Large Cardiac Muscle Patches Engineered From Human Induced-Pluripotent Stem Cell-Derived Cardiac Cells Improve Recovery From Myocardial Infarction in Swine.

BACKGROUND: Here, we generated human cardiac muscle patches (hCMPs) of clinically relevant dimensions (4 cm × 2 cm × 1.25 mm) by suspending cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human induced-pluripotent stem cells in a fibrin scaffold and then culturing the construct on a dynamic (rocking) platform. METHODS: In vitro assessments of hCMPs suggest maturation in response to dynamic culture stimulation. In vivo assessments were conducted in a porcine model of myocardial infarction (MI). Animal groups included: MI hearts treated with 2 hCMPs (MI+hCMP, n=13), MI hearts treated with 2 cell-free open fibrin patches (n=14), or MI hearts with neither experimental patch (n=15); a fourth group of animals underwent sham surgery (Sham, n=8). Cardiac function and infarct size were evaluated by MRI, arrhythmia incidence by implanted loop recorders, and the engraftment rate by calculation of quantitative polymerase chain reaction measurements of expression of the human Y chromosome. Additional studies examined the myocardial protein expression profile changes and potential mechanisms of action that related to exosomes from the cell patch. RESULTS: The hCMPs began to beat synchronously within 1 day of fabrication, and after 7 days of dynamic culture stimulation, in vitro assessments indicated the mechanisms related to the improvements in electronic mechanical coupling, calcium-handling, and force generation, suggesting a maturation process during the dynamic culture. The engraftment rate was 10.9±1.8% at 4 weeks after the transplantation. The hCMP transplantation was associated with significant improvements in left ventricular function, infarct size, myocardial wall stress, myocardial hypertrophy, and reduced apoptosis in the periscar boarder zone myocardium. hCMP transplantation also reversed some MI-associated changes in sarcomeric regulatory protein phosphorylation. The exosomes released from the hCMP appeared to have cytoprotective properties that improved cardiomyocyte survival. CONCLUSIONS: We have fabricated a clinically relevant size of hCMP with trilineage cardiac cells derived from human induced-pluripotent stem cells. The hCMP matures in vitro during 7 days of dynamic culture. Transplantation of this type of hCMP results in significantly reduced infarct size and improvements in cardiac function that are associated with reduction in left ventricular wall stress. The hCMP treatment is not associated with significant changes in arrhythmogenicity.

Sensor spacing affects the tissue impedance spectra of rabbit ventricular epicardium.

This study was designed to test the hypothesis that a complex composite impedance spectra develops when stimulation and recording of cardiac muscle with sufficiently fine spatial resolution in a four-electrode configuration is used. With traditional (millimeter 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 remain 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 4,000 Hz range. With all separations, we measured comparable spectra with 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 4,000 Hz that lowered from 395 Ω-cm to 236 Ω-cm, respectively, with maximal mean χt of 160 Ω-cm. This experimental confirmation of spectra development in whole heart experiments is important because such development is central to achieve measurements of intracellular and interstitial passive electrical properties in cardiac electrophysiological experiments using only interstitial access.

Fabrication and characterization of a thick, viable bi-layered stem cell-derived surrogate for future myocardial tissue regeneration.

Cardiac tissue surrogates show promise for restoring mechanical and electrical function in infarcted left ventricular (LV) myocardium. For these cardiac surrogates to be usefulin vivo, they are required to support synchronous and forceful contraction over the infarcted region. These design requirements necessitate a thickness sufficient to produce a useful contractile force, an area large enough to cover an infarcted region, and prevascularization to overcome diffusion limitations. Attempts to meet these requirements have been hampered by diffusion limits of oxygen and nutrients (100-200 µm) leading to necrotic regions. This study demonstrates a novel layer-by-layer (LbL) fabrication method used to produce tissue surrogates that meet these requirements and mimic normal myocardium in form and function. Thick (1.5-2 mm) LbL cardiac tissues created from human induced pluripotent stem cell-derived cardiomyocytes and endothelial cells were assessed,in vitro, over a 4-week period for viability (<5.6 ± 1.4% nectrotic cells), cell morphology, viscoelastic properties and functionality. Viscoelastic properties of the cardiac surrogates were determined via stress relaxation response modeling and compared to native murine LV tissue. Viscoelastic characterization showed that the generalized Maxwell model of order 4 described the samples well (0.7

A biophysical model for cardiac microimpedance measurements.

