Assessment With Potential Mapping Protective Effect of a Drug. Example
P. d’ALcHi,
P.
CLAUSER,
is a computerized
SATAPEC
multiplexed
channels
The
are printed
results
color)
showing
activation, The
placed
serving mesh
around
unipolar
before
and then
are plotted
calculated
aligning
are
versus
all of the individual
extent
of the
(2.5 mg/kg)
Key Words:
epicardial
injured
Potential
area
a beneficial
mapping;
of
tracings
Cardiac
With
Once
1
the
instants
is are
of the two groups maps,
obtained
by
these maps the location
on ischemia
ischemia;
the 240
ST variation
at different curves
is
starting
terminal)
ligation.
can be visualized.
effect
from
at Wilson
potential
An
electrodes
is made
eliminated,
ZST/240
electric
As an
with TMZ).
wire
are taken
taken
ST variations
maps, are then printed.
has shown
silver
min for 8 min following
Mean
SATAPEC.
pretreated
Recordings
and the mean
time.
with
A ligature
potential
any aberrant
memory
plotted
group
chlorided
(AIV).
Files containing
in the computer
of rabbits
artery every
or in
duration
using 2 groups of 12 rabbits
thoracotomy.
(reference
and
automatically.
is examined
spaced
following
electrograms
in black and white repolarization,
may be assessed
and the second
regularly
over 256 potentials.
data.
(TMZ)
interventricular
ligation
electrograms stored
240
the ventricles
epicardial
of maps (either
of a drug
as a control
of cardiac electrical
depolarization,
analog-digital
ability
with
from the anterior
and
distributions,
other
GAUTHIER
processing
out in the form
potential
V.
in the Laboratory for recording
built
the effect of trimetazidine
(one group
TMZ
system
cardioprotective
elastomer
AND
MOREL,
and for automatic
or various
example,
min
M.
of the Cardiac of Trimetazidine
Pretreatment
with
injury.
Trimetazidine
INTRODUCTION SATAPEC Electriques
(Systgme
d’Acquisition
Cardiaques)
et de Traitement
is a computerized
Automatique
system entirely
des Potentiels
developed
in our Labo-
ratory (Gaudron and d’Alchb,1977) that automatically records and processes cardiac electrical potentials (Figure 1). It consists of different electrode arrays, an acquisition chain with 256 multiplexed channels, a logic control and its own 512 k x 10 bit memory,
and a software
73 minicomputer,
library.
This library
a 40 megabyte
is presently
hard disk, a Tektronix
running
on with a PDP ll-
4209 color
terminal
and a
Sharp printer. With SATAPEC, it is possible to acquire up to 256 unipolar electrograms at the epicardial, endocardial or intraparietal levels during a single systole.
From the Laboratoire and the lnstitut Address Esplanade Received
de Physiologie
de Recherches
reprint
requests
to: Pr. P. d’Alch6,
de la Paix, 14000 Caen, May 1990;
et Bioinformatique
lnternationales
revised
Servier
(P.d’A, (P.L.),
Laboratoire
M.M.,
V.G.),
Neuilly-sur-Seine,
de Physiologie
Universite,
Caen,
France,
France.
et Bioinformatique,
Universit6,
France.
and accepted
January 1991.
43 Journal
of Pharmacological
0 1991
Elsewer
Science
Methods Publishing
26. 43-51 Co.,
Inc.,
(1991) 655 Avenue
016@5402/911$3.50 of the Americas,
hkw
pork,
NY 10010
44
P. d’Alchk et al.
::.. . . ..:. ; y;: 6 ::: : : : : :,:::
:.,:::: : :.,.: ‘.::.z;: ;
24x10
points
4ld-h 612kxlObii
4-24 -
P*
Electrode
XY
Colour printer
256 channels acquisition system
arrays
0
n
Computer system
plotter FIGURE 1.
