termination of equine atrial fibrillation by quinidine: an optical...

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Journal of Veterinary Cardiology (2008) 10, 87e102

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Termination of equine atrial fibrillation byquinidine: An optical mapping study*

Flavio H. Fenton, PhD a, Elizabeth M. Cherry, PhD a,Bruce G. Kornreich, DVM, PhD b,*

a Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USAb Department of Clinical Sciences, Cornell University, Ithaca, NY 14853, USA

Received 31 July 2008; received in revised form 30 September 2008; accepted 8 October 2008

KEYWORDSAtrial fibrillation;Alternans;Quinidine;Restitution;Optical mapping

pect of the Journal of Ved diagnostics. These imom/science/journal/1e Summary Plus link. Te several minutes. Reaenjoy the content. Anpaper which is indicateing author.ess: [email protected]

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Abstract Objective: To perform the first optical mapping studies of equine atriumto assess the spatiotemporal dynamics of atrial fibrillation (AF) and of its termina-tion by quinidine.Animals: Intact, perfused atrial preparations obtained from four horses withnormal cardiovascular examinations.Materials and methods: AF was induced by a rapid pacing protocol with or withoutacetylcholine perfusion, and optical mapping was used to determine spatial domi-nant frequency distributions, electrical activity maps, and single-pixel opticalsignals. Following induction of AF, quinidine gluconate was perfused into the prep-aration and these parameters were monitored during quinidine-induced termina-tion of AF.Results: Equine AF develops in the context of spatial gradients in action potentialduration (APD) and diastolic interval (DI) that produce alternans, conduction block,and Wenckebach conduction in different regions at fast pacing rates. Quinidineterminates AF and prevents subsequent reinduction by reducing the maximalfrequency and increasing frequency homogeneity.Conclusions: Heterogeneity of APD and DI promote alternans and conduction blockat fast pacing rates in the equine atrium, predisposing to the development of AF.

terinary Cardiology is the emphasis of additional web-based images permitting the detailing ofages can be viewed (by those readers with subscription access) by going to http://www.

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(B.G. Kornreich).

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88 F.H. Fenton et al.

Quinidine terminates AF by reducing maximum frequency and increasing frequencyhomogeneity. Our results are consistent with the hypothesis that quinidineincreases effective refractory period, thereby decreasing frequency. 2008 Elsevier B.V. All rights reserved.

Introduction

Atrial fibrillation (AF) is the most commonly diag-nosed cardiac arrhythmia in horses, with preva-lence ranging as high as 2.5% depending upon studypopulation.1e4 Although AF is commonly diagnosedas an incidental finding in horses, it remainsa significant cause of poor performance in equineathletes, and as such constitutes an importantdisease entity in equine patients.5e7 Horses arepresumably predisposed to AF primarily due totheir high vagal tone and large atrial mass,4,8e11

which combine to promote the maintenance ofmultiple reentrant circuits, or spiral waves, withinthe atria of affected individuals. While thespatiotemporal behavior of these spiral waves hasbeen the subject of considerable research anddebate, it is generally accepted that they areresponsible for the rapid, apparently irregularatrial depolarizations that are characteristic ofAF.10e18

The ideal treatment of human patients with AFis conversion to normal sinus rhythm (NSR), aschronic AF is associated with increased risk ofthromboembolism, stroke, and cardiovasculardeath in people.19e22 While these associationshave not been documented in equine patients, AFis a well documented cause of poor performance inthe horse.22e24 For this reason, although long-standing AF or significant structural heart diseasemay preclude successful conversion, many equinepatients with AF of relatively short duration (i.e.,less than four months) and no significant structuralcardiac pathology (termed lone AF) are convertedto NSR using either pharmacologic or electricalmodalities.23,24 The requirement for anesthesia,specialized equipment, and expertise for elec-trical cardioversion, however, combined with therelatively high success rate of pharmacologic car-dioversion, precludes electrical cardioversion inmost equine patients. Given these considerations,the vast majority of equine AF patients are con-verted pharmacologically using orally adminis-tered drugs.

Quinidine is the most commonly used drug toconvert equine patients with AF to NSR.7,28 ThisClass IA antiarrhythmic drug decreases themaximum rate of rise of phase 0 depolarization via

sodium channel blockade and prolongs repolari-zation via blockade of a number of potassiumchannels (most notably Ikr) in myocardial tissue.

29e37

The net effects of these actions are to decreaseconduction velocity and to prolong action potentialduration and effective refractory period in atrial,ventricular, and Purkinje myocytes.37e42 Theseactions are believed to form the basis for theireffectiveness in interrupting reentrant circuitsthat underlie AF, and a number of studies haveshown that quinidine is efficacious at converting themajority of equine fibrillation patients with loneAF to NSR.7,28,43,44

In spite of its relatively high clinical efficacy atconverting horses in AF to NSR, the definitivespatiotemporal mechanism by which quinidineachieves this conversion in horses remains unclear.Given the prevalence and clinical impact of equineAF, the frequency at which quinidine is adminis-tered to equine patients with AF, and the potentialrole that horses may play as a viable animal modelof human AF, an understanding of the mechanismby which quinidine terminates AF in horses is likelyto provide information that not only will be usefulto clinical veterinarians, but also will improve ourunderstanding of AF in other species, includinghumans.

