Optical mapping of repolarization and refractoriness from intact hearts
`IR Efimov, DT Huang, JM Rendt and G Salama
` 1994;90;1469-1480
`Circulation
`Circulation is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX
`72514
`Copyright © 1994 American Heart Association. All rights reserved. Print ISSN: 0009-7322. Online
`ISSN: 1524-4539
`
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`

`1469
`
`Optical Mapping of Repolarization and
`Refractoriness From Intact Hearts
`
`Igor R. Efimov, PhD; David T. Huang, MD; James M. Rendt, PhD;. Guy Salama, PhD
`
`Heterogeneities of repolarization (R) across
`Background
`the myocardium have been invoked to explain most reentrant
`arrhythmias. The measurement of refractory periods (RPs)
`has been widely used to assess R, but conventional electrode
`and extrastimulus mapping techniques have not provided
`reliable maps of RPs.
`Guinea pig hearts were stained with a
`Methods and Results
`voltage-sensitive dye to measure fluorescence (F) action po-
`tentials (APs) from 124 sites with a photodiode array. AP
`duration (APD) was defined as the time between depolariza-
`tion (dF/dt)max and R time points (ie, the time when AP returns
`to baseline or some percent thereof). However, R time points
`are difficult to determine because AP downstrokes are often
`encumbered by drifting baselines and motion artifacts, which
`make this definition ambiguous. In optical and microelectrode
`recordings, the second derivative of AP downstrokes is shown
`to contain an easily detected, unique local maximum. The
`
`S patial heterogeneities in repolarization have been
`proposed to explain the initiation of arrhythmias
`in cardiac tissue.' Theoretically, a unidirectional
`block can arise in regions containing abrupt variations
`of repolarization.2 Under certain conditions, these vari-
`ations can lead to wavefront fractionation and reentry.
`However, reliable experimental tests of this hypothesis
`have not been achieved.
`The major obstacle to testing this hypothesis is the
`lack of a practical technique to measure distributions of
`repolarization with sufficient spatial and temporal res-
`olution. The most common approach to map repolar-
`ization pathways is indirect, through measurements of
`refractory periods.3 Maps of refractoriness are conven-
`tionally measured by combining multiple extracellular
`electrode recordings with premature extrastimulus tech-
`niques.4 The heart is paced at a basic rate (Si), and a
`premature stimulus (S2) is applied with a variable
`coupling interval (S2-Sl). A disadvantage of this ap-
`proach is that the extrastimulus must be tested with
`variable delays, S2-S1, to determine the refractory pe-
`riod at each recording site and the process must be
`repeated at each electrode site to measure distributions
`of refractory periods. Maps of refractory periods de-
`rived by this approach are time-consuming and assume
`that the refractory period measured at one site did not
`vary while the measurements were repeated at other
`
`Received January 31, 1994; revision accepted May 23, 1994.
`From the Department of Cell Biology and Physiology, Univer-
`sity of Pittsburgh (Pa) School of Medicine.
`Correspondence to Guy Salama, Department of Cell Biology
`and Physiology, University of Pittsburgh School of Medicine,
`Pittsburgh, PA 15261.
`© 1994 American Heart Association, Inc.
`
`correlation between the position of this maximum (d'F/dt')m,
`and R has been tested during altered AP characteristics
`induced by changes in cycle length, ischemia, and hypoxia.
`Under these various modifications of the AP, the time points
`of (d F/dt )mM fell at 97.0±2.1% of recovery to baseline.
`Extrastimulus techniques applied to (1) isolated myocytes, (2)
`intact hearts, and (3) mathematical simulations indicated that
`(d2V/dt2)mua coincided with the effective RPs of APs. The
`coincidence of RPs and (d2V/dt )max was valid within 5 milli-
`seconds, for resting potentials of -75 to -90 mV and extra-
`stimuli three times threshold voltage.
`Thus, optical APs and (d2F/dt2)max can be used
`Conclusions
`to map activation, R, and RPs with AP recordings from a
`single heartbeat. (Circulation. 1994;90:1469-1480.)