Colour terminal
General diagram of SATAPEC
After analog-digital conversion and data processing, the results are displayed on the color terminal and then printed out (either in black and white or in color) as maps showing electrical potential distributions, depolarization, repolarization, durations of activation, or other types of data (e.g., A-H or H-V intervals). One of SATAPEC’s applications is the automated measurement of ST variations during the coronary ligation of an animal (dog, rabbit, etc.). Results are displayed in the form of evolution curves plotting mean ST variation vs. time and as electrical potential maps that visualize the location and the extent of the epicardial injured area. SATAPEC can thus be used to assess the cardioprotective effect of a drug. The technique used differs according to whether animals have been pretreated or not. In non pretreated animals, a repetitive ligation is performed in situ on the heart (each animal being its own control), but in pretreated animals a single ligation is used. The example discussed in this article is that of the single ligation technique used on animals pretreated with trimetazidine (TMZ). Trimetazidine (TMZ) or I-(2,3,4-trimethoxybenzyh-piperazine dihydrochloride (VASTAREL* 20 mg) was first used by Schmitt in 1964 as a treatment for angor. Since then, many researchers (Harpey et al., 1989) have demonstrated its cellular antiischemic activity by using it to reduce the deleterious effects of free radicals (Maridonneau-Parini, and Harpey, 1985; Guarnieri and Muscari, ‘l988), to reduce acidosis (Renaud, 1988; Lavanchy et al., 1987), and to obtain a more rapid reconstitution of energetic compounds (Lavanchy et al., 1987). Its ability to protect the heart has been underlined by electrophysiological findings (Honore et al., 1986). The clinical ben-
Mapping of Drug-Induced
Cardiac Protection
efits of TMZ can be seen during an exercise testing in a reduction of ST depression and in a lengthening of the time to onset of this ST depression (Sellier et al., 1987;. Passeron, 1986). METHODS Experimental
Procedure
The experiments were carried out using 2 groups of 12 New Zealand rabbits of either sex. One group served as a control and the other was pretreated with TMZ, receiving 2.5 mg/kg of TMZ through a catheter twice daily for a week. The last dose was administered 2 hours before ligation of the anterior interventricular artery (AIV). Following anesthesia (30 mg/kg of pentobarbital sodium) and thoracotomy, an elastomer mesh containing 240 evenly spaced chlorided silver electrodes is placed around the ventricles. A ligation is performed at the root of the AIV. Recordings are taken during one entire systole from the 240 unipolar epicardial electrograms (reference potential taken at Wilson terminal) at the following times: 1 min before ligation and every min for 8 min after ligation During the acquisition, the 240 signals are amplified individually by means of wide bandwidth amplifiers (0.05 Hz-2 kHz). The analogue input from each channel is stabilized by a “sample and hold” device before being simultaneously digitalized to 10 bit resolution. The sampling rate of 1 kHz corresponds to a total transfer rate of 240 kHz into the memory of SATAPEC. This implies that at the end of each millisecond, the 240 values are memorized. As it is necessary to start the record before the beginning of the systole, the QRS complex of one of immediately previous 240 electrograms is used for triggering the acquisition chain. An adjustable delay allows shifting of the recordings. In a second step (taking about 10 set), the values of the entire systole are transferred into the computer memory, in order to be processed. At the end of each experiment, data processing is carried out as follows: the 240 electrograms (Figure 21, with their recording electrode number, are plotted on paper using an XY table. Aberrant electrograms are eliminated and replaced by interpolation. all electrograms are displayed on the terminal one by one and the automatic positioning of the base line on the diastolic isoelectric segment is checked and, if necessary, corrected. Thus with this interactive software, the operator can eliminate artifacts. The measurement of ST amplitude of the 240 electrograms is carried out automatically. To this end, the instant of measurement is chosen after displaying the electrograms on the screen of the computer terminal with a simultaneously adjustable vertical bar for each tracing. The operator places the vertical bar at mid-
45
46
P. d’Alch6 et al.
6
‘36 _-k
1
100 MS.
FIGURE 2. Examples of unipolar epicardial electrograms from the 240 recorded simultaneously with SATAPEC. A: 1 min before the ligation, B: 8 min after the ligation.
plateau of the ST elevation on the more suitable tracings and verifies that this position is correct for the other tracings. At this instant-the same for all the tracingsthe ST amplitudes of the 240 electrograms are measured from the baseline, taken as the zero potential. Amongst the 240 potential values obtained, only the values superior or equal to 1 mV are taken into consideration. As a control, ST values are printed according to the position of the measuring electrode, which is designated by its number. Once the files containing ST variations at the different instants (0, 1, 2, 3, 4, 5, 6, 7, 8 min) are stored, the EST/240 curve is plotted vs. time for each animal. The color potential maps corresponding to the instants (0, 1, 2, 3, 4, 5, 6, 7, 8 min) of the ligation are drawn. When all the experiments have been treated, the mean variation and standard deviation of each group of rabbits are calculated. Then the evolution curve for these means during the 8 min ligation period is plotted automatically. After the alignment of the individual maps with respect to the location of the ligation, the mean potential maps for the control and the pretreated groups are plotted
and compared.
Statistical Analysis The statistical analysis of ST variation of more than 1 mV in the control and pretreated groups during the 8 min ligation period involves a two-way ANOVA (treatment x time) with repeated measurements on time (0, 2, 4, 6, 8 mn).