Optical mapping using voltage-sensitive dyeshas been used previously to investigate bothmechanisms of arrhythmogenesis and of pharma-cologic and electrical termination of arrhyth-mias.45e53 This technique has proven valuable inthe study of both atrial and ventricular arrhyth-mias and has been applied to a wide variety ofspecies including human, dog, sheep, swine,rabbit, guinea pig, mouse, and rat.46,54e64 Opticalmapping has not been applied to the study of atrialfibrillation in horses, to our knowledge.

We designed a series of optical mapping exper-iments using intact equine atrial preparationsperfused with the voltage-sensitive dye di-4-ANEPPS16,65e69 to test the hypotheses thatquinidine terminates AF, and that this terminationis associated with a measurable change in AFdominant frequency. To our knowledge, this is thefirst application of optical mapping to study themechanism of an antiarrhythmic drug in isolatedequine cardiac preparations.

Termination of equine atrial fibrillation by quinidine 89

Animals, materials and methods

Tissue harvesting and perfusion

All experiments were carried out in accordancewith IACUC guidelines in AAALAC-approved facili-ties. Immediately following euthanasia of horses ofeither gender for non-cardiac reasons ranging fromcareer-ending musculoskeletal abnormalities toblindness, hearts were removed (Fig. 1A) andplaced in 22e25 C Tyrode solution containing (inmM): MgCl2 2, NaH2PO4 0.9, CaCl2 2, NaCl 137,NaHCO3 24, KCl 4, Glucose 5.5.

70 All Tyrode solutionwas equilibrated with 95% oxygen/5% CO2. The atriawere then dissected from the heart (Fig. 1B) and thecoronary vasculature was cannulated with flexibletubing (1/4 inch ID, 1/16 inch wall thickness; FisherScientific Co, Pittsburgh, PA) that was sutured intothe coronary ostium immediately distal to theaortic valve leaflets using silk suture material ina simple continuous suture pattern (Ethicon Inc.,Piscataway, NJ). Perfusion of the excised atrium viathe coronary vasculature with 37 C Tyrode solutionwas instituted at a rate of 40 ml/min. This flow ratewas extrapolated from rates established for caninecardiac preparations, accounting for increasedtissue mass. Viability of the preparation wasassessed by visual inspection for normal contractionat a rate of approximately 30 beats per minute (seeSupplemental movie). Leakage from the cut edgesof the preparation was prevented by suturing usingsilk suture material in a simple continuous pattern.

Optical mapping

The preparations were placed in a plexiglaschamber that kept the tissue submerged in Tyrode

Figure 1 Equine heart and optical mapping setup. (A)Equine heart. (B) Equine left atrium. (C) Equine leftatrial preparation in optical mapping setup.

and at 37 C, as shown in Fig. 1C. To stain thetissue for optical mapping, the tissue was perfusedwith a single administration of 2 mM di-4-ANEPPS(Invitrogen Corporation, Carlsbad, CA) in 200 mlTyrode solution; if necessary, staining wasrepeated halfway through the experiment. Prepa-rations were perfused with Tyrode solution (withor without ACh or quinidine as required) and noperfusate was recirculated. Illumination wasprovided by nine high-power LEDs (Luxeon III star,LXHL-FM3C) at an excitation frequency of 530 20nanometers (nm). Fluorescence emission light wascollected by a Navitar lens (DO-2595, focal length25 mm, F/# 0.95, distance 60 cm), passed througha long-pass filter ( 90%). The signalwas digitized with a 16-bit A/D converter at framerates of 511 Hz (full frame, 128 128 pixels). ThePCI interface provides high-bandwidth uninter-rupted data transfer to the host computer. Opticalrecordings were performed in episodes lasting 16e20 s and were obtained continuously during quini-dine administration.

To prevent contraction and resultant motionartifact, 10e15 mM of blebbistatin, a myosin IIinhibitor (BIOMOL International, PlymouthMeeting, PA), was added to Tyrode solution andperfused into the preparation for approximately60 min prior to data collection. This concentrationof blebbistatin has been used previously to preventcontraction of cardiac preparations in our labora-tory and in other studies and has been shown toexert minimal effects on the electrophysiologicalproperties of cardiac tissue.71 Microelectroderecordings (Fig. 2) show that blebbistatin hasminimal effects on equine ventricular actionpotential amplitude, morphology, and durationand for a broad range of CLs.