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.

Linear electrode arrays for stimulation and recording within cardiac tissue space constants.

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.

Shock-induced changes of Ca(i)2+ and Vm in myocyte cultures and computer model: Dependence on the timing of shock application.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Interactions between paced wavefronts and monomorphic ventricular tachycardia: implications for antitachycardia pacing.

OBJECTIVES: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Effects of electrical shocks on Cai2+ and Vm in myocyte cultures.

Changes in intracellular calcium concentration (DeltaCa(i)2+) induced by electrical shocks may play an important role in defibrillation, but high-resolution DeltaCa(i)2+ measurements in a multicellular cardiac tissue and their relationship to corresponding Vm changes (DeltaVm) are lacking. Here, we measured shock-induced DeltaCa(i)2+ and DeltaV(m) in geometrically defined myocyte cultures. Cell strands (width=0.8 mm) were double-stained with Vm-sensitive dye RH-237 and a low-affinity Ca(i)2+-sensitive dye Fluo-4FF. Shocks (E approximately 5 to 40 V/cm) were applied during the action potential plateau. Shocks caused transient Ca(i)2+ decrease at sites of both negative and positive DeltaV(m). Similar Ca(i)2+ changes were observed in an ionic model of adult rat myocytes. Simulations showed that the Ca(i)2+ decrease at sites of DeltaV+m was caused by the outward flow of I(CaL) and troponin binding; at sites of DeltaV-m it was caused by inactivation of I(CaL) combined with extrusion by Na-Ca exchanger and troponin binding. The important role of I(CaL) was supported by experiments in which application of nifedipine eliminated Ca(i)2+ decrease at DeltaV+m sites. Largest DeltaCa(i)2+ were observed during shocks of approximately 10 V/cm causing simple monophasic DeltaV(m). Shocks stronger than approximately 20 V/cm caused smaller DeltaCa(i)2+ and postshock elevation of diastolic Ca(i)2+. This was paralleled with occurrence of biphasic negative DeltaVm that indicated membrane electroporation. Thus, these data indicate that shocks transiently decrease Ca(i)2+ at sites of both DeltaV-m and DeltaV+m. Outward flow of I(CaL) plays an important role in Ca(i)2+ decrease in the DeltaV+m areas. Very strong shocks caused smaller negative DeltaCa(i)2+ and postshock elevation of diastolic Ca(i)2+, likely caused by membrane electroporation.

Contributions of Purkinje-myocardial coupling to suppression and facilitation of early afterdepolarization-induced triggered activity.

Electrical loading by ventricular myocardium modulates conduction system repolarization near Purkinje-ventricular junctions (PVJs). We investigated how that loading suppresses and facilitates early afterdepolarizations (EADs) under conditions where there is a high degree of functional coupling between tissue types, which is consistent with the anatomic arrangement at the peripheral conduction system-myocardial interface. Experiments were completed in eight rabbit right ventricular (RV) free wall preparations. Free-running Purkinje strands were locally superfused, and action potentials were recorded from strands. RV free walls were bathed in normal solution. Surface electrograms were recorded near strand insertions into downstream free wall myocardium. Detailed histology was performed to assemble a computer model with interspersed Purkinje and ventricular myocytes weakly coupled throughout the region. Delays from Purkinje upstrokes to downstream peripheral conduction system and myocardial activation were comparable between experiments and simulations, supporting model node-to-node electrical coupling, i.e., the functional coupling. Purkinje action potential duration (APD) prolongation with localized isoproterenol in experiments and calcium current enhancement in simulations failed to establish EADs. With myocardial APD prolongation by delayed rectifier potassium current inhibition or L-type calcium current enhancement accompanying Purkinje APD prolongation in simulations, however, EAD-induced triggered activity developed. Collectively, our findings suggest competing contributions of the myocardial sink when there is a high degree of functional coupling between tissue types, with the transition from suppression to facilitation of EAD-induced triggered activity depending critically upon myocardial APD prolongation.

Spontaneous activity induced in rabbit Purkinje myocytes during coupling to a depolarized model cell.

