© 1995 American Heart Association, Inc.
Articles |
Optical Mapping Reveals That Repolarization Spreads Anisotropically and Is Guided by Fiber Orientation in Guinea Pig Hearts
From the University of Pittsburgh (Pa) School of Medicine, Department of Cell Biology and Physiology.
Correspondence to Dr Anthony J. Kanai, Department of Pharmacology, Box 3845, Duke University Medical Center, Durham, NC 27710.
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Abstract |
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Abstract Guinea pig hearts were stained with a voltage-sensitive dye and imaged on a photodiode array to record fluorescent action potentials (APs) from 124 sites. Activation and repolarization patterns were recorded from the epicardium during stimulation at different loci and correlated with the underlying fiber architecture. Endocardial APs were recorded by inserting a light guide into the ventricular cavity or by dissecting out the ventricular free wall to expose the endocardium. In hearts paced on the right atrium to simulate sinus rhythm, activation emerged synchronously over a large area of the ventricular epicardium and spread laterally in 5 to 7 ms. The apparent longitudinal and transverse velocities were 2.66±0.11 and 1.65±0.09 m/s (n=12). In contrast, repolarization began near the apex on the endocardium and spread transmurally in 6±1.3 ms (n=12) and then anisotropically along the epicardium in 25 to 30 ms with apparent maximum (0.53±0.11 m/s) and minimum (0.31±0.10 m/s) repolarization velocities that aligned with the longitudinal and transverse axes of epicardial fibers. When paced on the epicardium, activation of intact hearts (n=12) and perfused sheets (n=8) was anisotropic, with longitudinal (0.85±0.05 m/s) and transverse (0.44±0.04 m/s) conduction velocities that aligned with the epicardial fiber orientation. When activation was initiated at different sites on the epicardium, repolarization always began near the apex and exhibited patterns similar to those obtained under right atrial pacing, but with slower longitudinal (0.41±0.09 m/s) and transverse (0.23±0.07 m/s) repolarization velocities (n=18). In sheets stretched parallel to the longitudinal axis of surface fibers, AP durations (APDs) increased as a function of fiber length, from the length at zero developed tension to 120% of the length at maximum developed tension (Lmax). Spatial distributions of APDs did not change during stretches along the rising phase of the length-tension curve. In sheets stretched to 50% of Lmax, APDs were shorter and more homogeneous on the endocardium (mean APD, 188 ms;
APD, 195-186=9 ms) than on the epicardium (mean APD,
204 ms; APD, 212-186=26 ms; n=8). In guinea pig hearts, activation
is rapid; therefore, repolarization depends primarily on intrinsic
spatial heterogeneities of APDs. Consequently, repolarization begins at
endocardial cells with the shortest APDs and spreads transmurally and
then anisotropically on the surface according to the epicardial cell
orientation.
Key Words: myocardial fiber orientation • action potential duration • optical mapping • repolarization patterns • activation patterns
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation and repolarization patterns in mammalian hearts are important in understanding the mechanisms of normal and abnormal cardiac rhythms. However, although activation has been extensively characterized by use of extracellular electrodes,1 repolarization remains incompletely understood because of the poor resolution of T waves measured by surface electrograms. Measurements of repolarization to assess spatial heterogeneities of APDs have relied on (1) potential gradients,2 3 (2) refractory period determinations,4 5 (3) monophasic APs,6 (4) ARIs,7 8 or (5) intracellular microelectrodes9 by using isolated sheets of ventricular tissue, papillary muscles, or enzymatically dispersed myocytes. Each method offers advantages but also has limitations.
Potential gradients measured with 120 unipolar electrodes on the ventricles have suggested that repolarization is spread anisotropically during epicardial stimulation. However, during endocardial or midmural stimulation, potential gradients did not convey a three-dimensional view of repolarization patterns.10 Refractory periods were determined by recording the time of reexcitation after the application of extrastimuli at the recording site, but the method is time consuming, since a set of premature stimuli must be tested for each site.4 ARIs use an array of extracellular electrodes. The activation time point at each site is detected at the maximum negative point of the QRS complex [(dV/dt)min] and the recovery time point at the maximum positive slope of the T wave [(dV/dt)max].8 ARIs correlate with APDs measured with intracellular electrodes and with refractory periods measured by programmed stimulation.7 8 ARIs predicted similar patterns for activation and repolarization in dog hearts.8 Regrettably, substantial errors can occur in the estimation of activation and recovery, particularly under nonideal conditions, including alterations in recording sites relative to the activation sequence, nonuniform coupling resistances, and anisotropic membrane properties.11 Contact (suction or pressure) electrodes record monophasic APs, which closely resemble APs recorded with intracellular electrodes, and have been used to study epicardial and endocardial repolarization in vitro and in vivo. But, at most, 8 to 10 contact electrodes can be simultaneously used, and there arc concerns regarding cell depolarization and tissue damage under the recording electrode.12 Monophasic APs recorded with a single contact electrode suggested that APDs were inversely related to activation time.6 Intracellular microelectrode recordings from tissue patches and enzymatically dissociated guinea pig myocytes indicated that APDs are shorter at the apex than the base of the left ventricle.9 However, a concern with this approach is that APDs are modified by the lack of stretch and might not have the same characteristics as cells in intact hearts. Furthermore, intracellular electrodes offer limited sampling because of the difficulty of maintaining them in place on a beating heart.