Microelectrode recordings

Microelectrode recordings were performed on thinslices of ventricular tissue from the epicardiummounted in a Plexiglas chamber and superfusedwith normal Tyrode solution at a rate of 15 ml/min. The tissue was then stimulated using rectan-gular pulses of 2 ms duration and two to threetimes the diastolic threshold (0.1e0.3 mA) deliv-ered through Teflon-coated bipolar silver elec-trodes using a computer-controlled stimulator.Transmembrane action potentials were recordedfrom the epicardium with machine-pulled glasscapillary electrodes filled with 3 M KCl. Recordings

Figure 2 Use of blebbistatin to suppress contraction. With the application of blebbistatin, contraction and theresulting motion that can distort optical mapping signals can be avoided without affecting action potential and rateadaptation properties. Top: action potentials obtained from microelectrode recordings in equine ventricular tissue(epicardial) from three cycle lengths (240, 300, and 500 ms) before (black) and after (red) administration of 10e15 mMblebbistatin are nearly indistinguishable. Bottom: Rate adaptation curves obtained over a range of cycle lengthsbefore (black) and after (red) blebbistatin administration in equine ventricular tissue (epicardial) show excellentagreement. Action potential durations (APDs) are computed as 90 percent of repolarization.


were obtained from sites located within 3e4 mmof the bipolar stimulating electrode. Restitutioncurves were obtained using protocols as describedby Cherry and Fenton.72

Initiation of fibrillation

The intact atrial preparation was paced at a cyclelength (CL) of 500 ms using a bipolar stimulatingelectrode connected to an external pulse generator(Isostim A320, WPI Inc, Sarasota, Fla) deliveringbetween 0.3 and 0.9 mA of current at a pulseduration of 3 ms until consistent capture was veri-fied. For all experiments, the pacing electrode wasplaced on the epicardium of the free wall of theatrium, anatomically distant from the junction withthe pulmonary veins, and the region mapped wasalong a line with end points at the pacing electrodeand the opposite border of the atrium, defined asthe border farthest from the pacing site. Fibrilla-tion was initiated by a rapid pacing protocolbeginning with a CL of 250 ms and decreasing byincrements of 50 ms until fibrillation was initiated,as assessed by real-time monitoring of single pixels(similar to atrial electrograms) for the onset of

rapid, irregular signals. If rapid pacing alone did notinitiate fibrillation, 1 mM acetylcholine (ACh)(acetylcholine chloride, SigmaeAldrich, Milwau-kee, WI) was added to the Tyrode perfusate and thepacing protocol reinstituted until sustained fibril-lation was initiated. If 1 mM ACh did not initiatefibrillation, serial increases in ACh concentrationwere carried out (in mM: 2, 4, 6, 8, 10, 20) untilfibrillation was initiated and sustained.

Non-inducible, non-sustained, andsustained AF

Sustained AF was defined as fibrillation that lastedlonger than 3 min. Non-inducible AF was defined asfibrillation that lasted three seconds or less. Atrialfibrillation that lasted between 3 s and 3 min wasdefined as non-sustained.

Quinidine use and AF termination orprevention

Once fibrillation was initiated, the preparation wasperfused with quinidine gluconate (Eli Lilly andCo., Indianapolis, IN) in Tyrode solution beginning


with a concentration of 1.2 mM and progressingthrough 6, 12, and 24 mM concentrations untilfibrillation was terminated or 10 min of perfusionwith a given quinidine concentration had elapsed.These concentrations were chosen to determinedose response based upon previously documentedserum quinidine levels in horses given intravenousquinidine gluconate at clinically relevant doses(2e5 mg/ml), correcting for 20% protein binding ofquinidine that has been established in horseserum.28,73 A minimum of 4 rapid pacing protocolswas applied to a particular preparation at a givenquinidine concentration before characterizing thatconcentration as preventive of AF. A trial wasdefined as either the initial pacing protocolapplied to each preparation or to that appliedafter a change in ACh or quinidine concentration.

Data analysis

The dominant frequency of single pixels wascalculated in real time during the experiments.Offline, optical action potentials were determinedat every pixel by inverting the recorded fluores-cence signal (as the fluorescence is inverselyproportional to the membrane potential). Signaldrift for each pixel was removed by subtracting theline connecting successive resting membranepotentials immediately preceding action potentialupstrokes unless minima were not easily detect-able (during some episodes of AF), in which casea low-pass filter was used instead. For each pixel,action potentials were then normalized to thesignal maximum and minimum of the time series toobtain a normalized voltage (NV) signal. Signalswere subsequently averaged in time with a near-est-neighbor method and in space using a Gaussianfunction with a 3-pixel radius to reduce noise.

Action potential durations (APD) were measuredto 90 percent repolarization. Isochrones werecalculated from the activation times computed ateach pixel by determining when an action poten-tial upstroke reached 50 percent of the actionpotential amplitude and by using a spatial filterconsisting of a weighted average of nearestneighbors to smooth outlying points. Spatialfrequency maps were determined by computingthe power spectrum of each pixel within a 1e8 swindow and selecting the maximum frequency asthe dominant frequency. We verified that thefrequency maps did not change significantly bychoosing longer or shorter windows when thearrhythmia was stable, and we chose shorterwindow lengths to illustrate changes in arrhythmiacharacteristics during termination.

