Optical Imaging of the Heart Igor R. Efimov, Vladimir P. Nikolski...

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DOI: 10.1161/01.RES.0000130529.18016.35 2004;95;21-33 Circ. Res.

Igor R. Efimov, Vladimir P. Nikolski and Guy Salama Optical Imaging of the Heart

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This Review is part of a thematic series on Imaging of Cardiovascular Cells and Tissues, which includes the followingarticles:

Use of Chimeric Fluorescent Proteins and Fluorescence Resonance Energy Transfer to Monitor Cellular Responses

Imaging Microdomain Ca2� in Muscle Cells

Optical Imaging of the Heart

Examining Intracellular Organelle Function Using Fluorescent Probes

Two-Photon Microscopy of Cells and Tissue Brian O’Rourke, Guest Editor

Optical Imaging of the HeartIgor R. Efimov, Vladimir P. Nikolski, Guy Salama

Abstract—Optical techniques have revolutionized the investigation of cardiac cellular physiology and advanced ourunderstanding of basic mechanisms of electrical activity, calcium homeostasis, and metabolism. Although opticalmethods are widely accepted and have been at the forefront of scientific discoveries, they have been primarily appliedat cellular and subcellular levels and considerably less to whole heart organ physiology. Numerous technical difficultieshad to be overcome to dynamically map physiological processes in intact hearts by optical methods. Problems ofcontraction artifacts, cellular heterogeneities, spatial and temporal resolution, limitations of surface images, depth-of-field, and need for large fields of view (ranging from 2�2 mm2 to 3�3 cm2) have all led to the development of newdevices and optical probes to monitor physiological parameters in intact hearts. This review aims to provide a criticaloverview of current approaches, their contributions to the field of cardiac electrophysiology, and future directions ofvarious optical imaging modalities as applied to cardiac physiology at organ and tissue levels. (Circ Res. 2004;94:21-33.)

Key Words: optical mapping � fluorescent probes � electrophysiology � arrhythmia � defibrillation

Mammalian physiology has an ingrained hierarchy withmolecular and cellular physiology at its base, followed

by the interactions of large populations of cells and organsystems, and finally the integration of multiple organ func-tions of an entire animal. For the past 4 decades, cardiovas-cular physiology has been dominated by a “reductionist”approach, focusing on cellular mechanisms. Major strideshave been accomplished in our understanding of cellularmechanisms, including metabolism, intracellular signaling,trafficking, ion channel structure, function, and expression.With a greater understanding of cellular mechanisms camethe growing realization that organs such as the heart arecomposed of several types of interacting cells with significantand important heterogeneities of properties, cell-to-cell cou-pling, and function within each group. Thus, an understand-ing of molecular and cellular mechanisms must still beintegrated to explain the more complex organ system while

taking into account spatial and temporal heterogeneities ofcell functions throughout the organ.

Unfortunately, experimental methodologies available forstudies at the organ level are not as abundant as at the cellularscale. Nonoptical imaging modalities, including positronemission tomography, magnetic resonance, and ultrasoundimaging have only started to bridge molecular and organphysiology using novel contrast agents.1 On the other hand,optical modes of imaging, in combination with parameter-sensitive probes have already demonstrated their ability toovercome the problem of spatiotemporal resolution in twodimensions for a wide range of applications from singlemolecular events to in vivo whole animal physiology.

Fluorescence has been used to measure a wide range ofphysiological parameters in cells and tissues. For instance,cellular metabolic state can be monitored through (1) theintrinsic fluorescence changes of NADH or flavoproteins,2

Original received February 2, 2004; revision received April 16, 2004; accepted April 20, 2004.From the Case Western Reserve University (I.R.E., V.P.N.), Cleveland, Ohio; and the University of Pittsburgh (G.S.), Pittsburgh, Pa.Correspondence to Dr Igor R. Efimov, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106-7207. E-mail [email protected]© 2004 American Heart Association, Inc.

Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000130529.18016.35

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(2) the differential absorption changes of mitochondrialcytochromes, which report on the oxidative phosphorylationredox state, or (3) the oxygen content of blood and cardiacmuscle through the absorption changes of oxy to deoxyhemoglobin and myoglobin, respectively.3,4 More recent hasbeen the development of probes to selectively measurefunctional parameters such as membrane potential, intracel-lular concentrations of free calcium, magnesium, sodium andpotassium, pH, nitric oxide, oxygen tension, and sulfhydrylredox sate. Of these, optical probes of membrane potentialand intracellular free calcium ([Ca2�]i) indicators have had themost impact in cardiovascular physiology. The developmentof optical recordings of membrane potential was driven bythe need to overcome many obstacles in electrophysiologyand the promise of a technology “for measuring membranepotential in systems where, for reasons of scale, topology, orcomplexity, the use of electrodes is inconvenient or impos-sible.”5 Specific to cardiac electrophysiology, there was alsothe need to record transmembrane voltage changes during orimmediately after the firing of electric shocks used fordefibrillation. Optical mapping techniques and potentiometricprobes have now made major contributions to our under-standing of nerve network behavior and cardiac electrophys-iology in ways that could not have been accomplished byother approaches. Advances in neuroscience driven by opticalmapping have been extensively reviewed.6,7 Although there isconsiderable overlap in the instruments used for opticalmapping of neuronal and cardiac tissues, there are alsoimportant differences, and this review will focus on instru-mentation, optical probes, major findings, and future direc-tions of optical imaging as it applies to the heart.