Voltage-sensitive dyes and imaging techniques have been previously
used to measure APDs and repolarization patterns in "working"
guinea pig hearts.13 14 However, these earlier studies
were complicated by movement artifact.15 Since the
upstrokes of APs always precede force generation, they are not subject
to motion artifact that might distort activation patterns. On the other
hand, the downstrokes of APs are in phase with peak myocardial force
development and the onset of relaxation. The downstrokes of optical APs
in frog ventricular muscle were readily measured in zero
Ca2+ Ringer's solution, which stopped contractile
movement,14 but removal of extracellular Ca2+
prolongs APDs and may alter repolarization patterns and cause rhythm
disturbances in mammalian hearts. Movement could also be abated
by perfusing hearts with negative inotropic agents, such as
2,3-butanedione monoxime16 17 or
verapamil,18 but the concentrations needed
could alter upstroke velocities and APDs and interfere with
mechanoelectrical feedback.19 20 21 Novel chamber designs and
perfusion techniques now make it possible to place a
Langendorff-perfused or a working heart in a chamber with a glass
window or to pin a perfused working ventricular sheet on a
rack to adjust muscle length and thus record APs essentially free
of movement artifacts, without resorting to negative inotropic
agents.22 23 24 25 26 Newer dyes like di-4-ANEPPS27 28
and RH-42129 also offer improved signal-to-noise
ratios by yielding larger fractional changes in fluorescence
per unit change of membrane potential and by fluorescing at long
wavelengths (650 to 750 nm), where the quantum efficiency of
photodiodes is >90%. For optical APs that are distorted by mechanical
artifacts (
25% of the APs), repolarization time points can still be
uniquely identified through the maximum second derivative
[(d2F/dt2)max] of the
fluorescence downstroke.22 23 24 In Beeler-Reuter
simulations of the ventricular AP and in experiments on
guinea pig myocytes and intact perfused hearts,
(d2F/dt2)max was found to fall at
97±2% of recovery to baseline.30 The coincidence of
(d2F/dt2)max and AP repolarization
was maintained during changes in APD elicited by frequency,
hypoxia, and ischemia.30 Moreover, under
physiological conditions at normal resting
potential, (d2F/dt2)max was shown
to be coincident to the refractory period and could be used to map
repolarization and refractoriness in a single heart
beat.26 The close correlation between optically
recorded APs and those recorded with an intracellular
microelectrode has been well documented.15
In the present study, activation and repolarization waves from the epicardium and endocardium of guinea pig hearts were measured under different stimulation protocols by using a voltage-sensitive dye (RH-421), a light guide, and a photodiode array. The guinea pig heart offers several advantages: (1) The size of the heart made it possible to image electrical activity and analyze fiber architecture (through serial sectioning) from the entire left or right ventricular free wall. (2) The excellent collateral circulation made it possible to develop an isolated ventricular free wall preparation perfused and superfused in blood-free oxygenated Ringer's solution for several hours with no significant metabolic deficiencies. (3) The AP has been well characterized, with a long APD and a classical APD versus frequency relation. Unlike other small rodents, the electrogram waveform of the guinea pig heart is similar to that in the human heart, even though the guinea pig AP is devoid of Ito.30
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Materials and Methods |
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Intact Heart Preparation
Guinea pigs (350 to 450 g, either sex) were anesthetized with sodium pentobarbital (30 mg/kg IP), and their hearts were rapidly excised, cannulated at the aorta, and retrogradely perfused in a modified Langendorff setup. A key feature of Langendorff preparations is that the hearts do not perform mechanical work because of the absence of left ventricular filling and diastolic fiber stretch. This ensured that spatial heterogeneities of fiber stretch did not alter APDs to produce nonuniformities of APDs. The hearts were then stained with a voltage-sensitive dye (RH-421) and placed in a fluid-filled Plexiglas chamber with an optical window for monitoring fluorescence as previously described.13 23 24
Several bipolar stimulating electrodes (250-µm-diameter polytetrafluoroethylene [Teflon]–coated Ag+/AgCl wires with a 50 µm interelectrode distance) were simultaneously positioned on the intact heart: (1) For right atrial pacing, an electrode was glued to the right atrium with cyanoacrylate adhesive. (2) For endocardial pacing, an electrode was inserted into the ventricular chamber and implanted on the endocardium. (3) For epicardial pacing, one to five stimulating electrodes could be simultaneously positioned at the apex, base, anterior, posterior, or center of the epicardium.
Ventricular Sheet Preparation
The left ventricular free wall was isolated with its
coronary perfusion intact by excising the right ventricle, the
septum, and left papillary muscles from a Langendorff-perfused
heart. The removal of a ventricle and the septum decreased perfusion
pressure. The ligation of three to five cut coronary artery
branches supplying the excised tissue raised the perfusion pressure
above its initial value of 80 mm Hg, which was reestablished by
decreasing the flow rate. Typically, if two thirds of the tissue mass
was removed, the flow rate needed to be reduced by approximately two
thirds.
Perfused sheets were pinned horizontally on a two-piece Delrin
platform (Fig 1A
). One half of the platform was fixed,
whereas the other could slide on a set of stainless steel tracks. The
movable platform was connected to a tension transducer (Gould, model
M602) mounted on a micromanipulator. Heated Ringer's solution
(35±1°C) was delivered to both the aortic cannula and the tissue
chamber inlet to respectively perfuse and superfuse the preparation.
Ventricular free walls were pinned with either the
epicardium or endocardium facing up, with up to five sets of bipolar
stimulating electrodes (250-µm-diameter Teflon-coated
Ag+/AgCl with a 50 µm interelectrode distance)
placed on the top surface and one set placed on the bottom surface. A
No. 2 glass coverslip (22x22 mm) was placed at the
fluid-to-air interface above the tissue to prevent fluid
vibrational noise during optical recordings.
Ventricular sheets were passively stretched by displacing
the movable section of the rack by increments of 200, 400, or 800 µm.
Equilibrium length (Lo) was defined as the distance between
the fixed and movable pins when further stretches registered an
increase in passive and developed tension as measured by the
transducer. Stretches produced systematic increases in passive and
developed tension until developed tension reached a maximum, at length
Lmax. The length-tension curves indicated that
Lmax was 30% to 38% greater than Lo. The
coronary perfusion of isolated ventricular sheets
was of critical importance when attempting to measure APs with
negligible motion artifacts and to avoid tissue ischemia, which
decreased APDs and enhanced spatial inhomogeneities of APDs.
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EX=520±20 nm), and the
epifluorescence from the voltage-sensitive dye
(
645 nm) was refocused on a 12x12 photodiode array.
Photocurrents from 124 diodes were passed through
current-to-voltage converters, amplified, digitized, and stored
in computer memory. In addition to 124 APs being recorded from a
12x12-mm region of the epicardium, a bifurcated light guide was
inserted in the left ventricular cavity to monitor a single
AP from a 1-mm-diameter region of the endocardial surface. The same
optical setup, with a different source of illumination, was used to
record 124 APs from a 6x6-mm region of either the epicardium or
endocardium of ventricular sheet preparations.
Optical Apparatus
Intact hearts were illuminated with light from a 200-W
mercury-xenon arc lamp (PTI, Fig 1B
), which was collimated with a
parabolic reflector and cooled with a heat filter (water-filled
4-in optical path length). The excitation beam was controlled with an
electronic shutter (Melles Griot) to illuminate the heart only during
optical recordings. The beam was passed through an interference
filter (520±20 nm, Omega Optical), reflected by a 45° dichroic
mirror (600-nm long-wave pass, Corion), and focused on the stained
heart with an epi-illumination lens (50 mm, 1:1.8 E series,
Nikon). Epifluorescence was collected and passed through a
645-nm cutoff filter (Schott Glass, RG-645) and focused onto a 12x12
photodiode array (Centronics). The same optical apparatus
was also used to record signals from myocardial sheets, but the
optical axis (normal to the surface of the tissue and the photodiode
array) was vertically oriented. In this instance, light from two 100-W
tungsten-halogen lamps (PTI) was collimated, passed through
520±20-nm interference filters (Omega Optical), and refocused on the
preparation at a 45° angle of incidence. Epifluorescence
from stained tissue was collected, passed through a 645-nm cutoff
filter, and focused on the array. The physical dimensions of the
photodiode array were 18x18 mm (1.4x1.4 mm per diode with 0.1 mm of
dead space between diodes), but the area of the tissue image that was
focused on each array diode was either 1x1 mm (intact hearts) or
0.5x0.5 mm (isolated sheets), with a depth of field of 174 and 44
µm, respectively.