`Key Words * mapping *
`electrophysiology
`potentials
`
`action
`
`*
`
`sites. As a result, such mapping measurements cannot
`be practically repeated under different physiological
`conditions without compromising both spatial and tem-
`poral resolution. Another difficulty is that the definition
`of refractory period depends on the amplitude, dura-
`tion, and polarity of the stimulating current. Michelson
`et al5 studied refractory periods in normal and ischemic
`myocardium and pointed out the difficulties inherent in
`choosing a standardized threshold current for the defi-
`nition of refractory periods.
`Intracellular microelectrodes provide accurate mea-
`surements of repolarization but are not practical for
`simultaneous measurements from multiple sites be-
`cause of technical difficulty of maintaining multiple
`stable recordings.
`Suction electrodes have been used to measure action
`potential (AP) durations (APDs) and refractory periods
`by monitoring conduction time delays between two
`sites.6 Premature stimuli delivered near the relative
`refractory period increased conduction time delays be-
`cause the extra AP propagated at slower conduction
`velocity. Refractory periods could thus be accurately
`determined and were shown to coincide with AP repo-
`larizations. However, suction electrodes are not practi-
`cal for simultaneous recordings of refractory periods at
`multiple sites and may produce unreliable recordings
`caused by tissue damage.6
`Signal processing techniques have been proposed to
`estimate repolarization by use of unipolar electrograms
`by correlating the most rapid increase in voltage (dV/
`dt)max near the peak of the T wave to the repolarization
`of the local AP.7 Millar et a18 defined an "activation-
`recovery interval" as the time difference between the
`most rapid decrease in voltage in the QRS complex
`
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`

`1470
`
`Circulation
`
`Vol 90, No 3
`
`September 1994
`
`(dV/dt)min (activation time) and (dV/dt)ma near the peak
`of the T wave (repolarization time). Experimental8 and
`theoretical9 studies suggested that refractory periods
`were correlated with activation-recovery intervals.
`However, this correlation remained empirical, was
`strongly dependent on the shape, polarity, and kinetics
`of the T wave, and failed for multiphasic T waves.
`Ultimately, it proved to be of limited use because the
`amplitudes and kinetics of T waves were too variable to
`determine refractory periods.
`We have previously used voltage-sensitive dyes and
`imaging technique to simultaneously measure 124 APs
`from the epicardium of perfused hearts.10 In preliminary
`reports, we described signal processing techniques to
`uniquely identify the repolarization time points of fluo-
`rescence (F) APs through (d2F/dt2)max of their down-
`stroke."1 The algorithm was effective to measure repolar-
`ization time points even in optical AP recordings
`containing substantial distortions caused by movement
`artifacts. The method made it possible to measure het-
`erogeneities of repolarization and APDs in normal and
`hypoxic hearts during abrupt cycle length shortening.'2
`The present report investigates the physiological sig-
`nificance of this inflection point detected during AP
`downstrokes by theoretical and experimental ap-
`proaches. The data show that (d F/dtx)mx provides a
`reliable measurement of AP repolarization and is coin-
`cident with AP refractory period under physiological
`resting potentials. Preliminary reports of these findings
`have appeared in abstract form."1113
`Methods
`
`Preparations
`Guinea pigs of either sex weighing between 350 and 450 g
`were anesthetized with an intraperitoneal injection of sodium
`pentobarbital (Nembutal, 30 mg/kg). The hearts were rapidly
`excised, rapidly cannulated at the aorta (within 60 seconds),
`and retrogradely perfused in a modified Langendorif appara-
`tus. The Krebs-Ringer's perfusate consisted of (in mmol/L)
`NaCI 130, KCl 4.75, CaCl2 1.0, MgSO4 1.2, NaHCO3 12.5, and
`glucose 5.0. The solution was continuously gassed with 95%
`02/5% C02, pH adjusted to 7.4+0.1 with NaHCO3. Temper-
`ature was maintained at 35 + PC with a thermistor probe and
`a heat-exchange coil. A perfusion pressure of 80 to 90 cm H20
`was maintained from the beginning of the experiment with
`variable flow rate (oo2 mL. min-1 g-1 heart wt). More de-
`tailed methods describing the perfusion setup, the staining
`procedure, the heart chamber, optics, and computer hardware
`and software were previously reported'10"24 and are briefly
`restated below.