Results

Conduction in normal atria

Six equine atrial preparations were studied, withfour achieving adequate perfusion and staining withdi-4-ANEPPS to provide sufficient signal-to-noiseratio for data collection. Representative opticalaction potentials obtained during pacing to steadystate at different cycle lengths (CLs) are shown inFig. 3. Action potential durations measured at 90percent of repolarization were obtained at foursites in a single preparation over a range of CLs andare plotted in Fig. 3 as a function of CL (rateadaptation curve) and of diastolic interval (DI)(restitution curve). These curves show two impor-tant properties. First, the restitution curve is steep,with slope greater than one (slope> 1 forDIs< 70 ms, maximum slope of 3.2), indicatinga large decrease in action potential duration withsmall decreases in CL at rapid pacing rates. Despitethe steepness of the restitution curve, no alternanswere present close to the pacing site. Second, theminimum CL or DI for which action potentials couldbe obtained varied among the sites and constitutespatial heterogeneity in the tissue. Because of thisvariation, at short CLs part of the tissue witha higher minimum DI cannot support 1:1 propaga-tion, resulting in 2:1 conduction block in which onlyevery other beat propagates. This effect is furtherdemonstrated in Fig. 4, where isochrones are shownfor a long CL of 750 ms and for a short CL of 250 ms.At the long CL (Fig. 4A), although regions ofconduction slowing can be observed, the entiretissue responds to every paced beat in a 1:1 manner.At the short CL, however, the pacing cannot capturethe entire tissue, and a region in the upper right canconduct only every other beat (Fig. 4B). When thetissue in this region cannot conduct, the isochronescrowd together and form a line of block.

In between the 1:1 and 2:1 regions, electrotoniceffects (diffusive currents between cells) producea region of 2:2 conduction, or alternans. It isimportant to note that this alternans does not ariseas a direct consequence of rapid pacing,64,74,75 butstrictly because of the interaction between the 1:1and 2:1 regions. Fig. 5 shows optical actionpotentials from a series of sites spanning the 1:1region proximal to the pacing site and 2:1 regionsdistal to the pacing site. Alternans of both APD andaction potential amplitude (APA) develops in thetransitional area between regions of 1:1 and 2:1conduction. Action potential amplitude of alter-nating beats decreases in this region until block ofalternating beats occurs.

Figure 3 Optical action potentials and rate adaptation in equine atrial tissue. Representative action potentialsobtained from one preparation using optical mapping are shown for a range of cycle lengths. Action potentialdurations (APDs) are shown as a function of cycle length (CL, top) and diastolic interval (DI, bottom). The dotted linein the APD vs. DI plot has slope one and indicates that the steepest portion of the restitution curve has slope greaterthan one. Data from four sites are averaged for the longer cycle lengths (circles/solid line), while data for the second-and third-smallest cycle lengths (squares/dashed line) were obtained from only three sites because of the occurrenceof 2:1 block in the fourth site at these CLs. For the smallest cycle length (triangles), only one site did not exhibit 2:1block. Error bars indicate maximum difference between the average value and the individual values obtained. APDsare computed as 90 percent of repolarization. Optical action potentials (red) are shown together with microelectrodeaction potentials (black) obtained from a different equine atrial preparation; both show similar triangularmorphologies.


Induction of AF

The intrinsic dispersion of APD and DI can producenot only 2:1 conduction block, but also morecomplex Wenckebach patterns as the CL isdecreased further. As the conduction patternsbecome more complex, propagation can fail indifferent directions at different times and initiatefibrillation. Fig. 6 shows how the spatial distribu-tion of dominant frequencies changes with CL.Initially, only 1:1 propagation is present. Regionsof 2:1 conduction arise by a CL of 500 ms, regionsof 4:1 conduction occur by a CL of 250 ms, and

Wenckebach conduction appears between CLs of220 and 170 ms. By the time a CL of 160 ms isreached, AF has initiated in the region of Wenck-ebach conduction. Because pacing is continueddistal to this region, fibrillation is limited spatiallyand does not invade the rest of the tissue in thiscase.

Spatiotemporal dynamics of AF

Following cessation of rapid pacing (CL< 160 ms),fibrillation arises throughout the tissue. Fig. 7shows two examples of AF initiated in different

Figure 4 Activation isochrones at two different cycle lengths shown every 5 ms. (A) At a cycle length of 750 ms, theactivation proceeds rapidly through the tissue from the pacing site (white area obscuring the tissue at lower left) butwith slowing and crowding of isochrones in the upper right corner. (B) At a cycle length of 250 ms, not all of the tissueis activated on every beat, so two successive beats are shown, with gray indicating an area of block for the secondbeat. Conduction is slower relative to the longer cycle length, and there is more pronounced crowding of isochrones inthe upper right corner. Activation times are measured at each site as the time at which depolarization has reachedhalf of the full action potential amplitude.


preparations. Both examples show a wide variationin frequencies present, although both have largeregions with lower frequencies together withsmaller, higher-frequency areas. The electricaldynamics are complex with non-repeating patterns(also see movies in online Supplement).