Mapping of Drug-Induced The following
parameters
are taken
Cardiac Protection
into consideration:
mean elevation of ST segment in millivolts (mV) the extent of the lesion (number of values of more than 1 mV over the total number of potential Moreover, defined: limited
values
(240)).
in assessing
injury
the
gravity
Cl :ST elevation
of the
of between
lesion,
three
classes
of injury
were
1 and 3 mV
moderate injury G2:ST elevation of between 3 and 7 mV major injury G3:ST elevation of more than 7 mV. The groups can be considered to be homogeneous at the outset as all values are equal to 0. When group x time interaction is significant, supplementary analysis is performed
by the Newman-Keuls
test.
RESULTS Changes over time of mean ST variation show a significant difference (P = 0.017) between the two groups (control an pretreated), justifying supplementary analysis with the Newman-Keuls test. The Table 1 and the curves (Figure 3) show the significant effect of TMZ, the mean ST variation being lower in pretreated animals. This result is also displayed in the
ST elevation
0 FIGURE 3.
(mV)
2
4
6
Mean ST elevation in both groups after LAD ligation. -
Time (mid 8 Control _.__.__TMZ.
47
48
P. d’Alch6 et al.
POTENTIAL CONTROL
MAP N=12
C88TEM. ,
AIU
,
8
1 @I
MN
o1t -aa -08 .a0 .a0
-08 -00 -00
POTENTIAL
MQP
TMZ
,
N=12
AIU
C88TMZ. ,
8
101
MN
FIGURE 4. Map of mean potentials, measured 8 min after the beginning of the coronary ligation, at the ST plateau on the 12 control rabbits (top) and the 12 rabbits pretreated with TMZ (bottom).
form of maps of mean potentials at min 8 of the ligation period (Figure 4). These maps give information as to the location and the extent of lesions in both groups. In the pretreated group the ischemic area is reduced and interface between the ischemic and the healthy area, which is known to create electrical instability, is reduced.
Mapping of Drug-Induced
Cardiac Protection
DISCUSSION
The advantages of SATAPEC in assessing the cardioprotective as follows:
effect of a drug are
rapid display of results through the use of an epicardial mesh with 240 recording points, a multiplexing chain with analog-digital conversion and a computer. possibility of data checking and, if necessary, correction of aberrant data. output of results in the form of maps of epicardial potentials recorded during the ST plateau. This makes it possible to visualize the location and the extent of the injured area and thereby to assess the effects of a drug. The amplitude of ST segment increases quickly during the first minutes of the ligation. The main advantage of SATAPEC is to allow all the measurements of ST amplitude on the 240 electrograms during the same systole. In these experiments, the statistical analysis proves that 12 rabbits for the control and pretreated groups are enough for obtaining significant results. The fundamental question is: may the displacement of ST segment be considered to express ischemic phenomena? As early as 1920, Pardee observed that ST segment elevation in electrocardiograms is a clinical sign of myocardial ischemia. Wilson et al. (19331, Bailey et al. (19441, Rakita et al. (1954) demonstrated that following coronary ligation, there is a displacement of the ST segment on electrocardiograms. Thus, displacement of the ST segment (either as an elevation or as a depression) may be considered to be an index of ischemic lesion. ST segment displacement during ischemia is due to “injury currents” that form between ischemic and healthy areas during both systole and diastole. This has been confirmed by magnetocardiography, a technique that records variations in magnetic fields produced by cardiac ionic currents (Cohen and Kauffmann, 1975). An ischemic cell shows a drop in amplitude, a change in action potential plateau and a decrease in resting potential when compared to a healthy cell. These facts can account for the presence of systolic and diastolic “injury currents.” Is it possible that factors other than ischemia affect the ST segment? It is known that changes in pH levels, in the concentration of electrolytes in intra or extracellular media, changes in tem-
TABLE 1
Mean ST Elevation Expressed in mV. GROUP
X
TIME
N Control Treated
12 12
BEFORE
0.00 +- 0.00 0.00 k 0.00 p=l
2 MIN 2.86 2 0.24 2.53 2 0.16 p = 0.41 N.S.
4
MIN
3.71 2 0.36 2.59 t 0.18 p = 0.007 S.
6
MIN
4.09
” 0.50 3.01 k 0.18 p = 0.009 s.
8
MIN
4.21 2 0.43 3.19 k 0.30 p = 0.014 S.
INTERACTION
p = 0.017
Group effect
49
50
P. d’Alch6
et al.
perature
and the influence
segment. variation
A reduction in coronary perfusion pressure produces of the ST segment (Maroko et al., 19711, but all these
known
to be associated
resulting
of the cardiac
with ischemia.
from the enlargement
sympathetic
Another
all have an effect
of the QRS complex
in the
disturbances
critical factor is secondary
a phenomenon known as the QRS-ST-T gradient If such conduction disorders exist, the experiments
on the ST
a change
due to conduction
are
ST change disorders,
effect (Massie and Walsh, 1960). are not taken into consideration.