General PrinciplesOptical imaging modalities are based on physical principlesof wavelength-dependent light-tissue interaction, includingphoton scattering, total internal reflection, absorption, reflec-tance, and fluorescence. The physical interactions of photonswith tissue, namely, intrinsic tissue absorption and lightscattering, limit the depth of penetration and spatiotemporalresolution of images that can be obtained from bulk tissue.However, recent advances in optical contrast agents, opticalprobes of physiological parameters, light transport theory,light sources, and optical detectors have created conditionsfor major breakthroughs in optical imaging at the organ level.Novel imaging modalities with expanding areas of applica-tion have emerged, including optical diffusion tomography,8,9

optical coherence tomography,10 and various confocal fluo-rescence imaging approaches. These new technologies haveencouraged the development of new fluorescence or absorp-tion contrast agents and probes of physiological parameters,and the combination of new instruments and probes will mostlikely propel a new era of multidimensional and multipara-metric imaging in organ system physiology.

The ionic basis of cellular electrophysiology, intercellularcoupling, and electrical propagation are understood withconsiderable detail. Although intracellular and patch-clampmicroelectrode techniques have been used to elucidate thephysiology of excitable cells, an understanding of the behav-ior of cardiac tissues have been more difficult to attain by

conventional electrode techniques. More than 30 years ago,investigators discovered molecular probes that bind to theplasma membrane of neuronal11 and cardiac cells12 andexhibited changes in fluorescence and/or absorption thattracked changes in transmembrane potential.

Several optical properties of membrane-bound dyes havebeen used to measure membrane potential changes; namely,fluorescence, absorption, dichroism, birefringence, fluores-cence resonance energy transfer, nonlinear second harmonicgeneration, and resonance Raman absorption. However, moststudies in cardiac cells or tissues have relied on the fluores-cence mode that tends to yield higher fractional changes insignal compared with the other modes and because fluores-cence signals tend to be considerably less sensitive tomovement artifacts generated by muscle contractions.12,13

Several mechanisms have been proposed to explainvoltage-dependent changes in fluorescence and/or absorptionof dye molecules based on interactions of the electric fieldwith dye molecules resulting in intra- and extramolecularrearrangements of the dye in the membrane. Cohen andSalzberg7 introduced a simple classification of voltage-sensitive dyes into two groups, fast and slow dyes, based ontheir response times and presumed molecular mechanism ofvoltage sensitivity. Only the fast probes are used in cardiacelectrophysiology, due to their ability to follow voltagechanges on a time scale of microseconds.14 The precisemechanisms underlying the voltage-dependent spectroscopicproperties of fast voltage-sensitive dyes are still not fullyunderstood. Spectral shift in the properties of chromophore isthought to be related to the changes in excitation-inducedintramolecular relocation of electronic charge along theelectric field gradient (electrochromic theory15) or to electricfield–induced reorientation of the dye molecule in the plasmamembrane (solvatochromic theory16).

New Molecular Probes for Optical Recordingsof Electrical Activity

In tests of over 1500 different compounds, several usefulclasses of chromophores have emerged, including merocya-nine, oxonol, and styryl dyes. Styryl dyes represent the mostpopular family of dyes; RH-421 and di-4-ANEPPS being themost important members of this family. The spectroscopicproperties of these dyes have been shown to linearly changewith membrane potential changes in the normal physiologicalrange of transmembrane voltages.13,17,18

Initial studies were performed with a class of moleculescalled merocyanine dyes, which exhibited 1% fractionalchanges in fluorescence in cardiac tissue.12,13,17 As shown inFigure 1A, action potentials were simultaneously recordedthrough the fluorescence changes of the potentiometric dyeand with an intracellular microelectrode. The fluorescenceaction potentials were recorded from frog ventricular tissuestained with merocyanine 540 from a 2-mm diameter excita-tion spot and compared with microelectrode recording fromone of the cells excited by the incident light beam. The twotechniques show excellent correlation between the two sig-nals. In the intact heart, surface or volume electrograms arealso in excellent correlation with optical recordings. Butunlike electrode recordings, optical signals can be easily

22 Circulation Research July 9, 2004



recorded from different regions of the heart by simplydisplacing the optical paths. Figure 1B shows examples ofoptical action potentials from different regions of atrial andventricular rabbit myocardium.