Simultaneous APs could also be recorded from a
1-mm-diameter region of the endocardium by using a bifurcated light
guide (Dolan-Jenner Industries) and from the epicardium by using a
photodiode array (Fig 1B). One branch of the guide focused an
excitation beam on the endocardium; the second branch collected and
transmitted focused fluorescence emission to a photodiode (EG&G
Reticon). The source of the illumination was light from a 45-W
tungsten-halogen lamp (PTI) that was collimated and passed through
an interference filter (520±20 nm, Omega Optical). The tip of the
light guide, which was bent at 90° at 1 mm from its extremity, was
inserted into the left ventricular chamber through the
mitral valve. Light from the fiberoptics was detectable across the
ventricular wall, permitting the guide to be aligned
opposite a desired epicardial region of tissue monitored by one of the
photodiodes on the array.
Pacing Protocol
Intact hearts and isolated ventricular free walls
were typically paced at a cycle length of 300 ms (
200 bpm) with
square pulses of 1-ms duration at twice diastolic threshold
(twice capture voltage). This rate was within the
physiological range for guinea pigs (200 to 300 bpm
at 38°C). A constant SA nodal rhythm was obtained by pacing the right
atrium of intact hearts near the SA node at 200 bpm. Experiments were
carried out at a slightly lower temperature (35±1°C) to be able to
pace the heart reliably and overcome the intrinsic rate of the
pacemaker. There were no observable differences in activation or
repolarization patterns when hearts were maintained at 38°C. However,
APDs uniformly shortened with increasing temperature. Stimulation of
the endocardium was used as an alternative method to activate
the ventricles through the specialized conduction system. Epicardial
stimulation was used to avoid activation of the specialized conduction
system and permitted measurements of AP spread in
ventricular cells without the influence of Purkinje fibers.
During endocardial and epicardial stimulation of intact hearts, the
right atrium was simultaneously paced to prevent
out-of-phase SA nodal impulses from capturing the ventricles
and interfering with activation and repolarization patterns.
Data acquisition, signal analysis, and determination of conduction velocities were carried out as previously described.23 24
Histology
After electrophysiological
recordings were made, the region of tissue viewed by the array
was marked with fiducial points by impaling its edges with a
microelectrode dipped in india ink. The markings were placed so as to
correspond to the center of tissue patches viewed by diodes on the
corners of the array. The ventricular free walls were
excised and fixed in Bouin's solution with their epicardial surfaces
placed against a glass slide for 1 hour and then overnight without a
slide. All tissues were in diastole before the fixation
protocol. Fixation flat against a glass slide ensured that sections
were cut parallel to the imaged plane of the heart for accurate
correlation with maps of electrical activity. Fixed tissue was embedded
in paraffin, serial sections were cut every 5 µm, and every 10th
section was mounted on a glass slide. To ensure an accurate measurement
of the depth of all sections relative to the surface, care was taken to
identify the front surface of the epicardium. Mounted sections were
progressively stained with Mayer's hematoxylin, counterstained with
eosin, and placed under a glass coverslip.31
Stained sections were examined under a microscope between cross
polarizers to better visualize fiber orientation. An image of each
section was projected, magnified, and refocused on a 12x12
reticule, which conformed to the dimensions of the array and thus to
the tissue before fixation. Shrinkage caused by fixation (typically
35%) was automatically taken into account by aligning the fiducial
marks on the sections with their respective locations on the reticule.
The fiducial marks were also used to align the sections with respect to
orthogonal cartesian coordinate axes, where the base to apex axis was
parallel to the vertical y axis. The orientation of fibers
viewed by each photodiode was measured with a protractor mounted in an
eyepiece. One hundred twenty-four fiber orientations were averaged
to give the longitudinal fiber axis for the tissue imaged by the array.
The average fiber orientation (
F) of each section was
plotted as the function of its depth in the right and left
ventricular walls.
Tests and Precautions
APDs depend on physiological
parameters that include heart rate, temperature, resting
membrane potential, and the metabolic and contractile
states of the myocardium. Consequently, experimental
conditions were controlled to ensure valid measurements of spatial
heterogeneities of APDs. Heart rate was controlled by pacing the
preparations and continuously monitoring the cycle length to detect
possible rhythm disturbances. We verified that spatial
heterogeneities of temperature were negligible by measuring the
temperature at various sites on the surface of the epicardium with a
1-mm-diameter thermistor to verify that it was within ±1°C of
the set point (35°C). The thermistor was inserted between the heart
and the glass window of the chamber and was systematically displaced
along the heart's surface. Heat from the excitation beam raised the
temperature <1°C in 60 s and did not cause spatial variations of
temperature. Temperature effects caused by the excitation beam were not
significant, since the preparations were illuminated only during data
acquisition (10 to 15 s). Fluid accumulation and pressure in the
ventricular cavity could stretch the left ventricle and
thus alter APDs. This concern was addressed by cutting the mitral valve
to relieve fluid buildup in the ventricular cavity.
Mechanical contact and pressure between the window of the chamber and
the epicardial surface did not alter APDs or cardiac function. This
issue was verified by measuring optical APs in the absence and presence
of the chamber and with increasing pressure between the front and the
rear portion of the chamber to compress the heart. The compression of a
20-mm-diameter heart by 1, 2, 3, and 4 mm did not alter
coronary flow rate, pulse pressure, surface electrogram
recordings, or APDs. However, it increased
diastolic pressure respectively by 0, 2, 5, and 15 mm Hg.
Further compression of 5 and 6 mm increased diastolic
pressures by 30 and 50 mm Hg, reduced coronary flow, altered
electrogram recordings, and shortened APDs. The present
experiments were carried out with 2- to 3-mm compression, which reduced
motion artifact without changing AP characteristics, coronary
flow rates, or pulse pressure. Similar spatial heterogeneities of APDs
were also measured in isolated ventricular sheets stretched
along the rising phase of their length-tension curve. Thus, the use
of the chamber to restrain movement did not modify activation and
repolarization patterns.
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Results |
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To visualize patterns of activation and repolarization, maps were displayed as isochrones on the basis of gray scales. These maps were generated from the activation or repolarization times of n groups of the 124 APs detected by the photodiode array. Each group was assigned a gray tone, where light to dark shades represented early to late activation or repolarization times. Each diode location (box) on a symbolic map of the array was then filled with the appropriate gray tone representing that channel's activation or repolarization time group. Five gray tones allowed for the best visualization of the overall pattern while maintaining an excellent degree of detail.
Fig 4 shows the anatomic landmarks of the guinea pig heart superimposed
on a schematic of the array to depict the regions of the left (A) and
right (B) ventricles that were examined. The same regions of the left
and right ventricles were examined throughout this study. In Fig 2B, activation time delays were used to construct
gray-scale isochronal maps during the delivery of stimuli to
the center of the left ventricular epicardium. With stimuli
delivered every 350 ms, a 1.2-s scan recorded three cardiac beats.