`Hearts were paced at a fixed cycle length, typically 300 or
`350 milliseconds, with a bipolar Ag+ /AgCl electrode placed on
`the epicardium. The output of the pacer was adjusted to 1.5
`times the threshold voltage. A custom-designed perfusion
`chamber was used for studying intact guinea pig hearts. The
`preparation was held in place by a glass window at the front of
`the chamber and side and rear pads to minimize gross move-
`ment of the heart during contractions. Bipolar surface electro-
`gram recordings were measured by use of Teflon-coated silver
`wires (250-,um diameter), with Ag+/AgCl at the exposed tip of
`the wires and a 0.5-mm gap between the two wires. These
`sensing electrodes were sutured or glued to the epicardium to
`maintain stable recordings. The left epicardium was in contact
`with the glass of the front window to reduce the curvature of
`the ventricle and gross movement during contractions of the
`heart. The heart in the chamber was immersed in perfusate,
`which eliminated condensation on the glass window.
`
`Staining Procedures
`A styryl dye, RH-421 (S-1108, lot 2711-2, Molecular Probe),
`was used as the voltage-dependent fluorescent probe. When
`bound to the sarcolemma, RH-421 fluorescence was measured
`at wavelengths above the 645-nm cutoff filter when excited
`with a 520±20-nm interference filter. The dye exhibited a
`large fractional decrease in fluorescence of 6% to 9% per 100
`mV depolarization such that the voltage-dependent responses
`appeared as upside-down APs.14 The signals were automati-
`cally inverted under software to appear as rightside-up APs.
`This dye was chosen because it did not provoke detectable
`pharmacological effects, remained optically stable in solution,
`and exhibited optical APs with high signal-to-noise ratios (up
`to 250:1) for 2 to 4 hours.
`Hearts were stained by gradual injection of 50 to 100 IL
`from a 3 mmol/L stock solution of dye into a 5-mL bubble trap
`situated directly above the aortic cannula. The final dye
`concentration was approximately 2.5 ,mol/L, and 15 to 20
`minutes was allowed for the staining to be completed. The
`procedure resulted in homogeneous staining throughout the
`heart because the dye was efficiently delivered via coronary
`vessels. In experiments lasting more than 4 hours, photo-
`bleaching and/or dye washout reduced the signal amplitudes;
`in such cases, hearts were occasionally restained to restore the
`original signal-to-noise ratio.
`The experiments described in this study were based on a
`total of 20 guinea pig hearts. Six were used to map repolar-
`ization with voltage-sensitive dyes, 2 to prepare isolated myo-
`cytes for single-cell measurements of refractory periods, 6 for
`measurements during hypoxia, and 6 during ischemia. This
`investigation conformed with the Guide for the Care and Use
`of Laboratory Animals published by the US National Insti-
`tutes of Health (NIH publication No 85-23, revised in 1985).
`Instrument Setup
`Details of the optical and recording apparatus have been
`described elsewhere.10 The perfusion chamber containing the
`Langendorff-perfused heart was mounted on a micromanipu-
`lator and positioned along the optical axis of a photodiode
`array scanning apparatus. Light from two 45-W tungsten-
`halogen lamps was collimated, passed through a 520±20-nm
`interference filter, reflected off a 450 dichroic mirror, and
`focused on the left epicardium of the heart with a 35-mm
`camera lens (50 mm, f 1:1.4, Nikon). Epifluorescent light from
`the stained heart was collected, projected through a 645-nm
`cutoff filter, and focused to form an image of the heart on a
`12x 12-element photodiode array. The photodiode array con-
`sisted of 144 square diode elements, with each diode having
`dimensions of 1.4 x 1.4 mm separated by 0.1 mm of dead space.
`Of the 144 available diodes, 124 diodes were monitored; five
`elements from each corner were disregarded. The distances
`between the preparation, the lens, and the array could be
`varied and thus, the image of the heart falling on the array
`could be magnified by 1.5 to 6 times. In the present study, APs
`were recorded from a 12x 12-mm2 region of the epicardium.