Early studies faced marked phototoxic effects of potentio-metric probes in some preparations.11,17 These initial obsta-cles were resolved through the development of better probeswith reduced phototoxic effects and greater sensitivity topotential changes. A.S. Waggoner, L.A. Ernst, and G. Salama(unpublished data, 2003) have recently developed and testednew potentiometric dyes in search of probes with greaterfractional fluorescence changes per action potential andlonger peak excitation and emission wavelengths, with largeStoke shifts. The longer wavelengths would be particularlydesirable for optical recordings from deep inside the myocar-dial wall as light absorption and scattering diminishes withlonger wavelengths. One of these new dyes, PGH1 (Pitts-burgh 1), was found to yield excellent signal-to-noise ratioand stability when applied to the whole heart with anexcitation wavelength of 690 nm and an emission at 850 nm.Although initial tests of PGH1 by several groups were highlypositive in heart (B.-R. Choi, University of Pittsburgh, Pa,and R.A. Gray, University of Alabama), experiments onisolated cells reported up to 30% fractional fluorescencechange per action potential, but a high level of photobleach-ing or phototoxicity (E. Entcheva, Stony Brook University,G. Smith, University of Glasgow, personal communication).Further modifications of successful potentiometric probesand better staining procedures are likely to yield improve-ments in voltage sensitivity, depth penetration of the signals,and the opportunity to detect increasingly smaller potentialchanges from smaller areas of membrane stained with dye.

Another significant limitation of optical mapping of theheart is artifact introduced by the muscle contractions. These

“movement” artifacts distort optical action potentials, pre-venting accurate recordings of repolarization phase. Severalmethods have been used in the past to minimize the effect ofmovement artifact. Mechanical restriction of the movementcan successfully restrict the artifact without affecting physi-ology of the heart.19 This method works particularly well withsmall hearts such as mice, rats, and guinea pig. Alternativesinclude various pharmacological approaches, such as calciumchannel blockers,20 2,3-butanedione monoxime,21,22 or cy-tochalasin D.23 Another approach to reduce movement arti-facts is to apply a ratiometric technique.24,25 This method isbased on simultaneous measurements of fluorescent signal attwo different wavelength ranges: one where the dye exhibitsa largest voltage-dependent response and the other at awavelength where the potentiometric dye exhibits an invertedor no voltage-dependent response. The ratio or difference ofthese two signals would then result in an optical signal freeof, or with significantly reduced, motion artifacts.24–26 Un-fortunately, this method works well with relatively weakcontractions and with biphasic action spectra of fluorescentand/or absorption dyes.27

Calibration of Optical RecordingsIt is important to note that fast voltage-sensitive dyes do notprovide an absolute measurement of transmembrane potentialbut merely track the changes in membrane potential with hightemporal fidelity. Initial studies calibrated optical actionpotential by simultaneously recording fluorescence and mi-croelectrode signals from the same region of the heart.12 Thelinearity and kinetics of the voltage-dependent merocyaninedye signals were characterized in heart muscles undersucrose-gap voltage clamp conditions.13 More recent studieshave concentrated on application of ratiometry approach forquantitative measurements of transmembrane potential withnewer and better dyes. Initially these methods were devel-oped on neuroblastoma cells.28 The early experiments havenot been repeated to confirm the linearity of these dyes inartificial planar bilayers or cardiac cells. Simultaneous ratio-metric optical and microelectrode recordings of transmem-brane potential in perfused hearts confirmed excellent corre-lation and linearity of optical recordings.26

Need for Optical MappingThe spread of electrical activity is important for our under-standing of the mechanisms responsible for the normalcardiac rhythm and for the initiation and maintenance ofarrhythmias. Although much has been learned regarding theionic basis of the cardiac action potential using intracellularmicroelectrodes, single cell impalements cannot be practi-cally used to simultaneously record action potentials fromhundreds of recording sites. The generally used methods tomap activation and repolarization are based on surfaceunipolar and bipolar electrograms measured with arrays ofelectrodes.29,30 Although surface electrodes can describe thespread of excitation and repolarization, interpretation of datain some cases is uncertain.29,31 For instance, activationsequences were difficult to interpret during rapid synchro-nous depolarization, as after electric shock application, andduring slowly changing, low-level depolarization, as in ische-mia. Repolarization measured with an electrogram often does

Figure 1. Optical recordings of cardiac action potentials. A, Si-multaneous fluorescent (Vf) and microelectrode (Ve) recordingsof action potentials in the frog heart stained with Merocyanine-540. Recordings show a faithful reproduction of transmembranepotential by the fluorescence signals. Upstroke of optical actionpotential is typically slower than that of the microelectrode, dueto spatial averaging of a propagating wavefront. B, Optical re-cording of action potentials from different regions of the heartstained with di-4-ANEPPS: ventricular and atrial working myo-cardium, AV node, and Crista terminalis.