Three representative APs are shown in Fig 2 from early
(1), intermediate (2), and late (3) regions of tissue activation; the
fourth trace is a bipolar electrode recording. The complexity
of the electrogram waveform emphasizes the difficulty of defining
activation and repolarization time points from surface electrode
recordings. Gray-scale activation maps from three
successive beats (A through C) exhibited similar anisotropic patterns
of propagation, with activation times across the tissue of 19.2 (A) and
18.4 (B and C) ms, which were reproducible over 2 to 3 hours (data not
shown). The spread of APs was anisotropic and produced elliptical
isochrones, where the major and minor axes of the ellipse
represented the directions of fastest and slowest
conduction velocities.
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Detection of Repolarization Time Points
The time of depolarization has been routinely defined and detected
through the maximum first derivative of the AP upstroke,
(dV/dt)max, measured with intracellular
microelectrodes. For repolarization, there is no simple definition nor
standard algorithm. For example, values of 50%, 75%, and 90%
recovery of the downstroke to baseline have each been used as
definitions of the repolarization time point. With optical
recordings, the same approaches can be used to define
activation and repolarization by signal processing of the
voltage-dependent fluorescence signals. However, for
optical recording, the recovery to baseline is more susceptible
to movement artifacts that alter the kinetics of AP downstrokes.
Recently, the peak of the second derivative was shown to reliably
detect the repolarization time point of optical APs,22 23 24
because the downstroke of APs possesses an inflection point that can be
detected despite the presence of motion artifacts. In Fig 3, APs and phasic contractions were
simultaneously recorded from a 6x6-mm region of a
perfused left ventricular sheet stretched to 50% of
Lmax. One AP was chosen for its lack of movement artifact
(diode 52), and the next three APs were chosen because movement
artifacts altered their downstrokes in different manners (diodes 80,
81, and 124). The second derivatives,
(d2F/dt2)max values, of these
tracings (52'', 80'', 81'', and 124'') are shown along with
simultaneous recordings with a bipolar electrode,
developed tension, and its second derivative (BE, T, and T'',
respectively, in Fig 3). Despite the different AP downstrokes caused by
movement artifacts, the maximum second derivatives of AP downstrokes
occurred at 97±2% of return to baseline, which identified a set of
unique repolarization time points. APDs determined from the time
interval between (dF/dt)max and
(d2F/dt2)max predicted reproducible
APDs (±1.4 ms) from beat to beat at each diode. Adjacent diodes
(channels 80 and 81) had similar APDs, despite widely varying movement
artifacts. Movement artifacts did not shift
(d2F/dt2)max, most likely
because their kinetics are similar to the phasic contractions (T),
which had flat second derivatives during the repolarization phase of
APs (T''). The use of the
(d2F/dt2)max inflection point of AP
downstrokes provided a practical and reliable method to map APDs and
predicted the systematic spread of repolarization waves along the
epicardium.
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Fiber Orientation as a Function of Depth
Histological analysis was carried out
after optical recordings in order to correlate
ventricular fiber orientations at different depths in the
tissue with activation and repolarization patterns recorded under
various stimulation protocols. In hearts cannulated at the aorta and
hanging freely, fiber orientation was measured with respect to
arbitrary cartesian coordinates. The origin was at the center of the
ventricular free wall, the ordinate was aligned vertically
from base to apex, and the abscissa was aligned horizontally from
anterior to posterior. The longitudinal fiber axis of left ventricles
was oriented at a 135±5° (or 135°+180°=315°) angle on the
epicardium and rotated counterclockwise by 135° from epicardium to
endocardium where fibers aligned with the vertical axis (Fig 4, inset
A). The longitudinal axis of epicardial fibers on the
right ventricles was at a 15±5° (or 15°+180°=195°) angle and
rotated by 75° from epicardium to endocardium where fibers again
aligned with the vertical axis (Fig 4, inset B). Fig 4
represented the average of histological
studies from left (n=6) and right (n=6) ventricles. From serial
sections taken at different depths, fibers on the right epicardium (0.8
mm thick) were at 15±5° for the first 150 µm, were rotated
gradually counterclockwise by 10° per 100 µm for 500 µm and
more abruptly by 22° for 50 µm, and were then aligned with the
vertical axis for 100 µm of endocardial wall. In the thicker left
ventricles (1.5 mm), epicardial fibers were oriented at 135±5° for
the first 200 µm and rotated gradually counterclockwise at 5°
per 130 µm for 650 µm and then more abruptly by 10° in 65 µm
for 650 µm until the endocardial fibers aligned with the vertical
axis. It is important to note that the data shown in Fig 4 were
obtained on fixed tissue, which should be corrected for 35%
shrinkage. As a result, the actual thickness of the right ventricles
was 1.08±0.14 mm (n=6) compared with 0.8 mm for the fixed tissue; left
ventricles were 2.03±0.25 mm (n=6) compared with 1.5 mm.
Most noteworthy for both right and left ventricles was the vertical orientation of endocardial fibers and the consistent orientation of epicardial fibers at >250 µm from the surface. The orientation of epicardial fibers was uniform for a depth greater than the depth of field of the imaging system such that fiber rotation did not influence the present optical maps of electrical activation.
Superposition of Activation and Repolarization Patterns With
Fiber Orientation
Perfused guinea pig hearts stained with RH-421 were placed in a
fluid-filled chamber, and a 12x12-mm area of left or right
epicardium was focused on the array, as shown in Fig 4. In Fig 5, activation (top panels) and repolarization (middle
panels) patterns were recorded from the left ventricle during
various stimulation protocols (A, B, and C) and correlated with fiber
orientations at different depths of the tissue (bottom panels). During
right atrial pacing near the SA node, activation broke through at the
anterior epicardium and spread in 6.2 ms to the posterior epicardium
(Fig 5A). Activation spread anisotropically with the major axis
parallel to the longitudinal axis of endocardial fibers (Fig 5D).
Repolarization measured from the same set of APs began near the apex
and spread anisotropically across the tissue in 27.7 ms (Fig 5A').
During epicardial pacing near the center (Fig 5B) or upper left corner
of the tissue viewed by the array (Fig 5C), activation propagated
anisotropically with a major axis oriented (at 135°) parallel to the
longitudinal axis of epicardial fibers (Fig 5F). Repolarization
patterns elicited by right atrial pacing (Fig 5A') and epicardial
stimulation near the center (Fig 5B') or upper corner (Fig 5C') of the
array were also anisotropic, with major axes oriented 135° parallel
to the average orientation (F=135°, Fig 5F) of the
longitudinal axes of epicardial fibers. The major axis of
repolarization patterns was within experimental error (±5°) parallel
to the longitudinal axis of epicardial fiber (n=6).
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In Fig 6, a similar experiment was carried out on a
perfused right ventricle. During right atrial and endocardial
stimulation, the major isochrones for activation (Fig 6A and 6B)
were parallel to the long axis of endocardial fibers (Fig 6D). During
epicardial stimulation, the major axis of activation (Fig 6C) was
parallel to the long axis of epicardial fibers (Fig 6F). Irrespective
of the pacing protocol, repolarization spread anisotropically (Fig 6A',
6B', and 6C'), with major axes at 30°, indicating a slight rotation
of repolarization isochrones compared with the average longitudinal
fiber axis at 15° to 20° (Fig 6F).