Although fibrillation could be induced by rapidpacing, these arrhythmias were not sustained in allcases (

Figure 5 Development of alternans between regionsof 1:1 and 2:1 conduction. Because some regions of thetissue have a larger minimum diastolic interval, regionsthat conduct every beat (1:1) may be present withregions that conduct only every other beat (2:1) at somecycle lengths. In between these regions, alternans (2:2)can arise because of electrotonic effects. In this case,successive action potentials vary in amplitude andduration. Here odd beats (blue) propagate throughoutthe tissue, but a marked reduction in action potentialamplitude can be observed on even beats (red) asa wave propagates from a region of 1:1 conduction toa region of 2:1 conduction where block occurs.


reinitiated in this preparation under these condi-tions. These findings are summarized in Table 1.

Quinidine administration at concentrationsbetween 1.2 and 12 mM terminated sustained AF inall trials in which sustained AF was inducible. Fig. 9shows three examples of termination of sustainedAF in different preparations with different quini-dine dosages. In all trials in which quinidineterminated sustained AF, two phenomena wereobserved. The first was that the maximum domi-nant frequency decreased from 12e14 Hz (at high

ACh concentrations) or 7e11 Hz (at low AChconcentrations) gradually to 3e4 Hz immediatelyprior to AF termination, and the second was thatthe spatial distribution of frequencies becamemore homogenous, with larger regions of the tissueexhibiting the same dominant frequency.

Another important trend was that quinidineadministration made the reinduction of fibrillationmore difficult. Fig. 10 shows two examples of AFinduced in the presence of quinidine that termi-nated within two seconds of induction. Note thatthe same two phenomena observed with termina-tion of sustained AF by quinidine (Fig. 9) can beobserved: Fig. 10A shows a reduction in thedominant frequency over large parts of the domainas the fibrillation begins to terminate, whereasFig. 10B shows increased spatial homogeneity ofthe dominant frequency.

Discussion

Use of optical mapping to assess electricalactivity

We present here the first study of electricalactivity in equine cardiac preparations usingoptical mapping. The use of voltage-sensitive dyesto detect electrical activity in cardiac tissue is wellestablished.16,45e64 The ANEP (Amino-NaphthylEthenylPyridium) group of dyes in partic-ular has been used extensively to detect actionpotentials in excitable tissue due to its favorablesignal-to-noise ratio and high fluorescence changewith cellular depolarization.69 The molecularstructure of these compounds allows them tobecome anchored in the extracellular aspect ofthe membrane via two hydrophobic carbon chainsof variable length, with the stability of thisanchoring process being proportional to hydro-carbon chain length. Upon excitation of the dye bythe appropriate wavelength (excitationfrequency), the positive charge that rests in thepyridinium nitrogen is passed via resonance tothe aniline nitrogen. This increases the dipole onthe molecule, and when the cation moves backtoward the anion sulphur moiety bound to thepyridinium nitrogen during the return to groundstate, the absorbed energy is released as light(emission frequency).76

Since the excitation frequency is higher thanthe emission frequency in these compounds, filterscan be used to quantify relative changes in fluo-rescence. The amplitude of this shift in fluo-rescence upon excitation can be affected by theelectric field that is induced by the passage of an

Figure 6 Spatial frequency distributions obtained for a range of CLs. As the CL is decreased, an area of 2:1 blockdevelops distal to the pacing site (white area obscuring the tissue at lower left), in the upper right corner. The area ofblock grows as the CL is further decreased and more complex patterns of block develop, such as the presence of both2:1 and 4:1 regions of block at a CL of 250 ms. At the shortest CLs, the frequency pattern becomes more complex withWenckebach conduction. Dominant frequencies were determined during 6 s windows for each CL.


action potential along the membrane. The result-ing signal is inversely proportional to themembrane potential and can be monitored withCCD cameras, and the output of this change can beused to track the propagation of electrical wavefronts in excitable tissue.

Figure 7 Spatiotemporal dynamics of non-sustained atrpreparations. Spatial frequency distribution, normalized volduring the arrhythmia, and the optical signal from a singleself-terminated after less than one minute. Dominant frewindows. NV maps are shown at times (A) 1.5 and 1.8 s and (Bis tissue obscured by the pacing electrode.

