiological papers in bioengineering arising from questions in several organ systems to see where Bayesian electrophysiological models or ROC curves were important to the results that were achieved.
[Show abstract][Hide abstract] ABSTRACT: In this paper, we document a fabrication process that yields linear arrays of rectangular platinum black electrodes spaced 25 mum apart with edge-to-edge separation of 20 microm. The spatial arrangement is therefore sufficiently fine to insure stimulation and recording within cardiac tissue space constants, as six electrodes with dimensions of either 5 x 100 microm2, 5 x 250 microm2, or 5 x 500 microm2 were positioned in a 130-microm2 span in the arrays. Despite the small electrode sizes and available surface areas, favorable impedance characteristics were identifed. Averages ranged from 111 kOmega to 146 kOmega at 0.5 Hz and from 14 kOmega 39 kOmega at 500 Hz. Differences in impedances between the electrode sizes tested were small. Potential differences (deltaphis) recorded using the two central electrodes during stimulation with combinations at separations of only 75 microm, 100 microm, and 125 microm had low signal noise. As a preliminary test of the use of these arrays for possible application to impedance measurements in cardiac tissue, the deltaphis recorded during stimulation were compared to deltaphis obtained from finite-difference simulations using an isotropic volume conductor model. Anticipated decays in deltaphi with widening electrode separation identified in those simulations matched the decays in the recorded deltaphis closely. These findings are significant because they suggest intracellular and interstitial microimpedance mesurements in heart experiments will be straightforward.
[Show abstract][Hide abstract] ABSTRACT: Catecholamines are known to provoke cardiac arrhythmias, but important aspects such as localization of the arrhythmia source in multicellular tissue and exact ionic mechanisms are not well-known. In this work, a multicellular model of arrhythmias caused by local epinephrine application was developed; V (m) and Ca(i)(2+) changes at the arrhythmia source were measured using fluorescent dyes and high-resolution optical mapping. Cultured strands of neonatal rat myocytes (width approximately 0.4 mm) were produced by patterned growth. Epinephrine (1 micromol/l) was applied over an area of 0.3-0.6 mm via two micropipettes, and strands were stimulated by burst pacing. Local epinephrine application caused triggered arrhythmias with cycle lengths of 202-379 ms and duration of >10 s in 9 out of 16 preparations. Optical V(m) mapping demonstrated that in 78% of cases, the source of arrhythmia was located at the boundary of the locally perfused area. Staining with Ca(i)(2+)-sensitive dye Fluo-4 prevented arrhythmia induction in most cases (85%) likely due to Ca(2+) buffering by the dye. Optical Ca(i)(2+) mapping revealed non-propagated Ca(i)(2+) oscillations at the boundary of the locally perfused area in 45% cases. In conclusion, we developed a new model of catecholamine-dependent arrhythmias allowing mapping of V(m) and Ca(i)(2+) at the arrhythmia source with microscopic resolution. The arrhythmias typically originated from the boundary of the epinephrine-perfused area. The location of the arrhythmia source correlated with localized Ca(i)(2+) oscillations suggesting that arrhythmias were caused by Ca(i)(2+) overload at these locations.
Pflügers Archiv - European Journal of Physiology 04/2007; 453(6):871-7. DOI:10.1007/s00424-006-0162-6 · 4.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The resistivities of microscale components of excitable tissue include the longitudinal intracellular and interstitial resistivities and the membrane resistivity. Measurements of these tissue micro impedances have rarely been obtained, mainly because of the lack of a satisfactory measurement system. Here we evaluate a possible strategy for obtaining such measurements, and begin with a simulation. In the model, a one-dimensional fiber was stimulated with closely space interstitial electrodes at four frequencies, and the voltage differences that occurred in response were recorded. We then considered the inverse question, asking if tissue micro impedances could be found from the voltage measurements plus additive noise. In so doing, we used a Bayesian interpretation of the measured data to find the probability that each of the longitudinal and transmembrane resistivity sets was their origin. The Bayesian procedure proved better suited for interpreting the measurements than was conventional least-squares analysis. It was better because all known data, including realistic noise specifications and a priori probabilities, were included in the defined procedure. The results show that the micro impedances were found satisfactorily using realistic parameters and noise levels. The overall quantitative evaluation is promising for future experimental measurements.
Conference proceedings: ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference 02/2007; 2007:423-9. DOI:10.1109/IEMBS.2007.4352314
[Show abstract][Hide abstract] ABSTRACT: Responses of Ca(i)2+ to electrical shocks are believed to be important in defibrillation but measurements of shock-induced Ca(i)2+ changes during different phases of the action potential (AP) are lacking. The effects of shocks on Ca(i)2+ and Vm were investigated in geometrically defined cell cultures and in a computer model.
Uniform-field shocks (E = 10.4+/-0.9 V/cm) were applied 15-300 ms after AP upstroke in strands of cultured neonatal rat myocytes. Optical mapping was used to measure shock-induced Ca(i)2+ and Vm changes. A rat ionic model was used to elucidate ionic mechanisms of Ca(i)2+ responses.