OBJECTIVE: The development of an "injury current" secondary to heterogeneous ion accumulation and cellular uncoupling across the ischemic border zone has been implicated as a trigger for arrhythmias arising during acute ischemia. The purpose of the present study was to determine the effects of injury current across the Purkinje-ventricular interface in the development of abnormal automaticity. METHODS: Patch clamp and electronic cell coupling techniques were used to record action potentials from and to apply injury current to isolated rabbit Purkinje myocytes. Injury current was dependent upon: (1) a coupling resistance, which was varied to simulate different degrees of cellular uncoupling, and (2) the difference in Purkinje membrane potential and depolarized ischemic myocardium, which was represented by a passive resistor-capacitor circuit with initial voltages of -70, -60, or -50 mV. RESULTS: During coupling to the moderately depolarized cell (-60 or -50 mV), Purkinje myocytes developed repetitive, spontaneous activity within a window of coupling resistances. This abnormal automaticity was dependent upon L-type calcium current, as cadmium or nifedipine completely suppressed coupling-induced spontaneous activity. CONCLUSIONS: Our results demonstrate that injury current alone can induce spontaneous activity in normal Purkinje myocytes. The level of myocardial depolarization and the degree of cellular uncoupling required to induce this activity suggest spontaneous Purkinje activity induced by injury current as a potent trigger for acute ischemic arrhythmias.

Modulation of triggered activity by uncoupling in the ischemic border. A model study with phase 1b-like conditions.

OBJECTIVE: Triggered beats during regional ischemia may depend upon the electrical source and sink charge interactions between adjacent regions of normal and ischemic cardiac tissue that are partly controlled by electrical coupling. METHODS: To study these relationships, we modified parameters in the Luo-Rudy dynamic membrane equations to reflect physiologic conditions associated with phase 1b arrhythmias. Superthreshold delayed afterdepolarizations (DADs) formed after pacing. Coupling contributions were then examined using: (i) a single phase 1b myocyte connected via a variable resistance to a single normal myocyte, and (ii) a multicellular fiber with a 1-cm segment of phase 1b myocytes connected to a 1-cm normal segment having resistance changes that were confined to the ischemic segment. Integration of ionic, capacitive and coupling currents during DAD initiation allowed charge quantification. RESULTS: In cell pairs, phase 1b myocyte DADs were suppressed at resistances where normal myocyte pacing resulted in phase 1b myocyte excitation. Coupling charge requirements limited capacitive charging in the phase 1b myocyte, which occurred in combination with diastolic hyperpolarization that shifted transmembrane potential from threshold. In multicellular fiber simulations, DADs were suppressed with strong coupling in the phase 1b segment. Moderate uncoupling of that segment allowed superthreshold DAD formation away from the border that initiated action potential propagation in the normal segment. With severe uncoupling, propagation failed at the border. CONCLUSIONS: These findings support the clinical and experimental observation that intermediate uncoupling is an important contributor to phase 1b arrhythmogenesis.

A structural framework for interpretation of four-electrode microimpedance spectra in cardiac tissue.

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.

Novel methods of automated quantification of gap junction distribution and interstitial collagen quantity from animal and human atrial tissue sections.

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.

A new approach for resolution of complex tissue impedance spectra in hearts.

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 10-4000 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 ρt from rabbit ventricular epicardium. Simultaneous acquisition of a signal proportional to the supplied current (Vstim) with uPD allowed identification of the V- I ratio needed for ρt 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 SNRs between 10 (1) and 10 (3) measured with supplied currents as low as 10 nA. Comparable performance characteristics were identified during calibration of nine 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 ρt = 134 ± 53 Ω· 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 species-dependent intercellular uncoupling in hearts.

A new optrode design for intramural optical recordings.

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).

A novel approach to dual excitation ratiometric optical mapping of cardiac action potentials with di-4-ANEPPS using pulsed LED excitation.

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.

Bayesian quantitative electrophysiology and its multiple applications in bioengineering.

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.

Stimulatory current at the edge of an inactive conductor in an electric field: role of nonlinear interfacial current-voltage relationship.

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.

Bayesian analysis of fiber impedance measurements.

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.

Optical mapping of V(m) and Ca(i)(2+) in a model of arrhythmias induced by local catecholamine application in patterned cell cultures.

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.