In spite of these restrictions the measuring STvariations remains the basic technique for assessing ischemia both experimentally and clinically. The measurement of ST variation produced by SATAPEC is in fact the combination of TQ depression and ST elevation or, in other words, the variation measured from the baseline (and not from the true zero line, which is unknown). To determine the zero
line and thereby
evaluate
the exact role of the diastolic
and systolic injury
currents, it is necessary to use amplifiers with a direct current (DC) input. Given the large number of electrodes (240), the use of DC amplifiers would cause baseline stability
problems
that would
be very difficult
are made over short time intervals. to determine the zero line. The ST measurements
were
In order
only taken
to overcome
when
to assess &hernia,
during
measurements
it is not necessary
the first 8 min of ligation
as most
major biochemical changes in intra or extracellular media occur as occlusion time goes beyond 10 min. Schomig et al. (19841, for example, have observed that the level of plasmatic noradrenaline rises if occlusion time exceeds 10 min. In addition, Rakita et al. (1954) have shown that the amplitude of ST variation increases within a few min of the occlusion of an artery and reaches a plateau within 5 to 7 min. Another problem concerns the location and the number of electrodes ensure a reliable assessment of ST variation. In animal experimentation, done
at the epicardial
level have proven
to be the most accurate
cordial thoracic derivations, many potentials an ischemic zone is completely surrounded heterogeneous conduction distort recorded plottings.
needed to recordings
method.
In pre-
are “cancelled out,” particularly when by a healthy zone. In the latter case,
media interposed between the heart and the thorax The number of epicardial electrodes has to be high be-
cause current density is at a maximum at the junction of ischemic and healthy zones, but decreases rapidly beyond this point. Generally an insufficient number of electrodes
is used in epicardial
plotting
of ST variations.
CONCLUSION With SATAPEC it is possible taneously, i.e., during a single siderable epicardial cantly
to record up to 256 unipolar electrograms systole. Automatic data processing results
simulin con-
time saving. The antiischemic properties of a drug are clearly revealed by potential maps, as shown with trimetazidine, which was able to signifi-
reduce
the extent
of the ischemic
lesion.
REFERENCES Bailey RH, Ladue JS, York graphic
changes
DJ (1944)
(local ventricular
Electrocardioischemia
and
injury)
produced
sion of a coronary
in the dog by temporary artery,
showing
occiu-
a new stage
Mapping of Drug-Induced in the evolution
of myocardial
infarction.
Am
Heart / 27:164.
Cohen D, Kauffmann LA (1975) Magnetic determination of the relationship between the S-T segment shift and the injured current produced by coronary artery occlusion. Circ Research 36:414424. Dalla Volta S (1990) Comparison of trimetazidine with nifedipine in effort angina: a double-blind, cross-over study. Cardiovasc Drug and Therapy, in press. Gaudron J, d’Alche P (1977) An instrument for simultaneous collection of 256 electrocardiograms associated with an on-line computer. Adv in Cardiology
19:187-191.
Guarnieri C, Muscari C (1988) Beneficial effects of trimetazidine on mitochondrial function and superoxide production in the cardiac muscle of monocrotaline-treated rats. Biochemical Pharmacol37(24)
:4685-4688.
Harpey C, Clauser P, Labrid C, Freyria JL, Poirier JP (1989) Trimetazidine, a cellular antiischemic agent. Cardiovasc Drug Rev 6(4):292-312. Honore E, Adamantidis MM, Challice CE, Dupuis BA (1986) Cardioprotection by calcium antagonists, piridoxilate and trimetazidine. /RSC Medical Science
14:938-939.
Lavanchy N, Martin J, Rossi A (1987) Anti-ischemic effects of trimetazidine: 31 P-NMR Spectroscopy in the isolated rat heart. Arch intern Pharmacodyn Ther 286:97-110.
Maridonneau-Parini I, Harpey C (1985) Effect of trimetazidine on membrane damage induced by oxygen free radicals in human red cells. Brl C/in Pharmac
20:148-151.
Cardiac Protection
Maroko AR, Kjekshus JK, Sobel BE, Watanabe T, Covell JW, Ross J, Braunwald E (1971) Factors influencing infarct size following experimental coronary artery occlusion. Circulation 43:67. Massie E, Walsh JJ (1960) Clinical vectorcardiography and electrocardiography. Yearbook Medical Publ Chicago. Pardee HE6 (1920) An electrocardiographic sign of coronary artery obstruction. Arch intern Med 26: 244-249.