In experiments and simulations, shocks applied with short delays (15-40 ms) caused a transient decrease of Ca(i)2+ at sites of both DeltaV(+)m and DeltaV(-)m. Simulations indicated that the Ca(i)2+ decrease at DeltaV(+)m sites was caused by reversed outward flow of L-type Ca2+ current (I(CaL)), while the Ca(i)2+ decrease at DeltaV(-)m sites was due to the NaCa exchanger (NCX). At intermediate delays (40-150 ms), shocks caused a Ca(i)2+ decrease at sites of DeltaV(-)m and an increase at sites of DeltaV(+)m. Simulations indicated that the Ca(i)2+ increase at DeltaV(+)m sites was caused by transient reactivation of I(CaL) combined with a reverse-mode operation of NCX. Shocks applied at long delays (150-300 ms) caused a Ca(i)2+ increase at DeltaV(+)m and no change at DeltaV(-)m sites.
Effects of shocks on Ca(i)2+ depend on the timing of shock application. Shocks applied during the early AP cause a transient Ca(i)2+ decrease, while later in AP shocks induce a Ca(i)2+ increase at sites of DeltaV(+)m. Shock-induced Ca(i)2+ changes in different AP phases are primarily determined by combination of I(CaL) and NCX.
Cardiovascular Research 02/2007; 73(1):101-10. DOI:10.1016/j.cardiores.2006.10.028 · 5.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interactions between paced wavefronts and monomorphic ventricular tachycardia (VT) dictate antitachycardia pacing outcomes. We used optical mapping to assess those interactions during single and dual site pacing of rabbit ventricular epicardium.
Monomorphic VTs were initiated in six isolated rabbit hearts that were endocardially cryoablated to limit viable tissue to visible epicardium and establish apical tissue as the anatomic anchor. Preparations were optically mapped during single (n = 39) and dual (n = 43) site pacing at 50%-90% of VT cycle length (CL) with eight pulses per trial. Overall, we found six pulses that abruptly terminated VT. This occurred because the VT wavefront collided with the antidromic portion of the paced wavefront and the orthodromic portion of paced wavefront blocked in the VT's refractory region. When effective, dual site pacing that captured tissue at both leads simultaneously terminated the VT immediately, while single site pacing or dual site pacing that captured tissue at only one lead terminated the VT after resetting advanced the orthodromic wavefront. We found 12 pulses that induced polymorphic VT, with 11 of those pulses occurring during capture at only one lead. Expansion of the combined antidromic-VT wavefront around one or both ends of the arc of conduction block formed by the interaction of the orthodromic wavefront with the VT's refractory region initiated functional reentry. Six of these polymorphic VTs were nonsustained because the underlying wavefronts self-terminated. The wavefronts did persist for 4.2 +/- 3.5 cycles before self-terminating in these trials, and the post-pacing cycles presented a 146% increase in CL variability, compared with the variability prior to pacing. These temporal characteristics are similar to those of delayed termination in patients with ICDs.
The main difference between pulses that terminated abruptly and pulses that induced polymorphic VT was the effective separation of the antidromic and orthodromic portions of the paced wavefront from one another.
[Show abstract][Hide abstract] ABSTRACT: We analyzed central interstitial potential differences during multisite stimulation to assess the feasibility of using those recordings to measure cardiac microimpedances in multidimensional preparations. Because interstitial current injected and removed using electrodes with different proximities allows modulation of the portion of current crossing the membrane, we hypothesized that multisite interstitial stimulation would give rise to central interstitial potential differences that depend on intracellular and interstitial microimpedances, allowing measurement of those microimpedances. Simulations of multisite stimulation with fine and wide spacing in two-dimensional models that included dynamic membrane equations for guinea pig ventricular myocytes were performed to generate test data ( partial differentialphio). Isotropic interstitial and intracellular microimpedances were prescribed for one set of simulations, and anisotropic microimpedances with unequal ratios (intracellular to interstitial) along and across fibers were prescribed for another set of simulations. Microimpedance measurements were then obtained by making statistical comparisons between partial differentialphio values and interstitial potential differences from passive bidomain simulations (Deltaphio) in which a wide range of possible microimpedances were considered. Possible microimpedances were selected at 25% increments. After demonstrating the effectiveness of the overall method with microimpedance measurements using one-dimensional test data, we showed microimpedance measurements within 25% of prescribed values in isotropic and anisotropic models. Our findings suggest that development of microfabricated devices to implement the procedure would facilitate routine measurement as a component of cardiac electrophysiological study.
[Show abstract][Hide abstract] ABSTRACT: Cardiac electrical activity is significantly affected by variations in the conductance of gap junctions that connect myocytes to one another. To better understand how intrinsic (single cell) electrical activity is modulated by junctional conductance, we used a two-myocyte coupling system in which physically separate cells were electrically coupled via a variable resistance set by the investigator. This brief review summarizes our findings regarding: (1) the effect of the early phase of action potential repolarization (phase 1) and transient outward current (Ito) on action potential conduction, and (2) the effect of coupling on the action potential plateau (late repolarization). We found that inhibition of Ito markedly increased the ability of action potentials to propagate from cell-to-cell when junctional conductance was low. Electrically coupling two myocytes together also suppressed their beat-to-beat variability in action potential duration and contraction. Similarly, early afterdepolarizations (EADS) were readily suppressed by connecting a normal myocyte to one generating EADs. This high sensitivity of the plateau to variations in junctional interactions arises from the large increase in membrane resistance that occurs during this phase of the action potential.