`The depth of field of the optics restricted the detection of dye
`fluorescence to a depth of 144 ,um from the surface of the
`epicardium.14
`Signal Acquisition
`A scan of data acquisition consisted of 128 simultaneously
`recorded traces: 124 optical plus 4 instrumentation channels.
`The multiplexed instrumentation channels monitored the
`stimulus pulses, two surface electrogram signals located on the
`atrium and ventricle, and in some experiments, left ventricular
`pressure by use of a latex balloon inserted in the left ventric-
`ular cavity and a Statham pressure transducer (model P-10). A
`scan consisted of a continuous recording of these 128 channels
`for 1.2 to 34 seconds. The photocurrents from 124 diodes were
`fed to a current-to-voltage converter, amplified, digitized (1.3
`
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`

`Efimov et al
`
`Optical Mapping
`
`1471
`
`A B C D E
`
`X, <~
`
`600 ms
`400
`200
`0
`Tracings showing signal processing of optical action
`FIG 1.
`potentials (APs) to resolve depolarization and repolarization time
`points. A, An optical AP was recorded (at 1.3 kHz) from a
`lx1-mm area of left epicardium from a perfused guinea pig
`heart. The voltage-dependent optical signal corresponded to a
`fractional decrease of fluorescence of 8%. B, The fluorescence
`(F) AP shown in (A) was filtered with a boxcar of 8 milliseconds'
`duration. C, Its first time derivative was obtained after filtration
`from trace B. D, Trace C was filtered with a 23-millisecond
`boxcar, and the time of depolarization was taken at the time point
`of (dF/dt)ma, of the AP upstroke (arrows on trace D). E, The time
`derivative of trace D is shown; note the unique peak that
`coincides with the end of the repolarization phase of the AP. The
`repolarization time point was taken at (d2F/dt2)ma
`(arrows on
`trace E).
`
`photodiode array (Fig 1A). The diode recorded the sum
`of voltage-dependent optical response of a group of
`cells (viewed by the diode) contained in a 1 x 1-mm2 area
`of left epicardium and a depth of 144 ,um. The recording
`contained high-frequency noise (Fig 1A), which was
`filtered with a boxcar of 8 milliseconds' duration to
`obtain Fig 1B. The depolarization time point of an AP
`recorded with an intracellular electrode is convention-
`ally defined at the maximum first derivative of the AP
`upstroke, (dV/dt)ma. For optical APs, the time point of
`depolarization was also defined as the maximum first
`derivative of the fluorescence AP upstroke (dF/dt)ma,
`because it corresponded to the time when most of the
`cells viewed by a diode were depolarizing. The depolar-
`ization time point was detected by taking the first
`derivative of Fig 1B to obtain Fig 1C (dF/dtma,X
`at
`arrows). The first derivative trace (Fig 1C) was filtered
`with a 23-millisecond boxcar to generate Fig 1D before
`taking the second derivative to generate Fig 1E. The
`repolarization time point is typically defined as the
`recovery of the AP back to baseline, which provides an
`accurate method to measure AP duration whenever the
`downstroke has a large negative slope and returns
`abruptly to its resting potential. When the downstroke
`returns gradually to baseline, repolarization is alterna-
`tively defined as a percentage of recovery to baseline.
`These definitions are often not suitable for optical
`recordings because movement artifacts distort the repo-
`larization phase (see Fig 2). Consequently, the repolar-
`ization time point was taken as the time of maximum
`
`kHz per channel, 8 bits per sample), and stored in a memory
`buffer of a Digital Equipment Corp PDP 11-73 computer.
`
`Experiments on Isolated Myocytes
`Guinea pig myocytes were isolated with collagenase and
`pronase as previously described."5 Current clamp measure-
`ments were performed with an Axoclamp 1D. Data were
`collected at a 5-kHz sampling rate. Patch pipettes were filled
`with a solution of the following composition (in mmol/L): KCl
`140, HEPES 10, MgATP 5, EGTA 10 at pH 7.25. Cells were
`superfused with an external solution containing (in mmol/L):
`NaCl 130, KCl 5, MgCl2 1, CaCl2 2, HEPES 10, glucose 5, pH
`7.35 at 37°C.