Passeron J (1986) Efficacite de la trimetazidine dans I’angor d’effort stable de I’insuffisant coronarien chronique. La Presse Medicale 15:1775-1778. Rakita L, Borduas JL, Rothman S, Prinzmetal M (1954) Studies on the mechanism of ventricular activity. Early changes in the RS-T segment and QRS complex following acute coronary artery occlusion; experimental study and clinical applications. Am Heart / 48:351. Renaud JF (1988) Internal pH, Nat and Ca*+ regulation by trimetazidine during cardiac cell acidosis. Cardiovasc Drugs Therap 1:677-686. Schomig A, Dart AM, Dietz R, Mayer E, Kubler W (1984) Release of endogenous catecholamines in the ischemic myocardium of the Rat. Part A: Locally mediated release. Circ Research 55:689701.
Sellier P, Audouin P, Payen B, Corona P, Duong TC, Ourbak P (1987) Acute effects of trimetazidine evaluated by exercise testing. Eur / C/in Pharmacol33:205-207.
Wilson FM, MacLeod AC, Johnston FD, Hill GW (1933) Monophasic electrical response produced by the contraction of injured heart muscle. Proced
Sot
Exp Biol30:
797-798.
51
P. d’ALcHi,
P.
CLAUSER,
is a computerized
SATAPEC
multiplexed
channels
The
are printed
results
color)
showing
activation, The
placed
serving mesh
around
unipolar
before
and then
are plotted
calculated
aligning
are
versus
all of the individual
extent
of the
(2.5 mg/kg)
Key Words:
epicardial
injured
Potential
area
a beneficial
mapping;
of
tracings
Cardiac
With
Once
1
the
instants
is are
of the two groups maps,
obtained
by
these maps the location
on ischemia
ischemia;
the 240
ST variation
at different curves
is
starting
terminal)
ligation.
can be visualized.
effect
from
at Wilson
potential
An
electrodes
is made
eliminated,
ZST/240
electric
As an
with TMZ).
wire
are taken
taken
ST variations
maps, are then printed.
has shown
silver
min for 8 min following
Mean
SATAPEC.
pretreated
Recordings
and the mean
time.
with
A ligature
potential
any aberrant
memory
plotted
group
chlorided
(AIV).
Files containing
in the computer
of rabbits
artery every
or in
duration
using 2 groups of 12 rabbits
thoracotomy.
(reference
and
automatically.
is examined
spaced
following
electrograms
in black and white repolarization,
may be assessed
and the second
regularly
over 256 potentials.
data.
(TMZ)
interventricular
ligation
electrograms stored
240
the ventricles
epicardial
of maps (either
of a drug
as a control
of cardiac electrical
depolarization,
analog-digital
ability
with
from the anterior
and
distributions,
other
GAUTHIER
processing
out in the form
potential
V.
in the Laboratory for recording
built
the effect of trimetazidine
(one group
TMZ
system
cardioprotective
elastomer
AND
MOREL,
and for automatic
or various
example,
min
M.
of the Cardiac of Trimetazidine
Pretreatment
with
injury.
Trimetazidine
INTRODUCTION SATAPEC Electriques
(Systgme
d’Acquisition
Cardiaques)
et de Traitement
is a computerized
Automatique
system entirely
des Potentiels
developed
in our Labo-
ratory (Gaudron and d’Alchb,1977) that automatically records and processes cardiac electrical potentials (Figure 1). It consists of different electrode arrays, an acquisition chain with 256 multiplexed channels, a logic control and its own 512 k x 10 bit memory,
and a software
73 minicomputer,
library.
This library
a 40 megabyte
is presently
hard disk, a Tektronix
running
on with a PDP ll-
4209 color
terminal
and a
Sharp printer. With SATAPEC, it is possible to acquire up to 256 unipolar electrograms at the epicardial, endocardial or intraparietal levels during a single systole.
From the Laboratoire and the lnstitut Address Esplanade Received
de Physiologie
de Recherches
reprint
requests
to: Pr. P. d’Alch6,
de la Paix, 14000 Caen, May 1990;
et Bioinformatique
lnternationales
revised
Servier
(P.d’A, (P.L.),
Laboratoire
M.M.,
V.G.),
Neuilly-sur-Seine,
de Physiologie
Universite,
Caen,
France,
France.
et Bioinformatique,
Universit6,
France.
and accepted
January 1991.
43 Journal
of Pharmacological
0 1991
Elsewer
Science
Methods Publishing
26. 43-51 Co.,
Inc.,
(1991) 655 Avenue
016@5402/911$3.50 of the Americas,
hkw
pork,
NY 10010
44
P. d’Alchk et al.
::.. . . ..:. ; y;: 6 ::: : : : : :,:::
:.,:::: : :.,.: ‘.::.z;: ;
24x10
points
4ld-h 612kxlObii
4-24 -
P*
Electrode
XY
Colour printer
256 channels acquisition system
arrays
0
n
Computer system
plotter FIGURE 1.