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Analyses of repolarization patterns measured from left and right epicardia indicated that repolarization was not random but traveled systematically across the heart's surface. When measured during epicardial pacing, repolarization traveled anisotropically, with maximum and minimum velocities of 0.41±0.09 and 0.23±0.07 m/s (n=12) that aligned with the longitudinal and transverse axes of epicardial fibers, respectively. The repolarization patterns were similar to activation maps measured under the same pacing protocol but had slower repolarization velocities compared with 0.85±0.05 and 0.44±0.04 m/s for activation. These velocities represented intrinsic myocardial fiber velocities, since activation and repolarization patterns were guided by the orientation of surface fibers and had similar eccentricities (ratios of longitudinal to transverse velocities) of 1.93 and 1.78, respectively. During right atrial or endocardial pacing, activation of the epicardium was driven by the specialized conductile system (Purkinje fibers), resulting in maximum and minimum apparent velocities of 2.66±0.11 and 1.65±0.09 m/s, which aligned with the longitudinal and transverse axes of endocardial fibers, respectively. In contrast, repolarization first broke through on the epicardial surface from the apex (bottom of the array) to one third of the way up the wall of the ventricle (Figs 5A' and 6A') and spread with maximum and minimum apparent velocities (0.53±0.11 and 0.31±0.1 m/s, respectively; n=12), which remained predominantly aligned with the longitudinal and transverse axes of epicardial fibers.
The location of the first sites to repolarize did not significantly
vary with changes in the stimulation site. During epicardial
stimulation at the center of the left ventricle (Fig 5B), the first
sites to repolarize were typically located one third of the distance up
from the apex (Fig 5B'). When the stimulus site was shifted to the
anterior corner, near the base (Fig 5C), the first site to repolarize
shifted slightly up, toward the base (Fig 5C'). With all three
stimulation protocols (A through C), APDs were shorter at the apex than
the base (n=12). Note that in Fig 5, APs near the apex (Fig 5C, bottom
channels) depolarized after APs near the base (Fig 5C, top right), yet
the apex (Fig 5C', bottom) repolarized before the base (Fig 5C', top
right). Under right atrial (Figs 5A and 6A) or endocardial (Fig 6B)
stimulation, activation emerged at the center of the epicardium, guided
by the endocardial fiber orientation that had faster conduction
velocities compared with those measured during direct epicardial
stimulation. The orientations of maximum and minimum velocities during
atrial pacing were aligned with the axes of endocardial fibers, which
are activated by Purkinje fibers lining the endocardial
surface. Thus, Purkinje-driven activation begins through the rapid
activation of the endocardium, followed by transmural propagation that
emerges to synchronously activate a large patch of
epicardium.
To determine whether repolarization began on the epicardium or traveled
to the surface from deeper cells, APs were simultaneously
recorded from opposite sides of the ventricular wall. A
light guide was inserted into the left ventricular chamber
to record an endocardial AP while recording 124 APs from
the epicardium by using the array (see "Materials and Methods").
During right atrial pacing, endocardial APs depolarized and repolarized
before the epicardial APs immediately opposite them on the other side
of the wall (Fig 7A and 7A'), which indicated that
repolarization traveled from endocardium to epicardium in 6±1.3 ms
(n=6). When paced on the epicardium, the epicardium depolarized before
the endocardium, but the endocardium still repolarized either at the
same time (Fig 7B and 7B', n=4) or before the epicardium (not shown,
n=4). During right atrial pacing, activation on the endocardium
preceded that of the epicardium by 3 to 5 ms (n=8).
|
For the 1.6- to 1.9-mm-thick ventricles, the average transmural activation velocity was 0.48±0.12 m/s (n=8), which is similar to transverse activation velocities seen on the epicardium (0.44±0.04 m/s). Transmural activation velocities were similar (within experimental error) when APs spread from endocardium to epicardium (the right atrium was paced) or epicardium to endocardium (the epicardium was paced). Since it was assumed that repolarization began on the endocardium and traveled across the thickness of the ventricles (1.6 to 1.9 mm), the average transmural repolarization velocity was calculated to be 0.27±0.04 m/s (n=8), a value close to the transverse repolarization velocity of 0.23±0.07 m/s.
The light guide made it possible to measure endocardial APs from intact hearts and (if a direct path from endocardium to epicardium is assumed) provided an estimate of transmural activation and repolarization velocities. Even though the transmural activation and repolarization time delays were brief, these values were reproducible and could be resolved, given the sampling rate of the data (0.7 ms per frame). Moreover, transmural delays were in line with our measurements of transverse activation and repolarization velocities, given the width of the guinea pig ventricular wall. However, the light-guide approach was limited to one endocardial AP and could not resolve the two-dimensional spread of activity on the inner surface. Consequently, ventricular walls were dissected and stretched as sheets to expose the inner and outer surfaces of the same heart to optical array recordings.
Electromechanical Properties of Ventricular
Sheets
In isolated tissue experiments, the image magnification was
adjusted such that the array viewed 6x6- instead of 12x12-mm of
tissue. The recording area was reduced because of the smaller
area of endocardium and in order to avoid muscle injury at the sheet
edges. Perfused sheets were stained with RH-421 and mounted on the
horizontal chamber (Fig 1A) to record APs at a known contractile
state along the length-tension curve of the myocardium.
Left ventricular sheets were mounted horizontally to view
the epicardium and were rotated to stretch the muscle parallel to the
longitudinal axis of the surface fibers (Fig 8). The
resting length (Lo) was determined by stretching the tissue
until the transducer detected phasic contractions elicited by
repetitive stimulation (cycle length, 350 ms). The tissue was stretched
by 800-µm increments from Lo and allowed to equilibrate
for 5 to 10 minutes to ensure stable contractions before
recording APs (Fig 8b). Stretches produced the expected
increase in phasic contractions until Lmax (length at which
maximum active tension is generated) was reached; further stretches
reduced active tension. Length-tension curves were measured from
three different preparations with 0.5 (Fig 8a), 1.5 (Fig 8b), or 2.5
(Fig 8c) mmol/L Ca2+ in the perfusate, and AP maps
were recorded as a function of stretch.
|
In Fig 9A and 9B, the quality of the
signals from the epicardium are shown for sheets exhibiting weak (0.5
mmol/L Ca2+ and 13.3% stretch) and strong (2.5 mmol/L
Ca2+ at Lmax) contractions,
respectively. Even when the tissue generated large phasic contractions
(18 g tension) in 2.5 mmol/L Ca2+ at
Lmax, AP upstrokes were readily detected, and
repolarization phases were not obscured by mechanical artifacts. Some
diodes detected distorted AP downstrokes (Fig 9B; channels 13, 38, 61,
62, 73, 74, etc) because of motion artifacts. However, the signal
processing technique described earlier identified distinct
(d2F/dt2)max inflections and
provided unambiguous repolarization time points for all except two APs
(channels 73 and 74).
|
Length-tension relations and changes in APDs as a function of
muscle length were analyzed for left ventricular
sheets continuously paced at 350-ms cycle lengths. Fig 10A shows the average length-tension curve of 10
muscle sheets perfused with 1 mmol/L Ca2+ Ringer's
solution. The curves were normalized with respect to
Lmax, which was typically 38% greater than
Lo. When stretched 25% beyond Lmax,
developed tension decreased by 70%.
|
), posterior (right) side (
), apex (
), and anterior
(left) side (
). APDs increased with increasing stretch along the
ascending limb of the length-tension curve. Moreover, stretch did
not alter heterogeneities of duration in different regions of
myocardium. The SEM was within 3% of the mean APD value;
each data point represented the average duration of 31
optical APs from these regions of left ventricle. D, APDs were plotted
as a function of muscle length for sheets perfused with 0.5, 1.0, 1.5,
or 2.5 mmol/L Ca2+ Ringer's (n=3, for each
Ca2+ concentration). APDs decreased with increasing
Ca2+ and increased as a function of muscle length. The SEM
was within 3% of the mean APD value; each data point
represented the average duration of 31 optical APs.