`Computer Simulation
`Computer simulations were performed with mathematical
`models of electrical activity in ventricular myocardial fiber.'6
`The Beeler-Reuter model is a system of first-order nonlinear
`differential equations of the Hodgkin-Huxley type. To solve
`the system, an implicit scheme with time step At=0.01 milli-
`second was used. Activation and inactivation parameters in a
`model of Hodgkin-Huxley type obey the usual first-order
`equation
`
`dy/dt=a,(1 -y)-,8y(y)
`where y is a parameter (m, h, etc), t is time (milliseconds), and
`a; and /ly are rate constants (milliseconds). If we call y the
`parameter value at the nth time step (At), the finite-difference
`equation for the time derivative yields
`(yn+l-yn)/,At =acy(l_yn+l) _ 8 (yn+l)
`
`which can be rewritten to
`y+ 1 = (yn + a,At)/ [1 + (a, +1y)At]
`The equation for membrane potential is
`dE/dt=Xi/C=Ig(E-Er)/C
`where E is the membrane potential (millivolts), i
`is ionic
`currents (I£A/cm2), and C is the membrane capacitance (,uF/
`cm2). The finite-difference equation for the time derivative in
`this case is
`
`(En+1-En)/,At=(l/Q1g(En+1 Er)
`which takes the form
`En+l=[En+ (At/C)z;gEr]/[1 +(lvt/C)g]1
`Stimulations of a model cell with normal Beeler-Reuter
`parameters were made by applying an external current pulse
`with amplitude ie,t==12 /LA/cm2 and duration At=5 millisec-
`onds. Computations were performed on IBM/PC-386 clone
`and the CRAY C90 of the Pittsburgh Supercomputing Center.
`Programs were written with Borland C+ + compiler.
`Results
`Second Derivative of the Cardiac AP
`A signal-processing technique was used to detect the
`activation (depolarization) and recovery (repolariza-
`tion) time points of fluorescence (F) APs. The activa-
`tion time point was defined as the maximum first
`derivative (dF/dt)max of the AP upstroke. The recovery
`time point was defined as the maximum second deriva-
`tive of the AP downstroke, (d2F/dt')mx, because the
`second derivative of the cardiac AP was found to exhibit
`a local maximum between the most negative slope of the
`AP downstroke and the return to baseline."1-'3 Fig 1
`illustrates the method. Fluorescence APs are first re-
`corded from the epicardium by one of the diodes on the
`
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`

`1472
`
`Circulation
`
`Vol 90, No 3
`
`September 1994
`
`56
`
`16
`
`16
`
`54
`
`54
`
`122
`
`122
`
`F
`
`F"
`
`:5: :
`
`| AF/F=%
`
`16
`
`.
`
`---j _:
`:.
`
`J
`
`-
`
`i
`
`.. v
`P.
`'I
`
`=
`
`2mg
`
`_
`
`100 ms
`
`Tracings show that (d2FIdt2),,
`of action potential
`FIG 2.
`(AP) downstrokes are not distorted by motion artifacts. Four
`of 124 simultaneously recorded APs are shown, and each is
`followed by its second derivative determined as described
`in Fig 1. The AP signal from diode 56 was chosen as an
`example of an optical AP with negligible motion artifact. APs
`from diodes 16, 54, and 122 were selected for their sub-
`stantial levels of motion artifacts. Their second derivatives,
`d2F/dt2, show prominent peaks at the repolarization time
`points of the APs. The computer algorithm automatically
`determined (d2F/dt2)ma and placed a "tick" mark at the
`calculated repolarization time point. The algorithm identi-
`fied unique repolarization time points for 95±2% of the APs
`recorded by the array. Developed force was measured with
`a balloon inserted into the left ventricular cavity. Peak
`developed force occurred during or just before the rapid
`phase of repolarization, and developed force was substan-
`tial during the repolarization phases, which accounted for
`the movement artifact. However, the second derivative of
`the force (and most likely movement artifacts) was relatively
`flat during repolarization.