Restitution and alternans in equine atrialtissue

We have found that equine atrial preparationshave steeply sloped restitution curves (maximumslope of 3.2). Although restitution curve slope

ial fibrillation without acetylcholine perfusion in twotage (NV) maps showing complex and changing patternspixel are shown for each preparation. In both cases, AFquencies were determined during (A) 4.4 s and (B) 4 s) 6.4 and 8.4 s. White area within the tissue region in (A)

Figure 8 Spatiotemporal dynamics of sustained atrial fibrillation with acetylcholine perfusion in two preparations.Spatial frequency distribution, normalized voltage (NV) maps showing complex and changing patterns during thearrhythmia, and the optical signal from a single pixel are shown for each preparation. The concentration of ACh was1 mM in both cases. Dominant frequencies were determined during 5 s windows. NV maps are shown at times (A) 1.8and 3.2 s and (B) 15.4 and 15.5 s.


greater than one has been suggested as a predictorfor alternans arising from rapid pacing,74,75 equineatrial tissue, like porcine ventricular tissue63 andbullfrog myocardium,75 shows no alternans at thepacing site despite steeply sloped restitutioncurves. Both electrotonic77 and memory77,78

effects may be responsible for the absence ofalternans.

In contrast, alternans was found to arise asa mediator between regions of 1:1 conduction andregions of 2:1 block. It is important to note that thistype of alternans differs from alternans induced by

Table 1 Concentrations of acetylcholine (ACh) and quinsustained (NS) and sustained (S) arrhythmias in equinefibrillation, concentrations of quinidine required to terminquent reinduction of sustained fibrillation ([Quinidine]prev,S

Group Horse Trial [ACh] (mM) [Qui

I 1 1 0 (NS)2 1 (S)

2 1 0 (NS)2 1 (S)3 2

II 3 1 1 (NS)2 4 (S)3 4 (S)4 6 (S)

4 1 8 (S)2 10 (S)3 20 (S)

rapid pacing, where alternans originates at thepacing site (producing a region of 2:2) andconduction velocity can convert alternans fromspatially concordant to spatially discordant,79e81

thereby providing nodes (regions of 1:1). In thisscenario, large alternans amplitudes can lead toconduction block80e82 distal to the pacing site ina region beyond the first node. Here, alternans doesnot arise at the pacing site as 1:1 conduction ismaintained. Block occurs distal to the pacing site,and spatially concordant alternans developsbecause of electrotonic effects. Each action

idine (Quin) during induction and termination of non-atrial preparations. For preparations with sustainedate fibrillation ([Quinidine]term) and to prevent subse-) or any fibrillation ([Quinidine]prev) are indicated.

n]term (mM) [Quin]prev,S (mM) [Quin]prev (mM)

n/a n/a n/a12 12n/a n/a n/a6 6

6n/a n/a n/a1.26 66 6 126 6

66 6

Figure 9 AF termination by quinidine. Spatial frequency distributions and the optical signal from a single pixel areshown. Maximum dominant frequencies decreased from 5e7 Hz to 3e4 Hz before arrhythmia termination. (A)Concentrations of ACh and quinidine were 2 and 12 mM, respectively. Dominant frequencies were determined duringconsecutive 2 s windows. White area at lower left is tissue obscured by the pacing electrode. (B) Concentrations ofACh and quinidine were 4 and 1.2 mM, respectively. Dominant frequencies were determined during 4 s windows, withthe last three windows beginning 20, 32, and 50 s after the first window. (C) Concentrations of ACh and quinidine were6 mM and 6 mM, respectively. Dominant frequencies were determined during consecutive windows, with the firstwindow 3.4 s and the remaining two windows 4 s in duration.


potential that propagates from the 1:1 region butblocks in the 2:1 region undergoes a gradualdecrease in amplitude, from full amplitude to zeroamplitude within the region of block, therebyproducing a 2:2 region. Although this mechanism foralternans development has been postulated theo-retically to occur in the presence of ischemia,83e85

it has not been demonstrated previously in normaltissue or in an experimental setting.

Mechanisms of equine atrial fibrillation

Atrial fibrillation is a significant cause of morbidityand poor performance in horses, with an incidenceof between 1 and 2% of the equine populationevaluated by veterinarians annually.7,28,43 Mostcommonly diagnosed as a lone abnormalitywithout associated gross cardiac structural

pathology, the mechanism of AF in horses previ-ously has been the subject of research, both as animportant veterinary clinical entity and as a natu-rally occurring model of human AF.86,88

We have shown that equine AF arising fromrapid pacing in vitro occurs in conjunction with thedevelopment of regions of conduction block andWenckebach conduction. These patterns resultfrom intrinsic or dynamic spatial variability in APDand DI. Variability in restitution curve properties,including maximal slope values and minimum DIs,has been shown to occur in human ventricles87 andtheoretical studies have indicated that such vari-ability may give rise to reentry, independent ofrestitution curve slope values.87,88

In this study, we have found that during AFthere is no single dominant frequency for theentire preparation. Instead, multiple frequencies