Efimov et al Optical Imaging of the Heart 23



not coincide with the actual repolarization at the recordingsite.31,32

Progress in Imaging Technology: Advances inLight Detectors

Current imaging technology presents several approaches forfast imaging, including photomultipliers (PMT), laser scan-ning,33 charge-coupled device (CCD) cameras, and photo-diode arrays (PDA). Currently, only CCD cameras and PDAdetectors are predominantly used in heart imaging applica-tions.19,34 –37 Complementary metal-oxide semiconductor(CMOS) cameras represent another emerging candidate.Combination of PMT devices with a laser scanning techniquefailed to overcome spatiotemporal limitations imposed bysequential nature of recordings.38,39

PDA technology was first developed by Centronix.40,41

Now PDAs are available from Hamamatsu. WuTech42 pro-duces custom arrays of arbitrary size and shape made ofindividual photodiodes.43

CCD technology has a significant advantage of higherspatial resolution due to the large number of pixels on a CCDsensor. However, the rate of data acquisition is usually lower.This can be increased by pixel binning; however, this defeatsthe major advantage of CCD technology because binningreduces spatial resolution. For example, a camera fromDALSA has 128�128 pixels at 490 frames/sec.

Theoretically, CCD technology should provide significantadvantages with respect to signal-to-noise ratio, particularlyat low-light applications, due to the right combination of highquantum efficiency and low background noise level. It isimportant to emphasize that an optical mapping experimenton a whole heart or a multicellular preparations tends not tobe light-limited but deals with conditions where the greaterthe fluorescence intensity from the preparation, the greaterthe absolute amplitude of the fractional fluorescence change.The dynamic range of CCD cameras is constrained by theaccuracy of A/D conversion and the saturation of the sensorat light levels readily detected from heart tissue; a dynamicrange of 103 is not easily achieved. Thus, for practicalpurposes the majority of single cell and cell culture opticalmapping studies has been done using PDA.43–45

Improvements in CMOS technology produced a family ofnovel image sensors with high-speed image acquisition whileretaining the quantum efficiency of CCD. MiCAM UltimaCMOS camera from SciMedia was recently tested on aperfused guinea pig heart at 10 000 frames/sec and 100�100pixels. High signal-to-noise ratio allowed detection of acti-vation times from the first derivative of optical signals duringventricular fibrillation without spatial averaging. CMOS cam-eras are more costly than CCD or PDA cameras. However,due to clear advantages these CMOS cameras will becomecompetitive soon.

Design of Optical Mapping SystemAny optical imaging system consists of a 2D optical sensorand a stable light source, such as a laser or DC-poweredtungsten-halogen lamp, mercury source, or light-emittingdiodes. Figure 2 describes a typical single-lens design. Itconsists of a PDA, 256-channel signal conditioner/amplifier,

analog-to-digital converter, and a computer. An excitationbeam is passed through 520�30-nm interference filters andfocused on the surface of a perfused preparation placed in atissue chamber. A camera lens collects the fluorescence light,directs it to a 610-nm long pass filter and focuses an image ofa chosen region of the heart on the surface of PDA.

Mapping of Activation and RepolarizationFigure 3 shows a map of action potentials optically recordedfrom the anterior surface of a Langendorff-perfused guineapig heart. An optical trace at the right illustrates that thequality of optical recordings approaches that of the goldstandard: microelectrode recordings. When movement arti-facts are a concern, activation and repolarization times can bestill be determined by calculating the maximum first deriva-tive of the action potential upstroke and the maximum secondderivative of the downstroke, respectively19 (Figure 3). Al-ternatively, these times could be determined from the timepoint at which the upstroke reaches 50% or recovers to 90%of its maximum amplitude,46,47 respectively. Recently, phase-mapping techniques were introduced for an analysis ofquasiperiodic electrical activity in the heart.48 These tech-niques determine the specific phase of electrical activity thatcorresponds to each moment of the action potential. Thealgorithms used are based on analysis of phase-space trajec-tories, which result from a plot of a measured variable(optical action potential) against a transform of the samevariable (time-delay,48 first derivative, Hilbert transforma-tion49). Activation and repolarization data are usually present-ed as isophasic or isochronal maps (see Figure 3)4,19,37,50–52 ora vector field of conduction in the cardiac muscle (see Figure3),53,54 allowing quantitative assessment of spread of activa-tion and repolarization in highly anisotropic heart muscleunder normal and pathological conditions.54–56

Figure 2. Schematics of a typical optical mapping system.




Conventional high-resolution multielectrode mapping wassuccessful in assessing activation sequences. However, repo-larization was generally beyond the capabilities of electrodemapping.32 Only a monophasic action potential (MAP) tech-nique57 was able to reliably measure repolarization and actionpotential duration. However, MAP cannot be used for multi-site mapping. Optical mapping provides a tool with theunique ability to overcome limitations of electrode-basedtechniques and to reliably and faithfully record action poten-tials. However, it typically requires the use of mechanical orpharmacological immobilization techniques to suppress themotion artifacts.