`
`A
`
`150
`
`E Sf
`a 125
`
`B
`
`140
`
`120
`
`100l
`
`O 100
`
`0
`
`b
`
`95
`
`90
`
`1 00
`
`80
`,,_.____X___100
`
`~
`
`400
`300
`200
`Cycle Length (ms)
`
`290
`
`50D
`
`1O
`
`250
`200
`Cycle Length (ms)
`
`FIG 3.
`Graphs showing that (d2F/dt2),m, coincides with action
`potential (AP) repolarization under vanous physiological and
`pathological conditions. The correlation between (d2F/dt2),ms
`and repolarization was tested in simulations of the AP by use of
`the Beeler-Reuter model and with optical recordings under
`various conditions. AP durations (APD) were defined as the time
`difference dF/dt,,-(d2F/dt2)m,,
`of AP upstrokes and down-
`strokes. APs recorded by 12 of the 124 diodes were chosen for
`their negligible levels of movement artifact and were analyzed to
`correlate their percent repolarization (or recovery back to base-
`line) to the time point of (d2F/dt2),n,. Percent of repolarization
`was measured at the time point of (d2F/dt2),,,. For mathematical
`simulations, APDs were defined in the same way; that is, as the
`difference between (dV/dt)max and (d2V/dt2),,ax. A, APs were
`generated from Beeler-Reuter simulations and their APDs mea-
`sured as a function of cycle length (CL) (top trace). The percent
`repolarization at the time point of (d2V/dt2),,, was determined for
`APs at each CL and plotted in the lower graph. The data show
`that as APDs increased from 115.46 to 141.71 milliseconds with
`CLs increasing from 120 to 500 milliseconds, the time point of
`(d2V/Idt)max coincided with 99.46±0.23% (mean+SD) repolariza-
`tion back to baseline. B, The equivalent analysis was repeated
`with optical APs from the left epicardium of guinea pig hearts
`~paced at different cycle lengths. A bipolar stimulating electrode
`was placed on the right atrium to pace the heart at faster rates
`than the intrinsic pacemaker rate. APDs from 12 central diodes
`were analyzed to test the correlation between (d2F/dt2),,. and
`the percent repolarization. Top plot, Each data point represents
`jthe mean APD±SD for 12 APs recorded at a given cycle length.
`APDs increased from 94.09 to 130.39 milliseconds with increas-
`ing cycle lengths from 170 to 250 milliseconds. Bottom plot,
`Each data point represents the averaged percent repolar-
`ization+SD at the times that correspond to (d2FIdt2),
`of the 12
`APs; (d2F/dt2)max
`occurred at 96.14±0.82% repolarization
`(mean±SD). C and D, Same analysis as in B except that APDs and percent repolarization were plotted as a function of time of ischemia
`(C) and hypoxia (D). Preparations were paced at CL=350 milliseconds. lschemia experiments were carried out at 350C, APDs decreased
`from 156.52 to 81.42 milliseconds in 5 minutes of ischemia, and (d2F/dt2),
`fell at 96.96±1.44% of repolarization. Hypoxia experiments
`were carried out at 23°C, APDs decreased from 215 to 192 milliseconds in 30 minutes, and (d2F/dt^), fell at a similar percent
`repolarization of 95.67±0.43%. The higher percent repolarization in theoretical compared with the experimental results was attributed
`to the origins of optical signals, which represent the summed response of hundreds of cells in a patch of myocardium viewed by a diode.
`Experiments shown in B through D were reproduced in six hearts.
`
`C
`
`200
`
`\
`
`100' °°~
`
`D
`
`220-
`
`200
`
`\
`
`P.
`
`50
`100
`
`90
`
`1180 _
`I100
`
`95
`
`90
`
`5
`3
`4
`2
`0
`1
`Time of Ischemia (min)
`
`30
`10
`20
`0
`Time of Hypoxia (min)
`
`l
`
`X
`
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`

`Efimov et al
`
`Optical Mapping
`
`1473
`
`Plots showing Beeler-Reuter simulation of ac-
`FIG 4.