Figure 10 Self-termination of induced AF in the presence of quinidine. Spatial frequency distributions and theoptical signal from a single pixel are shown. After initiation of AF, dominant frequency and spatial heterogeneity offrequencies decreased. (A) Concentrations of ACh and quinidine were 2 and 12 mM, respectively. Dominantfrequencies were determined during consecutive 2 s windows. Because fibrillation terminates abruptly, the lastspatial frequency map still includes frequencies present during fibrillation. White area at lower right in (A) is tissueobscured by the pacing electrode. (B) Concentrations of ACh and quinidine were 6 and 12 mM, respectively. Dominantfrequencies were determined during consecutive 4 s windows.


are present simultaneously due to the presence ofregions of conduction block. These frequenciesranged from 1 Hz to 8 Hz for AF induced by rapidpacing without ACh. Although the spatial distri-bution of frequencies may be interpreted assupport for a single reentrant source located in theregion with the highest dominant frequency, ouroptical mapping data show that multiple waves arepresent with non-repeating patterns. Reentrantwaves rarely lasted for a full rotation. In this case,the presence of multiple frequencies arosebecause different regions of the tissue could becaptured at different rates, and no single sourcecould be identified in the region with the highestfrequency, as shown in the movies in theSupplement.

The range of maximal frequencies observedfrom rapid pacing-induced AF was similar to thedominant frequencies reported by Gelzer et al.using surface ECGs and intraatrial electrograms in

conscious horses with naturally occurring AF(5.76e8.51 Hz).86 Although AF was not sustainedwhen initiated without ACh, the range offrequencies observed with sustained AF inducedwith ACh was similar, although slightly higher thanthat observed with non-sustained AF in theabsence of Ach, with frequencies as high as 15 Hzobserved with higher ACh concentrations. More-over, the spatial pattern of frequency domains wassimilar with and without ACh.

Quinidine as antiarrhythmic therapy forequine AF

Oral quinidine has been the cornerstone of anti-arrhythmic therapy for equine AF for decades andis successful at converting horses with lone AF inapproximately 85% of cases.7,28 Although the sideeffects of quinidine therapy in horses are nottrivial, the relatively high efficacy of quinidine, its


availability, the lack of well characterized phar-amacologic options, and cost and risk associatedwith electrical cardioversion have combined toestablish quinidine as the standard of care forequine atrial fibrillation. In spite of its broad use inclinical veterinary medicine, the definitive mech-anism of action of quinidine in the conversion of AFto NSR in horses has not been established.

Although it is known that Class I antiarrhythmicsproduce conduction slowing and refractory periodprolongation, the mechanism by which theyterminate AF has been the subject of debate forsome time. Viewed in light of the multiple waveletreentry hypothesis of Moe, AF is believed to be dueto multiple reentrant waves that travel throughthe atrial myocardium.15 The size of each of thesewavelets is determined by their wavelength, whichcan be defined as the product of the APD andconduction velocity (CV). In normal atrialmyocardium, APD and CV promote a relatively longwavelength, leading to the development of a smallnumber of relatively large, meandering reentrantwaves that can collide and eventually self-termi-nate. Reduction of wavelength in diseasedmyocardium can promote the development ofmultiple smaller waves that all fit in the domain,resulting in sustained AF. This forms the basis forthe hypothesis that an increase in wavelengthpromotes the development of larger reentrantwaves that are more likely to terminate becausethey cannot fit. However, this may not be themechanism by which quinidine terminates atrialarrhythmias. Quinidine is a Na channel blockerthat decreases CV, but it has been shown toslightly increase APD while preferentially pro-longing the effective refractory period (ERP) atshort cycle lengths.37 Therefore, the wavelengthmay remain relatively unaffected by quinidineadministration. This apparent contradiction maybe explained by the known increased ERPproduced by quinidine.37,42

Increased ERP may underlie the decrease inheterogeneity observed with quinidine perfusion,and suggests that the mechanism by which quini-dine terminates equine AF may not involve alter-ations of wavelength, but rather an increase inminimum DI, allowing only lower frequenciesthroughout the tissue, and subsequent abolition ofWenckebach conduction. This hypothesis is sup-ported by the finding that quinidine applicationresulted in a decrease in maximal dominantfrequency. In our studies, the optical mappingfrequency domains in all preparations mappedindicate that the termination of AF in the presenceof quinidine is accompanied by a decrease in themaximal dominant frequency and by an increase in

the spatial homogeneity of the frequenciespresent (see Fig. 9). In all trials, terminationoccurred when the frequency reached 3e4 Hz.Gelzer et al. reported a similar decrease in thefrequency upon quinidine administration, andtermination also occurred when the arrhythmiafrequency reached between 3 and 4 Hz inconscious horses with naturally occurring AF, sug-gesting that the source of the electrical impulses isslowed by quinidine in the intact horse.86 Onemajor limitation, however, was that the non-invasive nature of that study precluded determi-nation of whether the dominant frequency inequine AF was the result of a single pacemakerfocus, several synchronized spiral waves, or theactivity of one reentrant wave. In addition, serumquinidine concentrations were not determined,which prevented a correlation from being madebetween drug concentration and electrophysio-logic effects. Our studies suggest that equine AFcan result from multiple reentrant waves withinthe atria of affected horses, and that the domi-nant frequency of AF decreases to 3e4 Hzimmediately prior to AF termination at concen-trations of quinidine that approximate thoseachieved in horses given clinically relevant dosesof quinidine.