In particular, optical mapping has made a significantcontribution to our understanding of the role of intrinsicmyocardial heterogeneities in the normal repolarization pro-cesses in normal heart;19,55 of dynamic concordant anddiscordant alternans in the onset of ventriculartachyarrhythmias;58,59 and the role of the slope of the resti-tution curve in the transition from ventricular tachycardia tofibrillation.60–62

Mapping of Stimulation and DefibrillationOptical mapping is an especially powerful tool in studies ofelectrostimulation therapy. Due to overwhelming stimulus-induced artifacts, the conventional electrode techniques arenot able to record electrical activity during and after stimuli.In contrast, optical recordings provide an accurate account oftransmembrane potential changes during stimulation anddefibrillation. Dillon20 demonstrated the prolongation of ac-tion potential duration by strong electric shocks appliedduring the refractory period. Optical mapping explained themechanisms of epicardial unipolar63 and bipolar pacing.64

Optical mapping also provided the experimental basis for thenew theory of stimulus-induced arrhythmogenesis and therelated theory of success and failure of defibrillation based onvirtual electrode effects,34,65,66 which are also known assecondary source effects.44,67 Figure 468 presents experimen-tal evidence of a virtual electrode-induced phase singularitymechanism of shock-induced arrhythmogenesis.66 Opticalrecordings for the first time allowed faithful dynamic regis-tration of maps of transmembrane potential before, during,

Figure 3. Maps of optical action potentials and methods of analysis (left to right, top to bottom): schematic diagram of anterior epicar-dium of the guinea pig heart and the imaging field of view. Maps of optical action potentials: detection of activation and repolarizationtime points using first (dF/dt) and second (d2F/dt2) derivatives of fluorescence (F). Maps of activation: conduction velocity vectors andrepolarization. See text for detail.




and after shock application. Figure 4B illustrates the typicalpattern of virtual electrode polarization mapped as a distri-bution of transmembrane potential seen immediately after theapplication of a shock. Virtual electrode patterns are charac-terized by the presence of both positive and negative polar-izations, which are induced by the virtual cathode and anode,respectively. As a result, a shock-induced wavefront ofbreak-excitation can form a reentrant pattern around thevirtual electrode-induced phase singularity.66,69,70

Mapping of the Impulse Generation andConduction System of the Heart

Optical mapping has made a significant contribution to func-tional studies of the fundamental mechanisms of impulse gen-eration in the SA and AV nodes, and impulse propagation in theconduction system of the heart. Kamino’s group pioneeredoptical mapping techniques in the brain and heart, investigatingthe genesis of spontaneous electrical activity in the embryonicheart.71–74 Optical mapping was also instrumental in the identi-fication of nonradial spread of activation via preferential path-ways from the SA node toward the AV node.75,76 Our groupsapplied optical mapping to studies of AV nodal conduc-tion,22,76,77 AV nodal reentrant arrhythmias,78,79 and AV junc-tional rhythm.80 Finally, Morley and colleagues54,56 pioneeredthe application of optical mapping to investigate mechanisms ofconduction in the Purkinje system, which was unattainable byelectrode-based techniques.

In addition to contributing to our understanding of mech-anisms of impulse generation and conduction, these studiesdemonstrated the ability of optical techniques to assesselectrical activity in three dimensions. Figure 5 presents22,80

an example of optical (OAP) and microelectrode (MAP)action potential recordings and histology from the distal AVjunctional area of the rabbit heart. Figure 5B shows histologyof this area in another preparation from the apical area of thetriangle of Koch,80 which clearly shows multilayered mor-phology of the AV junction. One can see the thin layer of ANtransitional tissue (E), the compact AV node (F), and looselycoupled deeper layer of NH transitional cells. Optical actionpotentials (OAPs) recorded during conducted beats contained

two components, representing electrical activity in the twolayers. OAPs recorded during AV block contained only onecomponent. Microelectrode recordings conducted simulta-neously provides evidence that the first component of opticalrecording corresponds to the superficial transitional AN layer(see E, in Figure 5A and 5B) of the AV junction, whereas thesecond component represents electrical activity of the deeperNH layer (F in Figure 5A and 5B).

Mapping of Atrial andVentricular Tachyarrhythmias

Optical mapping techniques presented a unique opportunityto study the mechanisms of supraventricular and ventricular

Figure 4. Optical imaging of shock-inducedarrhythmogenesis and defibrillation. A, Preparation.B, Shock-induced polarization. C, Shock-inducedconduction pattern. D, Optical recording of trans-membrane potential during normal action potential,T-wave shock, and shock-induced arrhythmia.

Figure 5. Three-dimensional (multilayer) imaging of the conductionthrough the atrioventricular (AV) junction. AV junctional preparationwas paced at atrial septum with a cycle length S1S1�280 ms (A,top) and 274 ms (A, bottom), which caused Wenckebach AV block.Bipolar electrograms were recorded from Crista terminalis (CrT)and bundle of His. Optical traces (OAP) represent the superimposi-tion of signals from the septum and the distal AV nodal areas. B,Masson Trichrome stained section through the distal AV node.Places of microelectrode recordings (MAP) are marked with blackand white circles. See text for detail.




arrhythmias because of unprecedented spatiotemporal resolu-tion and the ability to map all phases of electrical activity,including activation and repolarization.