Colour terminal
General diagram of SATAPEC
After analog-digital conversion and data processing, the results are displayed on the color terminal and then printed out (either in black and white or in color) as maps showing electrical potential distributions, depolarization, repolarization, durations of activation, or other types of data (e.g., A-H or H-V intervals). One of SATAPEC’s applications is the automated measurement of ST variations during the coronary ligation of an animal (dog, rabbit, etc.). Results are displayed in the form of evolution curves plotting mean ST variation vs. time and as electrical potential maps that visualize the location and the extent of the epicardial injured area. SATAPEC can thus be used to assess the cardioprotective effect of a drug. The technique used differs according to whether animals have been pretreated or not. In non pretreated animals, a repetitive ligation is performed in situ on the heart (each animal being its own control), but in pretreated animals a single ligation is used. The example discussed in this article is that of the single ligation technique used on animals pretreated with trimetazidine (TMZ). Trimetazidine (TMZ) or I-(2,3,4-trimethoxybenzyh-piperazine dihydrochloride (VASTAREL* 20 mg) was first used by Schmitt in 1964 as a treatment for angor. Since then, many researchers (Harpey et al., 1989) have demonstrated its cellular antiischemic activity by using it to reduce the deleterious effects of free radicals (Maridonneau-Parini, and Harpey, 1985; Guarnieri and Muscari, ‘l988), to reduce acidosis (Renaud, 1988; Lavanchy et al., 1987), and to obtain a more rapid reconstitution of energetic compounds (Lavanchy et al., 1987). Its ability to protect the heart has been underlined by electrophysiological findings (Honore et al., 1986). The clinical ben-
Mapping of Drug-Induced
Cardiac Protection
efits of TMZ can be seen during an exercise testing in a reduction of ST depression and in a lengthening of the time to onset of this ST depression (Sellier et al., 1987;. Passeron, 1986). METHODS Experimental
Procedure
The experiments were carried out using 2 groups of 12 New Zealand rabbits of either sex. One group served as a control and the other was pretreated with TMZ, receiving 2.5 mg/kg of TMZ through a catheter twice daily for a week. The last dose was administered 2 hours before ligation of the anterior interventricular artery (AIV). Following anesthesia (30 mg/kg of pentobarbital sodium) and thoracotomy, an elastomer mesh containing 240 evenly spaced chlorided silver electrodes is placed around the ventricles. A ligation is performed at the root of the AIV. Recordings are taken during one entire systole from the 240 unipolar epicardial electrograms (reference potential taken at Wilson terminal) at the following times: 1 min before ligation and every min for 8 min after ligation During the acquisition, the 240 signals are amplified individually by means of wide bandwidth amplifiers (0.05 Hz-2 kHz). The analogue input from each channel is stabilized by a “sample and hold” device before being simultaneously digitalized to 10 bit resolution. The sampling rate of 1 kHz corresponds to a total transfer rate of 240 kHz into the memory of SATAPEC. This implies that at the end of each millisecond, the 240 values are memorized. As it is necessary to start the record before the beginning of the systole, the QRS complex of one of immediately previous 240 electrograms is used for triggering the acquisition chain. An adjustable delay allows shifting of the recordings. In a second step (taking about 10 set), the values of the entire systole are transferred into the computer memory, in order to be processed. At the end of each experiment, data processing is carried out as follows: the 240 electrograms (Figure 21, with their recording electrode number, are plotted on paper using an XY table. Aberrant electrograms are eliminated and replaced by interpolation. all electrograms are displayed on the terminal one by one and the automatic positioning of the base line on the diastolic isoelectric segment is checked and, if necessary, corrected. Thus with this interactive software, the operator can eliminate artifacts. The measurement of ST amplitude of the 240 electrograms is carried out automatically. To this end, the instant of measurement is chosen after displaying the electrograms on the screen of the computer terminal with a simultaneously adjustable vertical bar for each tracing. The operator places the vertical bar at mid-
45
46
P. d’Alch6 et al.
6
‘36 _-k
1
100 MS.
FIGURE 2. Examples of unipolar epicardial electrograms from the 240 recorded simultaneously with SATAPEC. A: 1 min before the ligation, B: 8 min after the ligation.
plateau of the ST elevation on the more suitable tracings and verifies that this position is correct for the other tracings. At this instant-the same for all the tracingsthe ST amplitudes of the 240 electrograms are measured from the baseline, taken as the zero potential. Amongst the 240 potential values obtained, only the values superior or equal to 1 mV are taken into consideration. As a control, ST values are printed according to the position of the measuring electrode, which is designated by its number. Once the files containing ST variations at the different instants (0, 1, 2, 3, 4, 5, 6, 7, 8 min) are stored, the EST/240 curve is plotted vs. time for each animal. The color potential maps corresponding to the instants (0, 1, 2, 3, 4, 5, 6, 7, 8 min) of the ligation are drawn. When all the experiments have been treated, the mean variation and standard deviation of each group of rabbits are calculated. Then the evolution curve for these means during the 8 min ligation period is plotted automatically. After the alignment of the individual maps with respect to the location of the ligation, the mean potential maps for the control and the pretreated groups are plotted
and compared.