It is interesting to note that of the eight trials inwhich sustained AF was induced, termination of AFwas achieved with concentrations of quinidine thatare markedly lower than those that correspond tothose that are considered therapeutic in the clin-ical setting in six of them (6 mM in five trials, 1 mMin one trial). Given this finding, it is tempting tospeculate that termination of equine AF may beachieved in clinical cases with lower doses ofquinidine or to question why conversion of AF hasnot been achieved with the administration oflower quinidine doses. A number of parameters,including the effects of the intact autonomicnervous system, stretch-activated receptors in theintact heart, and components of whole blood onthe electrophysiology of atrial tissue and bindingkinetics of quinidine may explain this discrepancybetween our findings and the management ofequine AF in clinical patients.

Limitations

One limitation of our study is that rate adaptationand restitution data were obtained only beforeadministration of ACh and quinidine. For thisreason, it was not possible to obtain quantitativeinformation about the electrophysiological effectsof either agent. Thus, a detailed mechanistic


explanation for the defibrillating effects of quini-dine is beyond the scope of this work. Anotherlimitation is that our work was by necessity per-formed in vitro and not in vivo. However, thesimilarities between the frequencies obtainedhere and those obtained in vivo by Gelzer et al.86

suggest that the systems behave similarly. Anotherlimitation of this study is that sharp electrodeexperiments designed to determine the effects ofblebbistatin on action potential duration, ampli-tude, and morphology were, by necessity, per-formed on ventricular tissue. Given the scarcity ofequine cardiac tissue, atria from all hearts har-vested were reserved for optical mapping experi-ments. Previous studies, however, have shown thatblebbistatin has minimal effects on these param-eters in intact rabbit atrial and ventricular prep-arations and in cardiac myocytes isolated from ratventricle.71 Given these findings, we are confidentthat blebbistatin is likely to have minimal effect onthe electrophysiologic properties of equine atrialtissue. Finally, the number of preparations map-ped was small, owing to the difficulty in procuringequine cardiac tissue suitable for mapping. Forthis reason, no detailed study of the effects oftime on the viability of the preparation was per-formed. Nevertheless, optical signal quality andmorphology did not appear to vary substantiallyover time.

Conclusions

Our study demonstrates, for the first time, thatrapid pacing-induced AF in intact equine atrialpreparations develops in conjunction with ofregions of conduction block and Wenckebachconduction resulting from spatial variability in theminimal DI for propagation. In addition, we foundthat AF is associated with multiple frequenciesthat are induced by regions of conduction block inintact equine atrial preparations, and that thesefrequencies correspond with those that have beendocumented in conscious horses with naturallyoccurring AF. We have also demonstrated for thefirst time experimentally the occurrence of a novelform of alternans that occurs as a result of elec-trotonic currents between regions with 1:1 and 2:1conduction.

Our results also show that the termination of AFby quinidine in intact equine atrial preparations isassociated with a decrease in the maximal domi-nant frequency and by an increase in spatialhomogeneity of frequencies present, and thatquinidine terminates AF in these preparations atconcentrations that correspond to those achievedin horses given clinically relevant doses of

quinidine. In all cases in which quinidine termi-nated AF, termination was preceded by a slowingof dominant frequency. These findings are consis-tent with the hypothesis that the quinidine-induced increase in ERP results in an increase inminimum DI with subsequent abolition of Wenck-ebach conduction.

Acknowledgments

This work was supported in part by the NationalInstitutes of Health under Grant no. HL075515-S03-S04 (F.H.F.) We thank Stefan Luther and EberhardBodenschatz for helpful suggestions and acknowl-edge their support from the Max Planck Society.We also thank Robert F. Gilmour, Jr. for helpfulsuggestions. We thank Patrick B. Burke and MichaelW. Enyeart for expert technical assistance.

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Available online at www.sciencedirect.com
Termination of equine atrial fibrillation by quinidine: An optical mapping studyIntroductionAnimals, materials and methodsTissue harvesting and perfusionOptical mappingMicroelectrode recordingsInitiation of fibrillationNon-inducible, non-sustained, and sustained AFQuinidine use and AF termination or preventionData analysisResultsConduction in normal atriaInduction of AFSpatiotemporal dynamics of AFEffects of quinidine on AFDiscussionUse of optical mapping to assess electrical activityRestitution and alternans in equine atrial tissueMechanisms of equine atrial fibrillationQuinidine as antiarrhythmic therapy for equine AFLimitationsConclusionsAcknowledgmentsReferences

termination of equine atrial fibrillation by quinidine: an optical...

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