Jalife’s group pioneered the application of optical mappingto study arrhythmogenesis81–84 and made numerous signifi-cant contributions to our understanding of mechanisms ofboth atrial83 and ventricular arrhythmias.48,84 Since that time,many groups presented optical mapping data supportingreentrant nature of ventricular tachycardia66,81,85,86 and fibril-lation.48,87,88 Furthermore, optical mapping presented evi-dence for 3D nature of ventricular reentry, which is sustainedby scroll waves.86,89

Despite the success of dynamic optical imaging of wave-fronts and phase singularities during arrhythmias, there is stillno agreement on the mechanisms that induce and sustainventricular and atrial fibrillation.90 Two competing dominanttheories are being tested: the so-called “mother rotor”91–93 and“break-up”94–98 hypotheses. According to the mother rotorhypothesis, ventricular fibrillation is maintained by a singleor limited number of leading centers of reentrant nature, ie,mother rotors. These centers occupy regions of myocardiumthat are capable of sustaining the highest possible frequencyof reentrant activity.91–93 Disorganized activity observedthroughout the heart, in regions beyond those of the motherrotor(s) results from conduction blocks between the regionsencompassed by the mother rotor and regions with longerrefractory periods. In contrast, the break-up theory proposesthat fibrillation is perpetuated by dynamic or anatomicheterogeneities in the myocardium sustained through constantcreation and annihilation of waves.94,96–98 Profound complex-ity of conduction patterns during fibrillation makes quantita-tive analysis of wavefronts and wavebreaks a formidabletask.35,99 Therefore, frequency analysis of optical data wasadopted98,100 following the pioneering work of Wiggers.101

As presented in Figure 6A, according to one school ofthought,100 during fibrillation, dominant frequencies of elec-trical activity are distributed in a stationary pattern. Thisappears to support the mother rotor hypothesis. On the otherhand, as presented in Figure 6B and 6C,98 another group ofinvestigators presents evidence of constantly changing pat-tern of frequencies of electrical activity without clear domi-nant frequency. This issue remains controversial as evidencein support of both theories continues to appear in theliterature.

Multiparametric Optical Mapping: Imaging ofVoltage and Intracellular Calcium

Calcium cycling is arguably the single most important com-ponent of cardiac excitation-contraction coupling. The actionpotential elicits an influx of Ca2� through the activation ofL-type voltage-gated Ca2� channels that trigger a release ofCa2� from intracellular stores called the sarcoplasmic reticu-lum (SR) resulting in a contraction.102 Ca2� release from theSR occurs via Ca2� release channels or ryanodine receptorsthat are activated by a local elevation of intracellular Ca2�

([Ca2�]i) by a process called Ca2�-induced Ca2� release(CICR).103 Normally, depolarization triggers [Ca2�]i tran-sients, but in pathological conditions, abnormalities in [Ca2�]i

handling may activate Ca2�-dependent currents that influence

the time course of the AP and trigger a spontaneous mem-brane depolarization.104,105 Abnormalities in [Ca2�]i handlinghave been implicated as the underlying mechanism in anumber of pathologies that promote arrhythmias such asischemia, reperfusion-arrhythmias, the generation of earlyand delayed afterdepolarizations, and torsades de pointes thatoccurs in patients with the long QT syndrome. [Ca2�]i

overload has been implicated in triggering electromechanicalalternans and in increasing the steepness of APD restitutioncurves, which are both associated with promotion of arrhyth-mias. Thus, one cannot overstate the importance of simulta-neous measurements of APs and [Ca2�]i transients in intacthearts to address fundamental questions regarding the spatio-temporal relationship of voltage and [Ca2�]i and their inter-play in arrhythmias.

The first techniques for measuring changes in cytosolic[Ca2�]i concentration of coronary-perfused hearts involved

Figure 6. Frequency analysis of optically recorded electricalactivity during ventricular fibrillation. A, FFT spectra with a singledominant frequency and maps with static frequency domains,100

support the mother-rotor hypothesis of fibrillation. B and C,Complex FFT spectra and dynamic frequency domains98 sup-port the multiple wave-breaks hypothesis of fibrillation. See textfor details.