Statistical Analysis The statistical analysis of ST variation of more than 1 mV in the control and pretreated groups during the 8 min ligation period involves a two-way ANOVA (treatment x time) with repeated measurements on time (0, 2, 4, 6, 8 mn).
Mapping of Drug-Induced The following
parameters
are taken
Cardiac Protection
into consideration:
mean elevation of ST segment in millivolts (mV) the extent of the lesion (number of values of more than 1 mV over the total number of potential Moreover, defined: limited
values
(240)).
in assessing
injury
the
gravity
Cl :ST elevation
of the
of between
lesion,
three
classes
of injury
were
1 and 3 mV
moderate injury G2:ST elevation of between 3 and 7 mV major injury G3:ST elevation of more than 7 mV. The groups can be considered to be homogeneous at the outset as all values are equal to 0. When group x time interaction is significant, supplementary analysis is performed
by the Newman-Keuls
test.
RESULTS Changes over time of mean ST variation show a significant difference (P = 0.017) between the two groups (control an pretreated), justifying supplementary analysis with the Newman-Keuls test. The Table 1 and the curves (Figure 3) show the significant effect of TMZ, the mean ST variation being lower in pretreated animals. This result is also displayed in the
ST elevation
0 FIGURE 3.
(mV)
2
4
6
Mean ST elevation in both groups after LAD ligation. -
Time (mid 8 Control _.__.__TMZ.
47
48
P. d’Alch6 et al.
POTENTIAL CONTROL
MAP N=12
C88TEM. ,
AIU
,
8
1 @I
MN
o1t -aa -08 .a0 .a0
-08 -00 -00
POTENTIAL
MQP
TMZ
,
N=12
AIU
C88TMZ. ,
8
101
MN
FIGURE 4. Map of mean potentials, measured 8 min after the beginning of the coronary ligation, at the ST plateau on the 12 control rabbits (top) and the 12 rabbits pretreated with TMZ (bottom).
form of maps of mean potentials at min 8 of the ligation period (Figure 4). These maps give information as to the location and the extent of lesions in both groups. In the pretreated group the ischemic area is reduced and interface between the ischemic and the healthy area, which is known to create electrical instability, is reduced.
Mapping of Drug-Induced
Cardiac Protection
DISCUSSION
The advantages of SATAPEC in assessing the cardioprotective as follows:
effect of a drug are
rapid display of results through the use of an epicardial mesh with 240 recording points, a multiplexing chain with analog-digital conversion and a computer. possibility of data checking and, if necessary, correction of aberrant data. output of results in the form of maps of epicardial potentials recorded during the ST plateau. This makes it possible to visualize the location and the extent of the injured area and thereby to assess the effects of a drug. The amplitude of ST segment increases quickly during the first minutes of the ligation. The main advantage of SATAPEC is to allow all the measurements of ST amplitude on the 240 electrograms during the same systole. In these experiments, the statistical analysis proves that 12 rabbits for the control and pretreated groups are enough for obtaining significant results. The fundamental question is: may the displacement of ST segment be considered to express ischemic phenomena? As early as 1920, Pardee observed that ST segment elevation in electrocardiograms is a clinical sign of myocardial ischemia. Wilson et al. (19331, Bailey et al. (19441, Rakita et al. (1954) demonstrated that following coronary ligation, there is a displacement of the ST segment on electrocardiograms. Thus, displacement of the ST segment (either as an elevation or as a depression) may be considered to be an index of ischemic lesion. ST segment displacement during ischemia is due to “injury currents” that form between ischemic and healthy areas during both systole and diastole. This has been confirmed by magnetocardiography, a technique that records variations in magnetic fields produced by cardiac ionic currents (Cohen and Kauffmann, 1975). An ischemic cell shows a drop in amplitude, a change in action potential plateau and a decrease in resting potential when compared to a healthy cell. These facts can account for the presence of systolic and diastolic “injury currents.” Is it possible that factors other than ischemia affect the ST segment? It is known that changes in pH levels, in the concentration of electrolytes in intra or extracellular media, changes in tem-
TABLE 1
Mean ST Elevation Expressed in mV. GROUP
X
TIME
N Control Treated
12 12
BEFORE
0.00 +- 0.00 0.00 k 0.00 p=l
2 MIN 2.86 2 0.24 2.53 2 0.16 p = 0.41 N.S.
4
MIN
3.71 2 0.36 2.59 t 0.18 p = 0.007 S.