`tion potential (AP) and (d2V/dt2)ma,. An AP was simu-
`lated for model guinea pig ventricular cells with a
`-83-mV resting potential, paced at 1 Hz. A, Superpo-
`sition of the AP and its second derivative. B, Time
`course of the inward currents carried by voltage-gated
`Na+ (INa+) and Ca2, (Is) channels. C, Time course of
`inward (IK1) and outward (l1,) rectifying K' channels. D,
`Activation (m) and inactivation (h) gates of voltage-
`gated Na+ channels.
`
`0.05
`mV/ms2
`
`0.00
`
`-0.05
`
`2
`
`0
`s A/cm
`-1
`
`-2
`
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`'Na13
`,|INa
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`SSS@~~~~~~~--------------------------
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`---
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`d2F/dt2 of the AP downstroke, a time point that fell
`between minimum dF/dt of the downstroke and the
`return to resting potential as in Fig 1E (arrows).
`The second derivative of the optical AP was a partic-
`ularly useful (although empirical) method to define
`repolarization because it identified a unique time point,
`even for AP downstrokes that are distorted by motion
`artifacts. Of 124 simultaneous AP recordings, approxi-
`mately one half of the signals had negligible movement
`artifact, and repolarization could be determined as a
`percentage of recovery to baseline. For the remaining
`half of the APs, the return to baseline was ambiguous to
`various degrees because of movement artifacts. Fig 2
`shows an example of an AP recording with negligible
`movement artifact (trace 1) followed by APs (traces 2
`through 4) selected for the different kinetics of their
`movement artifacts. Even though the time course and
`direction of movement artifacts was unpredictable, we
`found that the second derivatives (shown below each
`trace) detected unique maxima that seemed indepen-
`dent of the movement artifact and thus appeared to
`identify AP repolarization time points. A possible ex-
`planation for the stable (d2F/dt2)ma,, in the presence of
`movement artifacts may be the relatively slow rate of
`change of force (trace F) during the repolarization
`phase of the heart. For instance, the second derivative
`of the left ventricular pressure wave was constant during
`
`the repolarization phase (trace F") and was not likely to
`temporally displace the voltage-dependent inflection
`point.
`Correlation Between (d2F/dt2)max and
`Repolarization Time Points
`The second derivative of AP downstrokes served as a
`useful algorithm to identify repolarization even in the
`presence of movement artifacts. However, to generate
`maps describing propagation of repolarization across
`the epicardium, it is important to demonstrate that
`(d2F/dt2)max occurred reproducibly at the same percent
`recovery to baseline and that changes in AP character-
`istics (eg, shape and/or duration) did not shift (d2F/
`dt2)max relative to the percent recovery. The correlation
`between (d2F/dt2)m and percent recovery was therefore
`tested under various experimental conditions and pac-
`ing frequencies. APDs from Beeler-Reuter simulations
`of the AP were calculated from the depolarization time
`point defined at (dV/dt)m, of AP upstrokes minus the
`repolarization time point defined at (d2V/dt2)m,, of AP
`downstrokes. For optical APs, APDs were calculated by
`the same algorithm except that (dV/dt)ma, was substi-
`tuted by (dF/dt)ma, and (d2V/dt2)m by (d2F/dt2)max. Per-
`cent of AP recovery back to baseline was measured at
`the point of (d2V/dt2)ma, [or (d2F/dt2)ma.. and plotted
`along with changes in APDs.
`
`Downloaded from
`
`circ.ahajournals.org
`
` at Washington University on November 3, 2009
`
`AliveCor Ex. 2024 - Page 6
`
`

`

`1474
`
`Circulation
`
`Vol 90, No 3
`
`September 1994
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`Plots showing refractory period predicted by the
`FIG 5.