19F nuclear magnetic resonance,106,107 fluorescent Ca2� indi-cator Indo-1,108 and a bioluminescent Ca2� indicator ae-quorin.109 Later, other fluorescent Ca2� indicators (Fluo 3110

and Rhod 2111,112) were successfully used for heart imaging.Simultaneous Ca2� and voltage imaging of the heart has alsobeen achieved by costaining with RH421 and Rhod-2,113

RH237, and Rhod-2,59,114 with RH237 and Fluo-4/OregonGreen BARTA 1,25 with di-4-ANEPPS and Fluor3/4,115 andwith di-4-ANEPPS and Indo-1.116

Figure 7 shows the optical system for simultaneous record-ing voltage and [Ca2�]i signals from perfused hearts stainedwith RH237 and Rhod-2.59 It uses two PDAs (Figure 7A).RH237 and Rhod-2 dyes can be excited at the same wave-length but fluoresce at different wavelengths, allowing theseparation of the Vm and [Ca2�]i signals. Rhod-2 was found tobe an excellent Ca2� indicator for perfused hearts because ofits rapid association/dissociation with Ca2�, fast loading intomyocytes of perfused hearts, and long-term stability or lowlevels of exocytosis.59 The independence of Vm and [Ca2�]i

recordings is illustrated in Figure 7C through 7E. First,signals from both Vm and [Ca2�]i arrays were recorded whenthe heart was stained with one of the two dyes but not bothdyes. Second, after staining with both dyes, Vm and [Ca2�]i

signals were recorded before and after the addition ofryanodine, known to markedly inhibit [Ca2�]i transients.

Multiparametric optical mapping is still in its infancy.Other probes are being synthesized and evaluated for simul-taneous recordings from the same tissue, stained with multi-ple fluorescent probes. This exciting progress is likely tobring about a better understanding of cellular physiology at atissue and organ level.

Mapping Transgenic Mouse ModelsMolecularly engineered mice are increasingly used to genet-ically alter a specific component of a complex signalingprocess and to develop models of human diseases. Transgenicand knockout mice are used as models for various cardiacdiseases and offer an effective strategy to elucidate themechanisms underlying arrhythmias, metabolic diseases, thepathology of heart failure, and altered ion channel and gapjunction expression.117 A limitation of mouse models is therapid heart rate and small size of the heart, which makes itdifficult to study changes in contractility, electrophysiology,and arrhythmias vulnerability. The challenge of studyingcardiac phenotypes in mice has been effectively tackled byoptical mapping of action potentials and [Ca2�]i transients.

Dominant-negative transgenic mice that overexpress anN-terminal fragment of the K� channel Kv1.1 were shown toexhibit prolonged action potential duration due to the loss ofa slowly inactivating 4-aminopyrydine sensitive current, Islow,

Figure 7. Multiparametric optical imaging: simultaneous optical recordings of transmembrane potential and intracellular calcium tran-sients. A, Experimental setup with 2 16�16 photodiode arrays. Hearts were stained with voltage-sensitive dye RH 237 and Ca2� indica-tor Rhod-2. B, Optically recorded action potentials (AP) and calcium transients (Cai) at 252 points of mapped region. C and D, Effectsof Ryanodine on AP and Cai recordings. To verify that the Rhod-2 signal is a measure of Cai released from the sarcoplasmic reticulumvia ryanodine receptors and was impervious to changes in Vm, ryanodine (10 �mol/L) was added to the perfusate to test the selectivityof its actions on Cai versus the AP. E, Superposition of an AP and Cai transient recorded from the same region of myocardium. AP andCai were simultaneously recorded at a sampling rate of 4000 frames/sec for each array. AP upstroke preceded the rise of Cai by10.4�0.4 ms.




which is likely to be encoded by Kv1.5.118 These mice werefound to have long-QT intervals, spontaneous nonsustainedventricular tachycardia (VT) during ambulatory telemetrymonitoring and inducible polymorphic VT during pro-grammed stimulation in anesthetized open chest prepara-tions.118,119 Optical mapping demonstrated a 2-fold increasein action potential durations and enhanced dispersion ofrepolarization from apex to base in dominant-negative trans-genic mice.120 In dominant-negative transgenic mice, a pre-mature impulse applied at the apex of the heart producedsustained reentrant VT that did not occur with stimulation atany location in controls hearts.120 Direct injection of adeno-viral vectors expressing wild-type Kv1.5 (AV-Kv1.5) in themyocardium of these Kv1 dominant-negative transgenic miceresulted in the overexpression of Kv1.5, a shortening of APD,shortened the QT interval, decreased dispersion of repolar-ization, and increased the heart rate. These changes wereconsistent with a physiological reversal of the arrhythmo-genic phenotype by the adenoviral induced expression ofKv1.5 in Kv1 dominant-negative mice.121

Transgenic mice overexpressing the inflammatory cyto-kine tumor necrosis factor � (TNF-�) develop a progressiveheart failure (HF) phenotype characterized by biventriculardilatation, decreased ejection fraction, and ventricular ar-rhythmias on ambulatory telemetry and decreased survivalcompared with control liter mates. Optical maps of actionpotentials and [Ca2�]i transients showed that TNF-� heartshad prolonged action potential durations, no change indispersion of repolarization, elevated diastolic, depressedsystolic, and prolonged [Ca2�]i compared with controls.114

Premature beats had lower action potential amplitudes,slower conduction velocities, and elicited reentrant beats.Increasing heart rate produced [Ca2�]i alternans in TNF-� butnot in control hearts. In this model, anomalies of both AP and[Ca2�]i contributed to arrhythmias.