6
MIN
4.09
” 0.50 3.01 k 0.18 p = 0.009 s.
8
MIN
4.21 2 0.43 3.19 k 0.30 p = 0.014 S.
INTERACTION
p = 0.017
Group effect
49
50
P. d’Alch6
et al.
perature
and the influence
segment. variation
A reduction in coronary perfusion pressure produces of the ST segment (Maroko et al., 19711, but all these
known
to be associated
resulting
of the cardiac
with ischemia.
from the enlargement
sympathetic
Another
all have an effect
of the QRS complex
in the
disturbances
critical factor is secondary
a phenomenon known as the QRS-ST-T gradient If such conduction disorders exist, the experiments
on the ST
a change
due to conduction
are
ST change disorders,
effect (Massie and Walsh, 1960). are not taken into consideration.
In spite of these restrictions the measuring STvariations remains the basic technique for assessing ischemia both experimentally and clinically. The measurement of ST variation produced by SATAPEC is in fact the combination of TQ depression and ST elevation or, in other words, the variation measured from the baseline (and not from the true zero line, which is unknown). To determine the zero
line and thereby
evaluate
the exact role of the diastolic
and systolic injury
currents, it is necessary to use amplifiers with a direct current (DC) input. Given the large number of electrodes (240), the use of DC amplifiers would cause baseline stability
problems
that would
be very difficult
are made over short time intervals. to determine the zero line. The ST measurements
were
In order
only taken
to overcome
when
to assess &hernia,
during
measurements
it is not necessary
the first 8 min of ligation
as most
major biochemical changes in intra or extracellular media occur as occlusion time goes beyond 10 min. Schomig et al. (19841, for example, have observed that the level of plasmatic noradrenaline rises if occlusion time exceeds 10 min. In addition, Rakita et al. (1954) have shown that the amplitude of ST variation increases within a few min of the occlusion of an artery and reaches a plateau within 5 to 7 min. Another problem concerns the location and the number of electrodes ensure a reliable assessment of ST variation. In animal experimentation, done
at the epicardial
level have proven
to be the most accurate
cordial thoracic derivations, many potentials an ischemic zone is completely surrounded heterogeneous conduction distort recorded plottings.
needed to recordings
method.
In pre-
are “cancelled out,” particularly when by a healthy zone. In the latter case,
media interposed between the heart and the thorax The number of epicardial electrodes has to be high be-
cause current density is at a maximum at the junction of ischemic and healthy zones, but decreases rapidly beyond this point. Generally an insufficient number of electrodes
is used in epicardial
plotting
of ST variations.
CONCLUSION With SATAPEC it is possible taneously, i.e., during a single siderable epicardial cantly
to record up to 256 unipolar electrograms systole. Automatic data processing results
simulin con-
time saving. The antiischemic properties of a drug are clearly revealed by potential maps, as shown with trimetazidine, which was able to signifi-
reduce
the extent
of the ischemic
lesion.
REFERENCES Bailey RH, Ladue JS, York graphic
changes
DJ (1944)
(local ventricular
Electrocardioischemia
and
injury)
produced
sion of a coronary
in the dog by temporary artery,
showing
occiu-
a new stage
Mapping of Drug-Induced in the evolution
of myocardial
infarction.
Am
Heart / 27:164.
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Cardiac Protection
Maroko AR, Kjekshus JK, Sobel BE, Watanabe T, Covell JW, Ross J, Braunwald E (1971) Factors influencing infarct size following experimental coronary artery occlusion. Circulation 43:67. Massie E, Walsh JJ (1960) Clinical vectorcardiography and electrocardiography. Yearbook Medical Publ Chicago. Pardee HE6 (1920) An electrocardiographic sign of coronary artery obstruction. Arch intern Med 26: 244-249.
Passeron J (1986) Efficacite de la trimetazidine dans I’angor d’effort stable de I’insuffisant coronarien chronique. La Presse Medicale 15:1775-1778. Rakita L, Borduas JL, Rothman S, Prinzmetal M (1954) Studies on the mechanism of ventricular activity. Early changes in the RS-T segment and QRS complex following acute coronary artery occlusion; experimental study and clinical applications. Am Heart / 48:351. Renaud JF (1988) Internal pH, Nat and Ca*+ regulation by trimetazidine during cardiac cell acidosis. Cardiovasc Drugs Therap 1:677-686. Schomig A, Dart AM, Dietz R, Mayer E, Kubler W (1984) Release of endogenous catecholamines in the ischemic myocardium of the Rat. Part A: Locally mediated release. Circ Research 55:689701.
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