Stretches to Lmax (or beyond) produced increases in APDs
that did not reverse upon return of the muscle to its
resting-length. This effect is illustrated in Fig 10B, where APDs
increased with increasing muscle length from Lo to 25%
beyond Lmax and were held at 125% of Lmax for
30 minutes (open circle). When the sheets were slackened back to
Lo, APs did not return to their original duration
(170 ms) but remained at 194 ms. Stretching the muscle for a second
time (solid circle) did not change APDs.
In Fig 10C, APDs from four regions of the left ventricle were measured
as a function of muscle stretch. The 124 APs recorded by the array
were divided into four groups of APs from the base, apex, anterior
(right side), and posterior (left side) regions. APDs increased as a
function of stretch and leveled off at 25% beyond Lmax.
APDs were longest at the base and anterior (right side) region of the
ventricles and shortest on the posterior (left side) and apex regions.
APDs from all four regions of the left ventricle increased in a
stepwise manner as a function of muscle length, so that stretch did not
significantly alter the regional differences in APDs. Increases in APDs
as a function of stretch were measured with 0.5, 1.0, 1.5, or 2.5
mmol/L Ca2+ in the perfusate, and as expected, APDs
increased with decreasing [Ca2+]o (Fig 10D).
A major concern was that fibers at various depths of sheet were stretched to different extents and that at Lmax, fibers in the midwall or the endocardium may have become stretched beyond Lmax. To compare APDs from the endocardium and epicardium, care was taken to measure APs from both surfaces at similar levels of passive stretch and to avoid overstretching of the preparations. From a resting length (Lo) and under continuous pacing, the tissue was stretched by equal increments of length (100 to 200 µm), and Lmax was identified when a small stretch failed to elicit an increase in developed tension. The muscle was then slackened to 50% of Lmax and allowed to equilibrate for 10 to 20 minutes before recording APs.
Epicardial Versus Endocardial Repolarization Patterns
A sheet of perfused left ventricle was dissected, as described in
"Materials and Methods." Papillary muscles and free
trabeculae were removed from the endocardium to reduce the
curvature of the ventricle and thus obtain uniformly stretched fibers.
The sheet was stained with RH-421 in the perfusate and then
mounted on the horizontal chamber rack with the epicardium facing up
and the longitudinal axis of surface fibers parallel to the direction
of stretch. The sheet was stretched to 50% of Lmax and
allowed to equilibrate during continuous pacing at a 350-ms cycle
length before recording APs from the epicardium. In Fig 11, activation (A through D) and repolarization (A'
through D') patterns were measured from the epicardium during pacing at
different locations (square pulse symbols) on the edges of the 6x6-mm
ventricular surface. Activation began at the stimulus site
and propagated anisotropically with the major axis parallel to the
longitudinal axis of epicardial fibers (top panels). As the stimulus
site varied, the first sites to depolarize changed accordingly. As in
the intact heart, repolarization (bottom panels) began near the apex,
regardless of the initial site of depolarization. During stimulation at
the base or posterior edge of the epicardium, repolarization began at
breakthrough sites near the apex of the anterior side of the left
ventricle (lower right edge of Fig 11A' and 11B'). With stimulation at
the apex or anterior edge of the sheet, repolarization also began near
the apex but was initiated from both the anterior and posterior edges
of the left ventricle (Fig 11C' and 11D'). Maps of APDs were similar to
the repolarization patterns shown in Fig 11A' through 11D' (not
shown).
|
The same ventricular sheet was removed from the rack,
flipped over so that signals could be recorded from the endocardium
(Fig 12), and restretched to 50% of
Lmax, with the longitudinal axis of endocardial
fibers parallel to the direction of stretch (90°/270°). Activation
(top panels) and repolarization (bottom panels) maps were measured from
6x6 mm of endocardium while pacing at different edges of the
endocardium. Stimulation at the anterior (Fig 12A) or posterior (Fig 12D) edges of the endocardium produced activation patterns with major
axes aligned at 105°, approximately parallel to the endocardial fiber
axis. However, pacing at the base (Fig 12A) or apex (Fig 12C) caused
rotations of the activation patterns, with the major isochrones
oriented at 180°. From heart to heart, the orientations of
endocardial activation patterns were less predictable than those
measured from the epicardium because of the tortuosity of free
trabeculae lining the endocardium and because the layer of
endocardial fibers rotated rapidly as a function of depth in the wall
(10°/200 µm, Fig 4A). In addition, pacing electrodes most likely
stimulated several layers of fibers with different orientations.
Nevertheless, endocardial repolarization was typically initiated near
the first site to depolarize but did not travel in a systematic pattern
and appeared to be random (Fig 12A' through 12D'). The distribution of
APDs on the endocardium was random (not shown), unlike that measured on
the epicardium.
|
Activation and repolarization patterns were measured from the endocardium and epicardium of 10 left ventricles. Conduction velocities were similar (within experimental error) on both sides of the tissue. Measurements were begun from the endocardial and then the epicardial surfaces, or vice versa, to avoid systematic errors in APDs caused by stretches during length-tension analysis. Measurements from both surfaces were completed in 60 to 90 minutes, which avoided loss of signals from dye washout, photobleaching, and/or rundown of the preparations. In 8 of 10 sheets, APDs were shorter on the endocardium than the epicardium of the same ventricular sheet. APDs varied on the endocardium from 186 to 195 ms, with a mean of 188 ms, and on the epicardium from 186 to 212 ms, with a mean of 204 ms. In 2 of 10 sheets, mechanical artifacts were too large to measure APDs from the endocardium.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Voltage-sensitive dyes and imaging and signal-processing techniques were combined to record activation and repolarization wave fronts from the epicardium and endocardium of intact guinea pig hearts and arterially perfused ventricular sheets. Although the present data were limited to two-dimensional surface recordings of electrical activity, patterns measured under different pacing protocols were correlated with the underlying fiber histology to formulate a three-dimensional conceptual view of the activation and repolarization process.