Gap junction channels are essential for cell-to-cell cou-pling and impulse propagation. Changes in channel conduc-tance may underlie the development and maintenance oflethal arrhythmias in pathological conditions.122–124 Null mu-tations of connexin 43 (Cx43) in mice, the predominant gapjunction channel proteins in ventricular tissue, was lethal inhomozygous (Cx43�/�) mice but seemingly normal in het-erozygous (Cx43�/�) mice. Measurements of conductionvelocity from the same line of Cx43�/� mice producedconflicting results depending on the optical mapping tech-nique. Using a CCD camera, Morley et al125 found nosignificant difference in conduction velocity between het-erozygous Cx43�/� mice and controls, whereas Eloff et al126

found a 23% to 35% slowing of conduction velocity using aphotodiode array (PDA) to map action potentials. The differ-ent findings perhaps reflect the differences in temporalresolution of the CCD and PDA technology because the sameline of Cx43�/� mice was used in both studies.

Mapping Developing MyocardiumIn embryonic hearts and neonatal cell cultures, the ability tooptically record simultaneously from multiple sites is makingimportant contributions to our understanding of the develop-ing heart. Optical mapping was applied to study the pattern-

ing and organization of the conduction system and workingmyocardium in the developing avian127,128 and mammali-an129,130 hearts. Optical mapping of action potentials and[Ca2�]i in combination with patterned cultured neonatal rat131–

133 mouse134 and genetically modified mice is bound to makemajor contributions to our understanding of propagation inthe adult and developing heart. Optical mapping has contrib-uted to investigation of the important role of mechanoelec-trical coupling in shaping the working myocardium and theconduction system of chick heart.135 Combination of opticalmapping of electrical activity with structural 3D mapping ofdeveloping hearts is especially exciting new opportunity.136,137

Emerging Optical Imaging ModalitiesImaging with voltage-sensitive probes has several limitations.Pharmacological effects of the dye include phototoxicity138

and increased contractility.139 Yet, arguably, the major limi-tation of organ-level optical imaging is its restricted depth ofpenetration.

To overcome the depth limitation, Hooks et al140 developedan elegant optrode-based method. This method extends theidea of a plunge needle electrode.141 Plunge optrodes presenta unique possibility for multisite intramural recordings oftransmembrane potential,140,142 yet cause damage of themyocardium.

Depth restriction is due to light absorption and scatteringon intrinsic differences of tissue optical properties (endoge-nous contrast). Absorption occurs primarily at endogenouschromospheres of hemoglobin, melanin, fat, and water. Scat-tering is usually due to refractive index differences of extra-and intracellular structures. These properties are stronglywavelength-dependent. Baxter et al143 proposed an originalmethod that takes advantage of this wavelength dependenceto visualize excitation waves within the cardiac muscle. Lightin the 700- to 900-nm range is known as the “therapeuticwindow,” due to low intrinsic tissue absorbance and highscattering of photons dominates in this range of wavelengths.This window opened an opportunity for tomographic recon-struction of 3D structures using novel biophotonics imagingmodalities, such as optical coherence tomography.137,144

Optical coherence tomography (OCT) exploits the hearttissue scattering heterogeneity and at present can reconstructthrough 2- to 3-mm depth of cardiac tissue with up to 1-�mresolution. The latter characteristics make tomography amethod of choice for in situ analysis of embryonic heartmorphology,136,137 AV nodal multilayer structures (Figure8),145 or complex 3D geometry of trabeculated structures ofmyocardium and the Purkinje network146 (Figure 8). Pres-ently available OCT technology is limited to structuralimaging only. However, emerging second harmonic OCTtechnique147,148 could directly resolve the 2D limitations ofconventional optical mapping. Alternatively, 3D limitationscould potentially be addressed with confocal,149 Nipkow disk,or Ronchi grating150 approaches. Another promising directionrelies on the possibility of solving the inverse problem forlight diffusion/scattering in the tissue.9 This could numeri-cally separate the optical signal collected from the heartsurface into individual components related to signals from thedifferent layers of the tissue.




ConclusionOptical imaging of cardiac electrical activity at the tissue andorgan levels has emerged as powerful novel approach duringthe last decade and has made a significant contribution tocardiovascular research. Exciting new developments in bio-photonics suggest that the best is yet to come. The nextdecade is likely to yield (1) novel optical molecular probesfor multiparametric optical sensing of various biologicalparameters, processes, molecules, proteins, and their func-tional states in real time with submillisecond resolution and(2) novel optical imaging modalities for 3D optical interro-gation of molecular probes with precise anatomical localiza-tion of the signal origin with subcellular spatial resolution.

AcknowledgmentsThis work was supported by NIH R01 grants HL67322 and HL58808(to I.R.E.) and HL57929, HL59614, HL69097, and HL70722(to G.S.).

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