`Beeler-Reuter model using premature stimuli. A, Second
`derivative of simulated action potential (AP) shows a
`prominent local maximum. B, Simulations of APs elicited
`by a pacing stimulus (S,) followed by premature stimuli
`(S2). Stimulations were modeled as square pulses with
`an amplitude of 12 pA/cm2 (ie, 2 times threshold voltage)
`and a duration of 5 milliseconds. The basic cycle length
`(S,) was 1000 milliseconds, and premature beats were
`applied once every 10 basic stimuli, at variable S2-S1
`intervals. C, Plot of additional depolarization time in-
`duced by a premature pulse S2 as a function of S2-S1
`interval. The increase in depolarization time is negligible
`when S2 fires during the absolute refractory period, and
`the total depolarization time begins to increase when
`S2-S1 is longer than 279 milliseconds (arrows), that is,
`when S2 fires past the time point of (d2V/dt)ma,x, ie, the
`end of the refractory period. Simulations were done on
`an IBM PC/386 clone computer. Integration of the equa-
`tions was performed with an implicit scheme with a time
`step At=0.01 milliseconds.
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`The consistent correlation of (d2F/dt2)ma,,, with repo-
`larization is illustrated in Fig 3. In Fig 3A, APDs were
`calculated with the Beeler-Reuter model based on
`(d2V/dt2)m,,a
`and plotted as a function of basic cycle
`length (CL, in milliseconds). The simulation predicted
`the expected increase of APDs at longer CL and showed
`that (d2V/dt2)m fell in the range of 99% to 100%
`recovery to baseline (A, bottom plot). The longer CLs
`(120 to 500 milliseconds) caused significant increases in
`APDs from 115.46 to 141.71 milliseconds; yet, the
`simulation experiment predicted that (d2V/dt2)m fell at
`99 ±0.52% of recovery for this range of APDs. Note that
`there was a slight decrease in percent repolarization at
`(d2V/dt2)max, which reflected changes in AP shape at
`longer CLs. Microelectrode recordings of APs from
`isolated myocytes indicated that (d2V/dt2)ma,
`fell at
`99.46±0.23% (mean±SD) of recovery (n=12 APs),
`which was in excellent agreement with findings from the
`Beeler-Reuter model. In Fig 3B, optical APs were
`measured from intact guinea pig hearts stained with a
`voltage-sensitive dye and imaged on a photodiode array.
`APDs increased from 94.09 to 130.39 milliseconds with
`increasing CL from 170 to 250 milliseconds (top), and
`(d2F/dt2)m.a fell at 96.14±0.82% of recovery to baseline.
`In Fig 3B, each data point represents the mean±SD of
`APD from a total of 12 optical APs from the center of
`the array. APDs were also modulated by metabolic
`perturbations. In Fig 3C and 3D, 24 APDs from central
`diodes were averaged and plotted as a function of time
`
`of ischemia and hypoxia, respectively. Ischemia in-
`duced by stopping coronary flow to the heart induced
`dramatic decreases in APD, from 156.52 to 81.42
`milliseconds. These changes in APDs did not alter the
`correlation between (d2F/dt2)m,,. and percent of recov-
`ery. That is, (d2F/dt2)ma fell at 96.95±1.44% during
`ischemia. Hypoxia (30 minutes) induced by switching
`from oxygen- to nitrogen-saturated Ringer's solution
`decreased APDs from 215 to 192 milliseconds. Again,
`(d2FIdt2)ma
`fell at 95.67±0.43% recovery to baseline.
`Note that this hypoxia experiment was carried out at
`room temperature (23°C), resulting in longer APDs
`(Fig 3D) compared with the other studies run at 35°C
`(Fig 3A through 3C). For Fig 3C and 3D, each point
`represented the mean APD±SEM of 124 optical APs
`as a function of time of ischemia or hypoxia. Fig 3B
`through 3D were generated from optical AP record-
`ings with excellent signal-to-noise ratio and negligible
`movement artifact such that the percent recovery to
`baseline could be readily determined. In Fig 4A, an
`AP was simulated for a guinea pig ventricular cell
`based on the Beeler-Reuter model and the ionic
`currents of voltage-gated Na+ (iNa+) and Ca21 (i,)
`channels (Fig 4B) and inward (iKl) and outward (i.1)
`K' rectifying channels (Fig 4C). The AP downstroke
`exhibits a maximum rate of voltage change, (d2V/
`dt2)ma, on its return to baseline (Fig 4A). The occur-
`rence of (d2V/dt2)max is a direct consequence of the
`kinetics of the AP do