Fiber Orientation in Guinea Pig Hearts
Fiber orientation in guinea pig hearts has not been extensively
examined. Our histological analysis of guinea
pig hearts showed remarkable similarities to studies of dog hearts,
despite major size differences. Early studies of dog hearts first fixed
and then flattened the tissue and reported that left
ventricular epicardial fibers were oriented at 120°
instead of 135° and rotated counterclockwise by 115° instead of
135° from epicardium to endocardium.32 33 More recent
studies reversed the procedure by first flattening and then fixing the
tissue.34 35 This latter procedure was similar to the
present study and reported results virtually identical to those
described here. One study reported that right ventricular
epicardial fibers were oriented at 20° instead of 15° and rotated
counterclockwise by 70° instead of 75° from epicardium to
endocardium.34 The other study reported right and left
ventricular fiber orientations in dog hearts that were in
excellent agreement with our guinea pig data.35 Besides
differences in the manner in which earlier studies took into account
the curvature of the tissue, they also examined a smaller percentage of
the total ventricular area.32 33 In the
present study, tissue architecture and electrical mapping were
analyzed under similar conditions. Thus, it was necessary to
flatten and then fix the tissue during diastole and to
examine close to 100% of the free walls to reproduce the conditions
during optical measurements. Others have shown that fiber orientations
were similar during diastole and systole and thus
independent of the contractile state of the
tissue.32 33
Ventricular Repolarization Patterns
Physiological activation of the heart through
pacing of the right atrium or pacing at various sites on the ventricles
produced repolarization wave fronts that spread anisotropically along
the epicardium of right and left ventricles according to the
orientation of surface fibers (Figs 5 and 6). During epicardial pacing,
the major and minor axes of the repolarization wave fronts traveled at
velocities (0.41±0.09 and 0.23±0.07 m/s, respectively) that were
twice as slow as activation velocities (0.85±0.05 and 0.44±0.04 m/s,
respectively; Figs 5 and 6) and represented the intrinsic
repolarization velocities for the myocardium.
Repolarization patterns did not significantly change when the
stimulation of the ventricles occurred through the specialized
conductile system by pacing the atrium (Figs 5A and 6A) or the
endocardium (Fig 6B) instead of direct stimulation of the epicardium.
This demonstrates that the initiation of repolarization wave fronts was
not significantly altered by the specialized conductile cells and
originated at cells near the apex of the heart. During right atrial or
endocardial pacing, there was a small but consistent increase
in longitudinal and transverse repolarization velocities (0.53±0.11
and 0.31±0.1 m/s, respectively) compared with epicardial pacing. This
apparent increase in repolarization velocity was attributed to the
sequential appearance of multiple breakthrough sites of transmural
repolarization that appeared on the epicardium. Attempts to shift the
first site to repolarize away from the apex by pacing different regions
of the myocardium were marginally effective in that the
first site to repolarize was only slightly displaced by repositioning
the stimulus electrodes (Figs 5B', 5C', and 11A' through 11D'). These
data indicated that groups of cells near the apex had shorter APDs
compared with ventricular cells located elsewhere.
Moreover, short APDs in cells near the apex were an intrinsic feature
of these cells that was independent of the rate or the contractile
state of the myocardium (altered by varying extracellular
Ca2+).
Several lines of evidence suggested that repolarization of the epicardium was not a "phase" wave that appeared to spread because of the intrinsic repolarization of individual cells but was spread via intercellular electrical coupling, like activation. Precautions were taken to avoid spatial heterogeneities of myocardial fiber stretch and perfusion: (1) Repolarization spread anisotropically across the epicardium, generating elliptical isochronal lines with major and minor axes that were parallel to the longitudinal and transverse axes of the cardiac fibers. (2) Repolarization patterns exhibited highly reproducible orientations and velocities from beat to beat (not shown) and had different orientations on the right and left ventricles according to the underlying fiber orientations. (3) The slow spread of repolarization strongly suggested that repolarization velocities were driven by the rate of AP repolarization or -dV/dt of AP downstrokes by analogy with activation that is driven by dV/dt of AP upstrokes. (4) Stimulation of the heart by pacing the right atrium instead of the epicardium changed activation times and the orientation of activation patterns but did not change repolarization patterns. This implied that Purkinje fibers speed up ventricular activation but not repolarization. (5) Repolarization patterns were not due to systematic artifacts, since (a) random patterns could be observed as on the endocardium; (b) on the epicardium, repolarization could be reversibly transformed from an anisotropic spread to random repolarization events by hypoxic episodes25 ; and (c) although repolarization delays between adjacent diodes (2 to 3 ms) were close to the sampling rate (0.7 ms), these delays were significant because they progressed in a systematic and structured manner along the epicardium. Thus, the graded repolarization delays could not be interpreted as a phase wave that coincidentally appeared like spreading wave fronts but indicated that repolarization spread from cell to cell by a process analogous to activation.
Previous Models of Repolarization
There is no consensus to date regarding whether the repolarization
of the myocardium actually spread from cell to cell or only
appears to spread like a phase wave. Mathematical simulations were
developed to model repolarization patterns, since they could not be
resolved experimentally. The models were based on a repolarization
process of the myocardium, which depends on a complex
interaction of the intrinsic properties of individual cells and
electrotonic coupling between cells.36 37 Each cardiac
cell was presumed to have a unique set of electrical properties
(variable upstroke velocity and APDs) on the basis of its geometry
and distribution of ionic channels. Because the spatial distribution of
these intrinsic cellular properties was not known, models assumed
either a random two-dimensional distribution of APDs37
or a one-dimensional strand of cells with uniform APDs arranged
with an abrupt boundary where cells with short APDs were coupled to
cells with long APDs.36 In both cases, electrical coupling
between cells was shown to modulate the intrinsic APDs of cells. Under
normal conditions (low-resistance electrotonic coupling between
cells), the local variation of APDs was averaged out and was thus not
evident. Ischemia, which induces an uncoupling of
cells,38 was modeled as an increase in axial resistance
between cells. Simulations with high intercellular resistances produced
significant variations in APDs.36 37 These simulations
failed to predict repolarization patterns and heterogeneities in APDs
because they did not include spatial heterogeneities of APDs and of
intracellular coupling.
Thus, the present study obtained direct experimental evidence that repolarization spreads from cell to cell on the epicardium, whereas on the endocardium, it does not spread and patterns are irregular.
Origin(s) of Repolarization Wave Fronts
Whereas depolarization is normally initiated by the pacemaker
cells of the SA node and follows well-established pathways, little
is known regarding the origin(s) and spreading of repolarization.
Optical repolarization maps indicated that some unidentified cells
(with short APDs) repolarize first to drive the repolarization of
adjacent cells, which would otherwise exhibit longer APDs. On the left
and right ventricular epicardium, the first sites to
repolarize were located near the apex at one third of the distance
toward the base. These cells most likely represented
breakthrough sites rather than the origin(s) of repolarization wave
fronts. The possibility that repolarization began on the endocardium
was tested by using the light guide and the photodiode array to
simultaneously measure APs from the endocardium and
epicardium on opposite sides of left ventricles. APDs on the
endocardium were consistently shorter by 9±1.8 ms compared
with the epicardium. Delays between endocardial and epicardial
activation and repolarization were brief, as expected, given the wall
thickness of ventricles. These delays were nevertheless significant
because (1) the sampling rate of signals was sufficiently rapid (0.7 ms
per frame), (2) the signal processing techniques identified unique
activation and repolarization timepoints, and (3) these brief
activation and repolarization delays were reproducible from beat to
beat when we compared endocardial to epicardial delays or delays
between adjacent diodes on the epicardium.
In perfused ventricular sheets, APDs were heterogeneous on the epicardium, being shorter near the apex than the base by 26 ms (n=8). On the endocardium, APDs were consistently shorter and more homogeneous than on the epicardium, being shorter near the apex than the base by 9 ms (n=8). These findings on perfused sheets supported the equivalent measurements on intact hearts using the light guide and photodiode array to determine APDs simultaneously on the endocardium and epicardium.
On the endocardium, the first site to depolarize was the first to
repolarize, but repolarization appeared random. The random
repolarization on the endocardium was attributed to the greater
homogeneity of APDs. If all cells had identical APDs, one would predict
that the first cells to depolarize would be the first to repolarize,
and the spread of repolarization and activation would be similar.
However, slight heterogeneities in APDs would produce random
repolarization patterns that spread slightly slower (13.8 to 23.0 ms)
compared with activation (11.5 to 15.4 ms, Fig 12A through 12D). These
data suggested that repolarization in intact hearts was initiated on
the endocardium at a region that repolarized first because it
depolarized first. Repolarization could then spread transmurally, break
through to the epicardium, and spread anisotropically along the
epicardial surface (Figs 5 and 6).
Repolarization Patterns Measured From Dog and Guinea Pig
Hearts
Repolarization has been extensively mapped in dog hearts with
extracellular electrodes, but no equivalent studies exist in guinea pig
hearts. Repolarization measured by ARIs has indicated that
repolarization spreads passively and that once the heart is
activated, repolarization duration is independent of the
activation sequence.39 In isolated blood-perfused dog
hearts, repolarization patterns measured from the anterior and
posterior surfaces were largely independent of the stimulation
protocol.39 The present measurement in guinea pig
hearts demonstrates that in the epicardium there is a substantial
degree of independence between activation and repolarization, as in dog
hearts. A number of factors could account for the striking correlation
between repolarization and fiber orientation in guinea pig hearts,
which was not reported in dog hearts: (1) The present studies
examined the ventricular free walls where fiber
orientations are highly regular compared with the anterior and
posterior septal surfaces. (2) In the dog heart, the repolarization
sequence was highly dependent on the activation sequence. This was
mainly due to the long activation time of the dog ventricles (30 to 60
ms), which tended to be greater than the intrinsic heterogeneities of
APDs. In the smaller guinea pig heart, the repolarization sequence was
independent of the activation sequence, because inhomogeneities of APDs
were larger than the duration of time it took to depolarize the
ventricles (5 ms in sinus rhythm, 20 ms during epicardial
pacing). (3) There may be species differences in the expression and/or
distribution of ionic channels. For instance, in dogs there are known
heterogeneities in the distribution of Ito,
resulting in greater Ito and longer APDs in epicardial than
endocardial cells.40 In contrast, guinea pig myocytes lack
Ito, yet studies of isolated myocytes reported
longer APDs from cells isolated from the base than the
apex41 and longer APDs in intramural cells compared with
endocardial and epicardial cells.42 The present
optical maps are consistent with heterogeneities of APDs
between myocytes from the apex and the base. (4) In situ studies of dog
hearts using monophasic AP recordings demonstrated an inverse
relation between APDs and activation time. When activation patterns
were altered by pacing the epicardium, this inverse relation was lost
but returned 2 hours later. This phenomenon, called cardiac memory,
indicated that under certain conditions repolarization was modified by
activation after substantial time delays.6 (5) The latter
studies bring up another complexity, because APDs are known to depend
on the mode of contraction. As shown in papillary muscles, isotonic
shortening prolongs APDs, whereas isometric contractions shorten
APDs.43 Such a mechanism could produce different
disparities of APDs along ventricles and from endocardium to epicardium
and could contribute to the different results in dog hearts in situ
compared with Langendorff-perfused guinea pig and dog
(blood-perfused) hearts, which do not perform mechanical
work.39
Mapping of Repolarization by Electrode and Optical
Techniques
The independence of activation and repolarization could be
detected by optical and ARI techniques, since both methods measure
repolarization time points [optical,
(d2F/dt2)max of AP downstrokes;
ARI, (dV/dt)max of T waves] that are coincident with the
refractory period of APs.8 25 30 In contrast with
epicardial repolarization, the first site to repolarize on the
endocardium was the first to activate, and APDs were more
uniform on the endocardium compared with the epicardium. ARI
measurements were not reported for the dog endocardium.
Optical techniques offer advantages as well as disadvantages compared with extracellular electrodes: (1) The voltage-dependent response of the dye is equivalent to an intracellular recording, dependent on the local transmembrane potential, and does not detect potential changes beyond the molecular distance of the dye. In contrast, unipolar and, to a lesser extent, bipolar recordings integrate potential changes beyond their physical dimensions. (2) Optical signals are independent of the direction of spread, whereas in bipolar recordings the shape and kinetics of the signals depend on the direction of spread. (3) The dyes' responses are not influenced by electrical artifacts. (4) Optical maps are readily obtained at high density, with virtually no dead space between recording sites; consequently, the electrical activity of the entire surface of the epicardium is investigated with equal weight. With optical recordings, there are concerns regarding phototoxic effects and motion artifact. Some of these problems have been overcome with better dyes, chamber designs, and signal processing to measure repolarization time points; however, optical methods have not been successfully applied in situ (blood-perfused hearts) or in the clinical setting or to measure intramural APs, as can be done with plunge electrodes.
Put together, optical maps of APs provide a set of rules that guide activation and repolarization pathways in guinea pig hearts under physiological sinus rhythm or under stimulation at a number of different epicardial sites. From these guidelines, three-dimensional patterns of propagation can be predicted for activation and repolarization in intact hearts.
|
Selected Abbreviations and Acronyms |
|---|
|
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|
Acknowledgments |
|---|
This study was supported by grants from the Whitaker Research Foundation and the American Heart Association (No. 87-1065) to Dr Salama and the Western Pennsylvania Affiliate of the American Heart Association to Dr Kanai. The authors would like to thank William Hughes for machining chambers and optical components, Gregory Szekeres and James VonHedemann for manufacturing electronic components, Gregory Shander for his assistance with the histology, William Bolish for software support, Richard Lombardi for his expertise in computer hardware and software, and Anne Crews, Dr Madison Spach, and Dr Harold Strauss for their critical review of the manuscript.
|
Footnotes |
|---|
Previously published as preliminary results in abstract form (Circulation. 1988;78:II-413 and 1990;82:III-649).
Received December 16, 1993; accepted June 13, 1995.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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3.
Spach MS, Barr RC. Analysis of
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